International Doctoral School

Rubén Agregán Pérez

DOCTORAL DISSERTATION

“Seaweed extract effect on the quality of meat products”

Supervised by the PhD:

José Manuel Lorenzo Rodríguez, Daniel José Franco Ruiz and Francisco Javier Carballo García

Year: 2019

“International mention”

International Doctoral School

JOSÉ MANUEL LORENZO RODRÍGUEZ, Researcher and Head of the Area of Chromatography, DANIEL JOSÉ FRANCO RUIZ, Researcher, both from The Centro Tecnolóxico da Carne, and FRANCISCO JAVIER CARBALLO GARCÍA, Professor of the Area of Food Technology of the University of Vigo,

DECLARES that the present work, entitled “Seaweed extract effect on the quality of meat products”, submitted by Mr RUBÉN AGREGÁN PÉREZ, to obtain the title of Doctor, was carried out under their supervision in the PhD programme “Ciencia y Tecnología Agroalimentaria” and he accomplishes the requirements to be Doctor by the University of Vigo.

Ourense, 25 February 2019

The supervisors

José Manuel Lorenzo Rodríguez, PhD Daniel José Franco Ruiz, PhD

Francisco Javier Carballo García, PhD

ACKNOWLEDGMENTS

First, I would like to thank my directors, José Manuel Lorenzo Rodríguez, PhD, Daniel José Franco Ruiz, PhD and Francisco Javier Carballo García, PhD for all the knowledge provided to me and for all the advice I received, specially to José Manuel Lorenzo for guiding me during this stage of my professional career. I also want to express my gratitude to the Centro Tecnolóxico da Carne and its manager, Mr Miguel Fernández Rodríguez, for having allowed me to do my Doctoral Thesis in its facilities, and to INIA for granting me a predoctoral scholarship for that purpose.

Second, I would like to thank all my laboratory colleagues for all the assistance I received and their companionship during this time, especially my laboratory supervisor, Laura Purriños Pérez, PhD, for providing me with all the required materials and equipment needed.

Finally, I give thanks to my family, my parents Juan Carlos and Sara, and my brother Pablo, for their concern and support for me. I extend this gratitude to the rest of my family and friends.

ABSTRACT

This Docthoral Thesis tried to contribute knowledge in the field of natural antioxidants applied to meat and meat products. Three brown macro-algae, Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcata and two micro-algae, Chlorella vulgaris and Spirulina platensis were selected to carry out the experiments of this research work, whose aims were, on the one hand, the study of the antioxidant potential of the previous algae extracts and the evaluation of the inhbitory effect of the macro-algae extracts in an oily food matrix, such as the canola oil and in a meat product such as the pork liver paté, and on the other hand, the study of the efficiency of several extraction conditions to provide extracts with good antioxidant properties, in order to select one of the extracts, from the macro or micro algae chosen for this thesis, to prove its efficacy in delaying oxidation reactions in pork patties.

The macro-algae were analysed for their nutritional contribution before experimenting with their extracts. They showed a good nutritional profile, with high amount of PUFAs and proteins with a remarkable biological value. Then, the antioxidant potential of the aqueous extracts of the macro-algae was assessed and their phenolic compounds tentatively identified, higligting the F. vesiculosus extract, which showed the highest antioxidant activity, and the presence of as major phenolic compounds in the three extracts analysed.

The aqueous extracts showed to be efficient in delaying both primary and secondary oxidation of lipids when they were applied in canola oil under accelerated storage conditions. Nevertheless, this antioxidant power could not be seen in pork liver pâtés. This unexpected result could be caused by the absence of contact among the product and the oxygen.

The low amounts of phenolic compounds found in the aqueous extracts of the macroalgae led to make the decision to analyse several extraction conditions to improve the antioxidant properties of the extracts. The UAE along with water/ethanol (50:50 v/v) was found the one that provided the best results. Once tested in both the macro and micro-algae it was decided to select the extract from the alga F. vesiculosus to be applied in pork patties, since its antioxidant activity per amout of extract was the highest of the extracts tested.

The addition of 1000 ppm of the F. vesiculosus extract to the pork patties protected them against oxidative phenomena. However, this protection was limited, thus the use of this extract in commercial meat or meat products is unviable.

The most relevant results extracted from this thesis are that aqueous extracts of macroalgae successfully protected canola oil from lipid oxidation, and that the UAE along with water/ethanol (50:50 v/v) provided an extract capable of delaying oxidation reactions in pork patties, although in a limited way.

RESUMEN

Esta tesis doctoral trató de contribuir al conocimiento dentro del campo de los antioxidantes de origen natural con aplicación en alimentos, en concreto en carne y productos cárnicos. Para ello se seleccionaron tres algas pardas, Ascophyllum nodosum, Fucus vesiculosus y Bifurcaria bifurcata, muy extendidas en toda la costa gallega y en la europea atlántica en general, para llevar a cabo los experimentos que conformaron esta tesis, cuyos objetivos de forma resumida fueron los siguientes:

1 Estudio de la composición química de las macro-algas A. nodosum, F. vesiculosus y B. bifurcata con el fin de conocer su contribución nutricional como alimentos.

2 Estudio del potencial antioxidante de los extractos acuosos de las macro-algas anteriores.

3 Evaluación del efecto inhibitorio de los extractos acuosos anteriores en los procesos de oxidación, los cuales tienen lugar en aceite (canola) y en una matriz compleja de carne como paté de hígado.

4 Evaluación de varias condiciones de extracción con el fin de seleccionar la más eficiente para valorar el potencial antioxidante de los extractos de A. nodosum, F. vesiculosus and B. bifurcata y el de las micro-algas Chlorella vulgaris and Spirulina platensis.

5 Estudiar la capacidad de protección contra la oxidación en hamburguesas de cerdo ejercida por el extracto con el mayor potencial mostrado in vitro.

Antes de realizar cualquier experimento con los extractos, se llevó a cabo un estudio de la composición química de las algas con el objetivo de conocer su aporte como nutriente en la dieta. Después de este estudio previo, las algas se extrajeron con agua, dando extractos acuosos, que fueron empleados en los sucesivos experimentos. Estos extractos se caracterizaron composicional y funcionalmente, llevándose a cabo analíticas para determinar tanto su composición química como su potencial antioxidante.

Antes de usar los extractos sobre material cárnico, se hicieron pruebas sobre un aceite poliinsaturado como el aceite de canola, con el fin de evaluar la eficacia antioxidante de dichos extractos sobre este tipo de matrices. El aceite se sometió a condiciones de almacenamiento aceleradas para poder así valorar la acción antioxidante de los extractos en un tiempo relativamente reducido. A continuación, se procedió a elaborar un paté de hígado de cerdo con adición de los extractos sustituyendo parcialmente la grasa saturada por aceites vegetales (aceites de canola y girasol alto oleico). La finalidad de este experimento fue evaluar la capacidad de los extractos para frenar la oxidación tanto lipídica como proteica durante el almacenamiento, y adicionalmente mejorar el perfil lipídico del producto final, aumentando los ácidos grasos mono (AGMs) y poliinsaturados (AGPs).

Los resultados de los análisis llevados a cabo sobre los extractos acuosos revelaron un contenido fenólico total bastante inferior a lo teóricamente esperable, de modo que ante la posibilidad de mejorar este parámetro, el cual podría aumentar considerablemente la capacidad antioxidante de los extractos, se procedió a valorar varias condiciones de extracción, escogiendo la más adecuada y probándola sobre las algas A. nodosum, F. vesiculosus, B. bifurcata y sobre dos micro algas, C. vulgaris y S. platensis.

El extracto con mayor actividad antioxidante de los anteriores fue seleccionado y añadido a hamburguesas de cerdo para probar su capacidad de retrasar los procesos de oxidación lipídica y proteica a lo largo del almacenamiento.

Se llevaron a cabo un total de ocho experimentos, los cuales se pueden dividir en dos grandes grupos, uno donde el objeto de estudio fueron las algas y/o los extractos, y otro en donde lo fue la capacidad de los extractos para proteger varias matrices alimentarias (aceite de canola, paté de cerdo y hamburguesa de cerdo) del enranciamiento oxidativo, evaluando su idoneidad como antioxidantes.

El primer grupo de experimentos tuvieron el objetivo, por una parte, de determinar la composición química de la matriz del alga y de su respectivo extracto, y por otra, la de determinar la aptitud de los extractos para eliminar radicales libres. En las algas se realizaron analíticas, como contenido en proteína, en grasa, carbohidratos totales, cenizas, perfiles de ácidos grasos, aminoácidos y minerales, y en los extractos analíticas, como contenido en sólidos totales, proteína, carbohidratos y compuestos fenólicos totales, capacidad antioxidante mediante ensayos de ORAC; ABTS, DPPH y FRAP, y rendimiento de extracción según el experimento. Adicionalmente, se realizó un análisis dirigido al intento de caracterizar los extractos fenólicamente (únicamente aquellos acuosos) usando la técnica de cromatografía líquida-diodo array-ionización por electrospray-masas/masas, cuyas siglas en inglés son LC- DAD-ESI-MS/MS.

Las analíticas pertenecientes al segundo grupo de experimentos en los que se dividió la tesis comprendieron, según el experimento, ensayos para medir la oxidación de la grasa, como los índices de anisidina, peróxidos, TBARS y oxidación total (TOTOX), dienos conjugados, un ensayo para medir la oxidación proteica y ensayos microbiológicos (contaje en placa) para hacer un seguimiento de los principales grupos microbianos (viables totales, bacterias ácido- lácticas, Pseudomonas spp, mohos y levaduras) en el producto durante su almacenamiento. De acuerdo también con el experimento, los valores correspondientes a la oxidación del producto fueron complementados con una compilación de los compuestos volátiles más destacados originados durante el almacenamiento, tanto para el aceite de canola como para el paté y la hamburguesa de cerdo. Por otra parte, para estos dos últimos productos se llevaron a cabo analíticas destinadas a determinar la humedad, la proteína y la grasa (paté) y la humedad, la proteína, la grasa y la ceniza (hamburguesa). También se determinaron propiedades físico- químicas, como el pH y el color sobre ambas matrices. Adicionalmente, en el caso de la hamburguesa se efectuó un análisis sensorial para, por una parte, probar la posible influencia de los extractos sobre los atributos organolépticos olor y sabor en la hamburguesa cocinada, y por otra parte, para juzgar desde un punto de vista subjetivo los atributos olor, color y decoloración superficial durante el almacenamiento del producto.

La composiciones químicas de las algas presentaron resultados dispares. B. bifurcata mostró valores de humedad significativamente (P < 0.05) inferiores a A. nodosum y F. vesiculosus. Sin embargo, su contenido en lípidos fue superior. Por otra parte, la especie F. vesiculosus presentó contenidos considerablemente superiores de proteínas y carbohidratos pero inferiores de cenizas con respecto a las especies A. nodosum y B. bifurcata. El mineral potasio resultó ser el más elevado en las tres algas, seguido por el sodio y el calcio, coincidiendo en mayor o menor grado con otro estudios. En cuanto al contenido de aminoácidos, éste fue de 7,48, 11,90 y 7,32 g/100 g alga seca para las algas A. nodosum, F. vesiculosus y B. bifurcata, respectivamente. Por otra parte, el ratio aminoácidos esenciales/aminoácidos totales de las tres algas sugirió que más del 40% de los aminoácidos son esenciales, siendo la leucina el más abundante. Los scores de casi todos los aminoácidos esenciales en las tres algas fueron superiores a 100 cuando se compararon con el patrón proteico propuesto por la FAO/OMS/UNU en el año 2007, lo que demuestra el elevado valor biológico de las proteínas de las algas estudiadas.

En lo tocante al perfil de ácidos grasos, las tres algas presentaron valores dispares (P < 0.001), si bien, también presentaron similitudes como el contenido de AGPs, los cuales resultaron ser los más abundantes (48,19-46,91% del total de ácidos grasos analizados). Sin embargo, fue un AGM como el ácido oleico (C18:1n-9 cis) el más abundante, salvo en el alga B. bifurcata, donde lo fue el ácido palmítico (C16:0).

El ratio n-6/n-3 presentado por las algas, inferior a 10, las sitúa como un alimento con un perfil graso saludable según las recomendaciones de la Organización Mundial de la Salud (OMS). Después de obtener la información nutricional sobre las tres algas de estudio, se extrajeron usando agua por motivos relativos a la seguridad alimentaria. Los extractos así obtenidos fueron usados en posteriores experimentos, previo análisis de su composición química, contenido y perfil fenólico, y actividad antioxidante.

El contenido de proteína fue significativamente inferior en el extracto de A. nodosum (ANE) (26,08 g/100 g extracto) respecto al de F. vesiculosus (FVE) y B. bifurcata (BBE). Sin embargo su contenido en carbohidratos (63,60 g glucosa/100 g extracto) fue superior. Los extractos presentaron una relación inversa entre el contenido total de sólidos y el contenido fenólico total, sugiriéndose que hay una cantidad nada desdeñable de sustancias diferentes de polifenoles.

Respecto a la actividad antioxidante presentada por los extractos, el ensayo de DPPH reveló que el FVE exhibió la actividad más alta (135,3 μmol Trolox (TE)/g extracto). El contenido tan elevado de proteínas en este extracto pudo haber influido en el resultado. Por otra parte, el ensayo de ORAC mostró datos discordantes respecto al de DPPH, ya que en este caso fue el BBE el que presentó el valor de actividad antioxidante más alto (979,9 μmol TE/g extracto). Esta diferencia puede deberse a la distinta base química de los diferentes ensayos.

La aplicación de la técnica LC-DAD-ESI-MS/MS sobre los extractos reveló la presencia de una serie de sustancias que podrían ser de naturaleza fenólica. El cromatograma correspondiente al ANE presentó 22 picos pertenecientes a posibles compuestos fenólicos, el correspondiente al FVE 19 y al del BBE 18. Sin embargo, no todos los picos pudieron ser identificados.

Una vez conocidos ciertos parámetros de los extractos, como la composición química y la actividad antioxidante, junto con una ligera idea de su perfil fenólico, se procedió a usarlos en una matriz oleosa como el aceite de canola, con el fin de probar su capacidad para extender la vida útil de matrices alimentarias similares.

Se llevaron a cabo dos experimentos en aceite de canola. En uno se añadieron concentraciones crecientes de BBE (200, 400, 600, 800 y 1000 ppm), y en otro 500 ppm de los extractos de las tres algas de estudio, A. nodosum, F. vesiculosus y B. bifurcata. Los experimentos fueron conducidos a 60 °C durante 16 días. En el primer experimento, los aceites añadidos con BBE y BHT (usado como control positivo) mostraron una oxidación lipídica primaria menos marcada, medida mediante el valor de peróxido y el de dienos conjugados, encontrándose una correlación positiva entre estos dos parámetros de oxidación (r = 0,93; P < 0,01, n = 120). Por lo tanto, se puede decir que el BBE tiene un papel destacado a la hora de inhibir las reacciones iniciales de la oxidación lipídica. De la misma forma que para la oxidación primaria, los aceites con BBE y BHT exhibieron un mejor control de la oxidación secundaria, medida mediante el valor de anisidina, deduciéndose que el BBE es capaz de controlar eficazmente la oxidación secundaria.

Al final del almacenamiento del aceite se identificaron una serie de compuestos volátiles usando cromatografía de gases acoplada a espectrometría de masas (GC-MS). Estos compuestos resultaron ser cuatro aldehídos (hexanal, heptanal, octanal y 2-octenal), un alcano (hexano), un alqueno (1-octen-3-ol) y un benceno (p-xileno). Los aldehídos fueron el grupo más amplio y también el más interesante, ya que son responsables de una amplia gama de sabores y olores característicos de la autoxidación de lípidos. Estos presentaron valores significativamente (P < 0.05) más bajos al término de los 16 días de almacenamiento en las muestras de aceite con extracto de alga respecto a las del control negativo (aceite sin antioxidantes). Este resultado demuestra la efectividad de los extractos para inhibir la producción de olores desagradables como consecuencia de la oxidación de la fracción lipídica.

En el segundo experimento, donde se añadieron 500 ppm de extracto de cada una de las algas de estudio, también se observó una protección de la matriz oleosa contra la oxidación. De hecho, la oxidación primaria resultó más inhibida al usar los extractos que al usar el BHT.

Después de haber corroborado que los extractos acuosos de las algas frenan adecuadamente los fenómenos de autoxidación en el aceite de canola, se procedió al estudio de su comportamiento en una matriz cárnica como el paté de hígado de cerdo.

Los extractos se añadieron a una concentración de 500 ppm a la masa de un paté de cerdo con sustitución parcial de grasa del mismo animal por aceite de canola y girasol alto oleico, más ricos en AGMs y AGPs. El paté se almacenó 180 días a refrigeración. Durante este tiempo se percibió una razonable estabilidad del producto a la oxidación lipídica primaria. En cuanto a la oxidación secundaria, los valores de TBARS no fueron demasiado concluyentes. En general, el paté elaborado demostró ser un producto muy estable lipídicamente a lo largo del almacenamiento. Esta estabilidad podría ser debida, por una parte, a la ausencia de espacio de cabeza entre el producto y la cubierta de la lata, y por otra, al cerrado hermético, impidiendo se de estas dos maneras el contacto del oxígeno con el producto.

En cuanto a la estabilidad de las proteínas frente a la oxidación, se observó un ligero incremento de los carbonilos a los 180 días de almacenamiento, percibiéndose un efecto protector de los extractos sobre la matriz proteica del paté.

Durante el almacenamiento se detectaron una serie de compuestos volátiles (30), que comprendieron los grupos de compuestos, hidrocarburos (20), ésteres (2), alcoholes (2), aldehídos (4) y cetonas (2). Los hidrocarburos fue el grupo químico más abundante tanto al comienzo del almacenamiento, representando entre el 87,5 y el 96,3% del total de los compuestos, como al final del mismo, variando entre el 83,9 y el 86,9%. El heptano, 2,2,4,6,6- pentamethyl fue el hidrocarburo de mayor concentración. El segundo grupo de compuestos mayoritarios fue el de los aldehídos, aunque a mucha distancia del primero, donde el hexanal fue el representante principal.

Las demás familias de compuestos volátiles representaron un porcentaje minoritario sobre el total.

Después de los 180 días de almacenamiento, los compuestos volátiles totales descendieron, atribuyéndose tal evento a la brusca reducción del compuesto heptano, 2,2,4,6,6- pentamethyl.

En lo referente a la composición química del paté, la adición de los extractos no provocó cambios ni en la humedad ni en el contenido de grasa. Por el contrario, la proteína sí parece haber sido afectada por la inclusión de extracto en la formulación.

Debido al bajo contenido de compuestos fenólicos encontrados en los extractos acuosos de las algas A. nodosum, F. vesiculosus y B. bifurcata usados en los anteriores experimentos, se decidió llevar a cabo un cribado de varias condiciones de extracción en el alga B. bifurcata, con el fin de seleccionar la que proporcionase una buena cantidad de compuestos fenólicos con una razonablemente elevada capacidad antioxidante, así como con un alto rendimiento de extracción.

Las condiciones de extracción fueron las siguientes:

 Método de extracción térmico a 121 °C y a una presión de 20 psi usando agua y agua/etanol (50:50 v/v) como disolventes. El tiempo de extracción fue de 30 minutos.  Método de extracción mediante la aplicación de ultrasonidos usando agua, etanol y agua/etanol (50:50 v/v) como disolventes. El tiempo de extracción fue de 30 minutos.

 Método de extracción mediante la aplicación de un 90 % de CO2 supercrítico (presión

de 35 MPa y temperatura de 40 °C) con un caudal de 25 g/CO2 min y un 10% de etanol. Los tiempos de extracción fueron de 30, 45 y 60 minutos.

El primer resultado salientable después de realizar las distintas extracciones, fue que el método de extracción con CO2 supercrítico (SC-CO2) proporcionó con mucho los peores resultados en cuanto a rendimiento y compuestos fenólicos, hecho que motivó el descarte de estos extractos para el análisis de su actividad antioxidante. La escasa extracción de compuestos fenólicos pudo tener su origen en la baja polaridad del CO2 bajo condiciones supercríticas. Otro dato interesante fue que las condiciones de extracción que proporcionaron un mayor rendimiento también proporcionaron extractos con un mayor contenido de compuestos de naturaleza fenólica. Estas condiciones fueron las que incluyeron como solvente agua/etanol (50:50 v/v). Por lo tanto, existe la posibilidad de que un incremento en el rendimiento de extracción favorezca un aumento de la liberación de compuestos fenólicos de la matriz del alga.

En lo referente a la actividad antioxidante, se destaca que la combinación de agua/etanol (50:50 v/v) y la aplicación de energía adicional (calor/presión o ultrasonidos) proporcionaron los mejores resultados.

Parece que los datos correspondientes al contenido fenólico siguen una tendencia correlativa con la actividad antioxidante, si bien, estos datos no son significativamente concluyentes como para poder aseverar dicha afirmación. Sin embargo, estudios previos apoyan esta hipótesis, sugiriendo que los polifenoles extraídos son unos de los mayores contribuidores de la capacidad antioxidante.

La condición de extracción que obtuvo los resultados más satisfactorios fue la que incluyó la extracción asistida con ultrasonidos (UAE) como método de extracción y el agua/etanol (50:50 v/v) como solvente, dando lugar a un rendimiento de 39,11 ± 1,54 g extracto/100 alga seca, un contenido fenólico de 5,46 ± 0,34 g equivalentes de floroglucinol (PGE)/100 g alga seca y una actividad antioxidante de 552,24 ± 72,81 µmol TE/g alga seca. Esta condición de extracción se aplicó a las macro-algas A. nodosum, F. vesiculosus y B. bifurcata y a dos micro-algas, S. platensis y C. vulgaris, evaluándose los mismos parámetros que en el experimento anterior, rendimiento de extracción, contenido fenólico total y actividad antioxidante.

Al igual que sucedió al comparar diferentes condiciones de extracción, las algas que proporcionaron un mayor rendimiento dieron los extractos más ricos en compuestos fenólicos. Fue el alga B. bifurcata la que maximizó estos dos parámetros, consiguiendo un rendimiento de extracción de 35,85 g extracto/100 g alga seca, y proporcionando un extracto con un contenido fenólico de 5,74 g PGE/100 g alga seca. Por otra parte, las micro-algas mostraron unos niveles muy pobres tanto de rendimiento en el proceso de extracción como de compuestos fenólicos en los extractos. El escaso rendimiento de extracción podría achacarse a los altos niveles de carotenoides que presentan las micro-algas, ya que la apolaridad de estos compuestos hace difícil su interacción con solventes polares como el usado (agua/etanol). Los florotaninos pudieron ser el motivo de el mayor contenido fenólico de los extractos de macro- algas. La actividad antioxidante aumentó a medida que lo hizo el contenido fenólico.

El verdadero valor de la actividad antioxidante de los extractos viene condicionado por el rendimiento de extracción de las algas. De acuerdo con esto, el extracto de F. vesiculosus obtuvo los mejores resultados de actividad, a pesar de que nuestros datos muestren que lo fue el de B. bifurcata. Esto es debido a que nuestros datos fueron expresados por cantidad de alga. El incorporar extractos con alta capacidad antioxidante a los alimentos, además de la protección inherente que aportan, presentan la ventaja de reducir posibles problemas relacionados con la adición de sabores u olores anómalos, ya que cuanto mayor es la actividad de un extracto menor es la cantidad a añadir al producto para obtener una determinada protección. Llegados a este punto, se decidió probar el extracto de F. vesiculosus sobre una matriz cárnica como la hamburguesa de cerdo para conocer su capacidad real de retrasar los fenómenos oxidativos.

El extracto incorporado a una concentración de 1000 ppm presentó una mayor capacidad de protección contra los fenómenos oxidativos que el incorporado a 250 y 500 ppm. En general, se puede argumentar que el contenido fenólico del FVE probablemente protegió las hamburguesas del estrés oxidativo originado durante el almacenamiento, retrasando la aparición de productos de degradación.

El perfil lipídico de la hamburguesa reveló una riqueza en AGPs (25,74% del total de ácidos grasos analizados). Este resultado probablemente se debió a la sustitución parcial de la grasa por un oleogel de linaza. Los AGMs, con un porcentaje del 41,10% del total, fueron los ácidos grasos más abundantes, con el oleico como ácido graso más representativo, seguidos por los AGSs con un 31,95%.

En cuanto al color de las hamburguesas durante el almacenamiento, se observó una desviación progresiva del tono rojizo hacia uno marronáceo. Fenómeno reportado en otros estudios con carne en la bibliografía.

Los resultados obtenidos para la oxidación mediante los métodos analíticos se contrastaron con criterios subjetivos. Para ello, se llevó a cabo una evaluación sensorial, que se hizo extensiva para muestras cocinadas a tiempo cero. En concreto, se realizaron test de aceptación tanto para las muestras crudas durante el periodo de almacenamiento como para las cocinadas a tiempo inicial, y otro a mayores de preferencia para estas últimas. El panel de cata estuvo formado por dieciséis miembros pertenecientes al personal del Centro Tecnológico de la Carne (Ourense, España). Los resultados obtenidos no mostraron diferencias significativas (P < 0.05) entre los distintos lotes cocinados a tiempo inicial para los atributos olor y sabor. La misma conclusión se obtuvo para los atributos sensoriales olor, color y decoloración superficial a lo largo del almacenamiento, con lo que se puede concluir que el FVE no mejoró a las concentraciones añadidas ninguno los atributos sensoriales estudiados.

Las conclusiones alcanzadas en este trabajo de investigación fueron de forma resumida las siguientes:

1 Las algas mostraron un perfil lipídico saludable, siendo los AGPs los ácidos grasos más abundantes. De la misma forma mostraron un poseer proteínas con un alto valor biológico. 2 El extracto acuoso de F. vesiculosus mostró de forma general una mayor capacidad antioxidante que los extractos acuosos de A. nodosum y B. bifurcata. 3 Los extractos acuosos de las macro-algas protegieron de la autooxidación lipídica una matriz oleosa como es el aceite de canola. Sin embargo, esta protección apenas pudo ser vista en un producto cárnico como e el paté de hígado de cerdo, debido a la elevada estabilidad de éste a la oxidación. 4 El uso de ultrasonidos junto con una mezcla de agua/etanol (50:50 v/v) demostró ser la condición de extracción más idónea para extraer el alga B. bifurcata, proporcionando un extracto con un elevado potencial antioxidante. 5 El extracto de F. vesiculosus obtenido bajo la condición de extracción anterior proporcionó mejores propiedades antioxidantes que el resto de macro-algas (A. nodosum y B. bifurcata) y micro-algas (C. vulgaris y S. platensis) extraídas. 6 El extracto de F. vesiculosus mostró una protección limitada contra la oxidación en hamburgesas de cerdo, lo que hace inviable su uso en carne o productos cárnicos.

General index

GENERAL INDEX

I. INTRODUCTION ...... 1 I.1. THE ROLE OF MEAT THROUGH HISTORY OF MEN ...... 3 I.2. DEFINITION AND CHEMICAL COMPOSITION OF MEAT ...... 4 I.2.1. FACTORS AFECTING THE CHEMICAL COMPOSITION OF MEAT...... 4 I.2.2. CHEMICAL COMPONENTS OF MEAT ...... 6 I.2.2.1. Moisture (water) ...... 6 I.2.2.2. Proteins ...... 6 I.2.2.3. Lipids ...... 8 I.2.2.4. Carbohydrates ...... 10 I.2.2.5. Minerals ...... 10 I.3. COLOUR AND PIGMENTS IN MEAT ...... 11 I.3.1. MYOGLOBIN ...... 11 I.3.1.1. Lipid oxidation-induced myoglobin oxidation ...... 13 I.3.2. PIGMENTS IN FRESH MEATS ...... 13 I.3.2.1. Myoglobin redox forms in packaged fresh meats ...... 13 I.3.2.2. Cytochrome c ...... 14 I.3.2.3. Sulfmyoglobin ...... 14 I.3.2.4. Acid ferrimyoglobin peroxide ...... 15 I.3.2.5. Ferrimyoglobin peroxide ...... 15 I.3.2.6. Ferrocholemyoglobin ...... 15 I.3.3. PIGMENTS IN COOKED MEATS ...... 15 I.3.4. PIGMENTS IN CURED MEATS ...... 16 I.4. OXIDATION IN MEAT ...... 16 I.4.1. LIPID OXIDATION IN MEAT ...... 17 I.4.1.1. Non-enzymatic oxidation of lipids. “Autoxidation” ...... 17 I.4.1.2. Enzymatic oxidation of lipids ...... 18 I.4.2. PROTEIN OXIDATION IN MEAT ...... 19 I.4.3. PRODUCTS RESULTING FROM LIPID AND PROTEIN OXIDATION. EFFECTS IN HUMAN HEALTH ...... 20 I.4.3.1. Lipid oxidation products ...... 20 I.4.3.2. Protein oxidation products ...... 21

i General index

I.5. USE OF ANTIOXIDANTS TO PREVENT OXIDATION IN MEAT ...... 22 I.5.1. SYNTHETIC ANTIOXIDANTS ...... 23 I.5.2. ANTIOXIDANTS FROM NATURAL ORIGIN ...... 24 I.5.2.1. Hydrophobic antioxidants ...... 25 I.5.2.2. Hydrophilic antioxidants ...... 25 I.5.3. NATURAL ANTIOXIDANTS IN FOODS ...... 28 I.5.4. MARINE ALGAE AS A NOVEL SOURCE OF NATURAL ANTIOXIDANTS ...... 29 I.5.5. ADDITION OF MARINE ALGAE AND THEIR EXTRACTS TO FOOD AS ANTIOXIDANTS ...... 32 I.6. ANTIMICROBIAL PROPERTIES OF MARINE ALGAE ...... 33

II. JUSTIFICATION AND OBJECTIVES ...... 35

III. MATERIAL AND METHODS ...... 39 III.1. ALGAL MATERIAL ...... 41 III.1.1. ORIGIN OF ALGAE ...... 41 III.1.2. PREPARATION OF MACRO-ALGAE...... 41 III.1.3. PROXIMATE AND NUTRITIONAL COMPOSITION OF MACRO-ALGAE ...... 41 III.1.3.1. Experimental design ...... 41 III.1.3.2. Analytical methods ...... 41 III.1.3.2.1. Moisture content ...... 41 III.1.3.2.2. Protein content ...... 42 III.1.3.2.3. Ash content ...... 42 III.1.3.2.4. Lipid content ...... 42 III.1.3.2.5. Amino acid content ...... 42 III.1.3.2.6. Protein quality: Chemical score of amino acids ...... 43 III.1.3.2.7. Fatty acid profile ...... 43 III.1.3.2.8. Mineral profile ...... 44 III.1.4. ALGAE EXTRACTION ...... 44 III.1.4.1. Green extraction ...... 44 III.1.4.2. Other alternative extraction technologies ...... 44 III.1.4.2.1. Hydrothermal and ultrasound-assisted extraction ...... 45

III.1.4.2.2. Extraction technology with supercritical CO2...... 45 III.1.5. ALGAE EXTRACT CHARACTERIZATION ...... 46 III.1.5.1. Extracts from green extraction ...... 46

ii General index

III.1.5.1.1. Experimental design...... 46 III.1.5.1.2. Analytical methods ...... 46 III.1.5.1.2.1. Total solid content ...... 46 III.1.5.1.2.2. Protein content ...... 46 III.1.5.1.2.3. Total carbohydrate content ...... 46 III.1.5.1.2.4. Total phenolic content ...... 47 III.1.5.1.2.5. Antioxidant activity ...... 47 III.1.5.1.2.6. Phenolic profile ...... 49 III.1.5.2. Extracts from alternative extractions (hydrothermal, ultrasound-assisted

extraction and supercritical CO2) ...... 49 III.1.5.2.1. Experimental design ...... 49 III.1.5.2.2. Analytical methods ...... 50 III.1.5.2.2.1. Extraction yield ...... 50 III.1.5.2.2.2. Total phenolic content ...... 50 III.1.5.2.2.3. Antioxidant activity ...... 50 III.2. KINETICS OF CANOLA OIL WITH MACRO-ALGAE EXTRACT ADDITION ...... 50 III.2.1. KINETIC OF CANOLA OIL WITH BIFURCARIA BIFURCATA EXTRACT ADDED AT INCREASING CONCENTRATIONS ...... 50 III.2.1.1. Experimental design ...... 50 III.2.1.2. Procedure for obtaining the samples ...... 51 III.2.2. KINETIC OF CANOLA OIL WITH ASCOPHYLLUM NODOSUM, FUCUS VESICULOSUS AND BIFURCARIA BIFURCATA EXTRACT ADDITION ...... 51 III.2.2.1. Experimental design ...... 51 III.2.2.2. Procedure for obtaining the samples ...... 51 III.2.3. ANALYTICAL METHODS ...... 52 III.2.3.1. Determination of lipid oxidation...... 52 III.2.3.1.1. P-anisidine value ...... 52 III.2.3.1.2. Peroxide value ...... 52 III.2.3.1.3. Total oxidation value ...... 52 III.2.3.1.4. Conjugated dienes ...... 53 III.2.3.1.5. Thiobarbituric acid-reactive substances ...... 53 III.2.3.2. Inhibition of oil oxidation ...... 53 III.2.3.3. Analysis of volatile compounds ...... 54 III.3. KINETICS OF MEAT PRODUCTS WITH MACRO-ALGAE EXTRACT ADDITION ...... 55

iii General index

III.3.1. KINETIC OF A PORK LIVER PÂTÉ WITH ASCOPHYLLUM NODOSUM, FUCUS VESICULOSUS AND BIFURCARIA BIFURCATA EXTRACTS ADDITION AND PARTIAL FAT REPLACEMENT BY SEED OILS ...... 55 III.3.1.1. Experimental design ...... 55 III.3.1.2. Procedure for obtaining the samples ...... 55 III.3.2. KINETIC OF PORK PATTIES WITH FUCUS VESICULOSUS EXTRACT ADDITION AND PARTIAL FAT REPLACEMENT BY LINSEED OIL OLEOGEL...... 56 III.3.2.1. Experimental design ...... 56 III.3.2.2. Linseed oil oleogel obtaining ...... 56 III.3.2.3. Procedure for obtaining the samples ...... 57 III.3.3. ANALYTICAL METHODS ...... 58 III.3.3.1. Proximate composition ...... 58 III.3.3.2. Fatty acid profile ...... 58 III.3.3.3. Microbial analysis ...... 58 III.3.3.4. Physicochemical parameters (colour and pH) ...... 59 III.3.3.5. Determination of lipid oxidation (conjugated dienes and thiobarbituric acid- reactive substances) ...... 59 III.3.3.6. Determination of protein oxidation ...... 60 III.3.3.7. Analysis of volatile compounds ...... 60 III.3.3.8. Sensory evaluation of raw and cooked pork patties ...... 61 III.4. STATISTICAL ANALYSIS ...... 61

IV. RESULTS AND DISCUSSION ...... 63 IV.1. PROXIMATE AND NUTRITIONAL COMPOSITION OF MACRO-ALGAE ASCOPHYLLUM NODOSUM, FUCUS VESICULOSUS AND BIFURCARIA BIFURCATA...... 65 IV.1.1. PROXIMATE COMPOSITION OF MACRO-ALGAE ...... 65 IV.1.2. MINERAL CONTENT OF MACRO-ALGAE ...... 66 IV.1.3. AMINO ACID CONTENT OF MACRO-ALGAE ...... 67 IV.1.3.1. Nutritional quality of protein...... 68 IV.1.4. FATTY ACID PROFILE OF MACRO-ALGAE ...... 69 IV.2. PROXIMATE COMPOSITION, ANTIOXIDANT ACTIVITY AND PHENOLIC CHARACTERIZATION OF AQUEOUS EXTRACTS FROM MACRO-ALGAE ASCOPHYLLUM NODOSUM, FUCUS VESICULOSUS AND BIFURCARIA BIFURCATA...... 70 IV.2.1. PROXIMATE COMPOSITION, TOTAL SOLID CONTENT AND TOTAL PHENOLIC CONTENT OF EXTRACTS ...... 71 iv General index

IV.2.2. ANTIOXIDANT ACTIVITY OF EXTRACTS ...... 72 IV.2.3. PHENOLIC CHARACTERIZATION OF EXTRACTS ...... 73 IV.2.3.1. Tentative identification of phenolic compounds ...... 73 IV.2.3.2. Non identified peaks ...... 75 IV.3. STUDY OF THE ANTIOXIDANT EFFECT OF ASCOPHYLLUM NODOSUM, FUCUS VESICULOSUS AND BIFURCARIA BIFURCATA AQUEOUS EXTRACTS IN CANOLA OIL...... 79 IV.3.1. BIFURCARIA BIFURCATA EXTRACT ADDED AT INCREASING CONCENTRATIONS TO CANOLA OIL ...... 79 IV.3.1.1. Evolution of lipid oxidation ...... 79 IV.3.1.2. Volatile compounds formed during lipid oxidation ...... 82 IV.3.2. ADDITION OF ASCOPHYLLUM NODOSUM, FUCUS VESICULOSUS AND BIFURCARIA BIFURCATA EXTRACTS TO CANOLA OIL ...... 83 IV.3.2.1. Evolution of lipid oxidation ...... 83 IV.4. STUDY OF THE EFFECT OF ASCOPHYLLUM NODOSUM, FUCUS VESICULOSUS AND BIFURCARIA BIFURCATA AQUEOUS EXTRACTS IN THE SHELF-LIFE OF A LOW-FAT PORK LIVER PÂTÉ ...... 86 IV.4.1. PROXIMATE COMPOSITION AND FATTY ACID PROFILE OF PÂTÉ ...... 86 IV.4.2. EVOLUTION OF MICROBIAL COUNTS IN PÂTÉ ...... 87 IV.4.3. EVOLUTION OF PHYSICAL PROPERTIES (pH AND COLOUR) OF PÂTÉ ...... 88 IV.4.4. EVOLUTION OF LIPID AND PROTEIN OXIDATION IN PÂTÉ ...... 89 IV.4.5. VOLATILE COMPOUNDS FORMED IN PÂTÉ DURING STORAGE ...... 91 IV.5. EVALUATION OF DIFFERENT EXTRACTION CONDITIONS IN MACRO-ALGA BIFURCARIA BIFURCATA ...... 93 IV.5.1. EXTRACTION YIELD AND TOTAL PHENOLIC CONTENT OF DIFFERENT BIFURCARIA BIFURCATA EXTRACTS ...... 93 IV.5.2. ANTIOXIDANT ACTIVITY OF DIFFERENT BIFURCARIA BIFURCATA EXTRACTS ...... 97 IV.6. ANTIOXIDANT POTENTIAL OF EXTRACTS FROM MACRO-ALGAE ASCOPHYLLUM NODOSUM, FUCUS VESICULOSUS AND BIFURCARIA BIFURCATA, AND MICRO-ALGAE CHLORELLA VULGARIS AND SPIRULINA PLATENSIS OBTAINED BY ULTRASOUND-ASSISTED EXTRACTION ...... 97 IV.6.1. EXTRACTION YIELD AND TOTAL PHENOLIC CONTENT OF ALGAE EXTRACTS ...... 99 IV.6.2. ANTIOXIDANT ACTIVITY OF ALGAE EXTRACTS ...... 99 IV.7. STUDY OF THE EFFECT OF FUCUS VESICULOSUS EXTRACT IN THE SHELF-LIFE OF PORK PATTIES FORMULATED WITH LINSEED OLEOGEL ...... 100 IV.7.1. PROXIMATE COMPOSITION AND FATTY ACID PROFILE OF PORK PATTIES ...... 100

v General index

IV.7.2. EVOLUTION OF PHYSICAL PROPERTIES (pH AND COLOUR) OF PORK PATTIES ... 102 IV.7.3. EVOLUTION OF LIPID AND PROTEIN OXIDATION IN PORK PATTIES ...... 105 IV.7.4. SENSORY PROPERTIES OF PORK PATTIES ...... 106

V. CONCLUSIONS ...... 109

VI. REFERENCES ...... 113

VII. ANEXES ...... 131 VII.1. PUBLICATIONS THAT INCLUDE THE RESULTS OF THIS DOCTORAL THESIS ...... 133 VII.2. CRITERIA OF QUALITY OF THE JOURNALS WHERE THE RESULTS OF THE PRESENT DOCTORAL THESIS HAVE BEEN PUBLISHED ...... 135 VII.2.1. PUBLICATION Nº 1 ...... 135 VII.2.2. PUBLICATION Nº 2 ...... 135 VII.2.3. PUBLICATION Nº 3 ...... 136 VII.2.4. PUBLICATION Nº 4 ...... 136 VII.2.5. PUBLICATION Nº 5 ...... 136 VII.2.6. PUBLICATION Nº 6 ...... 137 VII.2.7. PUBLICATION Nº 7 ...... 137

vi Table index

TABLE INDEX

Table I.1. Variation of the chemical composition (%) of animal carcasses from birth to maturity age ...... 5 Table I.2. Carcass and skeletal muscle tissue composition of different raw meats ...... 5 Table III.1. Extraction conditions used with each alga species ...... 45 Table III.2. Ingredients and amounts used for the preparation of pork liver pâtés ...... 56 Table III.3. Ingredients and amounts used for the preparation of pork patties ...... 57 Table IV.1. Proximate composition of seaweeds studied (mean ± standard deviation value) (n = 5) ...... 65 Table IV.2. Mineral profile of seaweeds studied (mean ± standard deviation value) (n = 5) .... 66 Table IV.3. Amino acid profile of the three seaweeds studied (mean ± standard deviation value) (n = 5) ...... 67 Table IV.4. Nutritional quality of protein for seaweeds studied ...... 68 Table IV.5. Fatty acid profile of seaweeds studied (mean ± standard deviation value) ...... 69 Table IV.6. Proximate composition, total solid content, total content and in vitro antioxidant activity determined by ABTS radical cation decoloration, DPPH free radical scavenging activity, ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) of A. nodosum, B. bifurcata, F. vesiculosus aqueous extracts and BHT compound (mean ± standard deviation value) (n = 3) ...... 71 Table IV.7. Phenolic compounds in A. nodosum, B. bifurcata and F. vesiculosus extracts using LC-DAD-ESI-MS/MS ...... 76 Table IV.8. Features of the non-identified compounds in A. nodosum, B. bifurcata and F. vesiculosus extracts using LC-DAD-ESI-MS/MS ...... 78 Table IV.9. Effect of seaweed extracts on proximate composition and fatty acid profile of low- fat pork liver pâtés (mean ± standard deviation value) (n = 9) ...... 87 Table IV.10. Summary of extraction conditions and their nomenclature, as well as the values of extraction yield, total phenolic content and antioxidant activity of B. bifurcata extracts obtained (mean ± standard deviation value) (n = 2) ...... 96 Table IV.11. Extraction yield, TPC and antioxidant activity of A. nodosum, F. vesiculosus, B. bifurcata, C. vulgaris and S. platensis extracts obtained by UAE method using water/ethanol (50:50, v:v) as solvent...... 98 Table IV.12. Effect of F. vesiculosus extract on proximate composition and fatty acid profile of pork patties on day 0 (mean ± standard deviation value) (n = 4) ...... 101

vii Table index

Table IV.13. Effect of F. vesiculosus extract on evolution of pH and colour parameters (L*, a* and b*) of pork patties during refrigerated storage (mean ± standard deviation value) (n = 4) ...... 103 Table IV.14. Effect of F. vesiculosus extract on TBARS evolution during refrigerated storage (mean ± standard deviation value) (n = 4) ...... 105 Table IV.15. Effect of F. vesiculosus extract on protein oxidation evolution during refrigerated storage (mean ± standard deviation value) (n = 4) ...... 106

viii Figure index

FIGURE INDEX

Figure I.1. Formation of a peptide bond ...... 7 Figure I.2. Formation of a triacylglycerol ...... 8 Figure I.3. General structure of glycogen...... 10 Figure I.4. The myoglobin molecule structure is shown on the left. The heme group is shown in blue with the iron atom in orange. The heme group structure is shown on the right ...... 12 Figure I.5. Different derived myoglobin pigments and their corresponding conversions in packaged fresh meats ...... 14 Figure I.6. General scheme of the different phases in non-enzymatic lipid oxidation ...... 18 Figure I.7. Formation of 13(S)13-hydroperoxyoctadeca-9, 11-dienoate (9Z, 11E) (13-(E, Z)- HPODE) by the action of lipoxygenase ...... 19 Figure I.8. Free radical-mediated pathway of protein oxidation ...... 20 Figure I.9. Chemical structures of commercial synthetic antioxidants. BHA (A), BHT (B), TBHQ (C) and PG (D) ...... 24 Figure I.10. Examples of a brown (Bifurcaria bifurcata-A), green (Ulva rigida-B) and red (Cryptopleura ramosa-C) seaweed ...... 31 Figure I.11. group structures. Phloretol (A), fuhalol (B), fucol (C), (D) and carmalol (E) ...... 32 Figure IV.1. Representative chromatogram of phenolic compounds from A. nodosum extract obtained by LC-DAD ...... 73 Figure IV.2. Representative chromatogram of phenolic compounds from B. bifurcata extract obtained by LC-DAD ...... 74 Figure IV.3. Representative chromatogram of phenolic compounds from F. vesiculosus extract obtained by LC-DAD ...... 74 Figure IV.4. Effect of the addition of B. bifurcata aqueous extract at the concentrations of 200, 400, 600, 800 and 1000 ppm and BHT at 50 and 200 ppm on the evolution of peroxide values (A), p-anisidine values (B), conjugated dienes (C) and TOTOX values (D) in canola oil stored at 60 °C for 16 days. Plotted values are means ± standard deviations of six determinations ...... 81 Figure IV.5. Inhibitory effect of the B. bifurcata aqueous extract at the concentrations of 200, 400, 600, 800 and 1000 ppm on the peroxide values (A), p-anisidine values (B), conjugated dienes (C) and TOTOX values (D) in canola oil stored at 60 °C for 16 days ...... 81 Figure IV.6. Effect of the addition of B. bifurcata aqueous extract at the concentrations of 200, 400, 600, 800 and 1000 ppm and BHT at 50 and 200 ppm on the content of hexanal (A),

ix Figure index heptanal (B), octanal (C) and 2-Octenal (D) in canola oil after 16 days of storage at 60 °C. Plotted values are means ± standard deviations of six determinations. A-BMeans not followed by a common letter are significantly different (P < 0.05; Duncan test) ...... 82 Figure IV.7. Effect of the addition of A. nodosum, F. vesiculosus and B. bifurcata aqueous extracts at the concentration of 500 ppm and BHT at 50 ppm on the evolution of peroxide values (A), p-anisidine values (B), TOTOX values (C), TBARS values (D) and conjugated dienes (E) in canola oil stored at 60 °C. Plotted values are means ± standard deviations of six determinations. a-eMeans in the same oil treatment not followed by a common letter are significantly different (P < 0.05; Duncan test) (differences among sampling points). 1-3Means in the same sampling point not followed by a common number are significantly different (P < 0.05; Duncan test) (differences among treatments) ...... 84 Figure IV.8. Evolution of pH during refrigerated storage of low-fat pork liver pâté manufactured with added BHT at a concentration of 50 ppm and natural antioxidant extracts at 500 ppm from seaweeds A. nodosum, F. vesiculosus and B. bifurcata. Plotted values are means ± standard deviations of nine replicates ...... 88 Figure IV.9. Evolution of luminosity (A), index of red (B) and index of yellow (C) during refrigerated storage of low-fat pork liver pâté manufactured with added BHT at a concentration of 50 ppm and natural antioxidant extracts at 500 ppm from seaweeds A. nodosum, F. vesiculosus and B. bifurcata. Plotted values are means ± standard deviations of nine replicates ...... 89 Figure IV.10. Evolution of conjugated dienes (A), TBARS values (B) and carbonyls (C) during refrigerated storage of low-fat pork liver pâté manufactured with added BHT at a concentration of 50 ppm and natural antioxidant extracts at 500 ppm from seaweeds A. nodosum, F. vesiculosus and B. bifurcata. Plotted values are means ± standard deviations of nine replicates. a-cMeans in the same oil treatment not followed by a common letter are significantly different (P < 0.05; Duncan test) (differences among sampling points) ...... 90 Figure IV.11. Changes in percentage of chemical families of volatiles and in total content of volatiles during storage of low-fat pork liver pâté manufactured with added BHT at a concentration of 50 ppm and natural antioxidant extracts at 500 ppm from seaweeds A. nodosum, F. vesiculosus and B. bifurcata. Plotted values are means ± standard deviations of nine replicates ...... 92 Figure IV.12. Average sensory scores for pork patties with different concentrations of F. vesiculosus extract. Hedonic scale used: 1 = excellent; 2 = good; 3 = acceptable; 4 = hardly acceptable; 5 = not acceptable ...... 107

x Figure index

Figure IV.13. Evolution of odour, discoloration at surface and taste attributes in raw pork patties with different concentrations of F. vesiculosus extract during refrigerated storage. Hedonic scale used: 1 = excellent; 2 = good; 3 = acceptable; 4 = hardly acceptable; 5 = not acceptable ...... 108

xi

Acronyms

ACRONYMS

[M-H]-: Formation of a peptide bond AAPH: 2,20-Azobis (2-methylpropionamidine) dihydrochloride ABTS: 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ACS: American Chemical Society ALE: Advanced lipid oxidation end product ANE: Ascophyllum nodosum extract ANOVA: Analysis of variance AOAC: Association of Official Agricultural Chemists ATP: Adenosine triphosphate AV: P-anisidine value BBE: Bifurcaria bifurcata extract BHA: Butylated hydroxyanisole BHT: Butylated hydroxytoluene BSA: Bovine serum albumin CD: Conjugated dienes CFU: Colony forming units CON: Control CS: Chemical score DAD: Diode array detector DNA: Deoxyribonucleic acid DNPH: Dinitrophenylhydrazine DPPH: 2,2-Diphenyl-1-picrylhydrazyl DVB/CAR/PDMS: Divinylbenzene/carboxen/polydimethylsiloxane DW: Dry weight of algae EAA: Essential amino acid EC: Efficient concentration ESI: Electrospray ionization eV: Electronvolt FA: Fatty acid FAME: Fatty acid methyl ester FAO: Food and Agriculture Organization FNB: Food and Nutrition Board

xiii Acronyms

FRAP: Ferric reducing antioxidant power FVE: Fucus vesiculosus extract GC: Gas chromatograph GC-MS: Gas chromatograph-mass spectrometer HIV: Human immunodeficiency virus ICP-OES: Inductively coupled plasma-optical emission spectroscopy IEAA: Essential amino acid index IOM: American Institute of Medicine IP: Inhibition percentage ISO: International Organization for Standardization LAB: Lactic acid bacteria LC-DAD-ESI-MS/MS: Liquid chromatography-diode array detection-negative electrospray ionization-tandem mass spectrometry LDL: Low-density lipoprotein LSM: Least square means m/z: Mass/charge MAP: Modified atmosphere packaging MDA: Malondialdehyde Meq: Milliequivalents MUFA: Monounsaturated fatty acid MW: Molecular weight n.d.: Not determined n.q.: not quantified NEAA: Non-essential amino acid NIST: National Institute of Standards and Technology ns: Not significant OGYE: Oxytetracycline glucose yeast extract agar ORAC: Oxygen radical absorbance capacity P/S: Polyunsaturated/saturated PBS: Phosphate-buffered saline PCA: Plate count agar PDMS: Polydimethylsiloxane PG: Propyl gallate PGE: equivalents PUFA: Polyunsaturated fatty acid xiv Acronyms

PV: Peroxide value ROS: Reactive oxygen specie RP-HPLC: Reversed phase-high performance liquid chromatography RSA: Radical scavenging activity RT: Retention time SC: Supercritical SEM: Standard error of mean SFA: Saturated fatty acid Sig.: Significance SPME: Solid-phase microextraction TBA: Thiobarbituric acid TBARS: Thiobarbituric acid-reactive substances TBHQ: Tertiary-butylhydroquinone TCA: Trichloroacetic acid TE: Trolox equivalent TEP: 1,1-3,3 tetraethoxypropane TOTOX: Total oxidation TPC: Total phenolic content TPTZ: 2,4,6-tripyridyl-striazine TSC: Total solid content TV: TOTOX values TVC: Total viable counts UAE: Ultrasound-assisted extraction UNU: United Nations University UV: Ultraviolet VC: Volatile compound WHO: World Health Organization CAE: Código Alimentario Español (translated: Spanish Food Code)

xv

I. INTRODUCTION

I. Introduction

I.1. THE ROLE OF MEAT THROUGH HISTORY OF MEN

Animals as a food source have been used by men since 5 millions years ago. The use of meat for human consumption covers a long period of time which can be divided in four distinct stages.

 First stage (> 2 million years ago): Men survived hunting small and/or young animals and acting as scavengers, capturing animals killed by other species. The first primitive stone tools date from 2.5 million years ago in eastern Africa, what indicates that early humans probably had the ability of cutting and processing meat. Cut marks on the bones of animals made with stone tools found in Kenia and Ethiopia corroborated this fact (Larsen, 2003).

 Second stage (starts around 2 million years ago): Humans started to hunt together, cooperating with each other. In this way, they got meat from large animals. The acquisition of large prey provided human communities regular access to protein and micronutrients. This full-scale hunting may be related to a significant increase in human heights discovered at this period of time of human evolution (Larsen, 2003).

 Third stage (starts around 10,000 years ago): The domestication of animals and plant growing begin. Domestic animals were an important meat, milk and skin sources in some regions all over the world. Meat and milk provided nourishment, whereas skins provided clothes and housing materials. Species, such as cattle, sheep and goats, were domesticated around 8000-9000 years ago. Nevertheless, chickens and pigs were domesticated some time later. This change in the way of obtaining food led to a narrowing of diet and a reduction in meat consumption. The study of human remains from around the world revealed that in this stage of human history the men health suffered a decrease (Larsen, 2003).

 Fourth stage: In this last period, the excessive intake of animal tissues has influenced on human health, especially after the World War II (Larsen, 2003).

Nowadays, population who live in developed countries intake the same amount of fat than traditional hunter-gatherers. However, the latter group suffered fewer cardiovascular diseases than the first ones. This was probably due to the fact that traditional hunter-gatherers ate meat richer in MUFAs and PUFAs, as well as at that time in the history there was a higher physical activity. If population is more and more sedentary and increase their intake of high fat foods, an increment in heart diseases will be produced (Larsen, 2003).

3 I. Introduction

I.2. DEFINITION AND CHEMICAL COMPOSITION OF MEAT

Meat is defined by the Codex Alimentarius as “All parts of an animal that are intended for or have been judged as safe and suitable for human consumption”. On the other hand, the CAE defines meat as “the edible part of the muscles of healthy cattle, sheep, goat, pig, horse, and camel, slaughtered in hygienic conditions. It is also applicable to poultry and marine mammals”.

Meat tissues are composed of five main chemical components: moisture (water), proteins, lipids, carbohydrates and inorganic matter (ash or minerals). Other minor components are non-protein nitrogen compounds (e.g., nucleotides, peptides, creatine, creatine phosphate, urea, inosine monophosphate, nicotinamide-adenine, and dinucleotides) and non-nitrogenous compounds (e.g., vitamins, glycolytic intermediates and organic acids). Skeletal muscle tissue is composed of approximately 75% water, 19% protein, 2.5% lipid, 1.5% non-protein nitrogen compounds, 1% carbohydrate and nitrogenous components, and 1% inorganic matter (Dikeman & Devine, 2014).

The most abundant chemical components (water, protein and fat) in meat tissues and meat products suffer modifications according to factors, such as species, age, anatomical location of meat piece, amount of skin and bone, as well as to the inclusion of added non-meat ingredients, such as salt, alkaline phosphates, sodium nitrite/nitrate, sugars, , and seasonings. The increase of fat amount causes the decrease of water, protein and ash. However, the carbohydrate percentage remains quite constant as the fat amount increases (Dikeman & Devine, 2014).

The eleven primary chemical elements represent over 99% of an animal’s body composition. Despite this so high percentage, other 25 essential and non-essential microelements are also part of the tissues. The main elements ordered by weight are oxygen (65%), carbon (18%), hydrogen (10%), nitrogen (3%), calcium (1.5%), phosphorus (1.0%), potassium (0.35%), sulphur (0.25%), sodium (0.15%), chlorine (0.15%), and magnesium (0.05%) (Dikeman & Devine, 2014).

I.2.1. FACTORS AFECTING THE CHEMICAL COMPOSITION OF MEAT

Meat tissue composition is susceptible to modifications depending on species, chronological and physiological maturity at the slaughtering, nutrition, genetic predisposition, and anatomical location of cuts within a carcass (Dikeman & Devine, 2014; Table I.1).

4 I. Introduction

Table I.1. Variation of the chemical composition (%) of animal carcasses from birth to maturity age (Dikeman & Devine, 2014). Component Birth Market maturity Beefa Porkb Turkeyc Beefd Porke Turkeyf Water 73.5 81.7 73.0 52.7 47.3 60.0 Protein 19.2 9.8 23.6 23.7 15.3 21.2 Fat 2.5 1.8 2.8 19.1 36 17.6 Ash 4.8 3.1 0.6 4.8 2.2 1.3

Muscle, fat and bone are the most abundant carcass components, which can suffer modifications in their proportions from birth to mature age. At birth, muscle (~67%) is the major component of the beef carcass, followed by bone (~25%) and fat (~8%). At mature age, muscle may achieve around 55% of a carcass, whereas fat and bone percentages are around 28% and 15%, respectively. Therefore, as the animal grows, the percentages of muscle and bone decrease, whereas the fat percentage increases. Among species, these differences are also present when comparing carcass and skeletal muscles (Table I.2). Overall, whole carcasses present more variation, whereas individual muscles within each specie are less different in gross composition (moisture, protein and fat). Nevertheless, muscles differ in their proportion of specific components (e.g., collagen content, concentration of myoglobin and sarcoplasmic proteins) or fatty acids (e.g., SFAs, MUFAs and PUFAs) (Dikeman & Devine, 2014).

Table I.2. Carcass and skeletal muscle tissue composition of different raw meats (Dikeman & Devine, 2014). Component (%) Carcass composition (boneless) (%) Beefa Porkb Chickenc Turkeyd Lambe Vealf Moisture 58.21 49.83 65.99 70.40 59.80 72.84 Protein 17.48 13.91 18.60 20.42 16.74 19.35 Fat 22.55 35.07 15.06 8.02 22.74 6.77 Ash 0.82 0.72 0.79 0.88 0.92 1.04 Skeletal muscle (%) Beefg Porkh Chickeni Turkeyj Lambk Veall Moisture 69.29 73.21 75.46 74.16 76.13 74.80 Protein 22.18 21.20 21.39 21.77 20.82 20.98 Fat 7.68 4.82 3.08 2.86 3.28 3.08 Ash 1.08 1.01 0.96 0.97 1.22 1.12

5 I. Introduction

I.2.2. CHEMICAL COMPONENTS OF MEAT

I.2.2.1. Moisture (water)

Water may be around from 65% to 80% of the total mass in living muscle tissues, and operates as a main component of cellular metabolism. Similarly, water comprises around 75- 80% of the total cell mass in post-mortem muscle tissue. Water are present in a large part of the muscle sarcoplasm as well as surrounding myofibrillar proteins. These myofibrils constitute 75-92% of the volume of lean muscle. Factors, such as ionic environment (pH), availability of specific cations and anions, and contraction degree of myofibrillar proteins affect greatly to the retention or loss of water from muscle tissues. Approximately less than 0.1% of the total water covering the protein is strongly bound in the polar accessible sites of the native structure of proteins. This type of water is called “constitutional” (bound or monolayer). It is strongly bound within a protein and it is non-freezing at -40 °C. A hydration layer is formed around the proteins due to water layers interact with the charged and polar proteins. This water is slightly mobile and it is also not freezeable at -40 °C. Additional water outside the hydration layers of protein is called bulk-phase water, constituting around 95% of the water in a cell. Bulk-phase water may be removed aplying thermal processings. This water is susceptible of freezing and allows a high degree of molecular mobility (Dikeman & Devine, 2014).

I.2.2.2. Proteins

Proteins comprise 16-22% of skeletal muscle tissue and they are formed by 20 amino acids bound through peptide links (Figure I.1). According to their function they are classified in: myofibrillar (contractile), sarcoplasmic (metabolic) and stromal (connective or support). Meat proteins contain around 16% nitrogen (including non-protein nitrogen compounds) (Dikeman & Devine, 2014).

Myofibrillar or also called salt-soluble proteins constitute around 11.5% of the 19% total protein and are extractible with a KCl solution of more than 0.3 mol/L (Dikeman & Devine, 2014). Myosin (43%), actin (22%), titin (8%), tropomyosin (5%), troponin (5%), nebulin (3%), C- protein (2%), α-actinin (2%), M protein (2%), and desmin (< 1%) comprise around 93% of the 20 amino acids that compose the muscular myofibril. Depending on the function of myofibrillar proteins, they can be separated into three groups, contractile proteins (e.g., myosin and actin), regulatory proteins (e.g., tropomyosin and troponin), and cytoskeletal proteins (e.g., titin, nebulin and C-protein) (Pearson & young, 1989). Actin and myosin comprise around 65% (22% and 43%, respectively) of the total myofibrillar proteins. Myosin

6 I. Introduction has a molecular weight of 520 kDa, formed by 2 subunits of 220 kDa and 4 of 20 kDa, whereas actin has a molecular weight of 42 kDa. Both proteins form a complex during rigor mortis leading to the formation of actomyosin, a protein which affects to the water holding capacity of muscle tissue and to the intermolecular binding in a meat gel matrix (gelation) (Dikeman & Devine, 2014).

Figure I.1. Formation of a peptide bond (Yruela & Sebastian, 2014).

Sarcoplasmic or water-soluble proteins comprise around 5.5% of the 19% total protein and they are extractible with low-ionic strength KCl solutions (0.06 mol/L). This type of proteins are found in the sarcoplasm that surrounds myofibrils and are formed primarily by oxidative enzymes (cytochromes, the flavin nucleotides), various heme pigments (myoglobin), mitochrondrial oxidative enzymes, lysosomal enzymes, and nucleoproteins. Sarcoplasmic proteins are found in a concentration of approximately 55 mg/mL. They are listed in descending order according to their abundance (mg/g tissue): glyceraldehyde phosphate dehydrogenase (12), aldolase (6), enolase (5), creatine kinase (5), lactate dehydrogenase (4), pyruvate kinase (3), phosphorylase (2.5), and myoglobin (0.1). The concentrations of these enzymes range depending on the species, muscle fibre type, maturity and sex (Dikeman & Devine, 2014).

Myoglobin (16 kDa) is the main pigment responsible for the colour of muscle tissues. This colour is related to the myoglobin concentration, the oxidation state of the iron atom in the porphyrin ring, and the molecule linked to the sixth site on the iron molecule (Dikeman & Devine, 2014).

Stromal or connective tissue proteins usually account for around 2% of the 19% total protein in skeletal muscle and are quite insoluble. Connective tissue is a viscous solution of soluble glycoproteins (proteoglycans) with extracellular fibres of collagen and elastin embedded in the glycoprotein matrix. Collagen is a triple-helical molecule and it is the major protein forming tissues, such as skin, ligaments, tendons, cartilage and bone. Collagen is extractible using strong acid or alkaline solutions, and digested using pepsin and collagenase. It is composed by uncommon amino acids (33% glycine, 11% alanine, 9-10% proline and 13-14% hydroxyproline).

7 I. Introduction

Regarding elastin, it is a rubbery protein arranged in a β-pleated sheet which is part of ligaments, arterial walls and support structure of organs. Its hight content (~ 90%) content in non-polar amino acids and desmosine cross-links makes it strongly resistant to solubilisation, cooking or enzymatic digestion (Dikeman & Devine, 2014).

I.2.2.3. Lipids

Animal adipose tissue is constituted mostly of neutral lipids known as triacylglycerols and phospholipids ranging both from 1.5% to 13% in muscle tissue. Lipids are also present as sterols, sterol esters (cholesterol and cholesterol compounds) and cerebrosides. Cell uses several lipid forms for different functions such as: energy source, structural and functional component of the wall, for protection of vital organs, and as solubilizing agents for specific hormones and vitamins (A, D, E and K) (Dikeman & Devine, 2014).

Figure I.2. Formation of a triacylglycerol (Kalita & Netravali, 2017).

Neutral lipids (triacylglycerols) are esters of glycerol (one molecule of glycerol linked to three long-chain fatty acids with an even number of carbon atoms). Triacylglycerols are divided in simple (all fatty acids are the same) or mixed (two or more fatty acids are different) (Dikeman & Devine, 2014).

Fatty acids are classified as saturated (absence of double bonds along the carbon chain); mono-unsaturated (presence of one double bond along the carbon chain); and polyunsaturated (presence of two or more double bonds along the carbon chain). Differences in the number of carbon atoms, number of double bonds, melting point, fluidity, hardness, and the susceptibility to lipid oxidation of the fatty acids linked to a triacylglycerol give a fat their own physical characteristics. The melting point of fats depends on the carbon chain length and number of existing double bonds. As the carbon atoms increase, the melting point increases, whereas as the number of double bonds increases, the melting point decreases. Internal fats covering organs are usually composed by more SFAs with higher melting points than external subcutaneous fatty acids in order to protect them (Dikeman & Devine, 2014).

8 I. Introduction

Fatty acids are named according to the carbon atoms from the carboxyl (COOH) or methyl (CH3) end of the molecule. Using the first type of numbering method, oleic acid (C18:1n-9) is named cis-9-octadecenoic acid. In the other type of numbering, the fatty acids with the first double bond located on the third carbon from the methyl end (n-3) are called “omega-3” fatty acids. Most of unsaturated fatty acids in meat belong to the n-6 family (first double bound in the sixth carbon from the methyl end) (Dikeman & Devine, 2014).

The proportion of the fatty acids comprising a fat deposit varies according to species and diet. Generally, oleic acid, ranging from 20 to 47%, is the major fatty acid in lamb, cattle and pig, whereas palmitic (C16:0) (26%) is the major in poultry. The classification by species according to the amount of saturated fat is as follows: lamb > cattle > pig > poultry. Palmitic (25-30%) and stearic (C18:0) (7-27%) SFAs are the most abundant in cattle, lamb, pig and poultry. Other fatty acids, such as linoleic (C18:2n-6) (2-20%), palmitoleic (C16:1n-7-) (1-7%), linolenic (C18:3n-3) (0.2-0.6%), arachidonic (C20:4n-6) (0.2-2%), lauric (C12:0) (trace), myristic (C14:0) (0.1-5%) and arachidic (C20:0) (trace to 3%) acids, are also present. Phospholipids are the structural and functional constituents of cell membranes accounting 0.5-1% of the lipid in skeletal muscle. The most abundant phospholipids in muscle tissue are: phosphatidylethanolamine (cephalin) (33%), phosphatidylserine (6%), phosphatidylcholine (lecithin) (58%) and sphingomyelin (3%) (Dikeman & Devine, 2014).

The fat consumed in human diet contains very few amount of sterols. Cholesterol is the primary sterol in animals and it is the precursor of bile acids, provitamin B and steroid hormones. Cholesterol may be free or esterified with fatty acids of different chain lengths and saturation. The type of meat, the anatomical location and the culinary preparation conditions are factors which produce variations in cholesterol amount of meat and meat products (Dikeman & Devine, 2014).

Fat content is dependent on species, age (Tables I.1 and I.2) and diet. As animal grows fat is accumulated in the following order: around vital organs, in a subcutaneous position, intermuscularly (between muscles) and lastly intramuscularly (inside muscle as ‘marbling’). In monogastric animals, feed has influence in the fatty acid composition of carcass. Therefore, a diet with high levels of PUFAs causes an increment of fat in carcasses, became them oilier and softer. Ruminants, however, accumulate more saturated fats with higher melting points (Dikeman & Devine, 2014).

9 I. Introduction

I.2.2.4. Carbohydrates

Glycogen is the major carbohydrate in animal tissues. Structurally it is formed by α-D- glucose units linked by α-1,6 glucosidic and α-1,4 glucosidic bonds, and varies from 0.5% to 1.5% in living skeletal muscle tissue. The initial content of glycogen in muscle tissues just after the slaughter influences muscle colour, texture, firmness, water holding capacity, emulsifying capacity, and shelf life.

Anaerobic glycolysis, once the animal has been slaughtered, causes the glucose portion is metabolized to lactate. The final pH reached in muscle is dependent on the final amount of lactate, which is proportional to the initial amount of glycogen. The concentration of pre- slaughter glycogen in animals’ body is affected by previous comfort conditions, and specifically the levels of adrenaline and/or pre-slaughter exercise play a significant role in the glycogen conversion to lactate with possible negative consequences on meat quality (Dikeman & Devine, 2014).

Figure I.3. General structure of glycogen (Sturm, 2014).

Other carbohydrates present in animal tissues are glycosaminoglycans and proteoglycans which are associated with the extracellular matrix of connective tissues, as well as glycoproteins present in plasma and blood, and some hormones, glycolytic intermediates, nucleotides, nucleosides, and glycolipids. Of all these carbohydrates, D-glucose is present in greater quantity. Glycosaminoglycans are covalently bound to proteins to form complex proteoglycans. Different compounds, such as hyaluronic acid, chondroitin sulfates, dermatan sulfate, keratin sulfate, and heparin-like polysaccharides are components of glycosaminoglycans (Dikeman & Devine, 2014).

I.2.2.5. Minerals

Around 3.5% of the total body weight is comprised of inorganic matter (bones and teeth), and is usually analysed as ash percentage. Therefore, this percentage in meat products

10 I. Introduction is an estimation of the total minerals which compose cellular constituents (myoglobin, haemoglobin and enzymes), small fragmets of bone or ingredients (sodium chloride, potassium chloride, alkaline phosphates, lactate salts, spices and seasonings) used in processing meats. Bone is an important component of carcasses contributing calcium and phosphorous mainly. Muscle tissue is poor in calcium (3-6 mg/g), but rich in potassium (250-400 mg/g), phosphorous (167-216 mg/g), sodium (55-94 mg/g), magnesium (22-29 mg/g), zinc (1-5 mg/g), iron (1-3 mg/g), and copper (0.5-0.13 mg/g). The heme iron from meat is more easily bioabsorbed by the body and covers 40-60% of the total iron needs. Calcium, magnesium, sodium and potassium take part in the muscle contraction, whereas magnesium and calcium are involved in post mortem muscle contraction. Sulphur (2.5 mg/g) is present in sulphur containing amino acids that compose proteins, while chlorine (0.65 mg/g) is mainly an anion that is part of salts and intracellular fluids. On the other hand, iron, copper, zinc, iodine, manganese, molybdenum, cobalt, and selenium are essential microelements in diet, with specific functions in muscles. Aluminium, arsenic, boron, lead, lithium, nickel, rubidium, silicon, silver, strontium, titanium, and vanadium are also present in meat, although they may not have well-defined functions or be simply environmental pollutants (Dikeman & Devine, 2014).

I.3. COLOUR AND PIGMENTS IN MEAT

In the sale point, the meat colour is the most important attribute that consumer judges. Consumers often associate the cherry red colour of meat with freshness and healthiness. Nevertheless, the dull-brown colour is used as an indicator of cooked meat. On the other hand, cured meat products have a characterists pink colour. Myoglobin is the sarcoplasmic heme protein primarily responsible for the colour of meat. In addition, haemoglobin, cytochrome and other pigments may be possible components of meat, appearing at low concentrations and contributing to colour to a lesser extent. Generally, pigments other than myoglobin have more influence on the colour of poultry and game meat than on red meat (Dikeman & Devine, 2014).

I.3.1. MYOGLOBIN

The pigment myoglobin is formed by a protein residue (globin) and a heme prosthetic group. In living muscles, myoglobin is responsible for oxygen binding and transport functions. The globin polypeptide chain is composed of eight helical segments which form a coil around the heme moiety. The hydrophobic heme group is arranged that the vinyl groups are oriented

11 I. Introduction towards the hydrophobic inner and the propionic acid groups towards the outer face of the molecule. An iron atom is located in the heme group of myoglobin which can be in a reduced (ferrous/Fe2+) or oxidized (ferric/Fe3+) form. The globin molecule of the molecule contributes to water solubility upon the hydrophobic heme group and also protects heme iron against oxidation (Dikeman & Devine, 2014).

Figure I.4. The myoglobin molecule structure is shown on the left. The heme group is shown in blue with the iron atom in orange (Bajaj et al., 2004). The heme group structure is shown on the right (García-Sánchez et al., 2013).

Mammalian and avian myoglobin molecules are composed of a single polypeptide of 153 amino acids. Avian myoglobin is around 300-400 Da heavier than mammalian one. For example, myoglobin molecular mass from turkey, chicken, ostrich and emu are 17 291, 17 291, 17 297 and 17 380 Da, respectively, while in beef, pork, sheep, goat, and horse are 16 946, 16 953, 16 923, 16 896 and 16 952 Da, respectively. These variations in molecular mass are the result of the replacement of low molecular weight amino acids in red-meat myoglobins by heavier ones in poultry myoglobins, causing a mass increase. Although variations exist among mammalian and avian species in amino acid sequences, several biological, structural and functional properties of myoglobin are equal in both classes (Dikeman & Devine, 2014).

The primary structure of myoglobin has a direct effect on biochemical attributes, such as autoxidation, heme retention, structural stability, thermostability, and oxygen affinity, which influence in meat colour stability and in the protein function in living animals. Myoglobins from livestock raised to produce meat have 85-100% homology in their primary structure. The amino acid sequences of pig, cattle, sheep, goat, water buffalo, and horse myoglobins differ at 29 positions. Five mutations of ancestral mammalian myoglobin are necessary to achieve pig myoglobin, whereas 24, 19 and 13 mutations are required to get cattle, sheep and horse

12 I. Introduction myoglobin, respectively. Therefore, pork myoglobin is classified as the most ancestral of those belonging to domestic mammals (Dikeman & Devine, 2014).

I.3.1.1. Lipid oxidation-induced myoglobin oxidation

Aldehyde products are originated due to the lipid oxidation and they covalently bind to myoglobin molecule leading to a rapid oxidation of heme group and the metmyoglobin formation, causing meat discoloration. The relationship between lipid oxidation and meat discoloration is species-specific; beef oxymyoglobin is more sensitive to a nucleophilic attack by lipid oxidation products than pig oxymyoglobin. Higher oxidation rates are noted in oxymyoglobins with higher number of histidine molecules as it happens in beef and sheep (12 histidines) in comparison with pork and chicken (9 histidines) (Dikeman & Devine, 2014).

Within an animal species destined to produce meat, carboxymyoglobin has the same amino acid sequence than oxymyoglobin, and therefore, it is possible that it interacts with lipid oxidation products. similarly to oxymyoglobin. Despite the fact that horse carboxymyoglobin demonstrated to have higher opposition to browning than horse oxymyoglobin when they are exposed to 4-hydroxynonenal, the amino acids abducted by 4-hydroxynonenal were the same in both redox forms indicating the possible existence of analogous mechanistic interactions (Dikeman & Devine, 2014).

I.3.2. PIGMENTS IN FRESH MEATS

I.3.2.1. Myoglobin redox forms in packaged fresh meats

When fresh meats are packaged, myoglobin pigment can be in any of the following four redox states: deoxymyoglobin, oxymyoglobin, carboxymyoglobin and metmyoglobin (Figure I.5). Deoxymyoglobin, oxymyoglobin and carboxymyoglobin are the myoglobin molecule in the ferrous state. Deoxymyoglobin is purplish-red, whereas oxymyoglobin and carboxymyoglobin give a bright cherry-red colour to meat what could cause the rejection of consumers. Deoxymyoglobin has not any ligand linked with the heme iron atom, whereas oxygen and carbon monoxide (CO) are linked to heme iron at sixth coordinate in oxymyoglobin and carboxymyoglobin, respectively. The oxidation of ferrous to ferric state causes the formation of metmyoglobin, giving a brown colour to meat what the consumer associates with a discoloration. The sixth coordinate of the ferric heme group of metmyoglobin is occupied by a water molecule, hence it is physiologically inactive since it is not able to bind any oxygen molecule. Myoglobin tend to bind to carbon monoxide (CO), leading to the formation of bright

13 I. Introduction cherry-red carboxymyoglobin. Carboxymyoglobin is more stable than oxymyoglobin in oxidative environments, due to the high interaction of carbon monoxide to the iron-porphyrin site on the myoglobin molecule. Therefore, when using MAP in fresh meats it is positive the use of low amounts of CO to avoid the loss of the cherry-red colour. The CO is lost when exposing to light and heat, despite being strongly bound to myoglobin in fresh meat (Dikeman & Devine, 2014).

Figure I.5. Different derived myoglobin pigments and their corresponding conversions in packaged fresh meats (Dikeman & Devine, 2014).

I.3.2.2. Cytochrome c

Cytochrome c is an hemoprotein of low-molecular weight (13 000 Da) composed by 104 amino acids, which is involved in redox reactions to produce ATP in the mitochondria. Red meats have lower cytochrome c content than poultry meats. Nonetheless, cytochrome c is much more stable to heat than myoglobin. Therefore, it can help to maintain the pink colour in cooked turkey while other heme pigments have undergone a browning process induced by denaturation (Dikeman & Devine, 2014).

I.3.2.3. Sulfmyoglobin

Green pigment sulfmyoglobin is originated as a result of reactions of hydrogen sulphide with ferrous heme of myoglobin. Green color is attributed to the growth of sulfhydryl- producing bacteria Pseudomonas mephitica. This microorganism needs an oxygen tension bellow 1% and a pH higher than 6.0 to produce hydrogen sulphide from sulfur-containing amino acids. To protect meat from this alteration, it is recommended not to pack meats with high final pH values under low oxygen tensions. Sulfmyoglobin can be converted into the

14 I. Introduction pigment red metsulfmyoglobin by oxygen or ferricyanide, causing the disappearance of the absorption (Dikeman & Devine, 2014).

I.3.2.4. Acid ferrimyoglobin peroxide

The oxidation of myoglobin along with the histidine by hydrogen hydroxide under acidic conditions results in this green pigment, also known as hydroperoxymetmyoglobin. Lactic acid microorganisms, such as Lactobacillus viridescens, Leuconostoc and Pediococcus, are able to produce hydrogen peroxide in aerobic environments. These bacteria are salt-tolerant, catalase-negative and are capable to grow at low temperatures (Dikeman & Devine, 2014).

I.3.2.5. Ferrimyoglobin peroxide

This red pigment is also produced because of the oxidation of myoglobin by hydrogen peroxide. The action of peroxide on myoglobin initiates the reaction intermediate ferrimyoglobin peroxide with colour red as well. The red pigment accumulates under alkaline conditions (pH 8.0) and then is turned into green pigment (acid ferrimyoglobin peroxide) when meat is under slightly acidic conditions (Dikeman & Devine, 2014).

I.3.2.6. Ferrocholemyoglobin

Ferrocholemyoglobin is a green pigment, which is generated when the myoglobin oxidation causes breakage of the porphyrin ring. The strong reducing agent sodium dithionite previously diluted is used to measure the oxidation degree of myoglobin. Green pigments sulfmyoglobin and hydroperoxy-metmyoglobin are only slightly oxidized, therefore, they can be transformed back into myoglobin by the addition of a reducing agent (Dikeman & Devine, 2014).

I.3.3. PIGMENTS IN COOKED MEATS

Globin protein is easily denatured when meat is cooked since it is sensitive to heat. In this manner, globin is transformed into metmyoglobin, and this pigment is subsequently converted in globin hemichrome (also called ferrihemochrome), giving a dull-brown colour to cooked meats. Globin denaturation causes the appearance of ferrous myoglobin which in turn causes the appearance of pink/red ferrohemochrome, which is readily oxidized later to brown ferrihemochrome. Anaerobic environments at cooking meat also cause the appearance of pink

15 I. Introduction ferrohemochrome. Due to the denaturation of globin protein by heat, these pigments coagulate and become insoluble in buffers (Dikeman & Devine, 2014).

I.3.4. PIGMENTS IN CURED MEATS

Cured meat products are characterized by a stable pink colour. This colour is generated as a result of the reaction between meat pigments and nitrates/nitrites during the curing process or nitrogen dioxide (NO2) exposure if products are subjected to smoke. The agent responsible of this pink colour is sodium nitrite (NaNO2), which is spread onto the meat surface or injected into it (Dikeman & Devine, 2014).

Despite there being three curing pathways (nitrate, nitrite and nitrogen dioxide), the nitrous acid is the only that reacts with myoglobin. When nitrite interacts with water nitrous acid is formed. This acid oxidizes myoglobin to metmyoglobin, remaining reduced to heme-bound nitrogen monoxide (NO). Nitric oxide metmyoglobin has a brown colour and it can be reduced to a red pigment called nitrosyl myoglobin under an anaerobic environment. If meat with this pigment is cooked, nitrosyl myoglobin is turned into nitrosyl hemochrome by denaturation, which is unable to react, remaining stable. Despite being stable, nitrosyl hemochrome is sensitive to oxygen, temperature and light, causing the cured colour loss and increasing the gray-tan pigment, typical in cooked meats because of the globin hemichrome denaturation (Dikeman & Devine, 2014).

I.4. OXIDATION IN MEAT

Cutting, mincing, irradiation, handling, packaging, storage, and cooking procedures promote chemical and enzymatic processes in meat, making it a perishable product (Papuc et al., 2017). Muscle tissue has high concentrations of unsaturated lipids, heme pigments, metal catalysts and oxidizing agents promoting oxidative reactions (Falowo, Fayemi, & Muchenje, 2014). On the other hand, after slaughter, endogenous antioxidants are depleted fast contributing to this process (Falowo, Fayemi, & Muchenje, 2014). In addition, breed, muscle types and their anatomical location are factors which affect to meat oxidation (Min et al., 2008). These degradation reactions of lipids and proteins in meat promote the appearing of off-odours, off-tastes, colour changes and possible toxic compounds, decreasing considerably the consumer acceptance (Papuc et al., 2017). Indeed, several diseases, such as atherosclerosis and cancer, have been linked to oxidative reaction products (Papuc et al., 2017).

16 I. Introduction

I.4.1. LIPID OXIDATION IN MEAT

Lipids, such as triacylglycerides, phospholipids and sterols, are widely present in meat (Falowo, Fayemi, & Muchenje, 2014). Meat products are susceptible to suffer two types of lipid oxidation: non-enzymatic oxidation, called “autoxidation” (Guyon, Meynier, & Lamballerie, 2016) and enzyme-catalysed oxidation.

I.4.1.1. Non-enzymatic oxidation of lipids. “Autoxidation”

Autoxidation take place in three simultaneous phases (initiation, propagation and completion) (Guyon, Meynier, & Lamballerie, 2016). The initiation phase begins with the attack of a reactive species with high electron reduction potential to abstract a highly reactive hydrogen atom from a lipid molecule and generate a lipid radical (Papuc et al. 2017). The hydrogen atoms in the allylic positions are the most easily abstractable (Papuc et al., 2017). Venkataraman, Schafer, & Buettner (2004) suggesting that a free radical that possesses a one- electron reduction potential higher than 0.6 V might be the initiator of lipid peroxidation.

These class of free radicals would include hydroxyl (HO·), nitrogen dioxide (NO2·), alkoxy (RO·), peroxyl (ROO·) and perhydroxyl (HOO·) radicals (Papuc et al., 2017). Iron in the form of iron- oxygen complexes, such as perferryl ion and ferryl ion, is an alternative way to initiate lipid peroxidation instead of free radicals with reduction potential higher than 0.6 V. In addition to these two types of initiation of lipid oxidation, there is another way in which light, oxygen and the presence of a photosensitizer promote the generation of free radicals in meat (Papuc et al., 2017). Hydroxyl radical, singlet oxygen and iron-oxygen complexes are the main ways of autoxidation initiation. Some authors suggest that the latter are the major initiators (Papuc et al., 2017).

In the next phase of autoxidation (propagation), the fatty acid radical resulting from the initiation step reacts with dioxygen forming a hydroperoxy radical. This radical can take a hydrogen atom to generate another unsaturated fatty acyl group (LH) and thus produce a new lipid radical (L·) and fatty acyl hydroperoxide. The presence of non-heme Fe2+ induces the decomposition of lipid hydroperoxide to alkoxy radical (Papuc et al., 2017).

Fe2+ + LOOH → Fe3+ + LO· + HO- Fe3+ + LOOH → Fe2+ + LOO· + H+

The chain of oxidative reactions concludes when the radicals L·, LO· and LOO· react with each other or with other free radicals to generate non-radical stable compounds. This last phase is called completion (Papuc et al., 2017).

17 I. Introduction

Figure I.6. General scheme of the different phases in non-enzymatic lipid oxidation (Guyon, Meynier, & Lamballerie, 2016).

I.4.1.2. Enzymatic oxidation of lipids

Lipoxygenase are the responsible of the enzymatic reactions of lipids. These enzymes catalyse the stereospecific deoxygenation of PUFAs, containing at least one 1-cis, 4-cis- pentadiene system, such as linoleic, linolenic and arachidonic acid (Papuc et al., 2017).

The oxidation of fatty acids by lipoxygenases take place in 4 phases:

 First phase: Subtraction of a labile hydrogen from a bis-allylic methylene group and reduction of Fe3+ ion bound enzyme to Fe2+ (Papuc et al., 2017).

 Second phase: Delocalization of a double bound and formation of a conjugated diene (Papuc et al., 2017).

 Third phase: Deoxygenation of the lipid radical and formation of peroxy radical (Papuc et al., 2017).

 Fourth phase: Peroxy radical reduction via Fe2+ bound enzyme and the protonation of the peroxy anion (Papuc et al., 2017).

Some authors have reported the finding of a lipoxygenase in microsomal fractions from beef, pork and turkey muscle, which could explain the low oxidative stability of muscle tissues (Papuc et al., 2017).

18 I. Introduction

Figure I.7. Formation of 13(S)13-hydroperoxyoctadeca-9, 11-dienoate (9Z, 11E) (13-(E, Z)-HPODE) by the action of lipoxygenase (Papuc et al., 2017).

I.4.2. PROTEIN OXIDATION IN MEAT

Protein oxidation of muscle tissue after slaughter is a free radical chain reaction (Papuc et al., 2017). The oxidation pathways of proteins are very complex and a greater variety of products than in lipid peroxidation are generated (Papuc et al., 2017). The chain reaction starts with the subtraction of a hydrogen atom by a ROS generating a protein carbon-centered radical (P·). This (P·) formed in the previous phase is transformed into a peroxyl radical (POO·) in the presence of oxygen, and in an alkyl peroxide (POOH) by subtraction of an hydrogen atom from another molecule. Further reactions with perhydroxyl radical (HO2·) promote the formation of an alcoxyl radical (PO·) and its hydroxyl derivative (POH) (Lund et al., 2011).

The greater oxidation processes take place in different locations into proteins, at the side chains of amino acid residues and at the backbone of proteins. In the first position, oxidation causes solubility loss, essential amino acid loss, and an increment of susceptibility to aggregation. Oxidation of thiol groups or formation of carbonyl groups are some examples. On the other hand, the oxidation of the backbone of a protein promotes modifications in the spatial arrangement of the atoms of the polypeptide chain, fragmentation, aggregation and polymerization of the proteins (Papuc et al., 2017).

Myofibrillar proteins can suffer oxidation processes, being myosin the most affected protein, followed by troponin T (Lund et al., 2011). Among all amino acids, arginine, cysteine, histidine, lysine, methionine, phenylalanine, proline, tryptophan, and tyrosine have been indicated as especially sensitive to ROS (Davies & Dean, 2003).

19 I. Introduction

Figure I.8. Free radical-mediated pathway of protein oxidation (Soladoye et al., 2015).

I.4.3. PRODUCTS RESULTING FROM LIPID AND PROTEIN OXIDATION. EFFECTS IN HUMAN HEALTH

I.4.3.1. Lipid oxidation products

Products generated by lipid peroxidation can be divided in two groups, primary and secondary products (Min & Ahn, 2005). Toxicity of primary products is quite lower than that of secondary products (Papuc et al., 2017). Lipid hydroperoxides (primary products) have been found that they promote DNA synthesis and begin the ornithine decarboxylase activity in the colonic mucosa, indicating tumorigenesis improvement (Papuc et al., 2017). On the other hand, secondary products (ALEs) such as carbonyls, alcohols, hydrocarbons and furans, are related to possible cytotoxic and mutagenic effects (Papuc et al., 2017). It is believed that ALEs activate an inflammatory response after being absorbed by the small intestine. Once they are in the circulatory system, they may affect various organs, such as liver, kidneys, lungs and the intestine itself (Kanner, 2007). Kanazawa, Kanazawa, & Natake (1985) tested the effects of feeding with oxidation products in Wister rats, and they observed that ALEs could increase significantly the radioactivity in rat livers. In addition, they reported a mild increase of liver enzyme activity in the serum and damage in the hepatocytes. Studies based on animal experimentation suggest that the ingestion of oxidized foods containing ALEs may promote LDL oxidation in vivo along with an increase of atherogenicity and foam cell formation (Papuc et al., 2017). MDA is the main ALE (Papuc et al., 2017). This secondary product of lipid

20 I. Introduction oxidation is a bifunctional aldehyde capable of reacting with many biomolecules, such as DNA and proteins, to generate an assortment of adducts (Papuc et al., 2017). Some of these adducts, such as deoxyguanosine, deoxyadenosine and deoxycytidine, are the result of the reaction of MDA with DNA (Papuc et al., 2017). These compounds might be mutagenic in mammalian cells and carcinogenic in rat livers (Papuc et al., 2017). In addition, Tesoriere et al. (2002) reported the possibility that the presence of MDA in living organism causes a quickly oxidative stress of cells and generates dysfunction of red blood cells due to the generation of oxidative cascades.

I.4.3.2. Protein oxidation products

There is not much information available about oxidized protein toxicity (Papuc et al., 2017). The problem is due to the different structures of the compounds resulting from the oxidation (Papuc et al., 2017). Rutherfurd, Montoya, & Moughan (2014) studied the consequences of oxidized protein intake from seven dietary sources on the true amino acid digestibility. According to the results obtained, they alluded to the existence of a balance among two processes: a protein denaturation along with a digestion increase, and formation of non-digestible peptides along with a digestion decrease. The decrease of protein digestibility increased the amount of protein substrate available for microbial enzymes in the colon, indicating potential harm for human health. Indeed, Kim, Coelho, & Blachier (2013) found that the increase of amino acid-derived compounds resulting from bacterial degradation could have a negative effect on colon epithelial renewal and homeostasis. Researches suggest that protein fermentation prevails in colon, since there are studies which report that the amount of protein bacterial metabolites are more abundant. Some compounds resulting from protein oxidation, such as toxic ammonia, phenols, acyclic amines, cyclic amines, N-nitroso compounds, and sulphides, are formed in the colon as a consequence of the bacterial enzyme activities over non-digested substrates from diet. Ammonia generated due to amino acid deamination is suspected to promote tumour formation through several mechanisms, such as modification of the morphology and metabolism of intestinal cells, alteration on the pattern of DNA replication, and early death of intestinal cells (Papuc et al., 2017). Aromatic amino acids are the substrate used by bacteria from the colon for the formation of phenolic compounds. These compounds are absorbed in the colon, and after a detoxification in the liver they are excreted by the urine. Nonetheless, phenol was reported to react with nitrite-forming p- diazoquinone, catalogued as a mutagenic substance. Acyclic amines, such as tyramine, pyrrolidine, piperidine, cadaverine, putrescine, and histamine, released in the large intestine

21 I. Introduction by hydrolysis and decarboxylation, are precursors of N-nitroso compounds, whose health hazard is well-known, having been classified as potentially carcinogenics (Papuc et al., 2017). On the other hand, cyclic amines coming from heterocyclic amino acid decarboxylation showed carcinogenic properties. A clasical example of the formation of these amines is the cooking of meat at high temperature. N-nitrosation reactions of amines are also present in the stomach. The acidic pH of the stomach makes easier the reaction of nitrite with secondary amines to generate N-nitroso compounds (Papuc et al., 2017). Many of these compounds resulting from these reactions were reported to be carcinogenic and mutagenic. Hydrogen sulfide is another compound with toxic effects also found in the human large intestine. This compound origins from the resulting reactions catalysed by enzymes biosynthesized by sulfate-reducing bacteria. Some harmful effects related to this substance were observed in the organism, such as ulcerative colitis appearance or cancer. In addition, hydrogen sulfide possesses the faculty to alter cellular homeostasis, modulate gene expression involved in cell- cycle progression, and to launch inflammatory and DNA repair responses (Papuc et al., 2017). Rodgers & Shiozawa (2008) suggested that oxidized amino acids may deceive the cellular mechanism responsible for protein synthesis. They can be incorrectly integrated into the growing polypeptide chain during the lengthening of this, generating damaged proteins in mammalian cells. The inadequate integration of oxidized amino acids in the protein leads to structural problems in the molecule (Papuc et al., 2017).

I.5. USE OF ANTIOXIDANTS TO PREVENT OXIDATION IN MEAT

The oxidative phenomena in meat and meat products can be reduced by adding synthetic or natural additives with antioxidant properties (Lorenzo & Munekata, 2016; Munekata et al., 2017; Lorenzo et al., 2018a, 2018b, 2018c; Zamuz et al., 2018; Lorenzo et al., 2019). These “additives or ingredients” are known as antioxidants, and it can be defined as the substance present at low concentration in a product regarding the concentration of an oxidizable substrate, which is able to delay the oxidation of this substrate (Halliwell & Gutteridge, 1990).

Antioxidants can be separated in two groups: primary antioxidants or radical scavengers and secondary antioxidants or antioxidants that prevent oxidation. Primary antioxidants inhibit the initiation and propagation of oxidative reactions by inactivating free radicals (L·, LO· and LOO·) and their conversion to stable species. Regarding secondary antioxidants, they are compounds whose way of action is to prevent or decrease the free radical generation (Armenteros et al., 2012).

22 I. Introduction

I.5.1. SYNTHETIC ANTIOXIDANTS

Traditionally, meat industry has used a great amount of synthetic antioxidants as an efficient and economic system of reducing oxidative phenomena incidence (Lorenzo et al., 2018a, 2018b, 2018c; Zamuz et al., 2018; Lorenzo et al., 2019). Thus, synthetic antioxidants, such as BHA, BHT, PG and TBHQ, have been used for years in products from meat origin. These compounds are called phenolic antioxidants owing to their structures derived from phenol. However, their use presents some drawbacks, such as their high volatility and their easiness to decompose at high temperatures (Armenteros et al., 2012). In addition, their use in food is restricted to a maximum amount marked by the legislation that can not be exceed (Armenteros et al., 2012). This amount restriction is due to their potential toxic effects. For instance, BHA and BHT are suspected to cause liver damage and carcinogenesis when they are used at high doses in experimental animals (Shahidi, & Ambigaipalan, 2015). On the other hand, BHA, TBHQ or PG have been singled out as possibly responsible for forming molecular complexes with nucleic acid structure, causing damage to double helical structure of DNA (Shahidi, & Ambigaipalan, 2015). Therefore, the use of these antioxidants in foodstuff is discussed. In fact, synthetic antioxidants, such as BHA and BHT, are not usually used in cooked meat products such as cooked ham (Armenteros et al., 2012).

The characteristics of the synthetic antioxidants most used in food are given below.

 BHA: BHA is a monophenolic antioxidant, commercially sold as a resulting product of the mixture of the isomers 3-tertiary-butyl-4-hydroxyanisole (90%) and 2-tetiary- butyl-4-hydroxyanisole (10%). BHA is highly efficient controlling oxidation reactions in products with short chain fatty acids. The mixture of BHA with other synthetic antioxidants, such as BHT, TBHQ and PG offers better protection than using each one individually (Shahidi & Ambigaipalan, 2015).

 BHT: BHT is also a monophenolic antioxidant. Nonetheless, effectiveness of BHT is lower than BHA due to the presence of two tert-butyl groups, which provide a higher steric hindrance. As in the case of BHA, BHT can offer higher protection against oxidation if it is supplied along with BHA. The combination of 3-BHA and BHT displayed better protection than both separately in soybean oil, lard and methyl oleate (Shahidi & Ambigaipalan, 2015).

 TBHQ: TBHQ presents two para-hydroxyl groups in its structure. These chemical groups are responsible of the TBHQ antioxidant capacity (Shahidi & Ambigaipalan, 2015), higher than that of BHA and BHT (Khan & Shahidi, 2001). TBHQ is highly

23 I. Introduction

effective in unsaturated vegetable oils, many animal fats for human consume and meat products (Shahidi & Ambigaipalan, 2015). Its addition as an ingredient does not cause modifications of flavour and odour in the product (Kashanian & Dolatabadi, 2009). TBHQ can be used alone or together with BHA or BHT. However, their combined use is not common in products with animal fats (Shahidi & Ambigaipalan, 2015).

 PG: PG has been used as an additive since 1948 in food packaging materials, fat foods, oils, mayonnaise, shortening and baked products. PG prevents the microorganism’s development avoiding respiration and nucleic acid synthesis. The combination of PG with BHA and BHT provides a higher antioxidant capacity than PG as a single antioxidant. Nevertheless, it is not permitted the combination with TBHQ. Despite the recognized antioxidant capacitiy and cytoprotective properties of PG, sometimes it may work as a prooxidative, cytotoxic and genotoxic additive in the presence of Cu(II) according to some studies (Shahidi & Ambigaipalan, 2015).

Figure I.9. Chemical structures of commercial synthetic antioxidants. BHA (A), BHT (B), TBHQ (C) and PG (D) (Sigma-Aldrich, 2019).

I.5.2. ANTIOXIDANTS FROM NATURAL ORIGIN

In the last few years, consumers have been aware of the problem of using synthetic antioxidants due to their potential toxicity (Shahidi & Ambigaipalan, 2015). Thus, food market claims their replacement by natural antioxidants capable to reduce oxidative reactions in meat and meat products with high fat content (Tomović et al., 2017). Recently, scientific community has been using pure compounds, extracts and/or essential oils as natural antioxidants. Within the compounds from natural origin with antioxidant capacities, spices, fruits, vegetal extracts or products derived from oilseeds can be included (Fernandes et al., 2016; Lorenzo & Munekata, 2016; Gómez et al., 2018; Lorenzo et al., 2018a, 2018b, 2018c; Zamuz et al., 2018).

Natural antioxidants can be classified according to nutritional value or solubility. Depending on solubility, antioxidants are divided in hydrophobic (vitamin E and carotenoids) and hydrophilic (vitamin C and phenolic compounds) antioxidants (Armenteros et al., 2012).

24 I. Introduction

I.5.2.1. Hydrophobic antioxidants

 Vitamin E: Vitamin E is a set of phenolic compounds known as tocopherols and tocotrienols. Alpha-tocopherol is the one that has the greatest vitamin potential and is also the most common in nature. This antioxidant is of lipid nature working as a protector of these type of molecules. Thereby, it prevents the peroxidation of PUFAs from the cellular membrane and the peroxidation of LDLs. Likewise, vitamin E neutralizes oxygen and peroxides, and scavenges hydroxyl free radicals and superoxide anions (Armenteros et al., 2012).

 Carotenoids: Carotenoids are a family of around 600 compounds known. They are classified in two groups: carotenes, which are hydrocarbons, and xanthophylls, their oxygenated derivatives (Armenteros et al., 2012). The presence of carotenoids in vegetable foods gives yellowish, orange and reddish colours. The antioxidant power of carotenoids is being investigated currently, although it is already known that some of them can act as antioxidants against free radicals (Krinsky, 1989).

I.5.2.2. Hydrophilic antioxidants

 Vitamin C: Vitamin C, also known as ascorbic acid, is a remarkable antioxidant that acts enhancing the effect of other antioxidants such as vitamin E. Vitamin C acts mainly neutralizing oxygen, scavenging hydroxyl radicals and superoxide anions, and furthermore it recovers the oxidized form of vitamin E (Armenteros et al., 2012).

 Phenolic compounds: The antioxidant capacity of these natural compounds is mainly due to their redox capacity and to their chemical structure, since they can act as reducing agents scavenging free radicals, or as chelating agents. In addition, some phenolic compounds are capable to regenerate other antioxidants, acting in a synergic way with them. Plants, fruits and vegetables possess a great diversity of phenolic compounds (Fernandes et al., 2016; Lorenzo & Munekata, 2016; Gómez et al., 2018; Lorenzo et al., 2018b, 2018c; Zamuz et al., 2018).

Phenolic compounds from vegetables are classified in monomeric and polymeric compounds. On the other hand, monomeric and polymeric compounds can be also further divided in phenolic acids (hydroxybenzoic and hydroxycinnamic acids) and , respectively (Armenteros et al., 2012).

25 I. Introduction

 Monomeric compounds

- Phenolic acids (hydroxybenzoic and hydroxycinnamic acids): Phenolic acids are found in many vegetal species, being the substituted derivatives of hydroxy-benzoic and hydroxycinnamic acids the most abundant (Shahidi & Ambigaipalan, 2015). Hydroxy-benzoic acids possess a chemical structure derived from benzoic acid. The different compounds within this group are generated by hydroxylations and methylations of the aromatic ring. The most common hydroxy-benzoic acids are p-hydroxy-benzoic, vanillic, syringic and protocatechuic acids (Armenteros et al., 2012). Regarding hydroxycinnamic acids, they are located in the plant cell wall, and they are structurally constituted by an aromatic ring, an aliphatic group, and a carboxylic acid at the end of molecule. Their names are due to the hydroxyl group replacement in the aromatic ring. The main hydroxycinnamic acids in nature are caffeic, sinapic, ferulic and p-coumaric acids, being the last two the most abundant (Armenteros et al., 2012). Some vegetable foods contain phenolic acids in the bound form. In this sense, chlorogenic acid, resulted from the caffeic and quinic acid combination, may be the best- known bound hydroxycinnamic acid (Shahidi & Ambigaipalan, 2015).

The reactivity of the phenol moiety (hydroxyl substituent on the aromatic ring) is the responsible for the antioxidant power of phenolic compounds. There are several mechanisms of action, but probably, the main one is the radical scavenging through hydrogen atom donation. Phenolic acids are affected by substituents on the aromatic ring. Consequently, each acid has different antioxidant power (Shahidi & Ambigaipalan, 2015).

Caffeic acid, which is one of the most outstanding natural cinnamic acids, selectively prevents the biosynthesis of leukotrienes, compounds related with immunoregulation diseases, asthma and allergic reactions. In addition, caffeic acid and some of its esters were reported to have antitumour activity against colon cancer (Shahidi & Ambigaipalan, 2015).

- Flavonoids: These compounds have several hydroxyl groups on their structural ring, and they are found as glycosides commonly. Depending on the glycosylation position they can be classified into flavones,

26 I. Introduction

isoflavones and dihydroflavones (glycosylation position at 7-hydroxyl), flavonols and dihydroflavonols (glycosylation position at 3 and 7- hydroxyl), and anthocyanidins (glycosylation position at 3 and 5- hydroxyl) (Armenteros et al., 2012).

The different chemical structure of flavonoids and the relative orientation of some moieties in the molecules promote different biochemical activities. Usually, the antioxidant power of flavonoids depends on three factors: (1) the metal-chelating potential, (2) the existence of hydrogen-/electron-donating substituents capable to reduce free radicals, and (3) the capacity of the to delocalize the unpaired electron promoting the appearance of a stable phenoxyl radical. The antioxidant activity of flavonoids is often favoured by the increase in the number of hydroxyl groups and the decrease in glycosylation (Shahidi & Ambigaipalan, 2015).

Studies reported that flavonoids have beneficial health properties. Some of them have antilipoperoxidant, antitumoral, antiplatelet, anti- ischaemic, anti-allergic, and anti-inflammatory activities (Shahidi & Ambigaipalan, 2015). In addition, epidemiological studies have linked flavonoid intake with less mortality by coronary heart disease (Armenteros et al., 2012).

 Polymeric compounds

- Tannins: Tannins are metabolites widely spread in plant kingdom, with more than 500 Da of molecular mass. They can be organized in condensed and hydrolyzable. Condensed tannins, also called proanthocyanidins, are polymers from flavonoids (from 10 to 20) bound by C-C or C-O links (Armenteros et al., 2012), and hydrolysable tannins are glycosylated gallic acids (Shahidi & Ambigaipalan, 2015). The antioxidant power of tannins was tested in vitro, and results showed inhibition against lipid peroxidation and lipoxygenases. In addition, they are capable to scavenge free radicals (Shahidi & Ambigaipalan, 2015).

Several studies have reported beneficial properties for human health. Some hydrolysable tannins are responsible of the inhibition of the cytopathic effects of HIV, and the expression of HIV-antigen in human

27 I. Introduction

lymphotropic virus type I-positive MT-4 cells (Shahidi & Ambigaipalan, 2015). On the other hand, Amarowicz & Pegg (2013) reported that hydrolyzable tannins have anti-proliferative activities against five lines of carcinoma. The greater the amount of these tannins the greater this activity will be.

I.5.3. NATURAL ANTIOXIDANTS IN FOODS

 Legumes, nuts and oilseeds: Different studies in literature have countersigned the antioxidant properties of many legumes, such as yellow and green peas, lentils, chickpea, common beans, beach bean, fava beans, and yellow and black soybeans (Akbarirad et al., 2016). Phenolic acids, flavonoids and tannins are the main phenolic compounds in these vegetables (Campos-Vega, Loarca-Piña, & Oomah, 2010). The richest legumes in are the varieties with great amounts of dark pigments, such as black gram and red kidney beans (Shahidi & Ambigaipalan, 2015).

Nuts are a rich source of , such as phenolic compounds, flavonoids, isoflavones, terpenes, organosulphuric compounds, and alpha-tocopherol (Shahidi & Ambigaipalan, 2015). The highest phenolic and flavonoid contents in nuts are found in walnuts, peanuts and pistachios (Yang, Martinson, & Liu, 2009). On the other hand, Maguire et al. (2004) reported that the highest contents of vitamin E are in hazelnuts, peanuts and macadamias.

Oilseeds are the most important source of natural antioxidants in plants. They contain a diversity of phenolic compounds with multiple antioxidant activities (Shahidi & Ambigaipalan, 2015). Tocopherol is naturally present in vegetable oils (Akbarirad et al., 2016). For example, sunflower oil contains tocopherol with the 94% in the alpha-tocopherol (vitamin E) form. In addition, sunflower oil has other sterols, such as campesterol and stigmasterol (Akbarirad et al., 2016). On the other hand, soybean, flax, canola, and evening primrose oilseeds are gaining interest in the last few years due to their positive properties on health (Shahidi & Ambigaipalan, 2015).

 Cereals: Cereal grains have high amounts of phenolic acids, especially of ferulic acid. The phenolic acids, vanillic and p-coumaric are also important in grains, although they are in small quantity (Akbarirad et al., 2016). Phenolic acids can be found in the free and esterified forms in wheat, rice, corn and oat (Shahidi & Ambigaipalan, 2015),

28 I. Introduction

being corn which has the highest phenolic content among these cereals, followed by wheat, oat and rice (Adom & Liu, 2002). According to Naczk & Shahidi (2006), the aleurone layer of grains contains the highest concentrations of phenolic acids, although these are also present in the embryos and in the seed layer of grains. Molecules such as catechins are found in cereal grains, being seeds of buckwheat, oats, rye and wheat that have the highest amounts (Akbarirad et al., 2016). Barley kernels contain significant amounts of tocopherol and tocotrienols such as tocols in the germ. On the other hand, carotenoids are not present in most of cereals. However, corn is a source of these antioxidants, showing to be an exception (Akbarirad et al., 2016).

 Fruits and vegetables: Polyphenols are considered as the major antioxidants in fruits; vitamins A, B, C and E, and carotenoids can be found in some fruits, but in less amount (Shahidi & Ambigaipalan, 2015). Most of polyphenols are flavonoids and they are found mainly in the ester and glycoside forms. Small fruits such as berries contain high amounts of polyphenol antioxidants. Indeed, berry fruits are among the top natural sources of phenolic compounds with antioxidant capacity, with levels up to four times higher than other fruits, and even 10 and 40 times higher than in vegetables and cereals, respectively (Shahidi & Ambigaipalan, 2015). Fruits and vegetables are the food with the most levels of flavonoids (Loprinzi & Mahoney, 2015). For example, the amount of quercetin glycoside in the outer leaves of lettuce might reach 237 ppm fresh weight, and the amount of kaempferol glycoside in kale 250 ppm fresh weight (Akbarirad et al., 2016). The richest vegetables in antioxidants are tomato, red pepper, Brassica vegetables, onion, and red beet. Red pepper is a rich source in vitamin C and cryptoxanthin. On the other hand, tomato contains a carotenoid in the peel called lycopene, which is responsible for its red colour. Another case of tuber rich in antioxidants is potato, containing ascorbic acid, alpha- tocopherol and polyphenolic compounds (Akbarirad et al., 2016).

I.5.4. MARINE ALGAE AS A NOVEL SOURCE OF NATURAL ANTIOXIDANTS

The market of marine products is gaining more and more attention due to the consumer concern about the health benefits about having a healthy diet (Roohinejad et al., 2017). Within these products, seaweeds or macro-algae are perfectly appropriated for human consume and animal feed (Gupta & Abu-Ghannam, 2011a). In fact, they have been being used for a long time as dietary supplements and with medicinal purposes (Farvin & Jacobsen, 2013). Currently,

29 I. Introduction they are widely used in Asian cuisine. However, it is not so common their use as a food in Europe and America (Agregán et al., 2017a). Seaweeds are classified in brown algae (phaeophyta), green algae (chlorophyta) and (rhodophyta). The colours of these algae families are due to the presence of pigments. In brown algae the accumulation of xanthophyll and fucoxanthin pigments provides a brownish colour (Gupta & Abu-Ghannam, 2011b). On the other hand, chlorophyll a and b are responsible of the colour of green algae, whose content is in the same ratio than in higher plants. Regarding red algae, its colour is due to the dominance of phycoerythrin and phycocyanin pigments, which mask pigments such as beta-carotene and chlorophyll a. The chemical composition of seaweeds is less known than that of terrestrial plants. However, it is known that they are a good source of protein, carbohydrates and minerals and a remarkable source of vitamins, containing A, B (B1, B2, B3, B5, B9 and B12), C, D, and E vitamins, aside from minerals, with calcium, sodium, phosphorus and potassium (Gupta & Abu-Ghannam, 2011a). The fat amount of seaweeds is around 1-6 g/100 g DW, although some brown algae varieties, such as Hizikia spp. and possess fewer fat contents (0.7-0.9 g/100 g DW). In addition, bioactive compounds, such as polyphenols, carotenoids, terpenoids and tocopherols are also present in macro-algae (Gupta & Abu-Ghannam, 2011a).

Seaweeds are usually exposed to severe environmental conditions. Light, rapid fluctuations in osmotic stress, desiccation and temperatures can lead to form oxidizing agents such as free radicals (Gupta & Abu-Ghannam, 2011a). Despite this fact, seaweeds rarely suffer serious photodynamic damage, what indicate that they have some type of defence against these environmental factors. Indeed, marine macro-algae have been found to be one of the largest sources of natural antioxidants and antimicrobials (Gupta & Abu-Ghannam, 2011a). They synthesize and accumulate some compounds, such as carotenoids, phenolic compounds (e.g. phenolic acids, phlorotannins and catechins), vitamin C and polysaccharides, among others, capable to struggle against these stress conditions (Ospina, Castro-Vargas, & Parada-Alfonso, 2017). There are many examples in literature about the presence of antioxidant compounds in macro-algae. Gallic acid and polyphenols, such as catechin, epicatechin and epigallocatechin gallate, have been found in the green seaweed Halimada (Gupta & Abu-Ghannam, 2011a). López et al. (2011) found gallic, p-coumaric, caffeic, vanillic, ferulic, syringic, gentisic, chlorogenic, and protocatechuic acids in extracts from Stypocaulon scoparium. Fucoxanthin carotenoid and the polyphenols called phlorotannins were found in Hijika fusiformis and Sargassum kjellamanianum, respectively (Gupta & Abu-Ghannam, 2011a). Many authors

30 I. Introduction suggest that phenolic content and antioxidant activity are highly correlated (Roohinejad et al., 2017).

A B C

Figure I.10. Examples of a brown (Bifurcaria bifurcata-A), green (Ulva rigida-B) and red (Cryptopleura ramosa-C) seaweed (Guiry, 2000-2019).

Brown algae are the only organisms in nature that produce phlorotannins (Lopes et al., 2012). These compounds are polyphenols constituted by units of phloroglucinol (1,3,5 trihydroxybenzene) (Wang, Jonsdottir, & Ólafsdóttir, 2009) with a molecular weight between 126 Da and 650 kDa, reaching up to 15% of the dry weight of algae (Koivikko, 2008).

They are biosynthesized through the acetate-malonate pathway, also called polyketide pathway, and located in physodes, a highly refractive and colourless vesicles in marine algae (Kadam, Tiwari, & O’Donnell, 2013). Phlorotannins are dehydro-oligomers or dehydro- polymers of with a skeleton of more than 150 compounds (Koivikko, 2008). Phloroglucinol monomers are linked by aryl-aryl and diaryl ether bonds constituting different groups of phlorotannins. The group of fucols are formed when aromatic rings are linked purely through aryl-aryl bonds. When there are only aryl ether bonds phlorethols are formed. Fuhalols are constituted by phloroglucinol monomers linked by ether bridges arranged in ortho and para positions. An extra hydroxyl group is present in each third ring. Eckols are those that have at least one residue of three rings with a dibenzodioxin compound replaced by a phenoxy group in the fourth carbon. In general, they have a small molecular size, and until now, they have only found in the Alarieae family. The further derivatives of phlorethols which contain a dibenzodioxin moiety are called carmalols. Finally, endofucophlorethols and isofuhalols are specialized groups, whose molecules are small and different (Koivikko, 2008).

Phlorotannins highlight by their strong antioxidant activity, which may be related with their molecular structure. The antioxidant power of polyphenols is largely due to their phenolic rings, whose function is scavenge peroxy, superoxide-anions and hydroxyl radicals. While terrestrial plants have from 3 to 4 rings, phlorotannin skeleton has up to 8 interconnected phenol rings (Wang, Jonsdottir, & Ólafsdóttir, 2009), improving the antioxidant activity. An example of phlorotannins in algae are those produced by H. fusiformis, which are showed to

31 I. Introduction be potential radical scavengers, being therefore antioxidative nutraceuticals (Siriwardhana, Lee, & Jeon, 2005).

Figure I.11. Phlorotannin group structures. Phloretol (A), fuhalol (B), fucol (C), eckol (D) and carmalol (E) (Lopes, 2014).

I.5.5. ADDITION OF MARINE ALGAE AND THEIR EXTRACTS TO FOOD AS ANTIOXIDANTS

Examples of algae or their extracts added to foods in order to prolong their shelf-life are reported in the literature. For instance, the edible marine algae Spaghetti (Himanthalia elongata), Nori (Porphyra umbilicalis) and Wakame (Undaria pinnatifida) were used in the manufacture of low-salt meat emulsion model systems (López-López et al., 2009), providing soluble polyphenolic compounds to these systems, enhancing their antioxidant capacity and thus their shelf-life. Sasaki et al. (2008) reported that the fucoxanthin compounds decreased the TBARS values along storage of cooked ground chicken breasts, improving the colour of the final product. Wang et al. (2010) studied the phlorotannin effectiveness against lipid oxidation in a washed cod muscle system and cod protein isolates during ice storage. Phlorotannins exhibited higher antioxidant activity than crude 80% ethanol extracts, displaying that the

32 I. Introduction efficiency of a 300 ppm level of a phlorotannin enriched sub-fraction was similar to that of 100 ppm PG, which is very effective in muscle foods.

Seaweed extracts were also used in edible oils in order to increase their shelf life (Gupta & Abu-Ghannam, 2011a). Athukorala et al. (2003) assessed the antioxidant potential of an extract from the red alga Grateloupia filicina against lipid peroxidation in linoleic acid and fish oil with positive results. With the same purpose, Siriwardhana et al. (2004) used a methanol extract from the brown alga H. fusiformis, and the results showed a clear antioxidant effect, even higher than that exerted by BHA and BHT. Similarly, Santoso, Yoshie-Stark, & Suzuki (2004) tested methanol extracts from 7 Indonesian seaweeds in a fish oil emulsion system, showing a protective effect as well.

I.6. ANTIMICROBIAL PROPERTIES OF MARINE ALGAE

In addition to antioxidant power, different studies attributed antimicrobial properties to marine algae. Because of this, they are susceptible potentially to be used as an alternative treatment to many infectious diseases (Abu-Ghannam & Rajauria, 2013). The antimicrobial activity of marine algae is caused by the presence of terpene compounds, both of phenolic and lipophilic nature (Gupta & Abu-Ghannam, 2011a). Many examples of seaweed extracts with antimicrobial activity are reported in literature. Gupta, Rajauria, & Abu‐Ghannam (2010) found a 100% inhibition of common pathogenic bacteria in food spoilage using extracts from the brown seaweeds, H. elongata, Laminaria saccharina and Laminaria digitata. Rhimou et al. (2010) assessed the antibacterial activity of 26 species of red algae against three gram-positive and two gram-negative bacteria. The 96% of the extracts tested were effective in at least one of the five microorganisms used, being Staphylococcus aureus the most sensitive. In the same line, Kolsi et al. (2015) found that extracts from thirteen marine macro-algae collected in Tunisia coast showed high antimicrobial activities, specially the brown algae species, against the human pathogenic bacteria, Escherichia coli, Listeria monocytogenes, Salmonella enterica, Agrobacterium tumefaciens, Pseudomonas aeruginosa, S. aureus, and Micrococcus luteus, against the yeasts, Candida tropicalis and Saccharomyces cerevisiae, and against the fungi Aspergillus niger. El Wahidi et al. (2015) reported that several extracts from Morocco Atlantic coast showed inhibitory activities against the pathogenic bacteria, Bacillus subtilis, S. aureus, E. coli, and P. aeruginosa, and against two pathogenic yeasts, Candida albicans and Cryptococcus neoformans. The highest antimicrobial activities were exhibited by the algae, Cystoseira brachycarpa, Cystoseira compressa, Fucus vesiculosus, and Gelidium sesquipedale, which showed to have a broad inhibitory spectrum. Similarly, Eom et al. (2015) reported that the

33 I. Introduction brown alga E. cava is a rich source of antibacterial compounds, when observing that a methanolic extract of this alga exhibited growth inhibition of the microorganism Streptococcus parauberis, an antibiotic resistant specie.

In addition to antioxidative properties of phlorotannins, they also have antibacterial and antifungal activity. Lopes et al. (2012) found antibacterial and antifungal activity in purified extracts of phlorotannins from ten common brown macro-algae from Portugal coast. The Gram-positive bacteria and Staphylococcus epidermidis were the most sensitive bacteria to these extracts, and Trichophyton rubrum the most susceptible fungus. Lopes et al. (2013) also found purified extracts of phlorotannins with antifungal activity. These phlorotannins, belonging to three different brown algae, exhibited fungistatic activity against yeasts and fungicidal against dermatophytes. Although there are works about the antimicrobial properties of extracts from several seaweeds, studies where the efficiency of these extracts are tested in real food, to improve the food safety are barely available.

34

II. JUSTIFICATION AND OBJECTIVES

II. Justification and objectives

The present Doctoral Thesis tried to contribute to the knowledge in the field of natural antioxidants applied to food destined for human consuption. Nowadays, the study of natural extracts from terrestrial plants are much extended and it exists a wide knowledge about their antioxidant potential as scavenge free radicals. On the contrary, extracts from marine ambient are still little known, having few studies published in literature. Therefore, it was decided to explore this potential source of natural compounds with antioxidant power, studying the possible inhibitory effect of oxidation in meat products of three brown macro-algae species, Ascophyllum nodosum, F. vesiculosus and B. bifurcata. In addition, other further investigations about these extracts and from others two species of micro-algae were carried out to complement the previous study. Therefore, the objectives proposed in this Doctoral Thesis were the following:

1. Study of the proximate composition of the algae A. nodosum, F. vesiculosus and B. bifurcata species in order to know their nutritional contribution as foods.

2. Study of the potential antioxidant of aqueous extracts from the previous seaweeds, gaining knowledge about their phenolic contribution and antioxidant activity.

3. Evaluation of the inhibitory effect of the previous aqueous extracts in the oxidation processes which take place in oil (canola) and in a meat complex matrix such as liver pâté, also assessing the possible impact of these extracts in the physicochemical parameters of this latter product.

4. Evaluation of several extraction conditions in order to select the most efficient to assess the antioxidant potential of the extracts from A. nodosum, F. vesiculosus and B. bifurcata and from the microalgae Chlorella vulgaris and Spirulina platensis.

5. Study of the protection capacity against oxidation in pork patties, exerted by the extract with the most antioxidant potential showed in vitro. In addition, the possible impact of this extract in the physicochemical parameters of the pork patties was also assessed.

The achievement of these objectives led to the results presented in this research work, which derived in the publications attached in the annexe included at the end of present memoir, whose references are as follows:

1. Agregán, R., Munekata, P. E. S., Domínguez, R., Carballo, J., Franco, D., & Lorenzo, J. M. (2017). Proximate composition, phenolic content and in vitro antioxidant activity of aqueous extract of the seaweeds Ascophyllum nodosum, Bifurcaria bifurcata and Fucus vesiculosus. Effect of addition of the extracts on the oxidative stability of

37 II. Justification and objectives

canola oil under accelerated storage conditions. Food Research International, 99(Pt- 3), 986-994.

2. Agregán, R., Lorenzo, J. M., Munekata, P. E. S., Domínguez, R., Carballo, J., & Franco, D. (2017). Assessment of the antioxidant activity of Bifurcaria bifurcata aqueous extract on canola oil. Effect of extract concentration on the oxidation stability and volatile compound generation during oil storage. Food Research International, 99(Pt- 3), 1095-1102.

3. Agregán, R., Munekata, P. E. S., Franco, D., Domínguez, R., Carballo, J., & Lorenzo, J. M. (2017). Phenolic compounds from three brown seaweed species using LC-DAD- ESI-MS/MS. Food Research International, 99(Pt-3), 979-985.

4. Lorenzo, J. M., Agregán, R., Munekata, P. E. S., Franco, D., Carballo, J., Şahin, S., Lacomba, R., & Barba, F. (2017). Proximate composition and nutritional value of three macroalgae: Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcata. Marine drugs, 15(11), 360.

5. Agregán, R., Franco, D., Carballo, J., Tomasevic, I., Barba, F. J., Gómez, B., Muchenje, V., & Lorenzo, J. M. (2018). Shelf life study of healthy pork liver pâté with added seaweed extracts from Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcata. Food Research International, 112, 400-411.

6. Agregán, R., Munekata, P. E. S., Franco, D., Domínguez, R., Carballo, J., Muchenje, V., Barba, F. J., & Lorenzo, J. M. (2018). Phenolic content and antioxidant activity of extracts from Bifurcaria bifurcata alga, obtained by diverse extraction conditions

using three different techniques (hydrothermal, ultrasounds and supercritical CO2). Environmental Engineering and Management Journal. Manuscript accepted.

7. Agregán, R., Munekata, P. E. S., Franco, D., Carballo, J., Barba, F. J., & Lorenzo, J. M. (2018). Antioxidant potential of extracts obtained from macro- (Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcata) and micro-algae (Chlorella vulgaris and Spirulina platensis) assisted by ultrasounds. Medicines, 5(2), 33.

8. Agregán, R., Barba, F. J., Gavahian, M, Franco, D., Khaneghah, A. M., Carballo, J., Ferreira, I. C. & Lorenzo, J. M. Fucus vesiculosus extracts as natural antioxidants for improving the physicochemical properties and shelf life of pork patties formulated with oleogels.

38

III. MATERIAL AND METHODS

III. Material and methods

III.1. ALGAL MATERIAL

III.1.1. ORIGIN OF ALGAE

Three brown macro-algae (A. nodosum, F. vesiculosus and B. bifurcata) and two micro- algae (C. vulgaris and S. platensis) were kindly supplied by the companies Portomuiños (A Coruña, Spain) and AlgaEnergy (Madrid, Spain), respectively. The macro-algae species belong to Kingdom Chromista, Phylum Ochrophyta, Class Phaeophyceae and Order Fucales, and were collected in the area of Camariñas (Atlantic coast, A Coruña, Spain). On the other hand, the micro-algae belong to Kingdom Eubacteria, Phylum Cyanobacteria, Class Cyanophyceae and Order Spirulinales, they were not collected, but produced by in vitro culture under controlled conditions.

III.1.2. PREPARATION OF MACRO-ALGAE

Prior to analysis, the macro-algae were subjected to a minimal preparation in order to reduce and homogenize the particle size. They were crushed by a conventional mincer and subsequently passed through a 0.8 mm porous mesh, thus obtaining a uniform powder. This powdery material was packed in plastic bags with 75% vacuum and stored at freezing temperature until further use.

III.1.3. PROXIMATE AND NUTRITIONAL COMPOSITION OF MACRO-ALGAE

III.1.3.1. Experimental design

The macro-algae A. nodosum, F. vesiculosus and B. bifurcata were analysed for moisture, protein and amino acid composition, lipid and fatty acid composition, and for ash and mineral content. In addition, the nutritional quality of seaweed protein was studied. Five samples of each one of the seaweeds were used.

III.1.3.2. Analytical methods

III.1.3.2.1. Moisture content

Moisture content was determined following the ISO (International Organization for Standardization) recommendation ISO 1442:1997 (ISO, 1997). Three grams of sample were dried in an oven (Memmert UFP 600, Schwabach, Germany) at 105 °C until constant weight.

41 III. Material and methods

III.1.3.2.2. Protein content

Protein content was determined following the ISO recommendation ISO 937:1978 (ISO, 1978) based in the Kjeldahl total nitrogen method (total nitrogen content was multiplied x6.25). Five hundred milligrams of ground seaweed were reacted with sulphuric acid H2SO4

(CuSO4 5H2O was used as a catalyst) in a digester (Gerhardt Kjeldatherm KB, Bonn, Germany).

Then, the organic nitrogen was transformed into (NH4)2SO4 and distilled in alkali condition (Gerhardt Vapodest 50 carroused, Bonn, Germany).

III.1.3.2.3. Ash content

Ash content was determined following the ISO recommendation ISO 936:1998 (ISO, 1998). Three grams of seaweed were placed in a muffle furnace (Carbolite RWF 1200, Hope Valley, UK) and treated at a temperature of 600 °C until constant weight.

III.1.3.2.4. Lipid content

Lipids were determined using the method proposed by Ortiz et al. (2006) with some modifications. Twenty grams of seaweed were extracted with 300 mL CHCl3/CH3OH/H2O (1:2:0.8 v/v/v) overnight maintaining the mixture in darkness. Subsequently, 79 mL chloroform, 79 mL water and 5% NaCl were added, obtaining a solvent final ratio of 1: 1: 0.9 v/v/v. Then, the sample was centrifuged at 3000g for 10 min and the chloroform phase was collected and subjected to concentration by means of a rotavapor (Büchi R-200, Oldham, UK) applying vacuum. Fat recovery was measured gravimetrically.

III.1.3.2.5. Amino acid content

The protein hydrolysis was carried out on 100 mg of ground dried alga with 5 mL 6N hydrochloric acid in an ampoule glass sealed for 24 h at 110°C. Then, the solution was diluted with 200 mL of distilled water and filtered through a 0.45 μm filter (Filter Lab, Barcelona, Spain). An aliquot was kept at -20°C until further analysis. Amino acids were derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (Waters AccQ-Fluor reagent kit) and determined by HPLC (Waters 2695 Separations Module + Waters 2475 Multi Fluorescence Detector + Waters AccQ-Tag amino acids analysis column). The amino acid content was expressed as mg/100 g dry matter.

42 III. Material and methods

III.1.3.2.6. Protein quality: Chemical score of amino acids

The CS of the essential amino acids was determined using a protein pattern recommended by FAO/WHO/UNU (2007) as a reference protein, applying the next equation:

the EAA value was also assessed according to the next equation (Shahidi & Synowiecki, 1993):

√

where a, b, c, . . . , j is the content of Phe, Tyr, Val, Met, Thr, Lys, His, Ile and Leu in the seaweeds, ap, bp, cp, . . . , jp is the content of Phe, Tyr, Val, Met, Thr, Lys, His, Ile and Leu in the protein standard (FAO/WHO/UNU, 2007), and n is the number of amino acids used.

III.1.3.2.7. Fatty acid profile

Fifty milligrams of lipid extracted from seaweeds were used to determine the fatty acid profile. Fatty acids were transesterified as follows: 4 mL sodium methoxide (2%) solution were added to the fat vortexing every 5 min for 15 min at room temperature. Then, 4 mL

H2SO4:methanol solution (1:2 v/v) were added and the mixture vortexed a few seconds and again before adding 2 mL distilled water. Organic phase, which contains FAMEs, was extracted with 2.5 mL hexane. A gas chromatograph (GC-Agilent 6890 N; Agilent Technologies Spain, S.L., Madrid, Spain) with a flame ionization detector was used for the separation and quantification of the FAMEs, using the next chromatographic conditions: initial column temperature 120 °C, maintaining this temperature for 5 min, programmed to increase at a rate of 5 °C min up to 200 °C, maintaining this temperature for 2 min, then at 1 °C/min up to 230 °C, maintaining this temperature for 3 min. The injector and detector were maintained at 260 and 280 °C, respectively. Helium was used as the carrier gas at a constant flow-rate of 1.1 mL/min, with the column head pressure set at 35.56 psi. The split ratio was 1:50 and 1 mL of solution was injected. Nonadecanoic acid (C19:0) at 0.3 mg/mL was used as internal standard and added to the samples prior to methylation. Individual FAMEs were identified by comparing their retention times with those of authentic commercial standards (Supelco 37 component FAME Mix, Sigma-Aldrich, Madrid, Spain). Cis-11-Octadecenoic acid (C18:1n-7c) methyl ester (Supelco, Sigma-Aldrich), trans-11-Octadecenoic (C18:1n-7t) methyl ester (Supelco, Sigma- Aldrich) and cis-9, trans-11 conjugated linoleic (CLA) (C18:2n-7) (Matreya LLC, Barcelona,

43 III. Material and methods

Spain) were not included in the commercial mix. Data were expressed in percentage of total fatty acid analysed.

III.1.3.2.8. Mineral profile

The ash of seaweeds was dissolved in 10 mL 1M HNO3. Minerals such as calcium, iron, potasium, magnesium, manganese, sodium, phosphorous, zinc, and copper were determined by ICP-OES, using a Thermo-Fisher ICAP 6000 plasma emission spectrometer (Thermo-Fisher, Cambridge, UK). The standard solutions were prepared at a concentration of 1,000 mg/L. Preliminary tests were carried out to determine the recovery of different quantities of the different added minerals (which were always higher than 97%) and to establish the accuracy of the methods used.

III.1.4. ALGAE EXTRACTION

III.1.4.1. Green extraction

Macro-algae A. nodosum, F. vesiculosus and B. bifurcata were extracted using a green and safe solvent such as water. The ground material was mixed with water in a ratio 1:30 (w/v) and left to stand for 5 min in order to obtain a correct hydration. Then, 800 mL of the alga- water suspension were sonicated for 10 min in an ultrasonic Hielscher UIP1000HD homogenizer (Hielscher Ultrasonics GmbH, Teltow, Germany) equipped with a flow cell for a residence time of 30 s and 90% of amplitude and with continuous recirculation. The process was stopped in case of temperature was raised above 40 °C. Finally, the extract was centrifuged at 2000 ×g and filtered through a cellulose filter of 20-25 μm pore size (Filter-lab, Filtros Anoia, S.A., Barcelona, Spain).

III.1.4.2. Other alternative extraction technologies

Some less-common extraction methods were applied to macro- (A. nodosum, F. vesiculosus and B. bifurcata) and micro-algae (C. vulgaris and S. platensis). According to the alga species, different extraction methods and solvents, as well as extraction times, were applied. This information is presented in Table III.1.

44 III. Material and methods

Table III.1. Extraction conditions used with each alga species. Extraction conditions Alga Method Solvent Time A. nodosum UAE Water/ethanol (50:50 v/v) 30 min F. vesiculosus UAE Water/ethanol (50:50 v/v) 30 min Hydrothermal Water 15 min B. bifurcata UAE Water/ethanol (50:50 v/v) 30 min

SC-CO2 Ethanol 30, 45, 60 min C. vulgaris UAE Water/ethanol (50:50 v/v) 30 min S. platensis UAE Water/ethanol (50:50 v/v) 30 min

III.1.4.2.1. Hydrothermal and ultrasound-assisted extraction

The hydrothermal extraction method was used in the alga B. bifurcata. Firstly, the ground alga was put into a glass bottle with water or water/ethanol (50:50 v/v) in the ratio 1:10 (w/v). Then, the glass bottle was subjected to 121 °C for 30 min in an autoclave (Raypa Stericlav-S 150 L, Terrassa, Spain). On the other hand, the UAE method was used in the macro- algae A. nodosum, F. vesiculosus and B. bifurcata, and in the micro-algae C. vulgaris and S. platensis. In this method, the ground alga was mixed with water, ethanol or water/ethanol (50:50 v/v) in an Erlenmeyer flask in the ratio 1:10 (w/v). Then, the mixture was subjected to ultrasounds in a water bath (Branson ultrasonic M3800-E, Dietzenbach, Germany) for 30 min at room temperature. Once the extraction period was finished, both in the hydrothermal and in the UAE method, the mixture alga-solvent was centrifuged at 3000 ×g/10 min/4 °C and filtered through a cellulose filter of 20-25 μm pore size (Filter-lab, Filtros Anoia, S.A., Barcelona, Spain) to remove the alga solid residues. The resulting extract was collected and stored at -20 °C until further use. An aliquot of the resulting extract from the UAE method using water/ethanol (50:50 v/v) was subjected to evaporation in a rotavapor (Büchi R-200, Oldham, UK) at 40 °C to remove the ethanol fraction. The resulting powder extract was stored at -20 °C for further analysis.

III.1.4.2.2. Extraction technology with supercritical CO2

The protocol followed for the use of supercritical CO2 in the algae B. bifurcata was as follows: twenty five grams of the ground alga were packed with glass beads into an extractor (Thar Designs SFE-1000 F-2-C10, Pittsburgh, USA) with a 1 L cylinder extractor and two 500 mL separators. The CO2 gas was precooled by a circulating bath (PolyScience, model 9506, Niles, IL, USA) and subsequently pumped using a P-200A piston pump (Thar Design Inc) at a flow rate of 25 g CO2/min. Ethanol was used as a co-solvent fluid in the extraction, being pumped by a

45 III. Material and methods

HPLC pump (Scientific Systems, Inc., USA, model Series III) at a suitable flow rate to achieve modifier concentrations of 10%. The extraction was conducted at 40 °C and 35 MPa. The extracts were collected at 30, 45 and 60 min and stored at -20 °C for further analysis.

III.1.5. ALGAE EXTRACT CHARACTERIZATION

III.1.5.1. Extracts from green extraction

III.1.5.1.1. Experimental design

Extracts from the green extraction carried out in the seaweeds A. nodosum, F. vesiculosus and B. bifurcata were obtained in duplicate and analysed for total solids, proteins, total carbohydrates, TPC and antioxidant activity (ORAC, DPPH, ABTS and FRAP assays). BHT was used as a positive control. In addition, the phenolic profile of extracts was determined.

III.1.5.1.2. Analytical methods

III.1.5.1.2.1. Total solid content

The TSC of the extracts was quantified gravimetrically after evaporating the water at 105 °C until constant weight (Talpur et al., 2011).

III.1.5.1.2.2. Protein content

The protein content in the extracts was quantified following the method of Lowry et al.

(1951). One millilitre of extract and 4 mL of a solution of 2% Na2CO3 in 0.1 N NaOH: 0.5%

CuSO4·5H2O in 1% potassium sodium tartrate (50:1 v/v) were placed in a test tube. The mixture was kept for 15 min at darkness and subsequently added with 0.4 mL of the Folin-Ciocalteu reagent diluted with distilled water (1:2 v/v). The tube was stirred and allowed to stand for another 30 min in darkness. Finally, the absorbance was read at 500 nm and converted to concentration by interpolation in a calibration curve constructed using different concentrations of BSA (Sigma-Aldrich, St. Louis, MO, USA). The results were expressed as g protein/100 g dried seaweed extract.

III.1.5.1.2.3. Total carbohydrate content

The total carbohydrate content was assessed by the phenol-sulphuric method (Dubois et al., 1956). An aliquot of extract was placed in a tube and the final volume was made up to 2 mL

46 III. Material and methods

with distilled water. One millilitre of a solution of 5% phenol and 5 mL 95.5% H2SO4 were added to the mixture and subsequently kept at room temperature for 10 min. Then, the sample was stirred and placed in a water bath at 25 °C for 15 min. The absorbance of the mixture was read at 490 nm and converted to concentration using a calibration curve with different concentrations of glucose. The results were expressed as g glucose/100 g dried seaweed extract.

III.1.5.1.2.4. Total phenolic content

The TPC of extracts was determined according to Singleton, Orthofer, & Lamuela- Raventos (1999). One hundred microliters of extract were mixed with 0.75 mL of the Folin- Ciocalteu reagent (diluted 1:10 with water). The mixture was allowed to stand at room temperature for 90 min and subsequently the absorbance was read at 725 nm. A calibration curve was constructed using phloroglucinol (a basic structural unit of phlorotannins) as standard, and the results were expressed as g PGE/100 g dried seaweed extract.

III.1.5.1.2.5. Antioxidant activity

 ORAC assay: The original method of Ou, Hampsch-Woodill, & Prior (2001) was modified as described by Dávalos et al. (2003). The reaction was carried out in 75 mM phosphate buffer (pH 7.4), and the final reaction mixture was 200 mL. The mixture of antioxidant (20 mL) and fluorescein (120mL; 70 nM final concentration) was pre-incubated for 15 min at 37 °C. AAPH solution (60 mL; 12 mM, final concentration) was added rapidly using a multichannel pipette. Then, the plate was immediately placed in a reader and the fluorescence recorded every minute for 120 min with an excitation and emission wavelengths of 485 nm and 520 nm, respectively. The plate was automatically agitated prior each reading. Phosphate buffer was used as a blank reagent. In addition, eight calibration solutions with Trolox as antioxidant were carried out in each assay. The results were calculated on the basis of the differences in areas under the fluorescein decay curve between the blank and the sample and expressed as μmol TE/g dried seaweed extract.

 DPPH free radical scavenging activity assay: The RSA of seaweed extracts for DPPH free radical was determined according to Brand-Williams, Cuvelier, & Berset (1995) with a slight modification: five microliters of diluted samples were mixed with 195 μL of DPPH free radical solution (6 × 10−5 M in methanol) in a 96-well plate. The mixture

47 III. Material and methods

was shaken gently and left to stand at room temperature for 30 min. Thereafter, the absorbance of the samples was measured at 515 nm against methanol using a microplate reader. The DPPH free radical scavenging activity of extracts was calculated from the standard curve of Trolox and expressed as μmol TE/g dried

seaweed extract. The EC50, (amount of antioxidant necessary to decrease the initial DPPH free radical concentration by 50%) was determined by linear regression analysis of the dose response curve plotted between the RSA against extract

concentration. In the samples where it was not possible to calculate the EC50 value, the percentage of inhibition of the DPPH free radical was indicated.

 ABTS radical cation decoloration assay: The method of Re et al. (1999) was adapted to the use of a plate reader. ABTS radical cation was produced by reacting 7 mM ABTS stock solution with 2.45 mM potassium persulfate (final concentration) allowing the mixture to stand in darkness at room temperature for 12-16 h before use. In the next step ABTS radical cation solution was diluted with PBS (pH 7.4) to an absorbance of 0.70 at 734 nm and equilibrated at 30 °C. An aliquot of each sample (with appropriate dilution) was mixed with the solution of ABTS radical cation (sample: ABTS solution relation, 1:100), and the decrease of absorbance was measured after 6 min at 734 nm in a microplate spectrophotometer reader. Trolox was used as the reference standard and the results were expressed as μmol TE/g dried seaweed extract. The percentage inhibition of absorbance was calculated and

plotted as a function of extract concentration to obtain the EC50. In the samples

where it was not possible to calculate the EC50 value, the percentage of inhibition of the radical ABTS radical cation was indicated.

 FRAP assay: The ability to reduce ferric ions was measured using the method described by Benzie & Strain (1996). The FRAP reagent was freshly prepared from 300 mM acetate buffer (pH 3.6), 10 mM TPTZ made up in 40 mM HCl and 20 mM

FeCl3·6H2O solution. All three solutions were mixed together in the ratio of 10:1:1 (v/v/v), respectively. A total of 300 μL of freshly prepared FRAP reagent were added to 10 µL of properly diluted samples and 30 μL of distilled water in a 96-well plate. The mixture was incubated at 37 °C for 8 min. Then, the absorbance was read using a microplate reader at 593 nm against reagent blank. The FRAP value was calculated using a calibration curve plotted using Trolox as standard, and expressed as μmol TE/g dried seaweed extract.

48 III. Material and methods

III.1.5.1.2.6. Phenolic profile

Analysis of phenolic compounds from the seaweed extracts was carried out in an Agilent 1100 HPLC system equipped with G1312B binary gradient pump, G1379A degasser, G1316A column thermostat, G1329A auto-sampler and G1315C DAD (Agilent Technologies, Waldbronn, Germany). Chromatography separation was performed using a Zorbax SB C18 (Agilent Technologies, Inc., Santa Clara, CA, USA) (150 × 3.0 mm I.D., 3.5 μm particle size) column, operating at 25 °C. The mobile phase was composed by acetic acid (2.5% v/v) in water (solvent A), and methanol containing 2.5% acetic acid (solvent B). The extracts were diluted to obtain 2 mg/mL with mobile phase A. The chromatographic process was carried out at a flow rate of 1.0 mL/min with a gradient program as follows: 0 min 95:5 (A:B v/v), 15 min 85:15 (A:B v/v), 35 min 70:30 (A:B v/v), 40 min 60:40 (A:B v/v), 50 min 40:60 (A:B v/v), 55 min 10:90 (A:B v/v), 55.01 min 0:100 (A:B v/v), 75 min 0:100 (A:B v/v). The diferent fractions were measured at the wavelengths of 240 and 370 nm. When the absorbance at these wavelengths exceeded a predetermined value (1% with respect to the baseline), a full spectral data was collected in the range between 190 to 600 nm.

The Agilent 6410B triple quadrupole equipped with an ESI source (Agilent Technologies, Inc., Palo Alto, CA, USA) was used for mass spectrometric analysis. ESI conditions were as follows: temperature 350 °C, nebulizer pressure 35 psi, N2 drying gas flow rate 10 L/min, fragmentor voltage 135 V, and capillary voltage 4500 V. Full mass scan spectra were recorded in negative ionization mode over the range of m/z 100-1600 Da (5 scan/s). The Agilent MassHunter Qualitative Analysis B.04.00 software (Agilent Technologies, Inc., Santa Clara, CA, USA) was used for data acquisition and qualitative analysis.

III.1.5.2. Extracts from alternative extractions (hydrothermal, ultrasound-assisted extraction and supercritical CO2)

III.1.5.2.1. Experimental design

Brown macro-alga B. bifurcata was extracted by three methods (hydrothermal, UAE and

SC-CO2) and using three different solvents (water, ethanol and water/ethanol (50:50 v/v)) depending on the method. The extracts were obtained in duplicate and analysed for extraction yield, TPC and antioxidant activity (ORAC, DPPH, ABTS and FRAP). On the other hand, the macro-algae A. nodosum and F. vesiculosus, and the micro-algae C. vulgaris and S. platensis were extracted by the UAE method using the mixture water/ethanol (50:50 v/v) as solvent. The extracts were obtained in duplicate and analysed for the previously indicated parameters.

49 III. Material and methods

III.1.5.2.2. Analytical methods

III.1.5.2.2.1. Extraction yield

Five milliliters of extract were taken and evaporated in a drying oven at 100 °C overnight. The weight of the final residue was used to calculate the extraction yield by gravimetry. The results were expressed as g extract/100 g DW. The DW was calculated subtracting the moisture content to total weight. Moisture content was measured following the protocol ISO recommended standard ISO 1442:1997 (ISO, 1997).

III.1.5.2.2.2. Total phenolic content

The TPC of the extracts was determined based on the procedure described by Medina- Remón et al. (2009) as follows: fifteen microliters of each extract were mixed with 170 μL of

Milli-Q water, and 12 μL of Folin-Ciocalteu reagent and 30 μL of Na2CO3 were subsequently added. The mixture obtained was kept in darkness for 1 h at room temperature. Once the sample reacted, 73 µL of Milli-Q water were added with a multichannel pipette and the absorbance measured at 765 nm. The TPC was expressed as g PGE/100 g DW.

III.1.5.2.2.3. Antioxidant activity

ORAC, DPPH, ABTS and FRAP assays were already described in the point III.1.5.1.2.5.

III.2. KINETICS OF CANOLA OIL WITH MACRO-ALGAE EXTRACT ADDITION

III.2.1. KINETIC OF CANOLA OIL WITH BIFURCARIA BIFURCATA EXTRACT ADDED AT INCREASING CONCENTRATIONS

III.2.1.1. Experimental design

Canola oil was used to carried out a kinetic in accelerated storage conditions with B. bifurcata extract addition at concentrations of 200, 400, 600, 800 and 1000 ppm. Two positive controls with addition of BHT at 50 and 200 ppm and one negative control without antioxidant addition were used. Two kinetics were performed and samples were analysed in triplicate for p-anisidine value, peroxide value, conjugated dienes and TOTOX at all sampling times (0, 4, 8, 12 and 16 days), and for specific volatile compounds originated as a consequence of lipid oxidation after 16 days. In addition, inhibition of oil oxidation for the previous chemical indices was assessed at all sampling times.

50 III. Material and methods

III.2.1.2. Procedure for obtaining the samples

Canola oil was supplied by a local company (Aceites Abril, Ltd., Ourense, Spain). Composition of canola oil was determined according to CODEX Stan 210 normative (Codex Alimentarius, 2005) and provided by the supplier company as follows: acidity (0.04%), peroxide value (2.29 meq O2/kg oil), moisture (< 0.01%) and impurities (< 0.01%). The fatty acid profile of the oil was determined using standard official methods and also provided by the supplier as follows: myristic acid (0.05%), palmitic acid (4.57%), palmitoleic acid (0.26%), stearic acid (1.61%), oleic acid (63.09%), linoleic acid (19.68%), linolenic acid (8.25%), arachidic acid (0.83%), gadoleic acid (1.14%), behenic acid (0.31%) and lignoceric acid (0.09%). Before applying, B. bifurcata extract suitably weighted according concentration and BHT were separately mixed with 1.5 mL 96% ethanol (v/v) in order to ensure an appropriate dispersion in the oil. The mixture was subjected to ultrasounds in a Branson 8510 ultrasonic water bath (400 W, 100% amplitude) (Emerson Industrial Automation, Eden Prairie, MN, USA) for 10 min, and then added to 20 mL canola oil. The oil with the antioxidants was shaken vigorously for 10 min using a vortexmixer ZX3 (Velp Scientifica Srl, Usmate Velate, Italy) and vacuum evaporated to remove the ethanol. Control sample was prepared in the same way than samples with antioxidant addition. All samples were stored in glass containers at 60 °C and agitated continuously at 100 rpm in an orbital shaker.

III.2.2. KINETIC OF CANOLA OIL WITH ASCOPHYLLUM NODOSUM, FUCUS VESICULOSUS AND BIFURCARIA BIFURCATA EXTRACT ADDITION

III.2.2.1. Experimental design

Canola oil was used to carried out a kinetic in accelerated storage conditions with A. nodosum, F. vesiculosus and B. bifurcata extract addition at a concentration of 500 ppm. One positive control with addition of BHT at 50 ppm and other negative control without antioxidant addition were used. Two kinetics were performed and samples were analysed in triplicate for AV, PV, CD, TOTOX and TBARS at all sampling times (0, 4, 8, 12 and 16 days).

III.2.2.2. Procedure for obtaining the samples

The canola oil used was the same as that of the previous kinetic; accordingly, its origin and its composition, as well as its fatty acid profile were also the same. The obtaining procedure of samples was performed in the same way as in the previous kinetic.

51 III. Material and methods

III.2.3. ANALYTICAL METHODS

III.2.3.1. Determination of lipid oxidation

III.2.3.1.1. P-anisidine value

The AV of the oil samples was performed following an IUPAC method (Paquot & Hautfenne, 1987). Oil samples (0.5-2 g) were dissolved with isooctane in 25 mL volumetric flasks. The samples were then reacted with p-anisidine solution in acetic acid (0.25% w/v) for 10 in darkness to produce a coloured complex. Absorbances of the samples with and without p-anisidine solution were read using an UV-1800 UV spectrophotometer (Shimadzu Corporation, Kyoto, Japan) at 350 nm, and the parameter AV was calculated as:

( )

where Eb is the net absorbance of the oil-solution, Ea is the net absorbance of the oil- anisidine-solution, and W is the weight of the sample (g).

III.2.3.1.2. Peroxide value

The PV was determined following the AOAC procedure 965.33 (AOAC, 2000). Oil samples (0.5 g) were dissolved with 10 mL trichloromethane, and subsequently 15 mL acetic acid and 1 mL KI saturated aqueous solution were added. The samples were slightly agitated for 1 min and kept in darkness for 5 min. After this incubation time, the samples were added with 75 mL distilled water and vigorously shaken. Finally, liberated iodine was titrated with 0.01 N sodium thiosulfate in an automatic titrator. The PV, expressed as meq O2/kg oil, was calculated according to the formula:

where V is the volume (mL) of sodium thiosulfate consumed in the titration, N is the normality of the sodium thiosulfate solution, and W is the weight of the sample (g).

III.2.3.1.3. Total oxidation value

The overall oxidation state of the oil samples is given by the TOTOX value and was calculated according to the formula:

52 III. Material and methods

III.2.3.1.4. Conjugated dienes

The CD were quantified using an UV-1800 UV spectrophotometer (Shimadzu Corporation, Kyoto, Japan) at a wavelength of 233 nm. Before analysis, 20 μL of oil were diluted with hexane in 25 mL volumetric flasks. The CD, expressed as percentage of conjugated dienoic acid, were calculated as follows:

where As is the absorbance observed at 233 nm, b is the cell length in cm, c is the concentration of the diluted sample (g/L), and K0 is the absorptivity by acid or ester groups. In this case, c was considered as 0.5 g/L, the final concentration for most edible oils; and K0 was considered for acids (value of 0.03).

III.2.3.1.5. Thiobarbituric acid-reactive substances

The TBARS were determined following the method of Shahidi, Desilva, & Amarowicz (2003). Two hundred milligrams of sample were dissolved in 1-butanol (ACS grade) into a 25 mL volumetric flask and vortexed thoroughly. A portion of 5 mL of this solution was transferred to a dry test tube, and 5 mL of fresh TBA reagent (200 mg TBA in 100 mL 1-butanol) were added. Then, the tube was placed in a 95 °C water bath for 120 min where a colorimetric reaction took place. Finally, the sample was cooled at room temperature and the absorbance read at 532 nm. The TBARS value was calculated as follows:

where A is the absorbance at 532 nm, and m is the weight of the sample (g).

III.2.3.2. Inhibition of oil oxidation

The IP of oil oxidation was calculated according to the next formula:

where “Δindex in sample” was the increase of the value of the concrete index in the sample, and “Δindex in control” was the increase of the value of the same index in the control batch.

53 III. Material and methods

III.2.3.3. Analysis of volatile compounds

The extraction of volatile compounds was performed using SPME. A SPME device (Supelco, Sigma-Aldrich, Bellefonte, PA, USA) containing a fused-silica fibre (10 mm length) coated with a 50/30 μm thickness of DVB/CAR/PDMS was used. A portion of sample (0.36 g) was weighted into a 40 mL vial, which was subsequently screw-capped with a laminated teflon-rubber disk. Before the extraction, the fibre was conditioned by heating in a gas chromatograph injection port at 270 °C for 60 min. The extraction was carried out in an oven at 60 °C in two phases. In the first phase (15 min) the sample was equilibrated at the extraction temperature in order to ensure an homogeneous temperature for sample and headspace, and in the second, the fibre was inserted into the sample vial through the septum and then exposed to headspace. Once sampling was finished, the fibre was withdrawn into the needle and transferred to the injection port of the GC-MS system.

A gas chromatograph 6890 N (Agilent Technologies, Santa Clara, CA, USA) equipped with a mass selective detector 5973 N (Agilent Technologies) was used with a DB-624 capillary column of 30 m × 0.25 mm id, 1.4 μm film thickness (J&W Scientific, Folsom, CA, USA). The SPME fibre was desorbed and maintained in the injection port at 260 °C during 8 min. The sample was injected in splitless mode. Helium was used as a carrier gas with a linear velocity of 40 cm/s. The temperature program was firstly isothermal for 10 min at 40 °C, raised to 200 °C at a rate of 5 °C/min, then raised to 250 °C at a rate of 20 °C/min, and finally held for 5 min (total run time of 49.5 min). Injector and detector temperatures were both set at 260 °C. The mass spectra were obtained using a mass selective detector working in electronic impact at 70 eV, with a multiplier voltage of 1953 V and collecting data at a rate of 6.34 scans/s over the range m/z 40-300.

Compounds were identified by comparing their mass spectra with those contained in the NIST05 (NIST, Gaithersburg, MD, USA) library (> 80% of coincidence) and/or by calculation of retention index relative to a series of standard alkanes (C5-C14) (for calculating Kovats indexes, Supelco 44,585-U, Sigma-Aldrich, Bellefonte, PA, USA) and matching them with data reported in the literature.

54 III. Material and methods

III.3. KINETICS OF MEAT PRODUCTS WITH MACRO-ALGAE EXTRACT ADDITION

III.3.1. KINETIC OF A PORK LIVER PÂTÉ WITH ASCOPHYLLUM NODOSUM, FUCUS VESICULOSUS AND BIFURCARIA BIFURCATA EXTRACTS ADDITION AND PARTIAL FAT REPLACEMENT BY SEED OILS

III.3.1.1. Experimental design

A pork liver pâté with 50% of pork backfat replaced by canola and high-oleic sunflower oils was manufactured in triplicate. Three batches of pâté were elaborated with A. nodosum, F. vesiculosus and B. bifurcata extract at a concentration of 500 ppm. One positive control batch with addition of BHT at 50 ppm and other negative without antioxidant addition were used. Three samples per batch were analysed for proximate composition (moisture, protein and fat) and fatty acid profile at 0 day, for physicochemical parameters (colour and pH), lipid oxidation (TBARS and CD) and protein oxidation at all sampling times (0, 45, 90, 135 and 180 days), and for volatile compounds at the beginning and at the end of storage (180 days). In addition, microbial analysis was performed at all sampling times.

III.3.1.2. Procedure for obtaining the samples

Pâtés were manufactured with the ingredients and amounts presented in Table III.2. The protocol was as follows: firstly, the fat and liver were chopped for 2 min in a cutter (Talsa, mod K30, Valencia, Spain) at 4 °C, until obtaining a homogeneous meat batter. During this step, salt, sodium nitrite and sodium ascorbate were added slowly. The pre-batter obtained was kept in darkness for 24 h. On the other hand, the backfat was pre-cooked at 70 °C (core temperature) in a hot water bath for 15 min. This fat, canola and high-oleic sunflower oils (supplied by the company Aceites Abril, Ltd., Ourense, Spain), water, seaweed extracts (obtained using green extraction) or BHT according to the batch, and sodium caseinate were mixed in the cutter until forming a pre-emulsion (~1min), maintaining all the time at temperature above 40 °C. Then, this pre-emulsion and the pre-batter previously manufactured were mixed along with milk powder and potassium phosphate until obtaining a homogenous batter (~1-2 min). Pâtés were manually distributed into cans until full filling (~100 g) and hermetically closed. Then, the cans were subjected to a thermal treatment (78 °C/75 min), and subsequently immersed in a blast chiller (-21 °C/30 min). Finally, the cans were stored under refrigerated conditions (4 °C).

55 III. Material and methods

Table III.2. Ingredients and amounts used for the preparation of pork liver pâtés. Ingredient (g/100 g) Batch CON BHT ANE FVE BBE Pork liver 33 33 33 33 33 Pork lean meat 20 20 20 20 20 Pork backfat 15 15 15 15 15 Cold water 11.43 11.43 11.38 11.38 11.38 Canola oil 11.25 11.25 11.25 11.25 11.25 High-oleic sunflower oil 3.75 3.75 3.75 3.75 3.75 Seaweed extract 0 0 0.05 0.05 0.05 BHT 0 0.005 0 0 0 NaCl 2 2 2 2 2 Milk powder 2 2 2 2 2 Sodium caseinate 1 1 1 1 1 Potassium phosphate 0.5 0.5 0.5 0.5 0.5 Sodium nitrite 0.05 0.05 0.05 0.05 0.05 Sodium ascorbate 0.025 0.025 0.025 0.025 0.025

III.3.2. KINETIC OF PORK PATTIES WITH FUCUS VESICULOSUS EXTRACT ADDITION AND PARTIAL FAT REPLACEMENT BY LINSEED OIL OLEOGEL

III.3.2.1. Experimental design

Pork patties with the 50% of fat added in form of linseed oil oleogel were manufactured. Three batches of patties were manufactured with F. vesiculosus extract addition at three different concentrations (250, 500 and 1000 ppm). One positive control batch with addition of BHT at 200 ppm and other negative without antioxidant addition were used. Four samples per batch were analysed for proximate composition (moisture, protein, ash and fat) and fatty acid profile at 0 day, for physicochemical parameters (colour and pH), oxidation parameters (TBARS and protein oxidation) after 11, 15 and 18 days of storage. In addition, a visual and odour acceptance test and another preference test were carried out at 0, 7, 11, 15 and 18 days and at the beginning of storage, respectively.

III.3.2.2. Linseed oil oleogel obtaining

The oleogel used in the manufacturing of patties is constituted by an oil phase and a mixture of two structurants (ɣ-oryzanol and β-sitosterol). The oil phase used was a commercial linseed oil Vitaquell® with 72% PUFAs (~55% α-linoleic), 19% MUFAs and 9% SFAs. Ɣ-oryzanol and β-sitosterol were mixed in the ratio 60:40 (w/w). This ratio corresponds to 1:1 M of each component and was reported as the ratio that allows to obtain a firmest transparent gel. The

56 III. Material and methods

ɣ-oryzanol was purchased from Oryza Co. (Japan) and the β-sitosterol from Sigma-Aldrich (France). Both structurants were dispersed by stirring until solubilisation along with the linseed oil at a temperature of 80 °C for 30 min. Then, the mixture was left cooling at room temperature until gel formation.

III.3.2.3. Procedure for obtaining the samples

Pork patties were manufactured as follows: firstly, the meat was chopped in a mincer machine (La Minerva, Bologna, Italy) using a 6 mm plate under refrigeration conditions. After chopping, the meat was mixed with the linseed oil oleogel, salt and water according to amounts indicated in Table III.3. The amount of fat in the pork patties was 10% (w/w), with the 5% in the form of oleogel. BHT and seaweed extract (obtained using the UAE method and water/ethanol (50:50 v/v)) were previously re-dissolved in 100% ethanol and in water/ethanol (50:50 v/v), respectively. Specific amounts of these solvents were also added to all batches in order to balance their amounts, having the same amount of ethanol and water in all of them. The batches were covered completely and left to stand for 60 min at refrigeration temperature. After this time, 60 g of the meat preparation was taken, and with the help of a burger maker (A-2000, Gaser, Girona, Spain), patties were formed and subsequently packed in polystyrene trays of 300 mm of thickness with 80% O2 and 20% CO2, using a heater sealer (LARI3/Pn T-VG-R-SKIN, Ca. Ve.Co., Palazzolo, Italy). Polyethylene film of 74 mm of thickness and permeability < 2 mL/(m2 bar/day) appropriate for gas mixture (Viduca, Alicante, Spain) was used. The trays were placed on metal shelving in a room illuminated in order to simulate supermarket conditions, at a temperature of 2 ± 1 °C. Lux values varied in the range of 15-20 in function of the tray position (HT 306, Digital luxometer, Italy). On the other hand, the light source used was conventional, which means that no wavelength (UV range) was filtered.

Table III.3. Ingredients and amounts used for the preparation of pork patties. Ingredient (g/100 g) Batch CON BHT FVE-250 FVE-500 BBE-1000 Pork loin 87 87 87 87 87 Water 6.96 6.96 6.96 6.96 6.96 Linseed oil oleogel 5 5 5 5 5 Salt 1.04 1.04 1.04 1.04 1.04 Seaweed extract 0 0 0.025 0.05 0.1 BHT 0 0.02 0 0 0

57 III. Material and methods

III.3.3. ANALYTICAL METHODS

III.3.3.1. Proximate composition

Moisture, protein and ash contents were quantified following the ISO recommended standard protocols ISO 1442:1997 (ISO, 1997), ISO 937:1978 (ISO, 1978) and ISO 936:1998 (ISO, 1998), respectively. Fat was extracted according to the AOCS Official Procedure Am 5-04 (AOCS, 2005) using an extractor Akom XT10 (ANKOM Technology Corp., Macedon, NY, USA).

III.3.3.2. Fatty acid profile

Fat was extracted by crushing 10 g of sample with an IKA T25 digital ultra-turrax (IKA®- Werke GmbH & Co. KG, Staufen, Germany), using 18 mL chloroform and 20 mL methanol as solvents. Then, 10 mL (1% NaCl) were added to enhance the phase separation, and the sample was centrifuged at 4000 ×g/10 min. The chloroform fraction was recovered and subsequently evaporated under vacuum at 56 °C in a rotavapor (Büchi R-200, Oldham, UK). The fat extracted was stored at -80 °C until FAME analysis. Fifteen milligrams of fat were used to determine the fatty acid profile. Fatty acids were transesterified according to the point III.1.3.2.7. A GC- Agilent 6890 N (Agilent Technologies Spain, S.L., Madrid, Spain) with a flame ionization detector was used for the separation and quantification of the FAMEs, using the chromatographic conditions described in the point III.1.3.2.7. Nonadecanoic acid at 0.3 mg/mL was used as internal standard and added to the samples prior methylation. Individual FAMEs were identified according to the point III.1.3.2.7. Data were expressed as percentage of total fatty acids identified.

III.3.3.3. Microbial analysis

Ten grams of sample were aseptically placed into a sterile bag and homogenized with 90 mL sterile 0.1% peptone water in a masticator blender (IUL Instruments, Barcelona, Spain) for 2 min at room temperature. Serial decimal dilutions of each sample were prepared in 0.1% peptone water solution, and duplicate 1 mL or 0.1 mL of samples of the initial homogenate and of appropriate dilutions were poured or spread onto agar plates. TVC were enumerated on PCA (Oxoid, Unipath Ltd., Basingstoke, UK) after incubation at 30 °C for 72 h. LAB on the de Man- Rogosa-Sharpe agar (Oxoid, Unipath Ltd., Basingstoke, UK) (pH 5.6) after incubating at 30 °C for 5 days. Pseudomonas spp. in Pseudomonas agar base (Merck, Darmstadt, Germany after incubation at 25 °C for 48 h. Molds and yeasts on OGYE agar (Merck, Darmstadt, Germany)

58 III. Material and methods after being incubating at 25 °C for 5 days. After incubation, the plates with 30-300 colonies were counted, and the results were expressed as number of CFU/g.

III.3.3.4. Physicochemical parameters (colour and pH)

Colour parameters were measured using a portable colorimeter Chroma Meter Cr-600d (Konica Minolta Sensing, Inc., Osaka, Japan) with a pulsed xenon arc lamp filtered to illuminant D65 lighting conditions, and with 0° viewing angle geometry and 8 mm aperture size in the CIELAB space: lightness, (L*); redness, (a*); yellowness, (b*) (CIE, 1978). Before measuring, the colorimeter was adjusted using a white ceramic tile. The colour measures were performed in three different points on the sample surface. On the other hand, pH values were determined using a digital pH meter (model 710 A+, Thermo Orion, Cambridgeshire, UK) equipped with a penetration probe.

III.3.3.5. Determination of lipid oxidation (conjugated dienes and thiobarbituric acid-reactive substances)

Lipid stability was evaluated by studying the evolution of the CD (primary products of lipid oxidation) and the TBARS (secondary products of lipid oxidation).

The CD were quantified as follows: half gram of sample was weighted in a 15 mL centrifuge tube and added with 5 mL distilled water. The tube content was homogenized with an IKA T25 digital ultra-turrax (IKA®-Werke GmbH & Co. KG, Staufen, Germany) and 15 mL were taken and poured in a new tube. Then, 5 mL hexane:isopropanol (3:1 v/v) were added and vortexed. Finally, the mixture was centrifuged at 3000 ×g/5 min and the absorbance measured at 233 nm in a spectrophotometer (Shimadzu Corporation, Kyoto, Japan).

TBARS were calculated according to the method described by Pateiro et al. (2014) with slight modifications. Two grams of sample were dispersed in 10 mL 5% trichloroacetic acid and homogenized in an IKA T25 digital ultra-turrax (IKA®-Werke GmbH & Co. KG, Staufen, Germany) for 2 min. The homogenate was kept at refrigeration temperature for 10 min and subsequently centrifuged at 2360 ×g/10 min. Then, the supernatant was filtered through a Whatman No. 1 filter paper, and 5 mL of the filtrate was mixed with 5 mL 0.02 M TBA reagent in a dried test tube, letting the mixture react in a water bath at 96 °C for 40 minutes. After this incubation period, the tube was cool in running tap water and sonicated 5 min in a water bath (Branson ultrasonic M3800-E, Dietzenbach, Germany) in order to remove the gas formed

59 III. Material and methods during reaction. Finally, the absorbance was read at 532 nm. TBARS were quantified from a standard curve of MDA with TEP and expressed as mg MDA/kg sample.

III.3.3.6. Determination of protein oxidation

Protein oxidation was determined by measuring the carbonyl formation. Two and a half grams of sample were dispersed in 20 mL 0.6M NaCl using an IKA T25 digital ultra-turrax (IKA®- Werke GmbH & Co. KG, Staufen, Germany). Then, 100 µL of the homogenate were taken and poured in a 1.5 mL Eppendorf tube. This step was performed twice, having therefore two tubes per sample. Then, 1 mL 10% TCA was added in both tubes inducing protein precipitation. The tubes were vortexed 30 s and centrifuged at 5000 ×g/5 min. The supernatant was removed and 1 mL 2M HCl was added to one of the tubes and 1 mL DNPH 0.2% dissolved in 2M HCl to the another tube. Then, they were kept 1 h in the darkness, agitating every 20 min. After this time, 0.8 mL 10% TCA was added, leaving to stand the tubes 15 min at refrigeration temperature. After, the tubes were vortexed 30 s and centrifuged at 5000 ×g/5 min. The resulting pellet in each of the tubes was resuspended with 1 mL ethanol/ethyl acetate (1:1 v/v), left to stand 5 min and centrifuged at 10000 ×g/5 min. This step was repeated three times. After this washing process, the pellets were dried by applying N2 gas stream and dissolved in 1.5 mL 6M guanidine in 20 mM sodium phosphate buffer, centrifuging the tubes to remove the non-soluble fraction. The absorbance of the samples was read at 370 and 280 nm by an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Santa Clara, USA) using 6M guanidine in 20 mM sodium phosphate buffer as blank. Protein oxidation was quantified from a standard curve performed with BSA and the results expressed as nmol carbonyl/mg protein.

III.3.3.7. Analysis of volatile compounds

The extraction of volatile compounds was performed using solid-phase microextraction (SPME). A SPME device (Supelco, Sigma-Aldrich, Bellefonte, PA, USA) containing a fused-silica fibre (10 mm length) coated with a 50/30 μm thickness of DVB/CAR/PDMS was used. One gram of sample was weighted into a 40 mL vial, which was subsequently screw-capped with a laminated teflon-rubber disk. Both the extraction procedure and equipment characteristics were described in the point III.2.3.3. The volatile compounds were identified according to the point III.2.3.3.

60 III. Material and methods

III.3.3.8. Sensory evaluation of raw and cooked pork patties

Sensory analysis were carried out in raw and cooked patties with sixteen panelists selected from the Centro Tecnolóxico da Carne (Ourense, Spain). Acceptance and preference tests were performed. In the first test, panelists accept or reject samples while in the second one, panelists select the order of the samples according to their organoleptic preferences. Raw samples were sensory assessed at 0, 7, 11, 15 and 18 days of storage by an acceptance test, while cooked samples were only assessed at day 0 by an acceptance test and other of preference. In the acceptance test, the panelist scored samples based on colour, discoloration at surface and odour, using a 5-point hedonic scale (1 = excellent, 5 = not acceptable), and in the preference, panelists scored giving the number 1 to the favourite sample, the number 2 to the next one and so on.

Sensory tests with cooked samples were carried out in a cabin (one per panelist) illuminated with white and red light. Samples were cooked in an oven (Rational Combimasterplus CMP61, Germany) until they reached a core temperature of 70 °C. Afterward they were given on disposable plastic dishes encoded with 3-digit random numbers (Macfie et al., 1989). At the beginning of tests and among samples, water and unsalted toasted bread were provided in order to remove waste and residual flavours of mouth.

III.4. STATISTICAL ANALYSIS

The values were given in terms of mean values ± standard deviations when it was required. Statistical analysis of the results were conducted using the IBM SPSS Statistics 23.0 program (IBM Corporation, Somers, NY, USA). After verification of normal distribution and constant variance of data, significant differences (P < 0.05) were determined using one-way ANOVA. The LSM were separated using Duncan’s post-hoc test (significance level P < 0.05).

61

IV. RESULTS AND DISCUSSION

IV. Results and discussion

IV.1. PROXIMATE AND NUTRITIONAL COMPOSITION OF MACRO-ALGAE ASCOPHYLLUM NODOSUM, FUCUS VESICULOSUS AND BIFURCARIA BIFURCATA

Macro-algae A. nodosum, F. vesiculosus and B. bifurcata were employed as natural antioxidant materials for the development of this researching work. Before being used in experiments, they were characterized nutritionally in order to know their contribution as foods.

IV.1.1. PROXIMATE COMPOSITION OF MACRO-ALGAE

The proximate composition of seaweeds is presented in Table IV.1. The moisture content showed significant (P < 0.001) in the macro-algae studied. The lowest value (7.95%) was reached by the B. bifurcata. This result is similar to others reported in literature in edible seaweeds (Gómez-Ordóñez, Jiménez-Escrig, & Rupérez, 2010; Rodrigues et al., 2015).

F. vesiculosus was found the seaweed with the highest protein content (12.99 g/100 g DW), followed by B. bifurcata (8.92 g/100 g DW) and A. nodosum (8.70 g/100 g DW). These results are in agreement with those reported by Fleurence (1999) who alsofound low protein amounts (< 15 g/100 g DW) in most of the brown seaweeds industrially exploited (F. vesiculosus, A. nodosum, L. digitata and H. elongata). The values presented in this study were lower than those reported by the previous author Fleurence (1999) in other seaweed species, such as Porphyra tenera (47 g/100 g DW) and Palmaria palmata (35 g/100 g DW) but higher than those found by Sánchez-Machado et al. (2004) (5.46 g/100 g DW) in H. elongata.

Table IV.1. Proximate composition of seaweeds studied (mean ± standard deviation value) (n = 5). Seaweed SEM Sig. A. nodosum F. vesiculosus B. bifurcata Moisture (g/100 g algae) 11.08 ± 0.53a 11.23 ± 0.08a 7.95 ± 0.06b 0.41 *** Protein (g/100 g DW) 8.70 ± 0.07a 12.99 ± 0.04b 8.92 ± 0.09c 0.53 *** Lipid (g/100 g DW) 3.62 ± 0.17a 3.75 ± 0.20a 6.54 ± 0.27b 0.36 *** Carbohydrate (g/100 g DW) 56.79 ± 0.72a 62.55 ± 0.13b 52.86 ± 0.34c 1.07 *** Ash (g/100 g DW) 30.89 ± 0.06a 20.71 ± 0.04b 31.68 ± 0.41c 1.34 *** a-cMeans in the same row not followed by a common superscript letter are significantly different (P < 0.05; Duncan test). ***(P < 0.001).

The macro-algae estudied presented lipid contents significantly (P < 0.001) differents. The highest values were found in B. bifurcata (6.54 g/100 g DW) which were close to those found by Peinado et al. (2014), Gómez-Ordóñez, Jiménez-Escrig, & Rupérez (2010) and Alves et al.

65 IV. Results and discussion

(2016) in brown macro-algae. On the other hand, ash content ranged from 20.71% DW to 31.68% DW. These findings are in agreement with others reported in literature (Peinado et al., 2014; Gómez-Ordóñez, Jiménez-Escrig, & Rupérez, 2010; Alves et al., 2016).

IV.1.2. MINERAL CONTENT OF MACRO-ALGAE

The mineral content of seaweeds is presented in Table IV.2. Potassium (3745.35-9316.28 mg/100 g DW) was the main mineral in the three seaweeds studied, followed by sodium (1836.82-4575.71 mg/100 g DW) and calcium (984.73-1160.27 mg/100 g DW). A similar trend was reported by other authors (Chan & Matanjun, 2017; Kumar et al., 2011; Matanjun et al., 2009) who found that potassium was the most abundant mineral, followed by sodium. On the other hand, B. bifurcata presented a Na/K ratio lower than those observed in the other seaweeds, hence, it could be used to regulate the Na/K ratio of diets, since this ratio and the hypertension incidence are closely linked (Li, 2009).

The levels of magnesium in the three seaweed ranged from 528.04 mg/100 g DW to 867.82 mg/100 g DW for B. bifurcata and A. nodosum, respectively, being significantly (P < 0.001) different among species.

Table IV.2. Mineral profile of seaweeds studied (mean ± standard deviation value) (n = 5). Mineral (mg/100 Seaweed SEM Sig. g DW) A. nodosum F. vesiculosus B. bifurcata Calcium (Ca) 984.73 ± 47.26a 1160.27 ± 23.10b 996.42 ± 12.83a 23.63 *** Iron (Fe) 13.34 ± 0.90a 18.99 ± 0.32b n.q. 2.28 *** Potassium (K) 3781.35 ± 13.40a 3745.05 ± 36.01a 9316.28 ± 101.94b 738.43 *** Magnesium (Mg) 867.82 ± 12.01a 732.37 ± 5.35b 528.04 ± 8.25c 38.27 *** Manganese (Mn) 1,96 ± 0.69a 8,28 ± 1,07b n.q. 1009.00 *** Sodium (Na) 4575.71 ± 50.05a 2187.51 ± 36.90b 1836.82 ± 52.12c 324.02 *** Phosphorous (P) n.q. 193.57 ± 1.13a 169.54 ± 1.41b 22.92 *** Zinc (Zn) n.q. n.q. n.q. Copper (Cu) n.q. n.q. n.q. Total 10224.91 ± 64.32a 8045.96 ± 94.44b 12848.97 ± 142.01c 564.24 *** a-cMeans in the same row not followed by a common superscript letter are significantly different (P < 0.05; Duncan test). ***(P < 0.001).

On the other hand, calcium content also showed significant (P < 0.001) differences among seaweeds, being F. vesiculosus which presented the highest value (1160.27 mg/100 DW). Phosphorous, the macro-mineral with the lowest content, was also detected in F. vesiculosus and B. bifurcata, with 193.57 mg/100 g DW and 169.54 mg/100 g DW, respectively.

A. nodosum and F. vesiculosus also contained iron (13.34 mg/100 g DW and 18.99 mg/100 g DW, respectively) and manganese (1.96 g DW and 8.28 mg/100 g DW, respectively). In this

66 IV. Results and discussion regard, F. vesiculosus may be suitable in diets to provide the necessary daily intake of iron and thus prevent anemia (Allen et al., 2006).

IV.1.3. AMINO ACID CONTENT OF MACRO-ALGAE

The amino acid composition of seaweeds is summarized in Table IV.3.

Table IV.3. Amino acid profile of the three seaweeds studied (mean ± standard deviation value) (n = 5). Seaweed Amino acid (mg/100 g DW) SEM Sig. A. nodosum F. vesiculosus B. bifurcata EAAs Threonine 363.22 ± 17.12a 613.08 ± 33.62b 360.27 ± 38.25a 39.23 *** Valine 353.89 ± 32.95a 582.70 ± 36.73b 372.82 ± 49.05a 35.54 *** Methionine 147.59 ± 18.71a 218.21 ± 20.20b 178.41 ± 18.08a 11.09 ** Isoleucine 295.26 ± 25.73a 507.82 ± 32.42b 299.73 ± 37.74a 33.36 *** Leucine 537.37 ± 38.87a 862.14 ± 57.02b 524.59 ± 61.38a 52.48 *** Phenylalanine 340.13 ± 17.74a 541.53 ± 25.72b 330.05 ± 32.32a 32.21 *** Lysine 431.72 ± 38.40a 800.28 ± 74.20b 393.06 ± 56.57a 61.09 *** Histidine 126.46 ± 10.65a 194.59 ± 8.73b 138.76 ± 12.70a 10.20 *** Arginine 316.79 ± 14.05a 557.87 ± 38.44b 330.11 ± 42.41a 37.09 *** Total EAAs 2912.42 ± 204.93a 4878.22 ± 304.12b 2927.79 ± 346.84a 309.35 *** NEAAs Tyrosine 162.85 ± 24.50a 327.01 ± 30.59b 175.00 ± 30.90a 25.58 *** Asparagine 846.64 ± 38.87a 1677.01 ± 156.39b 800.84 ± 105.55a 133.19 *** Serine 378.62 ± 13.57ab 630.54 ± 47.00a 357.10 ± 36.87b 41.05 *** Glutamic acid 1714.55 ± 133.17a 1974.47 ± 150.67b 1504.53 ± 178.74a 74.18 * Glycine 417.70 ± 12.89a 651.24 ± 30.84b 390.14 ± 29.42a 38.27 *** Alanine 655.73 ± 34.75a 985.40 ± 69.50b 846.65 ± 82.87c 49.27 *** Proline 399.24 ± 11.70a 575.19 ± 39.15b 318.40 ± 40.96c 35.15 *** Cysteine 0.00 ± 0.00a 205.23 ± 25.43b 0.00 ± 0.00a 31.58 *** Total NEAAs 4575.33 ± 198.91a 7026.10 ± 512.60b 4392.67 ± 502.38a 403.29 *** Total AA 7487.76 ± 400.31a 11904.32 ± 816.67b 7320.46 ± 848.14a 711.84 *** Relative amount EAAs (%) 38.87 ± 0.71a 40.99 ± 0.26b 39.99 ± 0.31c 0.33 ** a-cMeans in the same row not followed by a common superscript letter are significantly different (P < 0.05; Duncan test). *(P < 0.05); **(P < 0.01); ***(P < 0.001).

The total amino acid contents were 7.48, 11.90 and 7.32 g/100 g DW (P < 0.001), for A. nodosum, F. vesiculosus and B. bifurcata, respectively. These values were comparable to corresponding crude protein levels (Table IV.1), thus showing that there hardly non-protein nitrogenous in the seaweeds analysed. The three seaweeds studied contained all the EAAs (excluding cysteine in the A. nodosum and B. bifurcata), presenting the seaweeds A. nodosum and F. vesiculosus EAAs contents significant (P < 0.05) different among them. On the other hand, the EAAs/total amino acid ratio suggests that more than 40% of the amino acids were EAAs. This ratio was lower than that reported by Chan & Matanjun (2017) in Gracilaria changii,

67 IV. Results and discussion but comparable to those found in P. umbilicalis (36.87%), U. pinnatifida (42.72%) and H. elongata (40.82%) reported by Cofrades et al. (2010).

In the fraction corresponding to EAAs, leucine was the one that presented the highest levels, while in the non-essential fraction were glutamic and aspartic acids, Finally, the protein quality of the seaweed F. vesiculosus was superior than those of the other ones, because cysteine is absent in the species A. Nodosum and B. bifurcata.

IV.1.3.1. Nutritional quality of protein

The nutritional quality of the seaweed protein is presented in Table IV.4. The CS for each essential amino acid with respect to the pattern protein proposed by FAO/ WHO/UNU (FAO/WHO/UNU, 2007) for humans (children > 1-year old and adults) was calculated.

Table IV.4. Nutritional quality of protein for seaweeds studied. Seaweed IOM/FNB FAO/WHO/UNU Amino acid A. nodosum F. vesiculosus B. bifurcata (2002) (2007) (CS) (CS) (CS) Histidine 1.8 1.5 96.8 99.8 103.7 Isoleucine 2.5 3.0 113.1 130.3 112.0 Leucine 5.5 5.9 104.7 112.5 99.7 Lysine 5.1 4.5 110.3 136.9 97.9 Met + Cys 2.5 1.6 105.9 203.8 125.0 Phe + Tyr 4.7 3.8 152.1 176.0 149.0 Threonine 2.7 2.3 181.5 125.0 175.6 Valine 3.2 3.9 104.3 115.0 107.2 IEAAs 118.4 133.9 118.8 Pattern proteins are expressed in g/100 g protein. Values of CS and IEAAs are referred only respect to FAO/WHO/UNU (2007) protein pattern.

The profile of the FNB (Institute of Medicine, 2002) is also shown for comparative purposes. The analysis of the CS allows the order of the restrictive amino acids to be determined. All amino acids displayed concentrations higher than those presented by the FAO/WHO/UNU (2007), except for histidine in the A. nodosum and F. vesiculosus and leucine and lysine in B. bifurcata. Thus, histidine was the most limiting amino acid found in A. nodosum and F. vesiculosus while lysine seemed to be it in B. bifurcata. These results agree with those reported by Cofrades et al. (2010) who observed that the most limiting amino acid in the brown seaweeds was lysine.

68 IV. Results and discussion

IV.1.4. FATTY ACID PROFILE OF MACRO-ALGAE

The fatty acid profile of seaweeds is shown in Table IV.5.

Table IV.5. Fatty acid profile of seaweeds studied (mean ± standard deviation value) (n = 5). Seaweed SEM Sig. A. nodosum F. vesiculosus B. bifurcata C14:0 9.40 ± 0.11a 11.38 ± 0.11b 4.52 ± 0.46c 0.83 *** C14:1n-5 0.28 ± 0.00a 0.10 ± 0.00b 0.00 ± 0.00c 0.03 *** C15:0 0.30 ± 0.00a 0.37 ± 0.00b 0.17 ± 0.01c 0.02 *** C16:0 13.42 ± 0.46a 14.66 ± 0.36b 17.35 ± 0.43c 0.47 *** C16:1n-7 2.24 ± 0.01a 1.18 ± 0.02b 2.51 ± 0.16c 0.17 *** C17:0 0.41 ± 0.14a 0.82 ± 0.15b 0.54 ± 0.02a 0.06 ** C17:1n-7 0.29 ± 0.00a 0.20 ± 0.00b 1.87 ± 0.07c 0.22 *** C18:0 0.76 ± 0.01a 1.06 ± 0.08b 1.75 ± 0.13c 0.12 *** C18:1n-11 trans 0.00 ± 0.00a 0.00 ± 0.00a 3.57 ± 0.13b 0.48 *** C18:1n-9 cis 27.83 ± 0.26a 19.94 ± 0.31b 12.61 ± 0.35c 1.73 *** C18:1n-7 cis 0.45 ± 0.05a 0.39 ± 0.04a 0.52 ± 0.03b 0.02 ** C18:2n-6 trans 0.11 ± 0.00a 0.06 ± 0.00a 5.68 ± 0.21b 0.75 *** C18:2n-6 cis 7.47 ± 0.12a 6.43 ± 0.08b 1.92 ± 0.06c 0.67 *** C20:0 0.22 ± 0.01a 0.39 ± 0.01b 1.89 ± 0.18c 0.21 *** C18:3n-6 0.54 ± 0.01a 0.56 ± 0.01a 0.42 ± 0.05b 0.02 *** C20:1n-9 0.07 ± 0.01a 0.53 ± 0.01b 4.18 ± 0.12c 0.52 *** C18:3n-3 4.45 ± 0.03a 7.59 ± 0.11b 3.97 ± 0.09c 0.48 *** C18:2n-7 (CLA) 0.00 ± 0.00a 0.00 ± 0.00a 0.87 ± 0.10b 0.12 *** C21:0 0.00 ± 0.00a 0.00 ± 0.00a 0.71 ± 0.07b 0.09 *** C20:2n-6 5.05 ± 0.02a 6.46 ± 0.09b 1.44 ± 0.01c 0.61 *** C22:0 0.22 ± 0.00a 0.22 ± 0.00a 0.34 ± 0.02b 0.02 *** C20:3n-6 0.74 ± 0.04a 0.69 ± 0.02b 0.42 ± 0.04c 0.04 *** C22:1n-9 0.00 ± 0.00a 0.00 ± 0.00a 0.73 ± 0.04b 0.10 *** C20:3n-3 0.33 ± 0.01a 0.21 ± 0.00b 0.00 ± 0.00c 0.04 *** C20:4n-6 17.25 ± 0.26a 15.86 ± 0.24b 15.24 ± 0.37c 0.25 *** C22:2n-6 0.29 ± 0.01a 0.39 ± 0.01b 1.76 ± 0.09c 0.19 *** C20:5n-3 7.24 ± 0.08a 9.94 ± 0.14b 4.09 ± 0.08c 0.70 *** C24:0 0.41 ± 0.00a 0.36 ± 0.01b 0.34 ± 0.03b 0.01 ** C24:1n-9 0.00 ± 0.00a 0.00 ± 0.00a 0.53 ± 0.06b 0.07 *** C22:6n-3 0.00 ± 0.00a 0.00 ± 0.00a 11.10 ± 1.13b 1.49 *** SFAs 25.14 ± 0.49a 29.26 ± 0.34b 27.62 ± 0.77c 0.51 *** MUFAs 31.15 ± 0.23a 22.33 ± 0.33b 26.51 ± 0.48c 1.06 *** PUFAs 43.47 ± 0.54a 48.19 ± 0.62b 46.91 ± 1.37b 0.62 *** n-3 12.02 ± 0.11a 17.74 ± 0.25b 19.16 ± 1.03c 0.88 *** n-6 31.45 ± 0.42a 30.44 ± 0.38b 26.87 ± 0.48c 0.56 *** n-6/n-3 2.62 ± 0.01a 1.72 ± 0.01b 1.41 ± 0.07c 0.14 *** Results expressed as percentage of total fatty acid analysed. a-cMeans in the same row not followed by a common superscript letter are significantly different (P < 0.05; Duncan test). **(P < 0.01); ***(P < 0.001).

69 IV. Results and discussion

The PUFAs were the most abundant fatty acids, ranging from 43.47% to 48.19% for A. nodosum and F. vesiculosus, respectively. This result agree with those found by other authors (Cofrades et al., 2010; Chan, & Matanjun 2017; Alves et al., 2016) who reported that the PUFAs were the main fatty acids in seaweeds. Percentages of fatty acids varied significantly (P < 0.001)according to the seaweed. In this regard, the highest oleic acid contents (27.83 and 19.94%) corresponded to A. nodosum and F. vesiculosus, respectively, while that of arachidonic acid (15.24%) to B. bifurcata. A similar trend was observed by Peinado et al. (2014) and Ortiz et al. (2006) who found that oleic acid was the main fatty acid in seaweed samples. On the contrary, Chan, & Matanjun (2017) and Alves et al. (2016) found that docosahexaenoic acid (C22:6n-3; DHA) and palmitic acid were the most abundant in G. changiii and B. bifurcata, respectively. These differences on the fatty acid profile could be due to differences among species, as well as other abiotic factors, such as light, salinity and nutrients (Schmid, Guihéneuf, & Stengel, 2014). Eicosapentaenoic acid (C20:5n-3; EPA) ranged from 4.09 to 9.94% of the total fatty acids, whereas DHA was only detected in B. bifurcata (11.10% of the total fatty acids). Similar amounts of EPA in brown seaweeds were reported in literature (Peinado et al., 2014; Alves et al., 2016; Sánchez‐Machado et al., 2004).

Regarding n-6/n-3 ratio, the WHO recommended values below 4 (Allen et al., 2006). In the present study, it was observed a n-6/n-3 ratio ranging from 2.62 to 1.41, which places the three macro-algae studied within WHO recommendations.

IV.2. PROXIMATE COMPOSITION, ANTIOXIDANT ACTIVITY AND PHENOLIC CHARACTERIZATION OF AQUEOUS EXTRACTS FROM MACRO- ALGAE ASCOPHYLLUM NODOSUM, FUCUS VESICULOSUS AND BIFURCARIA BIFURCATA

After knowing the proximal and nutritional composition of the macroalgae A. nodosum, F. vesiculosus and B. bifurcata, aqueous extracts of these were obtained for further experiments, previously analysing their proximal composition, antioxidant activity and phenolic profile.

70 IV. Results and discussion

IV.2.1. PROXIMATE COMPOSITION, TOTAL SOLID CONTENT AND TOTAL PHENOLIC CONTENT OF EXTRACTS

The proximate composition of seaweed extracts and their TSCs and TPCs are shown in Table IV.6. The TSCs in descending order were: ANE (1.15%) > BBE (0.75%) > FVE (0.42%). These differences in total solids among the three seaweeds could be explained by the variation in proximal composition and the polarities of different compounds present in the seaweeds. On the other hand, the presence of mucilaginous substances could be related with the lowest TSC of the water extracts from these algae.

Table IV.6. Proximate composition, total solid content, total polyphenol content and in vitro antioxidant activity determined by ABTS radical cation decoloration, DPPH free radical scavenging activity, ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) of A. nodosum, B. bifurcata, F. vesiculosus aqueous extracts and BHT compound (mean ± standard deviation value) (n = 3). Seaweed extract BHT ANE FVE BBE TSC (g solids/L extract) 11.5 ± 1.45c 4.19 ± 0.53a 7.50 ± 0.91b Protein (g/100 g extract) 26.08 ± 1.07a 62.05 ± 2.30c 53.33 ± 1.69b Total carbohydrates 63.60 ± 2.45c 34.53 ± 2.92b 15.71 ± 3.48a (g glucose/100 g extract) TPC (g PGE/100 g extract) 0.96 ± 0.03a 1.15 ± 0.02b 1.99 ± 0.23c Antioxidant activity ABTS (µmol TE/g 147.26 ± 0.70a 1046.79 ± 18.38c 728.29 ± 7.07b 5105,03 ± 60.81d ABTS extract) ABTS EC 11.5 ± 0.06e,c 50 1.54 ± 0.02a 2.78 ± 0.03b 0.28 ± 0.03d (mg extract/mL) (43.3%) DPPH (µmol TE/g 2.27 ± 0.70a 135.31 ± 4.24c 47.53 ± 2.12b 976.04 ± 65.04d

DPPH extract) e,c e,a e,c d DPPH EC50 11.5 ± 0.42 4.19 ± 0.71 7.5 ± 0.70 0.95 ± 0.06 (mg extract/mL) (4.34%) (38.88%) (23.58%) ORAC (µmol TE/g extract) 278.34 ± 3.53b 756.50 ± 45.96c 979.86 ± 16.26d 134.24 ± 24.04a FRAP (µmol TE/g extract) 7.52 ± 0.70a 51.66 ± 0.74c 26.93 ± 0.54b 578.50 ± 12.02d a-dMeans in the same column not followed by a common superscript letter are significantly different (P < e 0.05; Duncan test); In these samples it was not possible to calculate EC50, so radical percentage inhibition was indicated.

Water was the solvent chosen for extraction of antioxidant compounds due to safety concerns regarding the use of some organic solvent in foods. In addition, Farvin & Jacobsen (2013) and Wang, Jonsdottir, & Ólafsdóttir (2009) observed that the extraction yields of water extracts were higher than those from organic solvent extracts.

71 IV. Results and discussion

The protein content was 26.08, 62.05 and 53.33 g/100 g extract for ANE, FVE and BBE, respectively, whereas total carbohydrate content was 63.60, 34.53 and 15.71 g glucose/100 g for ANE, FVE and BBE, respectively.

TSC and TPC were inversely proportional (Table IV.6). This outcome suggests that the TSC of the extracts is not related to the polyphenolic compound content of the seaweeds. This might be caused by the types of phenolic compounds in algae matrix and the performance of the solvent used (Chan et al., 2015).

IV.2.2. ANTIOXIDANT ACTIVITY OF EXTRACTS

The values of DPPH, ORAC, FRAP and ABTS radical-scavenging activity of seaweed extracts and BHT compound are shown in Table IV.6. The FVE showed the highest DPPH free radical scavenging activity (135.3 μm TE/g extract), while the BBE presented the highest TPC but, on contrary, only showed intermediate values (47.5 μmTE/g extract) of antioxidant activity. These results suggest that other substances apart of polyphenols extracted from seaweeds, such as small molecular weight polysaccharides, pigments, proteins or peptides may be influencing in the free radical scavenging activity (Farvin & Jacobsen, 2013). According to protein content of FVE, the highest protein contents of this extract could be responsible for their high radical scavenging activity. This circumstance, together with the low protein content of the ANE, which showed the lowest antioxidant activity, seems to suggest a certain protagonism of proteins in the antioxidant activity of the aqueous seaweed extracts.

FVE (EC38.9 4.2 mg/mL) showed to be the most effective extract, followed by BBE (EC23.6 7.5 mg/mL) and ANE (EC4.3 11.5 mg/mL). This finding is in agreement with those reported by Farvin & Jacobsen (2013) who found that the most effective aqueous extracts were those from F. vesiculosus and F. serratus with an EC50 value of 8.3 μg/mL. The higher antioxidant activity of the FVE with respect to the other two seaweed extracts was confirmed by using of the in vitro ABTS and FRAP assays.

However, the ORAC method showed discordant data. Thus, the highest antioxidant activity was observed for the BBE (979.9 μmol TE/g extract), while the FVE showed only the intermediate value (756.5 μmoL TE/g extract), and the ANE the lowest (278.3 μmol TE/g extract) again.

The antioxidant activity of all of the seaweed extracts was lower than that of the BHT reference compound, determined by the same methods used for extracts (ABTS, DPPH and FRAP). However, when measured by the ORAC assay, the antioxidant activity of the seaweed

72 IV. Results and discussion extracts was higher than that of BHT. This discrepancy may be explained by the different chemical basis of the various measurement methods used. Indeed, this fact was reported in previous reports (Craft et al., 2012). Moreover, BHT is a single compound, while seaweed extracts comprise a mixture of compounds of different nature, which interact with each other.

IV.2.3. PHENOLIC CHARACTERIZATION OF EXTRACTS

IV.2.3.1. Tentative identification of phenolic compounds

The assessment of the ANE composition by HPLC-DAD-ESI-MS/MS (Figure IV.1) revealed the presence of 22 peaks (named from A1 to A22). The assessment of the BBE composition (Figure IV.2) exposed the presence of 18 peaks (named from B1 to B18), and the evaluation of the FVE (Figure IV.3) showed the presence of 19 peaks (name from C1 to C19). Some of these compounds were tentatively identified and their characteristics are presented in Table IV.7. Fourteen phenolic compounds were tentatively identified from the ANE. Phlorotannins (5 compounds, peaks A2, A4, A6, A8, and A9) and flavonoids (5 compounds, peaks A7, A10, A11, A15, and A22) were the main groups in the ANE, followed by phenolic acids (4 compounds, peaks A1, A16, A17 and A18). In the BBE, fourteen phenolic compounds were tentatively identified. Most of these compounds are phlorotannins (peaks B2, B3, B5, B9, B13, B16, B17, and B18). Phenolic acids (peaks B1, B12 and B14) and flavonoids (peaks B7, B8 and B11) were also observed. Finally, thirteen phenolic compounds were tentatively identified in the FVE. Phlorotannins were again the main group of compounds (peaks C2, C4, C12, C13, C16, C17, and C18), followed by phenolic acids (peaks C6, C11 and C15) and flavonoids (peaks C7, C9 and C14).

Figure IV.1. Representative chromatogram of phenolic compounds from A. nodosum extract obtained by LC-DAD.

73 IV. Results and discussion

Figure IV.2. Representative chromatogram of phenolic compounds from B. bifurcata extract obtained by LC-DAD.

Figure IV.3. Representative chromatogram of phenolic compounds from F. vesiculosus extract obtained by LC-DAD.

The phenolic compounds tentatively identified from the FVE extract in the present study agree with previous studies. Heffernan et al. (2015) reported that phenolic compounds from F. vesiculosus are basically composed by low molecular weight phlorotannin oligomers from three to eight phloroglucinol units. The ANE and BBE composition in the present study are in agreement with previous studies in literature that also found an important contribution of phlorotannins on brown seaweed composition (Li et al., 2011; Martínez & Castañeda, 2013). Comparatively, phlorotannins were tentatively identified as the main compounds in all the extracts in the present study. Particularly, oligomers belonging to the fuhalol sub-class, although some flavonoids and derivatives were also indicated.

74 IV. Results and discussion

IV.2.3.2. Non identified peaks

Eight peaks from the ANE, 4 peaks from the BBE and 6 peaks from the FVE could not be identified based on mass spectrum and/or UV-visible data (Table IV.8). Other authors (Montero et al., 2016; Munekata et al., 2016) also reported similar impediments when identifying phenolic compounds in several vegetable matrices.

75 IV. Results and discussion

Table IV.7. Phenolic compounds in A. nodosum, B. bifurcata and F. vesiculosus extracts using LC-DAD-ESI-MS/MS. ID # Proposed compound RT (min) [M - H]- (m/z) Product ions (m/z) UV data (λ, nm) % Ref Peaks from ANE A1 Hydroxybenzoic acid derivative 1.26 419 343, 201, 137 n.d. 5.74 a A2 Eckol derivative 1.48 541 401, 371 n.d. 41.79 b A4 Tetrafuhalol 2.21 513 3.85 n.d. 20.16 c A6 Pentafuhalol 2.77 637 633, 385, 247 n.d. 8.55 c A7 Acacetin derivative 3.25 455 327, 283 n.d. 28.04 a A8 Phloroglucinol octamer 3.76 993 373 n.d. 2.36 d A9 Trifuhalol 4.37 389 375 n.d. 0.87 c A10 Hispidulin 4.69 299 n.d. n.d. 32.78 e A11 Acacetin derivative 5.32 657 532, 283 n.d. 3.90 a A15 Acacetin derivative 9.59 900 819, 739, 283 n.d. 3.46 a A16 Quinic acid derivative 43.04 363 249, 191 n.d. 48.04 e A17 Rosmarinic acid derivative 45.32 913 694, 359 n.d. 2.44 a A18 Quinic acid derivative 45.83 957 555, 341, 249, 191 n.d. 8.62 e A22 Gallocatechin derivative 68.16 529 305 n.d. 24.66 f Peaks from BBE B1 Rosmarinic acid 1.26 359 179 n.d. 29.19 a B2 Phloroglucinol derivative 1.51 401 205, 125 n.d. 100 g B3 Phloroglucinol derivative 2.04 391 125 n.d. 76.44 g B5 Phloroglucinol dimer derivative 2.79 517 247 255, 280 17.85 d B7 Acacetin derivative 3.26 327 283 n.d. 7.00 a B8 Cypellocarpin C 3.98 519 335 n.d. 2.53 h B9 Eckol derivative 4.74 545 371 n.d. 68.82 b B11 Acacetin derivative 10.57 743 429, 283 255, 280 1.24 a B12 Quinic acid derivative 42.99 363 249, 191 255 42.41 e B13 Trihydroxyheptafuhalol 45.28 933 914 255 3.89 c B14 Quinic acid derivative 45.84 957 555, 249, 191 n.d. 12.46 e

76 IV. Results and discussion

B16 Tetrafuhalol 55.43 513 499 n.d. 2.48 c B17 Dihydroxytetrafuhalol 57.62 545 387 n.d. 52.19 c B18 Hydroxytetrafuhalol 68.18 529 387 n.d. 22.84 c Peaks from FVE C2 Eckol derivative 1.46 401 3.71 n.d. 23.72 b C4 Phloroglucinol dimer derivative 2.72 517 247 280 29.46 d C6 p-Coumaric acid derivative 4.56 299 255, 163 n.d. 2.07 a C7 Acacetin derivative 5.51 322 283 n.d. 3.81 a C9 Acacetin derivative 19.99 947 691, 383, 283 n.d. 1.39 a C11 Quinic acid derivative 42.92 363 249, 191 n.d. 17.87 e C12 Hydroxytetrafuhalol 45.30 529 387, 219 n.d. 1.64 c C13 Dioxinodehydroeckol derivative 45.79 463 369 n.d. 4.15 i C14 Acacetin derivative 51.49 356 283 n.d. 2.51 a C15 Ferulic acid derivative 54.41 375 293, 221, 193 n.d. 3.78 a C16 Dihydroxytetrafuhalol 55.43 545 385 n.d. 1.26 c C17 Hydroxyhexafuhalol 57.83 777 529, 387, 375 n.d. 17.97 c C18 Hydroxyhexafuhalol 59.22 777 529, 375 n.d. 7.94 c The most abundant ions observed in the mass spectra are shown in bold. a Tentatively identified based on the mass spectral data cited by Hossain et al. (2010). b Tentatively identified based on the mass spectral data cited by Nakamura et al. (1996). c Tentatively identified based on the mass spectral data cited by Montero et al. (2016). d Tentatively identified based on the mass spectral data cited by Pantidos et al. (2014). e Tentatively identified based on the mass spectral data cited by Nagy et al. (2011). f Tentatively identified based on the mass spectral data cited by de Quirós, Lage-Yusty, & López-Hernández (2010). g Tentatively identified based on the mass spectral data cited by Orrego-Lagarón et al. (2016). h Tentatively identified based on the mass spectral data cited by Boulekbache-Makhlouf et al. (2013). i Tentatively identified based on the mass spectral data cited by Jung et al. (2014).

77 IV. Results and discussion

Table IV.8. Features of the non-identified compounds in A. nodosum, B. bifurcata and F. vesiculosus extracts using LC-DAD-ESI-MS/MS. ID # Proposed compound RT (min) [M - H]- (m/z) Product ions (m/z) UV data (λ, nm) % Peaks from ANE A3 Unknown 2.04 391 217 n.d. 100 A5 Unknown 2.57 553 276, 196, 135 255, 280 56.01 A12 Unknown 5.59 1153 900, 657, 321, 242 n.d. 2.22 A13 Unknown 6.07 819 738, 657, 253 n.d. 2.04 A14 Unknown 6.56 1315 1144, 981, 819, 657 n.d. 9.60 A19 Unknown 54.41 375 293, 221, 141 n.d. 4.10 A20 Unknown 55.43 779 499, 355, 217 n.d. 8.19 A21 Unknown 57.59 1365 551, 463, 217, 141 n.d. 52.17 Peaks from BBE B4 Unknown 2.21 769 611, 384 n.d. 5.59 B6 Unknown 3.08 325 203 255, 280 4.98 B10 Unknown 5.83 485 231, 151, 107 n.d. 18.14 B15 Unknown 54.41 633 375, 293, 221 n.d. 2.98 Peaks from FVE C1 Unknown 1.26 627 485, 343, 201, 135 n.d. 7.50 C3 Unknown 2.04 611 391, 217 520 100 C5 Unknown 4.30 699 536, 341, 255 n.d. 1.06 C8 Unknown 19.38 533 403, 255, 113 n.d. 4.93 C10 Unknown 40.25 587 507, 365, 217 280 2.40 C19 Unknown 68.04 529 381 n.d. 18.39 The most abundant ions observed in the mass spectra are shown in bold.

78 IV. Results and discussion

IV.3. STUDY OF THE ANTIOXIDANT EFFECT OF ASCOPHYLLUM NODOSUM, FUCUS VESICULOSUS AND BIFURCARIA BIFURCATA AQUEOUS EXTRACTS IN CANOLA OIL

Once parameters, such as proximal composition and antioxidant activity of the aqueous extracts from the macro-algae A. nodosum, F. vesiculosus and B. bifurcata, were known, as well as a slight notion of their phenolic profile, they were used in an oily matrix as canola oil, to test their capacity to extent the shelf-life of edible oils or similar foods.

IV.3.1. BIFURCARIA BIFURCATA EXTRACT ADDED AT INCREASING CONCENTRATIONS TO CANOLA OIL

IV.3.1.1. Evolution of lipid oxidation

The oil was subjected to thermal treatment at 60 °C for 16 days, what corresponds to 4 months at room temperature (Warner, Frankel, & Mounts, 1989) due to the fast hydroperoxide decomposition at high temperatures (Frankel, 1998). Several oxidation indices, such as PV, AV, CD and TOTOX value, were determined to assess the BBE effectiveness. Its addition at different levels resulted to have influence in all the lipid oxidation indices (Figure IV.4A-D). The PV is used as an indicator for the primary oxidation, because hydroperoxides are the first products generated in lipid oxidation. For all samples, the thermal treatment caused oxidation in canola oil, leading to a significant increase (P < 0.001) with the storage time, although this effect was markedly reduced in samples with BHT or BBE. The control oil samples reached a maximum PV of 381 meq O2/kg oil after 16 days of storage, whereas the PV of the sample containing 1000 ppm of BBE was 108 meq O2/kg oil after this period. These data indicate a higher stability in the oil with BBE addition, reaching the highest inhibitory effect in the middle stages of oxidation (8 and 12 days, with regression coefficient of 0.98). However, the highest IP for the PVs was observed at early stages of oxidation (4 days) with IP higher than 80% for all concentrations assessed. These results agree with those found by Shaker (2006), and show that the antioxidant compounds of the BBE play an important role in the inhibition of the initial stages of lipid oxidation. In addition, we can confirm that BBE did not display pro- oxidative effects during the storage time.

During lipid oxidation, hydroperoxides are decomposed to generate secondary oxidation products, such as ketones, alcohols and aldehydes, which are more stable during heating process. In order to confirm the results of primary oxidation, the simultaneous detection of

79 IV. Results and discussion primary and secondary lipid products is necessary. For this, AV was also determined in the canola oil samples. Figure IV.4B shows the changes recorded in the AVs during the oxidation process. The AVs were affected by the oxidation time, what caused significant differences (P < 0.001) in all samples. The BHT and BBE resulted in significant decrease in the AVs (P < 0.05) with respect to the control at the end of storage time. Percentages of inhibition at different storage times and concentrations are shown in Figure IV.5B. At the end of the storage period, the AVs of the samples with BHT decreased by 62% with respect to the control samples, independently of the concentration employed, while addition of BBE resulted in various decreases in AVs depending on the concentration, in the range 55-68%, with regard to control sample. After analysing the results, it can be inferred that the polyphenolic compounds of BBE were highly effective inhibiting the oxidation of secondary lipids. The BBE at level of 600 ppm provided a similar protection to that showed by BHT against the secondary lipid oxidation, and the BBE at level of 800 or 1000 ppm induced an inhibitory activity higher than that showed by BHT, while the BBE at level of 200 and 400 ppm had a lower capacity to inhibit the secondary oxidation than that showed by BHT. However, in both situations there were no significant differences (P > 0.05) among the BHT and BBE samples.

The formation of conjugated dienes has been associated to the oxidation of PUFAs (Kim et al., 2013; Roman et al., 2013), being their measurement a good parameter for the assessment of oil oxidation (Shahidi & Wanasundara, 1997). As we can observe in Figure IV.4C, the absorbance values of the CD were gradually increased (P < 0.001) with the increase in the oxidation time. The CD offer a measure of the lipid alteration grade due to the double bond conjugation caused by primary oxidation. Indeed, a high positive correlation among the CD and PVs was noted (r = 0.93; P < 0.01, n = 120), which is in accordance with previous results reported by other authors in soybean oil (Kim et al., 2013). The canola oil samples with the highest dose of BBE showed the lowest amount of CD at all sampling times. The oil with added BBE added at 600 ppm provided the same effectiveness in reducing CD than that with added BHT at 200 ppm. As with the PVs, the inhibitory effect of the BBE on CD formation was concentration-dependent (Figure IV.5C).

Figure IV.4D depicts the changes in the TV. TV represents a deterioration oxidative index, because it accounts for both peroxides and aldehydes (Shahidi & Wanasundara, 2002). As it can be observed in Figure IV.4D, the kinetic trends were similar to those obtained in PV production. The highest inhibition percentage (71.3%) was achieved by the BBE at 1000 ppm (Figure IV.5D), and the highest values (> 80%) for all the samples were achieved at early stages

80 IV. Results and discussion

(4 days), mainly due to the weight of the PV parameter in the formula used to obtain the TV

(Figure IV.5D).

) l

i 5 0 0 8 0 o

C O N C O N

g k / B H T -5 0 B H T -5 0 2 4 0 0

e 6 0

u

l O

B H T -2 0 0 B H T -2 0 0

a

q

v

e 3 0 0 B B E -2 0 0 B B E -2 0 0

e

m

n

( i

4 0 d

e B B E -4 0 0 B B E -4 0 0

i

u s

l 2 0 0

i a

B B E -6 0 0 n B B E -6 0 0

v

A -

e 2 0

B B E -8 0 0 p B B E -8 0 0

d 1 0 0

i x

o B B E -1 0 0 0 B B E -1 0 0 0 r

e 0 0 P 0 4 8 1 2 1 6 0 4 8 1 2 1 6 T im e (d a y s ) T im e (d a y s )

3 0 0 0

) d

i C O N

5 c

a C O N 2 5 0 0

B H T -5 0

c

s i

e B H T - 5 0

o 4 B H T -2 0 0 e

n 2 0 0 0

n

u

e e

B H T - 2 0 0 l i

i B B E -2 0 0

a

d

d

3 v

B B E - 2 0 0 1 5 0 0

d

d x

e B B E -4 0 0

e

t t

B B E - 4 0 0 o

t

a

a o

g 2 1 0 0 0 B B E -6 0 0

g u

B B E - 6 0 0 T

u

j j

n B B E -8 0 0 n

o B B E - 8 0 0 5 0 0

o 1

C c

B B E -1 0 0 0

f B B E - 1 0 0 0 o

0

0 %

( 0 4 8 1 2 1 6 0 4 8 1 2 1 6

T i m e ( d a y s ) T im e (d a y s )

Figure IV.4. Effect of the addition of B. bifurcata aqueous extract at the concentrations of 200, 400, 600, 800 and 1000 ppm and BHT at 50 and 200 ppm on the evolution of peroxide values (A), p-anisidine values (B), conjugated dienes (C) and TOTOX values (D) in canola oil stored at 60 °C for 16 days. Plotted values are means ± standard deviations of six determinations.

1 0 0 2 8 0 R = 0 .5 8 4 d a y s 2 4 d a y s R = 0 .8 8

8 0 2 8 d a y s 8 d a y s

R = 0 .9 8 6 0 V V 2 1 2 d a y s 1 2 d a y s

R = 0 .9 3 A

P 2

6 0 R = 0 .8 9

1 6 d a y s n 1 6 d a y s

n 2 i

i R = 0 .0 8

) ) 4 0 2

R = 0 .9 8

%

% (

( 4 0

2

P

P I I R = 0 .0 7 2 0 2 0

0 0 0 5 0 0 1 0 0 0 1 5 0 0 0 5 0 0 1 0 0 0 1 5 0 0 B B E c o n c e n tra tio n (p p m ) B B E c o n c e n tra tio n (p p m )

8 0 1 0 0 2 R = 0 .8 5 2 4 d a y s R = 0 .8 0 4 d a y s 8 d a y s 8 0 8 d a y s 2

6 0 R = 0 .9 3 D

1 2 d a y s V 1 2 d a y s T

C 2

6 0 R = 0 .9 7

1 6 d a y s n 1 6 d a y s

n

i

i

2 )

) 4 0 R = 0 .0 4 2 2

R = 0 .9 5 R = 0 .9 7

% %

( 4 0

(

P

P I I 2 0 2 0 2 R = 0 .9 3

0 0 0 5 0 0 1 0 0 0 1 5 0 0 0 5 0 0 1 0 0 0 1 5 0 0 B B E c o n c e n tra tio n (p p m ) B B E c o n c e n tra tio n (p p m )

Figure IV.5. Inhibitory effect of the B. bifurcata aqueous extract at the concentrations of 200, 400, 600, 800 and 1000 ppm on the peroxide values (A), p-anisidine values (B), conjugated dienes (C) and TOTOX values (D) in canola oil stored at 60 °C for 16 days.

81 IV. Results and discussion

IV.3.1.2. Volatile compounds formed during lipid oxidation

During the canola oil oxidation several VCs responsible for off-odours and flavours were generated. These compounds, such as alcohols, aldehydes, ketones and esters, represent typical secondary oxidation products resulting from auto-oxidation of oleic, linoleic and linolenic acids (Frankel, Hu, & Tappel, 1989).

8 0 0 C O N A B H T -5 0 6 0

) 6 0 0 C O N s

t B H T -2 0 0 i

) B H T -5 0

n A

s U

B B E -2 0 0 t

i

a B H T -2 0 0 n 4 0

e 4 0 0 U

r B B E -4 0 0

A

a B B E -2 0 0

(

e l

B B E -6 0 0 r

a B B E -4 0 0

A

n

(

a 2 0 0

B B E -8 0 0 l x

a B B E -6 0 0

e 2 0 n

H B B B B B B B B B E -1 0 0 0 a

t B B E -8 0 0

p e

0 B B B B B B B B B E -1 0 0 0 H

N 0 0 0 0 0 0 0 0 O -5 0 0 0 0 0 0 C T -2 -2 -4 -6 -8 0 H T E E E E -1 B H B B B B E N 0 0 0 0 0 0 0 B B B B B B O 5 0 0 0 0 0 0 - 2 2 4 6 8 0 B C T - - - - - 1 H T E E E E - B H B B B B E B B B B B B B

6 0

C O N

)

s t

i B H T -5 0

A n

U B H T -2 0 0

4 0 a

e B B E -2 0 0

r A

( B B E -4 0 0

l

a 2 0 B B E -6 0 0

n a

t B B E -8 0 0 c

O B B B B B B B B B E -1 0 0 0

0

N 0 0 0 0 0 0 0 5 0 0 0 0 0 0 O - 2 2 4 6 8 0 C T - - - - - 1 T E E E E - H E B H B B B B B B B B B B B

4 0 0 C O N

B H T -5 0 )

s 3 0 0 t i B H T -2 0 0

n A U

B B E -2 0 0

a e

r 2 0 0 B B E -4 0 0

A

(

l

a B B E -6 0 0

n e

t 1 0 0 B B E -8 0 0

c O

- B B B B B B B B B E -1 0 0 0 2 0

N 0 0 0 0 0 0 0 O -5 0 0 0 0 0 0 C T -2 -2 -4 -6 -8 0 H T E E E E -1 B H B B B B E B B B B B B B

Figure IV.6. Effect of the addition of B. bifurcata aqueous extract at the concentrations of 200, 400, 600, 800 and 1000 ppm and BHT at 50 and 200 ppm on the content of hexanal (A), heptanal (B), octanal (C) and 2-Octenal (D) in canola oil after 16 days of storage at 60 °C. Plotted values are means ± standard deviations of six determinations. A-BMeans not followed by a common letter are significantly different (P < 0.05; Duncan test).

82 IV. Results and discussion

In total, 7 VCs were identified on the basis of mass spectra analysis for all the treatments and in all the stages. These VCs were four aldehydes (hexanal, heptanal, octanal and 2-octenal), one alkane (hexane), one alkene (1-octen-3-ol) and one benzene (p-xylene). Aldehydes were the chemical group with higher number of VCs identified and also the most interesting because they can produce a wide range of flavours and odours (Shahidi et al., 1986). Their contents in the canola oil samples with different treatments are depicted in Figure IV.6A-D. For all the aldehydes, at the end of the storage period, differences between the control and the other treatments were statistically significant (P < 0.05). Specifically, hexanal (Figure IV.6A) reached > 600 UA in the control samples at the end of storage, while samples containing antioxidants displayed lower amounts. Within the BBE, inhibition percentages in the hexanal production were increased as the extract concentration increased. The results indicated that the BBE was effective in the control of the production of volatile substances responsible for off-odours and off-flavours.

IV.3.2. ADDITION OF ASCOPHYLLUM NODOSUM, FUCUS VESICULOSUS AND BIFURCARIA BIFURCATA EXTRACTS TO CANOLA OIL

IV.3.2.1. Evolution of lipid oxidation

As in the previous kinetic, where the BBE was added at several concentrations to canola oil, the oil was subjected to accelerated storage conditions (60 °C) for 16 days. The ANE, FVE and BBE were added at a concentration of 500 ppm and the PV, AV, CD, TOTOX, and TBARS determined in order to follow the oxidation process. The effect of the extracts on the evolution of the primary and secondary lipid oxidation products is shown in Figures IV.7A-E. Oxidation was controled by the addition of the seaweed extracts and BHT. The PV in the oil samples with seaweed extracts and BHT were lower than that in the control samples during the entire storage period. At the end of storage, the PV of the sample with BHT decreased by approximately 29% compared to that of the control sample, whereas the PV in the samples with seaweed extracts decreased in the range 65-68% with respect to that of the control batch. This fact indicates that the seaweed extracts at the concentrations used are more effective than BHT as inhibitors of lipid primary oxidation in canola oil. No differences were observed among the performance of the three seaweed extracts measured by PV. These results are consistent with those reported by Farvin & Jacobsen (2013) and Kindleysides, Quek, & Miller (2012) who noted that the antioxidant compounds of seaweed extracts play an important role in the inhibition of free radical formation during the initiation phase of

83 IV. Results and discussion oxidation, interruption of the propagation of the free radical chain reaction by acting as an electron donor, or scavenging free radicals in oil samples.

d ,3

) l

i 4 0 0 d ,3 o

C O N 6 0 C O N

g 3 5 0 k

/ B H T B H T e

2 d ,2

u l

O 3 0 0 d ,2

A N E a 4 5 A N E

q

v

e 2 5 0

F V E e F V E

m

n

( i

2 0 0 c ,3 d e B B E

B B E i 3 0 u

s c ,3 l d ,1 i d ,1

a 1 5 0

c ,2 n v

c ,2

b ,3 A b c ,3 - e 1 0 0 c ,1

p 1 5

d c ,1

i b ,2 b ,2 a ,3

x a b ,3 5 0 b ,1 b ,1 o a ,2 a a ,2 r a a ,1 e a ,1 0

P 0 0 4 8 1 2 1 6 0 4 8 1 2 1 6 S to ra g e tim e (d a y s ) S to ra g e tim e (d a y s )

e ,3 0.25 )

C O N l 7 5 0 i CON

o b,2 B H T BHT

e ,2 g 0.20 k

6 0 0 / e A N E ANE

A b,2

u l

D b, 12

a F V E 0.15 FVE v

4 5 0 M

d ,3 a a,12 x B B E g a BBE

o d ,1 a t m 0.10 a,1

3 0 0 ( a o a d ,2

T a,1

c ,3 S R 1 5 0 c ,2 c ,1 0.05 b ,3 A

b ,1 B

a b ,2 T 0 a ,1 0.00 0 4 8 1 2 1 6 0 4 8 12 16 S to ra g e tim e (d a y s ) Storage time (days)

) 5 d i C O N

c c ,3

a

c

s 4 B H T

i

e

o n

n A N E

e

i

e

i

d d 3

F V E

d d ,2

d

e

e t

t B B E a a b ,2

g 2

g

u

u j

j c ,1

n a b ,3 c ,1 2

n o o a ,3

C c ,1 2

c 1 b ,2

f a

a ,2 3 b ,1 o

a ,1 2 %

( 0 0 4 8 1 2 1 6 S to ra g e tim e (d a y s )

Figure IV.7. Effect of the addition of A. nodosum, F. vesiculosus and B. bifurcata aqueous extracts at the concentration of 500 ppm and BHT at 50 ppm on the evolution of peroxide values (A), p-anisidine values (B), TOTOX values (C), TBARS values (D) and conjugated dienes (E) in canola oil stored at 60 °C. Plotted values are means ± standard deviations of six determinations. a-eMeans in the same oil treatment not followed by a common letter are significantly different (P < 0.05; Duncan test) (differences among sampling points). 1-3Means in the same sampling point not followed by a common number are significantly different (P < 0.05; Duncan test) (differences among treatments).

The AV relates to secondary oxidation products (carbonyls), reflecting the magnitude of aldehyde formation in oils (Zhang et al., 2010a). The effect of the seaweed extracts compared to the BHT compound on the evolution of the AVs during storage of the canola oil is shown in Figure IV.7B. A significant (P < 0.001) increase in the AVs was observed between 12 and 16 days of storage, corresponding to high PVs in this period of the storage. These results agree with those reported by Suja et al. (2004) who found a similar trend concerning the production of secondary lipid oxidation products when assessing the antioxidant capacity of cake extract in soybean, sunflower and safflower oils. The addition of BHT and especially of algae extracts caused a significant decrease (P <0.001) in the AV with respect to those of the control

84 IV. Results and discussion batch. Although differences were not significant (P > 0.05), the BBE seems to offer the best protection against the secondary oxidation in the canola oil during the entire storage period. These outcomes are in agreement with those reported by Franco et al. (2016) who observed that natural extracts exerted a significant inhibitory effect of thermal oxidation in soybean oil heated at 60 °C for 14 days.

Figure IV.7C depicts the changes in the TV of the canola oil under accelerated conditions. In general, the kinetic trends were similar to those obtained in peroxide production. The TOTOX index for the oil samples with seaweed extracts and BHT was significantly (P < 0.05) lower than that observed for the control group. The best IP (66.45%) was reached in the FVE batch. These outcomes proved that the overall oxidation in the canola oil was reduced by these seaweed extracts.

The effect of the seaweed extracts and BHT on the evolution of the TBARS values during storage period of the canola oil is summarized in Figure IV.7D. TBARS values increased until twelfth day in all the batches to decrease significantly (P < 0.001) since this day until the end of storage period in the samples with ANE, FVE and BBE, while, as expected, the TBARS values continued increasing in the control and BHT-added oils (Figure IV.7D). After 16 days of storage, the TBARS values of the samples with seaweed extracts decreased in the range 44-47% with respect to the control batch. It is well known that MDA, the compound quantified in the TBARS analysis, is very unstable and it can disappear via degradation or due to advanced reactions with protein residues (Kikugawa, Kato, & Hayasaka, 1991). The important reduction of the TBARS values after 12 days of storage in the oils fortified with the seaweed extracts could be due to the inhibition of the production of MDA by the extracts, but also because of reactions with protein residues. The high protein content of the seaweed extracts (Table IV.6) might ease this possibility. In addition, these reactions take place especially in conditions of low water activity, as in the oils of the present study.

Based on Figure IV.7E, it can be noticed that the rate of CD formation was higher than the decomposition rate, leading to their accumulation in the oil during storage period. Both the addition of the seaweed extracts and BHT resulted in a significant (P < 0.001) decrease of the CD in comparison with the control batch. The seaweed extracts showed more effectiveness reducing the CD appearance than the BHT compound. These findings are partially agree with those found by Poiana (2012) who observed that grape seed extracts at a level of 600 ppm showed the same effectiveness in reducing the accumulation of CD as BHT. At the end of storage time, the CD values of samples with the seaweed extracts decreased in the range 66- 68% with respect to the control group.

85 IV. Results and discussion

IV.4. STUDY OF THE EFFECT OF ASCOPHYLLUM NODOSUM, FUCUS VESICULOSUS AND BIFURCARIA BIFURCATA AQUEOUS EXTRACTS IN THE SHELF-LIFE OF A LOW-FAT PORK LIVER PÂTÉ

The ANE, FVE and BBE were included in the formulation of a pâté with partially fat replacing by canola and high-oleic sunflower oils, in order to study their capacity to extent the shelf-life in this type of cooked meat product.

IV.4.1. PROXIMATE COMPOSITION AND FATTY ACID PROFILE OF PÂTÉ

The proximate composition (moisture, fat and protein contents) and the fatty acid profile of the low-fat pork liver pâté are presented in Table IV.9. The addition of the seaweed extracts to the pâté did not affect its moisture and fat content. This result is in accordance with those reported by other authors when they assessed the proximate composition of similar liver pâté samples (Estévez et al., 2007; Estévez, Ventanas, & Cava, 2006). However, significant differences were found in the protein content between the different samples, since the batches made with the ANE and BBE achieved significant (P < 0.05) higher values than the control samples (without antioxidant addition) and samples with addition of BHT as an antioxidant. A plausible explanation for this outcome could be due the important protein content of the seaweed extracts. Indeed, the protein contents quantified in the A. nodosum, F. vesiculosus and B. bifurcata aqueous extracts were 26.08, 62.05 and 53.33 g/100 g extract, respectively (Table IV.6).

Regarding the fatty acid composition, there were no significant differences (P > 0.05) among the different seaweed extracts for any fatty acid. The MUFAs were the most abundant (55% of total fatty acids), followed by the PUFAs (22.75% of total fatty acids) and SFAs (21.82 %) of total fatty acids, respectively. The oleic acid (49.61% of total fatty acids) highlighted among the rest of fatty acids. These values are similar to those reported by several authors who worked with natural antioxidants in the formulation of liver pâtés (Estévez et al., 2007; Pateiro et al., 2014, 2015). On the other hand, the linoleic (17.47%), palmitic (13.04%) and stearic (7.28%) acids also presented amounts to take into account, specially the first one, which was also found in high amount in previous studies (Estévez et al., 2007; Pateiro et al., 2014, 2015).

As expected, the inclusion of rich-PUFA oils in the manufacture of pork liver pâtés in order to replace pork backfat significantly influenced in the fatty acid profile. This formulation led the

86 IV. Results and discussion reduction of SFAs and the increase of PUFAs, thus improving the P/S and n-6/n-3 ratios, satisfying the nutritional recommendations of the FAO for human diet (Anonymous, 2010).

Table IV.9. Effect of seaweed extracts on proximate composition and fatty acid profile of low-fat pork liver pâtés (mean ± standard deviation value) (n = 9). Batch SEM Sig. CON BHT ANE FVE BBE ab b a ab ab Moisture (%) 52.88 ± 1.07 53.45±0.76 52.05 ± 1.24 53.03 ± 1.31 52.65±1.39 0.18 ns a a a a a Fat (%) 23.98 ± 0.87 23.83±1.29 23.85 ± 1.38 23.59 ± 0.54 24.10±3.47 0.26 ns a a b ab b Protein (%) 13.89 ± 0.55 13.99±0.17 14.42 ± 0.32 14.22 ± 0.30 14.34±0.21 0.06 ** Fatty acid profile a a a a a C14:0 0.58 ± 0.03 0.58 ± 0.03 0.58 ± 0.03 0.58 ± 0.03 0.58 ± 0.03 0.00 ns a a a a a C16:0 13.04 ± 0.30 13.04 ± 0.40 13.05 ± 0.37 13.06 ± 0.39 13.05 ± 0.39 0.05 ns a a a a a C16:1n-7 1.18 ± 0.06 1.17 ± 0.08 1.18 ± 0.08 1.18 ± 0.08 1.17 ± 0.08 0.01 ns a a a a a C18:0 7.26 ± 0.24 7.30 ± 0.26 7.27 ± 0.25 7.29 ± 0.24 7.29 ± 0.23 0.04 ns a a a a a C18:1n-9c 49.63 ± 0.26 49.58 ± 0.32 49.63 ± 0.29 49.60 ± 0.30 49.65 ± 0.29 0.04 ns a ab ab b b C18:1n-7c 3.00 ± 0.05 3.03 ± 0.04 3.04 ± 0.04 3.05 ± 0.04 3.05 ± 0.05 0.01 ns a a a a a C18:2n-6c 17.51 ± 0.31 17.47 ± 0.35 17.48 ± 0.33 17.47 ± 0.32 17.45 ± 0.32 0.05 ns a a a a a C20:1n-9 0.89 ± 0.01 0.89 ± 0.01 0.89 ± 0.00 0.89 ± 0.01 0.89 ± 0.01 0.00 ns a a a a a C18:3n-3 3.66 ± 0.08 3.66 ± 0.10 3.66 ± 0.09 3.66 ± 0.10 3.66 ± 0.11 0.01 ns a a a a a C20:4n-6 0.85 ± 0.01 0.86 ± 0.03 0.84 ± 0.02 0.84 ± 0.02 0.84 ± 0.02 0.00 ns a a a a a SFAs 21.78 ± 0.50 21.86 ± 0.61 21.81 ± 0.59 21.85 ± 0.61 21.82 ± 0.59 0.08 ns a a a a a MUFAs 55.43 ± 0.17 55.39 ± 0.21 55.44 ± 0.21 55.42 ± 0.22 55.46 ± 0.20 0.03 ns a a a a a PUFAs 22.80 ± 0.35 22.75 ± 0.42 22.75 ± 0.39 22.74 ± 0.40 22.72 ± 0.40 0.06 ns a a a a a ∑n-3 3.80 ± 0.06 3.79 ± 0.09 3.79 ± 0.09 3.77 ± 0.11 3.79 ± 0.10 0.01 ns a a a a a ∑n-6 18.95 ± 0.32 18.92 ± 0.36 18.92 ± 0.34 18.91 ± 0.34 18.89 ± 0.34 0.05 ns a a a a a n-6/n-3 4.98 ± 0.08 4.99 ± 0.09 4.99 ± 0.10 5.01±0.12 4.99±0.12 0.02 ns Results expressed as percentage of total fatty acid analysed (only fatty acids > 0.5% were shown). a- bMeans in the same row not followed by a common superscript letter are significantly different (P < 0.05; Duncan test). **(P < 0.01).

IV.4.2. EVOLUTION OF MICROBIAL COUNTS IN PÂTÉ

Changes in microbial counts during chilled storage of the low-fat pork liver pâté are not shown. Significant differences (P > 0.05) in TVC among the batches with different added antioxidants were not observed. The TVC were very low on day 0 and during storage in all the batches (counts < 1 Log CFU/g for all the batches and sampling points). These values were even lower than those reported by other authors in pâté samples (Delgado-Pando et al., 2011, 2012; Lorenzo et al., 2014a). Regarding the LAB, their evolution followed the same trend, with values lower than 1 Log CFU/g.

On the other hand, Pseudomonas spp., molds and yeasts were not detected in any of the samples, independently of the batch and storage time (data not shown). This result is consistent with those obtained by Lorenzo et al. (2014a) who did not find Pseudomonas spp. in

87 IV. Results and discussion pâté samples after five months of storage. Overall, the results achieved in this study showed that the thermal treatment intensity applied was enough to avoid Pseudomonas spp., molds and yeast growth for all the batches assayed.

IV.4.3. EVOLUTION OF PHYSICAL PROPERTIES (pH AND COLOUR) OF PÂTÉ

The evolution of the pH and colour parameters of the low-fat pork liver pâté during refrigerated storage is presented in Figures IV.8 and 9, respectively. In general, no significant differences (P > 0.05) were found for the pH among the different batches, independently of the storage time. Within each batch, the same pH evolution pattern was observed, increasing after 45 days of storage, followed by a progressive decrease until 135 days, and a new increase at the end of the storage. However, variations in the pH values during storage were minimal, with only significant differences (P < 0.05) in the ANE and in the FVE batches.

6 .3

6 .2

H 6 .1 p

6 .0

5 .9 0 5 0 1 0 0 1 5 0 2 0 0

T im e (d a y s )

C O N B H T A N E F V E B B E

Figure IV.8. Evolution of pH during refrigerated storage of low-fat pork liver pâté manufactured with added BHT at a concentration of 50 ppm and natural antioxidant extracts at 500 ppm from seaweeds A. nodosum, F. vesiculosus and B. bifurcata. Plotted values are means ± standard deviations of nine replicates.

On the other hand, the colour parameters (L*, a*, b*) hardly varied significantly both among batches and along storage (Figure IV.9A-C). Differences were found in the L* value after 180 days of storage, which was significantly lower (P < 0.05) in the FV and BB batches compared to the other batches, and in the a* and b* values, which were significantly lower (P < 0.05) in the control batch than in the batches manufactured with antioxidant extract.

88 IV. Results and discussion

2 0 6 0

1 8

5 5 d

y

e

t

r

i

f

s

o o

1 6

n

x

i

e

m

d

u

n I L 5 0 1 4

4 5 1 2 0 5 0 1 0 0 1 5 0 2 0 0 0 5 0 1 0 0 1 5 0 2 0 0

T im e (d a y s ) T im e (d a y s )

C O N B H T A N E F V E B B E C O N B H T A N E F V E B B E

2 1 Figure IV.9. Evolution of luminosity (A), index of 2 0

red (B) and index of yellow (C) during w

o 1 9 l

l refrigerated storage of low-fat pork liver pâté

e

y

f 1 8 manufactured with added BHT at a

o

x

e concentration of 50 ppm and natural antioxidant

d 1 7 n I extracts at 500 ppm from seaweeds A. nodosum, 1 6 F. vesiculosus and B. bifurcata. Plotted values

1 5 are means ± standard deviations of nine 0 5 0 1 0 0 1 5 0 2 0 0 replicates. T im e (d a y s )

C O N B H T A N E F V E B B E

IV.4.4. EVOLUTION OF LIPID AND PROTEIN OXIDATION IN PÂTÉ

The lipid stability of the low-fat pork liver pâté was followed through the measurement of the appearance of oxidation products. For this, two trials were used to quantify both lipid primary oxidation (CD) and lipid secondary oxidation (TBARS) products. (Figure IV.10A-B). The CD values did not show significant differences (P > 0.05) among batches at any storage time. On the contrary, when the batches were studied individually, significant differences (P < 0.05) were found on their evolution along storage.

Although the differences were not significant (P < 0.05), the CD values in the CON batch (average values of 2.91 μmol/g pâté) were higher than those found in the pâtés manufactured with seaweed extract addition (average values between 2.19 and 2.53 μmol/g of pâté) at the end of the storage. In conclusion, it could be deduced that the values obtained showed a reasonable stability of the pâté for primary lipid oxidation in agreement with those previously reported by Karwowska, Wójciak, & Dolatowski (2015) in organic fermented sausages elaborated with mustard seed and without nitrites.

89 IV. Results and discussion

P < 0 .0 1 .

) P < 0 .0 5 P < 0 .0 0 1 . P < 0 .0 1 P < 0 .0 0 1 d

i 4 c

a c a b a c b

c b c b c i s b c

a a e o a

b b a b c n

n a a b e

e a b

i i

3 a a d

d a

d

d c c b b

e

e

t

t

a

a

g

g

u

u

j j

n 2

m

o

o

c

C

f o

A

% ( 1 0 4 5 9 0 1 3 5 1 8 0 0 4 5 9 0 1 3 5 1 8 0 0 4 5 9 0 1 3 5 1 8 0 0 4 5 9 0 1 3 5 1 8 0 0 4 5 9 0 1 3 5 1 8 0

T im e (d a y s )

C O N B H T A N E F V E B B E

P < 0 .0 0 1 P < 0 .0 1 0 .6 P < 0 .0 5 P < 0 .0 1 P < 0 .0 1

c

)

e

t

a S p a a a

b c b c

r R

e 0 .4 a a a A

v a

i a b a b

B l

a a a T

a b g f b b b c a b c

k a

o / b b

A a

x b

e D

d 0 .2

M

n

I

g

m ( B 0 .0 0 4 5 9 0 1 3 5 1 8 0 0 4 5 9 0 1 3 5 1 8 0 0 4 5 9 0 1 3 5 1 8 0 0 4 5 9 0 1 3 5 1 8 0 0 4 5 9 0 1 3 5 1 8 0

T im e (d a y s )

C O N B H T A N E F V E B B E

2 0

) P <0 .05 P <0 .0 01 P <0 .0 01 P <0 .05 P <0 .05 n

i a

e t

o a r b b a a a p 1 5

a a c c c c g

s b l

m a b a b a b / y a

s a

n b

l o

y 1 0 b a b

n b a a

r

o

a

b

C

r

a c

5

l

o m

n C ( 0 0 4 5 9 0 1 3 5 1 8 0 0 4 5 9 0 1 3 5 1 8 0 0 4 5 9 0 1 3 5 1 8 0 0 4 5 9 0 1 3 5 1 8 0 0 4 5 9 0 1 3 5 1 8 0

T im e (d a y s )

C O N B H T A N E F V E B B E

Figure IV.10. Evolution of conjugated dienes (A), TBARS values (B) and carbonyls (C) during refrigerated storage of low-fat pork liver pâté manufactured with added BHT at a concentration of 50 ppm and natural antioxidant extracts at 500 ppm from seaweeds A. nodosum, F. vesiculosus and B. bifurcata. Plotted values are means ± standard deviations of nine replicates. a-cMeans in the same oil treatment not followed by a common letter are significantly different (P < 0.05; Duncan test) (differences among sampling points).

Concerning the secondary lipid oxidation, the results indicated significant differences (P < 0.05) among batches at the end of the storage. The values observed in the CON batch (0.39 mg

90 IV. Results and discussion

MDA/kg pâté) were higher than those found in the BHT batch (0.21 mg MDA/kg pâté) and in the batches manufactured with seaweed extracts (values from 0.19 to 0.21 mg MDA/kg pâté). On the other hand, significant differences (P < 0.05) in the TBARS values were also found over time for each one of the batches. The trend observed was different depending on the batch. Thus, in the CON batch the amount of MDA increased after 180 days respect to the initial time. Nevertheless, in the BHT, ANE, FVE and BBE batches the MDA concentration after 180 days of storage was similar to that on day 0 or even lower. This result agree with those found by several authors (Estévez & Cava, 2004; Pateiro et al., 2014, 2015) who reported modifications in the TBARS values during storage of pâtés.

Both the primary and secondary oxidation were very little remarkable in the pâté, obtaining a very stable product lipidically. The fact that the pâté cans were completely filled and hermetically closed, thus avoiding the presence of a head space and the subsequent oxygen entrance, could explain this unexpected result.

On the other hand, the oxidation of proteins was quantified by measuring the appearance of carbonyl compounds (Figure IV.10C). Carbonyl compounds are formed as a result of an oxidative degradation of the side chains of lysine, proline, arginine and histidine residues (Stadtman & Levine, 2003). The different batches did not show significant differences (P > 0.05) between them, independently of the storage time, except at 180 days. Overall, a significant increase in the protein oxidation during storage was observed for all the batches manufactured, reaching values significantly higher (P < 0.05) in the CON batch (13.41 nmol carbonyl/mg protein) than in the batches manufactured with antioxidants (values around 11.45 nmol carbonyl/mg protein) after 180 days of storage. In this sense, some authors have reported a significantly increase (P < 0.05) in the protein oxidation during storage of pâtés from Iberian and white pigs (Estévez, Ventanas, & Cava, 2006; Estévez & Cava, 2004), finding the highest concentration of carbonyls at the end of the storage, as in the present study.

IV.4.5. VOLATILE COMPOUNDS FORMED IN PÂTÉ DURING STORAGE

A total of 30 VCs were detected in the headspace of the pâté at the beginning and at the end of the storage through the SPME-GC-MS technique. These compounds comprised 20 hydrocarbons, 4 aldehydes, 2 esters, 2 alcohols, and 2 ketones. Hydrocarbons were the main chemical group both at the beginning (between 87.5% and 96.3% of the total volatile compounds) and at the end (between 83.9% and 86.9%) of the storage. This outcome coincides with those reported in dry-cured lacón (Domínguez et al., 2016) and in dry-cured foal sausage (Lorenzo et al., 2016). However, it differs of those obtained in studies with liver pâtés

91 IV. Results and discussion

(Estévez, Ventanas, & Cava, 2005; Estévez et al., 2004). These different results may be due to the different volatile extraction methods used. The main hydrocarbon detected was heptane, 2,2,4,6,6-pentamethyl. The different bathes presented different amounts (P < 0.001) of total hydrocarbons at the end of the storage, with the CON batch reaching the highest content (110.81 AU×10-6/g dry matter). Along storage time, the total hydrocarbon content decreased significantly (P < 0.01) in all the batches, especially in the BB batch. This decrease is attributed to the disappearance of the compound heptane, 2,2,4,6,6-pentamethyl at the end of the

storage.

) e

l n .s . P <0 .0 01 P <0 .0 01 P <0 .0 01 P <0 .0 01 1 0 0 ) 1 5

i n .s . P <0 .0 01 n .s . n .s . P <0 .01

e

t

l

i

a

l

t

a

o

l

v

8 0 o

l

v

a

l

t a

o 1 0

t

t

o

f 6 0

t

o

f

o

%

(

4 0 %

s

(

n 5

s

o

e

b

d r

2 0 y

a

h

c

e

o

r

d

l d

0 A 0 y

H 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0

C O N B H T A N E F V E B B E C O N B H T A N E F V E B B E

5 3 P =0 .0 01 P =0 .0 01 P =0 .0 01 P =0 .0 01 P =0 .0 01

P =0 .01 P =0 .0 01 P =0 .0 01 P =0 .0 01 n .s . )

)

e

l

i

e

l

t

i

a t

4 l

a

l

o

v

o

l v

2

a

l t

a 3

t

o

t

o

f

t

o

f

o

2

%

(

% 1

(

s

l

s o

r 1

h

e

t

o

s

c

l E 0 A 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0

C O N B H T A N E F V E B B E C O N B H T A N E F V E B B E

) P <0 .01 P <0 .0 01 P <0 .0 01 P <0 .0 01 P <0 .0 01

1 .5 4 0 0 A

) n .s . P <0 .0 01 n .s . P <0 .0 01 P <0 .0 01

e

U

l

(

i

t

s

a

l e

l 3 0 0

o

i

t

v

a l

1 .0 l

a

o

t

V

o

t l

2 0 0

f

a

t

o

o

T

%

( 0 .5

n

i s

1 0 0

e

s

n

e

o

g

t

n

e

a K

0 .0 h 0 C 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0 0 1 8 0

C O N B H T A N E F V E B B E C O N B H T A N E F V E B B E

Figure IV.11. Changes in percentage of chemical families of volatiles and in total content of volatiles during storage of low-fat pork liver pâté manufactured with added BHT at a concentration of 50 ppm and natural antioxidant extracts at 500 ppm from seaweeds A. nodosum, F. vesiculosus and B. bifurcata. Plotted values are means ± standard deviations of nine replicates.

92 IV. Results and discussion

The second most predominant chemical group was the aldehyde compounds. This chemical family is probably the most interesting lipid-derived volatile compounds (Shahidi et al., 1986) because they can produce a wide range of flavours and odours (Pateiro et al., 2014). The pâté with BHT showed a significantly lower amount of aldehydes (P < 0.05) than the rest, showing a significant decrease (P < 0.001) at the end of the storage. Hexanal was found the most abundant aldehyde, result previously reported by other studies in liver pâtés (Pateiro et al., 2014, 2015).

The other three families of VCs (esters, alcohols and ketones) represented a minor percentage of the total volatile compounds (Figure IV.11).

Overall, after 180 days of storage, a significant decline of the total VCs was noticed in all the batches studied, mainly attributed to a strong reduction of the compound heptane, 2,2,4,6,6- pentamethyl, as commented above.

IV.5. EVALUATION OF DIFFERENT EXTRACTION CONDITIONS IN MACRO- ALGA BIFURCARIA BIFURCATA

Because of the low amount of phenolic compounds found in the aqueous extracts of the macro-algae A. nodosum, F. vesiculosus and B. bifurcata used in the previously commented experiments, we decided to carry out a screening of several extraction conditions to obtain those which provide a good amount of phenolic compounds with a reasonably high antioxidant capacity, as well as a high extraction yield.

IV.5.1. EXTRACTION YIELD AND TOTAL PHENOLIC CONTENT OF DIFFERENT BIFURCARIA BIFURCATA EXTRACTS

The extraction yield and TPC of the B. bifurcata extracts are presented in Table IV.10, as well as a summary of the extraction conditions and their nomenclature. The highest extraction yield was obtained by the HW50E50 extraction condition (41.82 g extract/100 g DW, respectively), followed by the UW50E50 and HW100 (39.11 and 37.48 g extract/100 g DW, respectively). The combination of ultrasound with pure solvents (UW100 and UE100 extraction conditions) provided extracts of reduced yield (22.27 and 14.67 g extract/100 g DW, respectively), while the lowest extraction yields were obtained by the SC-CO2 extraction method (average yields lower than 4 g extract/100 g DW). Analysing the results, it was interesting to verify that the use of 50% ethanol achieved a higher extraction yield than using only water both for the hydrothermal method at 121 °C and for the UAE at room temperature

93 IV. Results and discussion

(41.82 vs 37.48 g/100 g DW, respectively, and 39.11 vs 22.27 g extract/100 g DW, respectively). About this outcome, some contradictory data were found in literature. Hwang & Do Thi (2014) reported higher extraction yields using water at 37 °C than using 70% ethanol in the dried (25 vs 18 %, respectively), roasted (25 vs 18 %, respectively) and seasoned (21 vs 16 %, respectively) red seaweed P. tenera. The same result was observed in the extraction of the brown seaweeds A. nodosum, Pelvetia canaliculata, Fucus spiralis and in the green seaweed Ulva intestinalis collected all in the Irish coast (Tierney et al., 2013). Nevertheless, López et al. (2011) reported outcomes in concordance with those presented in this work, where the mixture of an organic solvent such as methanol with water improved the extraction yield in the brown seaweed Stypocaulon scoparium with respect to use of only water.

The low extraction yields observed in the SC-CO2 method may be explained by the balance between the interactions of the seaweed solutes with their matrix components and the CO2. This finding suggested that additional energy and mixture of solvents were necessary to facilitate the release of polar and high molecular weight compounds from the alga matrix under the studied conditions for SC-CO2 method (35 MPa at 40 °C).

Regarding the TPC, the highest values were obtained by the HW50E50 and UW50E50 extraction conditions (5.65 and 5.46 g PGE/100 g DW, respectively), followed by the HW100

(2.92 g PGE/100 g DW). The use of the SC-CO2 method to extract phenolic compounds was inefficient, since very low compounds were extracted (0-0.06 g PGE/100 g DW) compared to the other methods.

Interestingly, the highest TPCs were found in the samples in which the highest extraction yields were obtained. It is possible to suggest that an increased extraction yield contributed to an increased release of phenolic compounds from algal structure.

The extraction of phenolic compounds was favoured by the hydrothermal and ultrasound processing, particularly using the water/ethanol mixture as solvent. The UW100 and UE100 extraction conditions (2.28 and 2.57 g PGE/100 g DW, respectively) displayed reduced efficiency in the extraction of phenolic compounds compared to the HW50E50 and UW50E50 extraction conditions. Hwang & Do Thi (2014) also found a higher phenolic extraction using a mixture of water and ethanol in the seaweed P. tenera. On the other hand, Kuda et al. (2005) reported very high amounts of phenolic compounds using water as the only extraction solvent. However, the temperature used by the authors (121 °C) could affect to the phenolic compound extraction.

94 IV. Results and discussion

The high extraction level of phenolic compounds obtained by the HW50E50 and UW50E50 extraction conditions could be due to the high solubility of these compounds in polar organic solvents, such as acetone, ethanol and methanol (Farvin & Jacobsen, 2013; Wang et al., 2012). On the other hand, increased temperature could be associated to enhanced extraction of polyphenols in algae species. In addition, cell wall softening and degradation facilitate the release of trapped compounds such as polyphenols due to the increasing temperature and treatment with ultrasounds (Balboa et al., 2013; Parniakov et al., 2015).

The reduced extraction of phenolic compounds by the SC-CO2 extraction method might be due to the low CO2 polarity under supercritical conditions (Abbas et al., 2008), preventing a good interaction between the solvent and seaweed, thus making the extraction difficult.

95 IV. Results and discussion

Table IV.10. Summary of extraction conditions and their nomenclature, as well as the values of extraction yield, total phenolic content and antioxidant activity of B. bifurcata extracts obtained (mean ± standard deviation value) (n = 2). Extraction conditions Extraction yield (g TPC Antioxidant activity Pressure and Nomenclature Method Solvent Time Temperature extract/100 g DW) (g PGE/100 g DW) (µmol TE/g DW) flow

a a a 100% water HW100 37.48 ± 0.77 2.92 ± 0.05 372.87 ± 58.25 Hydrothermal 30 min 121 °C 20 psi a a a Water/ethanol (50:50 v/v) HW50E50 41.82 ± 2.30 5.65 ± 0.61 426.88 ± 80.13 SEM 1.44 0.81 32.57 Sig. ns * ns 100% water UW100 22.27 ± 0.77b 2.28 ± 0.16a 253.06 ± 0.08a Room UAE Water/ethanol (50:50 v/v) 30 min - UW50E50 39.11 ± 1.54c 5.46 ± 0.34b 552.24 ± 72.81b temperature 100% ethanol UE100 14.67 ± 0.77a 2.57 ± 0.24a 227.06 ± 29.91a SEM 4.58 0.65 67.53 Sig. *** ** ** a a 30 min SC-CO2-30 0.49 ± 0.11 0.00 ± 0.00 nd 35 MPa and 25 g a a SC-CO2 90% CO2 and 10% absolute ethanol 45 min 40 °C SC-CO2-45 1.05 ± 0.05 0.01 ± 0.00 nd CO2/min b b 60 min SC-CO2-60 3.10 ± 0.54 0.06 ± 0.01 nd SEM 0.51 0.01 Sig. ** ** a-cMeans in the same column not followed by a common superscript letter are significantly different (P < 0.05; Duncan test). *(P < 0.05), **(P < 0.01); ***(P < 0.001).

96 IV. Results and discussion

IV.5.2. ANTIOXIDANT ACTIVITY OF DIFFERENT BIFURCARIA BIFURCATA EXTRACTS

The antioxidant activity of the different B. bifurcata extracts measured by the ORAC method is presented in Table IV.10. In view of the low phenolic compound extraction by the

SC-CO2 extraction method, the decision of not measuring the antioxidant activity of the resultant extracts was taken. The UW50E50 extraction conditions produced the extract with the highest antioxidant activity (552.24 µmol TE/g DW), followed by the HW50E50 and HW100 extraction conditions (426.88 and 372.87 µmol TE/g DW, respectively). From these results, it is comprobed that the combination of solvents (water/ethanol) and the application of additional energy (hydrothermal and UAE methods) led to the production of natural extracts with increased antioxidant activity. Some studies reported higher antioxidant activities in extracts obtained with polar organic mixtures than with water using the DPPH assay (Hwang & Do Thi, 2014; Tierney et al., 2013). However, it was observed that different results were given by some authors when using different essays to measure the antioxidant capacity in the same extract.

Although the data are not significantly conclusive, a correlation between phenolic content and antioxidant activity seemed to be observed. Previous studies have reported a strong correlation between polyphenols and antioxidant activity in macro-algae, suggesting that polyphenols are some of the main contributors of antioxidant activity (Vinayak, Sabu, & Chatterji, 2011; Wang, Jonsdottir, & Ólafsdóttir, 2009; Zhang et al., 2010b).

IV.6. ANTIOXIDANT POTENTIAL OF EXTRACTS FROM MACRO-ALGAE ASCOPHYLLUM NODOSUM, FUCUS VESICULOSUS AND BIFURCARIA BIFURCATA, AND MICRO-ALGAE CHLORELLA VULGARIS AND SPIRULINA PLATENSIS OBTAINED BY ULTRASOUND-ASSISTED EXTRACTION

After analysing the results of the previous chapter, it was concluded that the UAE method in combination with the mixture water/ethanol (50:50 v/v) as solvent was the best extraction condition. The overall values obtained for the variables studied (extraction yield, TPC and antioxidant activity) in the B. bifurcata extract were higher than those obtained by the other extraction conditions tested. Therefore, the UAE method with along water/ethanol (50:50 v/v) was selected to extract the macro-algae A. nodosum, F. vesiculosus and B. bifurcata and the micro-algae C. vulgaris and S. platensis and to evaluate the antioxidant potential of the extracts obtained.

97 IV. Results and discussion

Table IV.11. Extraction yield, TPC and antioxidant activity of A. nodosum, F. vesiculosus, B. bifurcata, C. vulgaris and S. platensis extracts obtained by UAE method using water/ethanol (50:50, v:v) as solvent. Algae SEM Sig. A. nodosum F. vesiculosus B. bifurcata C. vulgaris S. platensis Extraction yield (g extract/100 g DW) 25.86 ± 0.00a 9.69 ± 0.16b 35.85 ± 0.00c 7.46 ± 0.45d 4.40 ± 0.22e 4.05 *** TPC (g PGE/100 g DW) 4.66 ± 0.00a 1.94 ± 0.03b 5.74 ± 0.00c 0.34 ± 0.01d 0.20 ± 0.00e 0.75 *** Antioxidant activity (µmol TE/g DW) *** ORAC 298.74 ± 2.19a 154.83 ± 0.81b 556.20 ± 26.62c 31.21 ± 2.63d 12.21 ± 0.13d 67.04 *** ABTS 554.02 ± 16.46a 204.53 ± 5.56b 543.48 ± 8.11a 15.14 ± 0.71c 6.63 ± 0.15c 80.84 *** DPPH 50.69 ± 0.00a 26.93 ± 0.31b 144.65 ± 2.28c 0.82 ± 0.05d 0.95 ± 0.08d 17.75 *** FRAP 4.53 ± 0.18a 3.63 ± 0.27a 66.50 ± 1.27b 0.62 ± 0.00c 1.01 ± 0.01c 8.56 *** a-eMeans in the same row not followed by a common superscript letter are significantly different (P < 0.05; Duncan test). ***(P < 0.01).

98 IV. Results and discussion

IV.6.1. EXTRACTION YIELD AND TOTAL PHENOLIC CONTENT OF ALGAE EXTRACTS

The extraction yield and TPC of the different algae extracts are presented in Table IV.11. The macro-algae A. nodosum and B. bifurcata showed higher extraction yields than the macro- alga F. vesiculosus and the micro-algae species. Specifically, B. bifurcata reached the highest value (35.85 g extract/100 g DW). C. vulgaris and S. platensis showed lower extraction yields than the macro-algae, being the second one that presented the lowest value (4.40 g extract/100 g DW). Farvin & Jacobsen (2013) also found a low extraction yield for the seaweed F. vesiculosus compared to most of the algae studied by them when using water as extraction solvent. There are factors, such as the chemical composition of the raw material and the polarity of the solvents used in the extraction, which can explain these variations in the extraction yield (Agregán et al., 2017b).

Micro-algae are a good source of carotenoids (Dufossé et al., 2005; Barba, Grimi, & Vorobiev, 2015). For instance, C. vulgaris is reported to contain high amounts of carotenoids, such as lutein and carotene (Cha et al., 2009; Plaza et al., 2012). This fact might be the reason of the low extraction yield obtained for these algae, since carotenoids are lipophilic compounds (Ahmed et al., 2014), making it difficult to extract them with polar solvents, such as ethanol, water or mixtures of both, as in the present study.

Regarding TPC of the different extracts, the ANE and BBE showed higher TPCs than the FVE and two micro-algae extracts. Specifically, the BBE presented the highest content (5.74 g PGE/100 g DW). It is important to note that the micro-algae presented much lower TPCs than the macro-algae (Table IV.11). These differences could be due to the high amount of phlorotannins present in the macro-algae. Chew et al. (2008) attributed to this hypothesis the higher TPC found in the brown alga Padina antillarum with respect to the green alga Caulerpa racemosa and the red alga Kappaphycus alvarezii. Analysing the extraction yield and TPC data, a correlation was noted, where the highest extraction yields corresponded to the highest TPCs.

IV.6.2. ANTIOXIDANT ACTIVITY OF ALGAE EXTRACTS

The antioxidant activity of the different algae extracts is presented in Table IV.11. The macro-algae showed higher antioxidant activities than the micro-algae for all the assays performed. As in the case of the TPC, phlorotannins could play an important role in the results obtained. Within the macro-algae, the BBE reached the highest values (556.20, 144.65 and 66.50 µmol TE/g DW in the ORAC, DPPH and FRAP assays, respectively) and the FVE the lowest. This indicates that the BBE is richer in compounds capable of scavenging free radicals than the

99 IV. Results and discussion rest of extracts. As expected, a positive correlation among the TPC and the antioxidant activity was observed. Thus, the BBE, which showed the highest TPC, also presented the highest antioxidant activity in almost all the assays performed, followed by the FVE, ANE and the micro-algae extracts. The same correlation was reported by other authors after using the DPPH radical scavenging assay (Chew et al., 2008). Concerning micro-algae, some studies provided certainly contradictory results. On the one hand, it was found that the antioxidant capacity of micro-algae is partly caused by polyphenols (Goiris et al., 2012). However, other study did not reported any correlation between phenolic content and antioxidant activity of ethanolic extracts resulting from nine micro-algae strains (Maadane et al., 2015).

IV.7. STUDY OF THE EFFECT OF FUCUS VESICULOSUS EXTRACT IN THE SHELF-LIFE OF PORK PATTIES FORMULATED WITH LINSEED OLEOGEL

Based on the data obtained in the previous chapter, the BBE resulted to be that with the highest antioxidant activity. Although this result seems to indicate that this extract is the most suitable to be used in future applications, it is necessary to specify the aims. A good extraction yield is a capital point from an industrial point of view, looking for the exploitation of resources, what results in lower production costs. From this perspective, the BBE is the best option to be used as a natural antioxidant among the extracts analysed, since it possesses the highest antioxidant activity with the highest extraction yield. On the other hand, at the time of adding these natural extracts as antioxidants in food products, excessive amounts could add foreign odours and flavours. If the data are analysed without taking into account the extraction yield, with the same amount of extract, the FVE showed a higher antioxidant potential. This means that this extract will be neccesary in a fewer amount than the rest of extracts to obtain the same antioxidant activity in the food, which makes it to decrease the possibility of adding foreign odours and flavours. Therefore, the FVE was the chosen to be added to the pork patties, with the aim to study its capacity to extent the shelf-life in this and similar products.

IV.7.1. PROXIMATE COMPOSITION AND FATTY ACID PROFILE OF PORK PATTIES

The nutritional composition (moisture, fat, protein and ash) and fatty acid profile of pork patties are shown in Table IV.12. No significant (P > 0.05) differences in the chemical composition were observed among the different formulations. Moreover, the different concentrations of the FVE in the patties did not influence the percentages of moisture, fat, protein and ash, being these results in agreement with those reported by Rodríguez-Carpena,

100 IV. Results and discussion

Morcuende, & Estévez (2011), where no changes in the proximate composition of raw pork meat patties with incorporation of Mediterranean berries and avocado by-products were noted.

Table IV.12. Effect of F. vesiculosus extract on proximate composition and fatty acid profile of pork patties on day 0 (mean ± standard deviation value) (n = 4).

Batch SEM Sig. CON BHT FVE-250 FVE-500 FVE-1000 Moisture (%) 64.55 ± 0.90 64.92 ± 0.47 65.17 ± 0.98 65.24 ± 0.20 64.63 ± 1.42 0.19 ns Fat (%) 13.98 ± 1.23 13.34 ± 0.69 13.52 ± 1.05 13.75 ± 0.46 13.51 ± 0.66 0.18 ns Protein (%) 17.22 ± 1.13 16.81 ± 0.44 16.70 ± 0.27 16.54 ± 0.28 16.60 ± 0.14 0.13 ns Ash (%) 2.05 ± 0.04 2.10 ± 0.03 2.03 ± 0.04 2.04 ± 0.04 2.09 ± 0.05 0.01 ns FA profile C14:0 1.03 ± 0.01b 0.98 ± 0.02a 1.00 ± 0.02a 1.01 ± 0.01a 1.00 ± 0.02a 0.01 * C16:0 19.99 ± 0.24b 19.24 ± 0.14a 19.65 ± 0.27b 19.70 ± 0.11b 19.64 ± 0.33b 0.07 ** C16:1n-7 1.86 ± 0.01b 1.79 ± 0.02a 1.79 ± 0.03a 1.85 ± 0.03b 1.82 ± 0.03a,b 0.01 ** C17:0 0.18 ± 0.00 0.18 ± 0.01 0.18 ± 0.01 0.18 ± 0.00 0.18 ± 0.01 0.00 ns C17:1n-7 0.15 ± 0.00 0.15 ± 0.00 0.15 ± 0.01 0.15 ± 0.00 0.15 ± 0.01 0.00 ns C18:0 11.05 ± 0.19 10.79 ± 0.18 10.97 ± 0.18 10.93 ± 0.12 10.93 ± 0.20 0.04 ns C18:1n-9t 0.18 ± 0.01 0.17 ± 0.00 0.18 ± 0.01 0.18 ± 0.00 0.18 ± 0.01 0.00 ns C18:1n-9c 36.18 ± 0.08c 35.38 ± 0.26a 35.76 ± 0.38a,b 36.06 ± 0.18b,c 35.68 ± 0.31a,b 0.08 ** C18:1n-7c 2.45 ± 0.07 2.46 ± 0.02 2.46 ± 0.13 2.47 ± 0.15 2.45 ± 0.14 0.02 ns C18:2n-6 12.92 ± 0.06 13.25 ± 0.21 13.08 ± 0.23 13.18 ± 0.48 13.38 ± 0.16 0.06 ns C20:0 0.20 ± 0.00 0.19 ± 0.00 0.20 ± 0.00 0.19 ± 0.01 0.20 ± 0.00 0.00 ns C20:1n-9 0.69 ± 0.03 0.70 ± 0.02 0.67 ± 0.02 0.67 ± 0.02 0.69 ± 0.03 0.01 ns C18:3n-3 11.28 ± 0.46 12.68 ± 0.63 11.94 ± 1.01 11.40 ± 0.31 11.63 ± 1.00 0.19 ns C20:2n-6 0.39 ± 0.00 0.39 ± 0.01 0.40 ± 0.02 0.40 ± 0.01 0.40 ± 0.02 0.00 ns C20:4n-6 0.36 ± 0.03a 0.41 ± 0.01b 0.39 ± 0.01a,b 0.40 ± 0.03b 0.40 ± 0.01b 0.01 * SFAs 32.44 ± 0.44b 31.38 ± 0.27a 31.99 ± 0.46a,b 32.01 ± 0.21a,b 31.95 ± 0.55a,b 0.11 * MUFAs 41.50 ± 0.14c 40.65 ± 0.28a 41.01 ± 0.36a,b 41.38 ± 0.27b,c 40.96 ± 0.35a,b 0.09 ** PUFAs 24.96 ± 0.39a 26.73 ± 0.51b 25.81 ± 0.77a,b 25.38 ± 0.39a 25.81 ± 0.84a,b 0.18 * ∑n-3 11.28 ± 0.46 12.68 ± 0.63 11.94 ± 1.01 11.40 ± 0.31 11.63 ± 1.00 0.19 ns ∑n-6 13.68 ± 0.07 14.05 ± 0.22 13.87 ± 0.26 13.98 ± 0.51 14.17 ± 0.17 0.07 ns n-6/n-3 1.21 ± 0.06 1.11 ± 0.07 1.17 ± 0.11 1.23 ± 0.07 1.23 ± 0.12 0.02 ns Results expressed as percentage of total fatty acid analysed. a-cMeans in the same row not followed by a common superscript letter are significantly different (P < 0.05; Duncan test). *(P < 0.05); **(P < 0.01).

The MUFAs were the most abundant fatty acids in all batches, with an average of 41.10% of the total fatty acids analysed, followed by the SFAs and PUFAs, with an average of 31.95% and 25.74%, respectively. As expected, the PUFA content was considerably higher than those obtained by other authors in previous studies with pork meat without vegetable oil addition (Pateiro et al., 2015).

The oleic acid was the most abundant MUFA and one of the main fatty acids in all batches. All patties contained important amounts of palmitic and stearic acids, with an average of 19.64%

101 IV. Results and discussion and 10.93%, respectively. Similar results were reported in some researches with porcine meat (Pateiro et al., 2015; Delgado-Pando et al., 2011).

The pork patties in the present study showed n-6/n-3 ratio values between 1 and 2 for all formulations without significant differences (P > 0.05). This outcome is in agreement with the recommendations given by Simopoulos (2010) who reported that the n-6/n-3 ratio in a healthy diet should be in the range between 1:1 to 2:1.

IV.7.2. EVOLUTION OF PHYSICAL PROPERTIES (pH AND COLOUR) OF PORK PATTIES

The results of the pH and colour parameters in the pork patties along refrigerated storage are shown in Table IV.13. The different patties formulations led to significant differences in the pH on day 0 (P < 0.001) and 18 (P < 0.01). The pH of all the patties suffered modifications during storage, except for the FVE-500 batch. A marked decrease was observed on day 11, except for the BHT batch. Then, the pH increased sharply until day 15. However, in the FVE-250 batch, the pH began to increase since that day. Similar results were observed during refrigerated storage of pork patties with natural extracts (Lorenzo et al., 2014b).

The different formulations, as well as the storage time, affected the colour parameters (L*, a* and b*). The L* value did not seem to follow any specific trend during storage, despite showing a significant (P < 0.05) change in the BHT, FVE-250 and FVE-1000 batches. Regarding the a* value, a progressive loss of red colour on the patties surface was noted along storage. A similar trend was also observed in other investigations with meats from different species (Fernandes et al., 2016). The reduction of the a* value on the patties surface along storage confirmed the appearance of a fading phenomenon, since this process is mainly marked by the loss of this color parameter (Lorenzo et al., 2014b). Finally, the b* value also decreased over time, although less markedly than the a* value. These results are in good agreement with those found by Fernandes et al. (2016) in sheep patties with added BHT and as antioxidants. The different formulations affected the b* value along refrigerated storage.

102 IV. Results and discussion

Table IV.13. Effect of F. vesiculosus extract on evolution of pH and colour parameters (L*, a* and b*) of pork patties during refrigerated storage (mean ± standard deviation value) (n = 4). Batch Day SEM Sig. CON BHT FVE-250 FVE-500 FVE-1000 pH 0 5.65 ± 0.02a,1 5.68 ± 0.02a,b,1 5.70 ± 0.02b,1,2 5.71 ± 0.02b 5.75 ± 0.02c,2 0.01 *** 7 5.73 ± 0.042,3 5.66 ± 0.101 5.74 ± 0.052 5.75 ± 0.08 5.74 ± 0.042 0.02 ns 11 5.68 ± 0.031,2 5.66 ± 0.011 5.67 ± 0.041 5.65 ± 0.01 5.65 ± 0.021 0.01 ns 15 5.74 ± 0.023 5.77 ± 0.052 5.66 ± 0.061 5.76 ± 0.09 5.72 ± 0.032 0.01 ns 18 5.74 ± 0.04a,b,3 5.78 ± 0.02c,2 5.76 ± 0.04b,c,2 5.71 ± 0.01a 5.72 ± 0.01a,2 0.01 ** SEM 0.01 0.02 0.01 0.01 0.01 Sig. ** ** * ns *** L* 0 62.09 ± 1.28 63.11 ± 1.841,2 62.01 ± 0.191 61.15 ± 0.51 60.06 ± 3.021 0.41 ns 7 61.61 ± 1.99 65.54 ± 0.202 62.31 ± 2.321 63.48 ± 3.81 61.51 ± 1.391,2 0.57 ns 11 59.80 ± 2.47 63.84 ± 3.431,2 63.51 ± 1.531,2 62.47 ± 1.72 64.94 ± 1.762 0.61 ns 15 64.02 ± 1.53 61.37 ± 1.581 65.72 ± 2.272 62.02 ± 2.35 63.43 ± 2.201,2 0.53 ns 18 61.52 ± 1.78 60.09 ± 3.161 61.59 ± 1.291 62.79 ± 2.41 61.99 ± 1.921,2 0.48 ns SEM 0.48 0.64 0.48 0.51 0.57 Sig. ns * * ns * a* 0 12.03 ± 0.41b,3 11.10 ± 0.96a,b,3 9.88 ± 0.64a,3 10.25 ± 0.77a,4 10.33 ± 1.36a,4 0.25 * 7 9.35 ± 1.402 8.91 ± 0.622 9.17 ± 2.272,3 8.35 ± 1.533 8.70 ± 1.073 0.31 ns 11 8.86 ± 1.402 8.19 ± 0.651,2 7.88 ± 0.682 8.08 ± 0.792,3 7.18 ± 0.952 0.22 ns 15 6.07 ± 0.871 7.19 ± 0.291 5.62 ± 0.861 6.63 ± 1.052 7.02 ± 0.592 0.21 ns 18 4.72 ± 0.58a,1 7.36 ± 1.30b,1 4.94 ± 0.52a,1 5.07 ± 0.78a,1 5.54 ± 0.65a,1 0.27 ** SEM 0.62 0.36 0.50 0.45 0.42 Sig. *** *** *** *** ***

103 IV. Results and discussion

b* 0 22.51 ± 0.64b,3 21.58 ± 0.51a,b,3 20.75 ± 0.77a,2 20.92 ± 1.07a 21.09 ± 0.81a 0.21 * 7 19.86 ± 1.072 19.62 ± 1.691,2 20.55 ± 1.622 19.81 ± 2.60 20.85 ± 1.99 0.39 ns 11 19.36 ± 0.442 20.71 ± 0.642,3 19.67 ± 0.701,2 20.33 ± 1.12 20.06 ± 1.30 0.21 ns 15 17.91 ± 1.65a,1 19.10 ± 0.76a,b,1 19.18 ± 1.17a,b,1,2 19.95 ± 1.17b 20.78 ± 0.91b 0.32 * 18 17.65 ± 0.301 18.91 ± 0.711 18.04 ± 1.471 18.65 ± 1.06 19.22 ± 0.71 0.23 ns SEM 0.44 0.30 0.33 0.35 0.29 Sig. *** ** * ns ns a-cMeans in the same row (different batches on the same storage day) not followed by a common superscript letter are significantly different (P < 0.05; Duncan test). 1- 4Means in the same column (same batch in different storage days) not followed by a common superscript number are significantly different (P < 0.05; Duncan test). *(P < 0.05); **(P < 0.01); ***(P < 0.001).

104 IV. Results and discussion

IV.7.3. EVOLUTION OF LIPID AND PROTEIN OXIDATION IN PORK PATTIES

The lipid oxidation was quantified in the patties through the TBARS assay (Table IV.14). All patties formulations showed a significant (P < 0.001) increase in the TBARS values during refrigerated storage. According to the results, the patties which contained BHT and 1000 ppm of seaweed extract had greater oxidative stability than those containing 250 and 500 ppm and the control without antioxidant addition (Table IV.14).

The TBARS slightly increased until day 11, after which an abrupt increase was noted up to day 15. Other authors, such as Lorenzo et al. (2014b) and Sánchez-Escalante et al. (2003), reported similar TBARS evolutions during refrigerated storage of patties with added natural antioxidants. The latter study found that the batch used as a negative control showed a sharp increment of the secondary oxidation since day 4 (~0.8 mg MDA/kg sample) until day 12 (~3.8 mg MDA/kg sample). The sharp increment found in the present study (between day 15 and 18) showed comparable TBARS values (0.87 and 3.92 mg MDA/kg sample). After this point, Sánchez-Escalante et al. (2003) observed a stabilization and even a slight decrease in the later days.

Table IV.14. Effect of F. vesiculosus extract on TBARS evolution during refrigerated storage (mean ± standard deviation value) (n = 4).

Batch Day SEM Sig. CON BHT FVE-250 FVE-500 FVE-1000 0 0.13 ± 0.00e,1 0.10 ± 0.00c,1 0.09 ± 0.00b,1 0.12 ± 0.00d,1 0.08 ± 0.00a,1 0.00 *** 7 0.87 ± 0.172 0.58 ± 0.112 0.74 ± 0.142 0.66 ± 0.131,2 0.76 ± 0.152 0.04 ns 11 1.44 ± 0.01d,2 0.78 ± 0.09a,3 1.07 ± 0.01b,2 1.19 ± 0.01c,2 1.09 ± 0.01b,2 0.05 *** 15 3.92 ± 0.63b,c,3 0.96 ± 0.15a,4 3.70 ± 0.59b,c,3 4.46 ± 0.72c,3 3.54 ± 0.57b,3 0.30 *** 18 4.09 ± 0.66b,c,3 1.00 ± 0.16a,4 3.87 ± 0.62b,c,3 4.66 ± 0.75c,3 3.69 ± 0.59b,3 0.31 *** SEM 0.38 0.08 0.37 0.46 0.35 Sig. *** *** *** *** *** a-eMeans in the same row (different batches on the same storage day) not followed by a common superscript letter are significantly different (P < 0.05; Duncan test). 1-4Means in the same column (same batch in different storage days) not followed by a common superscript number are significantly different (P < 0.05; Duncan test). ***(P < 0.001).

The protein oxidation evolution in the pork patties during storage is shown in Table IV.15. A significant (P < 0.01) increase in the protein oxidation during storage was found in all the batches. The protein oxidation began on day 7 and increased until the end of storage. The CON batch was the least stable to protein oxidation, reaching a value of 6.81 nmol carbonyl/mg protein on day 18. Conversely, the BHT batch was the most effective, showing lower carbonyl formation than the other batches. The incorporation of the FVE to patties slowed down the carbonyl formation at the last storage day, reducing the protein oxidation values among 0.77

105 IV. Results and discussion and 1.15 nmol carbonyl/mg protein. This result suggests that the addition of FVE was effective in softening the final carbonyl concentration, especially in the batch with 1000 ppm of the seaweed extract. Other natural extracts, such as peel and seed extracts from avocado (Rodríguez-Carpena, Morcuende, & Estévez, 2011) and oregano extracts (Fernandes et al., 2016) showed a time delay of carbonyl formation when they were added to pork, beef and sheep meats, respectively.

The phenolic content found in the FVE probably protected the patties against oxidative process, delaying the appearance of degradation products.

Table IV.15. Effect of F. vesiculosus extract on protein oxidation evolution during refrigerated storage (mean ± standard deviation value) (n = 4).

Batch Day SEM Sig. CON BHT FVE-250 FVE-500 FVE-1000 0 3.57 ± 0.101 3.51 ± 0.191 3.49 ± 0.501 3.53 ± 0.191 3.47 ± 0.141 0.06 ns 7 3.79 ± 0.311 3.52 ± 0.061 3.68 ± 0.051,2 3.55 ± 0.101 3.53 ± 0.151 0.04 ns 11 4.58 ± 0.32b,2 3.58 ± 0.04a,1 4.34 ± 0.18b,2 4.51 ± 0.44b,2 4.45 ± 0.51b,2 0.11 ** 15 5.94 ± 0.17c,3 3.79 ± 0.09a,1,2 5.40 ± 0.77b,c,3 5.31 ± 0.18b,c,3 5.14 ± 0.29b,3 0.19 *** 18 6.81 ± 0.61c,4 4.04 ± 0.39a,2 6.04 ± 0.39b,3 5.72 ± 0.27b,3 5.66 ± 0.54b,3 0.23 *** SEM 0.31 0.06 0.27 0.22 0.22 Sig. *** ** *** *** *** a-cMeans in the same row (different treatments on the same storage day) not followed by a common superscript letter are significantly different (P < 0.05; Duncan test). 1-4Means in the same column (same treatment on different storage days) not followed by a common superscript number are significantly different (P < 0.05; Duncan test). **(P < 0.01); ***(P < 0.001).

IV.7.4. SENSORY PROPERTIES OF PORK PATTIES

Figure IV.12 and 13 show the findings of the sensory evaluation of the cooked (day 0) and raw (at 0, 7, 11, 15, and 18 days) pork patties, respectively. According to the results, the cooked samples on day 0 did not show significant differences in the odour and taste among all the different formulations. The most significant results can be correlated with odour attribute, reached for the sample with the highest acceptation (FVE-500 batch), while no differences were found among the CON batch and the FVE 500 and 1000 batches. Bañón et al. (2007) reported similar findings with no appreciable differences in the odour and taste on day 0 of low-sulfite beef patties formulated with green tea and grape seed extracts. Regarding the sample preference, no consistent differences were observed in the samples by the panelists, suggesting that the small differences found in the acceptance test seemed not having had influence on the preference test.

Regarding the evolution of the sensory properties during refrigerated storage, the colour at the surface and the odour attributes in the raw samples from all formulations carried out

106 IV. Results and discussion showed acceptable values until day 11. Since that time, the acceptability of the patties decreased until reaching a score of “hardly acceptable” at the end of storage. The FVE-1000 batch achieved better colour punctuations on day 18 than the rest of batches, but always behind acceptability. In accordance with the results obtained, it may be concluded that the FVE did not improved any of the attributes studied (colour, discoloration at surface and odour)

at the concentrations used for the study.

Acceptable odour taste

1 2 3 4 5 Hedonic scale CON BHT FVE-250 FVE-500 FVE-1000

Figure IV.12. Average sensory scores for pork patties with different concentrations of F. vesiculosus extract. Hedonic scale used: 1 = excellent; 2 = good; 3 = acceptable; 4 = hardly acceptable; 5 = not acceptable.

COLOUR

5

4

3 Acceptable

2 Hedonic scale Hedonic 1

0 0 5 10 15 20 Storage days CON BHT FVE-250 FVE-500 FVE-1000

107 IV. Results and discussion

DISCOLORATION AT SURFACE

5

4

3 Acceptable

2 Hedonic scale Hedonic 1

0 0 5 10 15 20 Storage days CON BHT FVE-250 FVE-500 FVE-1000

ODOUR

5

4

3 Acceptable

2 Hedonic scale Hedonic 1

0 0 5 10 15 20 Storage days CON BHT FVE-250 FVE-500 FVE-1000

Figure IV.13. Evolution of odour, discoloration at surface and taste attributes in raw pork patties with different concentrations of F. vesiculosus extract during refrigerated storage. Hedonic scale used: 1 = excellent; 2 = good; 3 = acceptable; 4 = hardly acceptable; 5 = not acceptable.

108

V. CONCLUSIONS

V. Conclusions

1. B. bifurcata was found to have higher lipid and ash contents than A. nodosum and F. vesiculosus, while F. vesiculosus had the highest protein content. The seaweeds showed to be appropriate foods from a lipid point of view with ratios n-6/n-3 according to WHO recommendations, being the PUFAs the most abundant fatty acids, contributing with almost the half of total fatty acids. On the other hand, all the essential amino acids were present in the three seaweeds studied, being the glutamic and aspactric acid the most abundant, and almost all amino acids were above the chemical score established by FAO/WHO/UNU, showing to be an extraordinary source of protein of high biological value.

2. The aqueous extracts from seaweeds A. nodosum, F. vesiculosus and B. bifurcata contained different levels of total phenolic compounds and diverse antioxidant properties in vitro, with the F. vesiculosus extract presenting overall the highest antioxidant capacity and the A. nodosum extract the lowest.

3. The tentative analysis of phenolic composition of the brown seaweed species A. nodosum, F. vesiculosus and B. bifurcata by LC-DAD-ESI-MS/MS indicates the presence of phlorotannins as major phenolic compounds. The presence of these compounds indicates a promising antioxidant potential and health benefits of seaweeds as a functional ingredient in food.

4. The addition of aqueous extracts from seaweeds A. nodosum, F. vesiculosus and B. bifurcata to an oily matrix such as conola oil, delayed both primary and secondary oxidative changes. This finding proved that these seaweed extracts can be used as an easily accessible source of natural antioxidants to stabilize even more complex food systems.

5. The level of protection of the aqueous extracts from A. nodosum, F. vesiculosus and B. bifurcata against the lipid oxidation in a low-fat pork liver pâté was negigible. The strong stability showed by the final product prevented the evaluation of the antioxidant potential of the extracts in the conditions established in the study. On the other hand, the partial pork backfat replacement by canola and high-oleic sunflower oils improved the fatty acid profile, what constitutes an added value for a high-fat meat product such as pâté.

6. The ultrasound-assisted extraction along with the mixture water/ethanol (50:50 v/v) showed to be the most promising extraction conditions in the alga B. bifurcata, achieving an adequate yield with an increased amount of recovered polyphenols with antioxidant activity. This particular combination of extraction method and solvent favoured the extraction of phenolic compounds, showing to be an interesting alternative regarding other extraction conditions.

111 V. Conclusions

7. Overall, the F. vesiculosus extract obtained by the previously cited extraction condition presented greater antioxidant properties than the other two macro-algae (A. nodosum and B. bifurcata) and micro-algae (C. vulgaris and S. platensis), because of their higher concentration of bioactive compounds with antioxidant activity.

8. The previous F. vesiculosus extract incorporated into pork patties at the concentration of 1000 ppm successfully protected the samples against oxidation. Even so, the extract presented limited protection in general, what makes it unviable to be used in meats or meat products.

112

VI. REFERENCES

VI. References

Abbas, K. A., Mohamed, A., Abdulamir, A. S., & Abas, H. A. (2008). A review on supercritical fluid extraction as new analytical method. American Journal of Biochemistry & Biotechnology, 4(4), 345-353. Abu-Ghannam, N., & Rajauria, G. (2013). Antimicrobial activity of compounds isolated from algae. In Functional Ingredients from Algae for Foods and Nutraceuticals (pp. 287-306), Domínguez, H. Woodhead Publishing (Sawston, UK). Adom, K. K., & Liu, R. H. (2002). Antioxidant activity of grains. Journal of Agricultural and Food Chemistry, 50(21), 6182-6187. Agregán, R., Lorenzo, J. M., Munekata, P. E., Dominguez, R., Carballo, J., & Franco, D. (2017b). Assessment of the antioxidant activity of Bifurcaria bifurcata aqueous extract on canola oil. Effect of extract concentration on the oxidation stability and volatile compound generation during oil storage. Food Research International, 99(Pt 3), 1095-1102. Agregán, R., Munekata, P. E., Domínguez, R., Carballo, J., Franco, D., & Lorenzo, J. M. (2017a). Proximate composition, phenolic content and in vitro antioxidant activity of aqueous extracts of the seaweeds Ascophyllum nodosum, Bifurcaria bifurcata and Fucus vesiculosus. Effect of addition of the extracts on the oxidative stability of canola oil under accelerated storage conditions. Food Research International, 99, 986-994. Ahmed, F., Fanning, K., Netzel, M., Turner, W., Li, Y., & Schenk, P. M. (2014). Profiling of carotenoids and antioxidant capacity of microalgae from subtropical coastal and brackish waters. Food Chemistry, 165, 300-306. Akbarirad, H., Ardabili, A. G., Kazemeini, S. M., & Khaneghah, A. M. (2016). An overview on some of important sources of natural antioxidants. International Food Research Journal, 23(3), 928-933. Allen, L.; de Benoist, B.; Dary, O.; Hurrel, R. (2006). Guidelines on Food Fortification with Micronutrientes. World Health Organization (WHO) and Food and Agriculture Organization of the United Nations (FAO) (Quebec, QC, Canada). Alves, C., Pinteus, S., Simões, T., Horta, A., Silva, J., Tecelão, C., & Pedrosa, R. (2016). Bifurcaria bifurcata: a key macro‐alga as a source of bioactive compounds and functional ingredients. International Journal of Food Science & Technology, 51(7), 1638-1646. Amarowicz, R., & Pegg, R. B. (2013). Inhibition of proliferation of human carcinoma cell lines by phenolic compounds from a bearberry-leaf crude extract and its fractions. Journal of Functional Foods, 5(2), 660-667. Anonymous (2010). Fats and fatty acids in human nutrition. Food and Agriculture Organization of the United Nations (FAO) report of an expert consultation. FAO food and nutrition paper, vol 91 (Rome, Italy).

115 VI. References

AOAC (2000). Peroxide value of oils and fats. Official method 965.33. Official Methods of Analysis (17th ed.). Association of Official Analytical Chemist (Gaithersburg, MD, USA). AOCS (2005). Rapid determination of oil/fat utilizing high temperature solvent extraction. Official procedure am 5-04. AOCS (Ed.). American Oil Chemists Society (Urbana, IL, USA). Armenteros, M., Ventanas, S., Morcuende, D., Estévez, M., & Ventanas, J. (2012). Empleo de antioxidantes naturales en productos cárnicos. Eurocarne, 207, 63-73. Athukorala, Y., Lee, K. W., Shahidi, F., Heu, M. S., Kim, H. T., Lee, J. S., & Jeon, Y. J. (2003). Antioxidant efficacy of extracts of an edible red alga (Grateloupia filicina) in linoleic acid and fish oil. Journal of Food Lipids, 10(4), 313-327. Bajaj, C., Djeu, P., Siddavanahalli, V., & Thane, A. (2004). Texmol: Interactive visual exploration of large flexible multi-component molecular complexes. In Procedings of the conference on Visualization 2004 (pp. 243-250). IEEE Computer Society Press (Washington DC, USA). Balboa, E. M., Conde, E., Moure, A., Falqué, E., & Domínguez, H. (2013). In vitro antioxidant properties of crude extracts and compounds from brown algae. Food Chemistry, 138(2- 3), 1764-1785. Bañón, S., Díaz, P., Rodríguez, M., Garrido, M. D., & Price, A. (2007). Ascorbate, green tea and grape seed extracts increase the shelf life of low sulphite beef patties. Meat Science, 77(4), 626-633. Barba, F. J., Grimi, N., & Vorobiev, E. (2015). New approaches for the use of non-conventional cell disruption technologies to extract potential food additives and nutraceuticals from microalgae. Food Engineering Reviews, 7(1), 45-62. Benzie, I. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Analytical Biochemistry, 239(1), 70-76. Boulekbache‐Makhlouf, L., Meudec, E., Mazauric, J. P., Madani, K., & Cheynier, V. (2013). Qualitative and semi‐quantitative analysis of phenolics in Eucalyptus globulus leaves by high‐performance liquid chromatography coupled with diode array detection and electrospray ionisation mass spectrometry. Analysis, 24(2), 162-170. Brand-Williams, W., Cuvelier, M. E., & Berset, C. L. W. T. (1995). Use of a free radical method to evaluate antioxidant activity. LWT-Food Science and Technology, 28(1), 25-30. Campos-Vega, R., Loarca-Piña, G., & Oomah, B. D. (2010). Minor components of pulses and their potential impact on human health. Food Research International, 43(2), 461-482. Cha, K. H., Lee, H. J., Koo, S. Y., Song, D. G., Lee, D. U., & Pan, C. H. (2009). Optimization of pressurized liquid extraction of carotenoids and chlorophylls from Chlorella vulgaris. Journal of Agricultural and Food Chemistry, 58(2), 793-797.

116 VI. References

Chan, P. T., & Matanjun, P. (2017). Chemical composition and physicochemical properties of tropical red seaweed, Gracilaria changii. Food Chemistry, 221, 302-310. Chan, P. T., Matanjun, P., Yasir, S. M., & Tan, T. S. (2015). Antioxidant activities and polyphenolics of various solvent extracts of red seaweed, Gracilaria changii. Journal of Applied Phycology, 27(6), 2377-2386. Chew, Y. L., Lim, Y. Y., Omar, M., & Khoo, K. S. (2008). Antioxidant activity of three edible seaweeds from two areas in South East Asia. LWT-Food Science and Technology, 41(6), 1067-1072. CIE (1978). Recommendations on uniform color spaces: color-difference equations, psychometric color terms. Commission Internationale de l'Eclairage (Ed.) (Paris, France). Cofrades, S., López-Lopez, I., Bravo, L., Ruiz-Capillas, C., Bastida, S., Larrea, M. T., & Jiménez- Colmenero, F. (2010). Nutritional and antioxidant properties of different brown and red Spanish edible seaweeds. Food Science and Technology International, 16(5), 361-370. Craft, B. D., Kerrihard, A. L., Amarowicz, R., & Pegg, R. B. (2012). Phenol‐based antioxidants and the in vitro methods used for their assessment. Comprehensive Reviews in Food Science and Food Safety, 11(2), 148-173. Dávalos, A., Bartolomé, B., Suberviola, J., & Gómez-Cordovés, C. (2003). ORAC-fluorescein as model for evaluating antioxidant activity of wines. Polish Journal of Food and Nutrition Science, 12(1), 133-136. Davies, M. J., & Dean, R. T. (2003). Radical-mediated Protein Oxidation. Oxford Science Publications (Oxford, UK). de Quirós, A. R. B., Lage-Yusty, M. A., & López-Hernández, J. (2010). Determination of phenolic compounds in macro-algae for human consumption. Food Chemistry, 121(2), 634-638. Delgado-Pando, G., Cofrades, S., Rodríguez-Salas, L., & Jiménez-Colmenero, F. (2011). A healthier oil combination and konjac gel as functional ingredients in low-fat pork liver pâté. Meat Science, 88(2), 241-248. Delgado-Pando, G., Cofrades, S., Ruiz-Capillas, C., Triki, M., & Jiménez-Colmenero, F. (2012). Enriched n-3 PUFA/konjac gel low-fat pork liver pâté: Lipid oxidation, microbiological properties and biogenic amine formation during chilling storage. Meat Science, 92(4), 762-767. Dikeman, M., & Devine, M. (2014). Encyclopedia of Meat Sciences, 1 (2th ed.). Academic Press (Cambridge, MA, USA). Domínguez, R., Munekata, P. E., Cittadini, A., & Lorenzo, J. M. (2016). Effect of the partial NaCl substitution by other chloride salts on the volatile profile during the ripening of dry- cured lacón. Grasas y Aceites, 67(2), 128.

117 VI. References

Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28(3), 350- 356. Dufossé, L., Galaup, P., Yaron, A., Arad, S. M., Blanc, P., Murthy, K. N. C., & Ravishankar, G. A. (2005). Microorganisms and microalgae as sources of pigments for food use: a scientific oddity or an industrial reality?. Trends in Food Science & Technology, 16(9), 389-406. El Wahidi, M., El Amraoui, B., El Amraoui, M., & Bamhaoud, T. (2015). Screening of antimicrobial activity of macroalgae extracts from the Moroccan Atlantic coast. Annales Pharmaceutiques Francaises, 73(3), 190-196. Eom, S. H., Santos, J. A., Kim, J. H., Jung, W. K., Kim, D. H., & Kim, Y. M. (2015). In vitro antibacterial and synergistic activity of an Ecklonia cava extract against anti biotic- resistant Streptococcus parauberis. Fisheries and Aquatic Sciences, 18(3), 241-247. Estévez, M., & Cava, R. (2004). Lipid and protein oxidation, release of iron from heme molecule and colour deterioration during refrigerated storage of liver pâté. Meat Science, 68(4), 551-558. Estévez, M., Ramirez, R., Ventanas, S., & Cava, R. (2007). Sage and essential oils versus BHT for the inhibition of lipid oxidative reactions in liver pâté. LWT-Food Science and Technology, 40(1), 58-65. Estévez, M., Ventanas, S., & Cava, R. (2005). Physicochemical properties and oxidative stability of liver pâté as affected by fat content. Food Chemistry, 92(3), 449-457. Estévez, M., Ventanas, S., & Cava, R. (2006). Effect of natural and synthetic antioxidants on protein oxidation and colour and texture changes in refrigerated stored porcine liver pâté. Meat Science, 74(2), 396-403. Estévez, M., Ventanas, S., Ramírez, R., & Cava, R. (2004). Analysis of volatiles in porcine liver pâtés with added sage and rosemary essential oils by using SPME-GC-MS. Journal of Agricultural and Food Chemistry, 52(16), 5168-5174. Falowo, A. B., Fayemi, P. O., & Muchenje, V. (2014). Natural antioxidants against lipid–protein oxidative deterioration in meat and meat products: A review. Food Research International, 64, 171-181. FAO/WHO/UNU Expert Consultation (2007). Amino acid requirements of adults. Protein and Amino Acids Requirements in Human Nutrition (pp. 135-159). FAO/WHO/UNU (Rome, Italy). Farvin, K. S., & Jacobsen, C. (2013). Phenolic compounds and antioxidant activities of selected species of seaweeds from Danish coast. Food Chemistry, 138(2-3), 1670-1681.

118 VI. References

Fernandes, R. P. P., Trindade, M. A., Lorenzo, J. M., Munekata, P. E. S., & De Melo, M. P. (2016). Effects of oregano extract on oxidative, microbiological and sensory stability of sheep burgers packed in modified atmosphere. Food Control, 63, 65-75. Fleurence, J. (1999). Seaweed proteins: biochemical, nutritional aspects and potential uses. Trends in Food Science & Technology, 10(1), 25-28. Franco, D., Pateiro, M., Rodríguez Amado, I., López Pedrouso, M., Zapata, C., Vázquez, J. A., & Lorenzo, J. M. (2016). Antioxidant ability of potato (Solanum tuberosum) peel extracts to inhibit soybean oil oxidation. European Journal of Lipid Science and Technology, 118(12), 1891-1902. Frankel, E. N. (1998). Lipid oxidation. The Oily Press (Dundee, Scotland). Frankel, E. N., Hu, M. L., & Tappel, A. L. (1989). Rapid headspace gas chromatography of hexanal as a measure of lipid peroxidation in biological samples. Lipids, 24(11), 976-981. García-Sánchez, M. A., Rojas-González, F., Menchaca-Campos, E. C., Tello-Solís, S. R., Quiroz- Segoviano, R., Diaz-Alejo, L. A., Salas-Bañales, E., & Campero, A. (2013). Crossed and linked histories of tetrapyrrolic macrocycles and their use for engineering pores within sol-gel matrices. Molecules, 18(1), 588-653. Goiris, K., Muylaert, K., Fraeye, I., Foubert, I., De Brabanter, J., & De Cooman, L. (2012). Antioxidant potential of microalgae in relation to their phenolic and carotenoid content. Journal of Applied Phycology, 24(6), 1477-1486. Gómez, B., Strozzi, I., Barba, F. J., Vargas, F. C., Saraiva, J. A., Marszałek, K., do Amaral Sobral, P. J., & Lorenzo, J. M. (2018). Evaluation of the antioxidant capacity of a guarana seed extract on canola oil lipid stability using accelerated storage. European Journal of Lipid Science and Technology, 120(12). Gómez-Ordóñez, E., Jiménez-Escrig, A., & Rupérez, P. (2010). Dietary fibre and physicochemical properties of several edible seaweeds from the northwestern Spanish coast. Food Research International, 43(9), 2289-2294. Guiry, M. D. (2000-2019). Examples of a brown (Bifurcaria bifurcata), green (Ulva rigida) and red (Cryptopleura ramosa) seaweed. Recovered from http://www.seaweed.ie. Gupta, S., & Abu-Ghannam, N. (2011a). Recent developments in the application of seaweeds or seaweed extracts as a means for enhancing the safety and quality attributes of foods. Innovative Food Science & Emerging Technologies, 12(4), 600-609. Gupta, S., & Abu-Ghannam, N. (2011b). Bioactive potential and possible health effects of edible brown seaweeds. Trends in Food Science & Technology, 22(6), 315-326.

119 VI. References

Gupta, S., Rajauria, G., & Abu‐Ghannam, N. (2010). Study of the microbial diversity and antimicrobial properties of Irish edible brown seaweeds. International Journal of Food Science & Technology, 45(3), 482-489. Guyon, C., Meynier, A., & De Lamballerie, M. (2016). Protein and lipid oxidation in meat: A review with emphasis on high-pressure treatments. Trends in Food Science & Technology, 50, 131-143. Halliwell, B., & Gutteridge, J. M. (1990). Role of free radicals and catalytic metal ions in human disease: An overview. Methods in Enzymology, 186, (1-85). Heffernan, N., Brunton, N. P., FitzGerald, R. J., & Smyth, T. J. (2015). Profiling of the molecular weight and structural isomer abundance of macroalgae-derived phlorotannins. Marine Drugs, 13(1), 509-528. Hossain, M. B., Rai, D. K., Brunton, N. P., Martin-Diana, A. B., & Barry-Ryan, C. (2010). Characterization of phenolic composition in Lamiaceae spices by LC-ESI-MS/MS. Journal of Agricultural and Food Chemistry, 58(19), 10576-10581. Hwang, E. S., & Do Thi, N. (2014). Effects of extraction and processing methods on antioxidant compound contents and radical scavenging activities of laver (Porphyra tenera). Preventive Nutrition and Food Science, 19(1), 40-48. Institute of Medicine (2002). Dietary Reference Intakes for Energy, Carbohydrate, Fibre, Fat, Fatty Acids Cholesterol, Protein and Amino Acids. National Academy Press (Washington, DC, USA). ISO (1978). Determination of nitrogen content, ISO 937:1978 standard. International Standards Meat and Meat Products. International Organization for Standardization (Genéve, Switzerland). ISO (1997). Determination of moisture content, ISO 1442:1997 standard. International Standards Meat and Meat Products. International Organization for Standardization (Genéve, Switzerland). ISO (1998). Determination of ash content, ISO 936:1998 standard. International Standards Meat and Meat Products. International Organization for Standardization (Genéve, Switzerland). Jung, H. A., Jung, H. J., Jeong, H. Y., Kwon, H. J., Ali, M. Y., & Choi, J. S. (2014). Phlorotannins isolated from the edible brown alga exert anti-adipogenic activity on 3T3-L1 adipocytes by downregulating C/EBPα and PPARγ. Fitoterapia, 92, 260-269. Kadam, S. U., Tiwari, B. K., & O’Donnell, C. P. (2013). Application of novel extraction technologies for bioactives from marine algae. Journal of Agricultural and Food Chemistry, 61(20), 4667-4675.

120 VI. References

Kalita, D., & Netravali, A. N. (2017). Thermoset Resin Based Fiber Reinforced Biocomposites. In Textile Finishing: Recent Developments and Future Trends (pp. 423-484), Mittal, K. L., & Bahners, T. John Wiley & Sons (Haboken, NJ, USA). Kanazawa, K., Kanazawa, E., & Natake, M. (1985). Uptake of secondary autoxidation products of linoleic acid by the rat. Lipids, 20(7), 412-419. Kanner, J. (2007). Dietary advanced lipid oxidation end products are risk factors to human health. Molecular Nutrition & Food Research, 51(9), 1094-1101. Karwowska, M., Wójciak, K. M., & Dolatowski, Z. J. (2015). The influence of acid whey and mustard seed on lipid oxidation of organic fermented sausage without nitrite. Journal of the Science of Food and Agriculture, 95(3), 628-634. Kashanian, S., & Dolatabadi, J. E. N. (2009). DNA binding studies of 2-tert-butylhydroquinone (TBHQ) food additive. Food Chemistry, 116(3), 743-747. Khan, M. R., & Shahidi, F. (2001). Effects of natural and synthetic antioxidants on the oxidative stability of borage and evening primrose triacylglycerols. Food Chemistry, 75(4), 431- 437. Kikugawa, K., Kato, T., & Hayasaka, A. (1991). Formation of dityrosine and other fluorescent amino acids by reaction of amino acids with lipid hydroperoxides. Lipids, 26(11), 922- 929. Kim, E., Coelho, D., & Blachier, F. (2013). Review of the association between meat consumption and risk of colorectal cancer. Nutrition Research, 33(12), 983-994. Kim, T. S., Yeo, J., Kim, J. Y., Kim, M. J., & Lee, J. (2013). Determination of the degree of oxidation in highly-oxidised lipids using profile changes of fatty acids. Food Chemistry, 138(2-3), 1792-1799. Kindleysides, S., Quek, S. Y., & Miller, M. R. (2012). Inhibition of fish oil oxidation and the radical scavenging activity of New Zealand seaweed extracts. Food Chemistry, 133(4), 1624-1631. Koivikko, R. (2008). Brown algal phlorotannins: improving and applying chemical methods. Turku University Publications. serie A1, Astronomica-chemica-physica-mathematica (pp. 64). Kolsi, R. B. A., Frikha, D., Jribi, I., Hamza, A., Fekih, L., & Belgith, K. (2015). Screening of antibacterial and antifongical activity in marine macroalgae and magnoliophytea from the coast of Tunisia. International Journal of Pharmacy and Pharmaceutical Sciences, 7(3), 47-51. Krinsky, N. I. (1989). Antioxidant functions of carotenoids. Free Radical Biology & Medicine , 7(6), 617-635.

121 VI. References

Kuda, T., Tsunekawa, M., Goto, H., & Araki, Y. (2005). Antioxidant properties of four edible algae harvested in the Noto Peninsula, Japan. Journal of Food Composition and Analysis, 18(7), 625-633. Kumar, M., Kumari, P., Trivedi, N., Shukla, M. K., Gupta, V., Reddy, C. R. K., & Jha, B. (2011). Minerals, PUFAs and antioxidant properties of some tropical seaweeds from Saurashtra coast of India. Journal of Applied Phycology, 23(5), 797-810. Larsen, C. S. (2003). Animal source foods and human health during evolution. The Journal of Nutrition, 133(11), 3893S-3897S. Li, B. F. (2009). Marine Health Food. Chemical Industry Press (Beijing, China). Li, Y. X., Wijesekara, I., Li, Y., & Kim, S. K. (2011). Phlorotannins as bioactive agents from brown algae. Process Biochemistry, 46(12), 2219-2224. Lopes, G. L. L. (2014). Seaweeds from the Portuguese coast: chemistry, antimicrobial and anti- inflammatory capacity. Thesis for Doctor Degree in Pharmaceutical Sciences. University of Porto (Porto, Portugal). Lopes, G., Pinto, E., Andrade, P. B., & Valentão, P. (2013). Antifungal activity of phlorotannins against dermatophytes and yeasts: Approaches to the mechanism of action and influence on Candida albicans virulence factor. PloS One, 8(8), e72203. Lopes, G., Sousa, C., Silva, L. R., Pinto, E., Andrade, P. B., Bernardo, J., Mouga, T., & Valentão, P. (2012). Can phlorotannins purified extracts constitute a novel pharmacological alternative for microbial infections with associated inflammatory conditions?. PloS One, 7(2), e31145. López, A., Rico, M., Rivero, A., & de Tangil, M. S. (2011). The effects of solvents on the phenolic contents and antioxidant activity of Stypocaulon scoparium algae extracts. Food Chemistry, 125(3), 1104-1109. López-López, I., Bastida, S., Ruiz-Capillas, C., Bravo, L., Larrea, M. T., Sánchez-Muniz, F., Cofrades, S., & Jiménez-Colmenero, F. (2009). Composition and antioxidant capacity of low-salt meat emulsion model systems containing edible seaweeds. Meat Science, 83(3), 492-498. Loprinzi, P. D., & Mahoney, S. E. (2015). Association between flavonoid-rich fruit and vegetable consumption and total serum bilirubin. Angiology, 66(3), 286-290. Lorenzo, J. M., & Munekata, P. E. S. (2016). Phenolic compounds of green tea: health benefits and technological application in food. Asian Pacific Journal of Tropical Biomedicine, 6(8), 709-719.

122 VI. References

Lorenzo, J. M., Gómez, M., Purriños, L., & Fonseca, S. (2016). Effect of commercial starter cultures on volatile compound profile and sensory characteristics of dry‐cured foal sausage. Journal of the Science of Food and Agriculture, 96(4), 1194-1201. Lorenzo, J. M., Munekata, P. E., Sant'Ana, A. S., Carvalho, R. B., Barba, F. J., Toldrá, F., Mora, L., & Trindade, M. A. (2018a). Main characteristics of peanut skin and its role for the preservation of meat products. Trends in Food Science & Technology, 77, 1-10. Lorenzo, J. M., Pateiro, M., Domínguez, R., Barba, F. J., Putnik, P., Kovačević, D. B., Shpigelman, I., Granato, D., & Franco, D. (2018b). Berries extracts as natural antioxidants in meat products: A review. Food Research International, 106, 1095-1104. Lorenzo, J. M., Pateiro, M., Fontán, M. C. G., & Carballo, J. (2014a). Effect of fat content on physical, microbial, lipid and protein changes during chill storage of foal liver pâté. Food Chemistry, 155, 57-63. Lorenzo, J. M., Sineiro, J., Amado, I. R., & Franco, D. (2014b). Influence of natural extracts on the shelf life of modified atmosphere-packaged pork patties. Meat Science, 96(1), 526- 534. Lorenzo, J. M., Trindade, M. A., Ahn, D. U., & Barba, F. J. (2019). Natural antioxidants to reduce the oxidation process of meat and meat products. Food Research International, 115, 377-378. Lorenzo, J. M., Vargas, F. C., Strozzi, I., Pateiro, M., Furtado, M. M., Sant'Ana, A. S., Rocchetti, G., Barba, F. J., Domínguez, R., Lucini, L., & do Amaral Sobral, P. J. (2018c). Influence of pitanga leaf extracts on lipid and protein oxidation of pork burger during shelf-life. Food Research International, 114, 47-54. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193(1), 265-275. Lund, M. N., Heinonen, M., Baron, C. P., & Estévez, M. (2011). Protein oxidation in muscle foods: A review. Molecular Nutrition & Food Research, 55(1), 83-95. Maadane, A., Merghoub, N., Ainane, T., El Arroussi, H., Benhima, R., Amzazi, S., Bakri, Y., & Wahby, I. (2015). Antioxidant activity of some Moroccan marine microalgae: Pufa profiles, carotenoids and phenolic content. Journal of Biotechnology, 215, 13-19. MacFie, H. J., Bratchell, N., Greenhoff, K., & Vallis, L. V. (1989). Designs to balance the effect of order of presentation and first‐order carry‐over effects in hall tests. Journal of Sensory Studies, 4(2), 129-148. Maguire, L. S., O’Sullivan, S. M., Galvin, K., O’Connor, T. P., & O’Brien, N. M. (2004). Fatty acid profile, tocopherol, squalene and content of walnuts, almonds, peanuts,

123 VI. References

hazelnuts and the macadamia nut. International Journal of Food Sciences and Nutrition, 55(3), 171-178. Martínez, J. H. I., & Castañeda, H. G. T. (2013). Preparation and chromatographic analysis of phlorotannins. Journal of Chromatographic Science, 51(8), 825-838. Matanjun, P., Mohamed, S., Mustapha, N. M., Muhammad, K. (2009). Nutrient content of tropical edible seaweeds, Eucheuma cottonii, Caulerpa lentillifera and Sargassum polycystum. Journal of Applied Phycology, 21(1), 75-80. Medina-Remón, A., Barrionuevo-González, A., Zamora-Ros, R., Andres-Lacueva, C., Estruch, R., Martínez-González, M. A., Diez-Espino, J., & Lamuela-Raventos, R. M. (2009). Rapid Folin–Ciocalteu method using microtiter 96-well plate cartridges for solid phase extraction to assess urinary total phenolic compounds, as a biomarker of total polyphenols intake. Analytica Chimica Acta, 634(1), 54-60. Min, B. R., Nam, K. C., Cordray, J. C., & Ahn, D. U. (2008). Factors affecting oxidative stability of pork, beef, and chicken meat. Animal Industry Report, 654(1), 6. Min, B., & Ahn, D. U. (2005). Mechanism of lipid peroxidation in meat and meat products -A review. Food Science and Biotechnology, 14(1), 152-163. Montero, L., Sánchez-Camargo, A. P., García-Cañas, V., Tanniou, A., Stiger-Pouvreau, V., Russo, M., Rastrelli, L., Cifuentes, A., Herrero, M., & Ibáñez, E. (2016). Anti-proliferative activity and chemical characterization by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry of phlorotannins from the brown macro- alga Sargassum muticum collected on North-Atlantic coasts. Journal of Chromatography A, 1428, 115-125. Munekata, P. E. S., Domínguez, R., Franco, D., Bermúdez, R., Trindade, M. A., & Lorenzo, J. M. (2017). Effect of natural antioxidants in Spanish salchichón elaborated with encapsulated n-3 long chain fatty acids in konjac glucomannan matrix. Meat Science, 124, 54-60. Munekata, P. E. S., Franco, D., Trindade, M. A., & Lorenzo, J. M. (2016). Characterization of phenolic composition in chestnut leaves and beer residue by LC-DAD-ESI-MS. LWT-Food Science and Technology, 68, 52-58. Naczk, M., & Shahidi, F. (2006). Phenolics in cereals, fruits and vegetables: Occurrence, extraction and analysis. Journal of Pharmaceutical and Biomedical Analysis, 41(5), 1523- 1542. Nagy, T. O., Solar, S., Sontag, G., & Koenig, J. (2011). Identification of phenolic components in dried spices and influence of irradiation. Food Chemistry, 128(2), 530-534.

124 VI. References

Nakamura, T., Nagayama, K., Uchida, K., & Tanaka, R. (1996). Antioxidant activity of phlorotannins isolated from the brown alga Eisenia bicyclis. Fisheries Science, 62(6), 923- 926. Orrego-Lagarón, N., Vallverdú-Queralt, A., Martínez-Huélamo, M., Lamuela-Raventos, R. M., & Escribano-Ferrer, E. (2016). Metabolic profile of naringenin in the stomach and colon using liquid chromatography/electrospray ionization linear ion trap quadrupole- Orbitrap-mass spectrometry (LC-ESI-LTQ-Orbitrap-MS) and LC-ESI-MS/MS. Journal of Pharmaceutical and Biomedical Analysis, 120, 38-45. Ortiz, J., Romero, N., Robert, P., Araya, J., Lopez-Hernández, J., Bozzo, C., Navarrete, E., Osorio, A., & Rios, A. (2006). , amino acid, fatty acid and tocopherol contents of the edible seaweeds Ulva lactuca and Durvillaea antarctica. Food Chemistry, 99(1), 98-104. Ospina, M., Castro-Vargas, H. I., & Parada-Alfonso, F. (2017). Antioxidant capacity of Colombian seaweeds: 1. Extracts obtained from Gracilaria mammillaris by means of supercritical fluid extraction. The Journal of Supercritical Fluids, 128, 314-322. Ou, B., Hampsch-Woodill, M., & Prior, R. L. (2001). Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. Journal of Agricultural and Food Chemistry, 49(10), 4619-4626. Pantidos, N., Boath, A., Lund, V., Conner, S., & McDougall, G. J. (2014). Phenolic-rich extracts from the edible seaweed, Ascophyllum nodosum, inhibit α-amylase and α-glucosidase: Potential anti-hyperglycemic effects. Journal of Functional Foods, 10, 201-209. Papuc, C., Goran, G. V., Predescu, C. N., & Nicorescu, V. (2017). Mechanisms of oxidative processes in meat and toxicity induced by postprandial degradation products: A review. Comprehensive Reviews in Food Science and Food Safety, 16(1), 96-123. Paquot, C., & Hautfenne, A. (1987). IUPAC, Standard Methods for the Analysis of Oils, Fats and Derivatives (7th ed.). Blackwell Scientific (London, UK). Parniakov, O., Apicella, E., Koubaa, M., Barba, F. J., Grimi, N., Lebovka, N., Pataro, G., Ferrari, G., & Vorobiev, E. (2015). Ultrasound-assisted green solvent extraction of high-added value compounds from microalgae Nannochloropsis spp. Bioresource Technology, 198, 262-267. Pateiro, M., Lorenzo, J. M., Amado, I. R., & Franco, D. (2014). Effect of addition of green tea, chestnut and grape extract on the shelf-life of pig liver pâté. Food Chemistry, 147, 386- 394. Pateiro, M., Lorenzo, J. M., Vázquez, J. A., & Franco, D. (2015). Oxidation stability of pig liver pate with increasing levels of natural antioxidants (grape and tea). Antioxidants, 4(1), 102-123.

125 VI. References

Pearson, A. M., & Young, R. B. (1989). Muscle and meat biochemistry. Academic Press (Cambridge, MA, USA). Peinado, I., Girón, J., Koutsidis, G., & Ames, J. M. (2014). Chemical composition, antioxidant activity and sensory evaluation of five different species of brown edible seaweeds. Food Research International, 66, 36-44. Plaza, M., Santoyo, S., Jaime, L., Avalo, B., Cifuentes, A., Reglero, G., Reina, G. G. B., Señorans, F. J., & Ibáñez, E. (2012). Comprehensive characterization of the functional activities of pressurized liquid and ultrasound-assisted extracts from Chlorella vulgaris. LWT-Food Science and Technology, 46(1), 245-253. Poiana, M. A. (2012). Enhancing oxidative stability of sunflower oil during convective and microwave heating using grape seed extract. International Journal of Molecular Sciences, 13(7), 9240-9259. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology & Medicine, 26(9), 1231-1237. Rhimou, B., Hassane, R., Martínez, J., & Nathalie, B. (2010). The antibacterial potential of the seaweeds (Rhodophyceae) of the Strait of Gibraltar and the Mediterranean coast of Morocco. African Journal of Biotechnology, 9(38), 6365-6372. Rodgers, K. J., & Shiozawa, N. (2008). Misincorporation of amino acid analogues into proteins by biosynthesis. The International Journal of Biochemistry & Cell Biology, 40(8), 1452- 1466. Rodrigues, D., Freitas, A. C., Pereira, L., Rocha-Santos, T. A., Vasconcelos, M. W., Roriz, M., Rodríguez-Alcalá, L. M., Gomes, A. M. P., & Duarte, A. C. (2015). Chemical composition of red, brown and green macroalgae from Buarcos bay in Central West Coast of Portugal. Food Chemistry, 183, 197-207. Rodríguez-Carpena, J. G., Morcuende, D., & Estévez, M. (2011). Avocado by-products as inhibitors of color deterioration and lipid and protein oxidation in raw porcine patties subjected to chilled storage. Meat Science, 89(2), 166-173. Roman, O., Heyd, B., Broyart, B., Castillo, R., & Maillard, M. N. (2013). Oxidative reactivity of unsaturated fatty acids from sunflower, high oleic sunflower and rapeseed oils subjected to heat treatment, under controlled conditions. LWT-Food Science and Technology, 52(1), 49-59. Roohinejad, S., Koubaa, M., Barba, F. J., Saljoughian, S., Amid, M., & Greiner, R. (2017). Application of seaweeds to develop new food products with enhanced shelf-life, quality and health-related beneficial properties. Food Research International, 99, 1066-1083.

126 VI. References

Rutherfurd, S. M., Montoya, C. A., & Moughan, P. J. (2014). Effect of oxidation of dietary proteins with performic acid on true ileal amino acid digestibility as determined in the growing rat. Journal of Agricultural and Food Chemistry, 62(3), 699-707. Sánchez‐Escalante, A., Djenane, D., Torrescano, G., Beltrán, J. A., & Roncales, P. (2003). Antioxidant action of borage, rosemary, oregano, and ascorbic acid in beef patties packaged in modified atmosphere. Journal of Food Science, 68(1), 339-344. Sánchez‐Machado, D. I., Lopez‐Cervantes, J., Lopez‐Hernandez, J., Paseiro‐Losada, P., & Simal‐ Lozano, J. (2004). Determination of the uronic acid composition of seaweed dietary fibre by HPLC. Biomedical Chromatography, 18(2), 90-97. Santoso, J., Yoshie-Stark, Y., & Suzuki, T. (2004). Anti-oxidant activity of methanol extracts from Indonesian seaweeds in an oil emulsion model. Fisheries Science, 70(1), 183-188. Sasaki, K., Ishihara, K., Oyamada, C., Sato, A., Fukushi, A., Arakane, T., Motoyama, M., Yamzaki, M., & Mitsumoto, M. (2008). Effects of fucoxanthin addition to ground chicken breast meat on lipid and colour stability during chilled storage, before and after cooking. Asian- Australasian Journal of Animal Sciences, 21(7), 1067-1072. Schmid, M., Guihéneuf, F., & Stengel, D. B. (2014). Fatty acid contents and profiles of 16 macroalgae collected from the Irish Coast at two seasons. Journal of Applied Phycology, 26(1), 451-463. Shahidi, F, & Wanasundara, U. N. (2002). Methods for measuring oxidative rancidity in fats and oils. In Food Lipids: Chemistry, Nutrition and Biotechnology (2th ed.) (pp. 387-403), Akoh, C. C., & Min, D. B. CRC Press (Boca Raton, FL, USA). Shahidi, F., & Ambigaipalan, P. (2015). Phenolics and polyphenolics in foods, beverages and spices: Antioxidant activity and health effects-A review. Journal of Functional Foods, 18, 820-897. Shahidi, F., & Synowiecki, J. (1993). Nutrient composition of mechanically separated and surimi-like seal meat. Food Chemistry, 47(1), 41-46. Shahidi, F., & Wanasundara, U. N. (1997). Measurement of lipid oxidation and evaluation of antioxidant activity. In Natural Antioxidants: Chemistry, Health Effects, and Applications (pp. 379-396), Shahidi, F. AOCS Press (Champaign, IL, USA). Shahidi, F., Desilva, C., & Amarowicz, R. (2003). Antioxidant activity of extracts of defatted seeds of niger (Guizotia abyssinica). Journal of the American Oil Chemists' Society, 80(5), 443-450. Shahidi, F., Rubin, L. J., D'Souza, L. A., Teranishi, R., & Buttery, R. G. (1986). Meat flavor volatiles: a review of the composition, techniques of analysis, and sensory evaluation. Critical Reviews in Food Science and Nutrition, 24(2), 141-243.

127 VI. References

Shaker, E. S. (2006). Antioxidative effect of extracts from red grape seed and peel on lipid oxidation in oils of sunflower. LWT-Food Science and Technology, 39(8), 883-892. Sigma-Aldrich (2019). Chemical structures of commercial synthetic antioxidants BHA, BHT, TBHQ and PG. Recovered from https://www.sigmaaldrich.com. Simopoulos, A. P. (2010). The omega-6/omega-3 fatty acid ratio: health implications. Oléagineux, Corps gras, Lipides, 17(5), 267-275. Singleton, V. L., Orthofer, R., & Lamuela-Raventos, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods in Enzymology, 299, 152-178. Siriwardhana, N., Lee, K. W., & Jeon, Y. J. (2005). Radical scavenging potential of hydrophilic phlorotannins of Hizikia fusiformis. Algae, 20(1), 69-75. Siriwardhana, N., Lee, K. W., Kim, S. H., Ha, J. H., Park, G. T., & Jeon, Y. J. (2004). Lipid peroxidation inhibitory effects of Hizikia fusiformis methanolic extract on fish oil and linoleic acid. Food Science and Technology International, 10(2), 65-72. Soladoye, O. P., Juárez, M. L., Aalhus, J. L., Shand, P., & Estévez, M. (2015). Protein oxidation in processed meat: mechanisms and potential implications on human health. Comprehensive Reviews in Food Science and Food Safety, 14(2), 106-122. Stadtman, E. R., & Levine, R. L. (2003). Free radical-mediated oxidation of free amino acids and amino acid residues in proteins. Amino Acids, 25(3-4), 207-218. Sturm, N. (2014). General structure of glycogen. Recovered from http://chemistry.gravitywaves.com. Suja, K. P., Abraham, J. T., Thamizh, S. N., Jayalekshmy, A., & Arumughan, C. (2004). Antioxidant efficacy of sesame cake extract in vegetable oil protection. Food Chemistry, 84(3), 393-400. Talpur, M. A., Changying, J., Chandio, F. A., Junejo, S. A., & Mari, I. A. (2011). Application of oven drying method on moisture content of ungrounded and grounded (long and short) rice for storage. Journal of Stored Products and Postharvest Research, 2(12), 245-247. Tesoriere, L., D'Arpa, D., Butera, D., Pintaudi, A. M., Allegra, M., & Livrea, M. A. (2002). Exposure to malondialdehyde induces an early redox unbalance preceding membrane toxicity in human erythrocytes. Free Radical Research, 36(1), 89-97. Tierney, M. S., Smyth, T. J., Hayes, M., Soler‐Vila, A., Croft, A. K., & Brunton, N. (2013). Influence of pressurised liquid extraction and solid-liquid extraction methods on the phenolic content and antioxidant activities of Irish macroalgae. International Journal of Food Science & Technology, 48(4), 860-869.

128 VI. References

Tomović, V., Jokanović, M., Šojić, B., Škaljac, S., & Ivić, M. (2017). Plants as natural antioxidants for meat products. In IOP Conference Series: Earth and Environmental Science (p. 012030). IOP Publishing (Bristol, UK). Venkataraman, S., Schafer, F. Q., & Buettner, G. R. (2004). Detection of lipid radicals using EPR. Antioxidants & Redox Signaling, 6(3), 631-638. Vinayak, R. C., Sabu, A. S., & Chatterji, A. (2011). Bio-prospecting of a few brown seaweeds for their cytotoxic and antioxidant activities. Evidence-Based Complementary and Alternative Medicine, 2011, Article ID 673083. Wang, T., Jonsdottir, R., & Ólafsdóttir, G. (2009). Total phenolic compounds, radical scavenging and metal chelation of extracts from Icelandic seaweeds. Food Chemistry, 116(1), 240- 248. Wang, T., Jónsdóttir, R., Kristinsson, H. G., Thorkelsson, G., Jacobsen, C., Hamaguchi, P. Y., & Ólafsdóttir, G. (2010). Inhibition of haemoglobin-mediated lipid oxidation in washed cod muscle and cod protein isolates by Fucus vesiculosus extract and fractions. Food Chemistry, 123(2), 321-330. Wang, T., Jónsdóttir, R., Liu, H., Gu, L., Kristinsson, H. G., Raghavan, S., & lafsdóttir, G. (2012). Antioxidant capacities of phlorotannins extracted from the brown algae Fucus vesiculosus. Journal of Agricultural and Food Chemistry, 60(23), 5874-5883. Warner, K., Frankel, E. N., & Mounts, T. L. (1989). Flavor and oxidative stability of soybean, sunflower and low erucic acid rapeseed oils. Journal of the American Oil Chemists' Society, 66(4), 558-564. Yang, J., Martinson, T. E., & Liu, R. H. (2009). Phytochemical profiles and antioxidant activities of wine grapes. Food Chemistry, 116(1), 332-339. Yruela, I, & Sebastian, A. (2014). Macromoléculas biológicas: proteínas, DNA y RNA (Biological macromolecules: proteins, DNA and RNA). In Bioinformática con Ñ": a collaborative project of young Spanish scientists to write a complete book about Bioinformatics. 185- 210. Zamuz, S., López-Pedrouso, M., Barba, F. J., Lorenzo, J. M., Domínguez, H., & Franco, D. (2018). Application of hull, bur and leaf chestnut extracts on the shelf-life of beef patties stored under MAP: Evaluation of their impact on physicochemical properties, lipid oxidation, antioxidant, and antimicrobial potential. Food Research International, 112, 263-273. Zhang, J., Zhang, H., Wang, L., Guo, X., Wang, X., & Yao, H. (2010b). Isolation and identification of antioxidative peptides from rice endosperm protein enzymatic hydrolysate by consecutive chromatography and MALDI-TOF/TOF MS/MS. Food Chemistry, 119(1), 226- 234.

129 VI. References

Zhang, Y., Yang, L., Zu, Y., Chen, X., Wang, F., & Liu, F. (2010a). Oxidative stability of sunflower oil supplemented with carnosic acid compared with synthetic antioxidants during accelerated storage. Food Chemistry, 118(3), 656-662.

130

VII. ANEXES

VII. Anexes

VII.1. PUBLICATIONS THAT INCLUDE THE RESULTS OF THIS DOCTORAL THESIS

133

Food Research International 99 (2017) 986–994

Contents lists available at ScienceDirect

Food Research International

journal homepage: www.elsevier.com/locate/foodres

Proximate composition, phenolic content and in vitro antioxidant activity of aqueous extracts of the seaweeds Ascophyllum nodosum, Bifurcaria bifurcata and Fucus vesiculosus. Effect of addition of the extracts on the oxidative stability of canola oil under accelerated storage conditions

Rubén Agregán a, Paulo E. Munekata b, Ruben Domínguez a, Javier Carballo c,DanielFrancoa,JoséM.Lorenzoa,⁎ a Centro Tecnológico de la Carne de Galicia, Rúa Galicia No. 4, Parque Tecnológico de Galicia, San Cibrán das Viñas, 32900 Ourense, Spain b Department of Food Engeneering, Faculty of Animal Science and Food Engeneering, University of São Paulo, 225 Duque de Caxias Norte Ave, Jardim Elite, Postal Code 13.635-900 Pirassununga, São Paulo, Brazil c Area de Tecnologia de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain article info abstract

Article history: Extracts from three macroalgae species (Ascophyllum nodosum (ANE), Bifurcaria bifurcata (BBE) and Fucus Received 4 July 2016 vesiculosus (FVE)) were tested for proximate composition (total solid, protein and total carbohydrate contents), Received in revised form 2 November 2016 total phenols content (TPC), and for their antioxidant activities in vitro in comparison to that of BHT compound by Accepted 10 November 2016 using four different assays (ABTS radical cation decolouration, DPPH free radical scavenging activity, ferric reduc- Available online 12 November 2016 ing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC)). The inclusion of the extracts as oil stabilizers in canola oil in substitution of the synthetic antioxidant (BHT) was also evaluated by assessing lipid Keywords: Seaweed extract oxidation parameters (peroxide value (PV), p-anisidine value (AV), TBARS value, conjugated dienes (CD) and Antioxidant activity TOTOX index) under accelerated storage conditions (16 days, 60 °C). There was an inverse relationship between Phenolic content total solid content and total polyphenols content in the seaweed extracts. FVE showed an intermediate TPC Ascophyllum nodosum (1.15 g PGE/100 g extract), but it presented the highest in vitro antioxidant activity when measured using the Bifurcaria bifurcata ABTS, DPPH and FRAP tests. BBE, that displayed the highest TPC (1.99 g PGE/100 g extract), only showed the Fucus vesiculosus highest in vitro antioxidant activity when measured using the ORAC test. ANE showed the lowest TPC and the lowest antioxidant activity in all the tests performed. The seaweed extracts added in a 500 ppm concentration significantly reduced the oxidation during canola oil storage at 60 °C, being this antioxidant effect significantly higher than that of BHT added at 50 ppm. Results indicate that seaweed extracts can effectively inhibit the oxi- dation of canola oil and they can be a healthier alternative to the synthetic antioxidants in the oil industry. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction different countries and their reported carcinogenic effect. Moreover, the increased consumer preference for more natural products has led Oxidation is a principal cause of quality deterioration in oils and fats the food industry to be interested in developing ingredients based on during processing and storage, resulting in the production of rancid natural antioxidants in order to reduce or avoid the lipid oxidation odours and unpleasant flavours, changes of colour, and reduction of and preserve good nutritional and organoleptic quality of foods. nutritional value (Jittrepotch, Ushio, & Ohshima, 2006). Antioxidant Seaweeds have been consumed since ancient times, being currently compounds play an important role as protective factors. They can widely used as food in Asian countries, and to a lesser extent in Europe delay or inhibit lipid oxidation by inhibiting the initiation or propaga- and America. Many seaweed species are used in food and pharmaceuti- tion of oxidizing chain reactions, and are also involved in scavenging cal industries, principally for the extraction of phytocolloids such as agar free radicals (Piccolella et al., 2008). Synthetic antioxidants such as and carrageenan to be used as thickening agents, and also for phyto- butylatedhydroxytoluene (BHT), butylatedhydroxyanisole (BHA), and chemicals, which have pharmacological interest (Jiménez-Escrig & propyl gallate (PG) are commercially available and have been used as Sánchez-Muniz, 2000). In addition, using polar solvents like water, phe- food preservatives. However, there is nowadays an urgent need of alter- nolic compounds which are attached to sugars or proteins, glycosides, natives for these synthetic compounds due to legal limits of use in organic acids, tannins, salts and mucus can be extracted (Sabeena Farvin & Jacobsen, 2013). A group of phenolic compounds found ⁎ Corresponding author. in brown seaweed called phlorotannins, which are polymers of E-mail address: [email protected] (J.M. Lorenzo). phloroglucinol, have been reported to possess strong antioxidant

http://dx.doi.org/10.1016/j.foodres.2016.11.009 0963-9969/© 2016 Elsevier Ltd. All rights reserved. R. Agregán et al. / Food Research International 99 (2017) 986–994 987 activity and their free radical potential scavenging is more powerful The protein content in the extracts was quantified following the than that of other polyphenols derived from terrestrial plants method of Lowry, Rosebrough, Farr, and Randall (1951). One milliliter (Ahn et al., 2007). The antioxidant compounds detected in several of the extract was placed in a test tube, and 4 mL of a solution of 2% seaweed genera have potential anti-aging, dietary, anti-inflammatory, Na2CO3 in OHNa 0.1 N: 0.5% CuSO4·5H2O in 1% potassium sodium tar- antibacterial, antifungal, cytotoxic, anti-malarial, anti-proliferative, and trate (50:1, v:v) was added. The mixture was allowed to stand for anticancer properties (Cornish & Garbary, 2010; Zubia, Robledo, & 15 min in darkness before 0.4 mL of the Folin-Ciocalteu reagent diluted Freile-Pelegrin, 2007). In addition, many seaweed species grow very with distilled water (1:2, v:v) was added. The mixture was stirred and fast and efficiently, particularly in comparison to terrestrial plants, and allowed to stand for another 30 min in darkness. Finally, the absorbance in other cases they may be readily available as by-products of the was read at 500 nm. The readings were converted to concentrations by aquaculture industry (Kindleysides, Quek, & Miller, 2012). interpolation in a calibration curve constructed using different concen- In view of this situation, the present study was conducted to charac- trations of bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA). terize in terms of proximate composition and total phenolic content The total carbohydrate content was assessed by the phenol- (TPC) the aqueous extracts from three different seaweed species sulphuric method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Al- (Ascophyllum nodosum, Bifurcaria bifurcata and Fucus vesiculosus), and iquots of dilutions of the extracts were placed in tubes and the final vol- to quantify their antioxidant activities in comparison to that of a ume was made up to 2 mL with distilled water. One milliliter of a synthetic antioxidant compound (BHT) using five in vitro assays: ABTS solution of 5% phenol and 5 mL of 95.5% sulphuric acid were added radical cation decolouration, DPPH free radical scavenging activity, fer- and the solution was mixed. The mixture was allowed to stand at ric reducing antioxidant power (FRAP) and oxygen radical absorbance room temperature for 10 min, before being stirred and placed in a capacity (ORAC). Once the antioxidant activity was demonstrated in water bath at 25 °C for 15 min. The absorbance of the mixture thus ob- in vitro studies, the effect of seaweed extracts will be studied in compar- tained was read at 490 nm. A calibration curve was constructed using ison to that of BHT on the oxidative stability of canola oil under acceler- different concentrations of glucose. Results were expressed as g glu- ated conditions, by using peroxide value, p-anisidine value, TBARS cose/100 g extract. values, conjugated dienes and TOTOX index to assess antioxidant The total phenolic content (TPC) of seaweed extracts was deter- effectiveness. mined according to Singleton, Orthofer, and Lamuela-Raventos (1999).Briefly, an aliquot (100 μL) of the extract was mixed with 2. Material and methods 0.75 mL of the Folin-Ciocalteu reagent (diluted 1:10 with water). The mixture was allowed to stand at room temperature for 90 min before 2.1. Materials the absorbance was read at 725 nm. A calibration curve was constructed using phloroglucinol (a basic structural unit of phlorotannins) as stan- The brown seaweeds, A. nodosum, B. bifurcata and F. vesiculosus used dard, and the results were expressed as grams of phloroglucinol equiv- in the present study, were kindly supplied by the Portomuiños company alents (PGE)/100 g of dried seaweed extract. (A Coruña, Spain). They were collected in the months of August and September in the Atlantic Ocean, in the area of Camariñas (A Coruña, 2.4. Screening of BHT and extracts for antioxidant activity Spain). Canola oil was provided by Aceites Abril, S.L. (Ourense, Spain). Com- The antioxidant capacity of extracts and BHT was measured by dif- position of canola oil according to CODEX Stan 210 normative (Codex ferent methods, because the use of a single determination is not recom- Alimentarius, 2005) was: acidity (0.04%), peroxide value (2.29 meq mended (Müller, Fröhlich, & Böhm, 2011). Hence four different

O2/kg), moisture (b0.01%) and impurities (b0.01%). The fatty acid pro- methods based on two distinct modes of antioxidant action (hydrogen file of canola oil was: myristic acid (0.05%), palmitic acid (4.57%), atom transfer and electron transfer) were assessed. For each extract palmitoleic acid (0.26%), stearic acid (1.61%), oleic acid (63.09%), (or BHT) and method, measurement was made in duplicate. linoleic acid (19.68%), linolenic acid (8.25%), arachidic acid (0.83%), gadoleic acid (1.14%), behenic acid (0.31%) and lignoceric acid (0.09%). 2.4.1. ABTS radical cation decoloration assay The method of Re et al. (1999) was adapted to the use of a plate 2.2. Seaweed extraction procedure reader. ABTS radical cation (ABTS•+) was produced by reacting 7 mM ABTS stock solution with 2.45 mM potassium persulfate (final concen- Seaweeds were dried in a conventional oven at 40 °C and then tration) allowing the mixture to stand in the dark at room temperature grounded to obtain a particle size b500 μm. Extraction was performed for 12–16 h before use. In the next step ABTS•+ solution was diluted in a magnetic stirrer at room temperature, using water as a solvent in with PBS (pH 7.4) to an absorbance of 0.70 at 734 nm and equilibrated a liquid/solid ratio of 30 g/g for 5 min to obtain a correct hydration. at 30 °C. An aliquot of each sample (with appropriate dilution) was Then an aliquot of 800 mL from the seaweed/water suspension was son- mixed with the working solution of ABTS•+ (sample:ABTS solution rela- icated for 10 min in an ultrasonic Hielscher UIP1000HD homogenizer tion, 1:100), and the decrease of absorbance was measured after 6 min (Hielscher Ultrasonics GmbH, Teltow, Germany) equipped with a flow at 734 nm in a microplate spectrophotometer reader. Trolox was used cell with a residence time of 30 s and 90 percentage of amplitude, as the reference standard and the results were expressed as TEAC values with continuous recirculation. The process was stopped if the tempera- (μmol of Trolox equivalents/g extract). The percentage inhibition of ab- ture reached a value higher than 40 °C. Finally the extract was centri- sorbance at 734 nm is calculated and plotted as a function of extract fuged at 2000 ×g and filtered through a cellulose filter of 20–25 μm concentration to obtain the EC50, the amount of antioxidant necessary pore size (Filter-lab, Filtros Anoia, S.A., Barcelona, Spain). Extracts to decrease the initial ABTS concentration by 50%. In the samples were produced in triplicate and used to measure the proximate compo- whereitwasnotpossiblecalculateEC50 value, the percentage of inhibi- sition, the TPC, and the antioxidant activity. tion of the radical ABTS was indicated.

2.3. Determination of proximate composition and total phenolic content 2.4.2. DPPH free radical scavenging activity The radical scavenging activities (RSA) of seaweed extracts and BHT After the extraction, the total solid contents (TSC) of the extracts compound for DPPH free radical were determined according to Brand- were quantified by gravimetric methods after evaporating the solvent Williams, Cuvelier, and Berset (1995) with slight modification: 5 μLof at 105 °C until constant weight (Talpur, Changying, Chandio, Junejo, & appropriately diluted samples were added to 195 μLofDPPH• solution Mari, 2011). (6 × 10−5 M in methanol) in a well of 96-well plate. The mixture was 988 R. Agregán et al. / Food Research International 99 (2017) 986–994 shaken gently and left to stand at room temperature for 30 min. There- value and conjugated dienes. Besides these indices, the total oxidation after, the absorbance at 515 nm was measured against methanol using a (TOTOX) of the canola oil samples was calculated. microplate reader. The DPPH free radical scavenging activity of extracts This experiment was carried out in duplicate. was calculated from the standard curve of Trolox and expressed as μmol of Trolox equivalents/g extract. The results were expressed in term of 2.6. Determination of peroxide value (PV)

EC50 determined by linear regression analysis of the dose response curve plotted between the RSA against extract concentration. The EC50 The PV was determined following the AOAC procedure 965.33 is defined as the extract concentration (μg/mL) required to decrease (AOAC, 2000). Oil samples (0.5 g) were dissolved with 10 mL of the initial DPPH• concentration by 50%. In the samples where it was trichloromethane. Then, 15 mL of acetic acid and 1 mL of saturated not possible calculate EC50 value, the percentage of inhibition of the aqueous solution of potassium iodide were added. The sample was DPPH• was indicated. slightly agitated 1 min and kept 5 min in the dark. Once incubation was finished, 75 mL of distilled water were added and the sample was 2.4.3. Ferric reducing antioxidant power (FRAP) assay vigorously shaken. Finally, liberated iodine was titrated with sodium The ability to reduce ferric ions was measured using the method de- thiosulfate 0.01 N in an automatic titrator. The PV, expressed as scribed by Benzie and Strain (1996). The FRAP reagent was freshly pre- milliequivalents O2/kg, was calculated according to the formula: pared from 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-s- triazine (TPTZ) made up in 40 mM HCl, and 20 mM FeCl 6H O solution. V N 1000 3 2 PV ¼ ð1Þ All three solutions were mixed together in the ratio of 10:1:1 (v/v/v). A W total of 10 μL of properly diluted samples and 30 μL of distilled water μ were added to 300 L freshly prepared FRAP reagent in a well of 96- where, V is the volume (mL) of sodium thiosulfate consumed in the ti- well plate. The mixture was incubated at 37 °C throughout the reaction. tration, N is the normality of the sodium thiosulfate solution and W is After 8 min, the absorbance was read using a microplate reader at the sample weight of the sample (g). 593 nm against reagent blank. The FRAP value was calculated and μ expressed as mol of Trolox equivalents/g extract based on a calibration 2.7. Determination of p-anisidine value (AV) curve plotted using Trolox as standard. Determination of AV of the oil samples was performed following an 2.4.4. ORAC (oxygen radical absorbance capacity) assay IUPAC method (IUPAC, 1987). Oil samples (0.5–2 g) were dissolved in The original method of Ou, Hampsch-Woodill, and Prior (2001) was isooctane in a 25 mL volumetric flask. The sample was then reacted modified as described by Dávalos, Bartolomé, Suberviola, and Gómez- with p-anisidine solution in acetic acid (0.25% w/v) for 10 min to pro- Cordovés (2003). The reaction was carried out in 75 mM phosphate duce a coloured complex. Absorbances of the samples with and without buffer (pH 7.4), and the final reaction mixture was 200 mL. The mixture p-anisidine solution were measured using an UV-1800 UV spectropho- of antioxidant (20 mL) and fluorescein (120 mL; 70 nM final concentra- tometer (Shimadzu Corporation, Kyoto, Japan) at 350 nm, and the pa- tion) was preincubated for 15 min at 37 °C. AAPH (2,20-Azobis (2- rameter AV was calculated as: methylpropionamidine) dihydrochloride) solution (60 mL; 12 mM, fi nal concentration) was added rapidly using a multichannel pipette. 25 ½1:2 ðÞE −E fl AV ¼ b a ð2Þ The plate was immediately placed in the reader and the uorescence re- W corded every minute for 120 min (excitation wavelength 485 nm, emis- sion wavelength 520 nm). The plate was automatically agitated prior each reading. A blank using phosphate buffer instead of the antioxidant where, Eb is the net absorbance of the oil-solution, Ea is the net absorbance solution and eight calibration solutions using Trolox as antioxidant were of the oil-anisidine-solution and W is the weight of the sample (g). also carried out in each assay. Results are calculated on the basis of the differences in areas under the fluorescein decay curve between the 2.8. Determination of conjugated dienes (CD) blank and the sample, and are expressed as μmol of Trolox equiva- fi lents/g extract. The CD were quanti ed using an UV-1800 UV spectrophotometer (Shimadzu Corporation, Kyoto, Japan) at 233 nm. Before analysis, 20 μL of oil were diluted with hexane in a volumetric flask of 25 mL. 2.5. Oxidation stability of canola oil under accelerated conditions The CD, expressed as percentage of conjugated dienoic acid was calcu- lated as follows: In order to evaluate the effectiveness of seaweed extracts against lipid oxidation, the test of oxidation stability of canola oil under accelerated conditions was used. Freshly refined canola oil (5 kg), free ¼ : AS ð Þ CD 0 84 − 3 of synthetic antioxidants, was divided into 5 portions. Three of them, bc K0 were supplemented with 500 ppm of A. nodosum extract (ANE),

B. bifurcata extract (BBE) and F. vesiculosus extract (FVE). The fourth where, As is the absorbance observed at 233 nm, b is the cell length in portion was mixed with the synthetic antioxidant BHT at 50 ppm, and cm, c is the concentration of the diluted sample (g/L) and K0 is the ab- the last portion, without any antioxidant addition, was used as a control. sorptivity by acid or ester groups. In this case, c was considered as

Before applying, ANE, BBE, FVE and BHT were separately mixed with 0.5 g/L, the final concentration for most edible oils; and K0 was consid- 1.5 mL of ethanol 96% (v/v) (to ensure appropriate dispersion in oil) ered for acids (value of 0.03). in an ultrasonic water bath for 10 min and then added to 20 mL of canola oil, mixed for 10 min and vacuum evaporated. Control sample was pre- 2.9. Determination of total oxidation value (TOTOX) pared using the same amount of ethanol 96% (v/v) used to dissolve BHT and seaweed extracts, and later treated in the same way. Canola oil sam- The overall oxidation state of oil given by the TOTOX was calculated ples were stored in glass containers at 60 °C and agitated at 100 rpm in according to the formula: an orbital shaker, and analyzed in triplicate after 0, 4, 8, 12 and 16 days of storage. The progress of lipid oxidation was monitored by measuring the standard chemical indices peroxide value, p-anisidine value, TBARS TOTOX ¼ AV þ 2PV ð4Þ R. Agregán et al. / Food Research International 99 (2017) 986–994 989

2.10. Determination of thiobarbituric acid-reactive substances (TBARS) values

The TBARS were determined following the method of Shahidi,

mol TE/g extract) Desilva, and Amarowicz (2003), where each sample (200 mg) was μ FRAP ( weighed into 25 mL volumetric flask and then made up to the mark d with 1-butanol (ACS grade) and mixed thoroughly. A 5.0 mL portion c b a of this solution was transferred into a dry test tube, and 5.0 mL of fresh TBA reagent (200 mg TBA in 100 mL 1-butanol) were added to it. Next, the tube was placed in a 95 °C water bath for 120 min where

7.52 ± 0.70 51.66 ± 0.74 26.93 ± 0.54 578.50 ± 12.02 a colorimetric reaction takes place. Finally it was cooled to room tem- c d a

b perature, and the absorbance of the solution (pink color) was read at 532 nm. The TBARS value was calculated as follows:

uration, DPPH free radical scavenging activity, ferric re- ðÞA 0:415

mol TE/g extract) TBARS ¼ μ ( 278.34 ± 3.53 756.50 ± 45.96 979.86 ± 16.26 134.24 ± 24.04 m ,c ,a e e d

,c where, A = absorbance at 532 nm and m = sample weight. e 50 2.11. Statistical analysis DPPH EC (mg extract/mL) 11.5 ± 0.42 (4.34%) 4.19 ± 0.71 (38.88%) 7.5 ± 0.70 (23.58%) 0.95 ± 0.06 The differences in proximate composition, TPC and in vitro antioxi- d

c dant activity among extracts (and BHT in the corresponding cases) b a were examined using an ANOVA test. The effect of seaweed extracts on oxidative stability of canola oil measured through the peroxide value, p-anisidine value, TBARS value and conjugated dienes determina- mol TE/g extract) μ aqueous extracts and BHT compound (means ± standard deviation of three samples). DPPH ( 2.27 ± 0.70 135.31 ± 4.24 47.53 ± 2.12 976.04 ± 65.04 tion was examined using a mixed-model ANOVA, where these parame- ters were set as dependent variables, seaweed extracts as fixed effect, ,c

e a b d and replicate as random effect. Differences were considered significant

50 if P b 0.05. The values were given in terms of mean values ± standard deviations. All statistical analysis were performed using IBM SPSS Statis- antioxidant activity determined by ABTS radical cation decolo Fucus vesiculosus , tics 19 software. ABTS EC (mg extract/mL) 11.5 ± 0.06 (43.3%) 1.54 ± 0.02 2.78 ± 0.03 0.28 ± 0.03 c d in vitro

a b 3. Results and discussion

3.1. Proximate composition and total phenolic content Bifurcaria bifurcata , mol TE/g extract) μ ABTS ( ABTS DPPH ORAC 5105.03 ± 60.81 147.26 ± 0.70 1046.79 ± 18.38 728.29 ± 7.07 The proximate composition and the TPC of the three seaweed ex- tracts are shown in Table 1. The TSC in descending order was: ANE (1.15%) N BBE (0.75%) N FVE (0.42%). These differences in total solids 0.05). b a b c among the three seaweeds could be explained by variation in proximal P extract, BHT: butylatedhydroxytoluene. composition and the polarities of different compounds present in the

Ascophyllum nodosum seaweeds. It was noted that the water extracts of FVE were very viscous cantly ( fi and they were very difficult to pass through the filter paper. This could Total polyphenols (g PGE/100 g extract) 0.96 ± 0.03 1.15 ± 0.02 1.99 ± 0.23 be due to the presence of mucilaginous substances and might be the

Fucus vesiculosus reason for the lowest TSC of the water extracts from this species. Extractants have an impact on the TSC of the extracts. Because of

c b a safety concerns regarding the use of some organic solvent extracts in foods, we selected water for extraction of antioxidant compounds extract, FVE: from seaweeds. In addition, Sabeena Farvin and Jacobsen (2013) and Wang, Jonsdottir, and Ólafsdóttir (2009) noticed that the extraction , so radical percentage inhibition was indicated. Total carbohydrates (g glucose/100 g extract) 63.60 ± 2.45 34.53 ± 2.92 15.71 ± 3.48 50 yields of water extracts were higher than those of organic solvent ex-

a c b tracts which indicated that most of the soluble components in seaweeds were high in polarity. Bifurcaria bifurcata s content expressed as g of phloroglucinol equivalents (PGE)/100 g extract, and Protein contents were 26.08, 62.05 and 53.33 g/100 g of extract for ANE, FVE and BBE, respectively, whereas total carbohydrate contents Protein (g/100 g extract) 26.08 ± 1.07 62.05 ± 2.30 53.33 ± 1.69 were 63.60, 34.53 and 15.71 g glucose/100 g for ANE, FVE and BBE, respectively. extract, BBE:

c a b Data on proximate composition of aqueous seaweed extracts are not available in literature. Values of protein and carbohydrate contents in the present study widely varied among species in agreement with the variability of data reported for ethanolic extracts from different edible Total solids (g solids/L extract) seaweeds (Boisvert, Beaulieu, Bonnet, & Pelletier, 2015). The TPC obtained from the tree different seaweed species ranged Ascophyllum nodosum

In these samples it was not possible calculate EC from 0.96 to 1.99 g PGE/100 g extract (Table 1). Our values were : Mean values in the same column not followed by a common letter differ signi Seaweed extract FVEBBE 4.19 ± 0.53 BHT 7.50 ± 0.91 ANE 11.5 ± 1.45 e d – ducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) of Table 1 Proximate composition, total polyphenol ANE: a PGE = phloroglucinol equivalents; TE = Trolox equivalents. lower than those reported by Wang et al. (2009) who found TPC of 990 R. Agregán et al. / Food Research International 99 (2017) 986–994

13.8, 16.9 and 17.6 g PGE/100 g of water extract for A. nodosum, quantification. In the present study, phloroglucinol (the main phenolic F. serratus and F. vesiculosus, respectively. However, values in the pres- compound in seaweed) was used as the standard (data expressed as g ent work were higher than those reported by Sabeena Farvin PGE/100 g), whereas gallic acid is usually used as standard in the and Jacobsen (2013) who reported TPC ranging from 0.011 to 0.61 g analysis of the extracts from terrestrial plants (data expressed as g GAE/100 g of water extract in several seaweed species. GAE/100 g). However, the TPCs of extracts from terrestrial plants In the present study there was an inverse relationship between TSC (Table 2) are generally higher than in the seaweeds considered in the and TPC (Table 1). Indeed, the seaweed extract (ANE) that showed the present study. Similar values to those found for the three seaweeds highest TSC (1.15%) presented the lowest TPC (0.96 g PGE/100 g ex- under study were reported for leaf extracts from Eucalyptus citriodora tract). In the case of TPC, our results indicate that BBE exhibited 107% (Ali et al., 2016), some essential oils (Turan, 2014) and for extracts and 73% higher TPC than FVE and ANE, respectively. This finding is in from different parts of artichoke (Claus et al., 2015). However, even disagreement with those reported by Wang et al. (2009) who found lower values than determined in the seaweed species under study that A. nodosum extract showed 27% higher TPC than F. vesiculosus ex- were reported for wheat bran extracts (Shahid Chatha, Hussain, tract in water. These outcomes suggested that the TSC of the extract Bajwa, Hussain Sherazi, & Shaukat, 2011). did not link with the polyphenolic compound contents of the seaweed but might be influenced by the types of phenolic compounds present 3.2. Antioxidant activity and the performance of the solvent used in the preparation (Chan, Matanjun, Yasir, & Tan, 2015). Values of DPPH, ORAC, FRAP and ABTS radical-scavenging activity of In order to stablish a comparison with extracts obtained from terres- the three extracts and of the BHT compound are presented in Table 1. trial plants, information in literature regarding the TPC, antioxidant ac- The ability of a seaweed extract to scavenge the reactive metabolites tivity and antioxidant performance in oils of the extracts from plants of will inhibit the formation of primary and secondary oxidation products. terrestrial origin was summarized in Table 2. An exact comparison of In our study, FVE presented the highest DPPH free radical scavenging ac- the TPC of the seaweed extracts analyzed in the present study and the tivity (135.3 μm TE/g extract), whereas BBE that had the highest TPC TPCs previously reported for extracts from terrestrial plants is not pos- only showed the intermediate values (47.5 μm TE/g extract). These out- sible because of differences in the analytical standards used for comes suggest that substances other than polyphenols present in water

Table 2 Information in literature on the characteristics of extracts from terrestrial plants and their use in the oxidative stabilization of canola oil.

Extract origin Solvent TPC g Antioxidant Thermal Concentration Conclusions Reference GAE/100 g activitya conditions added to the oil

Celery (Apium graveolens) Ethanol 37.6 89 180 °C 1200 ppm extract can stabilize canola Maleki et al. ethanol:water 34.3 84 oil very effectively compared to (2015) (50:50) 35.8 95 conventional synthetic water antioxidant. Rice bran (Tarom Mahali) Ethanol 86.2 54 120 °C 100 ppm 100 ppm of this extract show the Farahmandfar et ethanol:water 90.9 63 800 ppm same effectivity as synthetic al. (2015) (50:50) 1200 ppm antioxidant in stabilization of canola oil. Rosa woodssi hip with seed Acetone:water:acetic 14–69 DPPH 65 °C 200 μGAE/g The natural phenolic mixture Aladedunye,

(Rosae pseudofrutus cum acid (70:29:5:0.5) IC50= 120 °C 500 μGAE/g from this plant offered significant Kersting, and fructibus) 40–400 μg/g 180 °C protection against deterioration of Matthäus (2014) edible oils both under storage and frying conditions, presenting an excellent natural alternative to synthetic antioxidants commonly used in food processing industry. Leaf of Eucalyptus citriodora Ethanol 5.23 70.56 65 °C 300 ppm E. citridora leaf ethanolic extract is Ali et al. (2016) a rich source of natural polyphenols (phenolics and flavonoids) that can be used as natural antioxidant for oil (blend of canola, rapeseed and sunflower) stabilization and its storage for a long duration in the food industry. (Thymus vulgaris L.) Essential oils from the 18.91 75 110 °C 500 ppm The thyme was the most effective Turan (2014) sage (Salvia officinalis L.) four origins were 0.83 12.5 1000 ppm essential oil in inhibiting the rosemary (Rosmarinus analyzed and tested 0.35 13.5 2000 ppm formation of primary and officinalis L.) 0.73 42.2 secondary oxidation products in bay (Laurus nobilis) canola oil. Artichokes (Cynara Methanol 0.7–4.2 DPPH 90 °C 500 ppm Artichoke spikes are strong Claus et al. cardunculus var. scolymus L.) ethanol 0.3–3.4 (μmol TE/g) candidates for use in the food (2015) water 0.2–2.5 1557–23,331 industry as natural additives 1291–14,388 against oxidation of foods such as 1388–10,675 canola oil. Wheat bran Methanol 0.26 80 50 °C 600 ppm The extracts derived from the Shahid Chatha et acetone:water 0.29 92 wheat bran were found to be very al. (2011) (80:20) 0.21 73 effective towards suppressing the acetone 0.24 79 primary and secondary oxidation methanol:water products in canola oil. (80:20)

a Inhibition of DPPH scavenging activity (%), except when another concrete units are indicated in the column. For the wheat bran expressed as % of inhibition of peroxidation in a linoleic acid system. R. Agregán et al. / Food Research International 99 (2017) 986–994 991 extracts of seaweed, such as small molecular weight polysaccharides, 3.3. Effect of addition of seaweed extracts on the oxidation stability of cano- pigments, proteins, or peptides may influence the free radical scaveng- la oil ing activity (Sabeena Farvin & Jacobsen, 2013). According to data on proximate composition of the extracts obtained in the present study The effect of seaweed extracts on the evolution of primary and sec- and reported in Table 1, the highest protein contents of FVE could be re- ondary lipid oxidation products during storage of canola oil is shown sponsible for their highest radical scavenging activity. This circum- in Figs. 1–5. Determination of PV can be used as oxidation index for stance, together with the lower protein contents of the ANE which at the early stage of lipid oxidation (Zhang et al., 2010). According to our the same time show the lowest antioxidant activity seems to suggest a outcomes it can be observed that storage time promoted oxidation in certain protagonism of the proteins in the antioxidant activity of the canola oil and the PV increased significantly (P b 0.001) during the en- aqueous seaweed extracts. tire period showing the control samples the highest PV (381 meq O2/ In addition, radical scavenging activity of polyphenols also depends kg oil) at the end of the storage (Fig. 1). Oxidation was significantly re- upon their unique phenolic structure and the number and location of duced by addition of seaweed extracts and BHT, and the PV measured in the hydroxyl groups (Brand-Williams et al., 1995). To this regards, oil samples with seaweed extracts and BHT were lower than in control Brand-Williams et al. (1995) noticed that caffeic acid with two hydroxyl samples during the whole storage period. This reduction was signifi- groups is a more efficient antiradical compound than its monohydroxyl cantly higher (P b 0.001) for the seaweed extracts than for BHT. At the counterpart coumaric acid, likewise gallic acid, a trihydroxyl phenol, is end of storage period, PV of samples with BHT decreased by approxi- more potent than protocatechuic and gentisic acid, its dihydroxyl mately 29% compared to the control sample, whereas the PV decreased counterparts. The most effective extract was FVE (EC38.9 4.2 mg/mL) in the range of 65–68% relative to the control treatment in samples with followed by BBE (EC23.6 7.5 mg/mL) and ANE (EC4.3 11.5 mg/mL). This seaweed extracts. This indicates that in the concentration used seaweed finding is in agreement with those reported by Sabeena Farvin and extracts are more performant than BHT in the inhibition of the primary Jacobsen (2013) who observed that the most effective aqueous extracts oxidation of canola oil. No differences were observed among the perfor- were those from F. vesiculosus and F. serratus with an EC50 value of mance of the three seaweed extracts measured by means of the PV. 8.3 μg/mL. The highest antioxidant activity of the FVE in relation to the These results are consistent with data reported previously by Sabeena other two seaweed extracts was corroborated using the ABTS and Farvin and Jacobsen (2013) and by Kindleysides et al. (2012). These au- FRAP in vitro assays. thors observed that the antioxidant compounds of seaweed extracts However, the ORAC method showed discordant data. Using this have an important role in inhibiting of free radical formation during assay, the highest antioxidant activity was observed for BBE (979.9 μm the initiation phase of oxidation, interruption of the propagation of of TE/g extract), whereas the FVE showed only the intermediate value the free radical chain reaction by acting as an electron donor, or scav- (756.5 μm of TE/g extract), and, once again, the lowest value was obtain- enging of free radicals in oil samples. ed for ANE (278.3 μm of TE/g extract). These findings are in agreement The p-anisidine value relates to secondary oxidation products (car- with those reported by Wang et al. (2009) who found that the highest bonyls), reflecting the magnitude of aldehydes formation in oils ORAC value was determined in the 70% acetone extract from F. (Zhang et al., 2010). The effect of seaweeds extracts compared to BHT vesiculosus (2567 μm TE/g extract), followed by F. serratus (2545 μm on the evolution of AV values during storage of canola oil is presented TE/g extract), A. nodosum (1417 μm TE/g extract) and old blades of L. in Fig. 2. A significant (P b 0.001) increase in AV values was observed be- hyperborea (975 μm TE/g extract). tween 12 and 16 days of storage, corresponding to high PV values in this The ORAC method is able to determine the antioxidant capacity of period of storage. This result is in agreement with those reported by seaweed extracts by evaluating their ability to scavenge certain Suja, Abraham, Thamizh, Jayalekshmy, and Arumughan (2004) who peroxyl-radicals that induce oxidation in the presence of fluorescein found a similar trend concerning the production of secondary lipid oxi- (Prior et al., 2003). dation products when evaluating the antioxidant activity of sesame The antioxidant activity of all of the seaweed extracts was lower cake extract in soybean, sunflower and safflower oils. Results displayed than that of the reference compound butylated hydroxytoluene (BHT), in Fig. 2 showed that the addition of BHT and seaweed extracts resulted as determined by quantification by the ABTS, DPPH and FRA methods. in a significant (P b 0.001) decrease in AV compared to the control However, when measured by the ORAC assay, the antioxidant activity group, and once again seaweed extracts proved to be more effective of the seaweed extracts was higher than that of BHT. This discrepancy than the BHT compound in preventing the secondary oxidation. may be explained by the different chemical basis of the various mea- surement methods. Indeed, previous reports show different trends in the values obtained when different methods of measuring the antioxi- d,3 400 dant activity were applied to the same samples (Craft, Kerrihard, Control 350 Amarowicz, & Pegg, 2012). Moreover, BHT is a single compound, where- BHT d,2 as seaweed extracts comprise a mixture of compounds of different na- /kg oil) 300 2 ANE ture and that interact with each other. 250 FVE Reported data on the antioxidant activity of extracts of terrestrial 200 c,3 plants were obtained by the DPPH scavenging method (Table 2). Values BBE 150 d,1 reported for aqueous extracts of different parts of the artichoke (1388– c,2 b,3 10,675 μmol TE/g) (Claus et al., 2015) are higher than those determined 100 c,1 b,2 a,3 in the present study for the three seaweed species. Most previously re- 50 b,1 Peroxide value (meq O a a,2 ported results are expressed as % of inhibition of DPPH scavenging activ- a,1 0 ity. Values reported for the extracts from terrestrial plants (generally 0481216 higher than 50%, Shahid Chatha et al., 2011; Maleki, Ariaii, & Fallah, Storage time (days) 2015; Farahmandfar, Asnaashari, & Sayyad, 2015; Ali et al., 2016) are higher than those obtained for the three extracts in the present study Fig. 1. Effect of addition of extracts from three different seaweed species (Ascophyllum (4.34%, 38.88% and 23.58% for ANE, FVE and BBE, respectively). In addi- nodosum, Fucus vesiculosus and Bifurcaria bifurcata) (500 ppm) and of BHT compound tion to the higher antioxidant activity of the extracts from terrestrial (50 ppm) on the evolution of peroxide values during storage of canola oil. Plotted values are means ± standard deviations of two determinations. a–dMeans in the same oil plants, this may also be due to the higher concentration (lower moisture treatment not followed by a common letter are significantly different (P b 0.05) content) of the extracts from terrestrial plants used in the different (differences among sampling points). 1–3Means in the same sampling point not followed studies. by a common number are significantly different (P b 0.05) (differences among treatments). 992 R. Agregán et al. / Food Research International 99 (2017) 986–994

d,3 0.25 60 Control Control BHT b,2 0.20 BHT 45 d,2 ANE ANE b,2 FVE 0.15 b,12 FVE

a a,12 30 BBE a BBE c,3 a d,1 0.10 a,1 a a bc,3 c,2 a,1 p-Anisidine value 15 b,2 c,1 0.05 ab,3 b,1 a a,2 a,1 TBARS (mg MDA /kg oil) 0 0.00 0481216 0481216 Storage time (days) Storage time (days)

Fig. 2. Effect of addition of extracts from three different seaweed species (Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcata) (500 ppm) and of BHT compound Fig. 4. Effect of addition of extracts from three different seaweed species (Ascophyllum (50 ppm) on the evolution of p-anisidine values during storage of canola oil. Plotted values nodosum, Fucus vesiculosus and Bifurcaria bifurcata) (500 ppm) and of BHT compound are means ± standard deviations of two determinations. a–dMeans in the same oil (50 ppm) on the evolution of TBARS values during storage of canola oil. Plotted values are a–b treatment not followed by a common letter are significantly different (P b 0.05) means ± standard deviations of two determinations. Means in the same oil treatment – fi b (differences among sampling points). 1 3Means in the same sampling point not followed not followed by a common letter are signi cantly different (P 0.05) (differences among 1–2 by a common number are significantly different (P b 0.05) (differences among treatments). sampling points). Means in the same sampling point not followed by a common number are significantly different (P b 0.05) (differences among treatments).

Although differences are not significant (P N 0.05), the BBE seems pro- vide the best protection against secondary oxidation of canola oil sam- inhibition percentage (66.45%) was achieved in FVE treatment while ples during the whole period. At the end of storage period, AV of BHT samples raised to a 25.16% inhibition. These results demonstrated samples with BHT decreased by approximately 27% compared to the that the overall oxidation in the canola oil was reduced by these sea- control treatment, while the addition of seaweeds extract resulted in weed extracts. decreases in PV in the range of 62–67% relative to the control group. The effect of seaweeds extracts and BHT on the evolution of TBARS From these results it can be seen that TPC found in seaweed extract values during the storage period of canola oil is summarized in Fig. 4. had a strong inhibitory effect on the secondary lipid oxidation. These The TBARS values of control samples significantly (P b 0.001) increased outcomes are in agreement with those reported by Franco et al. with the increase in storage period. However, a different pattern was (2016) who noticed that natural extracts displayed a significant inhibi- observed in oil samples with seaweed extracts when compared with tory effect against thermal oxidation of soybean oil heated at 60 °C dur- control and BHT treatments. In control and BHT-added oils, TBARS ing 14 days. values continued to increase from 12th to 16th day of storage, while The TOTOX index of the oil was determined to obtain an overall in- in ANE, FVE and BBE treatments a significant (P b 0.001) decrease was formation of the oxidative stability as it incorporates both primary and noticed in the TBARS values from the twelfth day to the end of storage secondary oxidation measurements (De Abreu, Losada, Maroto, & period (Fig. 4). After 16 days of storage, TBARS values of samples with Cruz, 2010). Fig. 3 depicts the changes in the total oxidation values BHT decreased by approximately 17% relative to the control samples, (TOTOX index) of the canola oil under accelerated conditions. In gener- whereas in samples with seaweed extract, the TBARS values decreased al, the kinetic trends were similar to those obtained in PV production. in the range 44–47% relative to the control treatment. It is well known There was also a marked increase in TOTOX index after 12 days of stor- that malondialdehyde, the compound quantified in the TBARS analysis, age (Fig. 3). After this period, TOTOX index increased faster in control is very unstable and it can disappear via degradation, or due to advanced samples than in samples treated with seaweed extracts. TOTOX index reactions with protein residues (Kikugawa, Kato, & Hayasaka, 1991). for oil samples with seaweed extracts and BHT were significantly The important reduction of the TBARS values after 12 days of storage (P b 0.05) lower than the values observed for control group. The best in the oils fortified with seaweed extracts could be due to the inhibition of production of malondialdehyde by the seaweed extracts, but also to

e,3 Control 750 5 BHT Control e,2 c,3 600 ANE BHT 4 FVE ANE 450 d,3 BBE 3 FVE d,2 d,1 300 BBE

Totox value d,2 c,3 2 b,2 c,1 c,2 c,1 ab,3 c,12 150 b,3

b,1 Conjugated dienes 1 a,3 c,12 a b,2 b,2 a a,1 a,23 b,1 0 a,12 0481216 (% of conjugated dienoic acid) 0 Storage time (days) 0481216 Storage time (days)

Fig. 3. Effect of addition of extracts from three different seaweed species (Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcata) (500 ppm) and of BHT compound Fig. 5. Effect of addition of extracts from three different seaweed species (Ascophyllum (50 ppm) on the evolution of TOTOX index during storage of canola oil. Plotted values are nodosum, Fucus vesiculosus and Bifurcaria bifurcata) and of BHT compound (50 ppm) on means ± standard deviations of two determinations. a–eMeans in the same oil treatment the evolution of conjugated dienes during storage of canola oil. a–dMeans in the same oil not followed by a common letter are significantly different (P b 0.05) (differences among treatment not followed by a common letter are significantly different (P b 0.05) sampling points). 1–3Means in the same sampling point not followed by a common (differences among sampling points). 1–3Means in the same sampling point not followed number are significantly different (P b 0.05) (differences among treatments). by a common number are significantly different (P b 0.05) (differences among treatments). R. Agregán et al. / Food Research International 99 (2017) 986–994 993 its disappearance due to reactions with protein residues. The high pro- References tein content of the seaweed extracts (Table 1) could facilitate this possi- bility. Moreover, these reactions occur specially in conditions of low Ahn, G. -N., Kim, K. -N., Cha, S. -H., Song, C. -B., Lee, J., Heo, M. -S., ... Jeon, Y. -J. (2007). An- tioxidant activities of phlorotannins purified from Ecklonia cava on free radical scav- water activity, a circumstance that takes place in the oils under study. enging using ESR and H2O2-mediated DNA damage. European Food Research and The measurement of CD is also a good way for oil oxidation assess- Technology, 226(1–2), 71–79. ment because they are stable and remain in the oil (Suleiman, El- Aladedunye, F., Kersting, H. J., & Matthäus, B. (2014). Phenolic extract from wild rose hip with seed: Composition, antioxidant activity, and performance in canola oil. European Makhzangy, & Ramadan, 2006); several authors have employed this Journal of Lipid Science and Technology, 116(8), 1025–1034. assay to evaluate the antioxidant activity of different extracts, such as Ali, S., Chatha, S. A. S., Ali, Q., Hussain, A. I., Hussain, S. M., & Perveen, R. (2016). Oxidative grapeseed(Poiana, 2012), potato peel (Franco et al., 2016)or stability of cooking oil blend stabilized with leaf extract of Eucalyptus citriodora. – blackcurrant seeds extracts (Samotyja & Małecka, 2010). Based on International Journal of Food Properties, 19(7), 1556 1565. AOAC (2000). Peroxide value of oils and fats. Official method 965.33. Official Methods of Fig. 5 it can be noticed that the rate of CD formation was higher than Analysis (17th ed.). Gaithersburg: Maryland (USA): Association of Official Analytical the decomposition rate, leading to their accumulation in canola oil Chemist. Benzie, I. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure during the storage period. As pointed out by other authors, CD are high- “ ” – fl of antioxidant power : The FRAP assay. Analytical Biochemistry, 239(1), 70 76. ly in uenced by the type of lipids and oxidation time (Kim, Yeo, Kim, Boisvert, C., Beaulieu, L., Bonnet, C., & Pelletier, E. (2015). Assessment of the antioxidant Kim, & Lee, 2013). The inhibition of CD production is important in the and antibacterial activities of three species of edible seaweeds. Journal of Food early stages of lipid peroxidation reactions, in order to prevent the sub- Biochemistry, 39(4), 377–387. Brand-Williams, W., Cuvelier, M. E., & Berset, C. L. W. T. (1995). Use of a free radical meth- sequent formation of reactive lipid radicals. The initial rate of formation od to evaluate antioxidant activity. LWT- Food Science and Technology, 28(1), 25–30. of CD was slow, and it increased after 8 days of storage (Fig. 5). Both the Chan, P. T., Matanjun, P., Yasir, S. M., & Tan, T. S. (2015). Antioxidant activities and poly- addition of seaweed extracts and BHT resulted in significant (P b 0.001) phenolics of various solvent extracts of red seaweed, Gracilaria changii. Journal of Applied Phycology, 27(6), 2377–2386. decrease in CD values compared to control treatment. Also in order to Claus, T., Maruyama, S. A., Palombini, S. V., Montanher, P. F., Bonafé, E. G., Junior, O. D. O. S., prevent the CD formation, the seaweed extracts were more effective ... Visentainer, J. V. (2015). Chemical characterization and use of artichoke parts for than the BHT compound. These findings are partially in agreement protection from oxidative stress in canola oil. LWT- Food Science and Technology, 61(2), 346–351. with those reported by Poiana (2012) who noticed that grape seed ex- Codex Alimentarius (2005). Norma del Codex para Aceites Vegetales Especificados. Codex tracts at level of 600 ppm displayed the same effectiveness in reducing Alimentarius. the accumulation of CD than BHT. At the end of treatment, CD of sam- Cornish, M. L., & Garbary, D. J. (2010). Antioxidants from macroalgae: Potential applica- – ples with BHT decreased by approximately 36% compared to the control tions in human health and nutrition. Algae, 25(4), 155 171. Craft, B. D., Kerrihard, A. L., Amarowicz, R., & Pegg, R. B. (2012). Phenol-based antioxidants treatment, whereas in samples with seaweed extract, the CD values de- and the in vitro methods used for their assessment. Comprehensive Reviews in Food creased in the range 66–68% relative to the control group. Science and Food Safety, 11,148–173. fl Comparison of the present findings and previous data on natural ex- Dávalos, A., Bartolomé, B., Suberviola, J., & Gómez-Cordovés, C. (2003). ORAC- uorescein fi as model for evaluating antioxidant activity of wines. Polish Journal of Food and tracts or essential oils from plants of terrestrial origin is dif cult owing Nutrition Science, 12(1), 133–136. to the different concentrations of extracts and incubation temperatures De Abreu, D. P., Losada, P. P., Maroto, J., & Cruz, J. M. (2010). Evaluation of the effectiveness used to determine the antioxidant activities of the extracts added to ca- of a new active packaging film containing natural antioxidants (from barley husks) fi that retard lipid damage in frozen Atlantic salmon (Salmo salar L.). Food Research nola oil. However, the present ndings are consistent with several re- International, 43(5), 1277–1282. ports of similar or even higher antioxidant activity of extracts of Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric terrestrial plants rather than of synthetic antioxidants (BHT or TBHQ) method for determination of sugars and related substances. Analytical Chemistry, 28(3), 350–356. added to vegetable oils (Table 2). Farahmandfar, R., Asnaashari, M., & Sayyad, R. (2015). Comparison antioxidant activity of Tarom Mahali rice bran extracted from different extraction methods and its effect on 4. Conclusions canola oil stabilization. Journal of Food Science and Technology, 52(10), 6385–6394. Franco, D., Pateiro, M., Rodríguez Amado, I., López Pedrouso, M., Zapata, C., Vázquez, J. A., & Lorenzo, J. M. (2016). Antioxidant ability of potato (Solanum tuberosum)peelex- The addition of seaweed extracts to canola oil stored under favorable tracts to inhibit soybean oil oxidation. European Journal of Lipid Science and oxidation conditions appears to be a valuable tool to assess the ability of Technology. http://dx.doi.org/10.1002/ejlt.201500419. these natural extracts to inhibit lipid oxidation. The extracts of the dif- IUPAC (1987). In C. Paquot, & A. Hautfenne (Eds.), Standard Methods for the Analysis of Oils, Fats and Derivatives (7th ed.). London: Blackwell Scientific. ferent seaweed species (A. nodosum, B. bifurcata and F. vesiculosus) Jiménez-Escrig, A., & Sánchez-Muniz, F. J. (2000). Dietary fibre from edible seaweeds: contained different levels of total phenolic compounds and in vitro pos- Chemical structure, physicochemical properties and effects on cholesterol metabo- – sessed diverse antioxidant properties. It was found that A. nodosum had lism. Nutrition Research, 20(4), 585 598. Jittrepotch, N., Ushio, H., & Ohshima, T. (2006). Oxidative stabilities of triacylglycerol and less antioxidant activity than B. bifurcata and F. vesiculosus based on as- phospholipid fractions of cooked Japanese sardine meat during low temperature says used. The addition of seaweed extracts at 500 ppm to canola oil storage. Food Chemistry, 99(2), 360–367. showed efficacy in delaying both primary and secondary oxidative Kikugawa, K., Kato, T., & Hayasaka, A. (1991). Formation of dityrosine and other fluores- fi cent amino acids by reaction of amino acids with lipid hydroperoxides. Lipids, changes during storage under favorable oxidation conditions. So, nd- 26(11), 922–929. ings in the present work prove that seaweed extracts can be used as Kim, T. S., Yeo, J., Kim, J. Y., Kim, M. J., & Lee, J. (2013). Determination of the degree of ox- an easily accessible source of natural antioxidants. They can be used idation in highly-oxidised lipids using profile changes of fatty acids. Food Chemistry, 138(2–3), 1792–1799. safely in the edible oil industry to delay oxidation instead of synthetic Kindleysides, S., Quek, S. Y., & Miller, M. R. (2012). Inhibition of fish oil oxidation and the antioxidants. Further studies, however, should be done in the future to radical scavenging activity of New Zealand seaweed extracts. Food Chemistry, 133(4), identify the concrete phenolic compounds of these seaweed extracts, 1624–1631. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement and to determine the correlation between antioxidant effect and chem- with the Folin phenol reagent. Journal of Biological Chemistry, 193(1), 265–275. ical composition. Maleki, M., Ariaii, P., & Fallah, H. (2015). Effects of celery extracts on the oxidative stability of canola oil under thermal condition. Journal of Food Processing and Preservation, – Acknowledgements 40(3), 531 540. Müller, L., Fröhlich, K., & Böhm, V. (2011). Comparative antioxidant activities of caroten- oids measured by ferric reducing antioxidant power (FRAP), ABTS bleaching assay The authors thank INIA (Instituto Nacional de Investigación y (αTEAC), DPPH assay and peroxyl radical scavenging assay. Food Chemistry, 129(1), – Tecnología Agraria y Alimentaria, Spain) for granting Ruben Agregán 139 148. Ou, B., Hampsch-Woodill, M., & Prior, R. L. (2001). Development and validation of an im- with a predoctoral scholarship (CPR2014-0128). The authors also proved oxygen radical absorbance capacity assay using fluorescein as the fluorescent thank Council for Scientific and Technological Development (Brazil) probe. Journal of Agricultural and Food Chemistry, 49(10), 4619–4626. (CNPq no. 248705/2013-0) for financial support, and Aceites Abril, S.L. Piccolella, S., Fiorentino, A., Pacifico, S., D'Abrosca, B., Uzzo, P., & Monaco, P. (2008). Anti- oxidant properties of sour cherries (Prunus cerasus L.): Role of colorless phytochem- (Ourense, Spain) for supplying the canola oil samples used in this icals from the methanolic extract of ripe fruits. Journal of Agricultural and Food research. Chemistry, 56(6), 1928–1935. 994 R. Agregán et al. / Food Research International 99 (2017) 986–994

Poiana, M. A. (2012). Enhancing oxidative stability of sunflower oil during convective and Suja, K. P., Abraham, J. T., Thamizh, S. N., Jayalekshmy, A., & Arumughan, C. (2004). Anti- microwave heating using grape seed extract. International Journal of Molecular oxidant efficacy of sesame cake extract in vegetable oil protection. Food Chemistry, Sciences, 13(7), 9240–9259. 84(3), 393–400. Prior, R. L., Hoang, H. A., Gu, L., Wu, X., Bacchiocca, M., Howard, L., ... Jacob, R. (2003). As- Suleiman, A. E. M., El-Makhzangy, A., & Ramadan, M. (2006). Antiradical performance and says for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance physicochemical characteristics of vegetable oils upon frying of French fries: A pre- capacity (ORACFL)) of plasma and other biological and food samples. Journal of liminary comparative. Electronic Journal of Environmental, Agricultural and Food Agricultural and Food Chemistry, 51(11), 3273–3279. Chemistry, 5(1), 1429–1441. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Anti- Talpur, M. A., Changying, J., Chandio, F. A., Junejo, S. A., & Mari, I. A. (2011). Application of oxidant activity applying an improved ABTS radical cation decolorization assay. Free oven drying method on moisture content of ungrounded and grounded (long and Radical Biology and Medicine, 26(9), 1231–1237. short) rice for storage. Journal of Stored Products and Postharvest Research, 2(12), Sabeena Farvin, K. H., & Jacobsen, C. (2013). Phenolic compounds and antioxidant activi- 245–247. ties of selected species of seaweeds from Danish coast. Food Chemistry, 138(2), Turan, S. (2014). Efficiency of various plant essential oils in stabilization of canola oil and 1670–1681. of its purified triacylglycerols. Journal of Essential Oil Research, 26(3), 166–176. Samotyja, U., & Małecka, M. (2010). Antioxidant activity of blackcurrant seeds extract and Wang, T., Jonsdottir, R., & Ólafsdóttir, G. (2009). Total phenolic compounds, radical scav- rosemary extracts in soybean oil. European Journal of Lipid Science and Technology, enging and metal chelation of extracts from Icelandic seaweeds. Food Chemistry, 112(12), 1331–1336. 116(1), 240–248. Shahid Chatha, S. A., Hussain, A. I., Bajwa, J. U. R., Hussain Sherazi, S. T., & Shaukat, A. Zhang, Y., Yang, L., Zu, Y., Chen, X., Wang, F., & Liu, F. (2010). Oxidative stability of sun- (2011). Wheat bran extracts: A potent source of natural antioxidants for the stabili- flower oil supplemented with carnosic acid compared with synthetic antioxidants zation of canola oil. Grasas y Aceites, 62(2), 190–197. during accelerated storage. Food Chemistry, 118(3), 656–662. Shahidi, F., Desilva, C., & Amarowicz, R. (2003). Antioxidant activity of extracts of defatted Zubia, M., Robledo, D., & Freile-Pelegrin, Y. (2007). Antioxidant activities in tropical ma- seeds of niger (Guizotia abyssinica). Journal of the American Oil Chemists' Society, 80(5), rine macroalgae from the Yucatan Peninsula, Mexico. Journal of Applied Phycology, 443–450. 19(5), 449–458. Singleton, V. L., Orthofer, R., & Lamuela-Raventos, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods in Enzymology, 299,152–178.

Food Research International 99 (2017) 1095–1102

Contents lists available at ScienceDirect

Food Research International

journal homepage: www.elsevier.com/locate/foodres

Assessment of the antioxidant activity of Bifurcaria bifurcata aqueous extract on canola oil. Effect of extract concentration on the oxidation stability and volatile compound generation during oil storage

Rubén Agregán a,JoséM.Lorenzoa, Paulo E.S. Munekata b, Ruben Dominguez a, Javier Carballo c, Daniel Franco a,⁎ a Centro Tecnológico de la Carne de Galicia, Avda. Galicia n° 4, Parque Tecnológico de Galicia, San Cibrao das Viñas 32900, Ourense, Spain b Department of Food Engineering, Faculty of Animal Science and Food Engineering, University of São Paulo, 225 Duque de Caxias Norte Ave, Jardim Elite, 13.635-900, Pirassununga, São Paulo, Brazil c Área de Tecnología de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004, Ourense, Spain article info abstract

Article history: In this research the antioxidant activity of water extracts of Bifurcaria bifurcata (BBE) at different dose against bu- Received 28 July 2016 tylated hydroxytoluene (BHT) was evaluated in canola oil. Water extracts were firstly characterized in terms of Received in revised form 14 October 2016 total solid and polyphenolic compound contents, and their antioxidant activity together with that of BHT was Accepted 16 October 2016 evaluated using several in vitro tests (DPPH, ABTS, ORAC and FRAP). Next, the progress of lipid oxidation was Available online 18 October 2016 assessed in canola oil added with five BBE concentrations (200, 400, 600, 800 and 1000 ppm) and two BHT con- centrations (50 and 200 ppm) using an accelerated oxidation test. The progress in lipid oxidation was monitored Keywords: Bifurcaria bifurcata extract by assessing some chemical indices (peroxide value, p-anisidine value, and conjugated dienes) during oil storage Antioxidant activity and some volatile compounds at the end of the storage period. BBE showed a significant antioxidant effect, being Canola oil this ability concentration-dependent. The extent of lipid oxidation was inversely related to BBE dose, specially Volatile compounds with regard to primary oxidation products. At the highest level of BBE, significant decreases of primary and sec- Primary and secondary oxidation ondary oxidation products, with respect to the control, were obtained with reduction percentages of 71.53%, Seaweed 72.78%, 68.17% and 71.3% for peroxides, conjugated dienes, p-anisidine and TOTOX values, respectively. A level of 600 ppm or higher concentration of the extract inhibits the lipid oxidation in a similar way than BHT at 200 ppm. Regarding the inhibition of the formation of volatile compounds, both BBE and BHT strongly inhibited the formation of volatiles during oil storage, being this inhibition similar for all the concentrations of BBE and BHT essayed. Overall, results indicated that BBE can be used as a potential natural additive for improving oxidative sta- bility of canola oil. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction deterioration in flavor, texture, color and nutritional value of food is cur- rently one of the biggest challenges for the food industry. In order to During storage and shelf life of fatty-foods, the oxidative processes limit these deterioration processes, synthetic antioxidants such as cause large economic losses for the food industry. Degradation of color BHA, BHT or TBHQ are currently added, but these compounds have parameters, lipids and proteins that, in turn, can contribute to the been questioned due to their potential toxic and carcinogenic effects (Moure et al., 2001). For this reason, consumers are demanding natural Abbreviations: BBE, Bifurcaria bifurcata extract; BHT, butylated hydroxytoluene; ABTS, plant-based alternatives to replace this type of antioxidants. In this 2, 2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid); DPPH, 1,1 diphenyl-2-picryl sense, natural extracts have been essayed with success in complex hydrazyl; FRAP, ferric reducing antioxidant power; ORAC, oxygen radical absorbance foods such as meat products e.g. patties, dry-cured sausages or liver capacity; TPC, total phenolic content; TS, total solids content; PGE, phloroglucinol equivalents; PBS, phosphate buffer solution; TEAC, Trolox equivalent antioxidant pate (Lorenzo, González-Rodríguez, Sánchez, Amado, & Franco, 2013; capacity; EC, efficient concentration; RSA, radical scavenging activities; TPTZ, 2,4,6- Lorenzo, Sineiro, Amado, & Franco, 2014; Pateiro, Lorenzo, Amado, & tripyridil-s-triazine; AAPH, 2, 20-azobis (2-methyl propionamidine) dihydrochloride; Franco, 2014). TOTOX, total oxidation value; PV, peroxide value; AV, p-anisidine value; CD, conjugated In the last decades, the marine environment has proved to be one of dienes; IP, inhibition percentage; SPME, solid phase microextraction; VC, volatile the most interesting and rich source of natural compounds, many of compounds; ANOVA, analysis of variance. ⁎ Corresponding author. which exhibit particular chemical characteristics that are not found in E-mail address: [email protected] (D. Franco). the terrestrial environment (Murray, Moane, Collins, et al., 2013).

http://dx.doi.org/10.1016/j.foodres.2016.10.029 0963-9969/© 2016 Elsevier Ltd. All rights reserved. 1096 R. Agregán et al. / Food Research International 99 (2017) 1095–1102

Seaweed is a source of excellent bioactive compounds with potential 2.3. Screening of the extracts and BHT for antioxidant activity antioxidant activity such as polyphenols, carotenoids, alkaloids, ter- penes and tocopherol (Heo et al., 2009, Hu, Lin, Lu, Chou, & Yang, 2.3.1. ABTS radical cation decoloration assay 2008). Indeed, polyphenols derived from seaweed may be more power- The method of Re et al. (1999) was adapted to the use of a plate ful that analogous polyphenols derived from terrestrial plant origin reader. ABTS radical cation (ABTS•+) was produced by reacting 7 mM due to the presence of phlorotanins (Koivikko, Loponen, Pihlaja, & ABTS stock solution with 2.45 mM potassium persulfate (final concen- Journalainen, 2007). Within the macroalgae species, three different tration) allowing the mixture to stand in the dark at room temperature classes (green, red and brown) are found. The genus Bifurcaria,belong- for 12–16 h before use. In the next step ABTS•+ solution was diluted ing to Sargassaceae family, comprises two known species: B. bifurcata R. with PBS buffer (pH 7.4) to an absorbance of 0.70 at 734 nm, and equil- Ross and B. galapegensis. B. bifurcata lives in rock pools on the lower and ibrated at 30 °C. An aliquot of each BBE or BHT sample (with appropriate middle tidal zones and at the upper limit of the subtidal shore, distribut- dilution) was mixed with the working solution of ABTS•+ (sample: ed from Morocco (southern limit) to north-western Ireland (northern ABTS solution relation 1:100; v/v), and the decrease of absorbance limit) (Le Lann, Rumin, Cerantola, Culioli, & Stiger-Pouvreau, 2014; was measured after 6 min at 734 nm in a microplate spectrophotometer Muñoz, Culioli, & Kock, 2013). This species has revealed to possess com- reader. Trolox was used as the reference standard and the results were pounds with interesting biological properties such as antimicrobial or expressed as TEAC values (μmol of Trolox equivalents/g extract). The in- antioxidant activities. However, information on the bioactive com- hibition percentage of absorbance at 734 nm is calculated and plotted as pound profile of Bifurcaria bifurcata extracts (BBE) is scarce in the liter- a function of extract concentration to obtain the EC50, the amount of an- ature and most of the works carried out used organic solvents in the tioxidant necessary to decrease the initial ABTS concentration by 50%. In extraction process (Alves et al., 2016; Chernane, Mansori, Latique, & El the samples where it was not possible calculate the EC50 value, the per- Kaoua, 2014). The first step to produce antioxidants from macroalgae centage of inhibition of the radical ABTS was indicated. for use in the food industry have to rest in an inexpensive and environ- mental respectful extraction process, thinking in a further development 2.3.2. DPPH• free radical scavenging activity at industry level (Tierney et al., 2013). In this sense water is the most The radical scavenging activities (RSA) of seaweed extracts and BHT universal solvent with the view to produce food grade extracts for ex- for DPPH• radical were determined according to Brand-Williams, ploitation by the food industry as ingredient with the function of retard Cuvelier, and Berset (1995) with slight modification: 5 μLofappropri- the lipid oxidative deterioration. ately diluted samples were added to 195 μLofDPPH• solution (6 × In such situation, the objective of the present work was to study the 10−5 M in methanol) in a well of 96-well plate. The mixture was shaken antioxidant capacity of the Bifurcaria bifurcata aqueous extracts by gently and left to stand at room temperature for 30 min. Thereafter, the assessing their total phenolic content and their in vitro antioxidant ac- absorbance at 515 nm was measured against methanol using a micro- tivity in comparison with that of BHT using a wide range of scavenging plate reader. The DPPH• radical scavenging activity of extracts and tests. Next, the antioxidant activity of these extracts against that of BHT BHT was calculated from the standard curve of Trolox and expressed was investigated by assessing some chemical indices (peroxide value, p- as μmol of Trolox equivalents/g extract. The results were expressed in anisidine value, and conjugated dienes) and some volatile compounds term of EC50 determined by linear regression analysis of the dose re- in canola oil submitted to accelerated oxidation. sponse curve plotted between the RSA against extract concentration.

The EC50 is defined as the extract concentration (μg/mL) required to de- crease the initial DPPH• concentration by 50%. In the samples where it 2. Materials and methods was not possible calculate the EC50 value, the percentage of inhibition of the radical DPPH• was indicated. 2.1. Seaweed material and extraction procedure 2.3.3. Ferric reducing antioxidant power (FRAP) assay The brown seaweed Bifurcaria bifurcata used in the present study The ability to reduce ferric ions was measured using the method de- was kindly supplied by the Portomuiños company (Coruña, Spain). Sea- scribed by Benzie and Strain (1996). The FRAP reagent was freshly pre- weeds were dried in a conventional oven at 40 °C and then grinded to pared from 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-s- obtain a particle size b500 μm. Extraction was performed in a magnetic triazine (TPTZ) made up in 40 mM HCl, and 20 mM FeCl 6H Osolution. stirrer, using water as a solvent in a liquid/solid ratio of 30 g/g for 5 min 3 2 All three solutions were mixed together in the ratio of 10:1:1 (v/v/v). A to obtain a correct hydration. Next, an aliquot of 800 mL from the algae/ total of 10 μL of properly diluted samples and 30 μL of distilled water water suspension was sonicated for 10 min in an ultrasonic Hielscher were added to 300 μL freshly prepared FRAP reagent in a well of 96- UIP1000HD homogenizer (1000 W power) (Hielscher Ultrasonics well plate. The mixture was incubated at 37 °C throughout the reaction. GmbH, Teltow, Germany) equipped with a flow cell with a residence After 8 min, the absorbance was read using a microplate reader at time of 30 s and 90 percentage of amplitude, with continuous recircula- 593 nm against reagent blank. The FRAP value was calculated and tion. The process was stopped when the temperature reached a value expressed as μmol of Trolox equivalents/g extract based on a calibration higher than 40 °C. Finally, the extract was centrifuged at 2000 ×g and curve constructed using Trolox as standard. filtered through a cellulose filter of 20–25 μm pore size (Filter-lab, Filtros Anoia, S.A., Barcelona, Spain). Extracts were produced in tripli- 2.3.4. Oxygen radical absorbance capacity (ORAC) assay cate and used to measure the total polyphenol content (TPC) and the The original method of Ou, Hampsch-Woodill, and Prior (2001) was antioxidant activity. modified as described by Dávalos, Bartolomé, Suberviola, and Gómez- Cordovés (2003). The reaction was carried out in 75 mM phosphate 2.2. Determination of total solid and total phenolic content buffer (pH 7.4), and the final reaction mixture was 200 mL. The mixture of antioxidant (20 mL) and fluorescein(120mL;70nMfinal concentra- After the extraction, the total solid content (TS) of the extracts were tion) was preincubated for 15 min at 37 °C. An AAPH (2, 20-azobis (2- quantified by gravimetric methods evaporating the solvent at 105 °C methyl propionamidine) dihydrochloride) solution (60 mL; 12 mM, until constant weight. The total phenolic content (TPC) of seaweed final concentration) was added rapidly using a multichannel pipette. extracts was determined according to Singleton, Orthofer, and The plate was immediately placed in the reader and the fluorescence re- Lamuela-Raventós (1999), using phloroglucinol (a basic structural corded every minute for 120 min (excitation wavelength 485 nm, emis- unit of phlorotannins) as a standard, and expressing the results as sion wavelength 520 nm). The plate was automatically agitated prior phloroglucinol equivalents (PGE)/100 g extract. each reading. A blank using phosphate buffer instead of the antioxidant R. Agregán et al. / Food Research International 99 (2017) 1095–1102 1097 solution, and eight calibration solutions (2.5–40 μmol) using Trolox as 25 mL volumetric flask. The sample was then reacted with a p-anisidine antioxidant were also tested in each assay. Results are calculated on solution in acetic acid (0.25% w/v) for 10 min to produce a colored com- the basis of the differences in areas under the fluorescein decay curve plex. Absorbances of the samples with and without p-anisidine solution between the blank and the sample, and are expressed as μmol of Trolox were measured using an 1800 UV spectrophotometer (Shimadzu Cor- equivalents/g extract. poration, Kyoto, Japan) at 350 nm, and the parameter AV was calculated as follows: 2.4. Oxidation stability of canola oil under accelerated conditions 25 ½1:2 ðÞE −E AV ¼ b a ð2Þ This test was conducted to evaluate the effectiveness of BBE against W lipid oxidation. The study was carried out on canola oil free of additives, where, E is the net absorbance of the oil-solution, E is the net absor- using five concentration levels for the BBE (i.e., 200, 400, 600, 800 and b a bance of the oil-anisidine-solution and W is the weight of the sample 1000 ppm), two levels of BHT (50 and 200 ppm), and a control without (g). any antioxidant addition. Canola oil was supplied by a local company (Aceites Abril, S.L., Ourense, Spain). Composition of canola oil deter- 2.7. Determination of conjugated dienes (CD) mined according to CODEX Stan 210 normative (Codex Alimentarius, 2005) was provided by the supplier company; it was: acidity (0.04%), The CD were quantified using an 1800 UV spectrophotometer peroxide value (2.29 meq O /kg), moisture (b0.01%) and impurities 2 (Shimadzu Corporation, Kyoto, Japan) at 233 nm. Before analysis, (b0.01%). The fatty acid profile of canola oil also provided by the suppli- 20 μL of oil was diluted with hexane in a volumetric flask of 25 mL. er and determined using standard official methods was: myristic acid The CD, expressed as percentage of conjugated dienoic acid was calcu- (0.05%), palmitic acid (4.57%), palmitoleic acid (0.26%), stearic acid lated as follows: (1.61%), oleic acid (63.09%), linoleic acid (19.68%), linolenic acid (8.25%), arachidic acid (0.83%), gadoleic acid (1.14%), behenic acid AS CD ¼ 0:84 ð3Þ (0.31%) and lignoceric acid (0.09%). Before applying, BBE and BHT bc−K0 were separately mixed with 1.5 mL of ethanol 96% (v/v) (to ensure ap- propriate dispersion in oil) in a Branson 8510 ultrasonic water bath where, As is the absorbance observed at 233 nm, b is the cell length in (400 W, 100% amplitude) (Emerson Industrial Automation, Eden Prai- cm, c is the concentration of the diluted sample (g/L) and K0 is the ab- rie, MN, USA) during 10 min, and then added to 20 mL of canola oil, sorptivity by the acid or ester groups. In this case, c was considered as fi mixed for 10 min using a vortex mixer ZX3 (Velp Scientifica Srl, Usmate 0.5 g/L, the nal concentration for most edible oils, and K0 was consid- Velate, Italy) and vacuum evaporated. Control sample was prepared ered for acids (value of 0.03). using the same amount of ethanol 96% (v/v) used to dissolve BHT and BBE, and later treated in the same way. Canola oil samples without an- 2.8. Determination of total oxidation value (TOTOX index, TV) tioxidant or after addition of the different BBE or BHT concentrations were stored in glass containers at 60 °C and agitated at 100 rpm in an The overall oxidation state of oil given by the TOTOX was calculated orbital shaker. They were analyzed in triplicate after 4, 8, 12 and according to the formula: 16 days of storage. The progress of lipid oxidation was monitored by TOTOX ¼ AV þ 2PV ð4Þ measuring the standard chemical indices peroxide value, p-anisidine value, and conjugated dienes. Besides these indices, the total oxidation value (TOTOX) was calculated. The volatile compounds in oil samples 2.9. Inhibition of oil oxidation during storage were also analyzed. Analyses in each oil sample were carried out in duplicate. For all indices employed to monitor the oxidation process, the per- centage of inhibition of oil oxidation (IP) was calculated according to 2.5. Determination of peroxide value (PV) the next formula:

The PV was determined following the AOAC procedure 965.33 1−Δindex in sample fi IP ðÞ¼% 100 ð5Þ (AOAC Of cial Method 965.33, 2007). Oil samples (0.5 g) were dis- Δindex in control solved with 10 mL of trichloromethane. Then, 15 mL of acetic acid and 1 mL of a saturated aqueous solution of potassium iodide were added. where “Δ index in sample” was the increase of the value of the concrete The sample was slightly agitated for 1 min and kept for 5 min in the index in the sample, and “Δ index in control” was the increase of the dark. Once the incubation was finished, 75 mL of distilled water were value of the same index in the control. added and the sample was vigorously shaken. Finally, liberated iodine was titrated with sodium thiosulfate 0.01 N in an automatic titrator. 2.10. Analysis of volatile compounds

The PV, expressed as milliequivalents O2/kg, was calculated according to the formula: Extraction of volatile compounds was performed using solid-phase microextraction (SPME). A SPME device (Supelco, Bellefonte, PA, USA) V N 1000 containing a fused-silica fibre (10 mm length) coated with a 50/30 μm PV ¼ ð1Þ W thickness of divinylbenzene/carboxen/polydimethylsiloxane (DVB/ CAR/PDMS) was used. For headspace SPME (HS-SPME) extraction, where, V is the volume (mL) of sodium thiosulfate consumed in the ti- 0.36 g of each sample was weighed into a sample vial. The fibre, previ- tration, N is the normality of the sodium thiosulfate solution and W is ously conditioned by heating in a gas chromatograph injection port at the weight of the sample (g). 270 °C for 60 min, was inserted into the sample vial and then exposed to the headspace. Extractions were carried out in an oven at 60 °C for 2.6. Determination of p-anisidine value (AV) 45 min, after sample equilibration for 15 min at the extraction temper- ature, ensuring a homogeneous temperature for sample and headspace. Determination of AV of the oil samples was performed following an Once sampling was finished, the fibre was withdrawn into the needle IUPAC method (International Union of Pure and Applied Chemistry, and transferred to the injection port of the gas chromatograph–mass IUPAC, 1987). Oil samples (0.5–2 g) were dissolved in isooctane in a spectrometer (GC–MS) system. 1098 R. Agregán et al. / Food Research International 99 (2017) 1095–1102

A gas chromatograph 6890 N (Agilent Technologies, Santa Clara, CA, The DPPH radical scavenging activity for the BBE was 47.53 μmol of USA) equipped with a mass selective detector 5973 N (Agilent Technol- Trolox equivalents (TE)/g extract. For DPPH, the determination of the ogies) was used with a DB-624 capillary column of 30 m × 0.25 mm id, EC50 was not possible, since no concentration of BBE was potent enough 1.4 μm film thickness (J&W Scientific, Folsom, CA, USA). The SPME fibre to scavenge N50% of the DPPH radical. BBE only scavenged DPPH radi- was desorbed and maintained in the injection port at 260 °C during cals up to 23.58% at 7.5 g/L. Similar DPPH inhibition percentages were 8 min. The sample was injected in splitless mode. Helium was used as found for methanolic BBE at level of 2 g/L (Chernane et al., 2014). This a carrier gas with a linear velocity of 40 cm/s. The temperature program fact indicates that our extract is 3 times less efficient. When comparing was firstly isothermal for 10 min at 40 °C, raised to 200 °C at a rate of with other species, a similar value (25.6%) of DPPH inhibition was re- 5 °C/min, then raised to 250 °C at a rate of 20 °C/min, and finally held ported with A. nodosum extracts at 5 g/L obtained with 60% methanol for 5 min (total run time of 49.5 min). Injector and detector tempera- (O'Sullivan et al., 2011). For the BHT compound, the DPPH radical scav- tures were both set at 260 °C. The mass spectra were obtained using a enging activity was 976.04 μmol TE/g and the EC50 value was 0.95 g/L. mass selective detector working in electronic impact at 70 eV, with a On the other hand, when ABTS test was used the antioxidant activi- multiplier voltage of 1953 V and collecting data at a rate of 6.34 scans/ ties determined were 728.29 μmol TE/g and 5105.03 μmol TE/g for BBE s over the range m/z 40–300. Compounds were identified by comparing and BHT, respectively. The ABTS EC50 values were 2.78 g/L and 0.28 g/L their mass spectra with those contained in the NIST05 (National Insti- for BBE and BHT, respectively. Regarding ABTS test, to the best of our tute of Standards and Technology, Gaithersburg, MD, USA) library knowledge, we did not find data of B. bifurcata in the literature to (N80% of coincidence) and/or by calculation of retention index relative make comparisons. to a series of standard alkanes (C5–C14) for calculating Kovats indexes The FRAP assay measures the capacity of a compound to reduce a (Supelco) and matching them with data reported in the literature. Re- ferric oxidant into a ferrous complex by electron transfer (O'Sullivan sults for each volatile compound in each sample were the mean value et al., 2011). Our results indicate a reducing capacity for BBE but a low of three replicates. activity with only 26.93 μmol TE/g extract. Indeed, Boisvert et al. (2015) reported values of 22.3 μmol TE/g extract in S. longicruris, ex- 2.11. Statistical analysis tracted using 100% ethanol by pressurized liquid procedure. However, because of different extraction conditions a direct comparison with All statistical analysis was performed using the IBM SPSS Statistics other studies is not feasible. In the present study, the antioxidant activ- 19 software (IBM, Chicago, IL, USA). After verification of normal distri- ity of BHT measured using the FRAP assay was 578.50 μmol TE/g. bution and constant variance of data, significant differences were deter- The ORAC assay is a standardized method based on the scavenging of mined using one-way analysis of variance (ANOVA). A Duncan's test peroxyl radicals generated by 2, 20-azobis (2-methyl propionamidine) was performed to compare the mean values for oxidation time and con- dihydrochloride (AAPH). Our results indicated a considerable peroxyl centration extract at a significance level of 95% (P b 0.05). Correlations radical scavenging activity of the BBE with 979 μmol TE/g extract, between variables were determined by correlation analyses using the while the value determined for the BHT was 134.24 μmol TE/g. Alves Pearson's linear correlation coefficient with the above statistical soft- et al. (2016) evaluated the peroxyl radical scavenging activity by ware package mentioned. ORAC test in the same seaweed than the present study using methanol and dichloromethane with values of 3151 and 589 μmol TE/g extract, re- 3. Result and discussion spectively. Once again, the results obtained using different solvents are hardly comparable together. The ORAC assay is more biologically rele- 3.1. Total phenolic content of BBE and in vitro antioxidant capacity of BBE vant than DPPH and it has been checked to be especially useful for and BHT crude plant extracts where different compounds coexist and complex reaction mechanisms are involved (Bentayeb, Vera, Rubio, & Nerin, The total solid content (TS) of the Bifurcaria bifurcata aqueous ex- 2014). tract was 0.75%. Boisvert, Beaulieu, Bonnet, and Pelletier (2015) report- When comparing the in vitro antioxidant activity of BBE with that of ed lower values for TS in Ulva lactuca extract (0.23%) using pure ethanol BHT, we can observe that the relation between the antioxidant power of as solvent. However, Chernane et al. (2014) noticed an extraction yield the two substances varied according to the method used for measure- for Bifurcaria bifurcata of 10.85% using methanol as solvent. These differ- ment. In general, the activity of the BHT was higher than that of BBE, ences in extraction yield among seaweeds could be explained by varia- being 7 times higher when measured following the ABTS radical tions in the chemical composition of raw material and in polarity of decoloration method and around 20 times higher when measured by solvents used. For the first trait, Wang, Jonsdottir, and Olafsdóttir using the FRAP or the DPPH free radical scavenging method. However, (2009) observed large differences in extraction yield among different when used the ORAC assay, the antioxidant activity was 7 times higher seaweed species (green red and brown algae). For the second trait of for BBE than for BHT. This apparent discrepancy could be due to the dif- variation, in several studies where the solvents used were water and ferent chemical basis of the different methods of measurement. In fact, ethanol, the highest extraction yields were always reported for water results reported in literature show a different trend in the values obtain- (Farvin & Jacobsen, 2013; Tierney et al., 2013). Indeed, water has ed when different methods of measurement of the antioxidant activity more ability to extract water-soluble components such as polysaccha- were applied to concrete samples (Craft, Kerrihard, Amarowicz, & rides, proteins and peptides which are the main seaweed components. Pegg, 2012). Note also that BHT is a single compound, whereas BBE is The TPC of BBE was 19.9 mg phloroglucinol equivalents (PGE)/g ex- composed by a mix of compounds having different nature, and complex tract. This result was lower than those reported by Alves et al. (2016) relations are stablished among them. who found values of 220 and 68.33 mg PGE/g extract obtained for the same seaweed, using methanol and dichloromethane as solvents. Previ- 3.2. Effect of addition of BBE on the oxidation stability of canola oil ous studies on brown seaweeds have reported the ability of these or- ganisms to produce antioxidant compounds. In this regard, Chernane The assay was performed at 60 °C during 16 days. Due to rapid hy- et al. (2014) working with brown seaweeds (B. bifurcata and Fucus droperoxide decomposition at elevated temperatures (Frankel, 1998), spiralis) and green seaweeds (Enteromorpha intestinalis and Ulva rigida) this time would correspond to a 4 months' time at ambient temperature indicated the highest amount of polyphenols in brown algae than in the (Warner, Frankel, & Mounts, 1989). To evaluate antioxidant effective- green ones. Brown algae have also revealed more antioxidant power ness of BBE and BHT in canola oil, PV, AV, CD and TV were determined than red algae (Lage-Yusty, Alvarado, Farraces-Casais, & López- as lipid oxidation indices. The addition of BBE at different levels to cano- Hernández, 2014). la oil affected all lipid oxidation indices (Fig. 1A to D). PV is used as R. Agregán et al. / Food Research International 99 (2017) 1095–1102 1099

500 80 control A B control 400 BHT-50 BHT-50 60 /kg oil) BHT-200 2 BHT-200 300 BBE-200 BBE-200 40 BBE-400 BBE-400 200 BBE-600 BBE-600

p- Anisidine value 20 100 BBE-800 BBE-800 BBE-1000 BBE-1000 Peroxide value (meq O 0 0 0481216 0481216 Time (days) Time (days) 5 control 3000 control BHT-50 D C 2500 BHT-50 4 BHT-200 BHT-200 2000 BBE-200 3 BBE-200 BBE-400 1500 BBE-400 2 BBE-600

Totox value 1000 BBE-600 Conjugated dienes BBE-800 1 BBE-800 500 BBE-1000

(% of conjugated dienoic acid) BBE-1000 0 0 0481216 0481216 Time (days) Time (days)

Fig. 1. Effect of the addition of Bifurcaria bifurcata aqueous extract (BBE) (200, 400, 600, 800 and 1000 ppm) and BHT (50 and 200 ppm) on the evolution of peroxide value (A), p-anisidine value (B), conjugated dienes (C) and TOTOX value (D) in canola oil stored at 60 °C.

indicator for the primary oxidation, because hydroperoxides are the pri- During lipid oxidation, hydroperoxides, the primary reaction com- mary products of lipid oxidation. They are odorless and colorless, but pounds, decompose to produce secondary oxidation products such as they are labile species that can produce a complex array of secondary ketones, alcohols, and aldehydes, which are more stable during the products. Hence, the PV can be used as oxidation index for the early heating process. These volatile compounds are related to off-flavors stages of lipid oxidation (Zhang et al., 2010). For all samples, the thermal and off-odors of edible oils. In order to confirm results for primary oxi- treatment has produced oxidation in canola oil leading to a significant dation, the simultaneous detection of primary and secondary lipid prod- increase (P b 0.001) with storage time but this effect was markedly re- ucts is necessary. For this AV was also determined in canola oil samples. duced in samples with BHT or BBE (Fig. 1A). The control oil samples This index relates to secondary oxidation products (carbonyls), reached a maximum PV of 381 meq O2/kg oil after 16 days of storage reflecting the magnitude of aldehyde formation in oils (Khan & whereas the PV of the sample containing 1000 ppm of BBE was 108 Shahidi, 2001). Fig. 1B shows the changes recorded in AV during oxida- meq O2/kg oil after this period. These data indicate a higher stability of tion process. As we can observe, the AV is highly influenced by oxidation the oil treated with BBE addition, being the inhibitory effect of BBE time (Kim, Yeo, Kim, Kim, & Lee, 2013) and in fact significant differences against primary oxidation of lipid concentration-dependent, specifically (P b 0.001) were found during storage for all samples. In control sam- in the middle stages of oxidation (8 and 12 days, with regression coeffi- ples, the net formation of carbonyls was higher than in samples with cient of 0.98). However, the highest inhibition percentage (IP) for PV BBE reaching a maximum of 59.2 (Fig. 1B). Addition of BHT and BBE re- was found at early stages of oxidation (4 days) with IP higher than sulted in significant decrease in AV (P b 0.05) with respect to the control 80% for all concentrations assessed, indicated for a lower regression co- at the end of oxidation process. Percentages of inhibition at different efficient of 0.58 (Fig. 2A). Our results are in agreement with data report- storage stages and concentrations are shown in Fig. 2B. At the end of ed previously by Shaker (2006) and show that the antioxidant the oxidation storage, AV of samples with BHT decreased by 62% with compounds of BBE have an important role in inhibiting the initial reac- respect to the control, independently of the concentration employed, tions of lipid oxidation acting as an electron donor or as scavenger of while addition of BBE resulting in various decreases in AV values de- free radicals. At the end of the accelerated oxidation, BBE at 600 ppm pending on the concentration, in the range 55–68%, with regard to con- had an inhibitory effect of primary lipid oxidation comparable to BHT trol sample. As expected, and contrary to the results previously at 200 ppm. This is, a concentration three times higher was needed to indicated for IP in the PV, the highest regression coefficients were ob- obtain the same inhibition than using this chemical additive. Using po- tained in the latest stages of oxidation (12 and 16 days) with values of tato peel extracts, Rehman, Habib, and Shah (2004) needed concentra- 0.89 and 0.88, respectively (Fig. 2B). From these results, it can be in- tions 8–12 times higher than those of synthetic antioxidants to control ferred that polyphenolic compounds found in BBE had a strong inhibito- oil oxidation during storage. On the contrary, Mohdaly, Hassanien, ry effect on the secondary lipid oxidation. At the end of induced Mahmoud, Sarhan, and Smetanska (2013), have obtained lower PV oxidation process, there was no significant differences (P N 0.05) be- values using 100 and 200 ppm of potato peel extracts than in samples tween canola oil samples treated with BHT and those treated with BBE with 200 ppm of added BHT or BHA. In addition, we can confirm that at levels higher than 400 ppm. This means that BBE at level of BBE did not show pro-oxidative effects during heat oxidation process. 600 ppm provided protection in a similar way to BHT against secondary According to Shaker (2006) in grape peel extract the pro-oxidative ef- lipid oxidation, and BBE at level of 800 or 1000 ppm induced inhibitory fect was proven by increasing the amount of oxidized products with activity higher than BHT, while BBE at level of 200 and 400 ppm had larger heating process. lower strength to inhibit the secondary oxidation than BHT. However 1100 R. Agregán et al. / Food Research International 99 (2017) 1095–1102

80

100 2 2 4 days R =0.58 4 days R = 0.88 8 days 8 days 80 2 60 R =0.98 12 days 2 12 days 2 R =0.93 R = 0.89 60 2 16 days 16 days R =0.08 40

2 R =0.98 40

IP (%) in AV 2 R =0.07 IP (%) in PV 20 20 A B

0 0 0 500 1000 1500 0 500 1000 1500 BBE concentration (ppm) BBE concentration (ppm)

80 100 2 4 days R =0.85 2 R = 0.80 4 days 8 days 80 8 days 2 60 R = 0.93 12 days 12 days 2 R = 0.97 16 days 60 16 days 2 40 R = 0.04 2 R = 0.95 2 R = 0.97 40 IP (%) in CD 20 IP (%) in TV 20 2 R =0.93 C D

0 0 0 500 1000 1500 0 500 1000 1500 BBE concentration (ppm) BBE concentration (ppm)

Fig. 2. Inhibitory effect of the Bifurcaria bifurcata aqueous extract (BBE) concentrations (200, 400, 600, 800 and 1000 ppm) on the peroxide value (A), p-anisidine value (B), conjugated dienes (C) and TOTOX value (D) in canola oil stored at 60 °C.

in both situations there were no significant differences (P N 0.05) be- Fig. 1D depicts the changes in the total oxidation (TOTOX) values tween BHT and BBE samples. (TV). TV represents a deterioration oxidative index, because it accounts The measurement of conjugated dienes is also a good parameter for for both peroxides and aldehydes (Shahidi & Wanasundara, 2002). In the assessment of oil oxidation (Shahidi & Wanasundara, 1997). The general, the kinetic trends were similar to those obtained in PV produc- formation of conjugated dienes has been associated to the oxidation of tion. There was also a marked increase in TV after 12 days. After this pe- PUFA (Kim et al., 2013; Roman, Heyd, Broyart, Castillo, & Maillard, riod, TV increased significantly (P b 0.05) faster in control samples than 2013). Indeed, the PUFA oxidation occurs with the formation of hydro- in samples treated with BHT or BBE. The highest inhibition percentage peroxides, and once peroxides have been formed, the non-conjugated (71.3%) was achieved by BBE at 1000 ppm (Fig. 2D). At that same sam- double bonds present in unsaturated lipids suffer a rearrangement gen- pling point, a 66.2% of inhibition was obtained with BHT at 200 ppm. Ex- erating conjugated dienes, which specifically absorb at 232 nm (Gertz, cept for 4 days, the regression coefficients were higher than 0.92 for the Klostermann, & Kochhar, 2000). Several authors have employed CD ab- different stages of the oxidation process. The highest values for IP sorbance to evaluate the antioxidant activity of different extracts, such (N80%) were obtained at early stages (4 days), mainly due to the weight as from garlic (Chatha, Anwar, Manzoor, & Bajwa, 2006)orMoringa of the PV parameter in the formula used to obtain TV (Fig. 2D). oleifera (Siddiq, Anwar, Manzoor, & Fatima, 2005). As we can observe in Fig. 1C the absorbance values of CD were grad- 3.3. Effect of addition of BBE on the volatile compound (VC) profile of canola ually increased (P b 0.001) with the increase in the oxidation time. As in oil the case of hydroperoxides, based on information in Fig. 1C, one can as- sume that the rate of CD formation was higher than the decomposition During canola oil oxidation several VC responsible for off-odors and rate, leading to the accumulation in the oil samples for all cases. The flavors are produced. These compounds such as alcohols, aldehydes, ke- values reported for CD are a measure of lipid alteration due to double tones and esters, represent typical secondary oxidation products bonds conjugation because of primary oxidation. Indeed, a highly posi- resulting from auto-oxidation of oleic, linoleic and linolenic acids tive correlation between CD and PV was observed (r = 0.93; P b 0.01, (Frankel, Hu, & Tappel, 1989), resulting their presence in a loss in palat- n = 120) which is in agreement with previous results reported by ability and nutritional value that are penalized by consumers and have other authors in soybean oil (Kim et al., 2013). By oil supplementation large economic consequences for oil manufacturers. with BHT and BBE, the accumulation of CD decreased in a significant In total, 7 VC were identified on the basis of mass spectra analysis for way for all treatments (P b 0.05), obtaining with the highest BBE level, all treatments and in all stages. These VC were four aldehydes (hexanal, the lowest amount of CD (1.08, Fig. 1C). The canola oil samples with heptanal, octanal and 2-octenal), one alkane (hexane), one alkene (1- the highest dose of BBE had the lowest amount of CD in all stages of Octen-3-ol) and one benzene (p-Xylene). The aldehydes were the heating oxidation process. At a level of 600 ppm of BBE we obtained major group of identified compounds and also the most interesting the same effectiveness in reducing CD values than adding BHT at lipid-derived VC because they can produce a wide range of flavors and 200 ppm. As happened with PV, the inhibitory effect of BBE on CD for- odors (Shahidi, Rubin, & D'Souza, 1986). Their contents in canola oil mation was concentration-dependent (Fig. 2C). As we can observe, at samples with different treatments are depicted in Fig. 3A to D. Indeed, the end of treatment the level of 1000 ppm reduced CD accumulation aldehydes are usually associated to oxidation in vegetable oils (Issaoui by 72.78% with respect to control samples. et al., 2009). For all aldehydes, at the end of the period of oxidation R. Agregán et al. / Food Research International 99 (2017) 1095–1102 1101 B 800 A 60 Control Control A BHT-50 A BHT-50 600 BHT-200 BHT-200 40 BBE-200 BBE-200 400 BBE-400 BBE-400 BBE-600 BBE-600 20 200 BBE-800 BBE-800 Heptanal (Area Units) Hexanal (Area Unit) BBE-1000 B BBB BBB B BBB BBB BBE-1000

0 0

Control BHT-50 Control BHT-50 BHT-200 BBE-200 BBE-600 BBE-800 BHT-200 BBE-200 BBE-600 BBE-800 BBE - 400 BBE-1000 BBE - 400 BBE-1000

60 C 400 D Control Control BHT-50 A BHT-50 BHT-200 300 40 BHT-200 BBE-200 A BBE-200 BBE-400 200 BBE-400 BBE-600 20 BBE-600 BBE-800 100 BBE-800 Octenal (Area Units) B BBB BBB BBE-1000 2 - Octenal (Area Units) B BBB BBB BBE-1000 0 0

Control BHT-50 BHT-200 BBE-200 BBE-600 BBE-800 Control BHT-50 BBE - 400 BBE-1000 BHT-200 BBE-200 BBE-600 BBE-800 BBE - 400 BBE-1000

Fig. 3. Effect of the addition of Bifurcaria bifurcata aqueous extract (BBE) (200, 400, 600, 800 and 1000 ppm) and BHT (50 and 200 ppm) on the content of hexanal (A), heptanal (B), octanal (C) and 2-Octenal (D) in canola oil after 16 days of storage at 60 °C. process, differences between the control and the other treatments were (Ourense, Spain) for supplying the canola oil samples used in this statistically significant (P b 0.05). Specifically, hexanal (Fig. 3A) at the research. end of oxidation time reached N600 UA in control samples, while sam- ples containing antioxidants showed lower contents of this aldehyde. References Within BBE, inhibition percentages of hexanal production increased with increasing the extract's concentration, reaching inhibition percent- Alves, C., Pinteus, S., Simões, T., Horta, A., Silva, J., Tecelão, C., & Pedrosa, R. (2016). ages of 94, 95 and 96% for BBE at 400, 800 and 1000 ppm, respectively. Bifurcaria bifurcata: A key macro-alga as a source of bioactive compounds and func- tional ingredients. International Journal of Food Science and Technology, 51, Canola oil with BHT exhibited percentage of inhibition of 93 and 95% for 1638–1646. 50 and 200 ppm, respectively. For the other three aldehydes (Fig. 3Bto AOAC Official Method 965.33 (2007). Peroxide value of oils and fats. In official methods of D) the highest inhibition percentages were always reached in oil with analysis (17th ed.)Gaithersburg, Maryland (USA): Association of Official Analytical BHT at 200 ppm with values of 94%. From these results, it is clear that Chemist. Bentayeb, K., Vera, P., Rubio, C., & Nerin, C. (2014). The additive properties of oxygen rad- BBE was effective in the control of the production of volatile substances ical absorbance capacity (ORAC) assay: The case of essential oils. Food Chemistry, 148, responsible for off-odors and off-flavors. 204–208. Benzie, I. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: The FRAP assay. Analytical Biochemistry, 239,70–76. 4. Conclusions Boisvert, C., Beaulieu, L., Bonnet, C., & Pelletier, É. (2015). Assessment of the antioxidant and antibacterial activities of three species of edible seaweeds. Journal of Food The results of the present study indicate that canola oil oxidation can Biochemistry, 39,377–387. Brand-Williams, W., Cuvelier, E., & Berset, C. (1995). Use of a free radical method to eval- be reduced with BBE from aqueous extraction process. Indeed, the ef- uate antioxidant activity. Lebensmittel-Wissenschaft und Technologie, 28,25–30. fects of BBE in stabilizing canola oil were comparable to those of BHT, Chatha, S. A. S., Anwar, F., Manzoor, M., & Bajwa, J. R. (2006). Evaluation of the antioxidant using only a dose three times higher. BBE had a strong antioxidant activ- activity of rice bran extracts using different antioxidant assays. Grasas y Aceites, 57, – fi 328 335. ity both in initial and nal steps of the lipid oxidation, and thereby it can Chernane, H., Mansori, M., Latique, S., & El Kaoua, M. (2014). Evaluation of antioxidant ca- be used safely in food industries as a natural antioxidant instead of syn- pacity of methanol extract and its solvent fractions obtained from four Moroccan thetic antioxidants. Results of this study allow us to think that BBE could macro algae species. European ScientificJournal, 10,35–48. Craft, B. D., Kerrihard, A. L., Amarowicz, R., & Pegg, R. B. (2012). Phenol-based antioxidants be used as a potent source for the stabilization of more complex food and the in vitro methods used for their assessment. Comprehensive Reviews in Food systems. However, further research studies are necessary to evaluate Science and Food Safety, 11,148–173. the BBE antioxidant activity over a wider range of food matrices, such Dávalos, A., Bartolomé, B., Suberviola, J., & Gómez-Cordovés, C. (2003). ORAC-fluorescein as meat products. as model for evaluating antioxidant activity of wines. Polish Journal of Food and Nutrition Science, 12,133–136. Farvin, K. H. S., & Jacobsen, C. (2013). Phenolic compounds and antioxidant activities of Acknowledgements selected species of seaweeds from Danish coast. Food Chemistry, 138,1670–1681. Frankel, E. N. (1998). Antioxidants: Lipid oxidation. Dundee, Scotland: The Oily Press, 1998. Frankel, E. N., Hu, M. L., & Tappel, A. L. (1989). Rapid headspace gas chromatography of The authors thank INIA (Instituto Nacional de Investigación y hexanal as a measure of lipid peroxidation in biological samples. Lipids, 24,976–981. Tecnología Agraria y Alimentaria, Spain) for granting Ruben Agregán Gertz, C., Klostermann, S., & Kochhar, S. P. (2000). Testing and comparing oxidative stabil- with a predoctoral scholarship (CPR2014-0128). The authors also ity of vegetable oils and fats at frying temperature. European Journal of Lipid Science – fi and Technology, 102,543 551. thank Council for Scienti c and Technological Development (Brazil) Heo, S. J., Ko, S. C., Cha, S. H., Kang, D. H., Park, H. S., & Choi, Y. U. (2009). Effect of (CNPq no. 248705/2013-0) for financial support, and Aceites Abril, S.L. phlorotanins isolated from Ecklonia cava on melanogenesis and their protective 1102 R. Agregán et al. / Food Research International 99 (2017) 1095–1102

effect against photooxidative stress induced by UV-B radiation. Toxicology In Vitro, 23, Ou, B., Hampsch-Woodill, M., & Prior, R. L. (2001). Development and validation of an im- 1123–1130. proved oxygen radical absorbance capacity assay using fluorescein as the fluorescent Hu, C. C., Lin, J. T., Lu, F. J., Chou, F. P., & Yang, D. J. (2008). Determination of carotenoids in probe. Journal of Agricultural and Food Chemistry, 49, 4619–4626. Danaliella salina cultivated in Taiwan and antioxidant capacity of the algal carotenoid Pateiro, M., Lorenzo, J. M., Amado, I. R., & Franco, D. (2014). Effect of addition of green tea, extract. Food Chemistry, 109,439–446. chestnut and grape extract on the shelf-life of pig liver pate. Food Chemistry, 147, International Union of Pure and Applied Chemistry (IUPAC) (1987,). Standard methods for 386–394. the analysis of oils, fats and derivatives, 7th revised and enlarged edited by C. Hautfenne, Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Anti- Blackwell Scientific, London: Paquot and A. oxidant activity applying an improved ABTS radical cation decolorization assay. Free Issaoui, M., Hassine, K. B., Flamini, G., Brahmi, F., Chehab, H., Aouni, Y., ... Hammami, M. Radical Biology & Medicine, 26,1231–1237. (2009). Discrimination of some Tunisian olive oils varieties according to their oxida- Rehman, Z. U., Habib, F., & Shah, W. H. (2004). Utilization of potato peels extract as a nat- tive stability, volatiles compounds and chemometric analysis. Journal of Food Lipids, ural antioxidant in soybean oil. Food Chemistry, 85,215–220. 16,164–186. Roman, O., Heyd, B., Broyart, B., Castillo, R., & Maillard, M. N. (2013). Oxidative reactivity Khan, M. A., & Shahidi, F. (2001). Effects of natural and synthetic antioxidants on the ox- of unsaturated fatty acids from sunflower, high oleic sunflower and rapeseed oils idative stability of borage and evening primrose triacylglycerols. Food Chemistry, 75, subjected to heat treatment, under controlled conditions. LWT- Food Science and 431–437. Technology, 52,49–59. Kim, T. S., Yeo, J., Kim, J. Y., Kim, M. J., & Lee, J. (2013). Determination of the degree of ox- Shahidi, F., & Wanasundara, U. N. (1997). Measurement of lipid oxidation and evalutation idation in highly-oxidised lipids using profile changes of fatty acids. Food Chemistry, of antioxidant activity. In F. Shahidi (Ed.), Natural antioxidants: Chemistry, health ef- 138, 1792–1799. fects, and applications (pp. 379–396). Illinois: AOCS Press. Koivikko, R., Loponen, J., Pihlaja, K., & Journalainen, V. (2007). High performance liquid Shahidi, F., & Wanasundara, U. N. (2002). Method for measuring oxidative rancidity in fats chromatographic analysis of phlorotanins from the brown alga Fucus vesiculosus. and oils. In C. C. Akoh, & D. B. Min (Eds.), Food lipids (pp. 465). New York: Marcel Phytochemical Analysis, 18,326–332. Dekker. Lage-Yusty, M. A., Alvarado, G., Farraces-Casais, P., & López-Hernández, J. (2014). Modifi- Shahidi, F., Rubin, L. J., & D'Souza, L. A. (1986). Meat flavour volatiles: A review of the com- cation of bioactive compounds in dried edible seaweeds. International Journal of Food position, technique of analysis and sensory evaluation. Critical Reviews in Food Science Science and Technology, 49,298–304. and Nutrition, 24,219–227. Le Lann, K., Rumin, J., Cerantola, S., Culioli, G., & Stiger-Pouvreau, V. (2014). Spatiotempo- Shaker, E. S. (2006). Antioxidative effect of extracts from red grape seed and peel on lipid ral variations of diterpene production in the brown macroalga Bifurcaria bifurcata oxidation in oils of sunflower. LWT- Food Science and Technology, 39,883–892. from the western coasts of Brittany (France). Journal of Applied Phycology, 26, Siddiq, A., Anwar, F., Manzoor, M., & Fatima, M. (2005). Antioxidant activity of different 1207–1214. solvent extracts of Molinga oleifera leaves under accelerated storage conditions of Lorenzo, J. M., González-Rodríguez, R. M., Sánchez, M., Amado, I. R., & Franco, D. (2013). sunflower oil. Asian Journal of Plant Sciences, 4,630–635. Effects of natural (grape seed and chestnut extract) and synthetic antioxidants Singleton, V. L., Orthofer, R., & Lamuela-Raventós, R. M. (1999). Analysis of total phenols (butylatedhydroxytoluene, BHT) on the physical, chemical, microbiological and sen- and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. sory characteristics of dry cured sausage “chorizo”. Food Research International, 54, In L. Packer (Ed.), Methods in enzymology, oxidants and antioxidants. Vol. 299. (pp. 611–620. 152–178). San Diego, CA: Academic Press. Lorenzo, J. M., Sineiro, J., Amado, I. R., & Franco, D. (2014). Influence of natural extracts on Tierney, M. S., Smyth, T. J., Hayes, M., Soler-Vila, A., Croft, A. K., & Brunton, N. (2013). In- the shelf life of modified atmosphere-packaged pork patties. Meat Science, 96, fluence of pressurised liquid extraction and solid–liquid extraction methods on the 526–534. phenolic content and antioxidant activities of Irish macroalgae. International Journal Mohdaly, A. A. A., Hassanien, M. F. R., Mahmoud, A., Sarhan, M. A., & Smetanska, I. (2013). of Food Science and Technology, 48,860–869. Phenolic extracted from potato, sugar beet, and sesame processing by- products. Wang, T., Jonsdottir, R., & Olafsdóttir, G. (2009). Total phenolic compounds, radical scav- International Journal of Food Properties, 16, 1148–1168. enging and metal chelation of extracts from Icelandic seaweeds. Food Chemistry, 116, Moure, A., Cruz, J. M., Franco, D., Domínguez, J. M., Sineiro, J., Domínguez, H., et al. (2001). 240–248. Natural antioxidants from residual sources. Food Chemistry, 72,145–171. Warner, K., Frankel, E. N., & Mounts, T. L. (1989). Flavor and oxidative stability of soybean, Muñoz, J., Culioli, G., & Kock, M. (2013). Linear diterpenes from the marine brown alga sunflower and low erucic acid rapeseed oils. Journal of the American Oil Chemists' Bifurcaria bifurcata: A chemical perspective. Phytochemistry Reviews, 12,407–424. Society, 66,558–564. Murray, P. M., Moane, S., Collins, C., et al. (2013). Sustainable production of biologically Zhang, Y., Yang, L., Zu, Y., Chen, X., Wang, F., & Liu, F. (2010). Oxidative stability of sun- active molecules of marine based origin. New Biotechnology, 30,839–850. flower oil supplemented with carnosic acid compared with synthetic antioxidants O'Sullivan, A. M., O'Callaghan, Y. C., O'Grady, M. N., Quguineur, B., Hanniffy, D., Troy, D. J., ... during accelerated storage. Food Chemistry, 118,656–662. O'Brien, N. M. (2011). In vitro and cellular antioxidant activities of seaweed extracts prepared from five brown seaweeds harvested in spring from the west coast of Ire- land. Food Chemistry, 126,1064–1070. Food Research International 99 (2017) 979–985

Contents lists available at ScienceDirect

Food Research International

journal homepage: www.elsevier.com/locate/foodres

Phenolic compounds from three brown seaweed species using LC-DAD–ESI-MS/MS

Rubén Agregán a, Paulo E.S. Munekata b, Daniel Franco a, Ruben Dominguez a, Javier Carballo c,JoséM.Lorenzoa,⁎ a Centro Tecnológico de la Carne de Galicia, Adva. Galicia n° 4, Parque Tecnológico de Galicia, San Cibrao das Viñas, 32900 Ourense, Spain b Department of Food Engineering, Faculty of Animal Science and Food Engineering, University of São Paulo, 225 Duque de Caxias Norte Ave, Jardim Elite, 13.635-900 Pirassununga, São Paulo, Brazil c Area de Tecnologia de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain article info abstract

Article history: The phenolic compounds of extracts from Ascophyllum nodosum (ANE), Bifurcaria bifurcata (BBE) and Fucus Received 13 July 2016 vesiculosus (FVE) from Galicia (NW Spain) were analyzed by liquid chromatography-diode array detection Received in revised form 22 March 2017 coupled to negative electrospray ionization-tandem mass spectrometry (LC-DAD–ESI-MS/MS) with the interest Accepted 23 March 2017 to evaluate their potential application as functional ingredients. Phlorotannins were tentatively identified as the Available online 24 March 2017 main phenolic compounds in the three extracts, followed by phenolic acids, and flavonoids. Fuhalols were pres- ent in ANE and BBE, while hydroxyfuhalols were identified in BBE and FVE. Eckol derivatives were present in the Keywords: fi Ascophyllum nodosum three extracts. Quinic acid derivatives were tentatively identi ed in the three seaweed species; in addition, ANE Bifurcaria bifurcata showed specifically hydroxybenzoic and rosmarinic acid derivatives, BBE showed rosmarinic acid, and FVE Fucus vesiculosus contained p-coumaric and ferulic acid derivatives. Regarding flavonoids, acacetin derivatives were tentatively Phenolic profile identified in the three extracts, hispidulin and a gallocatechin derivative were specifically detected in ANE, and Phlorotannins cypellocarpin C was present in BBE. In conclusion, all brown seaweed extracts studied could be exploited as Marine polyphenols sources of antioxidant phenolic compounds with potential applications in the food and health sectors. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction The chemical composition of brown seaweeds is still under constant investigation and recent few studies have elucidated, at Seaweeds are consumed as part of traditional food in Asian countries least in part, the phenolic composition of this seaweed family. The and more recently they have been included in the diet of Western coun- phenolic composition of brown seaweeds can be largely composed tries due to their contents of bioactive compounds such as polysaccha- by phlorotannins that are oligomers of phloroglucinol (1,3,5- rides (including dietary fiber), minerals, vitamins (B12, C and E), trihydroxybenzene). The oligomer phlorotannins can be classified polyphenols and carotenoids (Burtin, 2003; Garcia-Vaquero & Hayes, according to the intermolecular linkage of phloroglucinol units in 3 2016; Gómez-Ordóñez, Jiménez-Escrig, & Rupérez, 2010). Brown sea- major groups that are known as fucols (phenyl/aryl linkages), weeds (phaeophyta, e.g. Ascophyllum nodosum, Himanthalia elongata, phlorethols (arylether bonds) and fucophlorethols (both types of Bifurcaria bifurcata, Fucus vesiculosus and Laminaria saccharina) are par- linkages) (Burtin, 2003; Martínez & Castañeda, 2013). ticular interesting sources of bioactive compounds such as soluble fiber, Phlorotannins are synthetized via the acetate-malonate pathway iodine and proteins (Garcia-Vaquero, Lopez-Alonso & Hayes, 2017). and stored in physodes (vesicles). They are the most researched group This group is also an interesting source of carotenoids since fucoxanthin of polyphenols in seaweeds. Their highest content is reported in is the compound associated with their characteristic yellowish or brown seaweeds, wherein it ranges from 5 to 15% of the dried weight brownish color. Brown algae bioactive compounds are also associated (Burtin, 2003). These tannins are highly hydrophilic with wide range with health benefits as anti-viral, anti-tumoral and anti-cancer activity of molecular sizes from 100 to more than 100,000 Da depending on (Gupta & Abu-Ghannam, 2011; Michalak & Chojnacka, 2015; the species as reported in previous studies (Arnold & Targett, 2000; Garcia-Vaquero, Rajauria, O'Doherty & Sweeney, 2017). Ferreres et al., 2012). In contrast to the terrestrial phenolic compounds (e.g. flavanols, flavones and phenolic acids), little knowledge exists about the potential health benefits and technological applications of ⁎ Corresponding author. marine polyphenols (phloroglucinol and phlorotannins) (Steevensz et E-mail address: [email protected] (J.M. Lorenzo). al., 2012).

http://dx.doi.org/10.1016/j.foodres.2017.03.043 0963-9969/© 2017 Elsevier Ltd. All rights reserved. 980 R. Agregán et al. / Food Research International 99 (2017) 979–985

Seaweeds contain complex mixtures of phenolic components, and flow rate 10 L/min, fragmentor voltage 135 V, and capillary voltage liquid chromatography-mass spectrometry (LC-MS/MS) has been dem- 4500 V. Full mass scan spectra were recorded in negative ionization onstrated to be a powerful analytical tool for rapid analysis of these mode over the range of m/z 100–1600 Da (5 scan/s). The Agilent polar, nonvolatile, and thermally labile constituents (Hossain, Rai, MassHunter Qualitative Analysis B.04.00 software (Agilent Technolo- Brunton, Martin-Diana, & Barry-Ryan, 2010). gies, Inc., Santa Clara, CA, USA) was used for data acquisition and quali- Thus, the aim of this study was to characterize the phenolic com- tative analysis. pounds of Ascophyllum nodosum, Bifurcaria bifurcata and Fucus vesiculosus extracts using liquid chromatography-diode array detection 3. Results and discussion coupled to negative electrospray ionization-tandem mass spectrometry (LC-DAD–ESI-MS/MS). This will give information on their potentiality 3.1. Extraction of the phenolic compounds as sources of natural antioxidants to be later used as functional ingredi- ents in the food industry. The stability of phenolic compounds in brown algae extracts and ex- traction conditions may influence the extraction of phenolic com- 2. Material and methods pounds from seaweeds. Phenolic compounds in algae seem to be particularly sensible to heating and UV radiation exposure as Le Lann, 2.1. Seaweed material and extraction procedure Jégou, and Stiger-Pouvreau (2008) pointed out. These authors reported significant reduction of phenolic content and antioxidant activity due to The brown seaweeds used in the present study were kindly supplied oven-drying (4 h at 50–60 °C) or greenhouse-drying (72 h at 15–30 °C) by Porto-muiños company (A Coruña, Spain). Three different samples in Sargassum muticum and Bifurcaria bifurcata extracts. from each seaweed species were collected in the months of August Regarding extraction conditions, an aqueous mixture of solvents and September in the Atlantic Ocean, at three different places in the may provide better yields of extraction than using only water as solvent. area of Camariñas (A Coruña, Spain). To obtain the Ascophyllum This outcome was reported by Koivikko, Loponen, Honkanen, and nodosum (ANE), Bifurcaria bifurcata (BBE) and Fucus vesiculosus (FVE) Jormalainen (2005) who evaluated several pure solvents and mixtures extracts, seaweeds were dried in a conventional oven at 40 °C and and they observed the highest extraction yield for acetone:water then they were grounded to obtain a particle size less than 500 μm. Ex- (70:30, v/v) compared to other aqueous mixtures and organic solvents traction was performed in a magnetic stirrer, using water as a solvent in in Fucus vesiculosus. Such considerations may explain, at least in part, a liquid/solid ratio of 30 g/g for 5 min to obtain a correct hydration. Then the reduced number of compounds tentatively identified in the extracts an aliquot of 800 mL from algae/water suspension was sonicated for obtained in the present study. Nevertheless, and despite of this, current- 10 min in an ultrasonic Hielscher UIP1000HD homogenizer (Hielscher ly water is preferred instead organic solvents to obtain extracts to be Ultrasonics GmbH, Teltow, Germany) equipped with a flow cell with a used in formulation of foods, due to economic, toxicological, and envi- residence time of 30 s and 90 percentage of amplitude and with contin- ronmental reasons. uous recirculation. The process was stopped if the temperature reached a value higher than 40 °C. Finally the extract was centrifuged at 2000 ×g 3.2. Tentative identification of phenolic compounds from seaweed extracts and filtered through a cellulose filter of 20–25 μm pore size (Filter-lab, Filtros Anoia, S.A., Barcelona, Spain). The evaluation of ANE composition by HPLC-DAD-ESI-MS (Fig. 1) revealed the presence of 22 peaks (named from A1 to A22). The 2.2. Phenolic profile by LC-DAD–ESI-MS/MS evaluation of BBE composition (Fig. 2) revealed the presence of 18 peaks (B1 to B18), and evaluation of the FVE (Fig. 3) showed the Phenolic compounds of seaweed extracts were separated and ana- presence of 19 peaks (C1 to C19). Some of these compounds were lyzed by UV/VIS DAD chromatography. Next, these compounds were tentatively identified and their characteristics are shown in Table 1. evaluated according to their m/z ratio in the ESI mass spectrometer. Fourteen phenolic compounds were tentatively identified from Tentative identification of phenolic compounds was performed by ANE. Phlorotannins (5 compounds, peaks A2, A4, A6, A8 and A9) matching UV/VIS and mass spectral data with already published data and flavonoids (5 compounds, peaks A7, A10, A11, A15 and A22)were in literature or by tentative based on mass spectra and/or UV data. the main groups in ANE, followed by phenolic acids (4 compounds, The analysis of phenolic compounds was carried out in an Agilent peaks A1, A16, A17 and A18). In the BBE extracts, fourteen phenolic com- 1100 HPLC system equipped with G1312B binary gradient pump, pounds were tentatively identified. Most of these compounds belong to G1379A degasser, G1316A column thermostat, G1329A auto-sampler the phlorotannins group (peaks B2, B3, B5, B9, B13, B16, B17 and B18). and G1315C diode array detector (DAD) (Agilent Technologies, Phenolic acids (peaks B1, B12 and B14) and flavonoids (peaks B7, B8 Waldbronn, Germany). Chromatography separation was performed and B11) were also observed. Finally, thirteen phenolic compounds using a Zorbax SB C18 (Agilent Technologies, Inc., Santa Clara, CA, USA) (150 × 3.0 mm I.D., 3.5 μm particle size) column, operated at 25 °C. The mobile phase was composed by acetic acid (2.5%, v/v) in water (solvent A), and methanol containing 2.5% acetic acid (solvent B). The extracts were diluted to obtain 2 mg/mL with mobile phase A. A flow rate of 1.0 mL/min was used with the following gradient program: 0 min 95:5 (A:B, v/v), 15 min 85:15 (A:B, v/v), 35 min 70:30 (A:B, v/v), 40 min 60:40 (A:B, v/v), 50 min 40:60 (A:B, v/v), 55 min 10:90 (A:B, v/v), 55.01 min 0:100 (A:B, v/v), 75 min 0:100 (A:B, v/v). The wavelengths of 240 and 370 nm were used to collect spectral data. When the absorbance at 240 nm and 370 nm exceeded a predetermined value (1% with respect to the baseline), a full spectral data was collected between 190 and 600 nm. The Agilent 6410B triple quadrupole equipped with an electrospray ionization (ESI) source (Agilent Technologies, Inc., Palo Alto, CA, USA) was used for mass spectrometric analysis. ESI conditions were as fol- Fig. 1. Representative chromatogram of phenolic compounds from Ascophyllum nodosum lows: temperature 350 °C, nebulizer pressure 35 psi, N2 drying gas extract (ANE) obtained by liquid chromatography-diode array detection (LC-DAD). R. Agregán et al. / Food Research International 99 (2017) 979–985 981

displayed a similar fragmentation pattern (molecular ion of m/z 777 and characteristics fragments of m/z 529 and 375). The proposed compound for the peak B13 (45.28 min) was a trihydroxyheptafuhalol with parent ion of m/z 933 and characteristic fragment of m/z 914. The presence of several phlorotannin oligomers was reported by Montero et al. (2016) in Sargassum muticum, a brown macroalgae collected on North-Atlantic coast (France, Portugal, Spain, Ireland and Norway). As in the present study, these authors reported the presence of several fuhalols and hydroxyfuhalols in the indicated seaweed species.

Fig. 2. Representative chromatogram of phenolic compounds from Bifurcaria bifurcata 3.2.2. Other phloroglucinol derivatives extract (BBE) obtained by liquid chromatography-diode array detection (LC-DAD). The proposed compound for peak A8 (RT 3.76 min) was a phloroglucinol octamer due to the [M − H]− of m/z 993 and character- istic fragment of m/z 373. This phloroglucinol oligomer was proposed by were tentatively identified in FVE. Phlorotannins was again the main Pantidos, Boath, Lund, Conner, and McDougall (2014) who indicated group (peaks C2, C4, C12, C13, C16, C17 and C18), followed by phenolic that this oligomer could be formed from C-C linked addition of acids (peaks C6, C11 and C15)andflavonoids (peaks C7, C9 and C14). phloroglucinol units to a core C-O-C linked dimer of phloroglucinol. Some other peaks remained not identified and their features are in- Phloroglucinol derivatives were identified in peaks B2 and B3. The dicated in Table 2. compound of peak B2 (RT 1.51 min) displayed molecular ion of m/z In this section, we will comment the characteristics of the com- 401 and product ions at m/z 205 and 125 [phloroglucinol − H]−. pounds positively identified. Another phloroglucinol derivative was observed in peak B3 (RT 2.04 min) that showed precursor ion of m/z 391 and fragment of m/z 125 [phloroglucinol − H]−. 3.2.1. Fuhalols The proposed candidate for peak B5 (2.79 min) was a phloroglucinol The proposed candidate for peak A4 (retention time (RT) 2.21 min) dimer derivative with [M − H]− of m/z 517 and fragment of m/z 247 is a tetrafuhalol with molecular ion of m/z 513 and fragment ion of m/z [phloroglucinol dimer − H]−. A phloroglucinol dimer derivative was 385. Tetrafuhalol was also the suggested compound for the peak B16 also proposed for the C4 peak (RT 2.72 min), due to [M − H]− of m/z (RT 55.43 min) wherein [M − H]− of m/z 513 and fragment of m/z 517 and fragment of m/z 247. This structure is possible due to the core 499 were observed. The proposed candidate for peak A6 (RT C–O–C linkage between two phloroglucinol units (Pantidos et al., 2.77 min) was pentafuhalol due to the parent ion of m/z 637 and char- 2014). This phlorotannin oligomer was reported by Nwosu et al. acteristic product ions of m/z 633, 385 and 247. Similarly, data evalua- (2011) in the composition of Ascophyllum nodosum and it was associat- tion of peak A9 (RT 4.37 min) indicated the presence of another ed with anti-diabetic activity. phlorotannin; trifuhalol was the proposed compound due to the [M Phloroglucinol is an ubiquitous secondary metabolite present in a − H]− of m/z 389 and fragment of m/z 375. free or polymerised state in brown seaweeds and higher plants The proposed candidate for peak B18 (RT 68.18 min) was (Rajauria, Foley, & Abu-Ghannam, 2016). It has a structural backbone hydroxytetrafuhalol due to the molecular ion of m/z 529 and the charac- of 1,3,5-trihydroxybenzene which is the monomeric building unit of teristic product ion of m/z 387. Hydroxytetrafuhalol is also the candidate phlorotannins, the phenolic compounds reported only in brown sea- for peak C12 (RT 45.30 min) since the precursor ion of m/z 529 and char- weeds (Quéguineur et al., 2012). Findings in the present study are in acteristic product ions of m/z 387 and 219 were observed. agreement with those reported by other authors (Lee et al., 2014; The peak B17 (RT 57.62 min) was tentatively identified as Rajauria et al., 2016) who noticed phloroglucinol derivatives in the dihydroxytetrafuhalol that displayed precursor ion of m/z 545 and brown seaweeds Ecklonia cava and Himanthalia elongata. fragment ion of m/z 387. The proposed compound for peak C16 The peaks A2, B9 and C2 were tentatively identified as eckol deriva- (RT 55.43 min) was also dihydroxytetrafuhalol; this compound tives. The compound of peak A2 (RT 1.48 min) was conceivably identi- showed precursor ion of m/z 545 and characteristic fragment of fied as an eckol derivative with molecular ion of m/z 541 and fragments m/z 385. of m/z 401 and 371 [eckol − H]−. Peak B9 (RT 4.7 min) was also identi- The peaks C17 (57.83 min) and C18 (59.22) were tentatively identi- fied as an eckol derivative; the molecular ion was observed with m/z fied as hydroxyhexafuhalol. In peak C17, the parent ion of m/z 777 and 545 and product ion of m/z 371 [eckol − H]−.ForpeakC2 (RT characteristics fragments of m/z 529, 387 and 375 were observed. C18 1.46 min) the suggested compound was an eckol derivative because of the molecular ion of m/z 401 and fragment of 371 ([eckol − H]−). Eckol is the common name of hexahydroxyphenoxydibenzo [1,4] dioxine, from the fucophlorethols group that is characterized by inter- molecular arylether and phenyl linkages between phloroglucinol mo- nomeric units (Martínez & Castañeda, 2013). The presence of eckol in brown seaweeds was previously reported by Kannan, Aderogba, Ndhlala, Stirk, and Van Staden (2013) in Ecklonia maxima (Osbeck) Papenfuss samples. The compound of peak C13 (RT 45.79 min) was tentatively identi- fied as dioxinodehydroeckol derivative, with molecular ion of m/z 463 and fragment of m/z 369. Jung, Hyun, Kim, and Choi (2006) isolated dioxinodehydroeckol and other phlorotannins from Ecklonia stolonifera, a brown algae species commonly found in Eastern and Southern of Korean coast. Dioxinodehydroeckol, also known as eckstolonol, is a Fig. 3. Representative chromatogram of phenolic compounds from Fucus vesiculosus common phlorotannin reported in literature from seaweed species be- extract (FVE) obtained by liquid chromatography-diode array detection (LC-DAD). longing to the Fucales and Laminariales orders (Ferreres et al., 2012). 982 R. Agregán et al. / Food Research International 99 (2017) 979–985

Table 1 Phenolic compounds in Ascophyllum nodosum (ANE), Bifurcaria bifurcata (BBE) and Fucus vesiculosus (FVE) extracts using LC-DAD—ESI-MS/MS.

ID # Proposed compound RT (min) [M − H]− (m/z) Product ions (m/z) UV data (λ, nm) % Ref

Peaks from ANE A1 Hydroxybenzoic acid derivative 1.26 419 343, 201, 137 n.d. 5.74 a A2 Eckol derivative 1.48 541 401, 371 n.d. 41.79 b A4 Tetrafuhalol 2.21 513 385 n.d. 20.16 c A6 Pentafuhalol 2.77 637 633, 385, 247 n.d. 8.55 c A7 Acacetin derivative 3.25 455 327, 283 n.d. 28.04 a A8 Phloroglucinol octamer 3.76 993 373 n.d. 2.36 d A9 Trifuhalol 4.37 389 375 n.d. 0.87 c A10 Hispidulin 4.69 299 n.d. n.d. 32.78 e A11 Acacetin derivative 5.32 657 532, 283 n.d. 3.90 a A15 Acacetin derivative 9.59 900 819, 739, 283 n.d. 3.46 a A16 Quinic acid derivative 43.04 363 249, 191 n.d. 48.04 e A17 Rosmarinic acid derivative 45.32 913 694, 359 n.d. 2.44 a A18 Quinic acid derivative 45.83 957 555, 341, 249, 191 n.d. 8.62 e A22 Gallocatechin derivative 68.16 529 305 n.d. 24.66 f

Peaks from BBE B1 Rosmarinic acid 1.26 359 179 n.d. 29.19 a B2 Phloroglucinol derivative 1.51 401 205, 125 n.d. 100 g B3 Phloroglucinol derivative 2.04 391 125 n.d. 76.44 g B5 Phloroglucinol dimer derivative 2.79 517 247 255, 280 17.85 d B7 Acacetin derivative 3.26 327 283 n.d. 7.00 a B8 Cypellocarpin C 3.98 519 335 n.d. 2.53 h B9 Eckol derivative 4.74 545 371 n.d. 68.82 b B11 Acacetin derivative 10.57 743 429, 283 255, 280 1.24 a B12 Quinic acid derivative 42.99 363 249, 191 255 42.41 e B13 Trihydroxyheptafuhalol 45.28 933 914 255 3.89 c B14 Quinic acid derivative 45.84 957 555, 249, 191 n.d. 12.46 e B16 Tetrafuhalol 55.43 513 499 n.d. 2.48 c B17 Dihydroxytetrafuhalol 57.62 545 387 n.d. 52.19 c B18 Hydroxytetrafuhalol 68.18 529 387 n.d. 22.84 c

Peaks from FVE C2 Eckol derivative 1.46 401 371 n.d. 23.72 b C4 Phloroglucinol dimer derivative 2.72 517 247 280 29.46 d C6 p-Coumaric acid derivative 4.56 299 255, 163 n.d. 2.07 a C7 Acacetin derivative 5.51 322 283 n.d. 3.81 a C9 Acacetin derivative 19.99 947 691, 383, 283 n.d. 1.39 a C11 Quinic acid derivative 42.92 363 249, 191 n.d. 17.87 e C12 Hydroxytetrafuhalol 45.30 529 387, 219 n.d. 1.64 c C13 Dioxinodehydroeckol derivative 45.79 463 369 n.d. 4.15 i C14 Acacetin derivative 51.49 356 283 n.d. 2.51 a C15 Ferulic acid derivative 54.41 375 293, 221, 193 n.d. 3.78 a C16 Dihydroxytetrafuhalol 55.43 545 385 n.d. 1.26 c C17 Hydroxyhexafuhalol 57.83 777 529, 387, 375 n.d. 17.97 c C18 Hydroxyhexafuhalol 59.22 777 529, 375 n.d. 7.94 c

RT: retention time in total ion chromatogram, [M − H]−: molecular ion, MW: molecular weight, n.d.: not determined. The most abundant ions observed in mass spectra are shown in bold. a Tentatively identified based on the mass spectral data cited by Hossain et al. (2010). b Tentatively identified based on the mass spectral data cited by Nakamura, Nagayama, Uchida, and Tanaka (1996). c Tentatively identified based on the mass spectral data cited by Montero et al. (2016). d Tentatively identified based on the mass spectral data cited by Pantidos et al. (2014). e Tentatively identified based on the mass spectral data cited by Nagy et al. (2011). f Tentatively identified based on the mass spectral data cited by de Quirós et al. (2010). g Tentatively identified based on the mass spectral data cited by Orrego-Lagarón, Vallverdú-Queralt, Martínez-Huélamo, Lamuela-Raventos, and Escribano-Ferrer (2016). h Tentatively identified based on the mass spectral data cited by Boulekbache-Makhlouf et al. (2013). i Tentatively identified based on the mass spectral data cited by Jung et al. (2014).

3.2.3. Phenolic acids and phenolic acid derivatives Peak A16 (RT 43.04 min) data suggested a quinic acid derivative as The A1 peak (RT 1.26 min) presented molecular ion of m/z 419 and probable compound due to the parent ion of m/z 363 and fragments of fragments of m/z 343, 201 and 137 [hydroxybenzoic acid − H]−,there- m/z 249 and 191 ([quinic acid − H]−). Another quinic acid derivative fore this compound was tentatively identified as a hydroxybenzoic acid was proposed for peak A18 (RT 45.83 min) wherein [M − H]− of m/z derivative. The presence of hydroxybenzoic acid and other phenolic 957 and product ions of m/z 555, 341, 249, and 191 were observed. acids in seaweeds was reported by Farvin and Jacobsen (2013), who Peak B12 (RT 42.99 min) was also tentatively identified as quinic acid evaluated the phenolic composition of selected algae species, including derivative wherein the precursor ion of m/z 363 and the fragments of brown seaweeds (Dictyota dichotoma and Saccharina latissima)of m/z 249 and 191 ([quinic acid − H]−) were observed. Similarly, another natural occurrence in Danish coast. In addition, Rajauria et al. (2016) quinic acid derivative was suggested for the peak B14 (RT 45.84 min, also detected four peaks corresponding to m-hydroxybenzaldehyde, with [M − H]− of m/z 957 and fragments of m/z 555, 249 and 191). A p-hydroxybenzaldehyde, gallic acid and gallic acid 4-O-glucoside quinic acid derivative was also suggested for peak C11 (RT 42.92 min) which were identified as hydroxybenzoic acid derivatives. wherein the [M − H]− of m/z 363 and fragments of m/z 249 and 191 R. Agregán et al. / Food Research International 99 (2017) 979–985 983

Table 2 Features of the non-identified compounds in Ascophyllum nodosum (ANE), Bifurcaria bifurcata (BBE) and Fucus vesiculosus (FVE) extracts using LC-DAD—ESI-MS/MS.

ID # Proposed compound RT (min) [M − H]− (m/z) Product ions (m/z) UV data (λ, nm) %

Peaks from ANE A3 Unknown 2.04 391 217 n.d. 100 A5 Unknown 2.57 553 276, 196, 135 255, 280 56.01 A12 Unknown 5.59 1153 900, 657, 321, 242 n.d. 2.22 A13 Unknown 6.07 819 738, 657, 253, n.d. 2.04 A14 Unknown 6.56 1315 1144, 981, 819, 657 n.d. 9.60 A19 Unknown 54.41 375 293, 221, 141 n.d. 4.10 A20 Unknown 55.43 779 499, 355, 217 n.d. 8.19 A21 Unknown 57.59 1365 551, 463, 217, 141 n.d. 52.17

Peaks from BBE B4 Unknown 2.21 769 611, 384 n.d. 5.59 B6 Unknown 3.08 325 203 255, 280 4.98 B10 Unknown 5.83 485 231, 151, 107 n.d. 18.14 B15 Unknown 54.41 633 375, 293, 221 n.d. 2.98

Peaks from FVE C1 Unknown 1.26 627 485, 343, 201, 135 n.d. 7.50 C3 Unknown 2.04 611 391, 217 520 100 C5 Unknown 4.30 699 536, 341, 255 n.d. 1.06 C8 Unknown 19.38 533 403, 255, 113 n.d. 4.93 C10 Unknown 40.25 587 507, 365, 217 280 2.40 C19 Unknown 68.04 529 381 n.d. 18.39

RT: retention time in total ion chromatogram, [M − H]−: molecular ion, MW: molecular weight, n.d.: not determined. The most abundant ions observed in mass spectra are shown in bold.

([quinic acid − H]−) were observed. This phenolic acid was reported by Nagy et al. (2011) in the composition of oregano (Folium origani cretici) Nagy, Solar, Sontag, and Koenig (2011) who assessed the phenolic com- subjected to irradiation at a dose of 10 kGy. position of some aromatic , and indicated the presence of such Peak A22 (RT 68.16 min) displayed molecular ion of m/z 529 and compound in oregano (Folium origani cretici), sage (Folium salviae fragment of m/z 305 that suggested a gallocatechin derivative as proba- officinalis)andthyme(Folium thymi). ble compound. The presence of flavan-3-ols was reported in four species The suggested compound for A17 (45.32 min) was a rosmarinic acid of seaweeds: Palmaria spp., Porphyra spp., Himanthalia elongata and the derivative with molecular ion of m/z 913 and fragments of m/z 694 and brown macroalga species Laminaria ochroleuca by (de Quirós, Lage- 359 ([rosmarinic acid − H]−). The proposed candidate for peak B1 Yusty, & López-Hernández, 2010). Among the flavan-3-ols tested, epi- (1.26 min) was rosmarinic acid with [M − H]− of m/z 359 and charac- gallocatechin was the predominant catechin and it was found in four teristic fragment of m/z 179. of the five seaweed species tested (de Quirós et al., 2010). The proposed compounds for peak C6 (RT 4.56 min) was a p- Cypellocarpin C is the proposed candidate for peak B8 (RT 3.98 min) coumaric acid derivative due to parent ion of m/z 299 and fragments that showed precursor ion of m/z 519 and fragment of m/z 335. This of m/z 255 and 163 ([p-coumaric acid − H]−). compound was also reported by Boulekbache-Makhlouf, Meudec, The peak C15 (54.41 min) was tentatively identified as a ferulic acid Mazauric, Madani, and Cheynier (2013) within the phenolic com- derivative due to the molecular ion of m/z 375 and fragments of m/z 293, pounds of Eucalyptus globulus leaves. 221 and 193 ([ferulic acid − H]−). 3.2.5. Non identified peaks 3.2.4. Flavonoids Eight peaks from ANE, 4 peaks from BBE and 6 peaks from FVE could The peak A7 (3.25 min) was tentatively identified as an acacetin de- not be identified based on mass spectrum and/or UV–Vis data. Similar rivative with [M − H]− of m/z 455 and fragments of m/z 327 and 283 difficulties for the identification of phenolic compounds were reported ([acacetin − H]−). The compound of peak A11 (5.32 min) was also ten- by other authors (Montero et al., 2016; Munekata, Franco, Trindade, & tatively identified as another acacetin derivative (molecular ion of m/z Lorenzo, 2016) in the characterization of the phenolic composition of 657 and fragments of m/z 532 and 283). A third acacetin derivative several vegetable matrices. was proposed for peak A15 (9.59 min) with parent ion of m/z 900 and fragments of m/z 819, 739 and 283. 3.3. Phenolic profile of the seaweed extracts The peak B7 (3.26 min) was tentatively identified as an acacetin de- rivative with parent ion of m/z 327 and a fragment of m/z 283 ([acacetin The phenolic compounds tentatively identified from FVE in the pres- − H]−). In a similar way, an acacetin derivative was also suggested for ent study are in accordance with previous studies. Heffernan, Brunton, compound B11 (RT 10.57 min) due to the molecular ion of m/z 743 FitzGerald, and Smyth (2015) reported that phenolics of Fucus and fragments of m/z 429 and 283. vesiculosus were predominantly composed by low molecular weight The suggest compound for peak C7 (RT 5.51 min) is an acacetin deriv- phlorotannin oligomers between 3 and 8 phloroglucinol units. ANE ative with parent ion of m/z 322 and fragments of m/z 283. Similarly, com- and BBE composition in the present study are in accordance with previ- pounds C9 (RT 19.99 min, [M − H]− of m/z 947 and fragments of m/z 691, ous studies in literature that also reported the major contribution of 383 and 283) and C14 (RT 51.49 min, [M − H]− of m/z 356 and fragment phlorotannins on brown seaweeds composition (Li, Wijesekara, Li, & of m/z 283) were also tentatively identified as acacetin derivative. Kim, 2011; Martínez & Castañeda, 2013). The simultaneous occurrence Hossain et al. (2010) reported the presence of acacetin and other fla- of phlorotannins with different linkage between phloroglucinol units vonoids (e.g. quercetin and apigenin) with elevated antioxidant poten- (e.g. fucol and fucophlorethol) was reported in the study performed tial in the phenolic composition of herbs as , oregano and rosemary. by Cérantola, Breton, Ar Gall, and Deslandes (2006) in brown seaweeds. Peak A10 (RT 4.69 min) was tentatively identified as hispidulin due In this study, the NMR analysis of isolated compounds by successive sol- to molecular ion of m/z 299. Such phenolic compound was reported by vent extraction revealed that compounds belonging to both fucol group 984 R. Agregán et al. / Food Research International 99 (2017) 979–985

(only aryl bond between phloroglucinol units) and fucophlorethol Ferreres, F., Lopes, G., Gil-Izquierdo, A., Andrade, P. B., Sousa, C., Mouga, T., & Valentão, P. (2012). Phlorotannin extracts from fucales characterized by HPLC-DAD-ESI-MSn:Ap- group (aryl and ether linkages between phloroglucinol units) are pre- proaches to hyaluronidase inhibitory capacity and antioxidant properties. Marine sented in Fucus spiralis. Drugs, 10(12), 2766–2781. Phlorotannins synthesis is associated with antifouling activity to in- Garcia-Vaquero, M., & Hayes, M. (2016). Red and green macroalgae for fish and animal feed and human functional food development. Food Reviews International, 32(1), crease the resistance to epibiosis development (organisms living on the 15–45. algal surface). Seaweed exudates composed by an insoluble agar matrix Garcia-Vaquero, M., Lopez-Alonso, M., & Hayes, M. (2017a). Assessment of the functional containing these compounds were reported for Fucus vesiculosus properties of protein extracted from the brown seaweed Himanthalia elongata (Lin- – (Jormalainen, Wikström, & Honkanen, 2008) and they may act as anti- naeus) S. F. Gray. Food Research International, 99,971 978. Garcia-Vaquero, M., Rajauria, G., O'Doherty, J. V., & Sweeney, T. (2017b). Polysaccharides fouling agents. Particularly for Ascophyllum nodosum, the synthesis of from macroalgae: Recent advances, innovative technologies and challenges in extrac- phenolic compounds may be affected by specific stimuli such as nutri- tion and purification. Food Research International, 99, 1011–1020. fi ents availability, season, presence of herbivore grazing, release of hydro- Gómez-Ordóñez, E., Jiménez-Escrig, A., & Rupérez, P. (2010). Dietary bre and physico- chemical properties of several edible seaweeds from the northwestern Spanish gen peroxide during summer due to photo-oxidation, heat stress and coast. Food Research International, 43(9), 2289–2294. exposure to ultra-violet radiation (Parys et al., 2009; Svensson, Pavia, Gupta, S., & Abu-Ghannam, N. (2011). Bioactive potential and possible health effects of & Toth, 2007). On the other hand, some stimuli may influence the exu- edible brown seaweeds. Trends in Food Science & Technology, 22(6), 315–326. Heffernan, N., Brunton, N. P., FitzGerald, R. J., & Smyth, T. J. (2015). Profiling of the molec- dation of phlorotannins and thus reducing their amounts in seaweed ular weight and structural isomer abundance of macroalgae-derived phlorotannins. tissues. The presence of herbivores was associated with increased re- Marine Drugs, 13(1), 509–528. lease of phlorotannins whereas nutrient availability seems not affect Hossain, M. B., Rai, D. K., Brunton, N. P., Martin-Diana, A. B., & Barry-Ryan, C. (2010). Char- acterization of phenolic composition in Lamiaceae spices by LC-ESI-MS/MS. Journal of this response in Fucus vesiculosus (Koivikko et al., 2005). Due to the Agricultural and Food Chemistry, 58(19), 10576–10581. marked effect of stimuli from the environment, the phenolic composi- Jormalainen, V., Wikström, S. A., & Honkanen, T. (2008). Fouling mediates grazing: tion widely varies within the same seaweed species and therefore the Intertwining of resistances to multiple enemies in the brown alga Fucus vesiculosus. Oecologia, 155(3), 559–569. phenolic composition of seaweeds must be characterized for each set Jung, H. A., Hyun, S. K., Kim, H. R., & Choi, J. S. (2006). Angiotensin-converting enzyme I of environmental conditions. inhibitory activity of phlorotannins from Ecklonia stolonifera. Fisheries Science, 72(6), Comparatively, phlorotannins were tentatively identified as the 1292–1299. main compounds in all extracts in the present study, particularly oligo- Jung, H. A., Jung, H. J., Jeong, H. Y., Kwon, H. J., Ali, M. Y., & Choi, J. S. (2014). Phlorotannins isolated from the edible brown alga Ecklonia stolonifera exert anti-adipogenic activity mers belonging to the fuhalol sub-class, although some flavonoids and on 3T3-L1 adipocytes by downregulating C/EBPα and PPARγ. Fitoterapia, 92, phenolic acid derivatives were also indicated. Although the results re- 260–269. vealed a variety of phenolic compounds in the analyzed seaweeds, addi- Kannan,R.R.,Aderogba,M.A.,Ndhlala,A.R.,Stirk,W.A.,&VanStaden,J.(2013). Acetylcholinesterase inhibitory activity of phlorotannins isolated from the tional analyses using other more performant techniques such as n-MS brown alga, Ecklonia maxima (Osbeck) Papenfuss. Food Research International, and NMR are necessary in order to support the proposed identifications 54(1), 1250–1254. in ANE, BBE and FVE. Koivikko, R., Loponen, J., Honkanen, T., & Jormalainen, V. (2005). Contents of soluble, cell-wall-bound and exuded phlorotannins in the brown alga Fucus vesiculosus, with implications on their ecological functions. Journal of Chemical Ecology, 31(1), 195–212. 4. Conclusion Le Lann, K., Jégou, C., & Stiger-Pouvreau, V. (2008). Effect of different conditioning treat- ments on total phenolic content and antioxidant activities in two Sargassacean spe- The tentative analysis of phenolic composition of selected brown cies: Comparison of the frondose Sargassum muticum (Yendo) Fensholt and the – seaweed species by LC-DAD-ESI-MS indicates the presence of cylindrical Bifurcaria bifurcata R. Ross. Phycological Research, 56(4), 238 245. Lee, J. H., Ko, J. Y., Oh, J. Y., Kim, C. Y., Lee, H. J., Kim, J., & Jeon, Y. J. (2014). Preparative iso- phlorotannins as major phenolic compounds. Presence of such com- lation and purification of phlorotannins from Ecklonia cava using centrifugal partition pounds indicates that brown seaweeds evaluated in the present study chromatography by one-step. Food Chemistry, 158,433–437. can constitute a new move in the understanding of the health benefits Li, Y. X., Wijesekara, I., Li, Y., & Kim, S. K. (2011). Phlorotannins as bioactive agents from brown algae. Process Biochemistry, 46(12), 2219–2224. of seaweeds as functional ingredient in food, pharmaceutical and cos- Martínez,J.H.I.,&Castañeda,H.G.T.(2013).Preparation and chromatographic analysis metic industry. Future research for purification of individual compound of phlorotannins. Journal of Chromatographic Science, 51(8), 825–838. and their mechanisms of action will provide a better understanding of Michalak, I., & Chojnacka, K. (2015). Algae as production systems of bioactive compounds. Engineering in Life Sciences, 15(2), 160–176. their nature and antioxidant activity. Montero, L., Sánchez-Camargo, A. P., García-Cañas, V., Tanniou, A., Stiger-Pouvreau, V., Russo, M., ... Ibáñez, E. (2016). Anti-proliferative activity and chemical characteriza- tion by comprehensive two-dimensional liquid chromatography coupled to mass Acknowledgements spectrometry of phlorotannins from the brown macroalga Sargassum muticum col- lected on North-Atlantic coasts. Journal of Chromatography A, 1428,115–125. The authors thank INIA (Instituto Nacional de Investigación y Munekata, P. E. S., Franco, D., Trindade, M. A., & Lorenzo, J. M. (2016). Characterization of phenolic composition in chestnut leaves and beer residue by LC-DAD-ESI-MS. LWT - Tecnología Agraria y Alimentaria, Spain) for granting Rubén Agregán Food Science and Technology, 68,52–58. with a predoctoral scholarship (CPR2014-0128). The authors also thank Nagy, T. O., Solar, S., Sontag, S., & Koenig, J. (2011). Identification of phenolic components fl – National Council for Scientific and Technological Development (Brazil) in dried spices and in uence of irradiation. Food Chemistry, 128(2), 530 534. fi Nakamura, T., Nagayama, K., Uchida, K., & Tanaka, R. (1996). Antioxidant activity of for nancial support (CNPq n.° 248705/2013-0), and Esteban Guitián Phlorotannins isolated from the brown alga Eisenia bicyclis. Fisheries Science, 62(6), from the University of Santiago de Compostela Mass Spectrometry Core 923–926. Facility for technical support in mass spectrometric analysis. Nwosu, F., Morris, J., Lund, A. V., Stewart, D., Ross, H. A., & McDougall, G. J. (2011). Anti- proliferative and potential anti-diabetic effects of phenolic-rich extracts from edible marine algae. Food Chemistry, 126(3), 1006–1012. References Orrego-Lagarón, N., Vallverdú-Queralt, A., Martínez-Huélamo, M., Lamuela-Raventos, R. M., & Escribano-Ferrer, E. (2016). Metabolic profile of naringenin in the stomach Arnold, T. M., & Targett, N. M. (2000). Evidence for metabolic turnover of polyphenolics in and colon using liquid chromatography/electrospray ionization linear ion trap quad- tropical brown algae. Journal of Chemical Ecology, 26(6), 1393–1410. rupole-Orbitrap-mass spectrometry (LC-ESI-LTQ-Orbitrap-MS) and LC-ESI-MS/MS. Boulekbache-Makhlouf, L., Meudec, E., Mazauric, J. -P., Madani, K., & Cheynier, V. (2013). Journal of Pharmaceutical and Biomedical Analysis, 120,38–45. Qualitative and semi-quantitative analysis of phenolics in Eucalyptus globulus leaves Pantidos, N., Boath, A., Lund, V., Conner, S., & McDougall, G. J. (2014). Phenolic-rich ex- by high-performance liquid chromatography coupled with diode array detection tracts from the edible seaweed, Ascophyllum nodosum,inhibitα-amylase and α-glu- and electrospray ionisation mass spectrometry. Phytochemical Analysis, 24,162–170. cosidase: Potential anti-hyperglycemic effects. Journal of Functional Foods, 10, Burtin, P. (2003). Nutritional value of seaweeds. Electronic Journal of Environmental, 201–209. Agricultural and Food Chemistry, 2(4), 498–503. Parys, S., Kehraus, S., Pete, R., Küpper, F. C., Glombitza, K. W., & König, G. M. (2009). Sea- Cérantola, S., Breton, F., Ar Gall, E., & Deslandes, E. (2006). Co-occurrence and antioxidant sonal variation of polyphenolics in Ascophyllum nodosum (Phaeophyceae). European activities of fucol and fucophlorethol classes of polymeric phenols in Fucus spiralis. Journal of Phycology, 44(3), 331–338. Botanica Marina, 49(4), 347–351. Quéguineur, B., Goya, L., Ramos, S., Martín, M. A., Mateos, R., & Bravo, L. (2012). Farvin, K. S., & Jacobsen, C. (2013). Phenolic compounds and antioxidant activities of se- Phloroglucinol: Antioxidant properties and effects on cellular oxidative markers in lected species of seaweeds from Danish coast. Food Chemistry, 138(2), 1670–1681. human HepG2 cell line. Food and Chemical Toxicology, 50(8), 2886–2893. R. Agregán et al. / Food Research International 99 (2017) 979–985 985 de Quirós, A. R. B., Lage-Yusty, M. A., & López-Hernández, J. (2010). Determination of phe- matography–high resolution mass spectrometry. Phytochemical Analysis, 23(5), nolic compounds in macroalgae for human consumption. Food Chemistry, 121(2), 547–553. 634–638. Svensson, C. J., Pavia, H., & Toth, G. B. (2007). Do plant density, nutrient availability, and Rajauria, G., Foley, B., & Abu-Ghannam, N. (2016). Identification and characterization of herbivore grazing interact to affect phlorotannin plasticity in the brown seaweed phenolic antioxidant compounds from brown Irish seaweed Himanthalia elongata Ascophyllum nodosum. Marine Biology, 151(6), 2177–2181. using LC-DAD–ESI-MS/MS. Innovative Food Science & Emerging Technologies, 37, 261–268. Steevensz, A. J., MacKinnon, S. L., Hankinson, R., Craft, C., Connan, S., Stengel, D. B., & Melanson, J. E. (2012). Profiling phlorotannins in brown macroalgae by liquid chro-

marine drugs

Article Proximate Composition and Nutritional Value of Three Macroalgae: Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcata

José M. Lorenzo 1,* ID , Rubén Agregán 1, Paulo E. S. Munekata 2, Daniel Franco 1 ID , Javier Carballo 3, Selin ¸Sahin 4, Ramón Lacomba 5 and Francisco J. Barba 6,* ID

1 Centro Tecnológico de la Carne de Galicia, Adva. Galicia n◦ 4, Parque Tecnológico de Galicia, San Cibrao das Viñas, 32900 Ourense, Spain; [email protected] (R.A.); [email protected] (D.F.) 2 Department of Food Engineering, Faculty of Animal Science and Food Engineering, University of São Paulo, 225 Duque de Caxias Norte Ave, Jardim Elite, Pirassununga, São Paulo 13.635-900, Brazil; [email protected] 3 Area de Tecnologia de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain; [email protected] 4 Department of Chemical Engineering, Engineering Faculty, Istanbul University, Avcilar, 34320 Istanbul, Turkey; [email protected] 5 Grupo Alimentario Citrus (GAC), Avda. dels Gremis, Parcela 28 Pol. Ind. Sector 13 del Túria, Riba-roja de Túria, 46394 València, Spain; [email protected] 6 Nutrition and Food Science Area, Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Universitat de València, Avda. Vicent Andrés Estellés, s/n, Burjassot, 46100 València, Spain * Correspondence: [email protected] (J.M.L.); [email protected] (F.J.B.); Tel.: +34-988548277 (J.M.L.); +34-963544972 (F.J.B.); Fax: +34-988548276 (J.M.L.); +34-963544954 (F.J.B.)

Received: 8 August 2017; Accepted: 8 November 2017; Published: 15 November 2017

Abstract: Proximate composition (moisture, protein, lipid and ash content) and nutritional value (fatty acid, amino acid and mineral profile) of three macroalgae (Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcate) were studied. Chemical composition was significantly (p < 0.001) different among the three seaweeds. In this regard, the B. bifurcata presented the highest fat content (6.54% of dry matter); whereas, F. vesiculosus showed the highest protein level (12.99% dry matter). Regarding fatty acid content, the polyunsaturated fatty acids (PUFAs) were the most abundant followed by saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs). On the other hand, the three seaweeds are a rich source of K (from 3781.35 to 9316.28 mg/100 g), Mn (from 8.28 to 1.96 mg/100 g), Na (from 1836.82 to 4575.71 mg/100 g) and Ca (from 984.73 to 1160.27 mg/100 g). Finally, the most abundant amino acid was glutamic acid (1874.47–1504.53 mg/100 dry matter), followed by aspartic acid (1677.01–800.84 mg/100 g dry matter) and alanine (985.40–655.73 mg/100 g dry matter).

Keywords: Ascophyllum nodosum; Fucus vesiculosus; Bifurcaria bifurcate; seaweeds; fatty acid profile; amino acid content; minerals; chemical composition

1. Introduction Seaweeds have been traditionally consumed as food in many cultures, and have been used as condiments, fertilizers, and as source of phycocolloids such as alginate, agar, and carrageenan for industrial applications [1–4]. Seaweeds are traditionally divided into three main groups corresponding to the phylum: green (Chlorophyta), red (Rhodophyta) and brown (Phaeophyta), depending on their chemical composition and nutritional value [5].

Mar. Drugs 2017, 15, 360; doi:10.3390/md15110360 www.mdpi.com/journal/marinedrugs Mar. Drugs 2017, 15, 360 2 of 11

Seaweeds are consumed in Asia as part of the daily diet. Nowadays, brown algae (66.5%) are the most consumed species, followed by red (33%) and green (5%) algae. Today, Japan, China and South Korea, are the countries with the greatest seaweed consumption [6]. Seaweeds are an excellent nutrient source, containing high amounts of macro- and micronutrients [7], as well as bioactive compounds (e.g., catechins such as gallocatechin, epicatechin and catechingallate, flavonols, and flavonol glycosides) with high antioxidant and health beneficial properties [8–10]. Food, pharmaceutical, and cosmetic industries have shown interest in the recovery of antioxidant compounds (isolated compounds and/or complex extract mixtures) assisted by conventional (solid-liquid or liquid-liquid extraction, Soxhlet, etc.) and innovative processing technologies (high-pressure, supercritical-CO2 (SC-CO2), electrotechnologies, microwave- and ultrasound-assisted extraction, among others) [3,9,11]. Several authors [12–15] have reported that the chemical composition of seaweeds varies according to maturity, habitats, environmental conditions, and species. A comprehensive study of nutritional (protein and amino acids, fat and fatty acids, carbohydrates, minerals, and vitamins) and bioactive compounds such as polyphenols, carotenoids, etc., from each seaweed, which can exert some beneficial properties on health, is necessary. There are many brown types of seaweeds found in the Spanish coast and the details of their chemical and nutritional composition are needed in order to fulfil the growing demand for Spanish seaweeds and their derived products. Thus, the aim of the present study was to evaluate the chemical and nutritional properties of three different brown seaweeds A. nodosum, F. vesiculosus and B. bifurcata from the Galician coast.

2. Results and Discussion

2.1. Chemical Composition of Seaweeds The proximate composition of the three seaweeds is summarized in Table1. As can be seen the moisture content showed significant (p < 0.001) differences among the three macroalgae, since the lowest value was observed in B. bifurcata (7.95%).

Table 1. Proximate composition of the three seaweeds studied (mean ± standard deviation values) (n = 5).

Seaweed Parameters A. nodosum F. vesiculosus B. bifurcata Moisture (g/100 g algae) 11.08 ± 0.53 a 11.23 ± 0.08 a 7.95 ± 0.06 b Protein (g/100 g DW) 8.70 ± 0.07 a 12.99 ± 0.04 b 8.92 ± 0.09 c Lipid (g/100 g DW) 3.62 ± 0.17 a 3.75 ± 0.20 a 6.54 ± 0.27 b Ash (g/100 g DW) 30.89 ± 0.06 a 20.71 ± 0.04 b 31.68 ± 0.41 c DW: dry weight of seaweed. a–c Means in the same row not followed by a common superscript letter are significantly different (p < 0.05; Duncan test).

This finding is in close agreement with the data reported by Rodrigues et al. [16], who also noticed that the moisture content of different edible seaweeds species ranged from 8.0 g/100 g of dry weight (DW) in dried Gracilaria gracilis to 11.8 g/100 g of DW in dried pinnatifida. In addition, Gómez-Ordoñez et al. [14] also reported similar moisture contents (between 6.64% and 9.86%) in several edible seaweeds from the northwestern Spanish coast. However, Chan & Matanjun [17] found lower moisture content (5.32%) in freeze-dried Gracilaria changii seaweed. F. vesiculosus specie presented the highest protein content (12.99 g/100 DW), followed by B. bifurcata (8.92 g/100 DW) and the A. nodosum (8.70 g/100 DW). These results are in agreement with the data reported by Fleurence [18], who also noticed low protein content (<15 g/100 DW) in most of the brown seaweeds industrially exploited (F. vesiculosus, A. nodosum, Laminaria digitata and Himanthalia elongata). Similar values were found by Gómez-Ordoñez et al. [14] and Alves et al. [19] in B. bifurcata (10.92 g/100 DW and 8.57 g/100 g DW, respectively) and by Chan & Matanjun [17] in G. changii Mar. Drugs 2017, 15, 360 3 of 11

(12.57 g/100 DW). However, these values were lower than those obtained by Rodrigues et al. [16] for brown (14.4–16.9 g/100 DW), red (20.2–23.8 g/100 DW) and green (18.8 g/100 DW) seaweed species. In addition, our values were lower than those observed by Fleurence [18] in other seaweed species such as Porphyra tenera (47 g/100 DW) and Palmaria palmata (35 g/100 DW). On the contrary, Sánchez-Machado, López-Cervantes, et al. [20] obtained lower protein content (5.46 g/100 DW) in H. elongata dried seaweed. According to Denis et al. [21], the protein content of seaweed changes during the year, having the maximum content during winter and the beginning of spring, and the minimum content during summer and early autumn periods. In addition, the protein level varied among different algal species, geographic areas, seasons, or environmental conditions [22]. In general, seaweeds exhibit low fat content (bellow 4%) [23], which varies significantly through the year [24]. Extractable lipid showed significant (p < 0.001) differences among seaweeds, since the highest levels were observed in B. bifurcata (6.54% DW). Our values were similar to those reported by Peinado et al. [12], who found the contents ranging from 3.95 to 4.64% DW in F. vesiculosus and by Gómez-Ordoñez et al. [14] and Alves et al. [19], who observed fat levels of 5.67% DW and 5.81% DW in B. bifurcata, respectively. On the other hand, ash contents were high and ranged from 20.71% DW to 31.68% DW for F. vesiculosus and B. bifurcata, respectively. These findings are in agreement with the data reported by Alves et al. [19] and Gómez-Ordoñez et al. [14] in B. bifurcata (34.31% DW and 30.15% DW, respectively) and by Peinado et al. [12] in F. vesiculosus (21–19% DW). The high ash levels constitute an important characteristic of seaweeds, and are higher than those observed in terrestrial vegetables [7]. It is known that high amounts of ash are linked with high levels of minerals.

2.2. Mineral Content of Seaweeds The mineral content of the three macroalgae is given in Table2. Among the macrominerals, K (3781.35–9316.28 mg/100 g DW) was the most abundant element in the three seaweeds studied, followed by Na (1836.82–4575.71 mg/100 g DW) and Ca (984.73–1160.27 mg/100 g DW). A similar trend was reported by other authors [17,25,26], who found that K was the main mineral element followed by Na. On the other hand, B. bifurcata presented a Na/K ratio lower than that observed in the other seaweeds (0.19 vs. 0.58 vs. 1.21, for the B. bifurcata, F. vesiculosus and A. nodosum, respectively), which is really interesting from the nutritional viewpoint, because high Na/K ratio diets and the hypertension incidence are closely linked [27]. Thus, B. bifurcata could be useful for the regulation of the Na/K ratio of diets. In addition, Rodrigues et al. [16] suggested that seaweeds with low ratios of Na/K are useful as salt replacers. The values of manganese in the three macroalgae ranged from 528.04 mg/100 g DW to 867.82 mg/100 g DW, for B. bifurcata and A. nodosum, respectively, differing significantly (p < 0.001) among species. These values were higher than those reported by Chan et al. [17] in G. changii (436.13 mg/100 g DW) and lower than the data previously found by Rodrigues et al. [16] in Sargassum muticum (1504 mg/100 g DW) and Codium tomentosum (1046 mg/100 g DW). On the other hand, Ca contents also showed significant (p < 0.001) differences among seaweeds, showing the highest Ca level in F. vesiculosus (1160.27 mg/100 DW). In this regard, Moreiras et al. [28] noticed that Wakame and Sea Spaghetti seaweed species contained approximately eight times more Ca than milk and they could be an excellent source of Ca for the prevention and treatment of osteoporosis, for growing children, and for pre- and post-menopausal women. Phosphorous, the least abundant macromineral, was also detected in F. vesiculosus and B. bifurcata, ranging from 169.54 mg/100 g DW to 193.57 mg/100 g DW for F. vesiculosus and B. bifurcata, respectively. A. nodosum and F. vesiculosus also contained iron (ranged from 13.34 mg/100 g DW to 18.99 mg/100 g DW) and Mg (from 1.96 mg/100 DW to 8.28 mg/100 g DW). Our Fe values were higher than those obtained by Rupérez [7] for Porphyra tenera (10.3 mg/100 g DW), but less than those found by Rao et al. [29] for Porphyra vietnamensis (33 mg/100 g DW). In this regard, F. vesiculosus can be a useful to provide the daily intake of iron and to prevent the anemia caused by iron deficiency [30]. Mar. Drugs 2017, 15, 360 4 of 11

Table 2. Mineral profile of the three seaweeds studied (mean ± standard deviation values) (n = 5).

Seaweed Minerals (mg/100 g DW) A. nodosum F. vesiculosus B. bifurcata Ca 984.73 ± 47.26 a 1160.27 ± 23.10 b 996.42 ± 12.83 a Fe 13.34 ± 0.90 a 18.99 ± 0.32 b n.q. K 3781.35 ± 13.40 a 3745.05 ± 36.01 a 9316.28 ± 101.94 b Mg 867.82 ± 12.01 a 732.37 ± 5.35 b 528.04 ± 8.25 c Mn 1.96 ± 0.69 a 8.28 ± 1.07 b n.q. Na 4575.71 ± 50.05 a 2187.51 ± 36.90 b 1836.82 ± 52.12 c P n.q. 193.57 ± 1.13 a 169.54 ± 1.41 b Zn n.q. n.q. n.q. Cu n.q. n.q. n.q. Total 10,224.91 ± 64.32 a 8045.96 ± 94.44 b 12,848.97 ± 142.01 c n.q. = not quantified. DW: dry weight of seaweed. a–c Means in the same row not followed by a common superscript letter are significantly different (p < 0.05; Duncan test).

2.3. Amino Acid Content of Seaweeds The amino acid (AA) composition of the three seaweeds evaluated is summarized in Table3. The total AA contents were 7.48, 11.90 and 7.32 g/100 g DW (p < 0.001), for A. nodosum, F. vesiculosus, and B. bifurcata, respectively; and these values were comparable to corresponding crude protein levels (Table1), thus showing that the amount of non-protein nitrogenous materials in these seaweeds was negligible.

Table 3. Amino acid profile of the three seaweeds studied (mean ± standard deviation values) (n = 5).

Seaweed Amino Acids (mg/100 g DW) A. nodosum F. vesiculosus B. bifurcata Essential amino acids Threonine 363.22 ± 17.12 a 613.08 ± 33.62 b 360.27 ± 38.25 a Valine 353.89 ± 32.95 a 582.70 ± 36.73 b 372.82 ± 49.05 a Methionine 147.59 ± 18.71 a 218.21 ± 20.20 b 178.41 ± 18.08 a Isoleucine 295.26 ± 25.73 a 507.82 ± 32.42 b 299.73 ± 37.74 a Leucine 537.37 ± 38.87 a 862.14 ± 57.02 b 524.59 ± 61.38 a Phenylalanine 340.13 ± 17.74 a 541.53 ± 25.72 b 330.05 ± 32.32 a Lysine 431.72 ± 38.40 a 800.28 ± 74.20 b 393.06 ± 56.57 a Histidine 126.46 ± 10.65 a 194.59 ± 8.73 b 138.76 ± 12.70 a Arginine 316.79 ± 14.05 a 557.87 ± 38.44 b 330.11 ± 42.41 a Total EAA 2912.42 ± 204.93 a 4878.22 ± 304.12 b 2927.79 ± 346.84 a Non-essential amino acids Tyrosine 162.85 ± 24.50 a 327.01 ± 30.59 b 175.00 ± 30.90 a Asparagine 846.64 ± 38.87 a 1677.01 ± 156.39 b 800.84 ± 105.55 a Serine 378.62 ± 13.57 ab 630.54 ± 47.00 a 357.10 ± 36.87 b Glutamic acid 1714.55 ± 133.17 a 1974.47 ± 150.67 b 1504.53 ± 178.74 a Glycine 417.70 ± 12.89 a 651.24 ± 30.84 b 390.14 ± 29.42 a Alanine 655.73 ± 34.75 a 985.40 ± 69.50 b 846.65 ± 82.87 c Proline 399.24 ± 11.70 a 575.19 ± 39.15 b 318.40 ± 40.96 c Cysteine 0.00 ± 0.00 a 205.23 ± 25.43 b 0.00 ± 0.00 a Total NEAA 4575.33 ± 198.91 a 7026.10 ± 512.60 b 4392.67 ± 502.38 a Total AA 7487.76 ± 400.31 a 11,904.32 ± 816.67 b 7320.46 ± 848.14 a Relative Amount EAA (%) 38.87 ± 0.71 a 40.99 ± 0.26 b 39.99 ± 0.31 c DW: dry weight of seaweed. a–c Means in the same row not followed by a common superscript letter are significantly different (p < 0.05; Duncan test). EAA: Essential Amino acids. Mar. Drugs 2017, 15, 360 5 of 11

The three seaweeds studied contained all the essential amino acids (EAAs) (excluding cysteine in the A. nodosum and B. bifurcata). The EAAs content ranged from 3075.28 mg/100 g DW to 5205.23 mg/100 g DW for the A. nodosum and F. vesiculosus, respectively, showing significant differences among species. The EAA/total AA ratio suggests that more than 40% of the AAs were EAAs. This ratio was lower than the data reported by Chan et al. [17], who observed the ratios (above 55%) in G. changii, but comparable to Porphyra umbilicalis (36.87%), Undaria pinnatifida (42.72%) and H. elongata (40.82%) reported by Cofrades et al. [13]. In the essential fraction, leucine was the most abundant, ranging from 524.59 mg/100 g DW to 862.14 mg/100 g DW for B. bifurcata and F. vesiculosus, respectively, followed by lysine (393.06–800.28 mg/100 g DW), threonine (360.27–613.08 mg/100 g DW) and valine (353.89–582.70 mg/100 g DW). These findings were not in agreement with those reported by Chan et al. [17], who observed that arginine was found to be the highest EAA in G. changii, representing 18.69% of the total AAs. On the other hand, glutamic and aspartic acids were the major amino acids found in the non-essential fraction and theses two AAs accounted between 30.67% and 34.20% of the total AAs, for the FV and AN species, respectively. The sum of aspartic and glutamic acids was higher than data reported by other authors [13,17] who found values below 25% in different seaweed species. According to Saini et al. [31], the special flavor and taste of seaweeds in linked to the glutamic and aspartic acids contents. The next highest NEEA were alanine > glycine > serine > proline. Finally, the protein quality of FV seaweed is better than those the other ones, because cysteine is lacking in the AN and BB species. The nutritional quality of the three seaweeds studied is shown in Table4. The chemical score (CS) for each of the essential amino acids with respect to the pattern protein, as proposed by Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO)/United Nations (UNU) [32] for humans (children > 1-year old and adults) was calculated.

Table 4. Nutritional quality of protein for the three seaweeds studied.

IOM/FNB FAO/WHO/UNU Seaweeds Amino Acid (2002) (2007) A. nodosum (CS) F. vesiculosus (CS) B. bifurcata (CS) Histidine 1.8 1.5 96.8 99.8 103.7 Isoleucine 2.5 3.0 113.1 130.3 112.0 Leucine 5.5 5.9 104.7 112.5 99.7 Lysine 5.1 4.5 110.3 136.9 97.9 Met + Cys 2.5 1.6 105.9 203.8 125.0 Phe + Tyr 4.7 3.8 152.1 176.0 149.0 Threonine 2.7 2.3 181.5 125.0 175.6 Valine 3.2 3.9 104.3 115.0 107.2 IAEE 118.4 133.9 118.8 Seaweeds: A. nodosum = Ascophyllum nodosum; F. vesiculosus = Fucus vesiculosus; and B. bifurcate = Bifurcaria bifurcata. Pattern proteins are expressed in (g/100 g protein). Values of CS and IEAA (Index Essential Amino Acids) are referred only respect to FAO/WHO/UNU (2007) protein pattern.

The profile of the Institute of Medicine, Food and Nutrition (FNB) [33] is also shown for comparative purposes. The analysis of the CS allows the order of the restrictive amino acids to be determined. Concentration of all the essential amino acids were above the FAO/WHO/UNU [32] except for histidine in the A. nodosum and F. vesiculosus and leucine and lysine in B. bifurcata. Thus, histidine was the most limiting AA found in A. nodosum and F. vesiculosus and lysine seemed to be the limiting AA in B. bifurcata. This is in agreement with the data found by Cofrades et al. [13], who found that the most limiting AA in the brown seaweeds was lysine. However, Chan et al. [17] observed that methionine was the most limiting AA found in G. changii.

2.4. Fatty Acid Profile of Seaweeds Table5 shows the fatty acid profile of the three seaweeds studied. The polyunsaturated fatty acids (PUFAs) were the most abundant, ranging from 43.47% to 48.19% for the A. nodosum Mar. Drugs 2017, 15, 360 6 of 11 and F. vesiculosus, respectively. This result is in agreement with the data previously reported by other authors [13,17,19], who found that PUFAs were the main fatty acids in seaweeds. However, Pen et al. [34] and Maehre et al. [15] observed higher saturated fatty acid (SFA) content in different seaweed species. In the present study, the percentage of fatty acid differed significantly (p < 0.001) among seaweeds. In this regard, the highest oleic acid (C18:1n-9) content (27.83–19.94%) was found in A. nodosum and F. vesiculosus, whereas B. bifurcata presented the highest arachidonic acid (C20:4n-6) level (15.24%). A similar trend was reported by Peinado et al. [12] and Ortiz et al. [35], who observed that oleic acid was the main fatty acid in seaweed samples. On the contrary, Chan et al. [17] and Alves et al. [19] reported that docosahexaenoic acid (C22:6n-3; DHA) and palmitic acid (C16:0) were the most abundant fatty acids in G. changgi and B. bifurcata, respectively. These differences on the fatty acid profile could be due to differences among species, as well as other abiotic factors such as light, salinity, and nutrients [36].

Table 5. Fatty acid profile of the three seaweeds studied (mean ± standard deviation values) (n = 5).

Seaweed Fatty Acids A. nodosum F. vesiculosus B. bifurcata C14:0 9.40 ± 0.11 a 11.38 ± 0.11 b 4.52 ± 0.46 c C14:1n-5 0.28 ± 0.00 a 0.10 ± 0.00 b 0.00 ± 0.00 c C15:0 0.30 ± 0.00 a 0.37 ± 0.00 b 0.17 ± 0.01 c C16:0 13.42 ± 0.46 a 14.66 ± 0.36 b 17.35 ± 0.43 c C16:1n-7 2.24 ± 0.01 a 1.18 ± 0.02 b 2.51 ± 0.16 c C17:0 0.41 ± 0.14 a 0.82 ± 0.15 b 0.54 ± 0.02 a C17:1n-7 0.29 ± 0.00 a 0.20 ± 0.00 b 1.87 ± 0.07 c C18:0 0.76 ± 0.01 a 1.06 ± 0.08 b 1.75 ± 0.13 c C18:1n-11 trans 0.00 ± 0.00 a 0.00 ± 0.00 a 3.57 ± 0.13 b C18:1n-9 cis 27.83 ± 0.26 a 19.94 ± 0.31 b 12.61 ± 0.35 c C18:1n-7 cis 0.45 ± 0.05 a 0.39 ± 0.04 a 0.52 ± 0.03 b C18:2n-6 trans 0.11 ± 0.00 a 0.06 ± 0.00 a 5.68 ± 0.21 b C18:2n-6 cis 7.47 ± 0.12 a 6.43 ± 0.08 b 1.92 ± 0.06 c C20:0 0.22 ± 0.01 a 0.39 ± 0.01 b 1.89 ± 0.18 c C18:3n-6 0.54 ± 0.01 a 0.56 ± 0.01 a 0.42 ± 0.05 b C20:1n-9 0.07 ± 0.01 a 0.53 ± 0.01 b 4.18 ± 0.12 c C18:3n-3 4.45 ± 0.03 a 7.59 ± 0.11 b 3.97 ± 0.09 c C18:2n-7 (CLA) 0.00 ± 0.00 a 0.00 ± 0.00 a 0.87 ± 0.10 b C21:0 0.00 ± 0.00 a 0.00 ± 0.00 a 0.71 ± 0.07 b C20:2n-6 5.05 ± 0.02 a 6.46 ± 0.09 b 1.44 ± 0.01 c C22:0 0.22 ± 0.00 a 0.22 ± 0.00 a 0.34 ± 0.02 b C20:3n-6 0.74 ± 0.04 a 0.69 ± 0.02 b 0.42 ± 0.04 c C22:1n-9 0.00 ± 0.00 a 0.00 ± 0.00 a 0.73 ± 0.04 b C20:3n-3 0.33 ± 0.01 a 0.21 ± 0.00 b 0.00 ± 0.00 c C20:4n-6 17.25 ± 0.26 a 15.86 ± 0.24 b 15.24 ± 0.37 c C22:2n-6 0.29 ± 0.01 a 0.39 ± 0.01 b 1.76 ± 0.09 c C20:5n-3 7.24 ± 0.08 a 9.94 ± 0.14 b 4.09 ± 0.08 c C24:0 0.41 ± 0.00 a 0.36 ± 0.01 b 0.34 ± 0.03 b C24:1n-9 0.00 ± 0.00 a 0.00 ± 0.00 a 0.53 ± 0.06 b C22:6n-3 0.00 ± 0.00 a 0.00 ± 0.00 a 11.10 ± 1.13 b SFA 25.14 ± 0.49 a 29.26 ± 0.34 b 27.62 ± 0.77 c MUFA 31.15 ± 0.23 a 22.33 ± 0.33 b 26.51 ± 0.48 c PUFA 43.47 ± 0.54 a 48.19 ± 0.62 b 46.91 ± 1.37 b n-3 12.02 ± 0.11 a 17.74 ± 0.25 b 19.16 ± 1.03 c n-6 31.45 ± 0.42 a 30.44 ± 0.38 b 26.87 ± 0.48 c n-6/n-3 2.62 ± 0.01 a 1.72 ± 0.01 b 1.41 ± 0.07 c Results expressed as percentage of total fatty acid analyzed. a–c means in the same row not followed by a common superscript letter are significantly different (p < 0.05; Duncan test). Saturated fatty acids: SFA. Monounsaturated fatty acids: MUFA. Polyunsaturated fatty acids: PUFA. Mar. Drugs 2017, 15, 360 7 of 11

Eicosapentaenoic acid (EPA) (C20:5n-3) represented from 4.09 to 9.94% of the total fatty acids, whereas docosahexaenoic acid (DHA) was only detected in B. bifurcata (11.10% of the total fatty acids). Other studies reported similar EPA percentages in brown algae [12,19,20]. In another work, Maehre et al. [15] found that none of the algae contained DHA, whereas the EPA content varied considerably among species. On the other hand, Western country diets are deficient in n-3 fatty acids due to the low seafood consumption versus the high intake of n-6 fatty acid from vegetable oil. In this regard, the World Health Organization (WHO) [30] recommended a n-6/n-3 ratio below 10. In our study, we observed n-6/n-3 ratio ranging from 2.62 to 1.41, placing the three macroalgae studied according WHO recommendations. This outcome is in agreement with those reported by other authors [17,19,35] who found n-6/n-3 ratios between 4.1 and 0.02.

3. Material and Methods

3.1. Algal Material The brown seaweeds, A. nodosum, F. vesiculosus and B. bifurcata used in the present study, were kindly supplied by Portomuiños Company (A Coruña, Spain). They were collected from August to September 2015, in the Atlantic Ocean, in the area of Camariñas (A Coruña, Spain). The samples were grinded to obtain powder with a particle size lower than 0.8 mm, using a conventional mincer. Then, the seaweeds were passed through a 0.8 mm mesh sieve and stored under vacuum in plastics bags at −20 ◦C until analysis.

3.2. Chemical Composition Moisture, protein, and ash were determined following the ISO recommendations (ISO 1442:1997 [37], ISO 937:1978 [38], and ISO 936:1998 [39], respectively). Moisture content was determined by measuring sample (3 g) weight loss at 105 ◦C in an oven (Memmert UFP 600, Schwabach, Germany), until constant weight. Kjeldahl total nitrogen method was used to determine protein percentage (total nitrogen content was multiplied ×6.25). Five hundred milligrams of seaweed were subjected to reaction with H2SO4 (CuSO4·5H2O was employed as a catalyst) in a digester (Gerhardt Kjeldatherm KB, Bonn, Germany), then the organic nitrogen was transformed into (NH4)2SO4, and distilled in alkali condition (Gerhardt Vapodest 50 carroused, Bonn, Germany). Ash content was assessed by determining seaweed (3 g) weight loss in a muffle furnace (Carbolite RWF 1200, Hope Valley, UK) at 600 ◦C until constant weight. Lipids were determined using the method proposed by Ortiz et al. [35] with some modifications. Lipids from each seaweed (20 g) were extracted with 300 mL of CHCl3/CH3OH/H2O (1:2:0.8), overnight under dark condition. Then, 79 mL of chloroform and 79 mL of water were added to each sample, obtaining a final solvent ratio of CHCl3/CH3OH/H2O of 1:1:0.9 by volume. NaCl (5%) was added and then, samples were centrifuged at 4000 rpm during 10 min. Chloroform phase was concentrated under vacuum condition in order to recover the lipids, which were gravimetrically measured.

3.3. Amino Acid Content Amino acids were extracted following the method proposed by Lorenzo et al. [40]. Amino acids were derived using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (Waters AccQ-Fluor reagent kit) and determined by RP-HPLC (Waters 2695 Separations Module + Waters 2475 Multi Fluorescence Detector + Waters AccQ-Tag amino acids analysis column). The amino acids content was expressed in mg/100 g of dry matter. Mar. Drugs 2017, 15, 360 8 of 11

3.4. Protein Quality: Chemical Score of Amino Acids The chemical score (CS) of the essential amino acids was determined using a protein pattern recommended by FAO/WHO/UNU [32] as reference protein applying the next equation (Equation (1)):

g EAA in tested protein CS = × 100 (1) g EAA in pattern protein

The essential amino acids index (EAA) value was also assessed according to the Equation (2) [40]: s a b c j EAA = 100 × n × × × ...... (2) ap bp cp jp where: a, b, c,..., j = content of Phe, Tyr, Val, Met, Thr, Lys, His, Ile and Leu in seaweeds. ap, bp, cp,..., jp = content of Phe, Tyr, Val, Met, Thr, Lys, His, Ile and Leu in protein standard [32]. n = number of amino acids used.

3.5. Fatty Acid Profile The lipids extracted (50 mg) were used to determine fatty acid profile. Total fatty acids were transesterified using the method previously by Domínguez et al. [41]. A GC equipment (GC-Agilent 6890 N; Agilent Technologies Spain, S.L., Madrid, Spain) with a flame ionization detector was used for the separation and quantification of the fatty acids methyl esters (FAMEs) using the chromatographic conditions proposed by Domínguez et al. [41]. Individual FAMEs were identified by comparing their retention times with those of authentic standards (Supelco 37 component FAME Mix, Sigma-Aldrich, Barcelona, Spain). C18:1n-7 cis (Supelco cis-11-Vaccenic methyl ester), C18:1n-11 trans (trans-11-vaccenic methyl ester) and C18:2n-7 (CLA) (Matreya LLC Methyl 9(z), 11 (E)-octadecadienoate) were not included in the commercial mix. In addition, nonadecanoic acid (C19:0) was used as internal standard, which was added to the samples prior to methylation. Data were expressed in g/100 g of FAME.

3.6. Mineral Profile The ash samples obtained by ISO recommended standard method [39] were dissolved in 10 mL of 1M HNO3. Mineral (Ca, Fe, K, Mg, Mn, Na, P, Zn and Cu) was determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), using a Thermo-Fisher ICAP 6000 plasma emission spectrometer (Thermo-Fisher, Cambridge, UK), following the method proposed by Lorenzo et al. [42]. All determinations were made in triplicate.

3.7. Statistical Analysis The differences in proximate composition, amino acid, fatty acid and mineral profiles among the three seaweeds studied were examined using an ANOVA test. Least-squares means were compared among seaweeds using the Duncan’s post hoc test (significance level p < 0.05). The values were given in terms of mean values ± standard deviations. All statistical analysis were performed using IBM SPSS Statistics® 21 software (IBM Corporation, Armonk, NY, USA).

4. Conclusions Among the three seaweeds studied (A. nodosum, F. vesiculosus, and B. bifurcata), B. bifurcata had the highest level of lipid and ash. It should also be noted that although B. bifurcata had the highest total mineral and K contents, F. vesiculosus presented the highest Ca, Fe, Mn, and P contents, while A. nodosum presented the highest Mg, and Na contents. This fact is of a great importance, Mar. Drugs 2017, 15, 360 9 of 11 especially when seaweeds are used to extract targeted minerals to be used in diets. F. vesiculosus had the highest protein content. The three seaweeds studied contained all the essential amino acids (excluding Cys in the A. nodosum and B. bifurcata). Glu and Asp acids were the predominant amino acids found in the non-essential fraction and theses two amino acids accounted between 30.67% and 34.20% of the total amino acids, for the F. vesiculosus and A. nodosum, respectively. Concentration of all the essential amino acids were above the chemical score established by FAO/WHO/UNU except for His in the A. nodosum and F. vesiculosus seaweeds and Leu and Lys in the B. bifurcata. Regarding fatty acids, polyunsaturated fatty acid (PUFA) were the predominant fatty acids in the three seaweeds evaluated, ranging from 43.47% to 48.19% for A. nodosum and F. vesiculosus, respectively. The highest oleic acid content (27.83–19.94%) was found in A. nodosum and F. vesiculosus, whereas B. bifurcata presented the highest arachidonic acid level (15.24%). Moreover, the n-6/n-3 ratio ranged from 2.62 to 1.41, placing the three macroalgae studied according to WHO recommendations (n-6/n-3 ratio < 10).

Acknowledgments: The authors thank INIA (Instituto Nacional de Investigaciones Agrarias y Alimentarias, Spain) for granting Ruben Agregán with a predoctoral scholarship (CPR2014-0128). Author Contributions: José M. Lorenzo, Rubén Agregán, Paulo E. S. Munekata, Daniel Franco and Javier Carballo conceived, designed and performed the experiments; Selin ¸Sahin,Ramón Lacomba and Francisco J. Barba supervised the study, wrote and reviewed the manuscript. All authors have read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest, and the founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

References

1. Peña-Rodríguez, A.; Mawhinney, T.P.; Ricque-Marie, D.; Cruz-Suárez, L.E. Chemical composition of cultivated seaweed Ulva clathrata (Roth) C. Agardh. Food Chem. 2011, 129, 491–498. [CrossRef] 2. Yaich, H.; Garna, H.; Besbes, S.; Paquot, M.; Blecker, C.; Attia, H. Chemical composition and functional properties of Ulva lactuca seaweed collected in Tunisia. Food Chem. 2011, 128, 895–901. [CrossRef] 3. Roohinejad, S.; Koubaa, M.; Barba, F.J.; Saljoughian, S.; Amid, M.; Greiner, R. Application of seaweeds to develop new food products with enhanced shelf-life, quality and health-related beneficial properties. Food Res. Int. 2016, 99, 1066–1083. [CrossRef][PubMed] 4. Barba, F.J. Microalgae and seaweeds for food applications: Challenges and perspectives. Food Res. Int. 2016, 99, 969–970. [CrossRef][PubMed] 5. Gupta, S.; Abu-Ghannam, N. Recent developments in the application of seaweeds or seaweed extracts as a means for enhancing the safety and quality attributes of foods. Innov. Food Sci. Emerg. Technol. 2011, 12, 600–609. [CrossRef] 6. Dawes, C.J. Marine Botany; Dawes, C.J., Ed.; John Wiley & Sons, Inc.: New York, NY, USA, 1998. 7. Rupérez, P. Mineral content of edible marine seaweeds. Food Chem. 2002, 79, 23–26. [CrossRef] 8. Agregán, R.; Munekata, P.E.S.; Franco, D.; Dominguez, R.; Carballo, J.; Lorenzo, J.M. Phenolic compounds from three brown seaweed species using LC-DAD-ESI-MS/MS. Food Res. Int. 2016, 99, 979–985. [CrossRef] [PubMed] 9. Poojary, M.M.; Barba, F.J.; Aliakbarian, B.; Donsì, F.; Pataro, G.; Dias, D.A.; Juliano, P. Innovative alternative technologies to extract carotenoids from microalgae and seaweeds. Mar. Drugs 2016, 14, 214. [CrossRef] [PubMed] 10. Roselló-Soto, E.; Galanakis, C.M.; Brnˇci´c,M.; Orlien, V.; Trujillo, F.J.; Mawson, R.; Knoerzer, K.; Tiwari, B.K.; Barba, F.J. Clean recovery of antioxidant compounds from plant foods, by-products and algae assisted by ultrasound processing. Modeling approaches to optimize processing conditions. Trends Food Sci. Technol. 2015, 42, 134–149. [CrossRef] 11. Zhu, Z.; Wu, Q.; Di, X.; Li, S.; Barba, F.J.; Koubaa, M.; Roohinejad, S.; Xiong, X.; He, J. Multistage recovery process of seaweed pigments: Investigation of ultrasound assisted extraction and ultra-filtration performances. Food Bioprod. Process. 2017, 104, 40–47. [CrossRef] 12. Peinado, I.; Girón, J.; Koutsidis, G.; Ames, J.M. Chemical composition, antioxidant activity and sensory evaluation of five different species of brown edible seaweeds. Food Res. Int. 2014, 66, 36–44. [CrossRef] Mar. Drugs 2017, 15, 360 10 of 11

13. Cofrades, S.; López-Lopez, I.; Bravo, L.; Ruiz-Capillas, C.; Bastida, S.; Larrea, M.T.; Jiménez-Colmenero, F. Nutritional and antioxidant properties of different brown and red Spanish edible seaweeds. Food Sci. Technol. Int. 2010, 16, 361–370. [CrossRef][PubMed] 14. Gómez-Ordóñez, E.; Jiménez-Escrig, A.; Rupérez, P. Dietary fibre and physicochemical properties of several edible seaweeds from the northwestern Spanish coast. Food Res. Int. 2010, 43, 2289–2294. [CrossRef] 15. Mæhre, H.K.; Malde, M.K.; Eilertsen, K.-E.; Elvevoll, E.O. Characterization of protein, lipid and mineral contents in common Norwegian seaweeds and evaluation of their potential as food and feed. J. Sci. Food Agric. 2014, 94, 3281–3290. [CrossRef][PubMed] 16. Rodrigues, D.; Freitas, A.C.; Pereira, L.; Rocha-Santos, T.A.P.; Vasconcelos, M.W.; Roriz, M.; Rodríguez-Alcalá, L.M.; Gomes, A.M.P.; Duarte, A.C. Chemical composition of red, brown and green macroalgae from Buarcos bay in Central West Coast of Portugal. Food Chem. 2015, 183, 197–207. [CrossRef] [PubMed] 17. Chan, P.T.; Matanjun, P. Chemical composition and physicochemical properties of tropical red seaweed, Gracilaria changii. Food Chem. 2017, 221, 302–310. [CrossRef][PubMed] 18. Fleurence, J. Seaweed proteins: Biochemical, nutritional aspects and potential uses. Trends Food Sci. Technol. 1999, 10, 25–28. [CrossRef] 19. Alves, C.; Pinteus, S.; Simões, T.; Horta, A.; Silva, J.; Tecelão, C.; Pedrosa, R. Bifurcaria bifurcata: A key macro-alga as a source of bioactive compounds and functional ingredients. Int. J. Food Sci. Technol. 2016, 51, 1638–1646. [CrossRef] 20. Sánchez-Machado, D.I.; López-Cervantes, J.; López-Hernández, J.; Paseiro-Losada, P.; Simal-Lozano, J. Determination of the uronic acid composition of seaweed dietary fibre by HPLC. Biomed. Chromatogr. 2004, 18, 90–97. [CrossRef][PubMed] 21. Denis, C.; Morançais, M.; Li, M.; Deniaud, E.; Gaudin, P.; Wielgosz-Collin, G.; Barnathan, G.; Jaouen, P.; Fleurence, J. Study of the chemical composition of edible red macroalgae Grateloupia turuturu from Brittany (France). Food Chem. 2010, 119, 913–917. [CrossRef] 22. Ito, K.; Hori, K. Seaweed: Chemical composition and potential food uses. Food Rev. Int. 1989, 5, 101–144. [CrossRef] 23. Herbreteau, F.; Coiffard, L.J.M.; Derrien, A.; De Roeck-Holtzhauer, Y. The fatty acid composition of five species of macroalgae. Bot. Mar. 1997, 40, 25–27. [CrossRef] 24. Manivannan, K.; Thirumaran, G.; Devi, G.K.; Hemalatha, A.; Anantharaman, P. Biochemical composition of seaweeds from Mandapam coastal regions along Southeast Coast of India. Am. J. Bot. 2008, 1, 32–37. 25. Kumar, M.; Kumari, P.; Trivedi, N.; Shukla, M.K.; Gupta, V.; Reddy, C.R.K.; Jha, B. Minerals, PUFAs and antioxidant properties of some tropical seaweeds from Saurashtra coast of India. J. Appl. Phycol. 2011, 23, 797–810. [CrossRef] 26. Matanjun, P.; Mohamed, S.; Mustapha, N.M.; Muhammad, K. Nutrient content of tropical edible seaweeds, Eucheuma cottonii, Caulerpa lentillifera and Sargassum polycystum. J. Appl. Phycol. 2009, 21, 75–80. [CrossRef] 27. Li, B.F. Marine Health Food; Chemical Industry Press: Beijing, China, 2009; pp. 51–72. 28. Moreiras, O.; Carbajal, Á.; Cabrera, L.; Cuadrado, C. Tablas de Composición de Alimentos, 7th ed.; Moreiras, O., Carbajal, Á., Cabrera, L., Cuadrado, C., Eds.; Ediciones Pirámide: Madrid, Spain, 2003. 29. Rao, P.V.S.; Mantri, V.A.; Ganesan, K. Mineral composition of edible seaweed Porphyra vietnamensis. Food Chem. 2007, 102, 215–218. [CrossRef] 30. Allen, L.; de Benoist, B.; Dary, O.; Hurrel, R. Guidelines on Food Fortification with Micronutrientes; World Health Organization and Food and Agriculture Organization of the United Nations: Quebec, QC, CA, 2006. 31. Saini, R.; Badole, S.L.; Zanwar, A.A. Bioactive Dietary Factors and Plant Extracts in Dermatology; Watson, R.R., Zibadi, S., Eds.; Humana Press: Totowa, NJ, USA, 2013; pp. 73–82. 32. FAO/WHO/UNU Expert Consultation. Amino acid requirements of adults. In Protein and Amino Acids Requirements in Human Nutrition; FAO/WHO/UNU: Rome, Italy, 2007; pp. 135–159. 33. Institute of Medicine. Dietary Reference Intakes for Energy, Carbohydrate, Fibre, Fat, Fatty Acids Cholesterol, Protein and Amino Acids; National Academy Press: Washington, DC, USA, 2002. 34. Peng, Y.; Xie, E.; Zheng, K.; Fredimoses, M.; Yang, X.; Zhou, X.; Wang, Y.; Yang, B.; Lin, X.; Liu, J.; et al. Nutritional and chemical composition and antiviral activity of cultivated seaweed Sargassum naozhouense Tseng et Lu. Mar. Drugs 2013, 11, 20–32. [CrossRef][PubMed] Mar. Drugs 2017, 15, 360 11 of 11

35. Ortiz, J.; Romero, N.; Robert, P.; Araya, J.; Lopez-Hernández, J.; Bozzo, C.; Navarrete, E.; Osorio, A.; Rios, A. Dietary fiber, amino acid, fatty acid and tocopherol contents of the edible seaweeds Ulva lactuca and Durvillaea antarctica. Food Chem. 2006, 99, 98–104. [CrossRef] 36. Schmid, M.; Guihéneuf, F.; Stengel, D.B. Fatty acid contents and profiles of 16 macroalgae collected from the Irish Coast at two seasons. J. Appl. Phycol. 2014, 26, 451–463. [CrossRef] 37. ISO. Determination of Moisture Content, ISO 1442:1997 Standard. International Standards Meat and Meat Products; International Organization for Standardization: Genéve, Switzerland, 1997. 38. ISO. Determination of Nitrogen Content, ISO 937:1978 Standard. International Standards Meat and Meat Products; International Organization for Standardization: Genéve, Switzerland, 1978. 39. ISO. Determination of Ash Content, ISO 936:1998 Standard. International Standards Meat and Meat Products; International Organization for Standardization: Genéve, Switzerland, 1998. 40. Shahidi, F.; Synowiecki, J. Nutrient composition of mechanically separated and surimi-like seal meat. Food Chem. 1993, 47, 41–46. [CrossRef] 41. Domínguez, R.; Borrajo, P.; Lorenzo, J.M. The effect of cooking methods on nutritional value of foal meat. J. Food Compos. Anal. 2015, 43, 61–67. [CrossRef] 42. Lorenzo, J.M.; Purriños, L.; Temperán, S.; Bermúdez, R.; Tallón, S.; Franco, D. Physicochemical and nutritional composition of dry-cured duck breast. Poult. Sci. 2011, 90, 931–940. [CrossRef][PubMed]

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Food Research International 112 (2018) 400–411

Contents lists available at ScienceDirect

Food Research International

journal homepage: www.elsevier.com/locate/foodres

Shelf life study of healthy pork liver pâté with added seaweed extracts from T Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcata ⁎ Rubén Agregána, Daniel Francoa, , Javier Carballob, Igor Tomasevicc, Francisco J. Barbad, Belén Gómeza, Voster Muchenjee, José M. Lorenzoa a Centro Tecnológico de la Carne de Galicia, Adva. Galicia n 4, Parque Tecnológico de Galicia, San Cibrao das Viñas, 32900 Ourense, Spain b Area de Tecnologia de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain c Department of Animal Source Food Technology, University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080 Belgrade, Serbia d Nutrition and Food Science Area, Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Universitat de València, Avda. Vicent Andrés Estellés, s/n, 46100 Burjassot, València, Spain e Department of Livestock and Pasture Science, University of Fort Hare, Private Bag, X 1314 Alice, South Africa

ARTICLE INFO ABSTRACT

Keywords: The effect of the addition of seaweed extracts from three brown algae species [Ascophyllum nodosum (AN), Fucus Oxidative stability vesiculosus (FV) and Bifurcaria bifurcata (BB)], which are a great source of natural antioxidants, on the oxidative Ascophyllum nodosum stability of refrigerated low-fat pork liver pâtés was studied. In the studied pâtés, half of pork fat was replaced Fucus vesiculosus with a mixture consisting of canola and high-oleic sunflower oil (75:25, v/v), thus improving their fatty acid Bifurcaria bifurcata profile in terms of polyunsaturated fatty acids (PUFA). In order to avoid the oxidation of PUFA in the new Healthy pâté samples, seaweed extracts (500 ppm) were added. In addition, some samples were formulated with a synthetic Volatile compounds Seaweed extracts antioxidant (BHT at 50 ppm) (BHT) and a control batch (CON) (without antioxidant) was also prepared, for comparison purposes. Thus, in total, five batches of liver pâté were prepared: CON, BHT, AN, FV and BB. Pâté samples were analyzed at 0, 45, 90, 135 and 180 days of refrigerated storage at 4 °C. The addition of seaweed extracts did not modify significantly (P > 0.05) the chemical composition or microbial characteristics of healthy pork liver pâté, except for the protein content, which resulted in a significant increase (≈2–3%) in the batches manufactured with addition of seaweed extracts compared to control samples. At the end of storage (180 days), L* values were significantly lower in the FV and BB batches than in the other batches. Moreover, the a* and b* values were also significantly lower in CON batches than in the samples with antioxidants added. Differences in oxidative parameters (conjugated dienes, TBARs and carbonyls) among batches were observed at the end of the storage time, showing samples with seaweed extracts a similar degree of protection against oxidation compared to BHT formulated samples. A decline of the volatile compounds was noted in all the batches during storage. The total volatile compounds at the end of the storage were significantly lower in BTH, AN, or BB batches than in control batches.

1. Introduction Cava, 2007). As a result, polyunsaturated fatty acids (PUFA) are de- graded, leading to the generation of lipid-derived volatiles, which Liver pâté is a traditional cooked meat product consumed in many promote detrimental effects on sensory and nutritional attributes countries, particularly in Europe, being Denmark, France, Germany and (Kanner, Hazan, & Doll, 1988). Spain the main consumers. This product is made of minced liver, fat and An alternative solution against lipid oxidation of pâtés is the in- meat mixed with water and different additives. After its manufacture, it clusion of antioxidants in their formulation. Synthetic antioxidants, is packaged into a glass container and a thermal treatment is carried out such as tert-butyl-4-hydroxy-toluene (BHT), have shown their effec- (Pateiro, Lorenzo, Amado, & Franco, 2014). tiveness as lipid oxidation inhibitors and are widely used in food in- Due to its high content in fat and non-heme iron, as well as the low dustry (Granato, Nunes, & Barba, 2017; Pateiro et al., 2014). However, content of natural antioxidants, liver pâté is very sensitive to lipid the use of these synthetic compounds has been linked to health risks. oxidation during manufacturing process (Estévez, Ramírez, Ventanas, & Therefore, an increased interest has been shown by food industry in

⁎ Correspondence to: Daniel Franco Ruiz, Centro Tecnológico de la Carne de Galicia, Adva. Galicia n 4, Parque Tecnológico de Galicia, San Cibrao das Viñas, 32900 Ourense, Spain. E-mail address: [email protected] (D. Franco). https://doi.org/10.1016/j.foodres.2018.06.063 Received 18 April 2018; Received in revised form 25 June 2018; Accepted 27 June 2018 Available online 28 June 2018 0963-9969/ © 2018 Elsevier Ltd. All rights reserved. R. Agregán et al. Food Research International 112 (2018) 400–411 natural antioxidants in order to replace the synthetic ones (Estévez, sunflower oil (3.75), sodium chloride (2), milk powder (2), sodium Ventanas, Ramírez, & Cava, 2004; Granato et al., 2017; Roselló-Soto caseinate (1), potassium phosphate (0.5), sodium nitrite (0.05) and et al., 2015). sodium ascorbate (0.025). Indeed, over the last years, the antioxidant properties of extracts The five pâté batches were manufactured in triplicate. A total of from different plants and vegetables (Aruoma, Halliwell, Aeschbach, & 2.2 kg of mix were used for each batch and manufactured as follows: Löligers, 1992; Juntachote, Berghofer, Siebenhandl, & Bauer, 2006; first, fat and liver were chopped in a cutter Talsa, mod K30 (Talsa, Lindberg Madsen & Bertelsen, 1995; Mc Carthy, Kerry, Kerry, Lynch, & Valencia, Spain) at 4 °C until obtaining a homogeneous meat butter Buckley, 2001), as well as edible seaweeds or marine macro- and mi- (2 min). During this process, salt, sodium nitrite and sodium ascorbate croalgae (Barba, Grimi, & Vorobiev, 2014;S.Gupta & Abu-Ghannam, were added slowly. The pre-butter obtained was kept under darkness 2011; Parniakov et al., 2015; Poojary et al., 2016; Roohinejad et al., during 24 h. The day after, the backfat was pre-cooked at 70 °C (core 2017; Zhu et al., 2017), have been shown. temperature) in a hot water bath for 15 min. The pre-emulsion of the fat These extracts are reported to possess high amounts of several source made of blanched pork backfat, canola and high-oleic sunflower natural antioxidants, such as polyphenols (eg. catechins, flavonols, and oils at the appropriate amounts, water, natural or synthetic antioxidants phlorotannins, among others), sulphated polysaccharides, and pig- according to the batch, and sodium caseinate was carried out in the ments, which have been demonstrated to possess an excellent anti- cutter for 1 min. Then, the pre-butter prepared the day before and the oxidant potential (Chojnacka, Saeid, Witkowska, & Tuhy, 2012). rest of ingredients (milk powder and potassium phosphate) were added For instance, seaweed extracts from different brown algae genera and mixed until obtaining a homogenous butter (1–2 min). such as Fucus, Ascophyllum or Bifurcaria, could be good candidates to be During the fat pre-emulsion, the temperature of the raw batter was included in liver pâté formulations as novel and natural antioxidants always above 40 °C, and during chopping the temperature was main- instead of synthetic ones (Holdt & Kraan, 2011). tained at ≈46 °C. The pâtés were manually distributed into cans until On the other hand, liver pâté is considered as a high-caloric food full filling (~100 g) and hermetically closed. Then, thermal treatment product with large amounts of saturated fats. The contribution of this (78 °C/75 min) was applied. Subsequently, the samples were immersed type of fat to the development of cardiovascular diseases, cancer, dia- in a blast chiller (−21 °C/30 min). The cans were stored under re- betes and other degenerative diseases (Morales-Irigoyen, Severiano- frigerated conditions in a room at 4 °C. Pork liver pâtés were analyzed Pérez, Rodriguez-Huezo, & Totosaus, 2012), has led to meat industry to at days 0, 45, 90, 135 and 180 days of storage. At each time of storage a seek strategies to reformulate these products with increasing levels of sample from each replicate of each batch was taken for analysis. PUFA (Martin, Ruiz, Kivikari, & Puolanne, 2008). A good option could be the replacement of animal fat with oils from seeds, rich in un- 2.2. Analytical methods saturated fatty acids. In this sense, canola oil with low levels of saturated fatty acids (SFA) 2.2.1. Chemical composition analysis (7%) and a substantial amount of monounsaturated fatty acids (MUFAs) Moisture, fat and protein were determined according to the ISO (61% oleic acid) and PUFAs (21% linoleic acid) (Johnson, Keast, & Kris- recommended standards. The moisture content was determined by Etherton, 2007) or high-oleic sunflower oil with 82% oleic acid (Gupta, drying at 105 ± 2 °C, until constant weight (ISO (International 2014), might be a useful tool to replace animal fat from liver pâté. Organization for Standardization), 1997); the protein content was es- Therefore, the aim of this study was to evaluate the effect of sea- timated by multiplying the nitrogen content, previously determined by weed aqueous extracts (from Ascophyllum nodosum (AN), Fucus vesicu- the Kjeldahl method, by 6.25 (ISO (International Organization for losus (FV) and Bifurcaria bifurcata (BB)) in pork liver pâté manufactured Standardization), 1978); and the fat content was assessed by the by partially replacing the pork fat with canola and high-oleic sunflower Soxhlet method according to the AOCS Official Procedure Am 5-04 oils. A dual goal was, therefore, pursued. On the one hand, it was (AOCS, 2005). evaluated how the addition of natural antioxidants into liver pâté can delay lipid oxidation, and on the other hand, the development of new 2.2.2. Analysis of fatty acid methyl esters meat products (liver pâté) with a healthier fatty acid profile was stu- The fat was extracted from 10 g of pâté using 18 mL of chloroform died. Moreover, the physicochemical, microbial, and nutritional prop- and 20 mL of methanol, using an IKA T25 digital ultra-turrax. Then, erties of all the samples were determined. 10 mL of NaCl 1% were added to enhance the phase separation, and the sample was centrifuged at 4000 ×g/10 min. The chloroform fraction 2. Materials and methods was recovered and the fat extracted through evaporation under vacuum at 56 °C in a rotavapor was stored at −80 °C until needed for fatty acid 2.1. Manufacture of the pâtés methyl esters (FAMEs) analysis. At this point, 50 mg of fat were used to determine the fatty acid profile according to the procedure described by The pâtés were prepared in the pilot plant of the Meat Technology Domínguez, Crecente, Borrajo, Agregán, and Lorenzo (2015). Separa- Center of Galicia (Ourense, Spain). The seaweed extracts were provided tion and quantification of the FAMEs was done using a gas chromato- by the company Portomuíños Ltd. (A Coruña, Spain). BHT was supplied graph (GC-Agilent 6890 N; Agilent Technologies Spain, S.L., Madrid by Sigma-Aldrich (Steinheim, Germany). Pork backfat and pork meat Spain) equipped with a flame ionization detector following the chro- were provided by a local slaughterhouse. The company Aceites Abril matographic conditions described by Domínguez et al. (2015). FAMEs Ltd. (Ourense, Spain) supplied canola and high-oleic sunflower (83% of composition was expressed as percentage of area compared to the total oleic acid) oils. In all bathes, 50% of pork fat was replaced by canola fatty acids identified. and high-oleic sunflower oils (75:25, v/v). Five batches of liver pâté were prepared: CON (control without 2.2.3. Microbial analysis addition of any antioxidant), BHT (with BHT added at 50 mg/kg), AN Ten grams of pâté were aseptically placed into sterile bags and (with Ascophyllum nodosum seaweed extract at 500 mg/kg), FV (with homogenized with 90 mL of sterile 0.1% peptone water in a masticator Fucus vesiculosus seaweed extract at 500 mg/kg) and BB (with Bifurcaria blender (IUL Instruments, Barcelona, Spain) for 2 min, at room tem- bifurcata seaweed extract at 500 mg/kg). perature. For each sample, serial decimal dilutions were prepared in An identical formula was used for all the batches, except for the peptone water solution (0.1%), and duplicate 1 mL or 0.1 mL samples of addition of the different antioxidants. The recipes used for the pre- the initial homogenate and of appropriate dilutions were poured or paration of pâtés (g/100 g) were as follows: subcutaneous fat (15), lean spread onto agar plates. Total Viable Counts (TVC) were enumerated in meat (20), liver (33), cold water (11.43), canola oil (11.25), high- oleic Plate Count Agar (PCA, Oxoid, Unipath Ltd., Basingstoke, UK) and

401 R. Agregán et al. Food Research International 112 (2018) 400–411 incubated at 30 °C for 72 h. Lactic Acid Bacteria (LAB) on the Man- withdrawn into the needle and transferred to the injection port of the Rogosa-Sharpe agar (Oxoid, Unipath Ltd., Basingstoke, UK) (pH 5.6) gas chromatograph–mass spectrometer (GC–MS) system. after incubation at 30 °C for 5 days. Pseudomonas spp. on Pseudomonas A gas chromatograph 6890 N (Agilent Technologies Spain, S.L., Agar Base (Merck, Darmstadt, Germany) after incubation at 25 °C for Madrid, Spain) equipped with a mass detector 5973 N (Agilent 48 h. Molds and yeasts on OGYE Agar (Merck, Darmstadt, Germany) Technologies Spain, S.L., Madrid, Spain) and with a DB-624 capillary incubated at 25 °C for 5 days. After incubation, for all the microbial column (J&W scientific: 30 m, 0.25 mm id, 1.4 μm film thickness) was groups evaluated, plates with 30–300 colonies were counted and results used. The SPME fibre was desorbed and maintained in the injection port were expressed as number of colony forming units (CFU)/g. at 260 °C for 5 min. The sample was injected in split-less mode. Helium was used as a carrier gas with a linear velocity of 40 cm/s. The tem- 2.2.4. Determination of colour parameters and pH values perature programmed was initially isothermal at 40 °C for 10 min, then Colour parameters were measured using a portable colorimeter raised to 200 °C at a rate of 5 °C/min, further raised to 250 °C at a rate of Chroma Meter Cr-400 (Konica Minolta Sensing, Inc., Osaka, Japan) 20 °C/min, and finally held at 250 °C for 5 min: total runtime 49.5 min. with a pulsed xenon arc lamp filtered to illuminant D65 lighting con- Injector and detector temperatures were both set at 260 °C. The mass ditions, with 0° viewing angle geometry and 8 mm aperture size in the spectra was obtained using a mass selective detector working in elec- CIELAB space: lightness, (L*); redness, (a*); yellowness, (b*) (CIE, tronic impact at 70 eV, with a multiplier voltage of 1953 V and col- 1978). Before measurements, the colorimeter was adjusted using a lecting data at a rate of 6.34 scans/s over the range m/z 40–300. white ceramic tile. The colour measures were done in three different The volatile compounds were identified using four different points on the pâté surface. The pH values were determined using a methods: a) comparing their mass spectra with those contained in the digital pH meter (model 710 A+, Thermo Orion, Cambridgeshire, UK) NIST05 (National Institute of Standards and Technology, Gaithersburg) equipped with a penetration probe. library, b) comparing their mass spectra and retention time with au- thentic standards (Supelco, Bellefonte, PA, USA), c) calculating the 2.2.5. Determination of lipid oxidation: Conjugated dienes (CD) and retention index relative to a series of standard alkanes (C5–C14) (for thiobarbituric acid-reactive substances (TBARs) calculating Kovats indexes, Supelco 44,585-U, Bellefonte, PA, USA), Lipid stability was evaluated by assessing the conjugated dienes and d) matching them with data reported in the available literature. − (primary products of lipid oxidation) and the thiobarbituric acid re- The results were expressed as area units (AU) × 10 6/g of dry matter. active substances (secondary products of the lipid oxidation). The conjugated dienes (CD) were quantified following the procedure de- 2.3. Statistical analysis scribed by Pateiro et al. (2014), and thiobarbituric acid reactive sub- stances (TBARs) were calculated from a standard curve of mal- In order to detect significant differences among different types of onaldehyde (MDA) produced from with 1,1–3,3 tetraethoxypropane batches as well as storage times, an analysis of variance (ANOVA) of (TEP) and expressed as mg MDA/kg sample (Vyncke, 1975). one way was performed for all the variables evaluated in the present study, using the IBM SPSS Statistics 23.0 program (IBM Corporation, 2.2.6. Determination of protein oxidation Somers, NY, USA). The least squares mean (LSM) were separated using Protein oxidation was determined by measuring the carbonyl for- Duncan's t-test. mation. A sample of pâté (2.5 g) was homogenized into 20 mL of NaCl 0.6 M using an IKA T25 digital ultra-turrax (IKA®-Werke GmbH & Co. 3. Results and discussion KG, Staufen, Germany). An aliquot of 100 μL was precipitated with 1 mL of TCA 10%, mixed for 30 s in a vortex and centrifuged at 3.1. Effect of extracts from seaweeds on the chemical composition of low-fat 5000 ×g/5 min. The resulting pellet was incubated with 1 mL of dini- pork liver pâtés trophenylhydrazine (DNPH) 0.2% in HCl 2 M under darkness for 1 h, shaking every 20 min. Then, it was washed three times with 1 mL of The nutritional composition (moisture, fat and protein) of low-fat ethanol/ethyl acetate 1:1 (v/v). Finally, the resulting pellet was dis- pork liver pâté batches is shown in Table 1. As can be seen, no sig- solved in 1.5 mL of guanidine 6 M in NaH2PO4 20 mM buffer, and the nificant differences (P > 0.05) were found among the different batches absorbance was measured at 370 and 280 nm against guanidine as regarding their moisture and fat contents. This result is in agreement blank using an Agilent 8453 UV–visible spectrophotometer (Agilent with those reported by other authors when they evaluated the prox- Technologies, Santa Clara, USA). The protein oxidation values were imate composition of similar liver pâté samples (Estévez et al., 2007; calculated from a standard curve performed with bovine serum albumin Estévez, Ventanas, & Cava, 2006). However, significant differences (BSA) and expressed as nmol carbonyl/mg protein. were detected in protein content among the different samples, since batches made with AN and BB extracts reached significant (P > 0.05) 2.2.7. Analysis of volatile compounds higher values than control samples CON (without antioxidant added) The extraction of volatile compounds was performed according to and BHT (with BHT added as antioxidant) batches. A possible ex- previous studies (Lorenzo & Fonseca, 2014; Lorenzo, Gómez, Purriños, planation for this fact might be the important protein content of sea- & Fonseca, 2016) using solid-phase microextraction (SPME). A SPME weed extracts. Indeed, (Lorenzo et al., 2017) quantified the protein device (Supelco, Bellefonte, PA, USA) containing a fused-silica fibre content in Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bi- (10 mm length) coated with a 50/30 μm thickness of DVB/CAR/PDMS furcata extracts being 26.08, 62.05 and 53.33 g/100 g of extract, re- (divinylbenzene/carboxen/polydimethylsilox-ane) was used for HS- spectively. SPME extraction. One gram of sample was weighted into a 40 mL vial Regarding fatty acid (FA) composition, there were no significant and the vial was screw-capped with a laminated teflon-rubber disk. The differences (P > 0.05) among the different batches for any FA, being fibre was inserted into the sample vial through the septum and then MUFA the predominant (55%) FA group. Within MUFA, oleic acid was exposed to headspace. The extractions were carried out in an oven to the main fatty acid (49.61%). These values are similar to those reported ensure a homogeneous temperature for the sample and headspace. Prior by several authors who worked with natural antioxidants in the for- to analysis, the fibre was conditioned by heating it into a gas chroma- mulation of liver pâtés (Estévez et al., 2007; Pateiro et al., 2014; tograph injection port at 270 °C for 60 min, following the manufacturer Pateiro, Lorenzo, Vázquez, & Franco, 2015). specifications. Extraction was performed at 35 °C for 30 min. Before Polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFAs) extraction, samples were equilibrated for 15 min at the temperature were the following FAs in quantitative importance, with mean values of used for the extraction. Once sampling was finished, the fibre was 22.75% and 21.82%, respectively. Within PUFAs, linoleic acid was the

402 R. Agregán et al. Food Research International 112 (2018) 400–411

Table 1 3.2. Effect of extracts from seaweed on the evolution of microbial counts of Effect of several antioxidants on the proximate and fatty acid composition (% of low-fat pork liver pâtés during storage total fatty acids, only FA > 0.5% were shown) of low-fat pork liver pâtés. Values are the mean of nine replicates. Changes in microbial counts during chilled storage of low-fat pork CON BHT AN FV BB SEM Sig liver pâtés are not shown. With regard to total viable counts (TVC), there were not observed significant differences (P > 0.05) among the Moisture (%) 52.88 53.45 52.05 53.03 52.65 0.18 ns batches with different antioxidants added. TVCs were very low at day 0 Fat (%) 23.98 23.83 23.85 23.87 24.10 0.26 ns Protein (%) 13.89a 13.99a 14.42b 14.17ab 14.34b 0.06 ** and during the whole storage in all batches (counts < 1 Log CFU/g for all batches and sampling points). These values were lower than those FA profile reported by other authors when they analyzed microbial contamination C14:0 0.58 0.58 0.58 0.58 0.58 0.00 ns C16:0 13.04 13.04 13.05 13.06 13.05 0.05 ns of pâté samples (Delgado-Pando et al., 2011; Delgado-Pando, Cofrades, C16:1n-7 1.18 1.17 1.18 1.18 1.17 0.01 ns Ruiz-Capillas, Triki, & Jiménez-Colmenero, 2012; Lorenzo, Pateiro, C18:0 7.26 7.30 7.27 7.29 7.29 0.04 ns Fontán, & Carballo, 2014). C18:1n-9c 49.63 49.58 49.63 49.60 49.65 0.04 ns During storage, the evolution of TVC was very similar in all batches, C18:1n-7c 3.00 3.03 3.04 3.05 3.05 0.01 ns C18:2n-6c 17.51 17.47 17.48 17.47 17.45 0.05 ns obtaining counts < 3 Log CFU/g at the end of the storage. These C20:1n-9 0.89 0.89 0.89 0.89 0.89 0.00 ns findings are in close agreement to those reported by Delgado-Pando C18:3n-3 3.66 3.66 3.66 3.66 3.66 0.01 ns et al. (2012), who observed values < 3 Log CFU/g after 85 days re- C20:4n-6 0.85 0.86 0.84 0.84 0.84 0.00 ns frigerated storage in pâtés enriched with n-3 PUFA and konjac gel SFA 21.78 21.86 21.81 21.85 21.82 0.08 ns added. As in the case of TVC, no significant differences (P > 0.05) MUFA 55.43 55.39 55.44 55.42 55.46 0.03 ns PUFA 22.80 22.75 22.75 22.74 22.72 0.06 ns among the batches were found for lactic acid bacteria (LAB) counts, Ʃn-3 3.80 3.79 3.79 3.77 3.79 0.01 ns which showed values < 1 Log CFU/g. This result is also similar to that Ʃn-6 18.95 18.92 18.92 18.91 18.89 0.05 ns reported by Delgado-Pando et al. (2012), who found counts < 1 Log n-6/n-3 4.98 4.99 4.99 5.01 4.99 0.02 ns CFU/g for LAB in all samples during the storage. On the other hand, Pseudomonas spp., molds and yeasts were not CON, control batch; BHT, batch manufactured with addition of tert-butyl-4- hydroxytoluene; AN, batch manufactured with addition of Ascophylum nodosum observed in any of the samples, independently of the batch and storage seaweed extract; FV, batch manufactured with addition of Fucus vesiculosus times (data not shown). This result is consistent with those obtained by seaweed extract; BB, batch manufactured with addition of Bifurcaria bifurcata Lorenzo et al. (2014), who did not find Pseudomonas spp. in pâté seaweed extract. samples after 5 months of storage. Overall, our results showed that the a–bMeans in the same row not followed by a common superscript letter are thermal treatment applied was effective to avoid Pseudomonas spp., significantly different (P < 0.05; Duncan test). molds and yeasts growth for all batches assayed, being enough to SEM: standard error of mean. Sig.: significance: **(P < 0.01); n.s. (not significant). control the growth of the microorganisms studied. SFA = Ʃ (C14:0 + C15:0 + C16:0 + C17:0 + C18:0 + C20:0 + C22:0 + C24:0). MUFA = Ʃ (C18:1n-9 t + C18:1n-9c + C18:1n-7c + C20:1n-9 + C22:1n-9 + 3.3. Effect of extracts from seaweed on physical properties of low-fat pork C24:1n-9). PUFA = Ʃ (C18:2n-6c + C18:3n-6 + C18:3n-3 + 9c,11 t-CLA + liver pâtés during storage C20:2n-6 + C20:3n-6 + C20:4n-6 + C22:5n-3 + C22:6n-3). Ʃn-6=Ʃ (C18:3n- 6 + C20:2n-6 + C20:3n-6 + C20:4n-6). Ʃn-3=Ʃ (C18:3n-3 + C22:5n-3 + C22:6n-3). The evolution of pH and colour parameters of low-fat pork liver pâté batches during refrigerated storage is shown in Fig. 1-A. In general, no significant differences (P > 0.05) were detected for pH values among predominant one (17.47%), followed by linolenic acid (3.6%). In this the different batches, independently of the storage time. For instance, line, Estévez et al. (2007) and Pateiro et al. (2014, 2015) found that Pateiro et al. (2015) also observed the same trend for pH values in pig linoleic acid was the predominant PUFA when they studied similar liver pâtés with natural antioxidants from grape and tea added, except samples, while linolenic acid values were lower than 1%. The differ- for the initial time. Within each batch, the same pH evolution pattern ences observed in this study compared to those found at the available was observed, showing an increase in pH values after 45 days storage literature are attributed to the use of different fat sources in pâté followed by a progressive decrease until 135 days, and a new increase manufacturing. Indeed, Delgado-Pando, Cofrades, Rodríguez-Salas, and at the end of storage. However, the variations in pH values during Jiménez-Colmenero (2011) reported higher percentages for linoleic storage were minimal and only significant differences (P < 0.05) acid (11.64%) due to the total replacement of animal fat with vegetable among storage times were found for AN and FV batches. oils. On the other hand, no significant differences (P > 0.05) were ob- Among SFAs, palmitic acid was the predominant compound served in colour parameters (L*, a*, b*) neither between batches nor (13.04%) followed by stearic acid (7.28%). The results are in close along the time (Fig. 1B–D), except for the samples analyzed at the end agreement to those previously reported by Estévez et al. (2007) and of the storage, where the L* values were significantly lower (P < 0.05) Pateiro et al. (2014, 2015) when they evaluated liver pâté samples, in the FV and BB compared to the other batches. Moreover, after although these authors found higher values for palmitic (~20%) and 180 days of storage, the a* and b* values were also significantly lower stearic (~11.5%) acids. (P < 0.05) in the CON batch than in the batches manufactured with As expected, the use of rich-PUFA oils replacing pork backfat in the antioxidants added. These results agree partially with the findings of manufacture of liver pâtés had a great significant influence in the FA other studies, in which with the exception of the last point (end of re- profile decreasing SFAs and increasing PUFAs, thus improving P/S and frigerated storage), no significant differences in luminosity (P > 0.05) n-6/n-3 ratios in order to satisfy the nutritional recommendations of the were detected in porcine liver pâtés elaborated with sage and rosemary FAO for human diet (Anonymous, 2010). These results contrast with essential oils as antioxidants among batches (Estévez et al., 2006). those found by Pateiro et al. (2014, 2015) who reported more un- However, in the referred study, the authors found significant differ- favorable ratios for P/S and n-6/n-3 in pork liver pâtés. However, in ences (P < 0.05) among sampling times during storage for each in- these studies the pork backfat was not replaced. dividual batch. On the other hand, Pateiro et al. (2014, 2015) reported significant differences (P < 0.001) among batches during storage of pâtés, but not among sampling times during refrigerated storage of batches elaborated with grape seed extract, which might be explained by the fact that antioxidants from extract provide a good stabilization of

403 R. Agregán et al. Food Research International 112 (2018) 400–411

Fig. 1. Evolution of pH (A), luminosity (B), index of red (C) and index of yellow (D) during refrigerated storage of low-fat pork liver pâté manufactured with added BHT and natural antioxidant extracts from seaweed. Plotted values are means ± standard deviations of nine replicates. CON, control batch; BHT, batch manufactured with addition of tert-butyl-4- hydroxytoluene; AN, batch manufactured with addition of Ascophyllum nodosum seaweed extract; FV, batch manufactured with addition of Fucus vesiculosus seaweed extract; BB, batch manufactured with addition of Bifurcaria bifurcata seaweed extract. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) colour. Ambrosiadis, Fletouris, and Papageorgiou (2014), who also reported an evolution of conjugated dienes content during storage of cooked pork 3.4. Effect of extracts from seaweed on the oxidative stability of low-fat meat patties with olive leaf extract added. On the contrary, the results pork liver pâtés during storage obtained in our study do not support the previous research of Karwowska, Wójciak, and Dolatowski (2015), who obtained a greater The evaluation of lipid stability of low-fat pork liver pâtés was in- lipid stability for organic fermented sausages elaborated with mustard vestigated by the determination of the primary and secondary lipid seed and without nitrites. oxidation products through measurements of conjugated dienes and Concerning secondary lipid oxidation, TBARs index was used as an TBARs, respectively (Fig. 2A–B). The values obtained for conjugated indicator of this reaction. The results of our study indicated significant dienes showed a reasonable stability of pâtés for primary lipid oxida- differences among the different batches at the end of the storage, being tion. the values observed in the CON batch (0.39 ± 0.12 mg MDA/kg pâté) In the present study, we did not find significant differences significantly higher (P < 0.01) than those observed for the batches (P > 0.05) among batches at any storage time. Regarding the differ- manufactured with BHT (0.21 ± 0.06 mg MDA/kg pâté) or when ences among storage times within each batch, these were significant for seaweed extracts (values from 0.19 to 0.21 mg MDA/kg pâté) were BHT (P < 0.05), CON (P < 0.01) FV (P < 0.001) and AN and BB used. Within each batch, a significant difference (P < 0.05) was ob- (P < 0.001) batches. A similar trend was observed for all the batches, served among storage times. increasing the values of conjugated dienes from 0 to 45 days. In this sense, the trend was different among the different batches. In Afterwards, the values of conjugated dienes significantly decreased CON batch the values after 180 days of storage were significantly from day 45 to day 135, and a new increase (generally not significant) higher (P < 0.05) than the values at 0 days of storage, while in BHT, was observed from day 135 to day 180 of storage. AN, FV and BB batches the values were similar or even lower than the At the end of the storage, the values of conjugated dienes were values observed after 180 days of storage and in samples at 0 days. higher for the CON batch (mean values of 2.91 μmol/g pâté) than for According to Karwowska et al. (2015), this decrease could be at- the batches manufactured with antioxidants (mean values ranging from tributed to the reaction of MDA with amino acids, sugars and nitrites in 2.19 to 2.53 μmol/g pâté), but these differences were not significant complex foods as liver pâté. This result is in agreement with those re- (P > 0.05). The use of antioxidants, therefore, did not significantly ported by several authors (Estévez & Cava, 2004; Pateiro et al., 2014, affect the evolution of the conjugated dienes during the storage. This 2015) who detected modifications in TBARs values during storage of finding is in agreement with the results obtained by Botsoglou, Govaris, pâtés. Overall, a strong stability with respect to lipid oxidation was

404 R. Agregán et al. Food Research International 112 (2018) 400–411

Fig. 2. Evolution of conjugated dienes (A), TBARs index (B) and carbonyls (C) during refrigerated storage of low-fat pork liver pâté manufactured with added BHT and natural antioxidants extracts from seaweed. Plotted values are means ± standard deviations of nine replicates. CON, control batch; BHT, batch manufactured with addition of tert-butyl-4- hydroxytoluene; AN, batch manufactured with addition of Ascophyllum nodosum seaweed extract; FV, batch manufactured with addition of Fucus vesiculosus seaweed extract; BB, batch manufactured with addition of Bifurcaria bifurcata seaweed extract. observed in pâtés for all storage time, even in the control batch. A a result of oxidative degradation of the side chains of lysine, proline, possible explanation for these results is related to the fact that the pâtés arginine and histidine residues (Stadtman & Levine, 2003). Except for were completely filled and hermetically closed, thus avoiding the pre- the values at 180 days of storage, no significant differences (P > 0.05) sence of a head space and the subsequent oxygen entrance. were found among the different batches, independently of the storage On the other hand, protein oxidation was followed by the carbonyl time. Overall, a significant increase in protein oxidation during storage compound measurement (Fig. 2-C). Carbonyl compounds are formed as was observed for all the batches elaborated, reaching values

405 R. Agregán et al. Food Research International 112 (2018) 400–411

Table 2 − − Effect of the addition of several antioxidants on the evolution of volatile compounds (AU × 10 6 g 1dry matter) in the headspace of low-fat pork liver pâtés. Values are the means of nine replicates.

Day R Batch SEM Sig

CO BHT AN FV BB

Hydrocarbons α-Pinene 0 ms 1.38 ± 0.19a 0.00 ± 0.00by 1.47 ± 0.05ay 0.00 ± 0.00by 0.88 ± 0.01cy 0.14 *** 180 ms 1.13 ± 0.32a 0.82 ± 0.16bz 0.00 ± 0.00cz 2.00 ± 0.31dz 1.53 ± 0.09ez 0.14 *** SEM 0.08 0.14 0.25 0.32 0.12 Sig ns *** *** *** *** 5-Undecene, 9-methyl-, (Z)- 0 ms 8.90 ± 1.88a 9.207 ± 5.52ay 9.33 ± 3.49a 8.11 ± 1.72a 9.31 ± 4.65a 0.65 ns 180 ms 8.86 ± 1.15a 6.03 ± 0.93bcy 5.54 ± 1.66b 7.68 ± 0.41ac 6.74 ± 1.59bc 0.33 ** SEM 0.46 1.29 1.03 0.38 1.08 Sig ns ns ns ns ns Cyclopentane, hexyl- 0 ms 1.92 ± 0.33ay 1.16 ± 0.07by 1.74 ± 0.50ab 2.13 ± 0.58a 1.23 ± 0.14b 0.11 ** 180 ms 0.00 ± 0.00az 0.00 ± 0.00az 1.21 ± 0.26b 1.57 ± 0.37c 1.36 ± 0.22bc 0.15 *** SEM 0.31 0.19 0.15 0.17 0.07 Sig *** *** ns ns ns Cyclopentane, pentyl- 0 ms 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ns 180 ms 8.90 ± 2.21az 5.65 ± 0.87bz 6.40 ± 1.10bz 8.46 ± 0.68az 6.57 ± 1.11bz 0.35 ** SEM 1.46 0.96 1.09 1.34 1.24 Sig *** *** *** *** *** Decane 0 ms 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ns 180 ms 6.86 ± 1.44abz 5.71 ± 1.16az 7.79 ± 0.85bz 9.56 ± 1.65cz 6.06 ± 0.57az 0.36 *** SEM 1.11 0.98 1.31 1.54 1.12 Sig *** *** *** *** *** Dodecane 0 ms 2.50 ± 0.35ay 2.47 ± 0.99ay 2.08 ± 0.12ay 11.08 ± 7.18b 2.82 ± 0.81ay 1.00 ** 180 ms 11.97 ± 3.09abz 10.11 ± 1.86az 9.22 ± 1.60az 13.73 ± 0.74b 12.25 ± 2.69abz 0.51 * SEM 1.61 1.35 1.24 1.59 1.87 Sig *** *** *** ns ** Eicosane 0 ms 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ns 180 ms 1.49 ± 0.43az 0.89 ± 0.13bz 0.73 ± 0.26bz 0.86 ± 0.23bz 0.38 ± 0.05cz 0.09 *** SEM 0.25 0.15 0.13 0.14 0.07 Sig *** *** *** *** *** Heptane, 2,2,4,6,6-pentamethyl- 0 ms 160.24 ± 45.53ay 206.42 ± 21.44aby 187.46 ± 50.43aby 155.50 ± 25.01ay 225.48 ± 7.19by 8.31 * 180 ms 11.36 ± 1.86az 7.78 ± 0.59bz 8.15 ± 1.29bz 13.07 ± 0.98cz 10.58 ± 0.96az 0.46 *** SEM 25.38 33.41 31.72 23.05 39.35 Sig *** *** *** *** *** Heptane, 3-ethyl- 0 ms 1.97 ± 0.48ab 1.55 ± 0.27a 2.61 ± 0.44c 2.39 ± 0.53bc 2.71 ± 0.22c 0.12 ** 180 ms 2.11 ± 0.67a 1.00 ± 0.57b 2.60 ± 0.41ac 3.15 ± 0.66c 2.20 ± 0.68a 0.18 *** SEM 0.16 0.16 0.13 0.21 0.21 Sig ns ns ns ns ns Heptane, 3-methylene- 0 ms 0.00 ± 0.00ay 0.00 ± 0.00ay 0.00 ± 0.00ay 0.00 ± 0.00ay 0.00 ± 0.00ay 0.00 ns 180 ms 2.03 ± 0.29az 0.00 ± 0.00by 3.68 ± 1.47cz 2.79 ± 0.97acz 4.14 ± 1.57cz 0.36 *** SEM 0.32 0.00 0.69 0.48 0.87 Sig *** ns *** *** ** Hexane 0 ms 18.76 ± 3.56ay 4.98 ± 0.50by 29.68 ± 5.07cy 18.97 ± 7.46ay 26.98 ± 1.70cy 1.91 *** 180 ms 26.75 ± 3.54az 2.37 ± 0.75az 13.50 ± 2.81az 7.41 ± 1.32bz 7.70 ± 2.16bz 1.76 *** SEM 1.62 0.47 2.96 2.43 3.59 Sig ** *** *** ** *** Nonane 0 ms 3.03 ± 0.26aby 2.55 ± 0.83ay 3.34 ± 0.49by 2.50 ± 0.20ay 3.44 ± 0.47by 0.12 * 180 ms 0.00 ± 0.00z 0.00 ± 0.00z 0.00 ± 0.00z 0.00 ± 0.00z 0.00 ± 0.00z 0.00 ns SEM 0.48 0.46 0.57 0.40 0.64 Sig *** *** *** *** *** Nonane, 3-methyl- 0 ms 1.25 ± 0.33a 0.00 ± 0.00by 1.25 ± 0.42a 0.00 ± 0.00by 0.68 ± 0.11cy 0.13 *** 180 ms 1.52 ± 0.45b 1.10 ± 0.21z 1.55 ± 0.15 1.78 ± 0.38z 1.78 ± 0.58z 0.09 ns SEM 0.12 0.19 0.11 0.29 0.25 Sig ns *** ns *** * Octane 0 ms 2.17 ± 0.25y 2.08 ± 0.36y 3.08 ± 1.41 1.73 ± 0.32 1.81 ± 0.71 0.16 ns 180 ms 1.71 ± 0.12az 0.78 ± 0.06bz 1.89 ± 0.28a 1.79 ± 0.54a 2.35 ± 0.94a 0.14 ** SEM 0.09 0.23 0.36 0.12 0.30 Sig ** *** ns ns ns Pentane, 2,3,3-trimethyl- 0 ms 0.00 ± 0.00y 0.00 ± 0.00ay 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ns 180 ms 3.32 ± 0.71az 3.81 ± 0.71az 3.22 ± 0.68az 3.36 ± 1.01az 6.81 ± 1.74bz 0.34 *** SEM 0.54 0.65 0.56 0.56 1.33 Sig *** *** *** *** *** Pentane, 2,3,4-trimethyl- 0 ms 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ns 180 ms 0.87 ± 0.10az 1.05 ± 0.20acz 0.84 ± 0.08az 2.90 ± 0.52bz 1.42 ± 0.30cz 0.18 *** SEM 0.14 0.19 0.14 0.47 0.30 Sig *** *** *** *** *** Tridecane 0 ms 0.00 ± 0.00ay 0.91 ± 0.24by 0.87 ± 0.27by 0.00 ± 0.00ay 1.06 ± 0.10 by 1.04 *** 180 ms 5.52 ± 1.52az 1.98 ± 0.75bz 3.13 ± 0.71bz 4.68 ± 0.31az 5.25 ± 1.03az 0.33 *** SEM 0.92 0.24 0.46 0.74 0.82 Sig *** * ** *** ** (continued on next page)

406 R. Agregán et al. Food Research International 112 (2018) 400–411

Table 2 (continued)

Day R Batch SEM Sig

CO BHT AN FV BB

Undecane 0 ms 2.82 ± 0.46ay 2.63 ± 0.83ay 2.84 ± 0.69ay 2.72 ± 0.26ay 2.63 ± 0.82ay 0.11 ns 180 ms 16.42 ± 4.33abz 13.88 ± 2.45az 19.79 ± 2.66bcz 22.21 ± 1.89cz 15.09 ± 2.07az 0.81 *** SEM 2.30 1.95 2.88 3.09 2.35 Sig *** *** *** *** *** Undecane, 3-methyl- 0 ms 0.00 ± 0.00y 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00y 0.00 ± 0.00 0.00 ns 180 ms 4.14 ± 1.45az 0.00 ± 0.00b 0.00 ± 0.00b 3.57 ± 0.30az 0.00 ± 0.00b 0.41 *** SEM 0.71 0.00 0.00 0.57 0.00 Sig *** ns ns *** ns Undecane, 3-methylene- 0 ms 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00y 0.00 ns 180 ms 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 1.29 ± 0.34bz 0.11 *** SEM 0.00 0.00 0.00 0.00 0.25 Sig ns ns ns ns *** Total hydrocrabons 0 ms 204.94 ± 46.57y 233.96 ± 25.02y 245.40 ± 55.00y 205.13 ± 27.66y 279.05 ± 11.15y 8.69 ns 180 ms 110.81 ± 16.36az 62.77 ± 2.77bz 89.23 ± 9.35cz 107.00 ± 6.35az 91.94 ± 7.02cz 3.88 *** SEM 18.11 29.02 28.56 16.58 34.35 Sig ** *** *** *** ***

Alcohols 2-Propanol, 1,1′-oxybis- 0 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ± 0.00y 0.00 ns 180 ms 1.50 ± 0.29az 1.55 ± 0.41az 1.83 ± 0.32az 2.31 ± 0.37bz 0.00 ± 0.00cz 0.17 *** SEM ms 0.24 0.27 0.31 0.37 0.00 Sig *** *** *** *** *** Cyclohexanol, 2,6-dimethyl- 0 ms 0.87 ± 0.10ay 0.97 ± 0.19aby 1.32 ± 0.40bcy 1.36 ± 0.24cy 1.68 ± 0.33cy 0.07 ** 180 ms 0.57 ± 0.12az 0.00 ± 0.00bz 0.00 ± 0.00bz 0.89 ± 0.13cz 0.00 ± 0.00bz 0.08 *** SEM 0.06 0.17 0.24 0.09 0.31 Sig ** *** *** ** *** Total alcohols 0 ms 0.87 ± 0.10ay 0.97 ± 0.19aby 1.32 ± 0.40bc 1.36 ± 0.24cy 1.68 ± 0.33dy 0.07 ** 180 ms 2.07 ± 0.36az 1.55 ± 0.41bz 1.83 ± 0.32ab 3.20 ± 0.36cz 0.00 ± 0.00dz 0.22 *** SEM 0.20 0.13 0.14 0.30 0.31 Sig *** * ns *** ***

Esters 2,2,4-Trimethyl-1,3-pentanediol 0 ms 1.19 ± 0.55ay 0.00 ± 0.00by 0.00 ± 0.00by 0.00 ± 0.00by 0.68 ± 0.06ay 0.12 *** diisobutyrate 180 ms 2.89 ± 1.28az 1.79 ± 0.54bz 1.97 ± 0.56abz 1.81 ± 0.56bz 0.00 ± 0.00cz 0.23 *** SEM 0.38 0.32 0.35 0.30 0.12 Sig * *** *** *** *** Cyclopentaneacetic acid, 3-oxo-2-pentyl-, 0 ms 0.55 ± 0.17ay 0.00 ± 0.00by 0.44 ± 0.15ay 0.00 ± 0.00by 0.88 ± 0.41c 0.07 *** methyl ester 180 ms 1.23 ± 0.38z 0.99 ± 0.28z 1.09 ± 0.43z 1.04 ± 0.23z 0.73 ± 0.47 0.08 ns SEM 0.13 0.17 0.15 0.17 0.15 Sig ** *** * *** ns Total esters 0 ms 1.75 ± 0.68ay 0.00 ± 0.00by 0.44 ± 0.15by 0.00 ± 0.00by 1.56 ± 0.45a 0.17 *** 180 ms 4.12 ± 1.44az 2.39 ± 0.80bz 3.07 ± 0.91abz 2.85 ± 0.56bz 0.73 ± 0.47c 0.28 *** SEM 0.49 0.43 0.48 0.46 0.22 Sig ** *** *** *** ns

Aldehydes Butanal, 3-methyl- 0 ms 7.42 ± 0.83ay 0.00 ± 0.00b 7.81 ± 0.27ay 7.36 ± 0.48ay 8.57 ± 0.49cy 0.64 *** 180 ms 2.18 ± 0.33az 0.00 ± 0.00b 2.33 ± 0.17acz 2.69 ± 0.19cz 2.71 ± 0.54cz 0.21 *** SEM 0.85 0.00 0.92 0.74 1.09 Sig *** ns *** *** *** Heptanal 0 ms 2.26 ± 0.15ay 1.16 ± 0.06b 2.60 ± 0.33cy 1.87 ± 0.17d 1.50 ± 0.06e 0.11 *** 180 ms 1.54 ± 0.13bcz 1.13 ± 0.16a 1.43 ± 0.17bz 1.73 ± 0.22c 1.70 ± 0.18c 0.06 *** SEM 0.12 0.04 0.21 0.06 0.06 Sig *** ns *** ns ns Hexanal 0 ms 11.63 ± 1.53acy 5.61 ± 0.28by 10.43 ± 0.93ay 10.01 ± 1.79ay 12.89 ± 0.20cy 0.53 *** 180 ms 7.15 ± 0.79az 3.54 ± 0.19bz 5.53 ± 0.40cz 6.96 ± 0.36az 7.39 ± 0.15az 0.30 *** SEM 0.79 0.35 0.84 0.62 1.01 Sig *** *** *** ** *** Pentanal 0 ms 3.01 ± 0.57ay 0.51 ± 0.10by 3.52 ± 0.30cy 2.98 ± 0.38ay 0.00 ± 0.00d 0.28 *** 180 ms 0.00 ± 0.00az 0.00 ± 0.00az 1.76 ± 0.37bz 0.00 ± 0.00az 0.00 ± 0.00a 0.15 *** SEM 0.49 0.09 0.31 0.48 0.00 Sig *** *** *** *** ns Total aldehydes 0 ms 24.32 ± 2.39ay 7.28 ± 0.22by 24.36 ± 0.48ay 22.22 ± 2.16ay 22.96 ± 0.33ay 1.37 *** 180 ms 10.87 ± 0.85az 4.66 ± 0.28bz 11.05 ± 0.30az 11.38 ± 0.53acz 11.80 ± 0.52cz 0.55 *** SEM 2.18 0.44 2.22 1.77 2.05 Sig *** *** *** *** ***

Ketones 2-Heptanone 0 ms 1.10 ± 0.10ay 0.76 ± 0.15by 1.11 ± 0.15ay 1.00 ± 0.05ay 1.09 ± 0.12ay 0.03 *** 180 ms 0.70 ± 0.06az 0.53 ± 0.05bz 0.77 ± 0.05adz 0.89 ± 0.09cz 0.80 ± 0.06dz 0.03 *** SEM 0.07 0.05 0.06 0.03 0.06 Sig *** * ** * ** (continued on next page)

407 R. Agregán et al. Food Research International 112 (2018) 400–411

Table 2 (continued)

Day R Batch SEM Sig

CO BHT AN FV BB

2-Pentanone 0 ms 1.25 ± 0.25ay 0.00 ± 0.00b 1.10 ± 0.22acy 0.97 ± 0.18cy 0.61 ± 0.12dy 0.10 *** 180 ms 0.67 ± 0.19az 0.00 ± 0.00b 0.38 ± 0.02cz 0.67 ± 0.02az 0.41 ± 0.05cz 0.05 *** SEM 0.11 0.00 0.13 0.06 0.04 Sig ** ns *** ** * Total ketones 0 ms 2.36 ± 0.21ay 0.76 ± 0.15by 2.21 ± 0.26acy 1.97 ± 0.17cy 1.71 ± 0.09dy 0.12 *** 180 ms 1.37 ± 0.18az 0.53 ± 0.05bz 1.16 ± 0.06cz 1.56 ± 0.08dz 1.21 ± 0.02cz 0.07 *** SEM 0.17 0.05 0.18 0.08 0.09 Sig *** * *** *** *** Total Volatile compounds 0 ms 234.22 ± 47.01y 242.97 ± 25.14y 273.73 ± 55.25y 230.68 ± 28.15y 306.96 ± 11.06 y 8.80 ns 180 ms 129.24 ± 15.87az 71.90 ± 2.25bz 106.34 ± 10.48cz 125.99 ± 6.73az 105.68 ± 7.02cz 4.50 *** SEM 19.57 29.00 30.31 17.59 36.94 Sig ** *** *** *** ***

CON, control batch; BHT, batch manufactured with addition of tert-butyl-4- hydroxytoluene; AN, batch manufactured with addition of Ascophylum nodosum seaweed extract; FV, batch manufactured with addition of Fucus vesiculosus seaweed extract; BB, batch manufactured with addition of Bifurcaria bifurcata seaweed extract. a–eMeans in the same row (different batches on the same storage day) not followed by a common superscript letter are significantly different (P < 0.05; Duncan test). y–zMeans in the same column (same batch in different storage days) not followed by a common superscript letter are significantly different (P < 0.05; Duncan test). SEM: standard error of mean. Sig.: significance: *(P < 0.05); **(P < 0.01); ***(P < 0.001); n.s. (not significant). AU: area units resulting of counting the total ion chromatogram (TIC) for each compound. R: Reliability of identification. ms: mass spectrum agreed with mass database (NIST05). significantly higher (P < 0.05) in CON batch (13.41 nmol carbonyl/mg heptane, 2,2,4,6,6-pentamethyl, which represented between 75.6% and protein) than in the batches manufactured using antioxidants (values 88.4% of total hydrocarbons at the beginning of the storage. ranging from 11.45 nmol carbonyl/mg protein) after 180 days of sto- The maximum value was achieved in BB batch at 0 − rage. The accumulation of carbonyls mainly occurred from day 45 to (225.48 AU × 10 6/g dry matter). The same compound was found in day 135 of storage, being stabilized from day 135 to day 180 in all dry-cured “lacón” (Domínguez et al., 2016) and in dry-cured ham batches. In this sense, some authors have reported a significantly in- (Bermúdez, Franco, Carballo, & Lorenzo, 2015) with a percentage crease (P < 0.05) in protein oxidation during storage of pâtés from ≈90% of total hydrocarbons. At the end of refrigerated storage, the Iberian and white pigs (Estévez et al., 2006; Estévez & Cava, 2004), values for heptane, 2,2,4,6,6-pentamethyl decreased dramatically, being the highest concentration of carbonyls detected and the end of reaching final levels between 9.1 and 12.5% of total hydrocarbons. storage, as in the present study. Total hydrocarbon amounts were similar (P > 0.05) among all batches at day 0, but significant differences (P < 0.001) were found at the end of storage with the highest values observed in the CON batch 3.5. Effect of extracts from seaweed on the volatile compound of low-fat − (110.81 AU × 10 6/g dry matter). A significant decrease (P < 0.01) pork liver pâtés during storage was noted in total hydrocarbons along the time in all batches, especially − for BB batch with a decrease from (279.05 to 91.94 AU × 10 6/g dry Table 2 summarizes GC-MS data obtained from the analysis of vo- matter), attributed to the strong decrease of the compound heptane, latile compounds from liver pâtés with different antioxidants added. A 2,2,4,6,6-pentamethyl at the end of storage. total of 30 volatile compounds were detected in the headspace of pâté The second most abundant group of volatiles were aldehydes. This at the beginning and at the end of storage by the SPME-gas chroma- chemical family is probably the most interesting lipid-derived volatile tography-mass spectrometer technique. compounds (Shahidi, Rubin, & Souza, 1986) because they can produce The volatile compounds grouped by chemical families, comprised a wide range of flavors and odors (Pateiro et al., 2014). Regarding the 20 hydrocarbons, 2 esters, 2 alcohols, 4 aldehydes, and 2 ketones. The total aldehyde contents, BHT batches reached a significant lower value analysis of the volatile compounds gives an indication of the chemical (P < 0.05) than the rest of batches at day 0 of storage (Table 2). A and metabolic processes that occur during manufacture (Lorenzo, significant decrease (P < 0.001) of the aldehyde content was found for Montes, Purriños, & Franco, 2012) and it is a useful indicator regarding all batches at the end of storage and the content in BHT batches was the oxidative stability and the aroma characteristics of the product significantly lower (P < 0.05) than in the other batches. Aldehydes (Estévez, Ventanas, & Cava, 2005). represented between 2.9% and 10.4% of total volatile compounds at Hydrocarbons were the predominant chemical group, representing day 0, and between 6.4% and 11.2% at day 180 (Fig. 3). between 87.5% and 96.3% of the total volatiles compounds found in the Hexanal, presumably derived from the n-6 fatty acids such as lino- samples at the beginning of the storage and between 83.9% and 86.9% leic and arachidonic acids (Wesierska, Szołtysik, & Rak, 2013), was the at the end (Fig. 3). This result disagree with those reported by other most abundant aldehyde, with values ranging between 42.8% and authors in the same type of product (Estévez et al., 2005, 2004). This 77.2% of total aldehydes at the beginning and values ranging from fact may be explained by the different volatile extraction methods used. 50.0% to 75.7% at the end of the storage. These findings are consistent For instance, there are many factors affecting SPME fibre performance, with those previously published in liver pâté elaborated with green tea, such as the election of the appropriate stationary phase and the ex- chestnut and grape seed extract (Pateiro et al., 2014, 2015). A sig- traction conditions. nificant reduction (P < 0.001) in the hexanal content was found in all In other meat products such as dry-cured lacón (Domínguez, batches after 180 days of storage (Table 2). Munekata, Cittadini, & Lorenzo, 2016) and dry-cured foal sausage The trend was similar in control batch and seaweed-added batches, (Lorenzo et al., 2016) hydrocarbons were the predominant volatile and different for BHT batches, which already showed lower values at compounds. Within hydrocarbons, the main compound detected was

408 R. Agregán et al. Food Research International 112 (2018) 400–411

Fig. 3. Changes in percentage of chemical families of volatiles and in total content of volatiles during storage of low-fat pork liver pâté manufactured with added BHT and natural antioxidants extracts from seaweed. Plotted values are means ± standard deviations of nine replicates. CON, control batch; BHT, batch manufactured with addition of tert-butyl-4- hydroxytoluene; AN, batch manufactured with addition of Ascophyllum nodosum seaweed extract; FV, batch manufactured with addition of Fucus vesiculosus seaweed extract; BB, batch manufactured with addition of Bifurcaria bifurcata seaweed extract.

− the beginning of the storage (5.61 AU × 10 6/g). A possible explana- meat products such as dry-cured “lacón” (Lorenzo & Fonseca, 2014). tion for this fact is that BHT had a strong antioxidant effect during liver Alcohols showed values in the range of 0.37–0.58% at the beginning of pâté heating process, an effect which was not shown by the natural the storage and from 0 to 2.54% at day 180. It was noted a significant antioxidants at this stage of the manufacture, hence the hexanal level increase (P < 0.001) in CON and FV batches at the end of storage. was lower in BHT batches at the beginning and ending of storage. This However, BB batch showed a contrary trend, decreasing from an initial reduction matches with those observed in earlier studies on liver-pâtés value of 0.54% to non-detected total volatile compounds. formulated with tea and grape seed extract after 24 weeks of storage In the present study, two ketones were detected in the headspace of (Pateiro et al., 2014, 2015). pâté, except in BHT batch, where only 2-heptanone was detected. The The other three families of volatiles (ester, alcohols and ketones) most abundant ketone both at the beginning and at the end of storage, represented a minor percentage of total volatile compounds. Esters was 2-heptanone (Table 2), which is in agreement with Pateiro et al. represented values between 0.1% and 0.76% of total volatile com- (2014, 2015), who also found 2-heptanone as the most abundant ketone pounds at day 0, increasing to 0.68% and 3.84% at day 180. (Fig. 3). in liver-pâté both at day 0 and after 24 weeks of storage. Very low amounts (< 0.02%) of esters have been reported in other In addition, this volatile compound was identified as the most

409 R. Agregán et al. Food Research International 112 (2018) 400–411 abundant ketone in dry-cured ham (Bermúdez et al., 2015) and dry- Delgado-Pando, G., Cofrades, S., Ruiz-Capillas, C., Triki, M., & Jiménez-Colmenero, F. cured “lacón” (Lorenzo & Fonseca, 2014). Following the same trend (2012). Enriched n-3 PUFA/konjac gel low-fat pork liver pâté: Lipid oxidation, mi- fi crobiological properties and biogenic amine formation during chilling storage. Meat than the aldehydes, a signi cant decrease was observed in total ketones Science, 92(4), 762–767. after the storage for all batches studied, especially for AN batch with a Domínguez, R., Crecente, S., Borrajo, P., Agregán, R., & Lorenzo, J. M. (2015). Effect of reduction of 47.5%. A similar behavior was observed by Pateiro et al. slaughter age on foal carcass traits and meat quality. Animal, 9(10), 1713–1720. Domínguez, R., Munekata, P. E., Cittadini, A., & Lorenzo, J. M. (2016). Effect of the (2015) in pâtés with 50 and 200 ppm of grape seed extract. Overall partial NaCl substitution by other chloride salts on the volatile profile during the after 180 days of storage, a significant decline of total volatile com- ripening of dry-cured lacón. Grasas y Aceites, 67(2), 128. pounds was noted in all batches studied, mainly attributed to a strong Estévez, M., & Cava, R. (2004). Lipid and protein oxidation, release of iron from heme reduction of heptane, 2,2,4,6,6-pentamethyl, as commented above. The molecule and colour deterioration during refrigerated storage of liver pâté. Meat Science, 68(4), 551–558. total volatile compounds at the end of the storage were significantly Estévez, M., Ramírez, R., Ventanas, S., & Cava, R. (2007). Sage and rosemary essential oils (P < 0.001) lower in BHT, AN, and BB batches than in CON batch, versus BHT for the inhibition of lipid oxidative reactions in liver pâté. LWT - Food – being also significantly (P < 0.001) lower in BHT batch than in AN Science and Technology, 40(1), 58 65. Estévez, M., Ventanas, S., & Cava, R. (2005). Physicochemical properties and oxidative and BB batches. stability of liver pâté as affected by fat content. Food Chemistry, 92(3), 449–457. Estévez, M., Ventanas, S., & Cava, R. (2006). Effect of natural and synthetic antioxidants 4. Conclusions on protein oxidation and colour and texture changes in refrigerated stored porcine liver pâté. Meat Science, 74(2), 396–403. Estévez, M., Ventanas, S., Ramírez, R., & Cava, R. (2004). Analysis of volatiles in porcine A strong lipid oxidation stability was observed in the pâtés along the liver pâtés with added sage and rosemary essential oils by using SPME-GC-MS. storage time in all the treatments studied, even in the pâtés without Journal of Agricultural and Food Chemistry, 52(16), 5168–5174. Granato, D., Nunes, D. S., & Barba, F. J. (2017). An integrated strategy between food added antioxidants. Nevertheless, the pâtés with antioxidants (seaweed chemistry, biology, nutrition, pharmacology, and statistics in the development of extracts and BHT) showed a slight degree of protection at the end of the functional foods: A proposal. Trends in Food Science and Technology, 62,13–22. storage. However, it was not possible to assess the antioxidant potential Gupta, M. K. (2014). Sunflower oil: History, applications and trends. Lipid Technology, 26(11−12), 260–263. of these extracts in the conditions established in the study due to the Gupta, S., & Abu-Ghannam, N. (2011). Recent developments in the application of sea- high oxidative stability of the samples. Therefore, subsequent studies weeds or seaweed extracts as a means for enhancing the safety and quality attributes have to be carried out in order to correct the shortcomings appeared in of foods. Innovative Food Science & Emerging Technologies, 12(4), 600–609. the present work, and thus being able to study the antioxidant effect of Holdt, S. L., & Kraan, S. (2011). Bioactive compounds in seaweed: Functional food ap- plications and legislation. Journal of Applied Phycology, 23(3), 543–597. the extracts reliably. ISO (International Organization for Standardization) (1978). Determination of nitrogen On the other hand, the partial pork backfat replacement by canola content, ISO 937:1978 standard. International standards meat and meat products. and high-oleic sunflower oils improved the fatty acid profile of the Switzerland: International Organization for Standardization.Genéve. ISO (International Organization for Standardization) (1997). Determination of moisture pâtés, reaching healthier P/S and n-6/n-3 ratios. This achievement content, ISO 1442:1997 standard. International standards meat and meat products. constitutes an added value for a product such as pâté, which is usually Switzerland: International Organization for Standardization.Genéve. made with a high content of animal fat. Johnson, G. H., Keast, D. R., & Kris-Etherton, P. M. (2007). Dietary modeling shows that the substitution of canola oil for fats commonly used in the United States would increase compliance with dietary recommendations for fatty acids. Journal of the Acknowledgments American Dietetic Association, 107(10), 1726–1734. Juntachote, T., Berghofer, E., Siebenhandl, S., & Bauer, F. (2006). The antioxidative properties of holy basil and in cooked ground pork. Meat Science, 72(3), The authors give thanks to INIA (Instituto Nacional de Investigación 446–456. y Tecnología Agraria y Alimentaria, Spain) for granting Ruben Agregán Kanner, J., Hazan, B., & Doll, L. (1988). Catalytic “free” iron ions in muscle foods. Journal with a predoctoral scholarship (CPR2014-0128). The authors also give of Agricultural and Food Chemistry, 36(3), 412–415. Karwowska, M., Wójciak, K. M., & Dolatowski, Z. J. (2015). The influence of acid whey thanks to Aceites Abril, Ltd. (Ourense, Spain) for supplying the canola and mustard seed on lipid oxidation of organic fermented sausage without nitrite. and high-oleic sunflower oils used in this research. José Manuel Journal of the Science of Food and Agriculture, 95(3), 628–634. Lorenzo is member of the MARCARNE network, funded by CYTED (ref. Lindberg Madsen, H., & Bertelsen, G. (1995). Spices as antioxidants. Trends in Food Science – 116RT0503). and Technology, 6(8), 271 277. Lorenzo, J. M., Agregán, R., Munekata, P. E. S., Franco, D., Carballo, J., Şahin, S., ... J, F. (2017). Proximate composition and nutritional value of three macroalgae: References Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcata. Marine Drugs, 15(11), 360. Lorenzo, J. M., & Fonseca, S. (2014). Volatile compounds of Celta dry-cured “lacón” as Anonymous (2010). Fats and fatty acids in human nutrition. FAO report of an expert affected by cross-breeding with Duroc and Landrace genotypes. Journal of the Science consultation. FAO food and nutrition paper. vol. 91. Rome, Italy: (FAO, Ed.). Food and of Food and Agriculture, 94(14), 2978–2985. Agriculture Organization of the United Nations. Lorenzo, J. M., Gómez, M., Purriños, L., & Fonseca, S. (2016). Effect of commercial starter ffi AOCS o cial procedure am 5-04. In AOCS (Ed.). Rapid determination of oil/fat utilizing cultures on volatile compound profile and sensory characteristics of dry-cured foal high temperature solvent extraction. Urbana, IL, USA: (AOCS, Ed.). American Oil sausage. Journal of the Science of Food and Agriculture, 96, 1194–1201. Chemists Society. Lorenzo, J. M., Montes, R., Purriños, L., & Franco, D. (2012). Effect of pork fat addition on Aruoma, O. I., Halliwell, B., Aeschbach, R., & Löligers, J. (1992). Antioxidant and pro- the volatile compounds of foal dry-cured sausage. Meat Science, 91(4), 506–512. oxidant properties of active rosemary constituents: Carnosol and carnosic acid. Lorenzo, J. M., Pateiro, M., Fontán, M. C. G., & Carballo, J. (2014). Effect of fat content on – Xenobiotica, 22(2), 257 268. physical, microbial, lipid and protein changes during chill storage of foal liver pâté. Barba, F. J., Grimi, N., & Vorobiev, E. (2014). New approaches for the use of non-con- Food Chemistry, 155,57–63. ventional cell disruption technologies to extract potential food additives and nu- Martin, D., Ruiz, J., Kivikari, R., & Puolanne, E. (2008). Partial replacement of pork fat by – traceuticals from microalgae. Food Engineering Reviews, 7(1), 45 62. conjugated linoleic acid and/or olive oil in liver pâtés: Effect on physicochemical fl Bermúdez, R., Franco, D., Carballo, J., & Lorenzo, J. M. (2015). In uence of type of characteristics and oxidative stability. Meat Science, 80(2), 496–504. muscle on volatile compounds throughout the manufacture of Celta dry-cured ham. Mc Carthy, T. L., Kerry, J. P., Kerry, J. F., Lynch, P. B., & Buckley, D. J. (2001). – Food Science and Technology International, 21(8), 581 592. Assessment of the antioxidant potential of natural food and plant extracts in fresh and ff Botsoglou, E., Govaris, A., Ambrosiadis, I., Fletouris, D., & Papageorgiou, G. (2014). E ect previously frozen pork patties. Meat Science, 57(2), 177–184. of olive leaf (Olea europea L.) extracts on protein and lipid oxidation in cooked pork Morales-Irigoyen, E. E., Severiano-Pérez, P., Rodriguez-Huezo, M. E., & Totosaus, A. meat patties enriched with n-3 fatty acids. Journal of the Science of Food and (2012). Textural, physicochemical and sensory properties compensation of fat re- – Agriculture, 94(2), 227 234. placing in pork liver pâté incorporating emulsified canola oil. Food Science and Chojnacka, K., Saeid, A., Witkowska, Z., & Tuhy, L. (2012). Biologically active compounds Technology International, 18(4), 413–421. — in seaweed extracts The prospects for the application. The open conference pro- Parniakov, O., Barba, F. J., Grimi, N., Marchal, L., Jubeau, S., Lebovka, N., & Vorobiev, E. ceedings journal. (2015). Pulsed electric field assisted extraction of nutritionally valuable compounds ff CIE (1978). Recommendations on uniform color spaces: color-di erence equations, psycho- from microalgae Nannochloropsis spp. using the binary mixture of organic solvents metric color terms. (Commission internationale de l'Eclairage, Ed.). Paris. France: and water. Innovative Food Science & Emerging Technologies, 27,79–85. Commission Internationale de l'Eclairage. Pateiro, M., Lorenzo, J. M., Amado, I. R., & Franco, D. (2014). Effect of addition of green Delgado-Pando, G., Cofrades, S., Rodríguez-Salas, L., & Jiménez-Colmenero, F. (2011). A tea, chestnut and grape extract on the shelf-life of pig liver pate. Food Chemistry, 147, healthier oil combination and konjac gel as functional ingredients in low-fat pork 386–394. – liver pâté. Meat Science, 88(2), 241 248. Pateiro, M., Lorenzo, J. M., Vázquez, J. A., & Franco, D. (2015). Oxidation stability of pig

410 R. Agregán et al. Food Research International 112 (2018) 400–411

liver pâté with increasing levels of natural antioxidants (grape and tea). Antioxidants, composition, techniques of analysis, and sensory evaluation. Critical Reviews in Food 4(1), 102–123. Science and Nutrition, 24(2), 141–243. Poojary, M. M., Barba, F. J., Aliakbarian, B., Donsì, F., Pataro, G., Dias, D. A., & Juliano, P. Stadtman, E. R., & Levine, R. L. (2003). Free radical-mediated oxidation of free amino (2016). Innovative alternative technologies to extract carotenoids from microalgae acids and amino acid residues in proteins. Amino Acids, 25(3–4), 207–218. and seaweeds. Marine Drugs, 14(11), 214. Vyncke, W. (1975). Evaluation of the direct thiobarbituric acid extraction method for Roohinejad, S., Koubaa, M., Barba, F. J., Saljoughian, S., Amid, M., & Greiner, R. (2017). determining oxidative rancidity in mackerel (Scomber scombrus L.). Fette, Seifen, Application of seaweeds to develop new food products with enhanced shelf-life, Anstrichmittel, 77(6), 239–240. quality and health-related beneficial properties. Food Research International, 99(part Wesierska, E., Szołtysik, M., & Rak, L. (2013). Physico-chemical, biochemical and mi- 3), 1066–1083. crobiological properties of traditional polish pork fermented products during ri- Roselló-Soto, E., Galanakis, C. M., Brnčić, M., Orlien, V., Trujillo, F. J., Mawson, R., ... J, pening. Food and Bioprocess Technology, 6(11), 2986–2995. F. (2015). Clean recovery of antioxidant compounds from plant foods, by-products Zhu, Z., Wu, Q., Di, X., Li, S., Barba, F. J., Koubaa, M., ... He, J. (2017). Multistage and algae assisted by ultrasounds processing. Modeling approaches to optimize recovery process of seaweed pigments: Investigation of ultrasound assisted extraction processing conditions. Trends in Food Science & Technology, 42(2), 134–149. and ultra-filtration performances. Food and Bioproducts Processing, 104,40–47. Shahidi, F., Rubin, L. J., & D'Souza, L. A. (1986). Meat flavor volatiles: A review of the

411

Environmental Engineering and Management Journal xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx http://www.eemj.icpm.tuiasi.ro/; http://www.eemj.eu

“Gheorghe Asachi” Technical University of Iasi, Romania

PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY OF EXTRACTS FROM BIFURCARIA BIFURCATA ALGA, OBTAINED BY DIVERSE EXTRACTION CONDITIONS USING THREE DIFFERENT TECHNIQUES

(HYDROTHERMAL, ULTRASOUNDS AND SUPERCRITICAL CO2)

Rubén Agregán1, Paulo E. S. Munekata2, Daniel Franco1, Rubén Domínguez1, Javier Carballo3, Voster Muchenje4, Francisco J. Barba5, José M. Lorenzo1

1Centro Tecnológico de la Carne de Galicia, Adva. Galicia n° 4, Parque Tecnológico de Galicia, San Cibrao das Viñas, 32900 Ourense, Spain 2Department of Food Engineering, Faculty of Animal Science and Food Engineering, University of São Paulo, 225 Duque de Caxias Norte Ave, Jardim Elite, postal code 13.635-900, Pirassununga, São Paulo, Brazil 3Area de Tecnologia de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain 4Department of Livestock and Pasture Science, University of Fort Hare, Private Bag X 1314, Alice, South Africa 5Nutrition and Food Science Area, Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Universitat de València, Avda. Vicent Andrés Estellés, s/n, Burjassot, 46100 València, Spain

Abstract

Extracts of Bifurcaria bifurcata seaweed were obtained by diverse conditions. Different extraction techniques, such as hydrothermal and ultrasounds, and three different solvents (water, ethanol and water/ethanol (50:50)) depending on technique were used. Moreover, supercritical CO2 (SC-CO2) with 10% of ethanol as co-solvent using different extraction times (30, 45 and 60 min) was also used as extraction technique. Extraction yield, phenolic content and antioxidant activity were measured for each extract. Hydrothermal extraction obtained better extraction yields than ultrasound extraction. Regarding the effect of solvent composition, water/ethanol (50:50) in hydrothermal treatment (HW50E50) and water/ethanol (50:50) in ultrasound treatment (UW50E50) showed the highest extraction yields. The worst extraction yields were shown by the extraction with SC-CO2. Water/ethanol (50:50) showed to be more efficient extracting phenolic compounds than water, although the highest extraction was achieved by ethanol. On the other hand, ultrasound-assisted extraction seemed to be more efficient extracting phenolic compounds than hydrothermal extraction. From the results obtained, it can be concluded that the use of ultrasound extraction technique and the use of water/ethanol as extracting solvent seemed to be the best extraction condition.

Key words: Bifurcaria bifurcata, hydrothermal, supercritical CO2, ultrasound

Received: January, 2018; Revised final: April, 2018; Accepted: July, 2018

 Author to whom all correspondence should be addressed: e-mail: [email protected]; Phone: +34 988548277; Fax: +34 988548276

PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY OF EXTRACTS FROM BIFURCARIA BIFURCATA ALGA, OBTAINED BY DIVERSE EXTRACTION CONDITIONS USING THREE DIFFERENT TECHNIQUES

(HYDROTHERMAL, ULTRASOUNDS AND SUPERCRITICAL CO2) Rubén Agregán1, Paulo E. S. Munekata2, Daniel Franco1, Rubén Domínguez1, Javier Carballo3, Voster Muchenje4, Francisco J. Barba5, and José M. Lorenzo1*

1 Centro Tecnológico de la Carne de Galicia, Adva. Galicia n° 4, Parque Tecnológico de Galicia, San Cibrao das Viñas, 32900 Ourense, Spain. 2 Department of Food Engineering, Faculty of Animal Science and Food Engineering, University of São Paulo, 225 Duque de Caxias Norte Ave, Jardim Elite, postal code 13.635-900, Pirassununga, São Paulo, Brazil. 3 Area de Tecnologia de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain. 4 Department of Livestock and Pasture Science, University of Fort Hare, Private Bag X 1314, Alice, South Africa. 5Nutrition and Food Science Area, Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Universitat de València, Avda. Vicent Andrés Estellés, s/n, Burjassot, 46100 València, Spain

Abstract

Extracts of Bifurcaria bifurcata seaweed were obtained by diverse conditions. Different extraction techniques, such as hydrothermal and ultrasounds, and three different solvents (water, ethanol and water/ethanol (50:50)) depending on technique were used. Moreover, supercritical CO2 (SC-CO2) with 10% of ethanol as co-solvent using different extraction times (30, 45 and 60 min) was also used as extraction technique. Extraction yield, phenolic content and antioxidant activity were measured for each extract. Hydrothermal extraction obtained better extraction yields than ultrasound extraction. Regarding the effect of solvent composition, water/ethanol (50:50) in hydrothermal treatment (HW50E50) and water/ethanol (50:50) in ultrasound treatment (UW50E50) showed the highest extraction yields. The worst extraction yields were shown by the extraction with

*Author to whom all correspondence should be addressed: E-mail: [email protected]; Phone: +34 988548277; Fax: +34 988548276

1 SC-CO2. Water/ethanol (50:50) showed to be more efficient extracting phenolic compounds than water, although the highest extraction was achieved by ethanol. On the other hand, ultrasound- assisted extraction seemed to be more efficient extracting phenolic compounds than hydrothermal extraction. From the results obtained, it can be concluded that the use of ultrasound extraction technique and the use of water/ethanol as extracting solvent seemed to be the best extraction condition.

Keywords: Bifurcaria bifurcata, hydrothermal, supercritical CO2, ultrasound

1. Introduction

The exploitation of antioxidant bioactive compounds is of great interest for consumers, industries and researchers due to the beneficial effects associated with their regular consumption and their potential use as food additives, in pharmaceutic and cosmetic industries to avoid oxidation processes (Patil et al., 2009). Marine sources of bioactive compounds have been gaining attention among the dietary and natural sources, particularly for its polyphenolic composition (Barba, 2017; Poojary et al., 2017). Phlorotannins is the main group of phenolic compounds in brown and other alga species. This group has been related to potential health benefits such as antidiabetic, antihypertensive and anti-inflammatory. Phlorotannins also exhibit antioxidant activity with promising application in pharmaceutical, food and chemical industry (Ibañez and Cifuentes, 2013; Stengel et al., 2011; Thomas and Kim, 2011). Brown seaweeds have high concentrations of nutrients (Lorenzo et al., 2017). In addition, they also possess high amounts of phenolic compounds (from 1 to 14 % dry weight (DW)), being Ascophyllum and Fucus the two genera with the highest contents (Holdt and Kraan, 2011). Bioactive compounds are isolated from algal biomass by different methods. Conventional extraction methods have been used to extract bioactive compounds from plant materials for a long time (Barba et al., 2014; Liza and Abdul, 2010). These conventional extraction methods (extraction in Soxhlet apparatus, solid-liquid extraction, and liquid-liquid extraction) have some disadvantages: demand of high volumes of solvent, difficult solvent separation after extraction, degradation of thermolabile compounds when extraction is done at high temperatures, time-consuming or energy intensive protocols (Dai and Mumper, 2010; Poojary et al., 2017). In contrast to the classic extraction methods, extraction assisted by innovative processing technologies is developing fast due to the ongoing consumer demands for clean and green extraction technics with minimal use of organic solvents and value addition to food processing by- products (Barbosa, 2005; Galanakis, 2013). The main advantage of these novel technologies is the

2 preservation, or at least the reduction of biological activity, after extraction (Michalak and Chojnacka, 2015). Ultrasound-assisted extraction is a non-conventional extraction technology widely used in the extraction of bioactive compounds from natural sources (Corbin et al., 2015; Roselló-Soto et al., 2015; Zhu et al., 2017). This technology increases the extraction efficiency and reduces energy requirements and solvent consumption compared to traditional methods (Macías et al., 2009). Ultrasound technology is based on the production of ultrasonic jet toward the solid surfaces, which induces cell disruption and particle size reduction. Thereby, ultrasound increases the contact area between solid and solvent and also facilitates the penetration of solvent and its contact with soluble compounds (Kentish and Feng, 2014). Other alternative extraction method to conventional procedures, and usually proposed, is the use of supercritical fluid extraction (SFE) (Barba et al., 2014; Michalak et al., 2015). Supercritical fluids have been gaining increasing attention because of its environmentally friend and improved reaction media character. Supercritical fluids are also cheap, non-toxic, non-flammable, non- explosive, and offer essential advantages compared to other substances (Lang and Wai, 2001). In the SFE, the solvent is conditioned at temperature and pressure above its critical point which improves transport properties: decreases viscosity and increases diffusivity. The use of CO2 as a supercritical fluid offers numerous advantages: it is non-toxic, noncorrosive, easily separated from the extract (Pan et al., 2012), cheap, available, inert to the product, non-flammable, and shows improved affinity to volatile compounds (Crampon et al., 2011). Although the use of total phenolic compounds and total antioxidant capacity have been reviled over the last years due to their non-specificity and the lack of scientific evidence regarding the beneficial effects of polyphenol-rich foods can be attributed to the antioxidant properties of these food, these parameters are still useful. For example, they can constitute a screening tool to predict the yield of antioxidant compounds, especially after extraction processes, which are for example used for the preparation of food additives, at the pharmaceutical and/or cosmetic level in the formulation of preparations to prevent their oxidation. In this study, different extraction conditions were tested on Bifurcaria bifurcata brown macro-alga. Two different extraction techniques, hydrothermal and ultrasounds, with three different extraction solvents, water, ethanol and water/ethanol (50:50) depending on the technique were used.

An extraction technique using supercritical CO2 with 10 % of ethanol at different times, 30, 45 and 60 min, were also used. The aim was to asses the different extraction conditions (technique-solvent or technique-time in case of supercritical CO2) according to the phenolic extraction and antioxidant activity of the extracts. In addition, extraction yield was also of interest.

3 2. Material and methods

2.1. Algal material

The brown seaweed Bifurcaria bifurcata used in the present study was kindly supplied by Portomuiños company (A Coruña, Spain). It was collected in the Atlantic Ocean, in the area of Camariñas (A Coruña, Spain). The seaweed was dried (40 °C) and grounded to obtain a powder with a particle lower than 0.8 mm, using a conventional mincer. Then, the seaweeds were passed through a 0.8 mm mesh sieve and stored under vacuum (75 %) in plastics bags at -20 °C until further analysis.

2.2. Obtaining of Bifurcaria bifurcata extracts using hydrothermal and ultrasonic techniques

The ground alga (5 g) was mixed with 50 mL of distilled water (HW100) and with 50 mL of water/ethanol (50:50) (HW50E50) in glass bottles (hydrothermal technique), and with 50 mL of distilled water (UW100), 50 mL of water/ethanol (50:50) (UW50E50) and ethanol (UE100) in Erlenmeyer flasks (ultrasound technique). In the hydrothermal technique, the bottles were introduced in an autoclave (Raypa Stericlav-S 150 L, Terrassa, Spain) and the extraction conducted at 121 ºC for 30 min. In the ultrasound technique the Erlenmeyer flasks were introduced in an ultrasonic bath (Branson ultrasonic M3800-E, Dietzenbach, Germany) and the extraction conducted at room temperature for 30 min. Then, the sets for both techniques were centrifuged at 4000 rpm for 10 min at 4º C, and filtered through a lab filter paper to remove the residue. The extracts obtained were stored at -20 ºC until needed for analysis.

2.3. Obtaining of Bifurcaria bifurcata extracts by extraction with supercritical CO2

A one liter cylinder extractor with two 500 mL separators (Thar Designs SFE-1000 F-2-

C10, Pittsburgh, USA) was used for the extraction experiments. The CO2 was precooled using a circulating bath (PolyScience, USA, model 9506) prior being pumped with a P-200A piston pump -1 (Thar Design Inc.) (flow rate: 25 g CO2 min ). The co-solvent, ethanol, was pumped by a HPLC pump (Scientific Systems, Inc., USA, model Series III). The flow rate was adjusted to achieve concentrations of a 10 % of ethanol. The extraction by SC-CO2 was conducted at temperature of 40

ºC and pressure of 35 MPa. The extracts were collected at 30, 45 and 60 min (SC–CO2–30; SC–

CO2–45; and SC–CO2–60, respectively), and stored at -20 ºC until needed for analysis analysis.

4 2.4. Measurement of the extraction yield

An aliquot of 5 mL from each extract was taken and evaporated in a drying oven at 100 ºC overnight. The weight of the final residue was used to calculate the extraction yield by gravimetry. Result was expressed as g extract 100 g-1 DW. The dry weight of Bifurcaria bifurcata seaweed was calculated subtracting its moisture content, which was measured by the International Organization for Standardization (ISO) recommendation for moisture content (ISO 1442:1997).

2.5. Determination of the phenolic content

Phenolic content was determined for all extracts obtained by all extraction conditions and solvents. This determination was based on a procedure described by Medina et al. (2009) as follows: 15 µL of samples were mixed with 170 µL of Milli-Q water, adding 12 µL of Folin- Ciocalteu reagent and 30 µL of sodium carbonate. The mixtures were incubated for 1 h at room temperature in the dark. After the reaction period, 73 µL of Milli-Q water were added with a multichannel pipette. Absorbance was measured at 765 nm. Phenolic content was expressed as g phloroglucinol equivalents (PGE) 100 g-1 DW.

2.6. Measurement of the antioxidant activity

Antioxidant activity was measured using the oxygen radical absorbance capacity (ORAC) assay described by Ou et al. (2001) and modified by Dávalos et al. (2003). In order to carry out the reaction, 75 mM phosphate buffer (pH 7.4) was used, being the final reaction volume of 200 mL. Twenty microliters of antioxidant were mixed with 120 mL of fluorescein (70 nM) and incubated for 15 min at 37 ºC. After that, 60 mL of 12 mM 2,2-azobis (2-methylpropionamidine) dihydrochloride (AAPH) were added. Then, the plate was placed into the reader and the fluorescence was recorded each minute for 120 min (excitation and emission wavelengths of 485 and 520 nm, respectively). The plate was automatically stirred before each measurement. A blank samples consisting of phosphate buffer instead of the antioxidant extract, and eight calibration solutions (using Trolox as antioxidant) were also determined. Results were calculated on the basis of the differences in areas under the fluorescein decay curve between the blank and the sample, and were expressed as µmol of Trolox equivalents (TE) g-1 DW.

5 2.7. Statistical analysis

The differences in extraction yield, phenolic content and antioxidant activity among the extraction conditions used in Bifurcaria bifurcata seaweed were examined using an ANOVA test. Least-squares means were compared among extraction conditions using the Duncan’s post hoc test (significance level P < 0.05). The values were given in terms of mean values ± standard deviations (n=2). All statistical analysis were performed using IBM SPSS Statistics® 21 software.

3. Results and discussion

3.1. Extraction yield

Yields of extracts from Bifurcaria bifurcata are presented in Table 1. The highest extraction yield was obtained by HW50E50 followed by UW50E50 and HW100 extraction conditions (41.82; 39.11 and 37.48 g extract 100 g-1 DW, respectively). The combination of ultrasound with pure solvents (UW100 and UE100 for water and ethanol, respectively) gave extracts of reduced yield (22.27 and 14.67 g extract 100 g-1 DW, respectively), while the lowest extraction yields were -1 obtained for SC-CO2 extraction technique (average yields lower than 4 g extract 100 g DW). In a similar way, Hwang and Do Thi (2014) found that solid-liquid extraction by hydrothermal aqueous extraction at 100 ºC enhanced extraction yield compared to 37 ºC in dried (41 vs 25 %, respectively), roasted (41 vs 32 %, respectively), and seasoned (26 vs 21 %, respectively) Porphyra tenera red alga. The extraction yield values reached by them in dried and roasted laver at 100 ºC was very similar to ours with a close temperature (37.48±0.77 vs 41.3-40.6 g 100g-1 DW, respectively). On the other hand, the use of ultrasounds with the same solvent by us achieved a lower value (22.27±0.77 g 100g-1 DW). However, when Hwang and Do Thi (2014) extracted with 100% water at 37 ºC, they reached extraction yield values similar to ours in all algae processed methods (dried, roasted and seasoned) (22.27±0.77 vs 25.5-32.1-21 g 100g-1 DW, respectively). Another interesting result observed by these authors is the effect of solvent composition on extraction yield. Porphyra tenera red alga extracts obtained from aqueous extraction at 37 °C had higher extraction yield than extracts with 70 % ethanol as solvent for dried (25 vs 18 %, respectively), roasted (32 vs 20 %, respectively), and seasoned (21 vs 16 %, respectively) alga. Tierney et al. (2013) reported the same finding on Ascophyllum nodosum, Pelvetia canaliculata, Fucus spiralis brown algae and Ulva intestinalis green alga collected in Irish coast (from 19 up to 30 % for water extracts and between 3 and 24 % for water/ethanol extracts). On contrary, our results showed that using 50% ethanol improved the yields than using 100 % water, both at 121 ºC

6 and at room temperature by ultrasounds (41.82±2.30 vs 37.48±0.77 g 100g-1 DW and 39.11±1.54 vs 22.27±0.77 g 100g-1 DW). López et al. (2011) also reported that the mixture of an organic solvent such as methanol and water in a solid-liquid extraction improved the extraction yield in the Stypocaulon scoparium brown alga in comparison to 100% water solvent. The solid-liquid extraction in several alga species from Danish coast using 100% water (14-51%) showed higher extraction yields than using 100% ethanol (3-28 %) at room temperature (Farvin and Jacobsen, 2013). This finding is in agreement with ours using the ultrasound technique (22.27±0.77 vs 14.67±0.77 g 100-1 DW).

The lower extraction yield observed for SC-CO2 treatments may be explained by the balance between the interactions of alga solutes with CO2 and alga matrix components. Our previous research about the proximate composition of Bifurcaria bifurcata and other alga species (Ascophyllum nodosum and Fucus vesiculosus) extracts revealed that most of the components in these algae were suggested to be of high polarity (Agregán et al., 2017). On the other hand, the characteristics of CO2 under supercritical conditions (7.2 MPa at 31 °C) are favorable for extraction of thermally labile and non-polar compounds (Abbas et al., 2008). Although the SC-CO2 treatments were performed in pressure (> 25 MPa) and temperature (< 50 °C) to facilitate the extraction of polar and high molecular weight compounds (Díaz-Reinoso et al., 2006), the results observed in the present study suggest that additional energy and mixture of solvents were necessary to facilitate the release of polar and high molecular weight compounds from alga matrix under the studied conditions for SC-CO2 (35 MPa at 40 °C).

3.2. Phenolic content

Phenolic contents from different extracts of Bifurcaria bifurcata are also presented in Table 1. The highest values for total phenolic content were obtained for HW50E50 and UW50E50 (5.65 and 5.46 g PGE 100 g-1 DW, respectively), followed by HW100 (2.92 g PGE 100 g-1 DW). The SC-

CO2 procedures were inefficient for all extraction times due to the lowest total phenolic content recovery in comparison to other extraction techniques, wherein average phenolic content in such extracts were in the range of 0-0.06 g PGE 100 g-1 DW. Interestingly, the highest total phenolic content was found for the samples where the highest extraction yield was obtained. It is possible to suggest that increased extraction yield also contributed to release of phenolic compounds from algal structure. It is also suggested that other non-phenolic compounds such as water soluble polysaccharides, proteins and organic acids were extracted (Chirinos et al., 2007).

7 The extraction of phenolic compounds was favored by hydrothermal and ultrasound processing, particularly for the water/ethanol mixture. The UW100 and UE100 treatments (2.28±0.16 and 2.57±0.24, respectively) displayed reduced efficiency in extraction of phenolic compounds in comparison to hydrothermal and ultrasound treatments (50:50). Hwang and Do Thi (2014) observed that extraction of phenolic compounds from Porphyra tenera were increased due to the change of water to 70 % ethanol solution as solvent. Auezoba et al. (2013) also found one of the highest phenolic contents in a solid-liquid extraction among all extractant solvents used, on Saccharina bongardiana brown alga, with aqueous mixtures of the organic polar solvents ethanol and methanol, in the ratio of 70% (0.619 g PGE 100 g-1 DW for water/ethanol and 0.708 g PGE 100 g-1 DW for water/methanol). These values were very lower than ours using water/ethanol (50:50) both in the hydrothermal and the ultrasound extraction technique (5.65±0.61 and 5.46±0.34 g PGE 100 g- 1 DW, respectively). However, some parameters, such as seaweed-solvent ratio, extraction time or agitation, all of them different among the studies, must be also taken into account. In a similar way, Tierney et al. (2013) observed that extraction of phenolic compounds from almost all Irish macroalgae species was increased by acetone/water solution (80:20) as solvent in comparison to water and even to ethanol/water solution (80:20). López et al. (2011) reported a higher total phenolic content using water/methanol (50:50) as extraction solvent than using 100% ethanol and methanol, on Stypocaulon scoparium alga during a solid-liquid extraction. Nevertheless, contrary to our study, they reported that 100% water extract reached the highest phenolic content, although parameters, such as solid-liquid ratio, agitation or temperature did not coincide in both studies. Kuda et al. (2005) used water at 121 ºC and ethanol at room temperature on seaweeds from Noto Peninsula (Japan), displaying very high phenolic contents when they used the water. However, temperature could have affected to the phenolic compounds extraction. The improved extraction of phenolic compounds in HW50E50 and UW50E50 can be explained by their higher solubility in polar organic solvents such acetone, ethanol and methanol (Farvin and Jacobsen, 2013; Wang et al., 2012). In addition, increased temperature has been associated to enhanced extraction of polyphenols in other algae species. Belda et al. (2016) reported that extraction of polyphenols from Himanthalia elongata brown alga was facilitated by increasing temperature (60 °C) in comparison to lower temperatures (25 and 40 °C) for 2 h. Moreover, cell wall softening and degradation facilitate the release of trapped compounds, such as polyphenols due to increasing temperature and ultrasound treatments (Balboa et al., 2013; Parniakov et al., 2015).

The reduced extraction of phenolic compounds by SC-CO2 treatments may be associated with reduced capacity of CO2 to extract hydrophilic compounds as observed for extraction yield results (Table 1) since CO2 has non-polar character under supercritical conditions (Abbas et al., 2008). Although the penetration and diffusion (crucial events for successful extraction of target

8 compounds) of SC-CO2 in algal matrix is suggested to occur during treatments applied in the present study, the level of interaction of SC-CO2 with phenolic compounds may be lower than the interaction of phenolic compounds with algal components and, therefore, impair the extraction of phenolic compounds by SC-CO2 extraction. In addition, the phenolic content of algal samples is mainly composed by phlorotannins. This group of phenolic compounds comprises more than 700 structures, is highly hydrophilic and has phloroglucinol as monomeric unit. Classification of phlorotannins can be done according to degree of polymerization: monomers, dimers, trimmers, tetramers and phlorotannins have a degree of polymerization of 1, 2, 3, 4 and ≥ 5, respectively. Another classification of phlorotannins is based in inter-molecular linkage: ether linkage (fuhalols/phlorethols); phenyl linkage (fucols); ether and phenyl linkage (fucophloroethols); and dibenzodioxin linkage (eckols). Such compounds can contribute to defences against stress and herbivores (Li et al., 2011; Singh and Bharate, 2006).

3.3. Antioxidant activity

Antioxidant activity measured by ORAC method from different extracts from Bifurcaria bifurcata is presented in Table 1. In view of the low polyphenol content obtained in the extraction with SC-CO2, the decision of not measuring its antioxidant activity was taken. The UW50E50 treatment produced the extract with the highest value of antioxidant activity followed by HW50E50 and HW100 (552.24, 426.88, and 372.87 µmol TE g-1 DW, respectively). From these results, it is also shown that the combination of solvents (water/ethanol) and the application of additional energy (hydrothermal and ultrasound) led to production of natural extracts with increased antioxidant activity. Hwang and Do Thi (2014) found that the use of 70 % ethanol instead of 100 % water improved the antioxidant activity of extracts from dried, roasted and seasoned Porphyra tenera red alga using DPPH radical (DPPH) scavenging assay. Nevertheless, using ABTS radical cation (ABTS+) decolorization assay only the 70 % ethanol extract from the seasoned alga showed higher antioxidant activity. In the same way, Tierney et al. (2013) reported higher DPPH scavenging activities in ethanol/water (80:20) and acetone/water (80:20) extracts than in water extracts. However, different results are given by the authors when they use ferric reducing antioxidant power (FRAP) and ferrous ion chelating (FIC) assays. Our results agree with those reported by Rajauria et al. (2013), who found that mixtures of methanol with water reached higher antioxidant activities than using water and methanol as pure solvents . The highest activities were achieved with mixtures around of the 50% of the solvents. On the other hand, López et al. (2011) noted higher antioxidant activity values in the water/methanol (50:50) extract than in the 100% methanol and ethanol extracts. Nonetheless, 100% water extract showed the highest antioxidant activity.

9 Lee et al. (2013) stated that ultrasound increased the antioxidant activity of Ecklonia cava extracts in comparison to conventional extraction (agitation in a shaking incubator), particularly for alkyl radical and H2O2 scavenging assays. Time of extraction was reduced in extracts submitted to ultrasound treatment from 24 h (shaking incubator) to 12 h (ultrasound) which was also considered as an important improvement by these authors. Differently, Hwang and Do Thi (2014) observed that increasing temperature during extraction stage was associated with reduced antioxidant activity. The authors obtained reduced antioxidant activity for extracts submitted to thermal processing at 100 °C with water as solvent in comparison to extraction at 37 °C with 70 % ethanol solution as solvent. Additionally, extraction temperature also influenced the type of compounds extracted from algal matrix and may lead to differences in antioxidant activity, as it was observed for total flavonoid content in that study.

Table 1. Extraction yield, phenolic content and antioxidant activity of Bifurcaria bifurcata extracts obtained by hydrothermal, ultrasound, and SC-CO2 extraction techniques.

Extraction conditions Extraction yield Phenolic content Antioxidant activity Technique Solvent/time (g 100 g-1 DW) (g PGE 100 g-1 DW) (µmol TE g-1 DW) 100% water 37.48±0.77a 2.92±0.05a 372.87±58.25a W/E (50:50) 41.82±2.30a 5.65±0.61a 426.88±80.13a Hydrothermal SEM 1.44 0.81 32.57 Sig. ns * ns 100% water 22.27±0.77b 2.28±0.16a 253.06±0.08a W/E (50:50) 39.11±1.54c 5.46±0.34b 552.24±72.81b Ultrasound 100% ethanol 14.67±0.77a 2.57±0.24a 227.06±29.91a SEM 4.58 0.65 67.53 Sig. *** ** ** 30 min 0.49±0.11a 0.00±0.00a nd 45 min 1.05±0.05a 0.01±0.00a nd b b Supercritical CO2 60 min 3.10±0.54 0.06±0.01 nd SEM 0.51 0.01 Sig. ** ** W/E: water/ethanol; DW: dry weight; PGE: phloroglucinol equivalent; nd: not determined; SEM: standard error of mean. a-cMeans in the same column not followed by a common superscript letter are significantly different (P < 0.05; Duncan test). Sig.: significance: *(P < 0.05); **(P < 0.01); ***(P < 0.001); ns (not significant).

Although the data are not significantly conclusive, a correlation among phenolic content and antioxidant activity seemed to be observed. Previous studies have reported a strong correlation between polyphenols and antioxidant activities in macroalgae, suggesting that polyphenols are some

10 of the main contributors to antioxidant activity (Vinayak et al., 2011; Wang, 2009; Zhang et al., 2010).

4. Conclusions

The results obtained in the present study indicated that the mixture water/ethanol (50:50) used as solvent has the ability to increase extraction yield, and particularly the amount of recovered polyphenols with antioxidant activity from Bifurcaria bifurcata extracts. Ultrasound is an interesting alternative to increase the extraction of antioxidant bioactive compounds from this algal matrix, while SC-CO2 is suggested to be avoided for antioxidant extraction purposes, even under optimized operation conditions. Testing the optimal extraction conditions obtained in this study in other brown seaweeds, ultrasound intensities, temperature and extraction period would be of interest for further research. In this way, extracts with high antioxidant activity could be obtained, minimizing their cost, and thus favoring the commercial exploitation of Bifurcaria bifurcata with significant benefits from an economical and environmental point of view.

Acknowledgements

The authors thank Instituto Nacional de Investigaciones Agrarias y Alimentarias (INIA) for granting Ruben Agregán with a predoctoral scholarship [CPR2014-0128]. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

References

Abbas K.A., Mohamed A., Abdulamir A.S., Abas H.A., (2008), A review on supercritical fluid extraction as new analytical method, American Journal of Biochemistry and Biotechnology, 4, 345-353. Agregán R., Munekata P.E., Dominguez R., Carballo J., Franco D., Lorenzo J.M., (2017), Proximate composition, phenolic content and in vitro antioxidant activity of aqueous extracts of the seaweeds Ascophyllum nodosum, Bifurcaria bifurcata and Fucus vesiculosus. Effect of addition of the extracts on the oxidative stability of canola oil under accelerated storage conditions, Food Research International, 99, 986-994. Auezova L., Najjar F., Selivanova O., Moussa E.H., Assaf M.D., (2013), Antioxidant activity of brown alga Saccharina bongardiana from Kamchatka (Pacific coast of Russia). A methodological approach. Journal of applied phycology, 25, 1189-1196.

11 Balboa E.M., Conde E., Moure A., Falqué E., Dominguéz H., (2013), In vitro antioxidant properties of crude extracts and compounds from brown algae, Food Chemistry, 138, 1764-1785. Barba F.J., Grimi N., Vorobiev E., (2014), New approaches for the use of non-conventional cell disruption technologies to extract potential food additives and nutraceuticals from microalgae, Food Engineering Reviews, 7, 45-62. Barba F.J., (2017), Microalgae and seaweeds for food applications: Challenges and perspectives, Food Research International, 99, 969-970. Reglero G., Senorans F.J., Ibañez E., (2005), Supercritical Fluid Extraction: An Alternative to Isolating Natural Food Preservatives, In: Novel Food Processing Technologies, Barbosa G.V., Tapia M.S., Cano M.P. (Eds.), CRC Press, Boca Raton London New York Washington D.C., 539-555. Belda M., Sanchez D., Bover E., Prieto B., Padrón C., Cejalvo D., Lloris J.M., (2016), Extraction of polyphenols in Himanthalia elongata and determination by high performance liquid chromatography with diode array detector prior to its potential use against oxidative stress, Journal of Chromatography B, 1033, 334-341. Chirinos R., Rogez H., Campos D., Pedreschi R., Larondelle Y., (2007), Optimization of extraction conditions of antioxidant phenolic compounds from mashua (Tropaeolum tuberosum Ruíz & Pavón) tubers, Separation and Purification Technology, 55, 217-225. Corbin C., Fidel T., Leclerc E.A., Barakzoy E., Sagot N., Falguiéres A., Renouard S., Blondeau J. P., Ferroud C., Doussot J., Lainé E., Hano C., (2015), Development and validation of an efficient ultrasound assisted extraction of phenolic compounds from flax (Linum usitatissimum L.) seeds, Ultrasonics Sonochemistry, 26, 176-185. Crampon C., Boutin O., Badens E., (2011), Supercritical carbon dioxide extraction of molecules of interest from microalgae and seaweeds, Industrial & Engineering Chemistry Research, 50, 8941- 8953. Dai J., Mumper R.J., (2010), Plant phenolics: extraction, analysis and their antioxidant and anticancer properties, Molecules, 15, 7313-7352. Dávalos A., Bartolomé B., Suberviola J., Gómez C., (2003), Orac-fluorescein as a model for evaluating antioxidant activity of wines, Polish Journal of Food and Nutrition Sciences, 12, 133- 136.

Díaz-Reinoso B., Moure A., Domínguez H., Parajó J.C., (2007), Supercritical CO2 extraction and purification of compounds with antioxidant activity, Journal of Agricultural and Food Chemistry, 54, 2441-2469. Farvin K.S., Jacobsen C., (2013), Phenolic compounds and antioxidant activities of selected species of seaweeds from Danish coast, Food Chemistry, 138, 1670-1681.

12 Galanakis C.M., (2013), Emerging technologies for the production of nutraceuticals from agricultural by-products: a viewpoint of opportunities and challenges, Food and Bioproducts Processing, 91, 575-579. Holdt S.L., Kraan S., (2011), Bioactive compounds in seaweed: functional food applications and legislation, Journal of Applied Phycology, 23, 543-597. Hwang N., Do Thi E.S., (2014), Effects of extraction and processing methods on antioxidant compound contents and radical scavenging activities of laver (Porphyra tenera), Preventive Nutrition and Food Science, 19, 40-48. Ibañez E., Cifuentes A., (2011), Benefits of using algae as natural sources of functional ingredients, Journal of the Science of Food and Agriculture, 93, 703-709. ISO, (1997), Determination of moisture content, ISO 1442:1997 standard. In: International Standards Meat and Meat Products, Genève, Switzerland: International Organization for Standardization. Kentish S., Feng H., (2014), Applications of power ultrasound in food processing, Annual Review of Food Science and Technology, 5, 263-284. Kuda T., Tsunekawa M., Goto H., Araki Y., (2005), Antioxidant properties of four edible algae harvested in the Noto Peninsula, Japan. Journal of Food Composition and Analysis, 18, 625-633. Lang Q., Wai C.M., (2001), Supercritical fluid extraction in herbal and natural product studies - a practical review, Talanta, 53, 771-782. Lee S.H., Kang M.C., Moon S.H., Jeon B.T., Jeon Y.J., (2013), Potential use of ultrasound in antioxidant extraction from Ecklonia cava, Algae, 28, 371-378. Li Y.X., Wijesekara I., Li Y., Kim S.K., (2011), Phlorotannins as bioactive agents from brown algae, Process Biochemistry, 46, 2219-2224. Liza M.S., Abdul R.A., Mandana B., Jinap S., Rahmat A., Zaidul I., Hamid A., (2010), Supercritical carbon dioxide extraction of bioactive flavonoid from Strobilanthes crispus (Pecah Kaca), Food and Bioproducts Processing, 88, 319-326. López A., Rico M., Rivero A., Tangil M.S., (2011), The effects of solvents on the phenolic contents and antioxidant activity of Stypocaulon scoparium algae extracts, Food Chemistry, 125, 1104- 1109. Lorenzo J.M., Agregán R., Munekata P.E.S., Franco D., Carballo J., Şahin S., Lacomba R., Barba F.J., (2017), Proximate composition and nutritional value of three macroalgae: Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcata, Marine Drugs, 15, 360 Macías M.D., Serrano C.M., Rodríguez M.R., de la Ossa E.M., (2009), Kinetics of the supercritical

fluid extraction of carotenoids from microalgae with CO2 and ethanol as cosolvent, Chemical Engineering Journal, 150, 104-113.

13 Medina A., Barrionuevo A., Zamora R., Andres C., Estruch R., Martínez M.A., Diez J., Lamuela R.M., (2009), Rapid Folin-Ciocalteu method using microtiter 96-well plate cartridges for solid phase extraction to assess urinary total phenolic compounds, as a biomarker of total polyphenols intake, Analytica Chimica Acta, 634, 54-60. Michalak I., Chojnacka K., (2015), Algae as production systems of bioactive compounds, Engineering in Life Sciences, 15, 160-176. Michalak I., Dmytryk A., Wieczorek P.P., Rój E., Łęska B., Górka B., Messyasz B., Lipok J., Mikulewicz M., Wilk R., Schroeder G., Chojnacka K., (2015), Supercritical algal extracts: a source of biologically active compounds from nature, Journal of Chemistry, 2015, ID 597140 Ou B., Hampsch-Woodill M., Prior R.L., (2001), Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe, Journal of Agricultural and Food Chemistry, 49, 4619-4626. Pan J.L., Wang H.M., Chen C.Y., Chang J.S., (2012), Extraction of astaxanthin from Haematococcus pluvialis by supercritical carbon dioxide fluid with ethanol modifier, Engineering in Life Sciences, 12, 638-647. Parniakov O., Apicella E., Koubaa M., Barba F.J., Grimi N., Lebovka N., Pataro G., Ferrari G., Vorobiev E., (2015), Ultrasound-assisted green solvent extraction of high-added value compounds from microalgae Nannochloropsis spp., Bioresource Technology, 198, 262-267. Patil B.S., Jayaprakasha G.K., Chidambara K.N., Vikram A., (2009), Bioactive compounds: historical perspectives, opportunities, and challenges, Journal of Agricultural and Food Chemistry, 57, 8142-8160. Poojary M.M., Barba F.J., Aliakbarian B., Donsì F., Pataro G., Dias D.A., Juliano P., (2017), Innovative alternative technologies to extract carotenoids from microalgae and seaweeds, Marine Drugs, 14, 214. Rajauria G., Jaiswal A. K., Abu-Gannam N., Gupta S., (2013), Antimicrobial, antioxidant and free radical‐ scavenging capacity of brown seaweed Himanthalia elongata from western coast of Ireland. Journal of Food Biochemistry, 37, 322-335. Roselló-Soto E., Galanakis C.M., Brnčić M., Orlien V., Trujillo F.J., Mawson R., Knoerzer K., Tiwari B.K., Barba F.J., (2015), Clean recovery of antioxidant compounds from plant foods, by- products and algae assisted by ultrasounds processing. Modeling approaches to optimize processing conditions, Trends in Food Science and Technology, 42, 134-149. Singh I.P., Bharate S.B., (2006), Phloroglucinol compounds of natural origin, Natural Product Reports, 23, 558-591.

14 Stengel D.B., Connan S., Popper Z.A., (2011), Algal chemodiversity and bioactivity: sources of natural variability and implications for commercial application, Biotechnology Advances, 29, 483-501. Thomas N.V., Kim S.K., (2011), Potential pharmacological applications of polyphenolic derivatives from marine brown algae, Environmental Toxicology and Pharmacology, 32, 325-335. Tierney M.S., Smyth T.J., Hayes M., Soler-Vila A., Croft A.K., Brunton N., (2013), Influence of pressurised liquid extraction and solid–liquid extraction methods on the phenolic content and antioxidant activities of Irish macro-algae, International Journal of Food Science & Technology, 48, 860-869. Vinayak R.C., Sabu A.S., Chatterji A., (2011), Bio-prospecting of a few brown seaweeds for their cytotoxic and antioxidant activities, Evidence-Based Complementary and Alternative Medicine, 2011, ID 673083. ang T., Jónsdóttir R., Liu H., Gu L., Kristinsson H.G., Ragha an S., la sdóttir G., 2 2 , Antioxidant capacities of phlorotannins extracted from the brown algae Fucus vesiculosus, Journal of Agricultural and Food Chemistry, 60, 2441-2469. Wang T., Jonsdottir R., Ólafsdóttir G., (2009), Total phenolic compounds, radical scavenging and metal chelation of extracts from Icelandic seaweeds, Food Chemistry, 116, 240-248. Zhang J., Zhang H., Wang L., Guo X., Wang X., Yao H., (2010), Isolation and identification of antioxidative peptides from rice endosperm protein enzymatic hydrolysate by consecutive chromatography and MALDI-TOF/TOF MS/MS, Food Chemistry, 119, 226-234. Zhu Z., Wu Q., Di X., Li S., Barba F.J., Koubaa M., Roohinejad S., Xiong X., He J., (2017), Multistage recovery process of seaweed pigments: Investigation of ultrasound assisted extraction and ultra-filtration performances, Food and Bioproducts Processing, 104, 40-47.

15

medicines

Article Antioxidant Potential of Extracts Obtained from Macro- (Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcata) and Micro-Algae (Chlorella vulgaris and Spirulina platensis) Assisted by Ultrasound

Rubén Agregán 1, Paulo E. S. Munekata 2, Daniel Franco 1 ID , Javier Carballo 3 ID , Francisco J. Barba 4 ID and José M. Lorenzo 1,* ID

1 Centro Tecnológico de la Carne de Galicia, Adva. Galicia No. 4, Parque Tecnológico de Galicia, San Cibrao das Viñas, 32900 Ourense, Spain; [email protected] (R.A.); [email protected] (D.F.) 2 Department of Food Engineering, Faculty of Animal Science and Food Engineering, University of São Paulo, 225 Duque de Caxias Norte Ave, Jardim Elite, Pirassununga 13635-900, São Paulo, Brazil; [email protected] 3 Area de Tecnologia de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain; [email protected] 4 Nutrition and Food Science Area, Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Universitat de València, Avda. Vicent Andrés Estellés, s/n, Burjassot, 46100 València, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-988-548-277

 Received: 15 March 2018; Accepted: 9 April 2018; Published: 10 April 2018 

Abstract: Background: Natural antioxidants, which can replace synthetic ones due to their potential implications for health problems in children, have gained significant popularity. Therefore, the antioxidant potential of extracts obtained from three brown macroalgae (Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcata) and two microalgae (Chlorella vulgaris and Spirulina platensis) using ultrasound-extraction as an innovative and green approach was evaluated. Methods: Algal extracts were obtained by ultrasound-assisted extraction using water/ethanol (50:50, v:v) as the extraction solvent. The different extracts were compared based on their antioxidant potential, measuring the extraction yield, the total phenolic content (TPC) and the antioxidant activity. Results: Extracts from Ascophyllum nodosum (AN) and Bifurcaria bifurcata (BB) showed the highest antioxidant potential compared to the rest of the samples. In particular, BB extract presented the highest extraction (35.85 g extract/100 g dry weight (DW)) and total phenolic compounds (TPC) (5.74 g phloroglucinol equivalents (PGE)/100 g DW) yields. Regarding the antioxidant activity, macroalgae showed again higher values than microalgae. BB extract had the highest antioxidant activity in the ORAC, DPPH and FRAP assays, with 556.20, 144.65 and 66.50 µmol Trolox equivalents (TE)/g DW, respectively. In addition, a correlation among the antioxidant activity and the TPC was noted. Conclusions: Within the obtained extracts, macroalgae, and in particular BB, are more suitable to be used as sources of phenolic antioxidants to be included in products for human consumption. The relatively low antioxidant potential, in terms of polyphenols, of the microalgae extracts studied in the present work makes them useless for possible industrial applications compared to macroalgae, although further in vivo studies evaluating the real impact of antioxidants from both macro- and micro-algae at the cellular level should be conducted.

Keywords: macroalgae; microalgae; extraction yield; total phenolic content; antioxidant activity

Medicines 2018, 5, 33; doi:10.3390/medicines5020033 www.mdpi.com/journal/medicines Medicines 2018, 5, 33 2 of 9

1. Introduction Free radicals may produce damage to lipids, proteins, cell membranes and nucleic acids, thus promoting the development of noncommunicable diseases [1]. Therefore, an increased interest in natural antioxidants to fight against free radicals has been shown. Consumers are particularly interested in natural antioxidants rather than synthetics [2] due to the problems of toxicity and carcinogenic effects that these may cause [3,4]. Brown seaweed species are rich in antioxidant polyphenols (from 1–14% dry solid), Ascophyllum and Fucus being two genera with the highest content [5]. Polyphenols are reported to possess benefits for heath, such as anticancer, antimicrobial, anti-inflammatory and antidiabetic activities [6]. Brown algae are rich in phlorotannins [5], polyphenols with multiple phenolic groups, which provide good antioxidant activities [7]. These compounds are exclusively from brown algae species [8,9]. In the same way as macroalgae, microalgae could represent an important source of antioxidant compounds [10,11]. They are a rich source of natural pigments with antioxidant properties, such as chlorophylls and carotenoids, thus giving an added value from a commercial point of view [12]. Moreover, their biodiversity, ease of cultivation and modulation of growth conditions has resulted in microalgae becoming among the most important resources in nature having antioxidant properties [13]. Conventional extraction has been traditionally used to recover antioxidants from algae. However, this presents some important drawbacks such long extraction time, high cost and degradation of product quality. In addition, the use of organic solvents as extractive compounds should be minimized since they may be harmful from a health and environmental point of view [14,15]. For this reason, non-conventional extraction methods were employed to recover bioactives in food and pharmaceutical applications, providing satisfactory results, such as reducing process time and cost and increasing yield [16,17]. Ultrasound-assisted extraction (UAE) is an innovative extraction approach, which is based on sound wave migration, thus promoting cavitation phenomena and leading to a disruption of cell walls and the subsequent release of intracellular compounds [18]. This extraction method offers many advantages, such as a better solvent penetration into cellular material, higher product yields and reproducibility, lower solvent consumption and higher processing throughput compared to the conventional extraction methods [19]. Actually, UAE is very often used in the extraction of natural antioxidant compounds [20]. It has been employed in the extraction of antioxidant compounds from seaweeds with satisfactory results [21–23]. Therefore, the aim of this work was to evaluate the potential use of Ascophyllum nodosum (AN), Fucus vesiculosus (FV) and Bifurcaria bifurcata (BB) macroalgae extracts and Chlorella vulgaris (CV) and Spirulina platensis (SP) microalgae extracts as antioxidants for the possible application in products intended to be used for human consumption. For this purpose, UAE will be used as the extraction technology to recover the antioxidant bioactive compounds.

2. Materials and Methods

2.1. Algal Material Brown macroalgae, Ascophyllum nodosum (AN), Fucus vesiculosus (FV) and Bifurcaria bifurcata (BB) (Chromista, Ochrophyta, Phaeophyceae, Fucales), were purchased to Portomuiños company (A Coruña, Spain). The collection was carried out in the Atlantic Ocean near the Camariñas area (A Coruña, Spain). Microalgae, Chlorella vulgaris (CV) (Plantae, Chlorophyta, Trebouxiophyceae, Chlorellales) and Spirulina platensis (SP) (Eubacteria, Cyanobacteria, Cyanophyceae, Spirulinales) were provided by AlgaEnergy (Madrid, Spain). Macroalgae samples were ground obtaining particles lower than 0.8 mm, by a conventional mincer. Then, algae were stored under vacuum (75%) at −20 ◦C until further use.

2.2. Obtaining Extracts from Macroalgae and Microalgae by UAE Method Each of the algae (5 g) was extracted using a mixture consisting of water/ethanol (50:50, v:v) (50 mL) in glass bottles. The extraction was performed in an ultrasonic bath (Branson ultrasonic Medicines 2018, 5, 33 3 of 9

M3800-E, Dietzenbach, Germany) at room temperature for 30 min. After extraction, the solvent was separated from the alga by centrifuging at 3000× g for 10 min at 4 ◦C and filtering with filter paper using a vacuum to remove the algal material. The supernatant was stored at −20 ◦C until analysis. Extraction yield, total phenolic content (TPC) and antioxidant activity (ORAC, ABTS, DPPH and FRAP assays) were evaluated for the obtained extracts, as described below. All experiments were performed in duplicate.

2.3. Measurement of the Extraction Yield Five milliliters of each extract were taken and evaporated in a drying oven at 100 ◦C overnight. The weight of the final residue was used to calculate the extraction yield by gravimetry. Results were expressed as g extract/100 g dry weight of algae (DW). The DW of each alga was calculated subtracting its moisture content to total weight of the algae. Moisture content was measured according to the method previously reported in the ISO recommendation [24].

2.4. Determination of the Total Phenolic Compounds TPC determination was based on a method described by Medina-Remón et al. [25]. Fifteen microliters of sample were mixed with 170 µL of Milli-Q water, adding later 12 µL of Folin-Ciocalteu reagent and 30 µL of sodium carbonate. The mixtures were incubated for 1 h at room temperature under darkness. Once the reaction was over, 73 µL of Milli-Q water were added with a multichannel pipette. The absorbance measurement was performed at 765 nm. TPC was expressed as g of phloroglucinol equivalents (PGE)/100 g extract and as g PGE/100 g DW.

2.5. Determination of the Antioxidant Activity

2.5.1. Oxygen Radical Absorbance Capacity Assay The original method of Ou et al. [26] modified by Dávalos et al. [27] was used. The reaction was carried out in 75 mM phosphate buffer (pH 7.4), 200 µL being the final reaction mixture. The mixture with antioxidant (20 µL) and fluorescein (120 mL; 70 nM final concentration) was pre-incubated for 15 min at 37 ◦C. AAPH (2,20-azobis (2-methylpropionamidine) dihydrochloride) solution (60 mL; 12 mM, final concentration) was added rapidly using a multichannel pipette. The plate was immediately placed in the reader, and the fluorescence was recorded every minute for 120 min (excitation wavelength 485 nm, emission wavelength 520 nm). Samples were stirred prior to each reading. Eight calibration solutions using Trolox as the antioxidant were used in each assay, and phosphate buffer was used as blank. Results were calculated on the basis of the differences in areas under the fluorescein decay curve between the blank and the sample and were expressed as µmol Trolox equivalents (TE)/g DW.

2.5.2. ABTS Radical Cation Decolorization Assay The method of Re et al. [28] was adapted to the use of a plate reader. ABTS•+ was produced by mixing 7 mM ABTS stock solution with 2.45 mM potassium persulfate (final concentration) leaving the mixture in the dark at room temperature for 12–16 h before use. In the following step, ABTS•+ solution was diluted with PBS (pH 7.4) to get an absorbance of 0.70 at 734 nm, being equilibrated at 30 ◦C. The working solution of ABTS•+ (sample: ABTS solution relation, 1:100) was added to an aliquot of each sample (with appropriate dilution). The decrease in absorbance was measured after 6 min at 734 nm in a microplate spectrophotometer reader. Trolox was used as the reference standard, and the results were expressed as µmol TE/g DW.

2.5.3. DPPH Radical Scavenging Assay The DPPH• scavenging method was performed with some modifications according to the procedure previously described by Brand-Williams et al. [29]. Five microliters of samples (previously Medicines 2018, 5, 33 4 of 9 diluted) were added to 195 µL of DPPH solution (6 × 10−5 M in methanol) in 96-well plates. The mixture was lightly shaken and left at room temperature for 30 min. Then, the absorbance at 515 nm was measured against methanol using a microplate reader. The DPPH• scavenging activity of extracts was determined using the standard curve of Trolox and expressed as µmol TE/g DW.

2.5.4. Ferric Reducing Antioxidant Power Ferric reducing antioxidant power was determined using the method described by Benzie and Strain [30]. The FRAP reagent was freshly prepared from 300 mM acetate buffer, pH 3.6, 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) made up in 40 mM HCl and 20 mM FeCl3·6H2O solution. The three solutions were mixed together in the ratio of 10:1:1 (v:v:v). Three-hundred microliters of freshly-prepared FRAP reagent were mixed with 10 µL of properly diluted samples and 30 µL of distilled water in 96-well plates. The mixture was heated at 37 ◦C and left at this temperature during the reaction. After 8 min, the absorbance was measured using a microplate reader at 593 nm against reagent blank. The FRAP value was calculated and expressed as µmol TE/g DW based on a calibration curve plotted using Trolox as the standard.

3. Results and Discussion

3.1. Extraction Yield Extraction yields from different algae are presented in Table1. AN and BB macroalgae showed higher extraction yields compared to FV and microalgae species. Specifically, BB achieved the highest value (35.85 and 35.85 g extract/100 g DW). CV and SP microalgae presented lower extraction yields than macroalgae, SP being the one that had the lowest value (4.56 and 4.24 g extract/100 g DW). Farvin and Jacbsen [31] also found a lower extraction yield for FV compared to most of the algae used in their study when using water as the solvent. They attributed this fact to the important viscosity found in these extracts, which made the filtering process through filter paper difficult. On the other hand, other authors also reported significant differences in extraction yield among several seaweed species using different extraction solvents, such as water, ethanol or diethyl ether [31,32]. Matanjun et al. [32] found a positive correlation among yield and solvent polarity when extracting antioxidant bioactive compounds from eight seaweed species from north Borneo. At the same time, they found differences between algae using the solvents separately. According to them, the variation in the yields from several extracts may be due to the different polarities of the compounds found in plants [31]. Other factors that can explain the modifications in extraction yields are (i) the chemical composition of the raw material and (ii) the polarity of the solvents used [33]. For instance, different solvents, such as ethanol, water or aqueous solutions of organic solvents, were tested to obtain the optimal solvent for improving extraction yields, obtaining the best results when ethanol was used [31,34]. Thus, taking into account the extraction solvent used (ethanol:water 50:50, v:v), it could be said that BB and AN macroalgae are richer in polar compounds than FV and both microalgae species. The observed lower content of polar compounds found in CV and SP and their extracts may be related to the amount of carotenoids present in microalgae. Microalgae are a good source of carotenoids [35,36]. For instance, Chlorella vulgaris is reported to contain high amounts of carotenoids, such as lutein and β-carotene [37,38]. As is well known, carotenoids are lipophilic compounds [39], making their extraction difficult with polar solvents, such as ethanol, water or mixtures of both, as in our study. Therefore, the extraction yields from CV and SP microalgae were probably affected by the use of water/ethanol (50:50, v:v) as the extraction solvent. Medicines 2018, 5, 33 5 of 9

Table 1. Extraction yields, TPCs and antioxidant activities from AN, FV, BB, CV and SP algae extracts obtained by the UAE method using water/ethanol (50:50, v:v) as the extraction solvent.

Extraction Algae TPC Antioxidant Activities Yield ORAC ABTS DPPH FRAP g PGE/100 g PGE/100 g/100 g DW µmol TE/g DW g Extract g DW AN 25.86 25.86 18 18 4.66 4.66 297.19 300.29 565.66 542.38 50.69 50.69 4.66 4.40 FV 9.58 9.80 20 20 1.92 1.96 155.41 154.26 200.60 208.46 26.72 27.15 3.45 3.82 BB 35.85 35.85 16 16 5.74 5.74 537.38 575.02 537.74 549.21 143.04 146.27 67.40 65.60 CV 7.78 7.14 4.5 4.7 0.35 0.34 33.07 29.35 15.64 14.64 0.86 0.79 0.62 0.62 SP 4.56 4.24 4.3 4.6 0.20 0.19 12.30 12.12 6.74 6.53 1.00 0.89 1.00 1.02 Algae: AN, Ascophyllum nodosum; FV, Fucus vesiculosus; BB, Bifurcaria bifurcata; CV, Chlorella vulgaris; SP, Spirulina platensis. UAE, ultrasound–assisted extraction. TPC, total phenolic content. DW, dry weight of alga. PGE, phloroglucinol equivalents. TE, Trolox equivalents.

3.2. Total Phenolic Content TPCs from the different algal extracts are presented in Table1. As can be seen in the table, AN and BB macroalgae showed higher TPC contents compared to FV and both microalgae. BB presented the highest content (5.74 and 5.74 g PGE/100 g DW). It should be noted that microalgae presented lower TPCs than macroalgae, SP being the one that reached the lowest content (0.20 and 0.19 g PGE/100 g DW). The differences (p < 0.05) observed between TPC in macro- and micro-algae could be due to the high content of our three macroalgae in phlorotannins. As already mentioned above, brown algae genera are rich in these compounds [6], composed of units of phloroglucinol joined to form polymers [31]. In addition, phlorotannins are bi-polar in nature [40]; therefore, they are soluble in polar solvents such as in the aqueous solution at 50% ethanol used in this study, ensuring their presence in the extract and strengthening our hypothesis. These compounds could help algae in their struggle against oxidative stress, as well as participate in the defense against grazers such as marine herbivores thanks to their plasticity [41]. Chew et al. [42] found a much higher TPC content in the Padina antillarum brown alga than in the Caulerpa racemosa green alga and in the Kappaphycus alvarezii red alga. They also attributed this fact to the phlorotannin presence in the brown algae. On the other hand, we did not only find that macroalgae had different TPCs (p < 0.05) than microalgae, but that all algae studied showed different total content (p < 0.05) in phenolic compounds. Connan et al. [43] reported that external-environmental factors, such as light, depth or salinity, and intrinsic factors, such as age or length, may affect the phenolic metabolic expressions of algae, generating great differences in the phenolic content [43,44]. Observing the extraction yield data, a correlation between extraction yields and TPC was noted, since the highest extraction yield values corresponded to the highest TPC values, the order being increased in the following way: BB > AN > FV > CV > SP. This correlation is related to the percentage of TPC in the extracts. Moreover, this percentage, as can be expected, was very similar for both macroalgae (16–20 g PGE/100 g extract) and for microalgae (4.3–4.7 g PGE/100 g extract). The differences observed for TPC in the different algae samples could be attributed to different factors, such as the period of the year or area in which they are collected. Hold and Kraan [6] reported that polyphenol content showed a correlation with the reproductive state of algae along time. Polyphenol content in Ascophyllum nodosum is minimum during May, the month with maximal fruit body shedding, and maximum in winter. However, March is the month in which the minimum of TPC was observed for Fucus vesiculosus, just before the period of maximum fertility [45]. On the other hand, Porphyra umbilicalis alga was affected by sun exposure and emersion. The authors noted that seaweeds exposed to air and water during the summer contained higher amounts of antioxidants than submerged seaweeds, submersion being a natural barrier for seaweeds against environmental stresses [46]. Medicines 2018, 5, 33 6 of 9

3.3. Antioxidant Activity Antioxidant activities of the different algae extracts are presented in Table1. Macroalgae showed higher antioxidant activities than microalgae for all the assays. As discussed above, brown algae are rich in phlorotannins showing high antioxidant activities. Ahn et al. [47] reported an interesting antioxidant capacity from three phlorotannins extracted from Ecklonia cava brown alga. Within macroalgae, BB had the highest values (537.38 and 575.02, 143.04 and 146.27, 67.40 and 65.60 µmol TE/g DW in the ORAC, DPPH and FRAP assays, respectively) and FV the lowest. This indicates that the BB algae are richer in compounds capable of scavenging free radicals. As expected, a positive correlation between TPC and antioxidant activity was noted. Thus, BB, which showed the highest TPC, also presented the highest antioxidant activity in almost all assays, followed by FV, AN and microalgae. The same correlation was reported by other authors after using the DPPH radical scavenging assay [42]. They found that when TPC content was higher, the IC50 decreased. According to these authors, the polyphenols present in seaweeds have the ability to scavenge free radicals. By using the DPPH assay, it was also found that seaweed extracts containing high levels of phenolic compounds also displayed potent antioxidant activities [31]. This correlation may mean that polyphenols are the compounds that contribute most to the antioxidant activity of our extracts. Other authors also came to the same conclusion with their extracts [31,42]. Focusing on microalgae, there are studies in which the results obtained were contradictory. On the one hand, it was found that the antioxidant capacity of microalgae is partly caused by polyphenols [48]. However, other authors did not find any correlation between the phenolic content and the antioxidant capacity of ethanolic extracts resulting from nine microalgae strains [49]. The determination of phenolic compounds as individual molecules in the extracts is, therefore, of great importance for radical scavenging activity [31]. This activity is also dependent on the structure of the compounds, as well as the amount and location of the hydroxyl groups in them [29]. For example, some studies found that caffeic acid, which has two hydroxyl groups, is a compound with greater antiradical activity than coumaric acid, a homolog of caffeic acid, but with only one hydroxyl group [29]. Therefore, the different phenolic combinations of the compounds will have an impact on the antioxidant activity of these [31].

4. Conclusions Extraction yield, TPC and antioxidant activity from macroalgae extracts obtained by the UAE method using water/ethanol (50:50, v:v) as the extraction solvent turned out to be higher than microalgae extracts obtained in the same way, meaning that macro-algae extracts, specially BB extract, are more suitable to be used as possible high-polyphenol antioxidants in products to be used for human consumption. The combined use of the aforementioned extraction method and solvent was inefficient to obtain microalgae extracts with good extraction yields and antioxidant potential. Therefore, these extracts obtained in this way are not interesting for possible industrial applications, presenting drawbacks, such as the cost or the amount that must be added to the product. Taking into account the data obtained, if additional research is carried out, it should be focused on BB algae extract. In addition, it would be interesting to assess the real impact of antioxidants from both macro- and micro-algae at a cellular level in further research.

Acknowledgments: The authors are grateful to the Instituto Nacional de Investigaciones Agrarias y Alimentarias (INIA) for the award of a predoctoral scholarship (CPR2014-0128) to Rubén Agregán. Author Contributions: Francisco J. Barba conceived of the experiments. Paulo E.S. Munekata designed the experiments. Rubén Agregán performed the experiments, analyzed the data and wrote the paper. Daniel Franco, Javier Carballo and José M. Lorenzo reviewed the paper before submitting. Conflicts of Interest: The authors declare no conflict of interest. Medicines 2018, 5, 33 7 of 9

References

1. Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M.T.; Mazur, M.; Telser, J. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 2007, 39, 44–84. [CrossRef] [PubMed] 2. Kranl, K.; Schlesier, K.; Bitsch, R.; Hermann, H.; Rohe, M.; Böhm, V. Comparing antioxidative food additives and secondary plant products—Use of different assays. Food Chem. 2005, 93, 171–175. [CrossRef] 3. Ito, N.; Hirose, M.; Fukushima, S.; Tsuda, H.; Shirai, T.; Tatematsu, M. Studies on antioxidants: Their carcinogenic and modifying effects on chemical carcinogenesis. Food Chem. Toxicol. 1986, 24, 1071–1082. [CrossRef] 4. Safer, A.M.; Al-Nughamish, A.J. Hepatotoxicity induced by the anti-oxidant food additive, butylated hydroxytoluene (BHT), in rats: An electron microscopical study. Histol. Histopathol. 1999, 14, 391–406. [PubMed] 5. Holdt, S.L.; Kraan, S. Bioactive compounds in seaweed: Functional food applications and legislation. J. Appl. Phycol. 2011, 23, 543–597. [CrossRef] 6. Fernando, I.S.; Kim, M.; Son, K.T.; Jeong, Y.; Jeon, Y.J. Antioxidant activity of marine algal polyphenolic compounds: A mechanistic approach. J. Med. Food 2016, 19, 615–628. [CrossRef][PubMed] 7. Koivikko, R.; Eränen, J.K.; Loponen, J.; Jormalainen, V. Variation of phlorotannins among three populations of Fucus vesiculosus as revealed by HPLC and colorimetric quantification. J. Chem. Ecol. 2008, 34, 57–64. [CrossRef][PubMed] 8. Li, Y.X.; Wijesekara, I.; Li, Y.; Kim, S.K. Phlorotannins as bioactive agents from brown algae. Process Biochem. 2011, 46, 2219–2224. [CrossRef] 9. Lopes, G.; Sousa, C.; Silva, L.R.; Pinto, E.; Andrade, P.B.; Bernardo, J.; Mouga, T.; Valentão, P. Can phlorotannins purified extracts constitute a novel pharmacological alternative for microbial infections with associated inflammatory conditions? PLoS ONE 2012, 7, e31145. [CrossRef][PubMed] 10. Rodriguez-Garcia, I.; Guil-Guerrero, J.L. Evaluation of the antioxidant activity of three microalgal species for use as dietary supplements and in the preservation of foods. Food Chem. 2008, 108, 1023–1026. [CrossRef] [PubMed] 11. Srivastava, A.K.; Bhargava, P.; Rai, L.C. Salinity and copper-induced oxidative damage and changes in the antioxidative defence systems of Anabaena doliolum. World J. Microbiol. Biotechnol. 2005, 21, 1291–1298. [CrossRef] 12. Vigani, M.; Parisi, C.; Rodríguez-Cerezo, E.; Barbosa, M.J.; Sijtsma, L.; Ploeg, M.; Enzing, C. Food and feed products from micro-algae: Market opportunities and challenges for the EU. Trend Food Sci. Technol. 2015, 42, 81–92. [CrossRef] 13. Giorgis, M.; Garella, D.; Cena, C.; Boffa, L.; Cravotto, G.; Marini, E. An evaluation of the antioxidant properties of Arthrospira maxima extracts obtained using non-conventional techniques. Eur. Food Res. Technol. 2017, 243, 227–237. [CrossRef] 14. Azmir, J.; Zaidul, I.S.M.; Rahman, M.M.; Sharif, K.M.; Mohamed, A.; Sahena, F.; Jahurul, M.H.A.; Ghafoor, K.; Norulaini, N.A.N.; Omar, A.K.M. Techniques for extraction of bioactive compounds from plant materials: A review. J. Food. Eng. 2013, 117, 426–436. [CrossRef] 15. Polshettiwar, V.; Varma, R.S. Aqueous microwave chemistry: A clean and green synthetic tool for rapid drug discovery. Chem. Soc. Rev. 2008, 37, 1546–1557. [CrossRef][PubMed] 16. Herrero, M.; Cifuentes, A.; Ibañez, E. Sub-and supercritical fluid extraction of functional ingredients from different natural sources: Plants, food-by-products, algae and microalgae: A review. Food Chem. 2006, 98, 136–148. [CrossRef] 17. Wang, L.; Weller, C.L. Recent advances in extraction of nutraceuticals from plants. Trends Food Sci. Technol. 2006, 17, 300–312. [CrossRef] 18. Hahn, T.; Lang, S.; Ulber, R.; Muffler, K. Novel procedures for the extraction of fucoidan from brown algae. Process Biochem. 2012, 47, 1691–1698. [CrossRef] 19. Roselló-Soto, E.; Galanakis, C.M.; Brnˇci´c,M.; Orlien, V.; Trujillo, F.J.; Mawson, R.; Knoerzer, K.; Tiwari, B.K.; Barba, F.J. Clean recovery of antioxidant compounds from plant foods, by-products and algae assisted by ultrasounds processing. Modeling approaches to optimize processing conditions. Trends Food Sci. Technol. 2015, 42, 134–149. [CrossRef] Medicines 2018, 5, 33 8 of 9

20. Corbin, C.; Fidel, T.; Leclerc, E.A.; Barakzoy, E.; Sagot, N.; Falguiéres, A.; Renouard, S.; Blondeau, J.P.; Ferroud, C.; Doussot, J.; et al. Development and validation of an efficient ultrasound assisted extraction of phenolic compounds from flax (Linum usitatissimum L.) seeds. Ultrason. Sonochem. 2015, 26, 176–185. [CrossRef][PubMed] 21. Kadam, S.U.; O’Donnell, C.P.; Rai, D.K.; Hossain, M.B.; Burgess, C.M.; Walsh, D.; Tiwari, B.K. Laminarin from Irish brown seaweeds Ascophyllum nodosum and Laminaria hyperborea: Ultrasound assisted extraction, characterization and bioactivity. Mar. Drugs 2015, 13, 4270–4280. [CrossRef][PubMed] 22. Moreira, R.; Chenlo, F.; Sineiro, J.; Arufe, S.; Sexto, S. Drying temperature effect on powder physical properties and aqueous extract characteristics of Fucus vesiculosus. J. Appl. Phycol. 2016, 28, 2485–2494. [CrossRef] 23. Rodrigues, D.; Sousa, S.; Silva, A.; Amorim, M.; Pereira, L.; Rocha-Santos, T.A.; Gomes, A.M.; Duarte, A.C.; Freitas, A.C. Impact of enzyme-and ultrasound-assisted extraction methods on biological properties of red, brown, and green seaweeds from the central west coast of Portugal. J. Agric. Food Chem. 2015, 63, 3177–3188. [CrossRef][PubMed] 24. ISO 1442. International Standards Meat and Meat Products—Determination of Moisture Content; International Organization for Standardization: Geneva, Switzerland, 1997. 25. Medina-Remón, A.; Barrionuevo-González, A.; Zamora-Ros, R.; Andres-Lacueva, C.; Estruch, R.; Martínez-González, M.Á.; Diez-Espino, J.; Lamuela-Raventos, R.M. Rapid Folin–Ciocalteu method using microtiter 96-well plate cartridges for solid phase extraction to assess urinary total phenolic compounds, as a biomarker of total polyphenols intake. Anal. Chim. Acta 2009, 634, 54–60. [CrossRef][PubMed] 26. Ou, B.; Hampsch-Woodill, M.; Prior, R.L. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J. Agric. Food Chem. 2001, 49, 4619–4626. [CrossRef][PubMed] 27. Dávalos, A.; Bartolomé, B.; Suberviola, J.; Gómez-Cordovés, C. Orac-fluorescein as a model for evaluating antioxidant activity of wines. Pol. J. Food Nutr. Sci. 2003, 12, 133–136. 28. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 1999, 26, 1231–1237. [CrossRef] 29. Brand-Williams, W.; Cuvelier, M.E.; Berset, C.L.W. T. Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci. Technol. 1995, 28, 25–30. [CrossRef] 30. Benzie, I.F.; Strain, J.J. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Anal. Biochem. 1996, 239, 70–76. [CrossRef][PubMed] 31. Farvin, K.S.; Jacobsen, C. Phenolic compounds and antioxidant activities of selected species of seaweeds from Danish coast. Food Chem. 2013, 138, 1670–1681. [CrossRef][PubMed] 32. Matanjun, P.; Mohamed, S.; Mustapha, N.M.; Muhammad, K.; Ming, C.H. Antioxidant activities and phenolics content of eight species of seaweeds from north Borneo. J. Appl. Phycol. 2008, 20, 367. [CrossRef] 33. Agregán, R.; Lorenzo, J.M.; Munekata, P.E.; Dominguez, R.; Carballo, J.; Franco, D. Assessment of the antioxidant activity of Bifurcaria bifurcata aqueous extract on canola oil. Effect of extract concentration on the oxidation stability and volatile compound generation during oil storage. Food Res. Int. 2016, 99, 1095–1102. [CrossRef][PubMed] 34. Tierney, M.S.; Smyth, T.J.; Hayes, M.; Soler-Vila, A.; Croft, A.K.; Brunton, N. Influence of pressurised liquid extraction and solid–liquid extraction methods on the phenolic content and antioxidant activities of Irish macroalgae. Int. J. Food Sci. Technol. 2013, 48, 860–869. [CrossRef] 35. Barba, F.J.; Grimi, N.; Vorobiev, E. New approaches for the use of non-conventional cell disruption technologies to extract potential food additives and nutraceuticals from microalgae. Food Eng. Rev. 2015, 7, 45–62. [CrossRef] 36. Dufossé, L.; Galaup, P.; Yaron, A.; Arad, S.M.; Blanc, P.; Murthy, K.N.C.; Ravishankar, G.A. Microorganisms and microalgae as sources of pigments for food use: A scientific oddity or an industrial reality? Trends Food Sci. Technol. 2005, 16, 389–406. [CrossRef] 37. Kwang, H.C.; Lee, H.J.; Koo, S.Y.; Song, D.G.; Lee, D.U.; Pan, C.H. Optimization of pressurized liquid extraction of carotenoids and chlorophylls from Chlorella vulgaris. J. Agric. Food Chem. 2009, 58, 793–797. 38. Plaza, M.; Santoyo, S.; Jaime, L.; Avalo, B.; Cifuentes, A.; Reglero, G.; Reina, G.G.B.; Señoráns, F.J.; Ibáñez, E. Comprehensive characterization of the functional activities of pressurized liquid and ultrasound-assisted extracts from Chlorella vulgaris. LWT-Food Sci. Technol. 2012, 46, 245–253. [CrossRef] Medicines 2018, 5, 33 9 of 9

39. Ahmed, F.; Fanning, K.; Netzel, M.; Turner, W.; Li, Y.; Schenk, P.M. Profiling of carotenoids and antioxidant capacity of microalgae from subtropical coastal and brackish waters. Food Chem. 2014, 165, 300–306. [CrossRef][PubMed] 40. Targett, N.M.; Arnold, T.M. Minireview—Predicting the effects of brown algal phlorotannins on marine herbivores in tropical and temperate oceans. J. Phycol. 1998, 34, 195–205. [CrossRef] 41. Van Altena, I.A.; Steinberg, P.D. Are differences in the responses between North American and Australasian marine herbivores to phlorotannins due to differences in phlorotannin structure? Biochem. Syst. Ecol. 1992, 20, 493–499. [CrossRef] 42. Chew, Y.L.; Lim, Y.Y.; Omar, M.; Khoo, K.S. Antioxidant activity of three edible seaweeds from two areas in South East Asia. LWT-Food Sci. Technol. 2008, 41, 1067–1072. [CrossRef] 43. Connan, S.; Goulard, F.; Stiger, V.; Deslandes, E.; Ar Gall, E. Interspecific and temporal variation in phlorotannin levels in an assemblage of brown algae. Bot. Mar. 2004, 47, 410–416. [CrossRef] 44. Amsler, C.D.; Fairhead, V.A. Defensive and sensory chemical ecology of brown algae. Adv. Bot. Res 2005, 43, 1–91. 45. Ragan, M.A.; Jensen, A. Quantitative studies on brown algal phenols. II. Seasonal variation in polyphenol content of Ascophyllum nodosum (L.) Le Jol. and Fucus vesiculosus (L.). J. Exp. Mar. Biol. Ecol. 1978, 34, 245–258. [CrossRef] 46. Sampath-Wiley, P.; Neefus, C.D.; Jahnke, L.S. Seasonal effects of sun exposure and emersion on intertidal seaweed physiology: Fluctuations in antioxidant contents, photosynthetic pigments and photosynthetic efficiency in the red alga Porphyra umbilicalis Kützing (Rhodophyta, Bangiales). J. Exp. Mar. Biol. Ecol. 2008, 361, 83–91. [CrossRef] 47. Ahn, G.N.; Kim, K.N.; Cha, S.H.; Song, C.B.; Lee, J.; Heo, M.S.; Yeo, I.K.; Lee, N.H.; Jee, Y.H.; Kim, J.S.; et al. Antioxidant activities of phlorotannins purified from Ecklonia cava on free radical scavenging using ESR and

H2O2-mediated DNA damage. Eur. Food Res. Technol. 2007, 226, 71–79. [CrossRef] 48. Goiris, K.; Muylaert, K.; Fraeye, I.; Foubert, I.; De Brabanter, J.; De Cooman, L. Antioxidant potential of microalgae in relation to their phenolic and carotenoid content. J. Appl. Phycol. 2012, 24, 1477–1486. [CrossRef] 49. Maadane, A.; Merghoub, N.; Ainane, T.; El Arroussi, H.; Benhima, R.; Amzazi, S.; Bakri, Y.; Wahby, I. Antioxidant activity of some Moroccan marine microalgae: PUFA profiles, carotenoids and phenolic content. J. Biotechnol. 2015, 215, 13–19. [CrossRef][PubMed]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Fucus vesiculosus extracts as natural antioxidants for improving

the physicochemical properties and shelf life of pork patties

formulated with oleogels

Running title: Fucus vesiculosus extracts as antioxidants to improve quality

in pork patties

Rubén Agregán1, Francisco J. Barba2, Mohsen Gavahian3, Daniel Franco1,

Amin Mousavi Khaneghah4, Javier Carballo5, Isabel C. F.R. Ferreira6,

Andrea Carla da Silva Barretto7 and José M. Lorenzo1*

1Centro Tecnológico de la Carne de Galicia, Adva. Galicia n° 4, Parque Tecnológico de Galicia, San Cibrao das Viñas, 32900 Ourense, Spain 2Nutrition and Food Science Area, Preventive Medicine and Public Health, Food Science, Toxicology and Forensic Medicine Department, Faculty of Pharmacy, Universitat de València, Avda. Vicent Andrés Estellés, s/n, 46100 Burjassot, València, Spain 3Product and Process Research Center, Food Industry Research and Development Institute, No. 331 Shih-Pin Rd., Hsinchu, 30062, Taiwan, ROC 4Department of Food Science, Faculty of Food Engineering, University of Campinas (UNICAMP), Campinas, São Paulo, Brazil 5Area de Tecnologia de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain 6Mountain Research Centre (CIMO), Polytechnic Institute of Bragança (IPB), Campus de Santa Apolonia, Bragança, Portugal 7Department of Food Technology and Engineering, Institute of Biosciences, Humanities and Exact Sciences (IBILCE), São Paulo State University, Rua Cristóvão Colombo, 2265, Zip Code 15.054-000 São José do Rio Preto, SP, Brazil * Corresponding author Jose M. Lorenzo, PhD E-mail: [email protected]

1 ABSTRACT

BACKGROUND: There are hardly information in literature about algae extracts as ingredients in meat to improve quality or increase shelf-life. For this reason, a Fucus vesiculosus extract (FVE) was used at the concentrations of 250 mg/kg (FVE-250), 500 mg/kg

(FVE-500) and 1000 mg/kg (FVE-1000) in the formulation of pork patties with linseed oil oleogel as a fat replacer to assess its effectiveness in improving the physicochemical properties and prolong the shelf life.

RESULTS: Total polyphenol content of FVE was determined as 20 g PGE/100 g extract, and the antioxidant values ranged from 37.5 µmol TE/g (FRAP assay) to 2111 µmol TE/g extract

(ABTS assay). Regarding oxidation stability, FVE-1000 and BHT treatments showed the lowest

TBARs values and carbonyls content. On the other hand, FVE did not improve color, surface discoloration, and odor attributes in patties over time. According to sensory evaluation, no significant differences among all cooked patties studied were observed.

CONCLUSION: The protection of the FVE was insufficient to guarantee a suitable oxidation inhibition, being its use in meat or meat products discarded.

Keywords: Seaweed extract; lipid and protein oxidation; physicochemical parameters; sensorial analysis; antioxidant activity

2 INTRODUCTION

Changes in muscle foods due to oxidative reactions both in lipids and proteins have a strong impact on the quality deterioration, causing a reduction on the shelf life as well as a considerable loss of nutrients.1 Oxidation reactions may occur in a variety of food products including meat and meat products as responsible phenomena for the deterioration of nutritional and sensory quality.1 The changes in the organoleptic attributes may result in the consumer dissatisfaction and product rejection. For instance, according to Carpenter et al.2 variations in color parameters could affect to purchase decision of consumers, since the color of product often regarded as an indicator of the product freshness and quality.3 Proteins can be also threatened by oxidation, leading to loss of amino acids and alterations in their functionality with negative consequences for the texture of product.4

Both scientific community and meat industry identify the spoiling of muscle foods resulting of oxidative reactions as a challenge leading them to develop strategies to slow down these processes.1 Therefore, to keep under control oxidation reactions in lipids and/or proteins, synthetic antioxidants, such as butylated hydroxyl toluene (BHT) and butylated hydroxyl anisole (BHA), are added to meat, fats, and oils.5 However, due to possible toxic effects on human’s health, notable concerns raised regarding the application of these substances in food products.6

In order to struggle against this inconvenient, as well as due to consumer trends to consume more natural products; some replacer for synthetic antioxidants were introduced.7

Plants represent an increasing alternative to the synthetic additives by using their compounds or extracts, which could contribute to delay oxidation and stabilize color and texture in foods.1

The marine environment showed to be very rich in natural compounds with chemical characteristics, being some of these absent in some terrestrial ecosystems. For example, macroalgae and their extracts are particularly rich in functional properties,8 being a valuable source for antioxidant agents, such as carotenoids, polyphenols, alkaloids, tocopherol and

3 terpenes.9 Brown algae Fucus vesiculosus is rich in phlorotannins, which are particular polyphenolic compounds of brown algae.10 Some phlorotannins have shown to possess higher antioxidant capacity than commercial antioxidants, being interesting alternatives to be used as a natural antioxidant.

The burger is a famous and very consumed product all over the world by all-age people, very joined to a rush lifestyle.11 However, consumers tend to perceive animal fats and meat as unhealthy.11 The replacement of these fats by vegetable oils turns out to be a successful strategy to improve the nutritional profile of muscle foods, thus recovering consumer confidence.

Linseed oil may be a good alternative to replace animal fat in these kinds of products. It is the top seed oil in α-linolenic acid content, with about 50-55%. Moreover, it has a percentage of 15-18% of α-linoleic acid, demonstrating a positive balance of polyunsaturated, monounsaturated and saturated FAs.12 Unfortunately, the replacement of hard fats with oils usually leads to a decrease on product quality, resulting in altered properties, such as yield, textural attributes and sensory characteristics.13 Consequently, the structuring of edible oils respecting their fat composition is actively researched. The use of gelling agents has recently proved to be an efficient approach for this purpose. These agents are organic liquids known as

“oleogels” encased in thermo reversible three-dimensional gel networks.14 These oleogels respect the FA profile of the oils, as well as the functionality and texture of the final product.

In this context, the aims of this investigation were to evaluate the effectiveness of Fucus vesiculosus extracts to extend the shelf life of pork patties manufactured with linseed oil oleogels and packaged under modified atmosphere conditions during refrigerated storage; the physicochemical properties and sensory attributes of the seaweed formulated pork patties were compared with control samples (manufactured with any antioxidant, or containing BHT, a chemical preservative).

4 MATERIAL AND METHODS

Algal material

The brown seaweed Fucus vesiculosus (Kingdom: Chromista, Phylum: Ochrophyta, Class:

Phaeophyceae and Order: Fucales) was supplied by Portomuiños company (A Coruña, Spain).

The algal material was collected in the Galician Atlantic coast, in the area of Camariñas, a village close to the town of A Coruña (NW of Spain). It was further subjected to milling by a conventional homemade mincer until obtaining particles lower than 0.8 mm in size using a porous mesh. After this treatment, the ground seaweed was packaged in plastic bags under vacuum at 75% and stored at -20 °C until further use.

Obtaining Fucus vesiculosus extracts

The algal material was kept in touch with a mixture of water/ethanol (50:50, v/v) in the proportion of 1:10 (w/v) for 30 min using an ultrasound bath at room temperature. Then, the solid residue was separated from the solvent by centrifuging for 10 min at 3000 x g and 4 °C.

The supernatant was collected and filtered under vacuum using a simple lab filter. The liquid extract obtained in this way was subjected to vacuum conditions at the temperature of 40 °C using a rotary evaporator to remove the ethanol of the used solvent. Finally, the aqueous extract was freeze-dried and kept at -20 °C until further use. The seaweed extract was reconstituted in water/ethanol (50:50, v/v) before its use.

Preparation of linseed oil oleogel

The oleogel used in the present study was constituted by an oil phase and a mixture of two structuring agents (ɣ-oryzanol and β-sitosterol). The oil phase used was a commercial linseed oil (Vitaquell®) with 72% polyunsaturated (approx. 55% of α-linoleic), 19% monounsaturated and 9% saturated fatty acids (FAs). The ɣ-oryzanol and β-sitosterol were mixed in a ratio of 60:40 (w/w). This ratio corresponded to 1:1 M of each component and was reported as the ratio that allows to obtaining the firmest transparent gel. The ɣ-oryzanol and the β-sitosterol were purchased from Oryza Co. (Japan) and Sigma-Aldrich (France),

5 respectively. Both structuring agents were dispersed by stirring until solubilization along with the linseed oil at a temperature of 80 °C for 30 minutes. Then, the mixture was left cooling at room temperature until gel formation.

Manufacture of pork patties

A total of 125 patties divided into 25 units per treatment (five treatments) were manufactured in the pilot plant from the Centro Tecnolóxico da Carne located in Ourense,

Spain. The different treatments were carried out as follows: patties with incorporated extract from the seaweed Fucus vesiculosus (FVE) at 250 (FVE-250), 500 (FVE-500) and 1000 (FVE-

1000) mg/kg as antioxidants, patties with added butylated hydroxytoluene (BHT) at the concentration of 200 mg/kg as conventional synthetic antioxidant (BHT), and finally, patties without antioxidant (CO) as control treatment. Patties were manufactured with primal cuts of pig loin purchased in a local market. The meat samples were chopped using a 6 mm plate under refrigeration conditions in a mincer machine (La Minerva, Bologna, Italy), and then mixed with linseed oil oleogel, salt, and water according to proportions indicated in Table 1.

Patties samples of 60 g were produced using a burger maker (A-2000, Gaser, Girona, Spain).

After production, the patties were wrapped in polystyrene trays of 300 mm of thickness with

80% O2 and 20% CO2, using a LARI3/Pn T-VG-R-SKIN heat sealer (Ca. Ve.Co., Palazzolo, Italy).

Polyethylene film of 74 mm of thickness and permeability <2 mL/(m2 bar/day) appropriate for gas mixture was used (Viduca, Alicante, Spain) and stored at 2 ± 1 °C under the light in order to simulate the typical storage condition of supermarkets. Lux values could vary in the range of

15-20 in function of the tray position (Digital luxometer HT 306, Faenza, Italy). The conventional light source was used in this study which means that no wavelength or range, such as UV was filtered. Pork patties were analyzed at initial day for chemical composition and

FA profile, and at 0, 7, 11, 15, and 18 days of storage, for physicochemical parameters (pH and color) and stability oxidation (TBARs values and protein oxidation). In addition, odor and visual acceptance were carried out in all sampling points, whereas preference tests were assessed at

6 the beginning of storage (5 units per treatment). In each sampling point, four patties (units) from each type of manufacture were taken for analysis.

Total Phenolic Content (TPC) and evaluation of the antioxidant activity of the seaweed extracts

TPC and total antioxidant capacity (ABTS, ORAC, DPPH and FRAP assays) were determined according to the methods previously described by the research group in algal samples. For a detailed description of the methods see Agregán et al.9

Chemical composition and fatty acid (FA) profile of pork patties

Protein, moisture, and ash contents were determined following ISO recommended standards ISO 937:1978,15 ISO 1442:1997,16 ISO 936:1998,17 respectively. The fat was extracted using an extractor (Ankom XT10, ANKOM Technology Corp., Macedon, NY, USA) and quantified following AOCS official procedure Am 5-04.18 The FA profile was determined according to the methods described by Fernandes et al.7 using a gas chromatograph equipped with FID detection (GC Agilent 7890N; Agilent Technologies Spain, S.L., Madrid, Spain).

Determination of physical parameters of pork patties

A portable digital pH-meter (Hanna Instruments, Eibar, Spain) equipped with a penetration probe was employed to measure the pH of pork patties.

The color parameters in the CIELAB color space system, including lightness (L*), redness

(a*) and yellowness (b*), were determined using a portable colorimeter device (Konica

Minolta CM-600d, Osaka, Japan) equipped with a pulsed xenon arc lamp filtered to D65 illuminant lighting conditions, with a zero-degree viewing angle geometry and aperture size of

8 mm. The color was measured in six different points on the patty surface after 10 min of exposure to the environmental atmosphere.

Determination of protein and lipid oxidations indexes

The protein oxidation was evaluated based on the procedure described by Mercier et al.19 and results were expressed as nmol of carbonyl/mg of protein. The stability pork patties lipid was assessed by thiobarbituric acid-reactive substance (TBARs) test, following the method

7 ascribed by Vyncke20 and expressing the results as mg of malonaldehyde (MDA)/kg of the sample.

Sensory evaluation of raw and cooked pork patties

Sensory analysis of raw and cooked patties was carried out with sixteen talented panelists selected from the Centro Tecnolóxico da Carne. An acceptance test was conducted for raw patties at 0, 7, 11, 15 and 18 days of storage, and an acceptance and preference test for cooked patties. In the acceptance test, panelist accepted or rejected the samples, and in the preference test, panelist classified the samples from the best to the worst according to their tastes. In the acceptance test, panelist scored raw samples based on color, discoloration at surface and odor, and in cooking based on odor and taste using a 5-point hedonic scale (1 = excellent, 5 = not acceptable). In the preference test for cooked samples, panelist scored giving the number 1 to favorite sample, the number 2 to the next one and so on. Patties were cooked in an oven (Rational Combimasterplus CMP61, Germany) equipped with a core temperature of

70 °C. Sensory tests were carried out in an individual cabin illuminated with white light.

Samples were given on disposable plastic dishes encoded with 3-digit random numbers.21

Water and unsalted toasted bread were provided at the beginning of testing and among samples in order to remove waste and residual flavors of mouth.

Statistical analyses

The statistical analysis of the results were conducted using the IBM SPSS Statistics® 23.0 program (IBM Corporation, Somers, NY, USA). A one-way analysis of variance (ANOVA) was applied to all the variables assessed in the study. Time and different treatments were defined as the fixed variables and the chemical composition (moisture, fat, protein and ash), the FA profile, the pH, the colour and the oxidation parameters (TBARS and protein oxidation) as the independents. The least square means (LSM) were separated using Duncan's post hoc test

(significance level P<0.05). The Friedman’s test (significance level α<0.05) was applied to cooked patties in the sensorial analysis to know the panelist preferences.

8 RESULTS AND DISCUSSION

Total phenolic content (TPC) and antioxidant capacity of Fucus vesiculosus extracts (FVE)

The TPC of the FVE was ≈20 g phloroglucinol equivalents (PGE)/100 g extract. These results contrast to those obtained by Agregán et al.8 who reported very low contents of phenolic compounds in water extracts of several brown seaweeds (Bifurcaria bifurcate, Fucus vesiculosus, and Ascophyllum nodosum) with values ranging from 0.96 and 1.99 g PGE/100 g extract. In another study, Wang et al.22 found 17.6 g PGE/100 g extract in a water extract from

Fucus vesiculosus, which is a value really close to the value found in the present work.

According to Wang et al.,22 phenolic compounds are more easily extractable using polar organic solvents than using water. Aqueous mixtures of methanol, ethanol, and acetone are recommended for this purpose,23 which resulted in a yield of 24.2 g PGE/100 g extract in a 70% acetone extract from Fucus vesiculosus, being this reported value not higher compared to the findings of the current investigation. Moreover, almost all the brown algae tested in the study carried out by Waterman and Mole23 had higher amounts of polyphenols than the red and green algae.

Similarly, according to Jiménez-Escrig et al.,24 the brown seaweeds tested showed higher

TPCs than the red seaweeds whereas the organic polar solvents displayed to be more efficient in extracting polyphenolic compounds than water. However, TPCs obtained for all seaweed extracts analyzed did not exceed the amount of 3.5 g PGE/100 g extract, which is considerably lower than the results obtained in the present study for the FVE, even using a combination of organic polar solvents, such as methanol and water, in the same ratio that applied in the current investigation. Jiménez-Escrig et al.24 and Farvin and Jacobsen25 reported high TPCs in all the Fucus species investigated. Generally speaking, brown algae are richer in phenolic compounds than other types of algae due to the high concentration of phlorotannins, a family of polyphenol compounds exclusively from brown algae found in special vesicles (physodes) within the cells.

9 On the other hand, the antioxidant value obtained via the ABTS test was 2111±22.63

8 µmol TE/g extract (IC50: 0.65±0.01). In a previous study, Agregán et al. reported lower values which ranged from 100 to 1100 µmol TE/g extract in aqueous extracts of three brown seaweeds (Ascophyllum nodosum, Fucus vesiculosus, and Bifurcaria bifurcata) using the same assay, while the Fucus extract showed the highest value. In this case, lower values could be related to the lower polyphenol content, since all these extracts displayed very lower concentrations of polyphenols than the extract used in the present study. Moreover, the positive correlation between polyphenol content and antioxidant capacity was well documented.22,25

Regarding ORAC assay, a value of 1598.5±34.65 µmol TE/g extract was observed for the

FVE, which is comparable to that reported by Wang et al.22 (1417 µmol TE/g extract for a 70% acetone extract) for Ascophylum nodosum. However, higher values were found by these authors when they used 70% acetone to obtain the extracts from Fucus vesiculosus and Fucus

Serratus (2567 and 2567 µmol TE/g extract, respectively), and also in water extracts of the

Ascophylum nodosum (2000 µmol TE/g extract). On the contrary, Agregán et al.8 reported a considerably lower value for the water extract from Fucus vesiculosus (756.5 µmol TE/g extract).

The DPPH assay of FVE gave values of 278±1.41 µmol TE/g extract (IC50: 3.47±0.01 g/L).

In this context, Agregán et al.8 also used the DPPH assay to measure antioxidant capacity, reporting a lower value in the water extract from Fucus vesiculosus (135.31 µmol TE/g extract) with a consequently higher value for its IC50 (4.19 g/L), since the lower IC50 value of the extract means higher radical scavenging activity. Farvin and Jacobsen25 found that the water extracts of two Fucus species, namely F. vesiculosus, and F. serratus, had the highest antioxidant activities (IC50 value of 0.0083 g/L for both) among other studied species. The ethanol extracts of these algal species also exhibit highest radical scavenging activities with the IC50 of 0.0099 g/L and 0.0092 g/L for F. vesiculosus and F. serratus, respectively. The reported IC50 values in

10 the study conducted by Farvin and Jacobsen25 were lower than those obtained for the FVE analyzed in the current study, thus indicating a higher antioxidant capacity of these extracts in the previously conducted research. In another study, Rajauria et al.26 also found lower antioxidant capacities in water and methanol extracts from Himanthalia elongata alga while compared with their mixtures. According to Connan et al.,27 environmental and intrinsic factors of algae, such as light or salinity and age or length could lead to big variations in the phenolic content of algae, what it may cause differences in their antioxidant capacities.

Based on the findings, a value of 37.5±2.12 µmol TE/g extract was recorded via FRAP assay for the FVE. This result was very close to those reported by Rajauria et al.26 who showed

41.15 µmol TE/g extract and 46.75 µmol TE/g extract for 40% and 60% methanol extracts, respectively, from Himanthalia elongata brown seaweed. The FRAP analytical method was also used by Agregán et al.8 to measure the antioxidant capacity of seaweeds and a slightly higher value in the water extract from F. vesiculosus (51.66 µmol TE/g extract) while compared to the other two extracts (Ascophyllum nodosum and Bifurcaria bifurcata), with values of 7.52 and 26.93 µmol TE/g extract, respectively, was noted.

Effect of FVE incorporation on the proximate composition and fa profile of pork patties

According to the Table 2, which shows the chemical composition (protein, fat, moisture, and ash) and FA profile of pork patties, no significant (P>0.05) differences in chemical composition were observed among the different formulations. Moreover, the different concentrations of FVE in patties did not influence the percentages of moisture, fat, protein and ash, being these results in close agreement with those reported by Rodríguez Carpena et al.,28 where no changes regarding the proximate composition of raw pork meat patties with incorporation of Mediterranean berries and avocado by-products were noted.

The monounsaturated FAs (MUFAs) were the most abundant type of FA among all formulations, with an average percentage of the area of 41.10% of the total FAs analyzed, followed by saturated (SFA) and polyunsaturated FA (PUFA) groups, with an average

11 percentage of area of 31.95% and 25.74%, respectively. As it was expected, the PUFA percentage was considerably higher than those obtained by other authors in previous studies regarding the pork meat without the addition of vegetable oils.29 This difference in FA profile may be attributed to the replacement of pork fat by linseed oil in the patties formulation since seed oils are well known to possess high amounts of PUFAs. Similar findings were reported by

Delgado-Pando et al.30 after replacing pork fat with olive, linseed and fish oils, and also by

Rodríguez-Carpena et al.31 when replacing pork fat with sunflower oil.

The oleic acid (C18:1n-9c), the most abundant MUFA and one of the main FA among all treatments, has higher amounts in CO treatment. All patties contained substantial amounts of palmitic (C16:0) and stearic acids (C18:0), as the most abundant SFAs, with an average percentage of 19.64 and 10.93, respectively. Similar results were shown in some researches on porcine meat. According to Pateiro et al.,29 who investigated the fat oxidation in enriched pâtés, percentages around 20-22% and 10-12% for palmitic and stearic acids, respectively were observed. On the other hand, linoleic (C16:2n-6) and linolenic (C18:3n-3) acids, the most abundant PUFAs were also present at a high percentage, with average values of 13.16% and

11.79%, respectively. These results were predictable taking into account that linseed oil is very rich in linoleic (15-18%) and linolenic (50-55 %) acids. In a low-fat pork liver pâté, Delgado-

Pando et al.30 reported a significant (P<0.05) increase in linoleic and linolenic acids when the pork fat was totally and partially replaced by a mixture of olive, linseed and fish oils.

Omega-6 and omega-3 FAs are essential fatty acids for humans due to the inability of the human body to synthesize them. Therefore, these FAs must be ingested through the intake of foods or supplements. Nevertheless, the consumption of diets with high amounts of omega-

6 PUFAs and very high n-6/n-3 ratios promotes several diseases including cardiovascular diseases and some certain types of cancers.32 On the other hand, high PUFA levels and low n-

6/n-3 ratios induce suppressive effects.32 Pork patties in the present study showed n6/n-3 ratio values between 1 and 2 for all treatments without significant differences (P> 0.05). These

12 results are in agreement with the recommendations given by Simopoulos32; the target n-6/n-3 ratio in a healthy diet should be balanced between 1:1 and 2:1.

Effect of FVE on physical properties of pork patties during storage

The results of the evaluation of pH and color parameters in pork patties along refrigerated storage are shown in Table 3. The different patty formulations led to significant differences in the pH at the day 0 (P<0.001) and 18 (P<0.01). In this regard, Lorenzo et al.3 reported the higher pH values during storage of pork patties formulated with chestnut and seaweed extracts when compared with patties manufactured with tea and grape seed extracts. On the other hand, in the current study, the refrigerated storage modified the pH values of patties in all formulations, except for the FVE-500 treatment; the pH values were significantly different in the CO and BHT (P<0.01) treatments, and in the FVE-250 (P<0.05) and

FVE-1000 (P<0.001) treatments. A marked decrease was observed at day 11, except for the

BHT treatment. Then, pH increased sharply until 15 days; however, in the FVE-250 treatment, pH began to increase at day 15. Similar results were observed in pork patties with added natural extracts during refrigerated storage.3

Different formulations of patties, as well as the storage time, also affected the color parameters (L*, a*, and b*). However, the lightness (L*) did not seem to follow any specific trend over storage time despite showing a significant (P<0.05) change in the BHT, FVE-250, and

FVE-1000 treatments. The L* values were not affected by different formulations as previously reported by Lorenzo et al.3 Regarding redness (a*), a progressive loss of red color on patties surface was noted along storage. Similar trends were also observed in other investigations with meats from different species.7 The reduction in redness on the patty surface along storage confirmed the appearance of a fading phenomenon since this process is mainly marked by the loss of a* values,3 which is attributed to the metmyoglobin produced through oxidation of myoglobin. From the beginning of storage patties showed a progressive brownish

13 appearance on the surface until the final of storage. Brown color in pork meat is reported that it is perceived with a* values ranging between 4.6 and 10.8.33

Overall, the addition of antioxidants to pork patties affected to redness along time, although differences were only significant at the beginning (P<0.05) and at the end (P<0.01) of the storage time. At day 0, patties manufactured with seaweed extract showed lower a* values than CO treatment. On the contrary, at day 18 the result was reversed, meaning that seaweed antioxidants helped to protect the red color on the patty surface. This stabilizing effect in a* values was also found by other authors using different natural antioxidants, such as grape seed,3 oregano,7,34 borage, rosemary,34 and avocado extracts.28 Finally, yellowness

(b*) also decreased over time, although less markedly than redness. These results were in good agreement with those found by Fernandes et al.7 in sheep patties with added BHT and oregano as antioxidants. The different formulations affected the b* values of patties along refrigerated storage. Better retention of yellow color was achieved in treatments with seaweed extract than in CO treatment.

Barbut et al.13 found that replacing of beef fat with canola oil oleogel made with different percentages of ethylcellulose and sorbitan monostearate, induced differences in lightness and redness of frankfurters. In this context, L* values were increased when replacing fat by oleogel in most of the treatments, although differences were not significant (P>0.05).

Conversely, a* values decreased significantly (P<0.05) in formulations with oleogel.

On the other hand, according to Barbut et al.,13 using of canola oil without being in gel form had a significant (P<0.05) effect on color, increasing and decreasing L* and a* values, respectively, regarding treatment formulated without beef fat replacement. Considering the addition of a seed (canola) oil generated changes in the surface color of the final product, it could be thought that the addition of linseed oil into our samples induced some modifications in patties color. The results reported by Utrilla et al.35 strengthen this hypothesis, finding that

14 higher olive oil contents in dry-ripened sausages produced higher and lower values of b* and a* values, respectively, at the beginning of ripening.

Oxidative stability during refrigerated storage of pork patties

Lipid oxidation was quantified in patties samples through the TBARs assay (Table 4). All treatments showed a significant (P<0.001) increase in TBARs values during refrigerated storage. According to the results in the present study, patties containing BHT (BHT treatment) and 1000 mg/kg of seaweed extract (FVE-1000) had higher oxidative stability than the CO, FVE-

250 and FVE-500 treatments (Table 4).

TBARs values slightly increased until day 11, after which an abrupt increase was noted up to 15 days. Other authors, such as Lorenzo et al.3 and Sánchez-Escalante et al.,34 reported similar evolutions of TBARs values in patties with added natural antioxidants during refrigerated storage. The latter found that the control treatment showed a sharp increment in the oxidation from day 4 (~0.8 mg MDA/kg sample) until day 12 (~3.8 MDA/kg sample). In the present study, oxidation showed the sharp increment with comparable TBARs values

(0.87±0.17 and 3.92±0.63 mg MDA/kg sample, respectively). After this point, Sánchez-

Escalante et al.34 observed a stabilization and even a slight decrease in the later days. On the other hand, they noted that TBARs values of some of the patties with added natural antioxidants, such as oregano, rosemary and rosemary with ascorbic acid, remained very stable until 16 days with values lower than 1 MDA/kg sample, increasing sharply until the end of storage reaching values between 1 and 3 mg MDA/kg sample, lower than those found by us at 18 days for patties with addition of seaweed extract.

Evolution of protein oxidation in pork patties during storage is shown in Table 5. A significant (P<0.01) increase in protein oxidation over time was found for all treatments.

Similar findings were reported by other authors.7,28 Protein oxidation began on day 7 and increased until the end of storage. The CO treatment was the least stable to protein oxidation, reaching a value of 6.81±0.61 nmol carbonyl/mg protein at day 18. The BHT treatment was the

15 most effective, showing the patties lower carbonyl values than in the other treatments. The treatments with the FVE significantly reduced the protein oxidation with respect to the control without antioxidant at the last storage day, reducing the carbonyl values between 0.77 and

1.15 nmol/mg protein. The FVE-1000 treatment also showed significantly (P<0.05) lower values than the CO treatment at day 15. This result suggests that the adding of FVE was effective in softening the final carbonyl concentrations, especially at the level of 1000 mg extract/kg sample. Other natural extracts, such as peel and seed extracts from avocado28 and oregano extracts7 showed a time delay of carbonyl formation when they were added to pork

(two first authors), beef and sheep meats, respectively. Nutritional quality or tenderness are appreciated attributes in meat which are affected by protein oxidation, for this reason, its inhibition is important to preserve raw material of these undesirable effects.7 The phenolic content found in the FVE probably protected patties against oxidative stress, delaying the appearance of degradation products.

Effect of FVE on sensory properties of pork patties

Figure 1 and 2 show the findings from the sensory evaluation of cooked (day 0) and raw

(at 0, 7, 11, 15, and 18 days) pork patties, respectively. According to the results in the present study, cooked samples at day 0 did not show any significant differences in odor and taste among all treatments. The most significant results can be correlated with odor attribute, reached for the sample with highest acceptation (FVE-500 sample) while no differences were found among the CO treatment and FVE 500 and 1000 treatments. Bañón et al.36 reported similar findings with no appreciable differences in odor and taste at day 0 in low sulfite beef patties formulated with green tea and grape seed extracts. Regarding the sample preference, no consistent differences were observed among the sample ordination by the panelists, wherewith the small differences found in the acceptance test seemed not having influence in the preference.

16 Regarding the evolution of the sensory properties during refrigerated storage, the color at the surface and odor attributes in raw samples from all groups studied showed acceptable values until day 11. From that time, the acceptability of patties decreased in all treatments until they reached a score of “hardly acceptable” at the end of storage. From day 11, the patties with BHT displayed color and discoloration at surface values close to acceptability. On the other hand, the FVE-1000 treatment reached better color punctuations at day 18 than the rest of treatments, but always behind acceptability. In accordance with results obtained, it may be concluded that FVE did not improve any of the attributes studied (color, discoloration at surface and odor) at the concentrations used during the shelf-life of pork patties.

CONCLUSIONS

In this study, Fucus vesiculosus extracts (FVE) were used, for the first time, as natural preservatives in pork patties. According to the results, the incorporation of FVE at the concentration of 1000 mg/kg into pork patties successfully protected the samples against oxidation, although this treatment was not as effective as the synthetic BHT at the concentration of 200 mg/kg. One of the key benefits of the proposed preservation technique in this study, i.e., using seaweed extract, was the absence of apparent differences in the sensory attributes studied in raw patties. Furthermore, the FVE-500 treatment obtained the best sensory scores for the odor attribute in the cooked product. Even so, the limited protection against oxidation of the extract obtained in the conditions reported in the present study makes it unviable to be used in meats or meat products. Therefore, increasing the antioxidant power of the extract seems to be the best way to improve the fat and protein protection in meat and meat products against oxidation over storage time. Further research may explore other potential benefits derived from the incorporation of FVE in meat products such as its potential nutritional benefits for the consumers.

17 ACKNOWLEDGEMENTS

The authors would like to thank Xunta de Galicia (grant number IN607B 2016/28).

Authors also thank INIA (Instituto Nacional de Investigaciones Agrarias y Alimentarias, Spain) for granting Ruben Agregán with a predoctoral scholarship (CPR2014-0128). Jose M. Lorenzo is a member of the MARCARNE network, funded by CYTED (ref. 116RT0503). Mohsen Gavahian wants to thank the support of the Ministry of Economic Affairs, project no. 107-EC-17-A-22-

0332, Taiwan (R.O.C). He also would like to declare that his main contribution to this work was related to the extraction and the extract antioxidant studies. Finally, Amin Mousavi Khaneghah wishes to thank the support of CNPq-TWAS Postgraduate Fellowship (Grant # 3240274290).

REFERENCES

1 Lorenzo JM, Pateiro M, Domínguez R, Barba FJ, Putnik P, Kovačević DB, Shpigelman A,

Granato D and Franco D. Berries extracts as natural antioxidants in meat products:

A review. Food Res. Int. 106: 1095-1104.

https://doi.org/10.1016/j.foodres.2017.12.005

2 Carpenter CE, Cornforth DP and Whittier D. Consumer preferences for beef color and

packaging did not affect eating satisfaction. Meat Sci. 57: 359-363.

https://doi.org/10.1016/S0309-1740(00)00111-X

3 Lorenzo JM, Sineiro J, Amado IR and Franco D. Influence of natural extracts on the shelf

life of modified atmosphere-packaged pork patties. Meat Sci. 96: 526-534.

https://doi.org/10.1016/j.meatsci.2013.08.007

4 Lund MN, Heinonen M, Baron CP and Estevez M. Protein oxidation in muscle foods: A

review. Mol. Nutr. Food Res. 55: 83-95. https://doi.org/10.1002/mnfr.201000453

5 Franco D, Rodríguez-Amado I, Agregán R, Munekata PES, Vázquez JA, Barba FJ and

Lorenzo JM. Optimization of antioxidants extraction from peanut skin to prevent

oxidative processes during soybean oil storage. LWT-Food Sci. Technol. 88: 1-8.

https://doi.org/10.1016/j.lwt.2017.09.027

18 6 Granato D, Nunes DS and Barba FJ. An integrated strategy between food chemistry,

biology, nutrition, pharmacology, and statistics in the development of functional

foods: A proposal. Trends Food Sci. Tech. 62: 13-22.

https://doi.org/10.1016/j.tifs.2016.12.010

7 Fernandes RPP, Trindade MA, Lorenzo JM, Munekata PES and De Melo MP. Effects of

oregano extract on oxidative, microbiological and sensory stability of sheep burgers

packed in modified atmosphere. Food Control 63: 65-75.

https://doi.org/10.1016/j.foodcont.2015.11.027

8 Agregán R, Munekata PE, Domínguez R, Carballo J, Franco D and Lorenzo JM. Proximate

composition, phenolic content and in vitro antioxidant activity of aqueous extracts of

the seaweed Ascophyllum nodosum, Bifurcaria bifurcata and Fucus vesiculosus.

Effect of addition of the extracts on the oxidative stability of canola oil under

accelerated storage conditions. Food Res. Int. 99: 986-994.

https://doi.org/10.1016/j.foodres.2016.11.009

9 Agregán R, Munekata PES, Franco D, Carballo J, Barba FJ and Lorenzo JM. Antioxidant

potential of extracts obtained from macro- (Ascophyllum nodosum, Fucus

vesiculosus and Bifurcaria bifurcata) and micro-algae (Chlorella vulgaris and Spirulina

platensis) assisted by ultrasound. Medicines. 5: 33.

https://doi:10.3390/medicines5020033

10 Liu X, Yuan W, Sharma-Shivappa R and van Zanten J. Antioxidant activity of phlorotannins

from phlorotannins brown algae. Int. J. Agr. Biol. Eng. 10: 184-191. http://doi:

10.25165/j.ijabe.20171006.2854

11 Afshari R, Hosseini H, Khaneghah AM and Khaksar R. Physico-chemical properties of

functional low-fat beef burgers: Fatty acid profile modification. LWT-Food Sci.

Technol. 78: 325-331. https://doi.org/10.1016/j.lwt.2016.12.054

19 12 Carter JF. Potential of flaxseed and flaxseed oil in baked goods and other products in

human nutrition. Cereal Food World

13 Barbut S, Wood J and Marangoni A. Potential use of organogels to replace animal fat in

comminuted meat products. Meat Sci. 122: 155-162.

https://doi.org/10.1016/j.meatsci.2016.08.003

14 Totosaus A, Gonzaléz-Gonzaléz R and Fragoso M. Influence of the type of cellulosic

derivatives on the texture, and oxidative and thermal stability of soybean oil oleogel.

Grasas Aceites 67: 152. https://doi.org/10.3989/gya.0440161

15 ISO 937:1978 standard. Determination of nitrogen content. International standards meat

and meat products. International Organization for Standardization (ISO). Genéve.

16 ISO 1442:1997 standard. Determination of moisture content. International standards

meat and meat products. International Organization for Standardization (ISO).

Genéve.

17 ISO 936:1998 Standard. Determination of ash content. International standards meat and

meat products. International Organization for Standardization (ISO). Genéve.

18 AOCS official procedure Am 5-04. Rapid determination of oil/fat utilizing high temperature

solvent extraction. Urbana: American Oil Chemists Society, 2005.

19 Mercier Y, Gatellier P and Renerre M. Lipid and protein oxidation in vitro, and antioxidant

potential in meat from Charolais cows finished on pasture or mixed diet. Meat Sci.

66: 467-473. https://doi.org/10.1016/S0309-1740(03)00135-9

20 Vyncke W. Direct determination of the thiobarbituric acid value in trichloracetic acid

extracts of fish as a measure of oxidative rancidity. Fett. Wiss. Technol. 72: 1084-

1087. https://doi.org/10.1002/lipi.19700721218

21 Macfie HJ, Bratchell N, Greenhoff K and Vallis LV. Designs to balance the effect of order to

of presentation and 1st-order carry-over effects in hall test. J. Sens. Stud. 4: 129-148.

20 22 Wang T, J nsd ttir R and Ólafsdóttir G. Total phenolic compounds, radical scavenging and

metal chelation of extracts from Icelandic seaweeds. Food Chem. 116: 240-248.

https://doi.org/10.1016/j.foodchem.2009.02.041

23 Waterman PG, Mole S (eds). Analysis of phenolic plant metabolites. Oxford: Blackwell

Scientific Publications, 1994.

24 Jiménez-Escrig A, Gómez-Ordóñez E and Rupérez P. Brown and red seaweeds as potential

sources of antioxidant nutraceuticals. J. Appl. Phycol. 24: 1123-1132. http://doi

10.1007/s10811-011-9742-8

25 Farvin KS and Jacobsen C. Phenolic compounds and antioxidant activities of selected

species of seaweeds from Danish coast. Food Chem. 138: 1670-1681.

https://doi.org/10.1016/j.foodchem.2012.10.078

26 Rajauria G, Jaiswal AK, Abu‐Gannam N and Gupta S. Antimicrobial, antioxidant and free

radical‐scavenging capacity of brown seaweed Himanthalia elongata from western

coast of Ireland. J. Food Biochem. 37: 322-335. https://doi.org/10.1111/j.1745-

4514.2012.00663.x

27 Connan S, Goulard F, Stiger V, Deslandes E and Gall EA. Interspecific and temporal

variation in phlorotannin levels in an assemblage of brown algae. Bot. Mar. 47: 410-

416. https://doi.org/10.1515/BOT.2004.057

28 Rodríguez-Carpena JG, Morcuende D and Estévez M. Avocado by-products as inhibitors of

color deterioration and lipid and protein oxidation in raw porcine patties subjected to

chilled storage. Meat Sci. 89: 166-173.

https://doi.org/10.1016/j.meatsci.2011.04.013

29 Pateiro M, Lorenzo JM, Vázquez JA and Franco D. Oxidation stability of pig liver paté with

increasing levels of natural antioxidants (grape and tea). Antioxid. 4: 102-123.

https://doi:10.3390/antiox4010102

21 30 Delgado-Pando G, Cofrades S, Rodríguez-Salas L and Jiménez-Colmenero F. A healthier oil

combination and konjac gel as functional ingredients in low-fat pork liver pâté. Meat

Sci. 88: 241-248. https://doi.org/10.1016/j.meatsci.2010.12.028

31 Rodríguez-Carpena JG, Morcuende D, Estévez M. Avocado, sunflower and olive oils as

replacers of pork back-fat in burger patties: effect on lipid composition, oxidative

stability and quality traits. Meat Sci. 90: 106-115.

https://doi.org/10.1016/j.meatsci.2011.06.007

32 Simopoulos AP. The omega-6/omega-3 fatty acid ratio: health implications. Ocl-Ol.

Corps.Gras Li. 17: 267-275. https://doi.org/10.1051/ocl.2010.0325

33 De Santos F, Rojas M, Lockhorn G and Brewer MS. Effect of carbon monoxide in

modified atmosphere packaging, storage time and endpoint cooking temperature

on the internal color of enhanced pork. Meat Sci. 77: 520-528.

https://doi.org/10.1016/j.meatsci.2007.04.031

34 Sánchez‐Escalante A, Djenane D, Torrescano G, Beltrán JA and Roncales P. Antioxidant

action of borage, rosemary, oregano, and ascorbic acid in beef patties packaged in

modified atmosphere. J. Food Sci. 68: 339-344. https://doi.org/10.1111/j.1365-

2621.2003.tb14162.x

35 Utrilla MC, Ruiz AG and Soriano A. Effect of partial replacement of pork meat with an

olive oil organogel on the physicochemical and sensory quality of dry-ripened

venison sausages. Meat Sci. 97: 575-582.

https://doi.org/10.1016/j.meatsci.2014.03.001

36 Bañón S, Díaz P, Rodríguez M, Garrido MD and Price. Ascorbate, green tea and grape seed

extracts increase the shelf life of low sulphite beef patties. Meat Sci. 77: 626-633.

https://doi.org/10.1016/j.meatsci.2007.05.015

22

Acceptable odour taste odour

1 2 3 4 5 Hedonic scale CON BHT FVE-250 FVE-500 FVE-1000

Figure 1. Average sensory scores for pork patties with different added extracts

DISCOLORATION AT SURFACE

5

4

3 Acceptabl e

2 Hedonic scale Hedonic 1

0 0 5 10 15 20 Storage days CON BHT FVE-250 FVE-500 FVE-1000

COLOR 5

4

3 Acceptabl e 2 Hedonic scale Hedonic 1 0 0 5 10 15 20 Storage days CON BHT FVE-250 FVE-500 FVE-1000

23 ODOR

5

4

3 Acceptabl e

2 Hedonic scale Hedonic 1

0 0 5 10 15 20 Storage days CON BHT FVE-250 FVE-500 FVE-1000

Figure 2. Evolution of color, discoloration of surface and odor attributes in raw pork patties with different added extracts during refrigerated storage. Hedonic scale used: 1 = excellent; 2 = good; 3 = acceptable; 4 = hardly acceptable; 5 = not acceptable. CO: control; BHT: tert-butyl-4-hydroxytoluene; FVE-250, 500 and 1000: Fucus vesiculosus extract at 250, 500 and 1000 mg/kg, respectively.

Table 1. Ingredient proportions used in the manufacture of pork patties with several added antioxidants

Treatment Ingredients (g/100 g) CO BHT FVE-250 FVE-500 FVE-1000 Pork loin 87 87 87 87 87 Linseed oil oleogel 5 5 5 5 5 Salt 1.04 1.04 1.04 1.04 1.04 Water 6.96 6.96 6.96 6.96 6.96 BHT 0 0.02 0 0 0 Seaweed extract 0 0 0.025 0.05 0.1 CO: control without any antioxidant; BHT: with addition of 200 mg/kg of tert-butyl-4-hydroxytoluene; FVE-250, FV-500 and FV-1000: with addition of Fucus vesiculosus extract at 250, 500 and 1000 mg/kg, respectively.

24 Table 2. Effect of the addition of Fucus vesiculosus extract and BHT on proximate composition and fatty acid (FA) profile (% of total FAs) of pork patties at = day (mean ± standard error)(n = 4)

Treatment SEM Sig. CO BHT FVE-250 FVE-500 FVE-1000 Moisture (%) 64.55±0.90 64.92±0.47 65.17±0.98 65.24±0.20 64.63±1.42 0.19 ns Fat (%) 13.98±1.23 13.34±0.69 13.52±1.05 13.75±0.46 13.51±0.66 0.18 ns Protein (%) 17.22±1.13 16.81±0.44 16.70±0.27 16.54±0.28 16.60±0.14 0.13 ns Ash (%) 2.05±0.04 2.10±0.03 2.03±0.04 2.04±0.04 2.09±0.05 0.01 ns FA profile C14:0 1.03±0.01 b 0.98±0.02 a 1.00±0.02 a 1.01±0.01 a 1.00±0.02 a 0.01 * C16:0 19.99±0.24 b 19.24±0.14 a 19.65±0.27 b 19.70±0.11 b 19.64±0.33 b 0.07 ** C16:1n-7 1.86±0.01 b 1.79±0.02 a 1.79±0.03 a 1.85±0.03 b 1.82±0.03 a,b 0.01 ** C17:0 0.18±0.00 0.18±0.01 0.18±0.01 0.18±0.00 0.18±0.01 0.00 ns C17:1n-7 0.15±0.00 0.15±0.00 0.15±0.01 0.15±0.00 0.15±0.01 0.00 ns C18:0 11.05±0.19 10.79±0.18 10.97±0.18 10.93±0.12 10.93±0.20 0.04 ns C18:1n-9t 0.18±0.01 0.17±0.00 0.18±0.01 0.18±0.00 0.18±0.01 0.00 ns C18:1n-9c 36.18±0.08 c 35.38±0.26 a 35.76±0.38 a,b 36.06±0.18 b,c 35.68±0.31 a,b 0.08 ** C18:1n-7c 2.45±0.07 2.46±0.02 2.46±0.13 2.47±0.15 2.45±0.14 0.02 ns C18:2n-6 12.92±0.06 13.25±0.21 13.08±0.23 13.18±0.48 13.38±0.16 0.06 ns C20:0 0.20±0.00 0.19±0.00 0.20±0.00 0.19±0.01 0.20±0.00 0.00 ns C20:1n-9 0.69±0.03 0.70±0.02 0.67±0.02 0.67±0.02 0.69±0.03 0.01 ns C18:3n-3 11.28±0.46 12.68±0.63 11.94±1.01 11.40±0.31 11.63±1.00 0.19 ns C20:2n-6 0.39±0.00 0.39±0.01 0.40±0.02 0.40±0.01 0.40±0.02 0.00 ns C20:4n-6 0.36±0.03 a 0.41±0.01 b 0.39±0.01 a,b 0.40±0.03 b 0.40±0.01 b 0.01 * SFA 32.44±0.44 b 31.38±0.27 a 31.99±0.46 a,b 32.01±0.21 a,b 31.95±0.55 a,b 0.11 * MUFA 41.50±0.14 c 40.65±0.28 a 41.01±0.36 a,b 41.38±0.27 b,c 40.96±0.35 a,b 0.09 ** PUFA 24.96±0.39 a 26.73±0.51 b 25.81±0.77 a,b 25.38±0.39 a 25.81±0.84 a,b 0.18 * ∑n-3 11.28±0.46 12.68±0.63 11.94±1.01 11.40±0.31 11.63±1.00 0.19 ns ∑n-6 13.68±0.07 14.05±0.22 13.87±0.26 13.98±0.51 14.17±0.17 0.07 ns n-6/n-3 1.21±0.06 1.11±0.07 1.17±0.11 1.23±0.07 1.23±0.12 0.02 ns CO: control without any antioxidant; BHT: with addition of 200 mg/kg of tert-butyl-4-hydroxytoluene; FVE-250, FV- 500 and FV-1000: with addition of Fucus vesiculosus extract at 250, 500 and 1000 mg/kg, respectively. a-cMeans in the same row not followed by a common superscript letter are significantly different (P < 0.05; Duncan test). SEM: standard error of mean. Sig.: significance: *(p < 0.05); **(p < 0.01); ns (not significant). SFA = Ʃ (C14:0 + C16:0 + C17:0 + C18:0 + C20:0). MUFA = Ʃ (C16:1n-7 + C17:1n-7 + C18:1n-9t + C18:1n-9c + C18:1n-7c + C20:1n-9). PUFA = Ʃ (C18:2n-6 + C18:3n-3 + C20:2n-6 + C20:4n-6). Ʃn-6 = Ʃ (C18:2n-6 + C20:2n-6 + C20:4n-6). Ʃn-3 = Ʃ (C18:3n-3).

25 Table 3. Effect of the addition of Fucus vesiculosus extract and BHT on evolution of pH and colour parameters (L*, a* and b*) during refrigerated storage of pork patties (mean ± standard error) (n = 4)

Treatment Day SEM Sig. CO BHT FVE-250 FVE-500 FVE-1000 pH 0 5.65±0.02 a,1 5.68±0.02 a,b,1 5.70±0.02 b,1,2 5.71±0.02 b 5.75±0.02 c,2 0.01 *** 7 5.73±0.04 2,3 5.66±0.10 1 5.74±0.05 2 5.75±0.08 5.74±0.04 2 0.02 ns 11 5.68±0.03 1,2 5.66±0.01 1 5.67±0.04 1 5.65±0.01 5.65±0.02 1 0.01 ns 15 5.74±0.02 3 5.77±0.05 2 5.66±0.06 1 5.76±0.09 5.72±0.03 2 0.01 ns 18 5.74±0.04 a,b,3 5.78±0.02 c,2 5.76±0.04 b,c,2 5.71±0.01 a 5.72±0.01 a,2 0.01 ** SEM 0.01 0.02 0.01 0.01 0.01 Sig. ** ** * ns *** L*

0 62.09±1.28 63.11±1.84 1,2 62.01±0.19 1 61.15±0.51 60.06±3.02 1 0.41 ns

7 61.61±1.99 65.54±0.20 2 62.31±2.32 1 63.48±3.81 61.51±1.39 1,2 0.57 ns

11 59.80±2.47 63.84±3.43 1,2 63.51±1.53 1,2 62.47±1.72 64.94±1.76 2 0.61 ns

15 64.02±1.53 61.37±1.58 1 65.72±2.27 2 62.02±2.35 63.43±2.20 1,2 0.53 ns

18 61.52±1.78 60.09±3.16 1 61.59±1.29 1 62.79±2.41 61.99±1.92 1,2 0.48 ns

SEM 0.48 0.64 0.48 0.51 0.57

Sig. ns * * ns *

a* 0 12.03±0.41 b,3 11.10±0.96 a,b,3 9.88±0.64 a,3 10.25±0.77 a,4 10.33±1.36 a,4 0.25 * 7 9.35±1.40 2 8.91±0.62 2 9.17±2.27 2,3 8.35±1.53 3 8.70±1.07 3 0.31 ns 11 8.86±1.40 2 8.19±0.65 1,2 7.88±0.68 2 8.08±0.79 2,3 7.18±0.95 2 0.22 ns 15 6.07±0.87 1 7.19±0.29 1 5.62±0.86 1 6.63±1.05 2 7.02±0.59 2 0.21 ns

26 18 4.72±0.58 a,1 7.36±1.30 b,1 4.94±0.52 a,1 5.07±0.78 a,1 5.54±0.65 a,1 0.27 ** SEM 0.62 0.36 0.50 0.45 0.42 Sig. *** *** *** *** *** b* 0 22.51±0.64 b,3 21.58±0.51 a,b,3 20.75±0.77 a,2 20.92±1.07 a 21.09±0.81 a 0.21 * 7 19.86±1.07 2 19.62±1.69 1,2 20.55±1.62 2 19.81±2.60 20.85±1.99 0.39 ns 11 19.36±0.44 2 20.71±0.64 2,3 19.67±0.70 1,2 20.33±1.12 20.06±1.30 0.21 ns 15 17.91±1.65 a,1 19.10±0.76 a,b,1 19.18±1.17 a,b,1,2 19.95±1.17 b 20.78±0.91 b 0.32 * 18 17.65±0.30 1 18.91±0.71 1 18.04±1.47 1 18.65±1.06 19.22±0.71 0.23 ns SEM 0.44 0.30 0.33 0.35 0.29 Sig. *** ** * ns ns CO: control without any antioxidant; BHT: with addition of 200 mg/kg of tert-butyl-4-hydroxytoluene; FVE-250, FV-500 and FV-1000: with addition of Fucus vesiculosus extract at 250, 500 and 1000 mg/kg, respectively. a-cMeans in the same row (different treatments on the same storage day) not followed by a common superscript letter are significantly different (p < 0.05; Duncan test). 1-4Means in the same column (same treatment in different storage days) not followed by a common superscript number are significantly different (p < 0.05; Duncan test). SEM: standard error of mean. Sig.: significance: *(p < 0.05); **(p < 0.01); ***(p < 0.001); ns (not significant).

27 Table 4. Change of TBARS values during refrigerated storage of pork patties manufactured with different antioxidants (mean ± standard error) (n = 4)

Treatment Day SEM Sig. CO BHT FVE-250 FVE-500 FVE-1000 0.13±0.00 e,1 0.10±0.00 c,1 0.09±0.00 b,1 0.12±0.00 d,1 0.08±0.00 a,1 0.00 *** 0 0.87±0.17 2 0.58±0.11 2 0.74±0.14 2 0.66±0.13 1,2 0.76±0.15 2 0.04 ns 7 1.44±0.01 d,2 0.78±0.09 a,3 1.07±0.01 b,2 1.19±0.01 c,2 1.09±0.01 b,2 0.05 *** 11 3.92±0.63 b,3 0.96±0.15 a,4 3.70±0.59 b,3 3.66±0.52 b,3 3.54±0.57 b,3 0.30 *** 15 4.09±0.66 b,3 1.00±0.16 a,4 3.87±0.62 b,3 3.76±0.75 b,3 3.69±0.59 b,3 0.31 *** 18 0.38 0.08 0.37 0.46 0.35 SEM *** *** *** *** *** Sig. 0.13±0.00 e,1 0.10±0.00 c,1 0.09±0.00 b,1 0.12±0.00 d,1 0.08±0.00 a,1 0.00 *** CO: control without any antioxidant; BHT: with addition of 200 mg/kg of tert-butyl-4-hydroxytoluene; FVE-250, FV-500 and FV-1000: with addition of Fucus vesiculosus extract at 250, 500 and 1000 mg/kg, respectively. a-eMeans in the same row (different treatments on the same storage day) not followed by a common superscript letter are significantly different (p < 0.05; Duncan test). 1-4Means in the same column (same treatment on different storage days) not followed by a common superscript number are significantly different (p < 0.05; Duncan test). SEM: standard error of mean. Sig.: significance: *** (P < 0.001), ns (not significant).

Table 5. Evolution of protein oxidation during refrigerated storage of pork patties manufactured with different antioxidants (mean ± standard error) (n = 4)

Treatment Day SEM Sig. CO BHT FVE-250 FVE-500 FVE-1000 3.57±0.10 1 3.51±0.19 1 3.49±0.50 1 3.53±0.19 1 3.47±0.14 1 0.06 ns 0 3.79±0.31 1 3.52±0.06 1 3.68±0.05 1,2 3.55±0.10 1 3.53±0.15 1 0.04 ns 7 4.58±0.32 b,2 3.58±0.04 a,1 4.34±0.18 b,2 4.51±0.44 b,2 4.45±0.51 b,2 0.11 ** 11 5.94±0.17 c,3 3.79±0.09 a,1,2 5.40±0.77 b,c,3 5.31±0.18 b,c,3 5.14±0.29 b,3 0.19 *** 15 6.81±0.61 c,4 4.04±0.39 a,2 6.04±0.39 b,3 5.72±0.27 b,3 5.66±0.54 b,3 0.23 *** 18 0.31 0.06 0.27 0.22 0.22 SEM *** ** *** *** *** Sig. 3.57±0.10 1 3.51±0.19 1 3.49±0.50 1 3.53±0.19 1 3.47±0.14 1 0.06 ns CO: control without any antioxidant; BHT: with addition of 200 mg/kg of tert-butyl-4-hydroxytoluene; FVE-250, FV-500 and FV-1000: with addition of Fucus vesiculosus extract at 250, 500 and 1000 mg/kg, respectively. a-eMeans in the same row (different treatments on the same storage day) not followed by a common superscript letter are significantly different (p < 0.05; Duncan test). 1-4Means in the same column (same treatment on different storage days) not followed by a common superscript number are significantly different (p < 0.05; Duncan test). SEM: standard error of mean. Sig.: significance: *** (p < 0.001), ns (not significant).

28 VII. Anexes

VII.2. CRITERIA OF QUALITY OF THE JOURNALS WHERE THE RESULTS OF THE PRESENT DOCTORAL THESIS HAVE BEEN PUBLISHED

VII.2.1. PUBLICATION Nº 1

“Proximate composition, phenolic content and in vitro antioxidant activity of aqueous extract of the seaweeds Ascophyllum nodosum, Bifurcaria bifurcata and Fucus vesiculosus. Effect of addition of the extracts on the oxidative stability of canola oil under accelerated storage conditions”

Agregán, R., Munekata, P.E.S., Domínguez, R., Carballo, J., Franco, D. and Lorenzo, J.M. (2017)

Food Research International, 99, 986-994.

Impact factor of the Food Research International journal (ISI Journal Citation Reports, 2017): 3.520

Relative position of the Food Research International journal within its categorie (ISI Journal Citation Reports, 2017):

 Food Science and Technology: 14/133 (Quartile Q1)

VII.2.2. PUBLICATION Nº 2

“Assessment of the antioxidant activity of Bifurcaria bifurcata aqueous extract on canola oil. Effect of extract concentration on the oxidation stability and volatile compound generation during oil storage”

Agregán, R., Lorenzo, J.M., Munekata, P.E.S., Domínguez, R., Carballo, J. and Franco, D. (2017)

Food Research International, 99, 1095-1102.

Impact factor of the Food Research International journal (ISI Journal Citation Reports, 2017): 3.520

Relative position of the Food Research International journal within its categorie (ISI Journal Citation Reports, 2017):

 Food Science and Technology: 14/133 (Quartile Q1)

135 VII. Anexes

VII.2.3. PUBLICATION Nº 3

“Phenolic compounds from three brown seaweed species using LC-DAD-ESI-MS/MS”

Agregán, R., Munekata, P.E.S., Franco, D., Domínguez, R., Carballo, J. and Lorenzo, J.M. (2017)

Food Research International, 99, 979-985.

Impact factor of the Food Research International journal (ISI Journal Citation Reports, 2017): 3.520

Relative position of the Food Research International journal within its categorie (ISI Journal Citation Reports, 2017):

 Food Science and Technology: 14/133 (Quartile Q1)

VII.2.4. PUBLICATION Nº 4

“Proximate composition and nutritional value of three macroalgae: Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcata”

Lorenzo, J.M., Agregán, R., Munekata, P.E.S., Franco, D., Carballo, J., Şahin, S., Lacomba, R. and Barba, F.J. (2017)

Marine Drugs, 15, 360.

Impact factor of the Marine Drugs journal (ISI Journal Citation Reports, 2017): 4.379

Relative position of the Marine Drugs journal within its categorie (ISI Journal Citation Reports, 2017):

 Chemistry, Medicinal: 5/59 (Quartile Q1)

VII.2.5. PUBLICATION Nº 5

“Shelf life study of healthy pork liver pâté with added seaweed extracts from Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcata”

Agregán, R., Franco, D., Carballo, J., Tomasevic, I., Barba, F.J., Gómez, B., Muchenje, V. and Lorenzo, J.M. (2018)

Food Research International, 112, 400-411.

136 VII. Anexes

Impact factor of the Food Research International journal (ISI Journal Citation Reports, 2018): 3.520

Relative position of the Food Research International journal within its categorie (ISI Journal Citation Reports, 2018):

 Food Science and Technology: 14/133 (Quartile Q1)

(The values of the criteria of quality given were those corresponding to the year 2017 since there are still no values for 2018).

VII.2.6. PUBLICATION Nº 6

“Phenolic content and antioxidant activity of extracts from Bifurcaria bifurcata algae, obtained by diverse extraction conditions using three different techniques (hydrothermal, ultrasounds and supercritical CO2)”

Agregán, R., Munekata, P.E.S., Franco, D., Domínguez, R., Carballo, J., Muchenje, V., Barba, F.J. and Lorenzo, J.M.

Environmental Engineering and Management Journal (Accepted for publication).

Impact factor of the Environmental Engineering and Management Journal (ISI Journal Citation Reports, 2018): 1.334

Relative position of the Environmental Engineering and Management Journal within its categorie (ISI Journal Citation Reports, 2018):

 Environmental Sciences: 171/242 (Quartile Q3)

(The values of the criteria of quality given were those corresponding to the year 2017, the lastest available).

VII.2.7. PUBLICATION Nº 7

“Antioxidant potential of extracts obtained from macro- (Ascophyllum nodosum, Fucus vesiculosus and Bifurcaria bifurcata) and micro-algae (Chlorella vulgaris and Spirulina platensis) assisted by ultrasounds”

Agregán, R., Munekata, P.E.S., Franco, D., Carballo, J., Barba, F.J. and Lorenzo, J.M. (2018)

Medicines, 5, 33.

Medicines journal is covered by following databases and archives:

137 VII. Anexes

- Indexing and Abstracting Services: Chemical Abstract (ACS), Directory of Open Access Journals (DOAJ), HINARI (WHO) and PubMed (NLM) - Full-text Archives: CLOCKSS (Digital Archive), e-Helvetica (Swiss National Library Digital Archive) and PubMed Central (NLM) - Content Aggregators: Academic OneFile (Gale/Cengage Learning), J-Gate (Informatics India), Science in Context (Gale/Cengage Learning) and WorldCat (OCLC)

(This journal does not appear in the Journal Citation Index (JCR) because there is still no citation history to assign an impact factor).

138