TESIS DE DOCTORAMIENTO Use of High Pressure and Ultrasound as a corrective measure of the pastiness in Dry-Cured Ham Cristina Pérez Santaescolástica 2020 Doctorado internacional Doctorado industrial
Cristina Pérez Santaescolástica TESIS of DOCTORAMIENTO: High Use DE Pressure and Ultrasound as a corrective measure of Dry-Cured pastiness in the Ham 2020 Internacional Doctoral School
Cristina Pérez Santaescolástica
DOCTORAL DISSERTATION
“Use of High Pressure and Ultrasound as a corrective measure of the pastiness in Dry-Cured Ham”
Supervised by the PhD:
José Manuel Lorenzo Rodríguez, Laura Purriños Pérez and Francisco Javier Carballo García
Year: 2020
“International mention”
“Industrial doctorate”
Internacional Doctoral School
JOSÉ MANUEL LORENZO RODRÍGUEZ, Researcher and Head of the Department of New Products Development and Food Packaging of the Centro Tecnolóxico da Carne, LAURA PURRIÑOS PÉREZ, Researcher and Head of the Department of Cromatography of 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 “Use of High Pressure and Ultrasound as a corrective measure of the pastiness in Dry-Cured Ham”, submitted by Ms CRISTINA PÉREZ SANTAESCOLÁSTICA to obtain the title of Doctor, was carried out under their supervision in the PhD programme “Ciencia y Tecnología Agroalimentaria” and it accomplishes the requirements to obtain the degree of Doctor by the University of Vigo.
Ourense, 5th of September 2020
The supervisors
José Manuel Lorenzo Rodríguez. PhD Laura Purriños Pérez, PhD
Francisco Javier Carballo García
To my mother, whom I miss so much and without whom I would not have achieved anything. Her love, understanding and confidence in me have made me what I am. Now, even when she is no longer by my side, I still listen to her words of encouragement that keep me going no matter how difficult.
AKNOWLEDGMENTS
First of all, I would like to thank my directors, José Manuel Lorenzo Rodriguez, PhD, Laura Purriños Pérez, PhD, and Francisco Javier Carballo García, PhD for all the help received, for the support obtained, for their patience and above all for sharing with me their knowledge and experience. I would also like to thank the Centro Tecnológico de la Carne and its manager, for allowing me to develop my thesis in their facilities and the INIA for granting me the scholarship that made it possible.
Secondly, I would like to thank everyone with whom I have had the pleasure of working and who have been part of this long journey, supporting, advising and motivating me. In particular, to my laboratory colleagues for always being available to clear up any doubt and to Manuel Juarez, PhD and Ilse Fraeye, PhD, for inviting me to collaborate in their facilities and who together with their teams made me feel at home despite the distance.
Finally, my close friends and my family without whom I would not have made it this far. My sister Elisa and my brother-in-law Jesus supporting me and giving me strength since I was born as if they were my parents. Jesus, nephew and at the same time brother who motivated me to be better every day so that I could be his reference and to guide him whenever he needed on his journey.
ABSTRACT
This doctoral thesis tried to contribute to solving one of the quality defects whose high incidence leads to great economic losses in the ham industry. In spite of an exhaustive control in the process, 10% of the finished hams present pasty textures, causing technological problems and quality reductions. For this, 200 hams were cured, in which the development of pasty textures was facilitated, being able to carry out the tests of this research work. The objectives were, in a first instance to study the relationship between the proteolysis index and the appearance of texture defects, identifying possible biomarkers associated with proteolytic activity, and also an instrumental determination of its organoleptic consequences. Secondly, from an analytical point of view, to study the effect of two selected non-invasive techniques applied as texture defects corrective measures (power ultrasound and high pressures) on protein structures and organoleptic attributes.
At first, adhesiveness and nitrogen fractions were determined to calculate the proteolysis index, being able to classify hams in three intervals: low (<32), medium (32-36) and high (> 36) proteolysis index. Subsequently, free amino acid profile, volatile compound profile and a proteomic study were determined. From the results, it was shown that a high proteolysis index was related to a more adhesiveness as much as an increment in bitter taste and important aromatic losses.
The use of power ultrasound could reduce the adhesiveness but it was observed that the taste of samples was turned sweeter, bitterer and more rancid, as well as the typical fatty odour of dry-cured ham could be decreased causing quality reductions. On the other hand, the treatments with high pressures in combination with high temperatures caused intense modifications in the profile of free amino acids and volatile compounds, especially at temperatures of 35 °C. These modifications gave rise to hams apparently with a greater sweet, acid and rancid taste, being the rancid aroma enhanced due to the increased content of aldehyde compounds (especially the hexanal content). However, it was observed that such impact can be minimized when the use of high pressures is combined with temperatures in the range of 0–20 °C.
Regarding the proteomic analysis, the main evidences that can be highlighted were the identification of MYH1 and MYH4 protein fragments as suitable proteolysis biomarkers as well as non-fragmented sarcoplasmic proteins namely FABP4 / H, PRDX6, SOD, CBR1 and ACY1 as independent candidate biomarkers of ultrasound treatment. Finally, it was observed that the application of high pressures could promote actin fragmentation.
RESUMEN
Esta tesis doctoral trató de contribuir a la mejora de la calidad del jamón crudo-curado, intentando solventar uno de los defectos de calidad cuya alta incidencia da lugar a grandes pérdidas económicas. A pesar de un exahustivo control en el proceso de elaboración, un 10% de los jamones curados presentan texturas pastosas, constituyendo un problema tanto para el consumidor, que demanda un producto de calidad, como para el comerciante, que se encuentra con una dificultad en el proceso de loncheado. Para ello se curaron 200 jamones en los que, a partir de variaciones en la temperatura y la humedad del proceso, se facilitó el desarrollo de texturas pastosas pudiendo así llevarse a cabo los estudios que forman parte de esta tesis y cuyos objetivos resumidos fueron los siguientes:
1. Estudio de la relación existente entre el índice de proteólisis y la aparición de defectos de textura, identificando posibles biomarcadores asociados a la actividad proteolítica. 2. Determinación instrumental de las consecuencias organolépticas derivadas de una alta actividad proteolítica. 3. Estudio del efecto a nivel estructural de la aplicación de temperaturas moderadas, asistidas con ultrasonidos de potencia, como medida correctora de la adhesividad. 4. Determinación instrumental de los cambios organolépticos provocados en el producto derivados del uso de temperaturas moderadas asistidas con ultrasonidos de potencia. 5. Estudio de las alteraciones proteicas derivadas del uso de altas presiones como medida correctora de adhesividad. 6. Evaluación instrumental del efecto de altas presiones en la calidad organoléptica del producto, identificando la temperatura óptima para su aplicación.
Una vez finalizada la curación de los jamones, éstos fueron deshuesados y loncheados. Para el estudio previo de las muestras se utilizaron cuatro lonchas de las que se extrajo el músculo biceps femoris por ser el más susceptible a sufrir reacciones proteolíticas. Dicho músculo se trituró consiguiendo una mejor homogeneización de la muestra y se envasó al vacío, conservándolo a temperatura de congelación hasta el momento de su análisis. En estas muestras, se determinaron, además de la adhesividad, las fracciones nitrogenadas, a partir de cuyo valor se calculó el índice de proteólisis, consiguiendo clasificar los jamones en tres intervalos: baja (<32), media (32-36) y alta (>36) proteólisis. A continuación, se estudió el perfil de aminoácidos libres y el perfil de compuestos volátiles, a fín de obtener una aproximación de las diferencias sápidas y aromáticas existentes entre los grupos. Paralelamente, dos muestras aleatorias de bajo índice y dos de alto índice de proteólisis fueron sometidas a un estudio proteómico a fín de determinar biomarcadores diferenciales de la actividad proteolítica.
Como era de esperar, los valores de nitrógeno no proteico fueron mayores cuanto mayor proteólisis presentaban las muestras. Además, se observó que un índice de proteólisis por encima de 36 % aumentaba de forma significativa (P<0,01) la adhesividad de las lonchas. Este efecto en la textura provocado por una intensa degradación de proteínas podría explicar el número de puntos notablemente mas alto (P <0,05) obtenido en las muestras de alto índice de proteólisis durante el estudio proteómico. Así mismo, cinco fragmentos de proteína estuvieron sobrerrepresentadas en el grupo de alto índice de proteólisis, observándose los mayores valores de cambio relativo (RC >0,4) para MYH1, ACTS y MYH4. Según esto, y teniendo en cuenta evidencias previas de una mayor degradación de la miosina en respuesta a la proteólisis, MYH1 y MYH4 podrian ser adecuados marcadores de proteólisis y, por tanto, dada su relación, de adhesividad.
En cuanto a los aminoácidos libres, el contenido total no mostró diferencias significativas entre grupos. Sin embargo, seis de los 18 aminoácidos analizados (serina, taurina, cisteína, metionina, isoleucina y leucina) mostraron diferencias significativas (P <0.05) entre los grupos con distintos niveles de proteólisis. En todos los grupos, la leucina fue el aminoácido mayoritario, siendo mayor su contenido cuanto mayor era el índice de proteólisis. Esta misma tendencia se observó para la serina, metionina e isoleucina, mientras que los valores de cisteína y taurina mostraron una tendencia opuesta. Debido a estas diferencias individuales, y al sabor de los aminoácidos involucrados, se observó que una alta degradación proteica podría incrementar el sabor amargo en los jamones.
Se identificaron un total de 39 compuestos volátiles que fueron clasificados como hidrocarburos lineales (6), hidrocarburos ramificados (14), cetonas (4), alcoholes (5), aldehídos (4), ésteres (2), ácidos (1), compuestos azufrados (1) y otros compuestos (2). La cantidad total de compuestos volátiles disminuyó de modo significativo (P<0,001) a medida que se incrementaba el índice de proteólisis, pasando de 1575 UA (unidades de area) x 106 / g de materia seca para jamones con un bajo índice de proteólisis a 997 UA x 106 / g de materia seca para jamones con un alto índice de proteólisis. De igual manera, el contenido total de cada una de las familias químicas en los que se clasificaron (excepto ésteres) presentó valores mayores en el grupo con menor índice de proteólisis. En todos los grupos estudiados, los hidrocarburos fueron los compuestos más abundantes, seguidos de los alcoholes, aldehídos, cetonas y ésteres. Individualmente, la abundancia de 36 de los 39 compuestos identificados fue significativamente menor (P <0,05) en el grupo de mayor índice de proteólisis. Entre los alcoholes, el etil alcohol fue el más abundante en los tres grupos estudiados (256 vs. 255 vs. 224 UA x 106 /g de materia seca, para los jamones de bajo, medio y alto índice de proteólisis, respectivamente) seguido por el 1-octen-3-ol y el benzilalcohol. Dentro de los aldehídos, el hexanal fue el más abundante en los tres grupos de jamones (104 vs. 79 vs. 43 UA x 106 / g de materia seca, para los jamones de bajo, medio y alto índice de proteólisis, respectivamente) seguido por el butanal, 3 metil. La cetona más abundante encontrada en las muestras de jamón fue la 2-butanona, 3-hidroxi (25,60 vs. 21,52 vs. 19,56 UA x 106 /g de materia seca, P<0,001, para los jamones clasificados como de bajo, medio y alto índice de proteólisis, respectivamente). Se encontraron cetonas de cadena lineal (2-pentanona, 2-heptanona y 3- heptanona), siendo la más abundante la 2-heptanona, que presentó los menores contenidos en las muestras de jamón clasificadas como de alto índice de proteólisis (9,93 UA x 106 /g de materia seca). Las 2-cetonas se encuentran frecuentemente en grandes cantidades aportando un peculiar aroma descrito como "verde", "fruta tropical", "nuez", "curado" o "tostado". A partir de estas diferencias, se podría asegurar que en los jamones con un alto índice de proteólisis existe una pérdida aromática importante, probablemente debido a que las reacciones proteolíticas limitan el desarrollo de reacciones lipoliticas, consideradas como el principal origen de los precursores aromáticos del jamón. Una vez comprobada la relación existente entre la intensidad de la proteólisis y la aparición del defecto de adhesividad, y tras una amplia revisión bibliográfica, se seleccionaron dos técnicas no invasivas como posibles medidas correctoras de tal defecto: los ultrasonidos de potencia y las altas presiones.
Para evaluar el posible uso de ultrasonidos de potencia como medida correctiva del defecto de adhesividad, 10 lonchas de 26 muestras pertenecientes al grupo de jamones de alto índice de proteólisis fueron envasadas al vacío y divididas en dos tratamientos que consistieron en la aplicación de temperaturas suaves (tratamiento convencional) o en combinación con ultrasonidos, con el objetivo de comprobar los efectos realmente debidos al uso de ultrasonidos. Sobre las muestras tratadas se analizaron los valores de adhesividad, perfil de aminoácidos libres y perfil de compuestos volátiles, comparándose los resultados con los datos obtenidos para esas mismas muestras antes del tratamiento. Así, se observó que ambos tratamientos provocaban una reducción mayor del 50% del valor inicial de adhesividad, pudiendo ser explicado este efecto por el hecho de que los enlaces de hidrógeno intramoleculares pudieron romperse debido a la vibración mecánica y los efectos de la cavitación térmica y ultrasónica, causando el aflojamiento de la estructura molecular y la reducción de las fuerzas de enlace moleculares.
El contenido total de aminoácidos libres se vio afectado por el tratamiento térmico, obteniéndose valores mayores en las muestras tratadas, independientemente de la aplicación de ultrasonidos. Así mismo, el contenido individual de todos los aminoácidos libres se vio aumentado después de la aplicación del tratamiento por ultrasonidos. Estos resultados mostraron que el producto final podría verse modificado sensorialmente, de forma que se obtendría un mayor sabor dulce, amargo y rancio.
Se identificaron 155 compuestos volátiles que se cuantificaron y clasificaron como parte de las principales familias químicas, obteniéndose 56 hidrocarburos, 24 alcoholes, 23 aldehídos, 21 cetonas, 16 ésteres y éteres, 6 ácidos carboxílicos, 4 compuestos nitrogenados y 5 compuestos azufrados. Se detectaron diferencias significativas (P <0,05) en el contenido total de compuestos volátiles entre las muestras control y las muestras tratadas por ultrasonidos, con una concentración mayor en el lote control (56662,84 UA x 103 / g de jamón curado). Individualmente, se encontraron diferencias significativas en el contenido de 94 compuestos volátiles (24 hidrocarburos, 15 alcoholes, 21 aldehídos, 15 cetonas, 10 ésteres y éteres, 4 ácidos carboxílicos, 2 compuestos nitrogenados y 3 compuestos azufrados). Los hidrocarburos fueron la familia química más numerosa con hasta 56 compuestos diferentes, representando un 30% del área total en las muestras control, y un 43% y 37% en las muestras tratadas con ultrasonidos y de forma convencional, respectivamente. El hidrocarburo alifático que se encontró en concentraciones más altas fue el 2,2,4,6,6-pentametil heptano, seguido del octano, y, con valores similares, pentano, hexano, undecano y dodecano. Cuantitativamente, la principal familia de compuestos volátiles en las muestras control fueron los aldehídos (aproximadamente el 41% del área total de los compuestos volátiles). Se observó una importante reducción del contenido total de aldehídos en las muestras tratadas con ultrasonidos, así como una mayor disminución en el grupo tratado de forma convencional (23509,08 vs. 10307,72 vs. 2381,68 UA x 103 / g de jamón curado, para los grupos control, ultrasonidos y convencional, respectivamente). El hexanal fue el aldehído lineal predominante en los grupos control y ultrasonidos, encontrándose el mayor valor en las muestras control (12264,83 vs. 5747,78 vs. 185,78 AU x 103 / g de jamón curado, para los grupos control, ultrasonidos y convencional, respectivamente). Sin embargo, las muestras del grupo convencional presentaron al propanal como el aldehído principal, cuya concentración fue mayor que en los otros dos grupos. Por otro lado, el 3-metil butanal fue el aldehído ramificado más abundante en todos los casos, presentando diferencias significativas (P <0,001) entre los grupos. Las muestras control mostraron la concentración más alta de este compuesto, mientras que las muestras del grupo convencional registraron la más baja. Asimismo, el contenido total de alcoholes mostró niveles más altos en las muestras del grupo convencional (6548,61 vs. 8599,43 vs. 12199,24 AU x 103 / g de jamón curado, para los grupos control, ultrasonidos y convencional, respectivamente). Este alto contenido de alcoholes totales encontrados en el grupo convencional es consecuencia de la mayor concentración individual de los alcoholes 2-metil butanol, 3-metil butanol y alcohol feniletílico. El incremento de 2- metilbutanol y 3-metilbutanol en el grupo tratado convencionalmente podría explicarse por la disminución observada en el 2-metilbutanal y el 3-metilbutanal, ya que los alcoholes ramificados pueden originarse, entre otras reacciones, por la reducción de aldehídos ramificados. El contenido total de cetonas se vio afectado significativamente (P <0,001) por el tratamiento, observandose la mayor concentración en las muestras pertenecientes al grupo convencional, y siendo la 2-heptanona la más abundante (427,95 vs. 664,14 vs. 980,43 AU x 103 / g de jamón curado, para los grupos control, ultrasonidos y convencional, respectivamente). También se encontraron otras 2-cetonas, como 2-butanona, 2-pentanona, 2-octanona y 2-nonanona. Todos estos compuestos presentaron los valores más altos en las muestras en las que se aplicó el tratamiento conventional. Los ésteres y éteres, los ácidos carboxílicos, los compuestos nitrogenados y los compuestos azufrados fueron las familias químicas que presentaron menores concentraciones. El contenido total de ésteres fue mayor en las muestras control (1906,99 vs. 1680,82 vs. 1385,33 AU x 103 / g de jamón curado, para los grupos control, ultrasonidos y convencional, respectivamente) pudiendo deberse estas diferencias a posibles pérdidas por volatilización. Con respecto al contenido total de ácidos carboxílicos, fue un 20% menor en el grupo asistido con ultrasonidos y un 70% menor en las muestras tratadas de forma convencional en comparacion con el grupo control, encontrándose las mayores diferencias en los contenidos de ácido pentanoico y ácido butanoico. Por otro lado, la 2,6-dimetil pirazina fue el principal compuesto nitrogenado, mostrando las lonchas del tratamiento convencional valores significativamente más altos (P ˂0,001) que los otros grupos, mientras que el lote de ultrasonidos no mostró diferencias en comparación con el grupo control, hecho que sugiere la aparición de cambios estructurales por la aplicación de ultrasonidos que puedan evitar reacciones entre los compuestos diceto y amino. Finalmente, la aplicación de temperatura también originó una disminución importante en los compuestos azufrados, siendo el disulfuro de dimetilo el compuesto más afectado (1740,04 vs. 206,48 vs. 738,87 AU x 103 / g de jamón curado, para los grupos control, ultrasonidos y convencional, respectivamente).
Para completar el conocimiento sobre los efectos de los ultrasonidos, se escogieron dos muestras al azar de cada uno de los tratamientos y se realizó un estudio proteómico en el que se observó una proteólisis significativamente mayor en las muestras de jamón sometidas a ultrasonidos que en las calentadas convencionalmente. En las muestras después del tratamiento con ultrasonidos, fueron más abundantes cinco proteínas sarcoplásmicas no fragmentadas (FABP4 / H, PRDX6, SOD, CBR1 y ACY1), lo que sugiere que podrían ser biomarcadores candidatos independientes de dicho tratamiento.
En el caso de las altas presiones se encontraron estudios previos que afirman su efectividad como medida correctora de defectos de textura. Además, existen múltiples usos para los cuales las altas presiones pueden ser muy útiles a lo largo del proceso de elaboración del jamón, por ejemplo, como medida para aumentar la seguridad microbiológica o como medio de monitorización de procesos. Teniendo en cuenta todo lo anterior, este bloque de experimentos se centró en ampliar el conocimiento sobre los efectos que provoca su utilización y sobre cómo dichos efectos varian en función de la temperatura de aplicación. Así, se seleccionaron 120 muestras que fueron divididas en cuatro grupos. Un grupo se mantuvo como control y los otros tres se trataron por altas presiones (600 Mpa durante 6 minutos) a tres temperaturas diferentes (0, 20 y 35 °C) con el fin de encontrar la combinación que tuviera menor impacto en la calidad organoléptica de las muestras. En todas las muestras se determinó el perfil de aminoácidos libres y el perfil de compuestos volátiles, obteniendo información sobre el posible impacto sensorial. Para comprobar los cambios en la estructura proteica derivados de la aplicación de altas presiones, se seleccionaron dos muestras y se realizó un estudio proteómico antes y después de su tratamiento a 0 °C, evitando así, cualquier efecto debido a la temperatura.
Del analisis del perfil de aminoácidos libres se observó que el tratamientoa 35 °C provocaba un aumento significativo (P <0,001) de la cantidad total de aminoácidos, hecho esperable ya que la temperatura afecta en gran medida a las proteínas, provocando su degradación. De acuerdo con esto, 13 de los 18 aminoácidos estudiados mostraron diferencias significativas entre los tratamientos, siendo las muestras tratadas a 35 °C las que presentaron los valores mayores de 12 de ellos: ácido aspártico, serina, glutamina, glicina, histidina, taurina, arginina, treonina, alanina, cisteína, valina y lisina. El aminoácido tirosina, por el contrario, fue el más abundante en las muestras control. De estos cambios en el perfil de aminoácidos libres provocados durante los tratamientos, se dedujo un aumento en la percepción de los sabores dulce, ácido y rancio, observándose un efecto más intenso en el grupo tratado a 35 °C.
Por otro lado, el contenido total de compuestos volátiles se vió afectado significativamente (P <0,001) por los tratamientos, siendo el grupo tratado a 35 °C el que mostró un mayor valor (78415,27 AU x 103 /g) y el grupo donde se aplicaron las altas presiones a 0 °C el de menor valor (28584,14 AU x 103 /g). En comparación con el grupo control, el tratamiento a 0 °C supuso una reducción del 55% en el contenido de hidrocarburos, 56% en aldehidos, 40% en alcoholes, 69% en ácidos carboxílicos, 85% en compuestos azufrados y 65% en compuestos clorados. El tratamiento a 20 °C mostró reducciones del 44%, 18%, 34%, 28% y 91% en el contenido de aldehídos, alcoholes, ácidos carboxílicos, compuestos nitrogenados y compuestos azufrados, respectivamente, mientras que el contenido de hidrocarburos, cetonas y compuestos clorados se incrementó en un 60%, 58% y 79%, respectivamente. Por otro lado, el tratamiento a 35 °C redujo un 22% la cantidad total de aldehídos, un 36% la de ácidos carboxílicos y un 82% la de compuestos azufrados, mientras que aumentaron un 109% los contenidos de hidrocarburos y cetonas, un 37% los de ésteres y éteres y un 69% los compuestos clorados. De forma individual, se encontraron diferencias significativas en el contenido de 147 compuestos volátiles, de un total de 149 identificados. Se observó que, a excepción del grupo tratado a 0 °C, el 80% del contenido total de compuestos volatiles tenia su origen en procesos lipolíticos, mientras que el 8-9% procedía de las reacciones de proteólisis. Sin embargo, en las muestras tratadas a 0 °C, el contenido de compuestos procedentes de la lipolisis era mucho menor (67%) mientras que el contenido de compuestos volátiles originados en la proteólisis se incrementó hasta un 20%. Estas diferencias podrían deberse al hecho de que las altas temperaturas promueven las reacciones lipolíticas, así como a que la aplicación de altas presiones puede inducir la desnaturalización de las proteínas.
Del estudio proteómico se deduce una mayor proteólisis en las muestras tratadas que en las muestras control debido al mayor número de puntos encontrados (116 vs. 123 en muestras control y tratadas con altas presiones a 0 °C, respectivamente). Se identificaron un total de 14 puntos con un alto Mascot (>60), entre los cuales 10 fueron actina y cuyo valor total de “relative change” fue 2,51, dato que indica que las actinas o sus fragmentos son más abundantes en las muestras que fueron tratadas a 0°C. A pesar de que la miosina es la proteína mayoritaria en el músculo de los animales, en este estudio sólo se encontró un punto identificado como miosina de cadena pesada en el grupo tratado. Sin embargo, la presencia de actina resultó ser de mayor relevancia según los valores obtenidos de “relative change” (2,51 vs. 0,05 para actina y miosina respectivamente). Debido a la presencia de varios fragmentos de actina en el gel correspondiente a las muestras tratadas, se puede deducir que la aplicación de altas presiones induce su fragmentación.
Finalmente, de todos los resultados anteriores, se pueden extraer las siguientes conclusiones:
1. Un mayor índice de proteólisis supuso un aumento en la adhesividad y en la cantidad de nitrógeno no proteico de las muestras. Sin embargo, los contenidos de nitrógeno básico volátil y de aminoácidos libres totales no se vieron afectados. 2. A medida que la proteólisis aumenta se incrementa la percepción sápida de amargor y se produce una importante pérdida olfativa debido a la reducción de compuestos volátiles. 3. Se identificaron un total de cinco proteínas miofibrilares y sarcoplasmáticas del músculo biceps femoris como candidatos de marcadores de proteólisis y de adhesividad. Además, dos isoformas distintas de miosina (miosina-1 y miosina-4) y la α-actina mostraron la mejor respuesta para las variables de proteólisis y adhesividad de acuerdo con la medida de RC, considerándose también biomarcadores potenciales para atributos de calidad ligados a la proteólisis, como la pastosidad. 4. El tratamiento térmico con temperaturas moderadas con o sin la aplicación de ultrasonidos de potencia en jamones loncheados y envasados al vacío reduce significativamente la adhesividad. Sin embargo, estos tratamientos modifican el perfil de aminoácidos, así como el de compuestos volátiles, afectando al sabor y al olor final del jamón. 5. Del estudio proteómico se dedujo que las muestras sometidas a ultrasonidos presentaban mayor proteólisis que las tratadas de forma convencional (tratamiento a temperaturas moderadas sin aplicación de ultrasonidos), así como mayor degradación miofibrilar en la proteína actina. FABP4/H, PRDX6, SOD, CBR1 y ACY1 podrían considerarse biomarcadores específicos de la aplicación de ultrasonidos debido a su mayor presencia. 6. Los tratamientos con altas presiones en combinación con temperaturas elevadas causaron intensas modificaciones en el perfil de aminoácidos libres y de compuestos volátiles, especialmente cuando se trataba a 35 °C. Estas modificaciones dieron lugar a jamones, aparentemente, con un mayor sabor dulce, ácido y rancio, siendo potenciado el aroma a rancio, debido al aumento de compuestos volátiles pertenecientes a la familia de los aldehídos (especialmente del contenido en hexanal). Sin embargo, se observó que dicho impacto se puede minimizar con el uso de altas presiones combinadas con temperaturas situadas en el rango de 0–20 °C. 7. Se constató, por el mayor número de puntos encontrados en el estudio proteómico, una mayor degradación proteica tras el uso de altas presiones. Se pudo comprobar que el uso de altas presiones fomenta la fragmentación de la proteína actina.
GENERAL INDEX
GENERAL INDEX
I. INTRODUCTION ------1
I.1. THE PRODUCT ------3 I.1.1. ECONOMICAL SIGNIFICANCE ------3 I.1.2. NUTRITIONAL VALUE ------4 I.1.3. HAM’S ANATOMY ------7 I.2. TECHNOLOGICAL ASPECTS ------8 I.2.1. MANUFACTURE PROCESS ------8 I.2.2. INVOLVED REACTIONS ------11 I.2.2.1. Lipolysis and lipid oxidation ------11 I.2.2.2. Proteolysis ------14 I.2.2.3. Secondary reactions ------16 I.2.3. DESICCATION PROCESS AND CURING SALT EFFECTS ------17 I.3. SENSORY ATTRIBUTES OF DRY-CURED HAM ------18 I.3.1. FLAVOUR ------18 I.3.2. TASTE DEVELOPMENT------19 I.3.3. ODOUR DEVELOPMENT ------20 I.3.4. TEXTURE ------22 I.4. POSSIBLE DEFECTS OF THE FINAL PRODUCT ------22 I.4.1. GENERAL DEFECTS ------22 I.4.2. TEXTURE DEFECTS ------24 I.5. NEW PROCESSING TECHNOLOGIES APPLIED TO HAM MANUFACTURE ------24 I.5.1. HIGH-PRESSURE TECHNIQUE APPLIED IN DRY-CURED HAM ------26 I.5.2. ULTRASOUNDS APPLIED IN DRY-CURED HAM ------29 II. JUSTIFICATION AND OBJECTIVES ------31 III. MATERIAL AND METHODS ------35
III.1. SAMPLES ------37 III.1.1. ELABORATION ------37 III.1.2. CORRECTIVE TREATMENTS ------37 III.1.2.1. Ultrasounds ------37 III.1.2.2. High-pressure ------38 III.2. EXPERIMENTAL DESIGN ------38 III.3. ANALYTICAL AND BIOCHEMICAL CHARACTERIZATION OF SAMPLES ------39 III.3.1. CHEMICAL ANALYSIS ------39 III.3.2. NITROGEN FRACTION ANALYSIS ------39 III.3.3. INSTRUMENTAL TEXTURE ------40 III.4. PROTEOMIC ANALYSIS ------40 III.4.1. PROTEIN EXTRACTION ------41 III.4.2. TWO-DIMENSIONAL ELECTROPHORESIS (2-DE) ------41 III.4.3. IMAGE ANALYSIS OF 2-DE GELS ------41 III.4.4. PROTEIN IDENTIFICATION BY MASS SPECTROMETRY (MS) ------41 III.5. INSTRUMENTAL EVALUATION OF ORGANOLEPTIC PRECURSORS ------42 III.5.1. FREE AMINO ACID ANALYSIS ------42 III.5.2. VOLATILE COMPOUND ANALYSIS ------43 III.6. STATISTICAL ANALYSIS ------44 III.6.1. PHYSICOCHEMICAL AND ORGANOLEPTIC PRECURSOR PARAMETERS------44 III.6.2. PROTEOMIC ANALYSIS ------44
i GENERAL INDEX
IV. RESULTS AND DISCUSSION ------47
IV.1. PRELIMINARY STUDY OF SAMPLES ------49 IV.1.1. SAMPLE CHARACTERIZATION ------49 IV.1.2. PROTEOMIC EVALUATION ------50 IV.1.3. INSTRUMENTAL EVALUATION OF THE ORGANOLEPTIC PRECURSORS ------57 IV.1.3.1. Free amino acids ------57 IV.1.3.2. Volatile compound profile------58 IV.2. EFFECTS OF THE APPLICATION OF ULTRASOUND AS CORRECTIVE MEASURE TO DECREASE THE ADHESIVENESS ------63 IV.2.1. BIOCHEMICAL EFFECTS ------63 IV.2.2. MICROSTRUCTURAL MODIFICATIONS ------64 IV.2.3. INSTRUMENTAL EVALUATION OF THE ORGANOLEPTIC PRECURSOR CHANGES ------71 IV.2.3.1. Effects on free amino acid profile and taste implications ------71 IV.2.3.2. Effects on the volatile profile and odour implications ------72 IV.3. EFFECTS OF THE APPLICATION OF HIGH-PRESSURE AS CORRECTIVE MEASURE TO DECREASE THE ADHESIVENESS ------83 IV.3.1. BIOCHEMICAL EFFECTS ------83 IV.3.1. MICROSTRUCTURAL MODIFICATIONS ------83 IV.3.3. INSTRUMENTAL EVALUATION OF THE ORGANOLEPTIC PRECURSOR CHANGES ------86 IV.3.3.1. Effects on free amino acid profile and taste implications ------86 IV.3.3.2. Effects on the volatile profile and odour implications ------88 V. CONCLUSIONS ------97 VI. REFERENCES ------101 VII. ANEXES ------117
VII.1. PUBLICATIONS THAT INCLUDE THE RESULTS OF THIS DOCTORAL THESIS ------119 VII.2. CRITERIA OF QUALITY OF THE JOURNALS WHERE THE RESULTS OF THE PRESENT DOCTORAL THESIS HAVE BEEN PUBLISHED------189 VII.2.1. PUBLICATION Nº 1 ------189 VII.2.2. PUBLICATION Nº 2 ------189 VII.2.3. PUBLICATION Nº 3 ------190 VII.2.4. PUBLICATION Nº 4 ------190 VII.2.5. PUBLICATION Nº 5 ------191 VII.2.6. PUBLICATION Nº 6 ------191
ii TABLE INDEX
TABLE INDEX
TABLE 1. VITAMINS PRESENT IN DRY-CURED HAM, RECOMMENDED DAILY ALLOWANCES, FUNCTIONS ON BODY METABOLISM AND CONSEQUENCES OF THEIR DEFICITS ------5 TABLE 2. MINERALS PRESENT IN DRY-CURED HAM, RECOMMENDED DAILY ALLOWANCES, FUNCTIONS ON BODY METABOLISM AND CONSEQUENCES OF THEIR DEFICITS ------6 TABLE 3. MAIN ENZYMES INVOLVED IN LIPOLYSIS PROCESS ------12 TABLE 4. MAIN ENZYMES INVOLVED IN PROTEOLYSIS PROCESS ------14 TABLE 5. HIGH-PRESSURE TECHNOLOGY APPLICATIONS ACROSS THE DRY-CURED HAM PROCESS SORTED BY METHOD PARAMETERS ------27 TABLE 6. ULTRASOUND TECHNOLOGY APPLICATIONS ACROSS THE DRY-CURED HAM PROCESS SORTED BY METHOD PARAMETERS ------30 TABLE 7.CHEMICAL COMPOSITION AND NITROGEN FRACTIONS OF DRY-CURED HAM SAMPLES SORTED BY THREE PROTEOLYSIS LEVEL (LOW<32 %; MEDIUM 32 -36 % AND HIGH>36%) ------49 TABLE 8. SPOT VOLUMES WITH STATISTICALLY SIGNIFICANT (P-VALUE < 0.05) DIFFERENTIAL ABUNDANCE IN DRY-CURED HAMS OF LOW AND HIGH PROTEOLYSIS LEVEL ------51 TABLE 9. PROTEIN IDENTIFICATION BY MALDI-TOF/TOF MS OF DIFFERENTIALLY (P-VALUE < 0.05) REPRESENTED 2-DE SPOTS IN DRY-CURED HAMS WITH LOW AND HIGH PROTEOLYSIS INDEX ------53 TABLE 10. FOLD CHANGE (FC) AND RELATIVE CHANGE (RC) OF DIFFERENTIALLY (P < 0.05) REPRESENTED PROTEIN FRAGMENTS IN DRY-CURED HAM WITH DIFFERENT PROTEOLYSIS INDICES ------55 TABLE 11. EFFECT OF PROTEOLYSIS INDEX ON FAA CONTENT (EXPRESSED AS MG/100 G DM) OF DRY-CURED HAM ------57 TABLE 12. VOLATILE COMPOUNDS PRESENT IN DRY-CURED HAM SAMPLES WITH DIFFERENT PROTEOLYSIS LEVELS (EXPRESSED 6 AS AREA UNITS OF THE TIC (AU-TIC)×10 /G DM) ------59 TABLE 13. DIFFERENTIALLY ABUNDANT (P <0 .05) 2-DE SPOT VOLUMES IN DRY-CURED HAM SUBJECTED TO CONVENTIONAL (CV) AND ULTRASOUND-ASSISTED (US) THERMAL TREATMENTS ------66 TABLE 14. LIST OF PROTEIN IDENTIFICATIONS BY TANDEM MASS SPECTROMETRY (MALDI-TOF/TOF MS) ------67 TABLE 15. EFFECT OF US AND TEMPERATURE TREATMENTS ON FAAS CONTENT (EXPRESSED AS MG/100 G DM) ------71 TABLE 16. EFFECT OF US AND THERMAL TREATMENTS ON VOLATILE PROFILE (EXPRESSED AS QUANTIFIED AREA UNITS OF THE 3 EIC (AU-EIC) X 10 /G DRY-CURED HAM) ------75 TABLE 17. IDENTIFICATION OF SELECTED PROTEIN SPOTS BY MALDI-TOF/TOF ------85 TABLE 18. SPOT VOLUMES WITH SIGNIFICANT DIFFERENCES BY THE EFFECT OF A HIGH-PRESSURE TREATMENT IN SLICED DRY- CURED HAM. FOLD CHANGE (FC) AND RELATIVE CHANGE (RC) OF THE SELECTED SPOTS ------86 TABLE 19. EFFECT OF DIFFERENT HPP TREATMENTS ON FAAS CONTENT (EXPRESSED AS MG/100 G DM) OF DRY-CURED HAM ------87 TABLE 20. LEVELS OF THE MAIN FAMILIES OF VOLATILE COMPOUNDS IDENTIFIED IN UNTREATED AND HPP AT 0° C, 20° C AND 3 35° C TREATED DRY-CURED HAM (EXPRESSED AS QUANTIFIED AREA UNITS OF THE EIC (AU-EIC) X 10 /G DRY-CURED HAM) ------88 TABLE 21. EFFECT OF HPP TREATMENTS ON VOLATILE COMPOUND CONTENT (EXPRESSED AS QUANTIFIED AREA UNITS OF THE 3 EIC (AU-EIC)×10 /G DRY-CURED HAM) ------91
iii
FIGURE INDEX
FIGURE INDEX
FIGURE 1. WORLD DISTRIBUTION OF DRY-CURED HAM CONSUMPTION ------4 FIGURE 2. DIFFERENT PARTS OF DRY-CURED HAM LEGS ------8 FIGURE 3. DIFFERENTIATION OF THE MAIN MUSCLES PRESENT IN A SLICE OF DRY-CURED HAM ------8 FIGURE 4. THE GENERAL PROCESS OF MANUFACTURE OF DRY-CURED HAM ------9 FIGURE 5. PRINCIPAL STEPS OF LIPOLYSIS ------12 FIGURE 6. LIPID OXIDATION PROCESS ------13 FIGURE 7. PRINCIPAL STEPS OF PROTEOLYSIS ------15 FIGURE 8. COLOUR GENERATION ------18 FIGURE 9. FLAVOUR DEVELOPMENT ------19 FIGURE 10. DIAGRAM OF HIGH-PRESSURE TREATMENTS ------26 FIGURE 11. EXPERIMENTAL DESIGN ------38 FIGURE 12. TEXTUROMETER ------40 FIGURE 13. REPRESENTATIVE 2-DE GEL IMAGES OF LOW (LP) AND HIGH PROTEOLYSIS (HP) PROTEOMES ------50 FIGURE 14. EFFECT OF TEMPERATURE TREATMENT ALONE (CV) OR US ASSISTED (US) ON INSTRUMENTAL ADHESIVENESS OF A-B DRY-CURED HAM. MEAN VALUES AND STANDARD DEVIATIONS. DIFFERENT LETTERS INDICATE SIGNIFICANT DIFFERENECS (P<0.001) ------63 FIGURE 15. EFFECT OF TEMPERATURE TREATMENT ALONE (CV) OR US ASSISTED (US-50) ON MOISTURE CONTENT OF DRY- CURED HAM. MEAN VALUES AND STANDARD DEVIATIONS ------64 FIGURE 16. 2-DE PROTEIN PROFILES OF SLICED DRY-CURED HAM AFTER CONVENTIONAL (CV) AND ULTRASOUND-ASSISTED (US-50) THERMAL TREATMENTS ------64 FIGURE 17. UPGMA-BASED CLUSTERING ANALYSIS USING RC-VALUES FOR DIFFERENTIALLY ABUNDANT SARCOPLASMIC PROTEINS IN CV AND US SAMPLES ------69 FIGURE 18. COMPARISON OF RC VALUES BETWEEN WELL-SEPARATED PROTEINS ACCORDING TO UPGMA DENDROGRAM: FABP4/4 VS. OTHER PROTEINS (SOD, CBR1, ACY1 AND PRDX6). MEAN VALUES AND STANDAR DEVIATIONS ------70 FIGURE 19. EFFECT OF HPP TREATMENTS ON MOISTURE CONTENT OF DRY-CURED HAM. MEAN VALUES AND STANDARD DEVIATIONS ------83 FIGURE 20. 2-DE GEL IMAGES OBTAINED FROM SLICED DRY-CURED HAM SAMPLES AFTER A STANDARD PROCESS (LEFT) AND AN HPP TREATMENT AT 0 °C (RIGHT) ------84
v
ACRONYMS
ACRONYMS
°C: Celsius degrees a*: Redness A6N8P5: Multiprotein bridging factor 1 AA: Amino acid ACTC1: α-Actin 1 ACTS: α-Actin, skeletal muscle ACY1: Aminoacylase ANOVA: Analysis of variance AOAC: Association of Official Agricultural Chemists AU: Area units aW: Water activity BF: Biceps femoris CAPZBF:-Actin-capping protein subunit beta CAT: Catalase CAZA2F:-Actin-capping protein subunit alpha-2 CBR1: carbonyl reductase CCNG1: Cyclin-G1 CHAPS: 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate CI: Confidence interval CL: Lower bounds of confidence intervals CO Control CU: Upper bounds of confidence intervals CV: Conventional thermal treatments DFD: Dark, firm and dry meat DM: Dry matter DTT: Dichloro diphenyl trichloroethane DVB/CAR/PDMS: Divinylbenzene/carboxen/polydimethylsiloxane EIC: Extraction ion chromatogram ENOB: β-Enolase EU: European Union eV: Electronvolt F1RQQ8: α-1,4-Glucan phosphorylase FAA: Free Amino Acid FABP4/H: Fatty acid-binding protein isoforms FC: Fold change FWHM: Full-width half mass g: Gram GC: Gas chromatograph GC–MS: Chromatograph–mass spectrometer GM: Gluteus medius GSHPx: Glutathione peroxidase HBB: Hemoglobin subunit beta
vii ACRONYMS
HDL: High-density lipoproteins HP: High proteolysis HPP: High pressure HPP-0: High pressure treatment at 0 °C HPP-20: High pressure treatment at 20°C HPP-35: High pressure treatment at 35°C HS71L: Heat shock 70kDa protein1-like IEF: Isoelectric focusing IMF: Intramuscular fat IPG: Immobilized pH gradient Kcal: Kilocalories kDa: Kilo Dalton kg: Kilogram KHz: Kilohertz L*: Lightness LD: Longissimus dorsi LDL: Low-density lipoproteins LP: Low proteolysis LRI: Lineal Retention Index m/z: Mass-to-charge ratio MALDI TOF/TOF MS: Matrix assisted laser desorption-ionization time of flight mass spectrometry mg: Milligram Min: Minute Mm: Millimetre MP: Medium proteolysis MPa: Mega Pascale Mr: Molecular mass MRI: Magnetic resonance imaging MS/MS: Tandem mass spectrometry MUFA: Monounsaturated fatty acids MYH1: Myosin-1 MYH4: Myosin-4 MYH7: Myosin-7 N: Nitrogen NIRS: Near Infrared spectroscopy NIST: National Institute of Standards and Technology No.: Number NPN: Non-protein nitrogen NT: Total nitrogen content Ob: Observed PEF: Pulse electric fields pI: Isoelectric point PI: Proteolysis index PRDX6: Peroxiredoxin-6 viii ACRONYMS
PSE: Pale, soft and exudative meat psi: Pounds-force per square inch PUFA: Polyunsaturated fatty acids RC: Relative change RDA: Recommended daily allowances RH: Relative humidity RP-HPLC: Reversed phase-high performance liquid chromatography SDS: Sodium dodecyl sulphate SE: Standard error SEM: Standard deviation of the mean SFA: Saturated fatty acids SM: Semimembranosus SOD: Superoxide dismutase SPME: Solid-phase micro extraction ST: Semitendinosus TFL: Tensor fasciae latae muscle TG: Triglycerides Th: Theoretical TIC: Total ion chromatogram TPIS: Triosephosphate isomerase TVBN: Total volatile basic nitrogen UPGMA: Unweighted Pair Group Method with Arithmetic mean US: Ultrasounds USA: United States VINC: Vinculin W: Watt Wcm−2: Watt per centimetre WHC: Water holding capacity
ix ACRONYMS
x
I. INTRODUCTION
I. Introducción
I.1. THE PRODUCT
Dry-cured ham refers to the product made with the hind limb, cut at the level of the ischiopubic symphysis, with foot and bone, which includes the whole musculoskeletal piece, from adult pigs, subjected to the corresponding process of salting and drying-ripening (Order 2484/1967). Its origin is not well-known but the historical references date from the antiquity. The Egyptians have already used salting techniques to preserve all kinds of food for long time but there is unknown the precise moment in which the dry-cured ham was manufactured for the first time. A curious legend relates that, in Roman time, a pig fell into a stream whose water had a high salt content, and then he drowned. Some shepherd found and picked the animal, to roast it for eating. At this moment, they observed that salted meat had a much more pleasant taste, especially the legs, and since then, when pigs were slaughtered, legs were salted. Thus, little by little, the pig slaughter was getting used to a social celebration in which families, friends and neighbours got together. Nowadays, specific organoleptic attributes turn dry-cured ham into a widely appreciable product around the world and an important economic source which has been the purpose of several studies for improving and optimizing the manufacturing process.
I.1.1. ECONOMICAL SIGNIFICANCE
As can be seen in fig. 1, cured ham is consumed by a wide range of people in more than 150 countries. The largest buyers outside Europe are Mexico and the United States (USA) and within the European Union (EU) are Italy, France and Germany. Although its consumption extends to practically the whole world, the production of cured ham is much more localized. Europe is the largest producer of ham in the world, highlighting Spain as the main producing country. In ten years, Spain has gone from exporting about 22 tonnes in 2007 to export near than 44 tonnes in 2017 to a total of 134 countries. Close to 20 % of the total production of dry- cured ham is exported, mainly to countries of the EU (82-84 %) due to the localization, proximity and closeness culture between countries. In this regard, Germany is in the first place with almost 12 million kg followed by France (around 11 million kg). Among non-European countries, it is underlined Mexico, which imports around 1300 tonnes of Spanish dry-cured ham per year, and USA and Australia both with close to 700 tonnes per year (Rodríguez Marín, 2018).
3 I. Introducción
Figure 1. World distribution of dry-cured ham consumption
I.1.2. NUTRITIONAL VALUE
From a nutritional point of view, dry-cured ham is a complete product considering that it has a great variety of required essential nutrients for human diets. Conversely to the popular thinking for its salt and fat contents, it can be considered a healthy food as it is detailed below (Jiménez-Colmenero et al., 2009).
Its water content represents values from up to 45 % and the caloric value is about 240-250 Kcal/100 g, but it varies in function of the fat content present in the piece. As a meat product, dry-cured ham has a very good proportion of proteins (30%) with high biological value since most of the essential amino acids (AAs) are presented. The high level of free amino acids (FAAs) is due to the intense proteolysis during manufacture process (Toldrá et al., 2000) and their presence helps to improve mineral absorption, as much as, its increments enhance the digestibility degree of proteins (Toldrá and Aristoy, 1993). Also, some AAs take part in important biological functions of the body such as helping metabolic processes, collaborating in mental fatigue recovery, intervening in nerve function and depressive states, regulating blood pressure and combating oxidative stress (Ventanas, 2006).
On the other hand, lipid fraction, which represents around 10-20 % (Jiménez-Colmenero et al., 2010), is not too high compared with other meat products and furthermore, it is underlined its fatty acid profile. From the total lipid amount, saturated fatty acids (SFA) entail 30-40 %, monounsaturated fatty acids (MUFA) represent about 45-55% and polyunsaturated fatty acids (PUFA) signify 10-15%. SFA consumption increases the content of low-density lipoproteins (LDL) in blood plasma which in high amount is related to cardiovascular risk. However, not all SFA have the same impact, the palmitic and stearic acids, the predominant saturated fatty acids in dry-cured ham, have low and non-existent effect, respectively. Whereas PUFAs reduce LDL levels and also the high-density lipoproteins (HDL) content, which have an effect against cardiovascular diseases, MUFAs present the capacity of reducing LDL levels, but HDL levels are kept intact (Rebollo et al., 1999). Besides that, hams show relative low values of cholesterol (around 55 mg of cholesterol / 100 g) compared to other daily foods such as egg or liver whose content are 600 mg and 360 mg of cholesterol per 100 g, respectively.
4 I. Introducción
Regarding vitamins, dry-cured ham is an important source of B-group vitamins. Table 1 shows a summary of the vitamins present in dry-cured ham, its recommended daily allowances (RDA) and its function in human metabolism together with deficit consequences. Dry-cured ham is considered the best source of thiamine and also has a great content in riboflavin, niacin, pyridoxine and cobalamin, all of them belong to group B of vitamins which are strongly involved in enzymatic reactions related to the proper functioning of the central nervous system (Jiménez-Colmenero et al., 2009). Fat-soluble vitamins are present in much less amount, so, vitamins A, D, K and E are only present in trace contents, just like Vitamin C. However, Vitamin E content is important from a sensorial point of view as much as nutritional due to its antioxidant capacity.
Table 1. Vitamins present in dry-cured ham, recommended daily allowances, functions on body metabolism and consequences of their deficits
Vitamin aAmount bRDA aFunction aDeficit per 100 g Water-soluble vitamins Thiamine (B1) 0.57-0.84 1.4 Coenzyme in enzymatic Beriberi disease. (mg) reactions. Riboflavin (B2) 0.20-0.25 1.6 Oxidizing agent in biological Optical diseases. (mg) system. Niacin (B3) (mg) 4.5-11.8 18 Part of metabolic reactions: Weakness, loss of anaerobic glycolytic appetite and, in pathway, oxidative extreme cases, phosphorylation and fatty- pellagra. acid biosynthesis and oxidation. Pyridoxine (B6) 0.22-0.42 2 Part of more than hundred Central nervous (mg) reactions about AAs system changes. metabolism. Cobalamin Tr-15.68 1 Coenzyme in metabolic Anaemia and physical (B12) (µg) reactions with an important and neurological role in normal function of all problems. cells. Folic acid (µg) Tr-13.49 200 Part of the polymeric Weakness, structure of glutamic acid. sleeplessness, etc. Vitamin C (mg) Tr 60 Cellular antioxidant. Hair losses, poor cicatrisation and haemorrhage. Fat-soluble vitamins Vitamin A (µg) Tr-5 800 To collaborate in teeth, Night blindness, tissues, mucous membranes conjunctive xerosis of
5 I. Introducción
Vitamin aAmount bRDA aFunction aDeficit per 100 g and skin development. the eyes, diarrhoea and respiratory diseases. Vitamin D (µg) Tr-0.3 5 To promote calcium and Skeleton deformation. phosphorous absorption. Vitamin K (µg) Tr-10 1 To collaborate in blood Rarely. clotting. Vitamin E (mg) 0.08-1.5 10 To prevent lipid oxidation. Neurologic, sensorial nervous and retina problems. aJiménez-Colmenero et al. (2010); Toldrá (2002). bCouncil Directive 90/496/CEE (1990).
Dry-cured ham not only constitutes a good source of heme iron but also enhance the bioavailability of non-heme iron from vegetable products when they are eaten together. It has a high concentration of zinc, whose bioavailability, similar to iron, seems benefited when animal proteins are ingested in conjunction with vegetables. It is interesting the content present of other minerals, which can be observed in Table 2. For instance, it can be cited the content of magnesium that is necessary for most of the metabolic reactions as a cofactor, as well as selenium which has a notable effect as antioxidant collaborating against cancer (Fleet and Cashman, 2003; Higgs, 2000).
Table 2. Minerals present in dry-cured ham, recommended daily allowances, functions on body metabolism and consequences of their deficits
Mineral a Amount b RDA a Function a Deficit per 100 g Calcium (Ca) 12-35 800 To maintain bones, blood Spasm, cramp, crawling (mg) clotting and to transmit sensation and osteoporosis. nervous impulse. Iron (Fe) 1.8-3.3 14 Take part in the process of Anaemia and fatigue. (mg) cellular breathing. Zinc (Zn) 2.2-3.0 15 To maintain cell Prasad-Halsted syndrome (mg) membrane, immune and hypogonadism. system and brain function. Magnesium 17-18 300 Cofactor in enzymatic and Cardiovascular deseases, (Mg) (mg) metabolic pathways. osteoporosis, diabetes, etc. Potassium 153-160 2000 Necessary for osmotic Spasm, cramp, fatigue and (K) (mg) balance. constipation. Phosphorus 157-180 800 To maintain cellular and Rickets, osteoporosis, (P) (mg) tissues. osteomalacia and muscular weakness. Selenium 29 55 To protect against free- Cardiomyopathy, (Se) (µg) radical injury. cardiovascular risk and liver
6 I. Introducción
Mineral a Amount b RDA a Function a Deficit per 100 g cancer. Sodium (Na) 1100-1800 2000 Necessary for osmotic Nausea, vomits, headache (mg) balance. and memory losses. aJiménez-Colmenero et al. (2010); Toldrá (2002). bCouncil Directive 90/496/CEE (1990)
The common amount of sodium present in dry-cured hams is around 11-18 mg / g of product, nearly half of the recommended daily allowance, and it is necessary to keep in mind that sodium is fundamental to maintain the correct electrolytic balance. Nevertheless, dry-cured ham has undergone a recent rejection by consumers due to the consciousness-raising about the negative consequences of excessive sodium consumption and the resulting change of lifestyle and eating habit modifications. It is well known that high salt intake is related to hypertension, stroke risk, different types of cancers and osteoporosis owing to a high level of calcium excretion (Karppanen and Mervaala, 2006). This evidence has caused that producers have to look for new strategies to reduce the use of salts being careful not to provoke important stroke risk technological consequences or quality damages (Armenteros et al., 2009) due to salt is involved in textural functions, organoleptic characteristics and water holding capacity (WHC) (Taormina, 2010).
Finally, the content of carbohydrates is not too relevant since they have a scarce concentration, only a small portion of free sugars such as glucose (0.3 %), fructose, maltose and ribose are present. Glycogen is the only interesting compound but its content falls after slaughtering due to its conversion into lactic acid.
I.1.3. HAM’S ANATOMY
In the leg, some different parts can be differentiated (Fig.2):
The knuckle, which includes the hind shin and the jarret areas, is located at the end of the leg and is composed of tibia and fibula as bone supports and biceps as ligamentous and tendinous intersections. The texture in these areas is harder and the taste differs to a large extent compared with other zones. The centre zone is considered the most important part. It is positioned over the femur bone and its muscular components are the end of the gluteus medius (GM), tensor fasciae latae (TFL) muscle, biceps femoris (BF), semitendinosus (ST) and semimembranosus (SM). Silverside and thick flank are included in it. The silverside is more valorised by consumer considering that the salt content is lower due to its intramuscular fat (IMF) contents. On the other hand, thick flank has less lean content and less juicy, the texture is more fibrous and the taste saltier being curing perception more pronounced. The butt end, including sharp end and rump areas, is made up of coxal bone and the top of GM and TFL muscles.
7 I. Introducción
Figure 2. Different parts of dry-cured ham legs
On the other hand, fig. 3 shows the muscles that are part of a ham slice. The organoleptic characteristics of the slice will be different based on the type of muscle since each muscle presents differences in the way that the drying stage is carried out. Salt diffusion and dehydration differ between external and internal muscles. In this regard, external muscles, that are not covered by fat or skin show higher and faster desiccation and tend to be less adhesive due to the lower proteolytic transformations, whereas deep muscles present higher water content (48-19 %) than external (41-45 %) muscles (Monin et al., 1997).
Figure 3. Differentiation of the main muscles present in a slice of dry-cured ham
Differences among muscles are important for the manufacturer to assure the desirable quality of the final product.
I.2. TECHNOLOGICAL ASPECTS
I.2.1. MANUFACTURE PROCESS
The general process comprises five principal stages, including selection and classification of the raw material, salting, post-salting, drying-ripening, and in most of the cases an ageing period to obtain extra quality products (fig.4). Sometimes, producers choose to expose hams to a smoking process like Country Ham style.
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Preparation, selection and classification of the raw material
Slaughter, quartering, outliner and bleed are included in this phase to prepare legs for the following process. At first, pH is measured to check the suitability of the meat for processing, allowing early detection of PSE (Pale, Soft and Exudative) and DFD (Dark, Firm and Dry) meats. To prevent future problems pH values 24 hours after slaughter must be situated between 5.6 and 6.2. Besides that, pieces which present petechiae or/and hematomas are removed due to it could be originated by anomalous stunning or bone fractures. Both can derive in spoiling problems due to the microbial growth together with the fact that external marks are negatively evaluated.
After selection, the piece is outlined removing part of the muscle, fat and skin to get the desired form. Then, bleed is made by either manual pressure or mechanical pressure removing every rest of blood possible considering that the remaining blood would deteriorate the piece in the following stage if it is decomposed. Finally, legs are kept in a refrigeration chamber at 0- 3 °C for 1 or 2 days to reduce the internal temperature.
Figure 4. The general process of manufacture of Dry-cured Ham
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Salting
The objective is to incorporate in the piece enough salt, curing salts and sometimes additives such as ascorbate and sugar, providing a microbial population inhibition, at the same time that the organoleptic characteristics are developed. Sodium chloride gives to the product the typical salty taste and nitrite helps to red colour development since it reacts with myoglobin and originates the typical pigment of raw cured meats called nitrosomyoglobin.
In the beginning, the pieces are classified by weight and fat level to get a better fitting of the salting time. Then the curing salts are incorporated, being two ways to provide them: by an unspecified amount or by an exact amount of curing salts. The first option, an unspecified amount of curing salts, consists of piling hams surrounded by dry salt into a chamber after rubbing them with a first layer of curing salts mix. In the chamber, hams are positioned alternating with curing salts and are temporally turned over to get a homogeneous distribution. The required time varies in function of the weight, fat content and temperature of the chamber being around 1 day per kilogram of weight. The second alternative, a determinate amount, consists of the addition of a calculated amount of salts, based on hams weight, using contents between 35 and 90 g per Kg of weight. This process is longer than the first one due to the fact that the process concludes when hams obtain the desired amount of salt, usually in 2- 4 weeks (Arnau, 1993). This method entails labour reductions and more standardization.
This phase occurs at 2-5 °C and 80-95 % of relative humidity (RH) to obtain the inhibition of undesirable microorganisms and to prevent the external desiccation of hams. During this phase, osmosis takes place and internal water leaves outside causing a weakening of 3-7 % and lead the salt diffusion to the internal muscles. At the end, the excess of salt is removed and pieces are brushed and cleaned. Having done that, legs are kept for a maximum time of 24 hours before the beginning of the post-salting stage.
Post-salting
In this step, salt is distributed uniformly into the muscles causing partial dehydration, that is, a significant water activity reduction which inhibits undesirable microbial growth. Additionally, both salt content and dehydration regulate proteolysis and lipolysis reactions which will lead to the characteristic flavour development. The temperature must be lower than 5 °C until the water activity reach 0.96 in all the piece for obtaining a microbiological stabilization, after that it can be established at around 5-8 °C. Like that, the RH needs to be enough high to prevent the crusting and enough low to remove the superficial humidity in a short time and thus prevent the development a defect named “remelo”. Usually, the fixed value is 85 %. Equally important is to maintain adequate air circulation to guarantee a good renewal of air. Legs are kept in these conditions for 60-75 days, approximately.
Drying-ripening
At this point, hams are transferred to a drying chamber that uses to be a room in which the hams are hanged in racks usually by the feet. At the beginning of the stage, the temperature starts at low values (below 15 °C) and moderate-high humidity (70 % approximately)
10 I. Introducción conditions, and by the time the temperature is increasing until 32 °C, occasionally reaching 35 °C. The objective is to continue dehydration and to intensify the biochemical reactions corresponding to proteolysis and lipolysis which influence to a great extent the characteristics of texture, odour and taste. Another important point in this time is the fat fusion which impregnates muscular fibres causing retention of a large part of odour compounds (Arnau, 1998).
Dry-ripening duration used to be not less than 3 months and can extend for more than 12 months, in which case the time becomes in the stage called ageing.
Ageing
The ageing happens in warehouses where the biochemical and enzymatic reactions that have begun previously continue for a minimum period of 10 months. At this moment, the action of microorganism, basically yeasts and moulds, has an important role in final flavour development. For that, temperatures around 15-20 °C together with values about 65-75 % of RH to facilitate microbial action are maintained. Hams keep under these conditions up to 4 years until their commercialization or preparation to be packed.
I.2.2. INVOLVED REACTIONS
During the manufacture process, several reactions take place. The way in which these reactions progress will define the organoleptic attributes at the end of the product manufacture. The main reactions consist of lipolysis and lipid oxidations, proteolysis and Maillard and Strecker reactions.
I.2.2.1. Lipolysis and lipid oxidation
Lipolysis is an enzymatic process in which the ester links establisehd between fatty acid and glycerol molecules are hydrolysed causing the release of free fatty acids (Fig. 5). These free fatty acids, in turn, will be substrates for other oxidative reactions that will originate aromatic volatile compounds in dry-cured products.
The fat present in hams could be divided into two types: IMF and adipose tissue (intermuscular fat). IMF includes mainly triglycerides (TG) (around 90 %) and phospholipids (Antequera and Martín 2001), meanwhile, adipose tissue is mainly TG (99%). The principal endogenous enzymes related to lipolysis reactions in IMF are lipases, including lysosomal acid lipase, neutral lipase, and phospholipases A1, A2, C and D (Table 3). While lipases work on TG at a pH between 5.5 and 6.2 (Motilva et al., 1992), phospholipases are responsible for hydrolysing phospholipids giving off fatty acids, SFA as much as MUFA and PUFA (Ripollés et al., 2011). On the other hand, in adipose tissue, the main acting enzyme is the hormone-sensitive lipase working over TG and generating glycerol and mono and diglycerides at the same time that a large number of fatty acids are given off. Also, triacylglycerides can be hydrolysed by lysosomal acid esterase and cytosol neutral esterase, although their activity is limited due to the lack of available substrate (Belfrage et al., 1984).
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Figure 5. Principal steps of lipolysis
The greater part of lipolysis takes place during the drying-ripening stage and, to a lesser extent, in the following ageing period. This fact could be due to the increment of temperature, being at the end of the drying so high, therefore, lipolysis reactions are reduced and oxidative reactions are getting more importance by the time. The evolution of lipolysis can be monitored by the evolution of free fatty acids along the process. SFA is the most stable fraction which shows a progressive increment during the process, meanwhile, MUFA and PUFA content drop along the curing stage, being PUFA one the most affected with a reduction from up to 88 % of the initial content. Both reductions, MUFA and PUFA, are attributed to oxidative reactions. However, it has been shown an increment of unsaturated fatty acids at the end of the curing process, mainly oleic and linoleic acids, probably due to their antioxidant characteristics (Antequera et al., 1993).
Table 3. Main enzymes involved in lipolysis process
Enzyme pH Target Active time Adipose Lipoprotein Basic Primary ester Active during tissue lipase salting and post- lipases Monoacylglycerol Basic 1- or 2-monoacylglycerols with salting no positional specificity. Hormone- Neutral Ester-bond in triacylglycerols Active during the sensitive lipase and the resulting ripening-drying diacylglycerols. Muscle Lysosomal acid Acid Hydrolyses tri-, di- and Stable during all lipases lipase monoacylglycerols, although it the process. has a marked preference for primary ester bonds of triacylglycerols. Toldrá et al. (1997)
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Lipid oxidation occurs and-or in consequence of lipolysis and it involves a group of chemical reactions called autoxidation including primary reactions in which lipid peroxides are formed as much as secondary reactions in which volatile compounds are originated. The molecular oxygen reacts with the free fatty acids created by enzymatic hydrolysis. The mechanism happens in three steps as can be observed in fig. 6: initiation, propagation and termination.
During the initiation, free radicals are created by the homolytic breakdown of the C-H links of fatty acids or by the abstraction of a hydrogen atom by catalysts (light, metallic ions or heat) or enzymatic (cyclooxygenase or lipoxygenase) action. Propagation is the phase in which free radicals from initiation can react with oxygen to form peroxide radicals (ROO●) and these, in turn, give rise to hydroperoxide (ROOH) by the hydrogen capture from a fatty acid. That is how the chain reaction is expanded. Hydroperoxides can be broken by heat or by catalysis induced by transition metals, causing new radicals which start again the oxidation process. These hydroperoxides are tasteless and odourless, and they are characterized by their instability being quickly decomposed in volatile compounds such as aldehydes, ketones or alcohols, all of them with high impact on sensory characteristics. Termination consists of the reaction between hydroperoxides and proteins, peptides or AAs, and their subsequent polymerization (Buscailhon et al., 1993) or break down into volatile compounds with a low molecular weight such as aldehydes, ketones, alcohols, hydrocarbons and acids.
Figure 6. Lipid oxidation process
The triglyceride composition consists in 49-51 % of MUFA, being oleic the main fatty acid with around 45% of the total, 7-15 % of PUFA and 36-41 % of SFA, being palmitic acid the main one. In the phospholipids, 40-48 % are PUFA, 35 % SFA and 18-25 % MUFA, being arachidonic acid, palmitic acid and oleic acid the main faty acids, respectively. Due to the fact that unsaturated fatty acids are oxidized faster than SFA, and phospholipids contain more unsaturated fatty acid than TG, oxidative reactions are initiated in phospholipidic fraction (Gray and Pearson, 1987). The number, position and geometry of double bonds will also influence on the speed of the process. In this sense, CIS configurations are oxidized more than the TRANS ones, and conjugated double bonds are more reactive than non-conjugated (Flores, 1997). Independently of unsaturations, fatty acids are oxidized faster when they are in free form than when they are linked to glycerol. In addition to that, oxidation is favoured by the presence of some pro-oxidant compounds such metallic ions, salt, globular proteins and oxygen, and at the same time, other compounds such as tocopherols, nitrites, ascorbic acid, FAAs, and peptides reduce it. The balance between pro-oxidant and anti-oxidant compounds varies in function of the moment along the curing process.
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The evolution of the oxidation process can be monitored by the assessment of peroxides and aldehydes since they are the primary and secondary products, respectively, of lipid oxidation. According to Martín et al. (1996), the highest increment in peroxides happens during the drying stage, but at the beginning of the ripening, peroxides as much as aldehydes show a reduction. In the latest month of the ripening/ageing, the increment of the temperature leads again to a large increment of peroxides. Anyway, lipid oxidation is a dynamic process in which peroxides are formed and at the same time, they are decomposed originating secondary products.
I.2.2.2. Proteolysis
Proteolysis refers to enzymatic reactions in which protein are broken into small peptides, AAs and other compounds that influence directly on texture and indirectly on odour development (Toldrá, 2006). The main enzymes implicated in the process are described in Table 4 and consist in endopeptidases, that are related to protein hydrolysis, and exopeptidases, that are associated with small peptides and FAAs generation (Toldrá et al., 2009).
Table 4. Main enzymes involved in proteolysis process
Enzyme pH Active against Observation Lysosomal Cathepsin B Acid Myosin and actin Stable and active proteinases Cathepsin L Acid Myosin heavy chain, all the process actin, tropomyosin, α- (5-10% of actinin and troponins recoveries after T and I 15 months of Cathepsin H Acid - processing) Cathepsin D Acid Myosin heavy chains, Disappears titin M and C proteins, between 6 and tropomyosin, 10 months of troponins T and I process Neutral Calpain Neutral Troponin T and I Requiring proteinases tropomyosin, C- calcium ions for protein, filamin, activity. desmin and vinculin Its activity is lost as well as titin and after the salting nebulin Muscle Aminopeptidase B Neutral Aromatic, aliphatic Activity still aminopeptidases Alanyl Neutral and basic aminoacyl- recovered after Pyroglutamyl Neutral bonds. more than 8 or Aminopeptidases Neutral even 12 months Leucyl Basic - aminopeptidase Toldrá et al. (1997).
The process consists of two steps. The first one is the hydrolysis of myofibrillar and sarcoplasmic proteins by action of muscular endopeptidases (cathepsins and calpains) that act
14 I. Introducción on myosin and troponin, enhancing tenderness and originating big peptides (Nagaraj et al., 2006). At the second step, the big peptides are broken down to smaller peptides and free AAs by exopeptidases (Fig. 7). Exopeptidases can mainly be included in two groups:
Aminopeptidases, dipeptidases and tripeptidases which break down the peptidic chain from the amino extreme giving off an AA, a dipeptide or a tripeptide, respectively. Carboxypeptidases, which are less known in meat products, act on the final carboxy group originating free AAs, peptidyldipeptidases that giving off dipeptides from the final carboxy of polypeptides and dipeptidases and tripeptidases that hydrolyse dipeptides and tripeptides, respectively (Toldrá et al., 1997).
Figure 7. Principal steps of proteolysis
Proteolysis reactions before the curing process. Endopeptidases begin protein degradations at post-mortem period favouring the tenderness of the meat. At the beginning of the curing process (salting and post-salting stages), the proteolytic reaction related to peptide formation predominate that turns into a progressive increment of peptide hydrolysis over time, that is, the AA generation gets longer. The increment of temperature and progressive desiccation help the hydrolysis of peptides which may be due to the tissue aminopeptidases activity since its optimal temperature is 37 °C (Toldrá et al., 1997). So, in the drying stage, it can be shown the most AA increment (Martuscelli et al., 2009) which slows down in the ageing stage. In this regard, the peptide generation decreases in the ageing stage probably due to peptidase inactivation due to the salt concentration. Some extrinsic factors will affect enzymatic activity, among which the drying level, pH, temperature and salt content can be highlighted. In this sense, cathepsins B, D and L are active at slightly acid pH meanwhile calpains I and II and cathepsin H act at neutral pH. On the other hand, the optimal temperature for cathepsin B is 30 °C and 37 °C for cathepsins H and L (Sánchez, 2003). During the drying, cathepsin activity is reduced due to the water activity decrease. The proteolytic activity is reduced when the salt content is increased (Antequera and Martín, 2001). However, residual activities of 5-10 % have been shown for Cathepsins B, H and L after 15 months but not for cathepsin D whose activity seems to disappear after 6 months of the process (Toldrá et al., 1993). Solubility losses of
15 I. Introducción proteins can influence the proteolytic process, stimulating or inhibiting it, and modifying some functional properties such as WHC that enhance the desiccation. Solubility changes are attributed essentially to the salt effect (Larrea et al., 2006) although heavy metals from contaminants in curing salts can catalyse oxidative reactions originating molecular links that give rise to resistant structures.
The level of AAs originated at the end of the process demonstrates the depth and extent of the proteolysis. The highest amounts in the final product correspond to glutamic acid, alanine and lysine (Jurado et al., 2007), AAs that are described as precursors of pleasant odours. Nevertheless, basic AAs, like histidine or arginine, show a minor increment due to the fact that they are involved in other subsequent degradative reactions such as generation of volatile compounds and amines. In addition to that, FAAs are the substrate of other reactions such as Maillard and/or Strecker degradation, having an indirect influence on the final odour. On the other hand, as a result of extreme proteolysis, total volatile basic nitrogen (TVBN) which is integrated by amines can appear. This fact is not desirable for normal dry-cured ham manufacture being this parameter considered as an alteration index. However, ammoniacal nitrogen fraction showing values below 1.1 mg/g of dry matter (DM) is considered as a not disturbing event (Martín et al., 1997). Since low molecular weight nitrogenous substances are generated, pH increments have been observed in different dry-cured products (Lorenzo et al., 2008; Lorenzo, 2014).
I.2.2.3. Secondary reactions
Maillard reactions are complex non-enzymatic glycosylation reactions of proteins in which an amine group from proteins, free AAs and/or amines, reacts with a reducing sugar giving rise to compounds that in turn, can react with each other or with another carbonyl groups from lipids generating volatile compounds. In general, these volatile compounds can be divided in three groups: i) sugar fragmentation products like furans, pyrazines, cyclopentane, carbonyls and acids; ii) AAs degradation products such as aldehydes and sulphur compounds; and iii) products from secondary reactions as pyrroles, pyridines, imidazoles, thiazoles and alcoholic condensation compounds. Processes in which the drying period is long and the water activity presents low values could favour these reactions (Flores et al., 2018). Into the mechanism of Maillard reaction, it is found the Strecker pathway which, also, takes part in volatile compound development (Cremer and Eichner, 2000). AAs experience oxidative deamination and decarboxylation in the presence of α—dicarbonyls to produce aminoketones, aldehydes and carbon dioxide (van Boekel, 2016; Hidalgo and Zamora, 2016). The volatile compounds that are formed can contribute to odour development. This is the case of 2-methyl propanal, 2-methyl butanal and 3-methyl butanal, which come from the AAs valine, isoleucine and leucine respectively, as well as, other sulphur compounds from sulphur AAs like methionine and cysteine.
The achievement of these secondary reactions depends on proteolysis, lipolysis and lipid oxidation extent, since compounds that are involved in all of them are widely interconnected, as well as processing conditions such as temperature, time and pH, which exercise a greater influence over the course of these reactions (Zamora et al., 2015).
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I.2.3. DESICCATION PROCESS AND CURING SALT EFFECTS
During ham manufacture, due to the addition of curing salts and dehydration, a desiccation process takes place and the water content decreases from initial values of 70-75 % to a final contents of 50-60 % (Armenteros et al., 2012). Water losses lead to increments in the percentage of fat and protein, creating an added-value because of the more protein content. The desiccation starts during salting due to osmotic dehydration and it continues during the following stages of the process. The intensity of the desiccation varies depending on the protection level of the piece (fat or skin area) and environmental RH. The process takes place practically only through muscles that are exhibited to air, hence a small area, which depends on the cut selected in the outlined time. In the post-salting stage, the desiccation is mainly by dragging of vapour and for salt diffusion into the piece, as well as, for water diffusion outside the leg. In this point, if the RH is too high or there is not enough ventilation, the microbial population can grow due to a great superficial humidity consequence of a slower drying. In contrast, if the water evaporation is higher than the arrival of the inner water (which happens with a very low RH or when the air velocity is not well adjusted), external muscle undergoes a deep desiccation that can give rise to crusted zones. Meanwhile, internal muscles seem like raw meat with higher salt taste in the final product (Ventanas and Cava, 2001). Drying-ripening stage is the step with the strongest dehydration due to both the high length of this stage and the environmental conditions which correspond to greater temperature and lesser RH than in the previous stages. The increment of temperature increases the dehydration of the piece that means that the internal water is thrown out faster and the evaporation is larger. Salt absorption and water losses lead to sodium chloride increments along de curing process, being the final amount from 5 to 12 % approximately (Virgili et al., 2007).
On the other hand, curing salts, which are mainly nitrites and nitrates together with sodium chloride, achieve diverse functions in the process. In the first place, the presence of curing salts protects the piece against microbial proliferation through a water activity reduction. Due to the pH of the raw material and the environmetal temperatures, hams are very susceptible to the microbial growth, so the stabilisation of the piece is needed to prevent sanitary risks. On the other hand, salt favours the desiccation processes and modulates the activity of the proteolytic enzymes. Also, it could play an oxidant role promoting lipid oxidations. So, the organoleptic characteristics of the product are closely related to the salt addition. In this regard, salt is also responsible for the colour development due to the nitric oxide originated from the nitrite (present as an impurity of the salt) decomposition that subsequently reacts with the myoglobin present in the muscles (Fig. 8). In the first step, nitrates are ionized in a watery environment to form nitrate ions, which are reduced to nitrite ions by the action of nitrate-reductase enzymes from bacteria like Lactobacillus, Micrococcus, Vibrio, etc. After that, the action of bacteria together a moderate acid medium (5.6-6.0) make that nitrite ions are degraded to nitric oxides which can react with the myoglobin. In nitrite presence, myoglobin is reduced to metmyoglobin which is characterized by a brown colour. Later, the metmyoglobin form reacts with nitric oxide giving rise to nitroso-metmyoglobin; however, this reaction does not occur completely (from 35 % to 75 % of metmyoglobin use to turn into nitroso- metmyoglobin) (Ranken, 2000). To make possible these events, it is necessary a reduced network which is possible by the action of enzymes that are naturally present in meat. Even so,
17 I. Introducción occasionally producers promote the action of enzymes by a chemical reducing agent. The most commonly used one is ascorbate that increases the colour formation and improves colour intensity and uniformity.
In contrast, in the absence of nitrification substances myoglobin is affected mainly by oxidation action of salt and it can be modified loading to changes in colour. In this sense, muscles show a lightly colour (pinky-red) due to the complex formed by Zn protoporphyrin (Moller et al., 2007). Recently, it is observed that this complex is correlated to the content in IMF. The formation of Zn protoporphyrin is greater with a good marbling condition, which may be due to the fact that fatty acids and phospholipids from IMF interact with the enzyme responsible for the complex formation (Bou et al., 2018).
Figure 8. Colour generation
I.3. SENSORY ATTRIBUTES OF DRY-CURED HAM
I.3.1. FLAVOUR
Flavour refers to the global sensation perceived when the product is consumed. This sensorial perception is due to the conjunction of odour and taste and it is generated by the union of volatile and non-volatile compounds and their interactions (Fig. 9). Structural components such as fat and proteins are implicated and determine the consistency of the product and its behaviour during mastication containing sapid and aromatic compounds. Since proteolysis and lipolysis are the main origins of flavour compounds, the type, number and amount of each flavour compound will be affected by the processing time. In this regard, it is shown that when the ripening process is longer, high flavour intensity is developed (Buscailhon et al., 1994).
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In the first place, taste contributes with some sensorial characteristics and after that, mastication allows for refining this sensorial impression due to flavour contribution by the retro oral cavity and nasal fossa. Mastication process, together with a slight increment of temperature in the mouth, favour the liberation of aromatic compounds in a specific amount and adequate velocity, promoting the production of salivary secretions and contributing to the odour and taste arrival to specific receptors. Besides that, IMF collaborates in juiciness acting as a lubricant when it gets in contact with the saliva.
Figure 9. Flavour development
In conclusion, flavour is the result of the contribution of some chemical compounds: volatile compounds from the degradation of components of raw meat and non-volatile substances such as AAs, peptides or nucleotides and other compounds which enhance tastes from other molecules. The origin of all these compounds is a global mechanism which includes lipolysis, proteolysis, Maillard and Strecker reactions that differ among products according to several factors like genetic characteristics, animal ages, level of muscular proteolytic and lipolytic enzymes present in raw meat, free fatty acids profile, and antioxidants contained in muscular tissues due to the animal feed.
I.3.2. TASTE DEVELOPMENT
There are five basic tastes, acid, sweet, salty, bitter and umami, which interactions will determine the global taste of the foods. Recently, a sixth taste called oleogustus has been proposed to define the fat sensation that is formed when animal products are ingested
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(Running et al., 2015). Sodium chloride contributes with salty taste when its concentration is up to 0,1 M. However, this taste perception can vary according to the specific characteristics of the product such as fat content or the formation of a complex between ions and proteins, which capture part of salt content reducing the level of the salty taste. The presence of AAs from proteolysis can act as enhancer or suppressive compounds besides the fact that AAs can provide their own taste. For instance, saltiness perception of the added salts during the manufacturing process could be influenced by glutamic and aspartic acid formation. According to that, the taste is mainly due to the presence of non-volatile peptides and AAs coming from biochemical processes that take place during the curing and which can vary according to the variable parameters of the raw material such as pH, water activity, enzymatic activity, captured salt amount and process time. Also, drying length will influence the amount and type of the AA at the end of the process.
Since each AA is related to a specific taste, the overall taste will be a function of the proteolysis intensity, that is, the amount of each AAs and their perception threshold. It has been described the relationship between the specific taste and the implicated AAs. In this sense, the sweet taste is originated by the presence of alanine, serine, proline, glycine and hydroxyl proline, as well as bitterness is derived from the phenylalanine, tryptophan, arginine, methionine, valine, leucine and isoleucine contribution. The configuration of the AA structures also has influence, being series D mainly sweet and series L bitter. It is shown that L-tryptophan and L-tyrosine are the bitterest AAs while D-tryptophan is the sweetest one (Solms, 1969). Glutamic acid and aspartic acid also collaborate with histidine and asparagine in sourness perception, as well as glutamic acid influence the umami taste at the same time that it can cover up bitter taste perception.
On the other hand, the bitter taste can be detectable due to the presence of peptides that contain one or two hydrophobic AAs (phenylalanine, leucine, isoleucine, valine or tryptophan), meanwhile the complex glutamic-tyrosine is related to mature taste and valine-glutamic and glycine-glutamic with sour taste (Sentandreu et al., 2003).
I.3.3. ODOUR DEVELOPMENT
There are many research focus on the relationship between the volatile compound and dry- cured ham odour identifying more than 200 compounds, but it is well known that not all of them have the same impact since each one is perceptible in a different amount depending on its threshold (Narváez-Rivas et al., 2012; García-González et al., 2008). Some factors derived from compounds belonging to the raw material and others formed during the curing process affect odour development. Lots of compounds ingested in the diet can be incorporated in animal bodies and can be kept in the raw material such as toluene, xylene isomers, terpenes and some ramified hydrocarbons. However, one-fifth part of odour compounds come from AAs and free fatty acids contribution. During salting and post-salting stage volatile compound derives from lipid oxidation, but whenever curing advances the AAs degradation increases. So, long curing processes lead to high odour intensity hams as a consequence of a great number of volatile compounds that are formed by lipid oxidation, as well as AA degradation (Buscailhon et al., 1993, 1994; Coutron-Gambotti et al., 1999).
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In general, aldehydes are one of the most important families as far as the odour is concerned due to their low threshold. Linear aldehydes derive from unsaturated fatty acid oxidation and contribute with either, strong and irritating odours (short-chain linear aldehydes) or citric notes (long-chain linear aldehydes) (Théron et al., 2010). Among linear aldehydes, the most important compound in hams is hexanal. Despite its presence in a very little concentration, it has a high effect on the aroma and it provides the ham with its characteristic grassy odour. It is also important the contribution of octanal for the aroma development due to its pleasant notes and its low threshold. In contrast, saturated aldehydes such as nonanal, 2,4-decadienal and 2-pentilfuran contribute with unpleasant notes. In this regard, pigs fed with pasture are characterized by high contents of oleic acid in the fat, whose oxidation entails high amounts of octanal and nonanal contributing with pleasant notes like greasy, oily woody and nutty notes. Whereas hams from pigs that have been fed with commercial feed present great amounts of hexanal and unsaturated aldehydes from linoleic acid oxidation (2,4-nonadienal and 2,4- decadienal) which contribute with unpleasant notes such as rancid or fry-like. It can show others aldehydes such as cyclic, ramified and sulphurous, and sulphur compounds which are originated by oxidative deamination-decarboxylation of AA by Strecker degradation (López et al., 1992).
Some characteristic odours of ham are due to compounds like 1-octen-ol and 1-penten-3-ol coming also from lipid oxidation. Especially important is the presence of 1-octen-3-ol due to its characteristic mushroom-like odour. Alcohols contribute greatly with peculiar odours and are mainly derived from lipid oxidation (Rivas-Cañedo et al., 2011), just as ketones, which come from lipid autoxidation and fermentation by microorganisms (Belitz et al., 1997). For instance, 2-heptanone, 2-decanoate and 2-undecanone show fruity notes, while 2-dodecanone contributes with oil-like odour. On the other hand, methyl-ketones are formed by decarboxylation of secondary products of hydroperoxide decomposition. Also, during lipid oxidation, enzymatic or non enzymatic esterification of free fatty acid and alcohols lead to esters compounds which contribute to appreciating notes. Esters appear in high amounts in the case of hams in which nitrites and nitrates are not incorporated due to these compounds inhibit lipid oxidation.
Other compounds that influence the odour of dry-cured ham are furans, responsible for cooked meat odour (Shahidi et al., 1986); nitrogenous compounds, especially pyrazines formed by Maillard and Strecker pathways due to its aromatic potential; and terpenes, which are derived from feeding and its posterior deposit in fatty tissues (Sabio et al., 1998). Lactones with a threshold between 0.007 and 0.1 ppm, created from hydroxy acids, contribute with a typical meat odour, as much as with fruity, fatty, oily and butter notes. In contrast, there are families of volatile compounds that have not much influence on the final odour due to their high threshold, among which, hydrocarbons are found. In spite of the high threshold that is observed in saturated linear hydrocarbons, the threshold is reduced in the presence of unsaturation. Their odour contribution varies greatly, being responsible for, among others, pine (pinene), wooden and lemon odour (limonene).
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I.3.4. TEXTURE
The texture is a global sensation that entails several characteristics such as juiciness, hardness, chewiness and cohesiveness. Its development starts immediately post-exsanguination due to rigor mortis process, in which changes in actin and myosin proteins succeeded turning the tender and soft muscle into tougher muscle. After that, tenderness is moulded as a consequence of the biochemical changes occurring in protein structures. These changes are mainly due to protein denaturalization and hydrolytic reactions. In this regard, soluble proteins (sarcoplasmic as much as myofibrillar) undergo a denaturalization process affecting texture characteristics. Myofibrillar proteins are also broken down into protein fragments and intermediate-size polypeptides in the first steps of proteolysis process increasing tenderness (Toldrá, 2002). Proteolysis effects are more frequent in the salting stage and are more pronounced in hams with less salt content due to the less inhibition of the enzymes responsible for the process.
On the other hand, several factors influence the texture characteristics. For instance, moisture and fat content play an important role in juiciness and chewiness perception, and IMF can be negatively related to dryness, fibrousness and hardness (Ruiz et al., 2000).
I.4. POSSIBLE DEFECTS OF THE FINAL PRODUCT
I.4.1. GENERAL DEFECTS
The manipulation of the living animals and/or the characteristics of the raw material are the key to prevent many problems which carry a detriment to the sensorial quality. In this regard, unpleasant notes from row material can be detected, especially sexual or faecal odour in hams from non-castrated males. Boar taint is due to the presence of androsterone hormone and also, it can be enhanced by the presence of indol and skatole from tryptophan. Like that, the way and time that the process takes place can transfer different odours like spicy, due to the long curing, or humid odour, due to the moulds or bacterial population. It is also possible that final products lack the odour when the time of the overall process is not enough long. During rearing, animal transportation to slaughtering or, even in the slaughter, can take place blows which can derive in bruises or veining on the surface. Poor treatments before the slaughter can generate the expulsion of synovial liquid which can emit a bad odour like cement during ripening. Similarly, fatty and connective tissues can present blood spots due to the use of electrical stunning. Also, rested blood in tissues could be oxidized and originate green, ochre or brown spots. The excessive oxidation of fat of muscles also generates darker colouring which diminishes consumer acceptability due to the appearance, especially the colour, which influences in a greater extent the consumer acceptability, not only in dry-cured ham but also in a high range of foods (Delwiche 2004). Besides that, an incorrect outliner (deficient skinning) can lead to result in problems like higher microbial growth and/or reductions in the velocity of salt diffusion. Most off-odours are generated by microorganisms that contaminate the product usually between slaughter and post salting stage and provoke alteration. Putrefaction odour could appear due to the bacterial growth which generates compounds such as ammonia, methanethiol and hydrogen sulphide accompanied by degradation of the muscle pigments
22 I. Introducción and, consequently, green or grey spots are displayed. Usually, this problem is a consequence of a deficient hygiene and contaminations, being bad refrigeration or non-homogeneous diffusion of salt some of the actions that could promote it. Other unpleasant odours that happen in presence of bacteria or moulds could be potato or earth odour due to Pseudomonas or phenol-like aroma from the action of Penicillium moulds. The penetration of microorganisms can originate gas into the veins provoking its inflammation. The presence of air inside the veins, in turn, can trigger mould growth with a resulting anomalous appearance and wrong flavour. Moreover, the use of feeds with a great amount of unsaturated fatty acids, which are susceptible to be oxidized, can cause an excessive oxidation and the apparition of off-flavours, commonly a rancid odour. Excessive lipid oxidation is connected with preservation problems of food and, in extreme, can provoke neuromyopathic diseases.
On the other hand, some practices can derive in burns, thus, it can be cited the addition of nitrite in the presence of oxygen, salt crystallization or freezing without protection. This later burn seems a white appearance and makes difficult the absorption of salt due to de area turns dryer. If the freezing and thawing make slowly the originated ice crystals can break the muscle fibres. Furthermore, if the velocity of the drying is very low, a slim can be formed on the surface. During drying it is originated a hard crust appearing a humid odour. In addition to that, the acid taste is possible when lactic bacteria are present in the product and a metallic taste should develop in hams which are subjected to short curing or due to an intense abrasion by metallic material. The enzymatic proteolysis, as well as the activity of some microorganisms, originates tyrosine which precipitates during the curing time producing crystals due to the water reduction and its high insolubility. Sometimes, in addition to crystals, tyrosine forms a white film, as same as the creatinine forms in vacuum packed or modified atmosphere hams. Excessive production of peptides and AAs can be associated with the development of bad tastes in the final product. Salty and bitter tastes are derived from anomalous proteolysis when salt is not well incorporated or when the protein breakdown generates peptides with bitter properties. Some treatments, like thermal treatments, high-pressure applications or vacuum packaging, can enhance the salty sensation or cause organoleptic modifications.
Due to an excessive RH, moulds also can growth taking place the development of spots of different colours (black, white, green, red or yellow). Other microorganisms that can generate spots are, for instance, Pseudomona libanensis, causing a blue colouring in the leg, and Pseudomonas fluorescens and Clostridium herbarum which provoke black spots. Mycotoxins could be formed by the action of fungi, in special from the genus Aspergillus (Udomkun et al., 2017). Among others, aflatoxin B1 is considered the most common, with a great toxic effect. Similarly, it has been reported high amounts of ochratoxin A in products with large content of salt like dry-cured ham, whose production is due to Aspergillus waterdijkiae and is linked to high sanitary risk (Vipotnik et al., 2017). Furthermore, there is a traditional defect called “Coxofemoral coquera”, which is characterized by crack formation inside the muscular tissues during drying showing an unstructured tissue and a brown and malodorous slim along the cracks. The appearance is very dry due to an intense drying and in some cases, there is a development of mites. Not only mites can grow into the ham but also insects, whose larvae can cause intestinal problems if they are ingested due to their resistance against acidity. As is the case of mites, they could provoke allergic symptoms. Ageing time has a high effect on
23 I. Introducción these infestations, it has been shown that hams with less than three months of ripening have absence or very low contamination while ageing up to five months increases the risk.
I.4.2. TEXTURE DEFECTS
An important matter to consider is the texture. In this regard, textures too soft, pastiness, superficial crust and too much fibrous texture must be prevented since consumers reject the product (Morales et al., 2008; 2013). It is possible the appearance of pastiness or unusually soft textures that entail technological problems to butchers in hams for slice commercialisation (Arnau 1991). This defect is associated with excessive proteolysis which causes the breakdown of tissue structures (Wu et al., 2014) probably due to the salt reduction tends to enhance enzymatic activity (Armenteros et al., 2012). Some causes provoke intense proteolysis, for instance, processing conditions, in which increments of temperature, as well as low levels of salt or short salting time, could favour the enzyme activity enhancing protein breakdown (Martín et al., 1997). In this regard, a hard texture can happen if the drying is very high due to the use of excessive temperature at the same time that RH is low and the air velocity is insufficient. These conditions are also associated with too fat exudation and, in extreme cases, mites can be penetrated and muscles fissures can appear. Moreover, it can be originated by greater proteolytic potentials of the raw meat, pH too high or, on the contrary, too low, leading to a sticky sensation of touch and elevated viscosity in the mouth when the product is put in contact with the saliva.
I.5. NEW PROCESSING TECHNOLOGIES APPLIED TO HAM MANUFACTURE
First of all, ham manufacture must provide a safe product. For that, fungi and microbial growth as much as pest must be controlled or removed. In this regard, there are some strategies against toxigenic fungi, chemical procedures such as the use of NaCl to inhibit the growth (Schmidt-Heydt et al., 2011) as much as biological techniques as the use of competing moulds (Andrade et al., 2014). Traditionally, methyl bromide was the main compound used to control pest due to its wide spectrum and fast action, but since 1992, due to the protective steps of the ozone layer, it was included into the substances that contribute to ozone depletion and it was proposed its complete reduction by 2005 (Osteen, 2003). Since that, some researchers have been focused on alternatives of methyl fluoride. In this context, phosphine, sulfuryl fluoride, carbon dioxide and ozone were the object of study, concluding that the use of the first three substances was not viable (Sekhon et al., 2010a; 2010b; Schilling et al., 2010), although phosphine was proposed as the most suitable alternative due to its high effectiveness, the lack of residues, low cost and the easiness of apply it (Fields and White, 2002). On the other hand, some physical methods have been proposed to control microbial growth. Among these methods, gamma irradiations (Jin et al., 2012), electron beam (Kong et al., 2017), high hydrostatic pressure (Rubio-Celorio et al., 2016), ultraviolet C irradiation (Lah et al., 2012), controlled atmosphere (Sanchez-Molinero et al., 2010), etc. can be found. Even though, some of them are not still suitable due to the excessive cost, the need of adapting the installations, poor effectiveness, sensorial quality losses or the scarcity of information available due to the lack of enough studies yet.
24 I. Introducción
Besides that, detection of defects of the final product is another application of the new technologies that can help to optimize and improve the process. In this regard, the most important aspect in the dry-cured ham process is to maintain rigorous control of the salting and curing stages, since all the reactions that give rise to the typical sensory properties of the product take place at these stages. To assure an adequate salt diffusion and appropriate water content for a better development of the process, the use of technologies such as irradiation (Fulladosa et al., 2010; 2015a), magnetic resonance imaging (MRI) (Fantazzini et al., 2009), infrared spectroscopy (Garrido-Novell et al., 2015) and ultrasounds (US) (de Prados et al., 2017) has been analysed. Irradiation techniques have been considered as one of the most effective preservation procedures (Alfaia et al., 2007; Kong et al., 2017). However, it is well known that lipid oxidation is its major consequence which effects on nutritional value and sensory characteristics are mainly off-odour production (Alfaia et al., 2007). MRI was evaluated as a method to quantify muscle and subcutaneous and intermuscular fat contents (Monziols et al., 2006), a task that is very important to improve the dry-cured ham process since penetration of salt and water loss are very influenced by the fat content (Ripollés et al., 2011). Besides that, Pérez-Palacios et al. (2011) proved its feasibility for the prediction of sensory properties by fat and texture characterization. On the other hand, near infrared spectroscopy (NIRS) can be used to classify raw pieces based on WHC at the beginning of the process, allowing for the early detection and disposal of meat that could result in defective products in the following process stages. Also, Ortiz et al. (2006) essayed NIR suitability in dry-cured hams classification according to other sensory parameters, such as pastiness, crusting or marbling.
On the other hand, less studied technologies including Pulse Electric Fields (PEF), microwaves and data mining should be explored in depth in the future. PEF application allows the reduction in the curing time since mass transfer is improved (Chauhan and Unni, 2015), however there are studies that proved PEF effects on muscle cell membranes influencing the interaction between fatty acids and cell membrane phospholipids with prooxidants in meat (Faridnia et al., 2015) which originates undesirable compounds which can cause off-flavours. Microwaves could be useful not only to online estimate salt, water and fat contents, but also to determine textural characteristics of the product (Damez and Clerjon, 2013; Fulladosa et al., 2018; García-Rey et al., 2005; Ortiz et al., 2006; Rubio-Celorio et al., 2016) in an easy way due to the availability of portable equipments. Electromagnetic waves have been widely used in meat products for investigating quality attributes like sensory and nutritional characteristics, chemical and physicochemical properties, and safety (Damez and Clerjon, 2013), as well as its potential for classifying slices based on different pastiness levels with a 93% probability of correct classification. The most promising discovery in recent years for detecting defects or for classifying products according to sensory attributes is data mining, which allows to make predictions based on an immense data file archive (Pérez-Palacios et al., 2014).
As can be observed above, the available information about non-invasive technologies is restricted to the preventive actions based on characterizing or monitoring the process, whereas the knowledge on their corrective uses is not large. From the few studies conducted so far, HPP and US techniques have demonstrated their potential use as texture defect correctives. As far as US implementation is concerned, it is an inexpensive technique but the
25 I. Introducción production line needs to be adjusted. Regarding HPP, it was shown the utility for reducing salt contents considering that the salty taste, after its application, is enhanced.
I.5.1. HIGH-PRESSURE TECHNIQUE APPLIED IN DRY-CURED HAM
The high-pressure technology is one of the most investigated techniques across the time. In 1899, Bert Hite designed a prototype of HPP system, which was used to pasteurize some foods, but in this time its viability in the food industry was limited due to the high cost involved. It was not until 1993 that the HPP technology was introduced in food manufacture with stabilizing purposes (Rivalain et al., 2010). The mechanism of action of HPP units resides in a chamber in which the food, previously packed, is positioned. After closing the vessel, the chamber is filled with a medium capable of transmitting the pressure (Fig. 10).
Figure 10. Diagram of High-Pressure treatments
The traditional main purpose of HPP treatments has been the microbial reduction to extend shelf life (Duranton et al., 2015). Concerning dry-cured ham, Table 5 shows the studies carried out in the last years. Likewise, the initial purpose of HPP use has also been microbial inactivation (Clariana et al., 2011; Garriga et al., 2004; Rubio-Celorio et al., 2016). However, HPP application implies modifications in the final products and the food could undergo physical, chemical, organoleptic, technical and functional alterations or all at once (Liu et al., 2008; Rivalain et al., 2010). However, these changes are not necessarily all negative. In this regard, dry-cured products showed an increase in saltiness perception caused by HPP (Clariana et al., 2012, 2011; Fulladosa et al., 2012; Rubio, Martinez et al., 2007), which is interesting in reduced salt product manufacture. Picouet et al. (2012) explained the increment in salty taste by the increase of the number of sodium ions released due to differences in salt and protein interactions after the application of pressure.
Application of high pressures (HPP) result in higher water losses (Fulladosa et al., 2009; Garcia-Gil et al., 2014; Serra et al., 2007a). In this regard, Rubio-Celorio et al. (2016) observed a 0.43% increase in water loss for every pressure increment of 100 MPa, while Picouet et al. (2012) observed even higher losses up to 0.82%. In addition to this, the pressure could induce reductions in the water holding capacity with treatments between 300 and 600 MPa (Fulladosa et al., 2009; Picouet et al., 2012). Previously, Serra et al. (2007a) showed a 24% increase in weight losses after salting in samples treated at 600 MPa before the salting process compared to samples treated at 400 MPa, and both HPP-treated samples showed lower NaCl content
26 I. Introducción than controls. This fact is attributed to an increase in protein denaturation caused by pressurization, in turn increasing weight losses during salting (Serra et al., 2007a). Due to this lower NaCl content, significantly higher values of water activity (aw) were observed in pressured samples from BF muscle, whereas no differences were found in SM muscle, maybe due to the large variability in the composition values of this muscle (Virgili and Schivazappa, 2002).
Table 5. High-pressure technology applications across the dry-cured ham process sorted by method parameters
MEASURE CONDITION Sample type PURPOSE OF THE REFERENCE Pressure Time Temperature RESEARCH 200 MPa 5 min 20°C Sliced vacuum- To reduce intrinsic Rubio-Celorio 400 MPa packed dry- water population et al. (2016) 600 MPa cured ham 600 MPa 6 min Variable Sliced, skin To reduce intrinsic Clariana et al. 900 MPa 5 min vacuum- microbial population (2011) packed dry- cured ham 600 MPa 6 min 31 °C Sliced, skin To inactivate microbial Garriga et al. vacuum- population (2004) packed dry- cured ham 400 MPa 10 min Variable Frozen To evaluate the effects Serra et al. 600 MPa vacuum- on physicochemical (2007a) packed ham parameters and Serra et al. antioxidant and (2007b) proteolytic enzyme activities at different stages of ham manufacture and on the final product characteristics 600 MPa 6 min 12 °C Sliced vacuum- To examine lipid and Fuentes et al. packed dry- protein oxidation and (2010) cured ham sensory properties 300 MPa 5 min 12 °C Sliced vacuum- To examine structural Picouet et al. 600 MPa packed dry- and molecular changes (2012) 900 MPa cured ham affecting sodium and water dynamics 500 MPa 3 min 3°C Sliced, skin To evaluate textural Garcia-Gil et vacuum- changes al. (2014) packed dry- cured ham 400 MPa 10 min 12°C Sliced vacuum- To investigate changes Rivas-Cañedo packed dry- in the volatile fraction et al. (2009) cured ham influenced by packaging material 600 MPa 6 min 21 °C Sliced, skin To evaluate the Martínez- vacuum- influence on the volatile Onandi (2017) packed dry- fraction Martínez- cured ham Onandi (2018)
27 I. Introducción
On the other hand, there are some studies on the effects of pressure treatments on enzymatic activity. These studies concluded that most of the enzyme activities are generally unaffected at 200 MPa and below (Masson et al., 2001), but inactivation begins to occur as the pressure increases, with differences depending on the nature of the enzyme (Rivalain et al., 2010). In this sense, it has been shown that pressures of 600 MPa reduce glutathione peroxidase (GSHPx), superoxide dismutase (SOD) and catalase (CAT) activity. Moreover, at 400 MPa the inactivation percentage of CAT increases. Based on this evidence, Serra et al. (2007a) concluded that the ageing conditions and the dry-curing process would have a greater effect on the enzymatic activity than the pressure application. However, modifications in protein bonds, protein solubility losses and decreases in cathepsin B, D and L activity have been observed in HPP-treatments above 400 MPa (Campus et al., 2008). The provided effect of HPP on both enzyme activity and protein denaturation, which are widely associated with the final texture, have caused an increased interest in its potential use in meat tenderization (Ichinoseki et al., 2006; Jung et al., 2000).
In spite of previous evidence in meat showing significantly higher shear force values in HPP- treated salted pork meat (Duranton et al., 2012) and in cooked beef meat (Jung et al., 2000), the textural effect on dry-cured hams has not been thoroughly studied. To date, only a few studies have been published about it, reporting higher hardness values in pressurized samples (Fuentes et al., 2010; Fulladosa et al., 2009; Rubio-Celorio et al., 2016). Fuentes et al. (2010) reported greater hardness and chewiness in pressurized samples, and they observed that the treated samples were less juicy and doughy than the controls. Likewise, colour parameters can also be affected by the application of pressure, as increments in reflectance and lightness (L*) and decreases in redness (a*) values have been reported (Andrés et al., 2004; Fulladosa et al., 2009; Hughes et al., 2014; Rubio-Celorio et al., 2016), modifications which could be explained by the fact that HPP promotes changes in protein structural conformations, which could have an undesirable impact on the organoleptic characteristics of final products (Serra et al., 2007b).
Since the final aroma is influenced by all the reactions occurring during the dry-curing process, mainly by proteolysis and lipid oxidation reactions, several researchers have focused on investigating the volatile profile changes after pressure application to evaluate the organoleptic impact (Clariana et al., 2011; Lorido et al., 2015). In this sense, since pressure promotes lipolysis reactions (Fuentes et al., 2010, 2014), mainly at pressures around 600 MPa (Andrés et al., 2004; Fuentes et al., 2010), the amount of some lipolysis-derived volatiles increases in HPP-treated samples. Martínez-Onandi (2018; 2017; 2016a) evaluated the changes in the volatile profile of dry-cured ham slices treated at 600 MPa and its evolution during 5 months of refrigerated storage. They observed higher levels of 12 volatile compounds in the treated samples, whereas, after the storage period, only 9 of the total volatile compounds were increased. On the other hand, at 400 MPa, Rivas-Cañedo et al. (2009) also observed increased amounts in volatile compounds, whereas after treatments between 200 and 600 MPa, Andrés et al. (2004) reported an important increase in hexanal which is considered the main compound derived from lipid oxidation and it contributes to the fatty distinctive matured ham flavour. Its amount was even more pronounced at 800 MPa. Similarly, Fuentes et al. (2010) noticed higher levels in 5 aldehydes after treatment at 600 MPa, which caused an increment of the rancid odour perception while differences in rancid flavour were not shown.
28 I. Introducción
However, enhanced saltiness, bitterness and cured flavour were observed in the HPP-treated samples. Besides that, HPP treatments could reduce the levels of some volatile compounds due to enzyme inactivation or matrix structure modifications (Garcia-Gil et al., 2014; Picouet et al., 2012). Thus, Martínez- Onandi et al. (2018) reported reductions in 23 volatile compounds, as described by Rivas-Cañedo et al. (2009) and Martínez-Onandi et al. (2016a) in 6 volatile compounds, and by Martínez-Onandi et al. (2017) in 31 volatile compounds.
In summary, the effects of HPP treatments depend on the pressure level, although the influence of factors such as the measuring conditions of the HPP treatment, packaging material or storage cannot be ignored (Martínez-Onandi et al., 2017).
I.5.2. ULTRASOUNDS APPLIED IN DRY-CURED HAM
Ultrasound technology is based on pressure fluctuations caused by sound waves that increase temperature and pressure leading to cavitation phenomena which could accelerate mass transfer (Jambrak et al., 2010) and favours extraction processes (Zhu et al., 2017a; 2017b). As shown in Table 6, the interest in the potential uses of the US has increased significantly in the last years. However, it is difficult to compare the different results obtained due to the high variability among studies, including many parameters that could influence the outcomes (Berlan and Mason, 1992). According to the intensity applied, US is classified into low-intensity, (frequencies above 100 kHz and intensities below 1 Wcm−2) and high-intensity ultrasound (frequencies from 18 to 100 kHz and intensities above 1 Wcm−2) (McClements, 1995). The first one can be considered as non-invasive due to the lower level of intensity. For this reason, it is useful for characterization purposes within food processing (Chandrapala et al., 2012; Koch et al., 2011a, 2011b). On the contrary, high-intensity ultrasound could induce physical, chemical and/or mechanical modifications (Jayasooriya et al., 2004) and this is why its traditional use in food manufacture has been for generating emulsions (Dolatowski et al., 2007).
Despite previous studies in meat which have not shown any effect on texture after US application, McDonnell et al. (2014a) found lower cohesiveness and gumminess values in treated hams. Moreover, hardness was found to increase upon treatments with high-intensity US, which could be due to the induced heating in the treated samples (Contreras et al., 2018).
29 I. Introducción
Table 6. Ultrasound technology applications across the dry-cured ham process sorted by method parameters
TECHNOLOGY MEASURE CONDITION PURPOSE OF THE REFERENCE INTENSITY TEMPERATURE (°C) RESEARCH Ultrasound (US) 1 MHz 2 °C To predict the fat De Prados et al. Through- content in dry-cured (2015) transmission hams for industrial classification To monitor dry De Prados et al. salting (2016) 2 °C and 15 °C To predict salt and Fulladosa et al. fat contents (2015b) 0, 2, 4, 6, 8, 10, 12, To characterize the Niñoles et al. 14, 20, 22 and 24 °C melting properties of (2010) subcutaneous fat 50 W 40, 45 and 50°C To shorten the hot Contreras et al. air mild thermal (2018) treatment applied to correct the texture 4.2, 11 and 19 Below 21°C To accelerate the McDonnell et al. W cm-2 curing stage (2014a) Ultrasound (US) 1 MHz 2 °C To monitor dry De Prados et al. Pulse-echo salting and to predict (2017) mode the final salt content Air-coupled 0.75 MHz 6 °C To characterize Corona et al. ultrasound (2013) Scanning 10 MHz 6 °C To characterize acoustic microscopy
30
II. JUSTIFICATION AND OBJECTIVES
II. Justification and objetives
The present Doctoral Thesis tried to contribute to the knowledge in the field of non-invasive technologies applied to the correction of textural defects in dry-cured ham. Current trends in the consumption of meat products are aimed at products with lower salt content, according to the NAOS strategy that recommends reducing salt intake in a heart-healthy diet. This trend entails significant technological problems, enhances the tendency of hams to present pasty textural problems, which make difficult the slicing process and promote greater adhesiveness between slices. This fact negatively influences the perceived qualit by the consumer.
Therefore, the objectives proposed in this Doctoral Thesis were the following:
1. Determination of the relationship between proteolysis index and the incidence of textural defects, especially adhesiveness, identifying potentials biomarkers. 2. Study of the possible sensorial consequences, from an instrumental perspective, caused by excessive proteolysis 3. Structural evaluation of moderate heat treatment assisted by power ultrasound as a corrective measure of adhesiveness defect in sliced ham. 4. Instrumental evaluation of organoleptic changes due to corrective treatment with power ultrasound. 5. Evaluation of the effect of high-pressure treatments on protein structures. 6. Instrumental study of organoleptic changes derived from a treatment based on high pressures at different pressurization temperatures, identifying the optimal treatment.
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 the present memoir, whose references are as follows:
1. López-Pedrouso, M., Pérez-Santaescolástica, C., Franco, D., Fulladosa, E., Carballo, J., Zapata, C., and Lorenzo, J. M. (2018). Comparative proteomic profiling of myofibrillar proteins in dry-cured ham with different proteolysis indices and adhesiveness. Food Chemistry, 244, 238-245. 2. Pérez-Santaescolástica, C., Carballo, J., Fulladosa, E., Garcia-Perez, J. V., Benedito, J., and Lorenzo, J. M. (2018). Effect of proteolysis index level on instrumental adhesiveness, free amino acids content and volatile compounds profile of dry-cured ham. Food Research International, 107, 559-566. 3. Pérez-Santaescolástica, C., Carballo, J., Fulladosa, E., José, V. G. P., Benedito, J., and Lorenzo, J. M. (2018). Application of temperature and ultrasound as corrective measures to decrease the adhesiveness in dry-cured ham. Influence on free amino acid and volatile compound profile. Food Research International, 114, 140-150. 4. López-Pedrouso, M., Pérez-Santaescolástica, C., Franco, D., Carballo, J., Garcia-Perez, J. V., Benedito, J., ... and Lorenzo, J. M. (2019). Proteomic footprint of ultrasound intensification on sliced dry-cured ham subjected to mild thermal conditions. Journal of Proteomics, 193, 123-130. 5. Pérez-Santaescolástica, C., Carballo, J., Fulladosa, E., Munekata, P. E. S., Campagnol, P. B., Gómez, B., and Lorenzo, J. M. (2019). Influence of high-pressure processing at
33 II. Justification and objectives
different temperatures on free amino acid and volatile compound profiles of dry-cured ham. Food Research International, 116, 49-56. 6. López-Pedrouso, M., Pérez-Santaescolástica, C., Franco, D., Carballo, J., Zapata, C., and Lorenzo, J. M. (2019). Molecular insight into taste and aroma of sliced dry-cured ham induced by protein degradation undergone high-pressure conditions. Food Research International, 122, 635-642.
34
III. MATERIAL AND METHODS
III. Material and methods
III.1. SAMPLES
III.1.1. ELABORATION
Two hundred raw hams with a pH value below 5.5, which were more prone to develop defective textures, were obtained from a commercial slaughterhouse. Hams were coming from pigs belonging to crosses of Large white and Landrace breeds (medium fat content). All animals (castrated males) were reared in the same conditions. The pigs were allowed ad libitum access to water and feed. The basal diet contained: barley (81.08%), lard (4.0%), soya (12.05%), methionine (0.08%), lysine (0.30%), threonine (0.11%), calcium carbonate (0.96%), dicalcium phosphate (0.66%), salt (0.33%) and minerals and vitamins (0.4%).
All hams (n=200) were weighted (11.9 kg ± 1.1 kg) and salted according to the traditional system. Hams were manually rubbed with the following mixture: 0.15 g of KNO3, 0.15 g of
NaNO2, 1.0 g of dextrose, 0.5 g of sodium ascorbate and 10 g of NaCl per kilogram of raw ham. The hams were next pile salted at 3 ± 2 °C and 85 ± 5% RH during 4 (n=50), 6 (n=50), 8 (n=50) or 11 days (n=50) according to their corresponding raw weight. After salting, hams were washed with cold water and post-salted at 3 ± 2 °C and 85 ± 5% RH during 45 days. Drying of hams was performed at 12 ± 2 °C and 70 ± 5% RH until reaching a weight loss of 29%, then, they were vacuum packaged and kept at 30 °C for 30 days to induce proteolysis. After this time, hams continued the drying process at 12 ± 2 °C and 65 ± 5% RH until reaching a weight loss of 34%, and next they were vacuum packaged again and kept at 30 °C during 30 days more. After this period, hams were dried again until the end of the drying process (weight loss of 36%). At the end of the process, hams were cut and boned and the cushion part containing BF and SM muscles was excised and sampled for a preliminary characterization of samples.
III.1.2. CORRECTIVE TREATMENTS
III.1.2.1. Ultrasounds
Ultrasound effect was evaluated from a total of 26 dry-cured hams previously classified with a high proteolysis index (PI>36%). At the end of the manufacture process, when hams were cut and boned and the cushion part containing the BF muscle was excised and sampled, ten slices of each ham (1.5 mm) were vacuum packed in individual plastic bags of polyamide/polyethylene (oxygen permeability of 50 cm3/m2/24 h at 23 °C and water permeability of 2.6 g/m2/24 h at 23 °C and 85% RH, Sacoliva® S.L., Spain) and divided into two treatments (13 samples per treatment): conventional thermal treatments (CV) and thermal treatment assisted by power ultrasound (US). In the thermal treatments assisted by power ultrasound (US), ultrasound was only applied during the heating stage, which was defined as the time needed to reach in the centre of the slice a temperature 5 °C below that in the heating medium, measured using a thermocouple. Thus, average ultrasonic treatment time was of 7.5 min, after samples were kept in a water bath (50 °C) to complete 5 h of treatment. These heating temperature and time were chosen to avoid the appearance of cooking flavours in the ham, as found in preliminary experiments. Thermal treatments were applied in an ultrasonic bath (600 W, 25 kHz, model GAT600W, ATU, Spain) using water as heating fluid. On
37 III. Material and methods the other hand, in the conventional thermal treatment (CV) samples were kept in a water bath for 5 h at 50 °C. All the samples were analysed before the application of the corresponding treatment (control -CO- group).
III.1.2.2. High-pressure
BF muscles of 120 raw hams were randomly divided into four treatments (30 hams per treatment). From each ham unit, three 1.5 mm-thick slices were vacuum packed in individual plastic bags of polyamide/polyethylene (oxygen permeability of 50 cm3/m2/24 h at 23 °C and water permeability of 2.6 g/m2/24 h at 23 °C and 85% RH, Sacoliva® S.L., Spain) and stored in a chamber at 4 °C ± 2 °C until the treatment application. The treatment of the packaged slices was applied using a NC Hyperbaric WAVE 6000/120 equipment (NC Hyperbaric, Burgos, Spain). Three different treatments were performed at 600 MPa during 6 min, each one accompanied by a different temperature: the first at 0 °C (HPP-0), the second at 20 °C (HPP-20) and the third at 35 °C (HPP- 35). In order to evaluate the effects of HPP treatments, a fourth group of samples was not treated and was used as a control (CO) batch.
III.2. EXPERIMENTAL DESIGN
For a better understanding, fig.12 shows a scheme of the global study, the samples used for each part of the study as much as the conducted analysis in each case.
Figure 11. Experimental design
III. Material and methods
The global study was carried out in three steps. Firstly, the purpose was to characterize the samples before to be treated in order to known the main characteristics and be able to classify them by their proteolysis index. Also, the relationship between proteolysis index and the adhesiveness incidence was evaluated and the consequences from adhesiveness development were studied. Secondly, samples with PI > 36 were subjected to US treatments for checking the efficacy of ultrasounds against the adhesiveness defect and for testing possible consequences of their application. For that, the adhesiveness as well as proteomic analysis, free amino acid and volatile compounds were determined after and before the treatments and compared among treatments. Finally, a mix of samples with different proteolysis levels were selected for HPP treatments since it has been probed that HPP could be used as corrective measure for texture defects. Three different temperatures were selected to apply the treatments and their organoleptic impact was evaluated from an analytical point of view. Similarly than in the study of the US effect, free amino acid and volatile compounds were measured after the HPP application and compared with the untreated group (control). Also, a proteomic study was carried out comparing between control and HPP treated at 0 °C samples, thus it could avoid any temperature effect and only the changes originated by HPP application were observed.
III.3. ANALYTICAL AND BIOCHEMICAL CHARACTERIZATION OF SAMPLES
III.3.1. CHEMICAL ANALYSIS
BF samples were minced and subjected to chemical analysis in triplicate. Water content was analysed by drying at 103 ± 2 °C until reaching a constant weight (AOAC, 1990), whereas the chloride content was analysed according to ISO 1841-2 (1996) using a potentiometric titrator 785 DMP Titrino (Metrohm, Herisau, Switzerland) and results were expressed as a percentage of NaCl.
III.3.2. NITROGEN FRACTION ANALYSIS
Total nitrogen content (NT) was determined according to the Kjeldahl method (ISO, 1978) using the Vapodest 50S analyser (Gerhardt, Königswinter, Germany). It concerns a semi-micro rapid routine method using block-digestion with copper catalyst, steam distillation into boric acid, and titration. A known quantity of the sample (1 ± 0.1 g) was analysed. The content of non-protein nitrogen was assessed as described by Lorenzo et al. (2008). Two and a half g of sample were homogenized in 25 mL of deionized water in an Ika Ultra-Turrax. Afterwards, 10 mL of 20% trichloroacetic acid (99.5% purity, Merck, Darmstadt, Germany) were added, and the mix was well stirred and let to stabilize for 60 min at room temperature. Next, it was centrifuged at 1734 x g for 10 min. After centrifugation, the supernatant was filtered, and 15 mL of the filtrate were used for determination of the nitrogen content, following the same method used for the total nitrogen (NT) determination (ISO, 1978). The proteolysis index (PI) was calculated as the ratio (non-protein nitrogen/total nitrogen) × 100 according to Ruiz- Ramírez et al. (2006). Total volatile basic nitrogen (TVBN) content was assessed according to the Commission Regulation (EC) No 2074/2005 (Commission Regulation, 2011). A 10 g sample of muscle was homogenized with 90 mL of perchloric acid, and the resulting suspension was
39 III. Material and methods centrifuged at 10000 x g for 10 min using an Allegra X-22 centrifuge (Beckman Coulter, California, EEUU). Fifty millilitres of the supernatant were analysed for the nitrogen content following also the Kjeldahl method using a Vapodest 50S analyser (Gerhardt, Königswinter, Germany). The TVBN values were expressed as mg nitrogen/100 g of DM. Finally, dry-cured hams were categorized in different proteolysis index level groups according to their proteolysis index: low proteolysis level (IP<32%) (LP), medium proteolysis level (32% < IP < 36%) (MP) and high proteolysis level (IP>36%) (HP).
III.3.3. INSTRUMENTAL TEXTURE
Textural analysis was performed using a texture analyser (TA-XT Plus; Stable Micro Systems, Godalming, UK) by carrying out a separation test using different load cells with a specific probe. Instrumental adhesiveness was measured in sliced ham samples (1 mm) by applying probe tests and calculating the negative area of a force-time curve in tension tests with a single-cycle. As can be shown in Fig.12, the texturometer was equipped with a probe connected to a special device that enables horizontal probe displacement. After separation of the slices, the probe returned to the initial position.
Figure 12. Texturometer
Previous tests were done to optimize the conditions for the measurement. For that, samples of commercial sliced dry-cured ham packaged under vacuum conditions were used, and different conditions were tested: different load cell (5 and 50 N), speeds (0.5, 4, 6 and 10 mm/s), distances (80, 100 and 130 mm) and slice thicknesses (0.5, 1, 1.5, 2, 3 and 4 mm). Finally, the best conditions obtained for the measurement of adhesiveness of dry cured ham slices were applied in this study and correspond to: load cell=5 N; speed=0.5 mm/s and distance=100 mm. From the obtained graph of force vs. distance, the adhesiveness was calculated. All the measurements were made in triplicate, at room temperature.
III.4. PROTEOMIC ANALYSIS
For proteomic analyses, four biological samples per study were lyophilized and frozen at −80 °C until the protein extraction.
III. Material and methods
III.4.1. PROTEIN EXTRACTION
Total protein from BF muscle was extracted from 50 mg of lyophilized dry-cured ham. Samples were homogenized with 1 mL of lysis buffer (7M urea; 2M thiourea; 4% CHAPS; 10mM DTT, and 2% Pharmalyte™ pH 3–10; GE Healthcare, Uppsala, Sweden) and sonicated (Sonifier 250; Branson Ultrasonics Corporation, Danbury, CT) in short pulses at 0 °C. Excess salts and other interfering substances were removed twice using the 2-D Clean-Up Kit (GE Healthcare) following manufacturer’s instructions. This method for selectively precipitating protein was carried out using 200 μL of sonicated sample and the resulting pellet was redissolved in 200 μL of lysis buffer. The protein concentration was assessed using a commercial CB-X protein assay kit (GBiosciences, St. Louis, MO) according to the manufacturer’s instructions in a Chromate® microplate reader (Awareness Technology, Palm City, FL).
III.4.2. TWO-DIMENSIONAL ELECTROPHORESIS (2-DE)
The 2-DE was performed according to Franco et al. (2015a). Briefly, 250 μg of protein in lysis buffer were mixed with rehydration buffer (7M urea, 2M thiourea, 4% CHAPS, 0.002% bromophenol blue), reaching 450 μL of total volume. Finally, 0.6% DTT and 1% IPG buffer (Bio- Rad Laboratories, Hercules, CA) were added. This protein extract was loaded into an immobilized pH gradient (IPG) strips (24 cm, pH 4–7 linear, Bio-Rad Laboratories). The isoelectric focusing (IEF) was carried out on a PROTEAN IEF cell system (Bio-Rad Laboratories). The IEF protocol was: Step-1 (rehydration), 50 V for 12 h; Step-2, 250 V for 30 min; Step-3, 00 V for 1 h; Step-4, 1000 V for 1 h; Step-5, 4000 V for 2 h; Step-6, 8000 V for 2 h; and Step-7, 10,000 V until to reach 70,000 V. In the equilibration step, the strips were washed in buffer I (50mM Tris pH 8.8, 6M urea, 2% SDS, 30% glycerol, 1% DTT) for 15 min and buffer II (50mM Tris pH 8.8, 6M urea, 2% SDS, 30% glycerol, 2.5% iodoacetamide) for another 15 min. The second dimension separation was performed using an Ettan DALTsix vertical gel system (GE Healthcare) with 12% SDS-PAGE gels at 16-18 mA/gel until the bromophenol blue dye front reached the end of the gels. The 2-DE gels were stained with SYPRO Ruby fluorescent stain (Lonza, Rockland, ME).
III.4.3. IMAGE ANALYSIS OF 2-DE GELS
Gels were visualized and digitalized using the Gel Doc XR+ system (Bio-Rad Laboratories). The detection and quantification of 2-DE spot volumes were performed with PDQuest Advanced software v. 8.0.1 (Bio-Rad Laboratories) after background subtraction. Spot volume normalization was performed using those validated across all replicate gels. Observed values of molecular mass (Mr) were determined across protein spots from standard Mr markers ranging from 15 to 200 kDa (Fermentas, Burlington, ON, Canada), whereas those of isoelectric point (pI) were established according to their position on the IEF-strips.
III.4.4. PROTEIN IDENTIFICATION BY MASS SPECTROMETRY (MS)
For matrix assisted laser desorption-ionization time of flight mass spectrometry (MALDI TOF/TOF MS) analysis, selected spots were excised from the gel and they were dehydrated
41 III. Material and methods with acetonitrile using a vacuum centrifuge. The gel piece was washed with Ambic buffer (50mM ammonium bicarbonate in 50% methanol). The proteins were reduced with 10mM DTT in 50mM ammonium bicarbonate and alkylated with 55mM acetamide in 50mM ammonium bicarbonate. Extracts were repeatedly rinsed with Ambic buffer, dehydrated by addition of acetonitrile and dried in a SpeedVac. Then, the proteins were hydrolysed with modified porcine trypsin (Promega, Madison, WI) at a final concentration of 20 ng/μL of trypsin in 20mM ammonium bicarbonate overnight at 37 °C. The total digest was incubated three times in 40 μL of 60% acetonitrile with 5% formic acid, concentrated in a SpeedVac and stored at −20 °C until analysis. Dried samples were dissolved in 4 μL of 0.5% acetic acid. Equal volumes (0.5 μL) of peptide and matrix solution, consisting of 3 mg of α-cyano-4-hydroxycinnamic acid dissolved in 1 mL of 50% acetonitrile and 0.1% trifluoroacetic acid, were deposited onto a 384 Opti-TOF MALDI plate (Applied Biosystems, Foster City, CA) using the thin-layer method (Vorm et al., 1994). Mass spectrometric data were obtained in an automated analysis loop using 4800 MALDI-TOF/TOF analyser (Applied Biosystems). Mass spectra were acquired in positive-ion reflector mode with an Nd:YAG laser operating at 355 nm and an average accumulation of 1000 laser shots. A minimum of three trypsin autolysis peaks were used for internal calibration, in order to decrease peptide mass errors for protein identification. All MS/MS spectra were performed by selecting the precursors with a relative resolution of 300 (FWHM, full-width half mass) and metastable suppression. Automated analysis of mass data was achieved using the 4000 Series Explorer Software v. 3.5 (Applied Biosystems). Peptide mass fingerprint and peptide fragmentation spectra data of each sample were combined using GPS Explorer Software v. 3.6 and Mascot software v. 2.1 (Matrix Science, Boston, MA) to search against UniProt/SwissProt database. Mascot search parameters were: precursor mass tolerance of 50 ppm, 0.6 Da MS/MS fragment tolerance, carbamidomethyl cysteine as a fixed modification, oxidized methionine as a variable modification and permitting one missed cleavage. Proteins with at least two matched peptides and statistically significant (p-value < 0.05) Mascot scores were selected as positively identified.
III.5. INSTRUMENTAL EVALUATION OF ORGANOLEPTIC PRECURSORS
III.5.1. FREE AMINO ACID ANALYSIS
The free amino acids were extracted following the procedure described by Lorenzo et al. (2015). Samples were prepared by homogenizing 5 g of sample with 25 mL of hydrochloric acid 0.1 N, in an Ika Ultra-Turrax for 8 min while cooled by submerging the extract in ice. The homogenized samples were centrifuged for 20 min at 10000 x g and the supernatant material was filtered through glass wool prior to further analyses. Two hundred µL of this extract was deproteinized by adding 20 µl of N-Leu (internal pattern) and 780 µL of acetonitrile. After 30 min of cooled resting, extract was centrifuged for 5 min at 10000 g and 4ºC. Amino acids were derivatized with 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate (Waters AccQ-Fluor reagent kit) following the manufacturer specifications, and then analysed by RP-HPLC using a Waters 2695 Separations Module with a Waters 2475 Multi Fluorescence Detector, equipped with a Waters AccQ-Tag amino acid analysis column. Finally, the results were expressed as mg of free amino acid/100 g of DM.
III. Material and methods
III.5.2. VOLATILE COMPOUND ANALYSIS
The extraction of the volatile compounds in preliminary study was performed using solid- phase microextraction (SPME). An SPME device (Supelco, Bellefonte, USA) containing a fused silica fibre (10mm length) coated with a 50/30 layer of divinylbenzene/ carboxen/polydimethylsiloxane (DVB/CAR/PDMS) was used. For chromatographic analyses, a gas chromatograph 6890N (Agilent Technologies, Santa Clara, CA, USA) equipped with a mass selective detector 5973N (Agilent Technologies) was used with a DB-624 capillary column (30 m, 0.25 mm i.d., 1.4 μm film thickness; JandW Scientific, Folsom, CA, USA). For extraction, 1 g of each sample was weighed in a 20 mL vial, after being ground using a commercial grinder. The vials were subsequently screw-capped with a laminated Teflon rubber disc. The fibre, previously conditioned by heating in a Fibre Conditioning Station at 270 °C for 30 min, was inserted into the sample vial. The extractions were carried out at 37 °C for 30 min, after equilibration of the samples for 15 min at the temperature used for extraction, which ensured a homogeneous temperature for both sample and headspace. Once sampling was finished, the fibre was transferred to the injection port of the gas chromatograph-mass spectrometer (GC– MS) system. The SPME fibre was desorbed and maintained in the injection port at 260 °C for 8 min. The samples were injected in splitless mode. Helium was used as a carrier gas with a flow of 1.2 mL/ min (9.54 psi). The temperature program was firstly isothermal for 10 min at 40 °C, then raised to 200 °C at 5 °C/min and next to 250 °C at 20 °C/min, and finally held for 5 min; total run time was 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, collecting data at 6.34 scans/s over the range m/z 40–300. Compounds were identified by comparing their mass spectra with those contained in the NIST14 (National Institute of Standards and Technology, Gaithersburg) library, and/or by comparing their mass spectra and retention time with authentic standards (Supelco, Bellefonte, PA, USA), and/or by calculation of retention index relative to a series of standard alkanes (C5–C14) (for calculating Kovats indexes, Supelco 44585-U, Bellefonte, PA, USA) and matching them with data reported in literature. The results are expressed as area units of total ion chromatogram (AU-TIC) × 106/g of sample.
On the other hand, when the effects of US and HPP treatments were studied, the extraction of the volatile compounds was performed, also, using solid-phase microextraction (SPME) with an autosampler Pal RTC-120 and separated, identified and quantified in a gas chromatograph 7890B GC-System (Agilent Technologies, Santa Clara, CA, USA) equipped with a mass selective detector 5977B MSD (Agilent Technologies). Autosampler parameters, gas chromatograph and mass spectrometer control and data acquisition were done with the software MassHunter GC/MS Acquisition B.07.05.2479 (Agilent Technologies, Santa Clara, CA, USA). An SPME device (Supelco, Bellefonte, PA, USA) containing a fused silica fibre (10mm length) coated with a 50/30 layer of DVB/CAR/PDMS was used. The fibre was conditioned by heating in a Fibre Conditioning Station at 270 °C for 30 min. For extraction, 1 g of each sample was weighed in a 20 mL vial, after being ground using a commercial grinder. The vials were subsequently screw-capped with a laminated Teflon rubber disc. The extractions were carried out at the same condition as the preliminary study, with the exception that after each injection, the SPME fibre was cleaned in the SPME Conditioning Station at 270 °C for 2 min to
43 III. Material and methods ensure that fibre is completely clean before the next extraction. The mass detector transfer line was maintained at 260 °C. The ion source used in the present study was an Extraction Source Xtr EI 350 (Agilent Technologies, Santa Clara, CA, USA).The mass spectra were obtained using the 5977B mass selective detector working in electronic impact at 70 eV, collecting data at 2.9 scans/s over the range m/z 40–550 in scan acquisition mode. The mass source was maintained at 230 °C while the mass quad was set at 150 °C.
All data were analysed with the software MassHunter Quantitative Analysis B.07.01. A new method from acquired scan data with library search was created. The integration was done with Agile2 algorithm, while peak detection was done with deconvolution. Volatile compounds were identified by comparing their mass spectra with those contained in the NIST14 (National Institute of Standards and Technology, Gaithersburg) library, being considered as correctly identified when their spectra presented a library match factor > 85% and were taken into account if they appear in at least the half out of the total samples of each group. The identification, also could be made depending on the case by comparing their mass spectra and retention time with authentic standards (pentane, octane, decane, undecane, dodecane, tridecane, propanal, butanal, pentanal, hexanal, heptanal, octanal, decanal, nonanal and pentadecanal) (Supelco, Bellefonte, PA, USA), and/or by calculation of retention index relative to a series of standard alkanes (C5–C14) (for calculating Kovats indexes, Supelco 44,585-U, Bellefonte, PA, USA) and matching them with data reported in literature. After integration, peak detection and identification of each compound, the extraction ion chromatogram (EIC) from the quantifier ion was obtained from each peak. The final results were expressed as area units of the EIC × 103 per gram of sample (AU-EIC × 103/g of sample).
III.6. STATISTICAL ANALYSIS
III.6.1. PHYSICOCHEMICAL AND ORGANOLEPTIC PRECURSOR PARAMETERS
The effect of proteolysis index group/level, as well as the effect of each following treatments on analytical and biochemical measures, were examined using a one-way ANOVA, where these parameters were set as a factor. The values were given in terms of mean values and standard error of the means (SEM). When a significant effect (P < 0.05) was detected, means were compared using the Tukey's test. Correlations between variables (P < 0.05) were determined by correlation analyses using the Pearson's linear correlation coefficient. All analyses were conducted using the IBM SPSS Statistics 24.0 program (IBM Corporation, 2016) software package.
III.6.2. PROTEOMIC ANALYSIS
Quantitative changes of 2-DE gel spot volumes in sample groups were assessed using the measures “fold change” (FC) and “relative change” (RC) (Franco et al., 2015a, 2015b). The measure FC is given by FC=V1/V2, where V1 and V2 are the mean volumes of each batch. FC values less than one were represented as their negative reciprocal. The relative change is provided by the relationship RC=DV/ |DVmax|, where DV=V1−V2 and DVmax is the maximum
III. Material and methods observed value of DV over spots. It is worth noting that FC is a measure traditionally used to quantify differential protein abundance between treatments. But it has the disadvantage that its range varies from -∞ to +∞ and range boundaries are achieved with the presence of unique spots independently of the existing differences in volume. In contrast, RC always ranges from −1.0 to +1.0. It provides, therefore, a more intuitive measure of the strength of change and maximum values of its range are not necessarily achieved with the mere occurrence of unique spots. Accordingly, RC is a particularly appropriate measure for the analysis of proteome profiles exhibiting a large number of unique spots. The UPGMA (Unweighted Pair Group Method with Arithmetic mean) clustering method was used to produce a dendrogram from distance matrix of pairwise values of RC over proteins with the XLSTAT v.2014.5.01 statistical software.
Statistical quantitative and qualitative differences in 2-DE spot volumes between treatments were assessed by bootstrap resampling methods. The non-parametric bootstrap confidence interval was obtained for the mean volume of each spot by the biascorrected percentile method as previously described (Efron, 1982). The 95% bootstrap confidence interval corrected by Bonferroni's method was constructed from 20,000 bootstrap samples of size N=4. Descriptive statistics and other statistical analysis were performed with IBM SPSS Statistic V21.0 (SPSS, Chicago, IL, USA) software package.
45
IV. RESULTS AND DISCUSSION
IV. Results and Discussion
IV.1. PRELIMINARY STUDY OF SAMPLES
IV.1.1. SAMPLE CHARACTERIZATION
The instrumental adhesiveness, chemical composition and nitrogen fractions of the samples of the different proteolysis levels (low, medium and high) are shown in table 7. In the present study, it was found that there is a negative correlation (r=−0.218, P < 0.01) between the PI and the salt concentration. Proteolytic activity in ham is highly correlated to salt content, the negative relationship has been extensively reported (Flores et al., 2006; Armenteros et al., 2009; dos Santos et al., 2015). Statistical analysis did not show significant differences in salt content among the three PI levels studied, presenting mean values of 11.63 g/ 100 g DM.
Table 7.Chemical composition and nitrogen fractions of dry-cured ham samples sorted by three proteolysis level (Low<32 %; Medium 32 -36 % and High>36%)
Parameters Groups SEM P-value LP MP HP Instrumental adhesiveness (g) 71.43a 77.20a 90.15b 1.580 0.005 % IP 31.10a 34.50b 38.59c 0.249 <0.001 Moisture (%) 58.98 58.83 58.86 0.071 0.065 Salt (% DM) 11.88 11.86 11.16 0.135 0.067 TN (% DM) 11.85 11.76 11.7 0.027 0.062 NPN (% DM) 3.76a 4.02b 4.42c 0.025 <0.001 TBVN (mg/ 100 g DM) 385.79 389.21 394.65 2.612 0.112 a–c Mean values in the same row (corresponding to the same parameter) not followed by a common letter differ significantly (P < 0.05; Tukey's Test). SEM: standard error of the mean.
No works related to the instrumental adhesiveness of dry-cured ham slices were found in the literature. Results from this study showed that significant (P < 0.001) differences between PI levels groups were found. Thereby, the higher the PI the higher the adhesiveness (71.43, 77.20 and 90.15 g, for LP, MP and HP groups, respectively). According to García -Garrido et al. (1999), hams with a defective texture exhibited high moisture/protein ratios as a result of both increased moisture and decreased protein contents related to hams with normal texture.
The PI is often used to describe the intensity of proteolysis during dry-cured ham processing and its optimal value varies from country to country. For instance, in Spain, the PI reflecting a good quality could be considered between 32 and 36, whereas in Italy is between 22 and 30 (Careri et al., 1993). Results in the present study are in agreement with data reported by other authors (García -Garrido et al., 1999; Pugliese et al., 2015; Zhao et al., 2008) who observed values between 17.23 and 35.2 % in dry-cured hams. These differences in PI among hams could be due to differences in raw materials, salting procedure, ripening process, duration of steps, and temperature and relative humidity used in the processing of dry-cured hams. Also, Ruiz-Ramírez et al. (2006) observed that the anatomic location of the muscle, the pH of the fresh piece, and the amount of added NaCl affected the PI at the end of the dry-cured ham process. Another noted relation is that established between the content of the nitrogen
49
IV. Results and Discussion compounds and the proteolysis reactions because proteolytic processes break down the proteins giving rise to smaller peptides and free amino acids (Armenteros et al., 2009). Besides, Petrova et al. (2016) noticed that the PI during dry-cured ham processing is directly related to the enzymatic activity. In this regard, in the present study, the non-protein nitrogen content also showed significant (P < 0.001) differences among ham groups, since the lowest values were observed in the LP batch (3.76 vs. 4.02 vs. 4.42 g/100 g of DM, for LP, MP and HP groups, respectively). This is an expected result since the hams have been classified according to their IP. Low activity values of proteolytic enzymes would result in low protein degradation and a smaller amount of NPN in samples (Petrova et al., 2016). This finding is in agreement with data reported by García -Garrido et al. (1999) who observed that the NPN levels were 30% higher in hams of defective texture than in normal pieces. In addition, Martín et al. (1997) noticed that the high temperatures during the drying stage stimulate the formation of non- protein nitrogen compounds as the enzymatic activity increases. Finally, the TVBN content was not affected by PI, showing this nitrogen fraction mean values of 389.88 mg/100 g of DM. These values were higher than those reported by other authors in dry-cured ham (values ranging from 50 to 240 mg/100 g of dry matter) (Martín et al., 1997; Ventanas et al., 1992), and also higher than data reported by Lorenzo et al. (2008) in dry-cured lacón (85.6–109.7 mg/100 g of DM).
IV.1.2. PROTEOMIC EVALUATION
High-quality 2-DE gels were obtained of low and high proteolysis being excluded from the further analysis the saturated, faint and non-reproducible spots over replicates. Representative 2-DE gel images proteomes are shown in Fig. 13.
Figure 13. Representative 2-DE gel images of low (LP) and high proteolysis (HP) proteomes
The total numbers of selected spots for proteomic analysis were 92 and 123 spots in low and high proteolysis groups, respectively. We found that proteomic profiles of low and high proteolysis samples were remarkably differentiated (Table 8).
50 IV. Results and Discussion
Table 8. Spot volumes with statistically significant (p-value < 0.05) differential abundance in dry-cured hams of low and high proteolysis level Spot Low proteolysis High proteolysis No. Mean (±SE) p(θB ⩽ θ) 95% bootstrap Mean (±SE) p(θB ⩽ θ) 95% bootstrap Volume CI (CL, CU) Volume CI (CL, CU) 1 684±31 0.57 617,746 280±75 0.53 79,409 2 741±150 0.53 353,962 1531±128 0.52 1259,1742 3 392±81 0.55 247,554 − − − 4 − − − 1360±215 0.54 815,1712 5 − − − 307±18 0.75 281,333 6 − − − 271±25 0.73 236,306 7 − − − 366±113 0.58 121,566 8 − − − 2010±419 0.6 1241,2904 9 − − − 2186±473 0.56 1320,3073 10 − − − 2360±500 0.53 1348,3212 11 − − − 1174±342 0.56 647,2156 12 − − − 688±95 0.49 520,881 13 − − − 667±219 0.54 53,1014 14 − − − 1302±257 0.58 976,1830 15 − − − 661±58 0.55 509,764 16 − − − 508±43 0.56 422,589 17 − − − 655±185 0.64 377,1074 18 − − − 619±194 0.6 229,1003 19 − − − 582±193 0.56 237,974 20 − − − 163±13 0.75 145,182 21 − − − 468±116 0.53 259,695 22 − − − 798±176 0.49 437,999 23 234±16 0.75 211,257 − − − 24 725±183 0.49 341,993 1801±212 0.68 1419,2259 25 − − − 1459±56 0.76 1379,1537 26 − − − 1980±327 0.75 1518,2443 27 − − − 477±112 0.51 248,602 28 − − − 3396±855 0.62 2016,5152 29 283±122 0.52 67,510 − − − 30 235±65 0.67 84,310 489±65 0.67 409,639 31 − − − 324±95 0.51 99,541 32 − − − 507±160 0.61 185,826 33 − − − 477±112 0.51 248602 34 1079±177 0.75 829,1329 443±178 0.62 318,652 35 524±99 0.77 394,674 − − − 36 − − − 387±16 0.61 359,422 37 255±6 0.76 246,263 333±40 0.64 284,426 38 − − − 142±66 0.67 37,289 39 252±29 0.54 172,302 455±98 0.58 338,658 40 − − − 266±47 0.53 158,358 41 1756±408 0.56 957,2485 3274±249 0.56 2990,3783
51
IV. Results and Discussion
Spot Low proteolysis High proteolysis No. Mean (±SE) p(θB ⩽ θ) 95% bootstrap Mean (±SE) p(θB ⩽ θ) 95% bootstrap Volume CI (CL, CU) Volume CI (CL, CU) 42 965±267 0.55 649,1511 2041±254 0.56 1577,2555 43 − − − 544±82 0.52 372,667 44 − − − 1103±113 0.74 943,1264 45 1145±197 0.56 814,1556 − − − 46 465±43 0.76 405,525 − − − 47 475±86 0.73 354,597 1469±302 0.56 722,1963 48 − − − 608±31 0.63 567,679 49 779±34 0.62 706,843 1517±312 0.58 1112,2441 50 − − − 1370±46 0.59 1277,1462 51 − − − 622±33 0.71 0.569,0.697 52 1089±344 0.66 543,1862 − − − 53 − − − 2544±665 0.62 1485,4037 54 1622±462 0.55 654,2496 − − − 55 − − − 313±116 0.58 46,537 56 − − − 661±292 0.61 28,1180 57 683±67 0.74 589,777 352±62 0.75 264,440 58 643±90 0.63 634,849 399±121 0.56 156,623 Gel position of spots is shown in Fig. 13. CL and CU are the lower and upper bounds, respectively. The bootstrap distribution was median biased if p (θB ⩽ θ) ≠ 0.50, where θB and θ are the bootstrap and sample mean estimates, respectively.
In total, 58 protein spots showed statistically significant differential abundance by the bootstrap re-sampling statistical method. It should be noted that Bonferroni-corrected 95% bootstrap confidence intervals (CIs) for means of spot volumes did not overlap in matched spots of different intensity or did not overlap zero in unique spots. It is important to highlight that only eight unique spots were observed in LP samples, whereas in HP samples there were 37 spots (P < 0.001, Fisher’s exact test). This difference probably reflects an increased protein fragmentation in samples with high proteolysis. In this regard, after the evaluation of protein fragmentation, it was found that most differentially abundant protein spots in LP and HP ham samples (40 out of 58 spots) were successfully identified (P < 0.05) by MALDI-TOF/TOF MS. The comparison of theoretical and observed molecular masses revealed that an important number (55%) of identified spots contained protein fragments. It is noteworthy, however, that most (86%) of these spots were unique spots present only in HP samples. Accordingly, the proteomic profile in HP samples showed increased levels of protein fragmentation. It also shows that PI scores can be good indicators of differential proteolysis over proteomes.
52 IV. Results and Discussion
Table 9. Protein identification by MALDI-TOF/TOF MS of differentially (P-value < 0.05) represented 2-DE spots in dry-cured hams with low and high proteolysis index Spot Protein Abbrev. Accession Mascot Sequence Number of pI Mr th/obs No. number score coverage matched th/obs (kDa) (Uniprot) (%) peptides 1 Vinculin VINC P26234 60 19 17 5.6/6.2 124.4/76.1 Fragment 2 Serum albumin ALBU P08835 144 21 13 6.1/6.1 71.6/72.9 3 Serum albumin ALBU P08835 125 21 14 6.1/6.3 71.6/73.2 4 Serum albumin ALBU P08835 601 42 19 6.1/6.5 71.6/70.7 5 Serum albumin ALBU P08835 56 10 7 6.1/6.1 71.6/66.3 9 Myosin-1 MYH1 Q9TV61 503 17 36 5.6/5.6 224.4/59.6 Fragment 10 Myosin-1 MYH1 Q9TV61 373 15 31 5.6/5.6 224.4/62.6 Fragment 11 Myosin-1 MYH1 Q9TV61 493 16 35 5.6/5.7 224.4/62.8 Fragment 12 Myosin-1 MYH1 Q9TV61 582 16 30 5.6/4.7 224.4/53.3 Fragment 13 Myosin-1 MYH1 Q9TV61 331 8 15 5.6/4.8 224.4/52.9 Fragment 14 Myosin-1 MYH1 Q9TV61 467 15 28 5.6/4.9 224.4/52.8 Fragment 15 Myosin-1 F1SS62 Q9TV61 287 24 34 5.5/5.1 171.0/61.2 Fragment 16 Myosin-4 MYH4 Q9TV62 249 11 19 5.6/5.1 224.0/60.8 Fragment 17 Myosin-1 MYH1 Q9TV61 249 15 25 5.6/5.2 224.4/59.4 Fragment 20 α-1,4-Glucan phosphorylase F1RQQ8 F1RQQ8 102 13 10 6.7/6.5 97.7/55.4 Fragment 21 α-Actin, skeletal muscle ACTS P68137 180 28 9 5.2/5.9 42.4/45.5 22 Heat shock 70kDaprotein1-like HS71L A5A8V7 66 6 4 5.6/6.7 70.7/45.1 Fragment 23 Myosin-7 MYH7 P79293 380 13 21 5.6/4.4 223.0/44.2 Fragment 24 α-Actin, skeletal muscle ACTS P68137 96 14 4 5.2/4.9 42.4/40.1 25 Myosin-4 MYH4 Q9TV62 241 12 21 5.6/4.9 224.0/43.4 Fragment 26 Myosin-4 MYH4 Q9TV62 701 15 30 5.6/5.1 224.0/43.8 Fragment 28 α-Actin, skeletal muscle ACTS P68137 255 34 10 5.2/5.6 42.4/40.1 29 α-Actin, skeletal muscle ACTS P68137 69 19 5 5.2/4.7 42.4/39.1 30 Desmin DESM P02540 87 10 4 5.2/4.4 53.6/38.0
53
IV. Results and Discussion
Spot Protein Abbrev. Accession Mascot Sequence Number of pI Mr th/obs No. number score coverage matched th/obs (kDa) (Uniprot) (%) peptides 31 α-Actin, skeletal muscle ACTS P68137 94 13 4 5.2/4.8 42.4/42.6 32 Myosin-4 MYH4 Q9TV62 424 11 22 5.6/4.9 224.0/39.3 Fragment 34 F-Actin-capping protein subunit alpha-2 CAZA2 Q29221 269 47 11 5.6/5.8 33.1/39.1 36 F-Actin-capping protein subunit alpha-2 CAZA2 Q29221 67 9 2 5.6/6.1 33.1/35.7 40 β-Enolase ENOB Q1KYT0 92 23 7 8.1/6.5 47.4/35.1 44 F-Actin-capping protein subunit beta CAPZB A0PFK7 395 46 13 5.5/4.9 31.6/31.0 45 α-Actin, skeletal muscle ACTS P68137 149 17 5 5.2/5.3 42.4/32.6 46 α-Actin, skeletal muscle ACTS P68137 159 30 8 5.2/4.5 42.4/25.5 Fragment 47 α-Actin, skeletal muscle ACTS P68137 117 12 4 5.2/5.3 42.4/25.4 Fragment 48 Myosin-1 MYH1 Q9TV61 415 15 32 5.6/5.5 224.4/62.5 Fragment 49 α-Actin, skeletal muscle ACTS P68137 180 17 5 5.2/5.6 42.4/25.4 Fragment 50 Peroxiredoxin-6 PRDX6 Q9TSX9 665 58 15 5.7/5.7 25.0/25.5 51 α-Actin, skeletal muscle ACTS P68137 126 14 4 5.2/5.3 42.4/24.0 Fragment 53 α-Actin, skeletal muscle ACTS P68137 180 14 4 5.2/5.5 42.4/24.2 Fragment 55 Multiprotein bridging factor 1 A6N8P5 A6N8P5 70 49 10 10.0/6.1 16.4/24.0 56 Triosephosphate isomerase TPIS Q29371 85 33 8 7.0/6.6 26.9/24.0 The Mascot baseline statistically significant (P < 0.05) score was 56. Sequence coverage (%): percentage of coverage of the entire amino acid sequence by matched peptides. Number of matched peptides: total number of identified spectra matched for the protein. Theoretical (Th) isoelectric point (pI) and molecular mass (Mr) were obtained from UniProtKB/Swiss-Prot databases. Observed (Ob) pI and Mr were obtained from the spot position on the gel. Protein fragments: Mr (Th)/Mr (Obs) ratio higher than 1.5.
54 IV. Results and Discussion
The remaining spots, with theoretical and empirical mass ratios below 1.5, were excluded from further analysis. It is not possible to assess whether they contain either entire or slightly degraded proteins at the level of resolution of 2-DE. The use of an internal standard in multiplexing methods such as two-dimensional difference gel electrophoresis (2-D DIGE) could reduce inter-gel variation, increasing statistical power (Chevalier, 2010). However, 2-DE could identify the strongest protein changes between sample groups, and therefore the most useful biomarkers for proteolysis and adhesiveness. All fragments detected in our study corresponded to seven non-redundant myofibrillar or sarcoplasmic muscle proteins: myosin-1 (MYH1), myosin-4 (MYH4), α-4 glucan phosphorylase (F1RQQ8), α- actin (ACTS or ACTA1), heat shock 70 kDa protein 1-like (HS71L), myosin-7 (MYH7) and vinculin (VINC). However, most fragments (86%) resulted from hydrolysis of myosin heavy chain and α-actin myofibrillar proteins: nine MYH1 spots, four MYH4 spots, one MYH7 spot and five ACTS spots (Table 9). It is noteworthy, however, that the amount of protein fragments does not provide determinant information by itself to reliably evaluate the extent of differential proteolysis over proteins and sample groups. A complete characterization of differential proteolysis not only requires determining the number of protein fragments, but also the quantification of their volumes.
On the other hand, quantitative differences in proteolysis intensity between low and high proteolysis ham batches were assessed by FC and RC statistics from protein fragment volumes. Table 10 shows FC and RC values for each protein found to be differentially affected by proteolysis. It can be seen that RC-values of proteins ranged between −0.04 and +1.0. Only five proteins (i.e. MYH1, ACTS, MYH4, HS71L and F1RQQ8) showed positive RC-values, indicating that their fragments were over-represented in high-proteolysis hams. In contrast, MYH7 and VINC proteins underwent decreased proteolysis in high-proteolysis samples given that their RC values were of negative sign. This result suggests that MYH7 and VINC proteins are not useful biomarkers of proteolysis intensity. MYH1, ACTS and MYH4 proteins showed the highest level of degradation in high proteolysis samples (RC-values > 0.40).
Table 10. Fold change (FC) and relative change (RC) of differentially (P < 0.05) represented protein fragments in dry-cured ham with different proteolysis indices Spot No. Protein (abbrev.) fragment FC RC 9–15, 17, 48 Myosin-1 (MYH1) +∞ +1.00 46,47,49,51,53 α-Actin, skeletal muscle (ACTS) +13.23 +0.60 16,25,26,32 Myosin-4 (MYH4) +∞ +0.43 22 Heat shock 70kDaprotein1-like (HS71L) +∞ +0.08 20 α-1,4-Glucan phosphorylase (F1RQQ8) +∞ +0.02 23 Myosin-7 (MYH7) −∞ −0.02 1 Vinculin (VINC) −2.44 −0.04 FC and RC values of MYH1, ACTS and MYH4 were computed from all fragments of the same protein on 2-DE gels.
Previous proteomic studies based on one-dimensional electrophoresis and 2-DE have systematically demonstrated that myosin heavy chain and α-actin are the main targets of proteolysis in the BF muscle, particularly at the end of ripening (Larrea et al., 2006; Tabilo et al., 1999; Théron et al., 2011; Toldrá et al., 1993). In 12-month-old Parma and S. Daniele dry-
55
IV. Results and Discussion cured ham, most isoforms of myosin and actin were found to be completely hydrolysed (Di Luccia et al., 2005). This study found that MYH1 (RC=+1) was a more sensitive biomarker for proteolysis than ACTS (RC=+0.60). This difference can be attributed to the fact that myosin is more sensitive to denaturation by salt content (Graiver et al., 2006). However, it is found that two specific isoforms of myosin heavy chain (MYH1 and MYH4) were intensively degraded in response to proteolysis. It suggests that these two myosin heavy chain isoforms might exhibit differential susceptibility to degradation by proteolytic enzymes during dry-cured ham processing. In this regard, Théron et al. (2011) reported differential MYH1 or MYH4 fragmentation in BF and SM muscles with different proteolytic activity, due to differences in salt and moisture content in the course of dry-cured ham processing. Specifically, fragments of these two myosin heavy chains isoforms were overrepresented in BF muscle, an internal muscle with lower NaCl concentration, higher water content and increased proteolytic activity. Taken together, the available evidence suggests that MYH1 and MYH4 can be suitable biomarkers for proteolysis under different scenarios.
Of the five fragmented proteins over-represented in HP samples, two were sarcoplasmic proteins: HS71L and F1RQQ8. They are proteins with a considerably lower relative representation in the proteome of BF muscle, which explains their low RC values (< 0.10). The HS71L protein is a molecular chaperone that appears to play a critical role in multiple cellular functions, including activation of proteolysis of misfolded proteins, controlling the targeting of proteins for subsequent degradation, and protection of the proteome in response to stress (Archibald et al., 2010; Radons, 2016; The UniProt Consortium, 2017). On the other hand, the F1RQQ8 protein is a phosphorylase that catalyses and regulates the breakdown of glycogen to glucose-1-phosphate for the generation of ATP during glycogenolysis (Archibald et al., 2010; Gautron et al., 1987; The UniProt Consortium, 2017). Fragments of F1RQQ8 resulting from proteolytic activity were also detected in post-mortem longissimus dorsi (LD) porcine muscle (Lametsch, Roepstorff and Bendixen, 2002), as well as in dry-cured BF and SM muscles (Théron et al., 2011). Specifically, the BF muscle showed more F1RQQ8 fragments than the SM muscle during the ripening of dry-cured ham, due to its higher proteolytic activity (Théron et al., 2011). It seems that FIRQQ8 is a good biomarker of proteolysis in agreement with our observations. In the present study, the instrumental adhesiveness seems to be dependent on the proteolytic activity in dry-cured ham. Therefore, the identified biomarkers also apply for the meat quality trait of adhesiveness. These biomarkers provide non-invasive tools, alternative to sensory analysis or mechanical measures, to assess variations in adhesiveness. The identified proteins can also be potential biomarkers for other proteolysis-related ham quality traits. It is particularly true in the case of pastiness, considering that pastiness variations are closely related with the extent of proteolysis and adhesiveness (Morales et al., 2008; Škrlep et al., 2011).
56 IV. Results and Discussion
IV.1.3. INSTRUMENTAL EVALUATION OF THE ORGANOLEPTIC PRECURSORS
IV.1.3.1. Free amino acids
The effect of PI on FAA content (expressed as mg/100 g DM) of dry-cured ham is shown in Table 11.
Table 11. Effect of proteolysis index on FAA content (expressed as mg/100 g DM) of dry-cured ham Amino Acid/ Groups SEM P-value Sensory attributes LP MP HP Aspartic acid 184 182 183 2.53 0.961 Serine 177a 194b 203b 2.92 <0.001 Glutamic acid 451 447 462 5.70 0.538 Glycine 199 194 196 2.54 0.788 Histidine 98.1 102 101 1.44 0.497 Taurine 97.5b 91.2a 86.0a 1.27 <0.001 Arginine 398 386 379 5.62 0.398 Threonine 210 220 224 2.83 0.117 Alanine 415 407 416 5.11 0.706 Proline 276 280 291 3.43 0.163 Cysteine 443b 287a 269a 9.93 <0.001 Tyrosine 189 194 198 2.78 0.485 Valine 384 389 400 4.42 0.291 Methionine 195a 207ab 217b 2.61 0.002 Lysine 267 251 248 3.70 0.094 Isoleucine 338a 350a 371b 4.20 0.004 Leucine 567a 586a 624b 6.89 0.002 Phenylalanine 375 392 401 4.62 0.061 Total Amino Acids 5399 5333 5406 62.8 0.878 Sweet 1 1236 1268 1299.09 12.35 0.102 Bitter 2 1860a 1924ab 2004b 21.42 0.019 Acid 3 719 729 737 7.78 0.613 Aged 4 632 621 623 5.85 0.712 a-b Mean values in the same row (corresponding to the same parameter) not followed by a common letter differ significantly (P < 0.05; Tukey's Test). SEM: standard error of the mean. 1 Sweet flavour=Σ of alanine, glycine, threonine, serine and proline; 2 Bitter flavour=Σ of leucine, valine, isoleucine, methionine and phenylalanine; 3Acid flavour=Σ of glutamic acid, aspartic acid and histidine; 4Aged flavour=Σ of lysine, tyrosine and aspartic acid.
No significant differences in the total amount of FAAs among the three different groups (mean values of 5370 mg/100 g of DM) were observed. The total FAA content observed in the present study was higher than those in previous studies on dry-cured ham (about 4000 mg/100 g DM; Córdoba et al., 1994; Martín et al., 2001; Ruiz et al., 1999). However, other studies showed higher total concentrations in dry-cured ham (about 12,500 g/100 g DM, Jurado et al., 2007; Zhao et al., 2005). In general, the FAA profile observed in the present study coincides with those reported in different types of dry-cured ham (Bermúdez et al., 2014; Jurado et al., 2007;
57
IV. Results and Discussion
Martín et al., 2001; Virgili et al., 2007; Zhao et al., 2005). The individual FAAs showed higher values in HP hams, except for taurine, arginine, cysteine and lysine that presented higher concentrations in dry-cured ham with low PI than in MP and HP groups. On the other hand, six of the 18 FAAs quantified in this study showed significant differences among PI levels (P < 0.05): serine, taurine, cysteine, methionine, isoleucine and leucine. Leucine was the major amino acid in all groups, showing a significant increase (P < 0.01) when PI increased (566.83, 586.15 and 623.75 mg/100 g of DM for LP, MP and HP batches, respectively). A similar trend was observed for serine, methionine and isoleucine, showing the highest levels in dry-cured hams with high PI. However, taurine and cysteine content presented an opposite behaviour, reaching the highest values in dry-cured hams with low PI. According to Bermúdez et al. (2014), the FAA content variations depend on the ratio between FAA formation and degradation. These differences in the individual FAA content among the three ham groups studied could induce differences in flavour. According to the FAA profile and the differences observed in the present study, our results seem to indicate that only bitter taste could be significantly (P < 0.05) affected by PI, showing the bitter AAs the highest values in dry-cured hams with high PI. This finding is in agreement with data reported by other authors (Careri et al., 1993; Parolari, Virgili and Schivazappa, 1994) who noticed that an excess of proteolysis is undesirable because it may confer a bitter or metallic aftertaste in dry-cured hams.
IV.1.3.2. Volatile compound profile
An increase in the relative abundance of total volatiles in the headspace of ham might suppose a more intense odour or flavour, or not, or it might have a negative or positive effect; this will depend on the type of volatile compounds involved. Thirty-nine volatile compounds were identified and quantified and they were classified into the following chemical families: hydrocarbons (14), alcohols (5), aldehydes (4), esters (2) ketones (4) acid (1), sulphur compounds (1) and other compounds (2) according to Lorenzo and Carballo (2015). Statistical analysis showed significant differences (P < 0.001) in the total volatile content between groups, with the highest concentration observed in the batch with low PI, and decreasing as the PI increased (1575.24 vs. 1337.81 vs. 997.49 AU×106/g of DM for LP, MP and HP batches, respectively).
As shown in Table 12, the main family of volatile compounds were the hydrocarbons, which, as discussed previously, are compounds derived from the oxidative decomposition of lipids, maybe catalysed by hemocompounds such as hemoglobin and myoglobin (Ramírez and Cava, 2007). Furthermore, Martín et al. (2006) suggested that methyl hydrocarbons could be synthesized by moulds as a product of secondary degradation of TG. It was observed a higher content of hydrocarbons in the batch with lower PI compared to the other ones (759 vs. 605 vs. 416 AU×106/g of DM for LP, MP and HP groups, respectively). These outcomes could be due to the greater lipid oxidation in the low PI ham group compared to the other two groups. However, at the sensory level, these differences do not have a great impact on the quality of the final product since the hydrocarbons are compounds that have little contribution to aroma because of their high odour threshold values (Wu et al., 2015). Among hydrocarbons, undecane was the most abundant in the three ham categories studied and this compound could be used to discriminate dry-cured hams according to their PI.
58 IV. Results and Discussion
Table 12. Volatile compounds present in dry-cured ham samples with different proteolysis levels (expressed as Area Units of the TIC (AU-TIC)×106/g DM) Volatile compound LRI R Groups SEM P-value LP MP HP Octane 754 ms, lri,s 57.49c 36.59b 26.07a 1.76 <0.001 Decane 1033 ms, lri,s 61.95c 47.44b 30.13a 1.75 <0.001 Undecane 1144 ms, lri,s 143.42c 123.26b 71.39a 4.01 <0.001 6-Tridecene 1223 ms 10.66b 10.11b 6.05a 0.44 <0.001 Dodecane 1243 ms.lri,s 84.90b 80.25b 46.84a 2.37 <0.001 Tridecane 1338 ms.lri,s 27.44b 26.79b 17.07a 0.73 <0.001 Total lineal hydrocarbons 377.69c 323.96b 194.36a 9.31 <0.001 Pentane, 2,3,4-trimethyl- 666 ms 13.92ab 15.54b 11.50a 0.53 0.006 Pentane, 2,3,3-trimethyl- 675 ms 33.08c 18.68a 24.44b 1.24 <0.001 Heptane, 3-methylene- 743 ms 30.73b 22.33a 19.37a 0.91 <0.001 Heptane, 3-ethyl- 866 ms.lri 24.88c 15.49b 9.48a 0.76 <0.001 2,3-Dimethyl-3-heptene, (Z)- 898 ms 8.69b 6.49a 5.83a 0.25 <0.001 Octane, 3-ethyl- 996 ms 23.39c 19.23b 16.15a 0.61 <0.001 Nonane, 3-methyl- 999 ms 16.97c 12.85b 8.94a 0.42 <0.001 Cyclohexane, 1,2-diethyl-1- 1041 ms 13.85c 11.27b 5.69a 0.45 <0.001 methyl- Cyclopentane, pentyl- 1082 ms 66.26c 50.50b 33.98a 2.15 <0.001 5-Undecene, 9-methyl-, (Z)- 1169 ms 78.70c 64.56b 34.22a 2.05 <0.001 Undecane, 3-methyl- 1215 ms 31.98c 27.08b 19.68a 0.84 <0.001 Undecane, 3-methylene- 1233 ms 12.58b 13.04b 8.69a 0.41 <0.001 5-Undecene, 3-methyl-, (E)- 1235 ms 12.46c 9.92b 5.77a 0.53 <0.001 10-Methylnonadecane 1293 ms 2.92b 2.68b 1.92a 0.12 <0.001 Total branched hydrocarbons 347.95c 283.77b 213.44a 9.29 <0.001 Total hydrocarbons 759.93c 605.28b 416.99a 21.71 <0.001 2-Pentanone 620 ms.lri 10.82b 7.94a 10.66b 0.31 <0.001 2-Butanone, 3-hydroxy- 711 ms.lri 25.60b 21.52a 19.56a 0.53 <0.001 3-Heptanone 940 ms 4.24c 2.97b 2.20a 0.15 <0.001 2-Heptanone 950 ms.lri 11.08 11.13 9.93 0.26 0.089 Total ketones 48.85b 43.22a 42.06a 0.65 <0.001 Ethyl alcohol 307 ms 256.05b 255.00b 223.95a 5.26 0.018 1-Butanol, 3-methyl- 737 ms 23.73c 17.61b 12.99a 0.92 <0.001 1-Hexanol 932 ms.lri 20.43b 17.67b 11.70a 0.81 <0.001 1-Octen-3-ol 1062 ms.lri 60.28c 47.67b 30.22a 2.21 <0.001 Benzyl Alcohol 1157 ms.lri 24.78c 21.97b 17.53a 0.44 <0.001 Total Alcohols 364.49b 357.68b 299.65a 6.09 <0.001 Butanal, 3-methyl- 537 ms.lri 82.17b 82.72b 68.65a 1.99 0.005 Hexanal 814 ms.lri,s 104.42c 79.42b 43.87a 3.59 <0.001 Heptanal 959 ms.lri,s 21.64c 17.02b 11.60a 0.55 <0.001 Benzeneacetaldehyde 1154 ms 22.85c 17.34b 14.55a 0.57 <0.001 Total Aldehydes 232.10c 195.75b 140.52a 5.97 <0.001 Acetic acid, ethyl ester 437 ms 35.57 31.22 34.97 0.91 0.128 Decanoic acid, ethyl ester 1442 ms 4.92c 4.13b 3.23a 0.12 <0.001 Total Esters 40.92 35.45 38.11 0.94 0.072 Acetic acid 571 ms 55.16b 40.46a 35.72a 1.52 <0.001 Total Acids 55.16b 40.46a 35.72a 1.52 <0.001 Disulfide, dimethyl 702 ms.lri 4.86a 6.06b 4.30a 0.18 <0.001 Total sulfur compounds 4.86a 6.06b 4.30a 0.18 <0.001 Pyrazine, 2,6-dimethyl- 964 ms.lri 15.44b 13.74a 14.21ab 0.26 <0.001
59
IV. Results and Discussion
Volatile compound LRI R Groups SEM P-value LP MP HP Ethanol, 2-butoxy- 974 ms 41.42c 31.17b 25.95a 1.19 0.029 Other Compounds 56.86b 44.91a 40.16a 1.33 <0.001 Total Compounds 1575.24c 1337.81b 997.49a 37.22 <0.001 a–cMean values in the same row (corresponding to the same parameter) not followed by a common letter differ significantly (P < 0.05; Tukey's Test). SEM: standard error of mean; LRI: Lineal Retention Index calculated for DB-624 capillary column (J and W scientific: 30m×0.25mm id, 1.4 μm film thickness) installed on a gas chromatograph equipped with a mass selective detector; R: Reliability of identification; lri: linear retention index in agreement with literature (Domínguez et al., 2014; Flores et al., 2005; Pateiro et al., 2015); ms: mass spectrum agreed with mass database (NIST14); s: mass spectrum and retention time identical with an authentic standard.
As can be shown in Table 12, significant differences were observed in the total content of alcohols (P < 0.001) among groups, as well as in all of individual alcohols. In all cases, the highest values corresponded to the hams with lower PI. Alcohols follow the same mechanism of generation as acids; straight-chain aliphatic alcohols can be generated by the oxidation of lipids, whereas branched alcohols are most likely derived from the Strecker degradation of amino acids through the reduction of their respective aldehydes (Pérez-Palacios et al., 2010). Alcohols, because of their low odour threshold, contribute to the aroma of ham, with fatty, woody and herbaceous notes (García and Timón, 2001). Among the alcohols, in the three ham groups, ethyl alcohol was the most abundant and represented about 72% of the total alcohols. On the other hand, high 1-octen-3-ol content was also found in the three groups (60.28 vs. 47.67 vs. 30.22 AU×106/g of DM for LP, MP and HP groups, respectively). This alcohol has low odour threshold and is associated with mushroom-like, earth, dust, fatty, sharp and rancid odours (García-González et al., 2008; Théron et al., 2010). In addition, it was found a positive correlation between 1-octen-3-ol and cysteine (r=0.766; P < 0.01).
Aldehydes are known as the major contributors to the unique flavour of dry-cured ham due to their rapid formation during lipid oxidation and their low odour thresholds (Ramírez and Cava, 2007). Statistical analysis showed that the total aldehyde content was significantly affected (P < 0.001) by PI, reaching the highest values in dry-cured hams with low PI (232.10 vs. 195.75 vs. 140.52 AU×106/g of DM for LP, MP and HP groups, respectively). Within aldehydes, hexanal was the most abundant compound, showing significant differences (P < 0.001) among batches (104.42 vs. 79.42 vs. 43.87 AU×106/g of DM for LP, MP and HP groups, respectively). Hexanal at low concentrations has a pleasant and grassy aroma (Aparicio and Morales, 1998), which turns fatty at medium concentration and extremely rancid and tallowy at high concentrations (Morales et al., 1997). At the concentrations observed in the analysed hams, hexanal contributes to grassy odour in hams with high PI, and, perhaps, to a fatty perception in the case of hams with low PI. It was found a positive correlation between cysteine and hexanal (r=0.599; P < 0.01), heptanal (r=0.516; P < 0.01) and benzene acetaldehyde (r=0.561; P < 0.01).
The low odour thresholds of ketones indicate that they have a great impact on ham aroma. In dry-cured ham, their origin can be diverse. Ramírez and Cava (2007) found that the majority of ketones originated from lipid oxidation, whereas a few others, such as 3-hydroxybutan-2- one, are formed through Maillard reactions and the methyl ketones are generated by microorganism esterification. Hams with low PI presented higher total ketones content than
60 IV. Results and Discussion those in the two other groups (48.85 vs. 43.22 vs. 42.06 AU×106/g of DM for LP, MP and HP groups, respectively). Although the concentration of 2-heptanone did not show significant differences among groups, this compound contributes to ham aroma with spicy/blue cheese/acorn sensory notes due to low odour thresholds.
Esters did not show significant differences among the three groups studied (40.92 vs. 35.45 vs. 38.11 AU×106/g of DM for LP, MP and HP groups, respectively). Esters are formed through the enzymatic esterification of fatty acids and alcohols during curing, mostly by the action of microorganisms such as lactic acid bacteria and Micrococcaceae (Purriños et al., 2011). Esters have low olfaction threshold values; however, taking into account that the analysed samples presented very low values of these compounds, it can be considered that they do not contribute to the aroma of dry-cured ham.
Only a sulphur compound, dimethyl disulphide, was found at very low concentrations in the three batches studied, and its presence could come from the degradation of sulphur amino acids through microbial deamination (Belitz and Grosch, 1999). However, a significant correlation between dimethyl disulphide and cysteine, taurine or methionine was not found.
Finally, acetic acid was the only acid identified in the headspace of the dry-cured ham samples, showing the highest content in hams with low PI (55.16 vs. 40.46 vs. 35.72 AU×106/g of DM for LP, MP and HP groups, respectively). This outcome is in agreement with data reported by Pérez-Juan et al. (2006) who observed that acetic acid was the most abundant acid detected in dry-cured ham. The main straight-chain carboxylic acids are derived from the hydrolysis of TG and phospholipids and mainly from the oxidation of unsaturated fatty acids (Pugliese et al., 2015). The origin of acetic acid in ham is not clear. According to some authors, this is originated from carbohydrate fermentation by microorganisms (Kandler, 1983) and from the Maillard reaction according to others (Martín et al., 2006).
In short, most of the volatile compounds detected in the present study come from the oxidation of lipids. Usually, the processing conditions that favour the lipid oxidation (e.g. increased salt content) inhibit the action of proteolytic enzymes. This is probably the reason by which hams having the low PI showed the highest amounts of most of the volatile compounds determined. Apart from the volatiles formed directly from the lipid oxidation, oxidized lipids formed during ripening could react with the FAAs converting them into Strecker aldehydes, α- keto acids and amines. The lipid oxidation products (free radicals and reactive carbonyls) can also influence the subsequent reactions suffered by these compounds: the formation of Strecker aldehydes and other aldehydes from α-keto acids, the formation of Strecker aldehydes and olefins from amines, the formation of shorter aldehydes from Strecker aldehydes, and the addition reactions suffered by the olefins produced from the amines (Hidalgo and Zamora, 2016). This could be the most probable origin of the butanal, 3-methyl (from leucine) and the benzene acetaldehyde (from phenylalanine) detected in the present study; the 1-butanol, 3-methyl also probably comes from the reduction of the butanal, 3- methyl having this same origin. The formation of these Strecker aldehydes from Maillard reactions is unlikely in hams, given the very low amounts of reducing sugars present in such food matrix. On the other hand, these compounds could also be formed by reactions between
61
IV. Results and Discussion protein carbonyls and amino acids (Estévez, Ventanas and Heinonen, 2011), but this origin in the present study is also unlikely, given that hams with a more intense proteolysis were those that presented the lowest values of these two compounds. Due to the non-polar character of most of the volatiles determined, the special structure of the hams with abundant fat infiltrated in muscle tissue could favour the retention of these compounds. Fat solubilizes and traps these compounds avoiding its loss in the prevailing environmental conditions during maturation.
62 IV. Results and Discussion
IV.2. EFFECTS OF THE APPLICATION OF ULTRASOUND AS CORRECTIVE MEASURE TO DECREASE THE ADHESIVENESS
IV.2.1. BIOCHEMICAL EFFECTS
Statistical analysis showed that both, US and CV treatments, significantly (P <0.001) decreased the instrumental adhesiveness of dry-cured hams from 85.27 g for CO to 40.59 g and 38.68 g for US and CV groups, respectively (Fig. 14). However, there were no significant differences between the US and CV treatments.
Figure 14. Effect of temperature treatment alone (CV) or US assisted (US) on instrumental adhesiveness of dry- cured ham. Mean values and standard deviations. a-b Different letters indicate significant differenecs (P<0.001)
The decrease of instrumental adhesiveness in dry-cured ham slices maybe due to the fact that the intramolecular hydrogen connections can break due to the mechanical vibration and also to the effects of thermal and ultrasonic cavitation causing loosening of the molecular structure and reduction of molecular nodes (Yang and Lian-sheng, 2003). Denaturation and structural changes of proteins due to thermal treatment could also decrease the instrumental adhesiveness of dry-cured ham slices (Tornberg, 2005). Finally, some changes such as the aggregation of the globular heads of myosin (Morales et al., 2008), cell membrane destruction (Rowe, 1989) and the transversal and longitudinal shrinkage of meat fibres (Tornberg, 2005) could take place during the thermal treatment. The findings in the present work are in agreement with data reported by Morales et al. (2008) who showed that the thermal treatment at 30 °C for 168 h on both sliced and whole dry-cured ham decreased softness, adhesiveness and pastiness in BF muscle, without increasing hardness in SM muscle or affecting their physicochemical parameters (moisture, aw and PI). Besides, Gou et al. (2008) observed a decrease of soft textures in whole dry-cured ham pieces without affecting the sensory properties after a treatment of 10 days ageing process at 30 °C.
Regarding US application, our outcomes are in agreement with data reported by Contreras et al. (2018) who did not find any significant difference in hardness and elasticity of dry-cured ham slices between ultrasonically assisted heated and conventionally heated samples. However, our results are in disagreement with those reported by Hu et al. (2014) who did not show a significant difference between control and US starch corn samples, but they found a lower hardness, elasticity and brittleness in the US treated samples. Taking into account that texture is one the most important sensory attributes of dry-cured ham, which affect its
63
IV. Results and Discussion acceptability by consumer, the application of both treatments, US and CV, could be used to reduce the instrumental adhesiveness of dry-cured ham slices by immersing the packaged samples in a water bath during a short period of time.
On the other hand, statistical analysis did not show significant differences (P > 0.05) in moisture content among groups, presenting mean values of 59.01, 58.68 and 58.57 g/100 g for CO, US and CV groups, respectively (Fig. 15). Our moisture values were in the range of data (48.3-65.2 g/100 g) reported by other authors (Bermúdez et al., 2014; Prevolnik et al., 2011; Pugliese et al., 2015) for dry-cured ham.
Figure 15. Effect of temperature treatment alone (CV) or US assisted (US-50) on moisture content of dry-cured ham. Mean values and standard deviations
IV.2.2. MICROSTRUCTURAL MODIFICATIONS
Representative 2-DE gel images of the proteome of dry-cured ham after CV and US thermal treatments are shown in Fig. 16. A total of 79 and 112 matched well-resolved and reproducible 2-DE spots were detected across biological replicates in CV and US sample groups, respectively.
Figure 16. 2-DE protein profiles of sliced dry-cured ham after conventional (CV) and ultrasound-assisted (US-50) thermal treatments
64 IV. Results and Discussion
The Bonferroni-corrected 95% bootstrap confidence intervals for means of spot volumes across replicates revealed that 40 protein spots showed significant (P-value < 0.05) differences between sample groups (Table 13). Most spots (i.e. 33 out of 40 spots) were unique spots present in only one of the sample groups. Specifically, most percentage (94%) of the unique spots was detected in the US sample group (P < 0.05, Fisher's exact test). It can be concluded, therefore, that the proteome of dry-cured hams subjected to CV and US thermal treatments was markedly different at the qualitative level. As it was previously pointed out, the commonly used FC measure does not provide useful information on quantitative differences among unique protein spots because of it gives −∞ or +∞ values. On the other hand, only seven non- unique protein spots (i.e. spots 4, 5 19, 20, 26, 36 and 37) showed statistically significant quantitative differences in volume, with FC expressed in absolute value ranging from 1.8 to 2.9. All spots with significant differential abundance were selected for protein identification by tandem mass spectrometry (MALDI-TOF/TOF MS). The list of proteins that were unambiguously identified is shown in Table 14. Only seven non-redundant myofibrillar and sarcoplasmic proteins were identified: ACTS, aminoacylase (ACY1), PRDX6, carbonyl reductase (CBR1), SOD, cyclin-G1 (CCNG1) and two fatty acid-binding protein isoforms (FABP4/H). It is noteworthy that most spots (76%) contained the myofibrillar protein actin.
Protein fragmentation for each spot was assessed through the difference between the observed Mr on 2- DE gel and its theoretical value obtained from UniprotKB/Swiss-Prot databases. A ratio between the theoretical and observed Mr above 1.5 was used as a validation criterion of protein fragments on 2-DE gels as previously indicated. Using this criterion, we found higher protein fragmentation after US thermal treatment (P < 0.05, Fisher's exact test). Most fragments (90%) detected on 2-DE gels corresponded to actins. It cannot be excluded that other spots with ratios between the theoretical and observed Mr close to 1.5 (e.g. spots 19, 20 and 21) contained partially degraded actins. This result can be understood taking into account that the most abundant muscle proteins (60–70%) belong to the myofibrillar fraction (Lana and Zolla, 2016). More specifically, the myofibrillar proteins actin and myosin account for ca. 29% and 13% of muscle proteins, respectively, after rigor mortis but before degradation changes post-mortem (López-Bote, 2017). Therefore, the major proportion of actin in muscle contributes to explain higher release, extraction and degradation of actin from ruptured myofibrils after the US treatment. Accordingly, Kang et al. (2017) found higher fragmentation of myofibrillar proteins by ultrasound application in beef loin during curing. Ultrasonic cavitation during salting of pork meat also enhanced the extraction of proteins such as actin, produced by the dissociation of actomyosin (Ozuna et al., 2013). On the other hand, chicken meat treated by ultrasound (150 W) was shown to generate electrophoretic patterns with a significantly increased intensity of actin bands as compared to control samples (Zou et al., 2018).
65
IV. Results and Discussion
Table 13. Differentially abundant (P <0 .05) 2-DE spot volumes in dry-cured ham subjected to conventional (CV) and ultrasound-assisted (US) thermal treatments Spot no. * Average volume ± SE Fold change (FC) CV US 1 – 530±86 +∞ 2 – 1687±299 +∞ 3 – 790±221 +∞ 4 1825±390 5295±645 +2.9 5 10802±1220 5500±830 −2.0 6 – 656±178 +∞ 7 – 2602±267 +∞ 8 – 396±93 +∞ 9 – 2581±1274 +∞ 10 – 613±231 +∞ 11 – 2710±367 +∞ 12 – 1005±204 +∞ 13 – 1763±100 +∞ 14 – 2647±411 +∞ 15 2386±186 – −∞ 16 – 1643±455 +∞ 17 – 1436±232 +∞ 18 – 405±146 +∞ 19 8081±881 3619±288 −2.2 20 6439±808 2787±307 −2.3 21 – 1080±420 +∞ 22 – 505±208 +∞ 23 1153±727 – −∞ 24 – 2460±582 +∞ 25 – 1391±312 +∞ 26 964±174 2538±259 +2.6 27 – 1775±769 +∞ 28 – 1979±78 +∞ 29 – 872±512 +∞ 30 – 882±304 +∞ 31 – 1178±324 +∞ 32 – 1206±231 +∞ 33 – 571±190 +∞ 34 – 853±349 +∞ 35 – 624±203 +∞ 36 6324±1070 11068±712 +1.8 37 376±102 1064±175 +2.8 38 – 582±241 +∞ 39 – 1769±1000 +∞ 40 – 1023±553 +∞ *Location of numbered spots on 2-DE gels is shown in Fig. 16.
66 IV. Results and Discussion
Table 14. List of protein identifications by tandem mass spectrometry (MALDI-TOF/TOF MS) Spot Protein (abbrev.) Accesion no. Mascot Sequence Number of pI Mr Protein no. (Uniprot) score coverage (%) matched Th/Obs Th/Obs (kDa) fragment peptides 4 Actin, alpha skeletal muscle (ACTS) P68137 142 16 5 5.2/5.3 42.4/45.3 − 5 Actin, alpha skeletal muscle (ACTS) P68137 48 11 3 5.2/5.5 42.4/45.1 − 7 Actin, alpha skeletal muscle (ACTS) P68137 88 31 9 5.2/5.9 42.4/42.9 − 8 Aminoacylase-1 (ACY1) P37111 63 25 10 5.6/6.1 42.2/43.3 − 9 Actin, alpha skeletal muscle (ACTS) P68137 111 30 9 5.2/6.2 42.2/43.4 − 10 Actin, alpha skeletal muscle (ACTS) P68137 46 22 6 5.2/6.2 42.2/40.4 − 11 Actin, alpha skeletal muscle (ACTS) P68137 165 19 6 5.2/5.7 42.2/40.6 − 12 Actin, alpha skeletal muscle (ACTS) P68137 52 30 9 5.2/5.9 42.2/41.1 − 13 Actin, alpha skeletal muscle (ACTS) P68137 50 24 7 5.2/5.5 42.2/36.0 − 14 Actin, alpha skeletal muscle (ACTS) P68137 123 24 7 5.2/5.7 42.2/37.3 − 15 Actin, alpha skeletal muscle (ACTS) P68137 71 39 10 5.2/5.9 42.2/36.2 − 19 Actin, alpha skeletal muscle (ACTS) P68137 76 27 10 5.2/4.8 42.2/31.7 − 20 Actin, alpha skeletal muscle (ACTS) P68137 87 20 6 5.2/4.9 42.2/31.8 − 21 Actin, alpha skeletal muscle (ACTS) P68137 50 8 3 5.2/5.1 42.2/30.4 − 22 Actin, alpha skeletal muscle (ACTS) P68137 45 24 7 5.2/5.1 42.2/28.4 + 23 Actin, alpha skeletal muscle (ACTS) P68137 58 33 9 5.2/5.3 42.2/28.4 + 25 Actin, alpha skeletal muscle (ACTS) P68137 89 23 6 5.2/5.8 42.2/29.0 + 26 Peroxiredoxin-6 (PRDX6) Q9TSX9 303 47 10 5.7/6.0 25.1/28.0 − 27 Actin, alpha skeletal muscle (ACTS) P68137 70 20 7 5.2/4.7 42.2/23.6 + 28 Actin, alpha skeletal muscle (ACTS) P68137 53 28 8 5.2/5.1 42.2/27.0 + 29 Actin, alpha skeletal muscle (ACTS) P68137 109 14 4 5.2/5.0 42.2/24.0 + 30 Actin, alpha skeletal muscle (ACTS) P68137 87 34 10 5.2/5.3 42.2/26.5 + 31 Actin, alpha skeletal muscle (ACTS) P68137 107 11 4 5.2/5.4 42.2/27.3 + 33 Carbonyl reductase (CBR1) Q28960 59 32 10 7.6/5.6 32.0/24.4 −
67
IV. Results and Discussion
Spot Protein (abbrev.) Accesion no. Mascot Sequence Number of pI Mr Protein no. (Uniprot) score coverage (%) matched Th/Obs Th/Obs (kDa) fragment peptides 35 Actin, alpha skeletal muscle (ACTS) P68137 49 24 7 5.2/6.4 42.2/22.0 + 37 Superoxide dismutase (SOD) P04178 102 41 4 6.0/6.6 16.0/16.0 − 38 Cyclin-G1 (CNG1) Q52QT8 49 27 9 9.1/6.4 34.6/14.5 + 39 Fatty acid-binding protein, O97788 59 40 6 6.3/5.9 14.8/12.3 − adypocites (FABP4) 40 Fatty acid-binding protein, heart O02772 89 44 6 6.1/6.1 14.8/13.4 – (FABPH) Proteins were positively identified (P < 0.05) according to Mascot scores. Sequence coverage (%) is the proportion of the overall amino acid sequence covered by matched peptides. Number of peptides matched is the total number of identified spectra matched for the protein. Theoretical (Th) pI and Mr values for each protein spot were obtained from UniProtKB/Swiss-Prot databases. Observed (Obs) pI and Mr values for each protein spot were assessed using lineal IPG strips and molecular mass markers, respectively. Protein fragments verify that the ratio between the theoretical and observed Mr is above 1.5.
68 IV. Results and Discussion
It is noteworthy that no fragment of the myofibrillar protein myosin was detected on 2-DE gels even though it is a highly represented protein in the muscle. Experimental observations obtained in brine-cooked ham assisted by ultrasound have demonstrated that the denaturation temperature of myosin is lower than actin using differential scanning calorimetry (McDonnell et al., 2014b). Also, Kang et al. (2017) reported that oxidized myosin by the ultrasound application was responsible for its protein aggregation. Overall, oxidation and polymerization processes may change the structure and conformation of the myosin increasing susceptibility to proteolysis. Therefore, US treatment could lead to a higher denaturation of myosin caused by molecular frictions and possible acoustic cavitation in salty meat. This cavitation could produce hot spots and pressure fluctuations facilitating the weakening of the myosin structure and contributing to its destabilization. However, there were no statistically significant differences in myosin degradation between CV and US thermal treatments in a range of Mr from 10 to 70 kDa on 2-DE gels. Therefore, it is not possible to determine whether myosin degradation was due to US or CV thermal treatment.
The observations showed that the proteolysis increased markedly in sliced dry-cured hams subjected to power ultrasound. Several authors have previously reported that proteolysis is intensified by ultrasound treatment (Jayasooriya et al., 2004; Kang et al., 2017; Wang et al., 2018). In the present study, proteolysis can be facilitated by the fact that dry-cured ham was cut into thin slices. In addition, US thermal treatment can produce an intensification of heat transfer by convention due to the vibrations (Legay et al., 2011). The sonication phenomenon triggers mechanical vibration, agitation, shear forces, turbulence, acoustic cavitation and free radicals which could change protein structure conducing to increased proteolysis. Degradation of myofibrillar proteins by power ultrasound alters the structural integrity of the myofibrils producing changes in textural properties (Zou et al., 2018). In the present study, it is found that the degradation of actin increased significantly with US thermal treatment. Interestingly, differentially degraded actins were detected on 2-DE gels. Therefore, the actin can be a useful candidate biomarker to monitor the level of proteolysis of dry-cured ham subjected to different power ultrasound and treatment times and the effects of these treatments on quality attributes.
Figure 17. UPGMA-based clustering analysis using RC-values for differentially abundant sarcoplasmic proteins in CV and US samples
69
IV. Results and Discussion
A total of five unfragmented sarcoplasmic proteins showed significant (P <0.05) differential abundance after US thermal treatment: ACY1, PRDX6, CBR1, SOD and FABP4/H. It is found that all RC-values were of a positive sign, which indicates that proteins were over-represented in the US sample group. In addition, change intensity was noticeably different among proteins, with RC-values ranging from +0.14 (ACY1) to +1.0 (FABP4/H). The UPGMA dendrogram based on the RC distance matrix showed that the value of RC was notably higher for FABP4/H isoforms than for the other proteins (Fig. 17). The difference was found to be statistically significant using 95% bootstrap confidence intervals (Fig. 18). FABP is a protein that exhibits a diversity of isoforms related to the transfer of fatty acids into cells and their combustion in the mitochondria (Nechtelberger et al., 2001).
Figure 18. Comparison of RC values between well-separated proteins according to UPGMA dendrogram: FABP4/4 vs. other proteins (SOD, CBR1, ACY1 and PRDX6). Mean values and standar deviations
The protein PRDX6 also showed a high value of RC (RC=+0.56). It is a member of the thiol- specific antioxidant protein family that protects cells against oxidative injury, prevents lipid oxidation and participates in the regulation of phospholipid turnover. It has been reported that the level of PRDX6 is correlated to meat tenderness in bovine muscle because of its relationship with μ-calpain activity and proteolysis (Jia et al., 2009). SOD also showed a relevant level of extraction after ultrasound treatment (RC=+0.25). It is an oxidative enzyme that leads to hydrogen peroxide and inactivates superoxide radicals, which are intermediate products of oxidative rancidity in dry-cured meat (dos Santos et al., 2017). Accordingly, a previous study has demonstrated that SOD has antioxidant activity in dry-cured ham (Hu et al., 2016). The proteins CBR1 (RC=+0.20) and ACY1 (RC=+0.14) were only present in dry-cured hams samples subjected to ultrasound treatment, but their effects on dry-cured ham quality are yet little known. The increased extraction susceptibility of FABP4/H and of the rest of sarcoplasmic proteins found to be differentially represented in dry-cured ham subjected to US thermal treatment suggests that they can be candidate biomarkers to assess the ultrasonic application.
70 IV. Results and Discussion
IV.2.3. INSTRUMENTAL EVALUATION OF THE ORGANOLEPTIC PRECURSOR CHANGES
IV.2.3.1. Effects on free amino acid profile and taste implications
Table 15 shows the effect of temperature treatment alone or US assisted on the FAAs of dry- cured ham. Statistical analysis displayed that total FAA content was significantly (P <0.001) affected by both treatments, presenting the higher values the samples from the US-50 group (6691.5 vs. 6067.5 vs. 5278.2 mg/100 g DM for US-50, CV and CO groups, respectively).
Table 15. Effect of US and temperature treatments on FAAs content (expressed as mg/100 g DM) Amino Acid/ Group SEM P-value Sensory attributes CO CV US-50 Aspartic acid 164.65a 149.32a 212.10b 5.12 <0.001 Serine 191.48a 204.82a 243.71b 5.82 <0.001 Glutamic acid 430.61a 463.93a 544.77b 12.37 <0.001 Glycine 187.99a 216.85b 245.58c 5.92 <0.001 Histidine 99.02a 113.51a 133.55b 3.64 <0.001 Taurine 80.95a 100.04b 102.75b 2.59 <0.001 Arginine 364.86a 361.99a 518.93b 14.68 <0.001 Threonine 218.46a 250.30b 281.96c 6.64 <0.001 Alanine 398.16a 459.75b 544.41c 12.95 <0.001 Proline 287.99a 330.99b 372.34c 8.80 <0.001 Cysteine 287.14a 417.09b 437.18b 17.04 <0.001 Tyrosine 181.33a 219.62b 228.49b 6.94 <0.001 Valine 385.79a 428.48a 484.95b 10.05 <0.001 Methionine 213.90a 250.63b 259.31b 6.07 <0.001 Lysine 247.69a 276.72a 351.95b 9.51 <0.001 Isoleucine 364.94a 421.89b 411.06b 8.20 <0.001 Leucine 608.59a 700.38b 750.85b 15.83 <0.001 Phenylalanine 391.01a 461.91b 495.85b 11.81 <0.001 Total Amino Acids 5278.18a 6067.45b 6691.53b 148.81 <0.001 Sweet 1 1328.43a 1499.88b 1705.69c 33.75 <0.001 Bitter 2 2014.89a 2256.99b 2289.93b 36.00 <0.001 Acid 3 699.95a 765.60a 904.94b 16.90 <0.001 Aged 4 601.69a 645.23a 767.19b 14.89 <0.001 a-cMean values in the same row (corresponding to the same parameter) not followed by a common letter differ significantly (P < 0.05; Tukey's Test). SEM: standard error of the mean. 1 Sweet flavour=Σ of alanine, glycine, threonine, serine and proline; 2 Bitter flavour=Σ of leucine, valine, isoleucine, methionine and phenylalanine; 3Acid flavour=Σ of glutamic acid, aspartic acid and histidine; 4Aged flavour=Σ of lysine, tyrosine and aspartic acid.
No significant differences were observed between US-50 and CV treatments. The values observed in the present study are within the range of FAA contents (from 4000 to 12,500 mg/100 g DM) described by other authors (Bermúdez et al., 2014; Jurado et al., 2007; Martín et al., 2001) in dry-cured ham. The higher total FAA content in samples submitted to
71
IV. Results and Discussion ultrasound at 50 °C could be due to the release of some FAAs from cell tissues that were destroyed by the US.
All the individual FAAs were influenced by US and temperature treatments, showing the highest content in sliced dry-cured ham submitted to ultrasounds at 50 °C, except for isoleucine which presented the highest level in samples from CV group. According to Jambrak et al. (2014), the ultrasound treatment can modify the protein structure due to partial cleavage of intermolecular hydrophobic interactions, rather than the breakdown of peptide or disulphide bonds increased the release of FAAs. It could be seen that leucine, glutamic acid and alanine were the most abundant FAA in the three studied groups and the sum of these three amino acids reached around 27% of the total FAAs. Both treatments (ultrasound and temperature) significantly increased the bitter taste of dry-cured ham. On the other hand, the use of temperature did not significantly modify the acid and aged taste, whereas these two tastes were significantly increased by using ultrasounds. The temperature significantly increased the sweet taste of hams and this taste was significantly further increased by the US treatment at 50 °C.
IV.2.3.2. Effects on the volatile profile and odour implications
A total of 155 volatile compounds were found in headspace of the dry-cured ham (Table 16). These volatile compounds were classified as part of some of the main chemical families according to Narváez-Rivas et al. (2012) and Purriños et al. (2011): 56 hydrocarbons, 23 aldehydes, 21 ketones, 16 esters and ethers, 24 alcohols, 6 carboxylic acids, 4 nitrogenous compounds and 5 sulphur compounds. Significant differences (P < 0.05) were detected in the total volatile compound content between CO and US-50 groups, with a higher concentration in the CO batch (56,662.84 AU×103/g of dry-cured ham) than in the US-50 treatment (45,848.47 AU×103/g of dry-cured ham), being the values in the CV treatment intermediate (48,497.25 AU×103/g of dry-cured ham). The fact that US had been used as a method to improve the food preservation (Knorr et al., 2011) together with the hypothesis that spoilage could originate higher concentrations of volatile compounds in the headspace (Carrapiso et al., 2010), could explain the less content of total volatile compounds in the US-50 group.
Regarding the different chemical families, except for hydrocarbons, the sum of the volatile compounds of each family showed significant differences among groups. Moreover, the levels of 94 individually volatile compounds were significantly influenced by the treatment (24 hydrocarbons, 15 ketones, 15 alcohols, 21 aldehydes, 10 ester and ethers, 4carboxilic acids, 3 sulphur compounds and 2 nitrogenous compounds). As shown in Table 16, hydrocarbons were the most numerous chemical family with up to 56 different compounds, 24 of them have already been identified in other previous studies in hams (Bermúdez et al., 2015; Narváez- Rivas et al., 2012). Hydrocarbons represented a percentage of 30% of the total area of the volatile compounds in CO samples, whereas in both US-50 and CV groups this chemical family was the most abundant (accounting for 43% and 37%, for US-50 and CV batches, respectively). The aliphatic hydrocarbon, that was found in higher concentration was 2,2,4,6,6-pentamethyl heptane, followed by octane, and then, with similar values, pentane, hexane, undecane and dodecane.
72 IV. Results and Discussion
Meanwhile, the main family of volatile compounds in CO group were the aldehydes (approximately 41% of the total area of volatile compounds). An important reduction of total aldehydes content in US-50 group was observed, as well as a higher decrease in CV batch (23,509.08 vs. 10,307.72 vs. 2381.68 AU×103/g of dry-cured ham for CO, US-50 and CV groups, respectively). According to previous studies in ham (Andrés et al., 2007; García et al., 1991; García-González et al., 2008; Jurado et al., 2009; Sánchez-Peña et al., 2005), hexanal was the predominant linear aldehyde in CO and US-50 groups, with the highest content presented in CO samples (12,264.83 vs. 5747.78 vs. 185.78 AU×103/g of dry-cured ham for CO, US-50 and CV groups, respectively). In contrast, CV batch presented propanal as the main aldehyde, whose concentration was higher than in the other two groups. On the other hand, 3-methyl butanal was the most abundant branched aldehyde determined in all cases, but presenting significant differences (P < 0.001) among the groups. CO samples showed the highest concentration of this compound, while CV group registered the lowest one. In this way, in the previous study it was found that high-proteolytic hams presented lower amounts of hexanal and 3-methyl butanal than low-proteolytic hams. Lower amounts of these aldehydes in both treatment groups than in control was expected since high temperatures promote protein degradation and enhance proteolytic reactions. According to Ramírez and Cava (2007), who proposed the degradation of isoleucine amino acid as the most probably origin of 2-methyl butanal, a negative correlation between these two compounds was found (r=−0.547; P < 0.01), as well as significant (P < 0.001) difference among the groups, observing higher levels in CV group than in the others ones.
Likewise, the total alcohol content showed higher levels in CV samples than in the other two groups (6548.61 vs. 8599.43 vs. 12,199.24 AU×103/g of dry-cured ham for CO, US and CV groups, respectively). This high content of total alcohols found in CV group is a consequence of the higher amounts of three specific individual alcohols: 2-methyl butanol, 3-methyl butanol and phenylethyl alcohol. The increment of 2-methyl butanol and 3-methyl butanol in CV group could be explained for the decrease observed in the 2-methyl butanal and 3- methyl butanal since that branches alcohols may be originated, among others reasons, from the reduction of branched aldehydes (Martín et al., 2006). Otherwise, the major alcohol detected in similar levels in all the groups was 1-octen-3-ol (3543.17 vs. 3818 vs. 3922.68 AU×103/g of dry-cured ham for CO, US-50 and CV groups, respectively).
In addition to aldehydes, Carrapiso et al. (2002) identified ketones as important compounds contributing to odour in dry-cured ham. In the present study, statistical analysis showed that the total ketones content was significantly (P < 0.001) affected by the treatment, observing the greatest level in CV group, and being the 2-heptanone and the acetoin the most abundant ones with higher amount in CV samples than in CO and US-50 groups (427.95 vs. 664.14 vs. 980.43 and 484.130 vs. 501.60 vs. 2031.51 AU×103/g of dry-cured ham for CO, US-50 and CV groups, respectively). In agreement with previous studies (Ramírez and Cava, 2007; Sabio et al., 1998), other 2-ketones were also found, such as 2-butanone, 2-pentanone, 2-octanone and 2-nonanone. All these compounds presented the highest values in the samples from CV treatment.
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IV. Results and Discussion
Esters and ethers, carboxylic acids, nitrogenous compounds and sulphur compounds were the chemical families that presented minor levels of volatile compounds. Esters are compounds distributed in the essential oils with a high flavouring effects, derived from the reaction of an alcohol or phenol with acids (Reineccius, 1991). Some studies reported low values of esters in volatile dry-cured ham profiles (Martín et al., 2006), whereas other studies carried out in cooked pork meat showed a greater content of these compounds (Gorbatov and Lyaskovskaya, 1980). According to this, it could be assumed that temperature affects the ester compound formation. However, this effect was not observed in the present study, since the CV samples showed the lowest total content of esters (1906.99 vs. 1680.82 vs. 1385.33 AU×103/g of dry- cured ham for CO, US-50 and CV groups, respectively). This fact may be explained because the high temperature produced losses by volatilisation. Regarding carboxylic acids, total content was 20% less in US-50 group and 70% in CV treatment than in CO group. The highest differences were found in pentanoic acid and butanoic acid contents.
On the other hand, 2,6-dimethyl pyrazine was found as the main nitrogenous compound. Pyrazines are usual compounds in meat and meat products cooked at high temperatures (Mussinan and Walradt, 1974) and their formation is a result of the reaction between diketones and amino compounds at high temperatures (Shibamoto and Bernhard, 1976). According to this, CV samples showed higher significant values (P ˂ 0.001) than the other batches, whereas US-50 batch did not show any difference compared with CO group. It is possible that the structural changes that were originated by US application can prevent reactions between diketones and amino compounds. The temperature application originated an important decrease in the sulphur compounds, being the dimethyl disulphide the most affected compound (1740.04 vs. 206.48 vs. 738.87 AU×103/g of dry-cured ham for CO, US-50 and CV groups, respectively). The sulphur amino acids showed a negative and significant (P < 0.01) correlation with dimethyl disulphide (r=−0.557, r=−0.614 and r=−0.512, for taurine, cysteine and methionine, respectively) and dimethyl trisulphide (r=−0.550, r=−0.599 and r=−0.493, for taurine, cysteine and methionine, respec vely), sugges ng that these compounds could be originated by the amino acids catabolism (Sabio et al., 1998).
With reference to the odour contribution, only five hydrocarbons were previously described as odour descriptors, octane, heptane, hexane, ethyl benzene and 2-ethyl furan, whose contribution is related with sweet notes. As mentioned previously, this chemical family has not very odorant impact due to its high threshold. Considering their low threshold, aldehydes are the most active compounds followed by ketones and esters, and to a lesser extent by alcohols. Hexanal and 3-methyl butanol are the most odour-active compounds identified in hams (Carrapiso et al., 2002) and were the main volatile compounds showed in CO samples, contributing principally with the characteristic greasy odour of ham and to a lesser extent with fruity notes. Significant lower levels of hexanal were found in treated groups, observing the lowest content in CV group. Lower contents in CV batch were also detected for nonanal, octanal, heptanal, 2-methyl butanal, 3-methyl butanal, 2,4-decadienal, 4-nonenal, 2-octenal 2- methyl propanal, methional and benzaldehyde. According to this, the application of high temperature without US could promote an important reduction, specially, on fatty and grassy notes.
74 IV. Results and Discussion
Table 16. Effect of US and thermal treatments on volatile profile (expressed as quantified area units of the EIC (AU-EIC) x 103/g dry-cured ham) Compound m/z LRI R Groups SEM P-value CO US-50 CV Pentane 43 500 ms, lri, s 883.71a 688.22a 1471.54b 94.96 0.005 Pentane, 2-methyl- 71 543 ms 2.57a 3.29ab 4.50b 0.29 0.023 1-Butene, 2,3-dimethyl- 57 571 ms 19.51a 10.68a 30.18b 1.73 <0.001 n-Hexane 69 600 ms, lri, s 810.40b 529.80a 1541.71c 61.77 <0.001 Heptane 71 700 ms, lri, s 802.78 514.56 879.78 68.82 0.103 Pentane, 2,3,4-trimethyl- 71 756 ms, lri 232.76a 365.58ab 437.24b 26.54 0.003 Pentane, 2,3,3-trimethyl- 71 763 ms, lri 319.34a 508.02b 620.06b 34.30 <0.001 Pentane, 3-ethyl- 70 770 ms, lri 51.97a 77.48ab 85.39b 5.22 0.015 1-Pentene, 3-ethyl-2-methyl- 83 774 ms 32.98 37.73 45.65 2.22 0.069 Hexane, 2,2,5-trimethyl- 57 799 ms 374.97a 655.05ab 705.58b 51.55 0.010 Octane 85 800 ms, lri, s 1942.31 1335.15 1731.67 154.33 0.257 2-Octene, (E)- 112 833 ms, lri, s 201.22 122.73 157.66 14.94 0.078 Heptane, 3,4,5-trimethyl- 85 842 ms 67.19a 110.46b 120.25b 7.11 0.002 3-Octene, (E)- 112 845 ms 84.68 59.41 70.66 6.16 0.217 Octane, 2-methyl- 71 899 ms 12.42 15.12 13.79 1.00 0.530 Hexane, 2,2,5,5-tetramethyl- 57 914 ms 301.96 409.36 394.91 26.67 0.168 4-Nonene 70 926 ms 130.55 148.11 173.08 7.24 0.057 Nonane 57 900 ms, lri, s 131.63a 167.86ab 193.45b 9.61 0.020 Heptane, 2-methyl-3-methylene- 69 930 ms 12.74a 14.51ab 17.80b 0.74 0.024 2-Octene, 4-ethyl- 70 982 ms 121.06 109.24 139.94 7.45 0.322 Octane, 3-methyl-6-methylene- 69 985 ms 204.18a 223.88ab 286.28b 12.68 0.028 Octane, 4-ethyl- 69 991 ms 72.43a 83.39ab 99.48b 4.11 0.026 Heptane, 3,3,4-trimethyl- 57 994 ms 6.01a 11.98b 3.49a 0.73 <0.001 Pentane, 3,3-dimethyl- 85 995 ms 6.14 5.74 7.14 0.43 0.483 Decane 71 1000 ms, lri, s 392.40 484.05 448.96 35.08 0.440 Nonane, 2,3-dimethyl- 56 1003 ms 62.32 61.17 73.08 3.76 0.252
75
IV. Results and Discussion
Compound m/z LRI R Groups SEM P-value CO US-50 CV 1-Octene, 2,6-dimethyl- 69 1010 ms 72.47 78.95 89.54 4.12 0.519 3-Octene, 4-ethyl- 70 1012 ms 23.62 22.29 26.35 1.30 0.068 Nonane, 3-methylene- 70 1022 ms 165.31a 193.91a 219.60a 9.67 0.023 Heptane, 2,2,4,6,6-pentamethyl- 57 1027 ms 3130.36 6386.68 2772.86 571.68 0.536 3-Ethyl-3-hexene 83 1042 ms 46.18a 68.29a 99.93b 5.40 <0.001 Undecane, 3,6-dimethyl- 57 1068 ms 247.95ab 333.34b 119.46a 31.54 0.042 Tridecane, 6-methyl- 57 1079 ms 241.55 296.61 296.67 18.19 0.326 Undecane, 2,5-dimethyl- 57 1085 ms 159.26 140.65 150.96 11.19 0.788 Decane, 2,3,5-trimethyl- 57 1099 ms 102.23b 56.83a 81.27ab 7.43 0.032 Undecane 57 1100 ms, lri, s 930.86 1346.47 1216.44 83.08 0.085 2,3-Dimethyl-3-heptene, (Z)- 83 1123 ms, lri 56.04b 25.71a 10.65a 4.09 <0.001 2-Undecene, 9-methyl-, (Z)- 70 1132 ms 368.85 345.35 367.91 22.50 0.900 5-Undecene, 6-methyl- 168 1144 ms 11.24 8.17 9.33 0.74 0.202 4,4-Dipropylheptane 85 1153 ms 51.23 43.30 50.12 3.10 0.548 2-Undecene, 3-methyl-, (E)- 70 1181 ms 60.96 55.41 61.11 3.49 0.774 4-Nonene, 5-butyl- 70 1197 ms 24.26 23.38 20.87 1.53 0.066 Dodecane 57 1200 ms, lri, s 664.51 948.13 849.77 53.50 0.678 Decane, 3-ethyl-3-methyl- 57 1228 ms 50.22 42.58 46.32 2.93 0.551 Dodecane, 2-methyl- 57 1233 ms 23.00a 38.36b 30.39ab 2.06 0.005 1-Tetradecene 97 1236 ms 31.84 30.42 28.93 2.10 0.857 Tridecane 71 1300 ms, lri, s 228.76 318.27 217.88 21.11 0.131 Tridecane, 3-methyl- 85 1304 ms 31.82 38.27 37.84 1.87 0.252 Total Aliphatic hydrocarbons 15578.28 19062.05 17144.10 1014.41 0.356 Furan, 2-ethyl- 81 703 ms 38.75ab 14.06a 60.00b 4.76 <0.001 Toluene 92 804 ms 122.47a 131.23a 178.32b 5.72 <0.001 Cyclobutane, 1,1,2,3,3-pentamethyl- 70 813 ms 247.78 268.52 288.93 13.91 0.490 Ethylbenzene 91 917 ms 17.64 18.84 17.70 0.81 0.811
76 IV. Results and Discussion
Compound m/z LRI R Groups SEM P-value CO US-50 CV Benzene, 1,3-dimethyl- 106 926 ms 19.44 21.44 21.39 0.60 0.267 2-n-Butyl furan 81 944 ms 35.70 32.04 42.78 2.85 0.383 Cyclopentane, 1-ethyl-3-methyl- 83 1123 ms 56.04b 25.71a 10.65a 4.09 <0.001 Cyclopentane, ethyl- 98 1148 ms 300.84c 173.68b 38.57a 20.28 <0.001 Total Aromatic and cyclic hycrocarbons 808.45 743.01 769.51 26.04 0.565 Total Hydrocarbons 16867.18 19912.67 17932.30 1045.39 0.479 Propanal 58 526 ms, lri, s 139.01a 102.85a 751.47b 43.60 <0.001 Propanal, 2-methyl- 72 557 ms 213.22b 173.69b 7.43a 16.50 <0.001 Butanal 72 584 ms, lri, s 23.16c 10.81b 1.45a 1.69 <0.001 Butanal, 3-methyl- 58 659 ms, lri 1968.06c 1240.06b 68.91a 142.21 <0.001 Butanal, 2-methyl- 57 671 ms 1139.71b 929.14b 43.06a 84.00 <0.001 Pentanal 57 728 ms, lri, s 951.76 640.68 697.89 65.64 0.090 2-Butenal, 2-methyl- 84 801 ms 104.37b 55.38a 27.29a 7.60 <0.001 Hexanal 56 865 ms, lri, s 12264.83c 5747.78b 185.13a 889.71 <0.001 Heptanal 70 974 ms, lri, s 853.54c 401.98b 25.49a 68.21 <0.001 Methional 104 999 ms 134.75b 134.52b 7.04a 12.33 <0.001 Benzaldehyde 106 1045 ms 352.12c 200.47b 67.03a 22.05 <0.001 Octanal 56 1066 ms, lri, s 370.02c 249.58b 98.19a 23.99 <0.001 5-Ethylcyclopent-1-enecarboxaldehyde 124 1099 ms 32.99b 17.82a 10.03a 2.31 <0.001 Benzeneacetaldehyde 91 1119 ms, lri 796.26c 356.03b 37.78a 52.71 <0.001 2-Octenal, (E)- 70 1123 ms 44.78b 17.22a 10.22a 3.11 <0.001 Decanal 81 1129 ms, lri, s 24.68 23.26 23.18 1.66 0.912 Nonanal 57 1148 ms, lri, s 614.70c 380.07b 133.97a 38.15 <0.001 4-Nonenal, (E)- 83 1201 ms 33.21b 23.96ab 23.29a 1.66 0.013 Benzaldehyde, 3-ethyl- 134 1209 ms 33.46b 27.15b 8.76a 2.53 <0.001 2-Decenal, (E)- 70 1272 ms 28.90b 19.66ab 13.75a 1.79 <0.001 2,4-Decadienal, (E,E)- 81 1315 ms 23.10b 8.08a 1.22a 2.20 <0.001
77
IV. Results and Discussion
Compound m/z LRI R Groups SEM P-value CO US-50 CV 2-Undecenal 95 1339 ms 6.56b 2.44a 2.76a 0.62 0.004 Pentadecanal- 82 1516 ms, lri, s 3.90a 9.02b 4.73a 0.68 0.003 Total Aldehyde 23509.08c 10307.72b 2381.68a 1562.86 <0.001 Acetone 58 528 ms 246.04a 438.13b 958.64c 50.42 <0.001 2,3-Hexanedione 41 562 ms 391.05b 226.53a 696.97c 30.69 <0.001 2-Butanone 72 596 ms 177.17a 264.28b 504.65c 22.63 <0.001 Cyclopentanone, 3-methyl- 56 667 ms 30.74ab 18.76a 34.05b 2.46 0.043 2-Pentanone 86 720 ms, lri 101.75a 78.17a 305.68b 25.87 <0.001 Acetoin 45 787 ms 484.13a 501.60a 2031.51b 153.68 <0.001 3-Heptanone 57 960 ms, lri 43.80 37.03 37.54 1.88 0.225 2-Heptanone 58 967 ms, lri 427.95a 664.14ab 980.43b 62.05 <0.001 Cyclohexanone, 2-ethyl- 69 972 ms 39.00a 42.78a 65.73b 3.25 0.002 2-Nonen-4-one 69 979 ms 13.48 14.36 17.24 0.94 0.272 2-Hepten-4-one, 6-methyl- 69 992 ms 72.65a 80.61ab 99.82b 3.86 0.015 4-Octanone, 5-hydroxy-2,7-dimethyl- 69 1042 ms 9.29a 18.03ab 21.64b 1.62 0.003 1-Octen-3-one 70 1046 ms 109.18 96.80 71.31 8.50 0.202 5-Hepten-2-one, 6-methyl- 69 1056 ms 104.35ab 93.37a 134.10b 5.81 0.026 2-Octanone 58 1059 ms 38.35a 95.71a 163.52b 12.65 <0.001 3-Nonanone 113 1134 ms 23.48 21.34 23.80 1.59 0.818 1-Hexanone, 5-methyl-1-phenyl- 105 1137 ms 15.19a 28.98b 24.08b 1.56 <0.001 2-Nonanone 58 1141 ms 16.85a 71.11b 56.62b 6.38 <0.001 2(3H)-Furanone, 5-ethyldihydro- 85 1158 ms 187.86 226.67 199.86 8.50 0.156 5-Hexen-3-one 57 1161 ms 48.92 38.56 53.49 3.65 0.298 2,6-Bis(1,1-dimethylethyl)-4-(1-oxopropyl)phenol 233 1448 ms 11.04b 0.00a 0.00a 1.50 <0.001 Total Ketone 2322.78a 3046.03b 6772.32c 265.18 <0.001 Acetic acid ethenyl ester 86 588 ms 25.62a 17.51a 50.61b 3.17 <0.001 Ethyl Acetate 61 598 ms 107.45 162.28 142.48 13.45 0.210
78 IV. Results and Discussion
Compound m/z LRI R Groups SEM P-value CO US-50 CV Methane, oxybis[dichloro- 83 611 ms 224.46 251.18 231.85 14.17 0.734 Propanoic acid, ethyl ester 57 737 ms 46.38b 15.79a 19.06a 3.40 <0.001 Butanoic acid, ethyl ester 71 855 ms 77.53c 53.05b 22.14a 4.57 <0.001 Butanoic acid, 2-methyl-, ethyl ester 102 908 ms 46.49 49.14 39.04 3.89 0.624 Butanoic acid, 3-methyl-, ethyl ester 88 913 ms 121.86ab 138.61b 67.83a 10.09 0.024 Oxalic acid, butyl propyl ester 57 936 ms 131.63a 167.86ab 193.45b 9.61 0.024 Ethanol, 2-butoxy- 57 985 ms, lri 394.15b 296.66ab 218.86a 22.78 0.004 Carbonic acid, bis(2-ethylhexyl) ester 112 1003 ms 25.20 25.06 28.09 1.61 0.736 Hexanoic acid, ethyl ester 88 1050 ms 184.39b 150.70b 79.11a 11.29 <0.001 2-Piperidinecarboxylic acid, 1-acetyl-, ethyl ester 84 1124 ms 30.54b 18.80a 15.15a 1.89 <0.001 Carbonic acid, tridecyl vinyl ester 57 1168 ms 210.11 163.66 189.81 15.26 0.447 Octanoic acid, ethyl ester 88 1204 ms 75.26b 77.21b 42.04a 4.19 <0.001 Decanoic acid, ethyl ester 88 1336 ms 33.57b 27.32b 12.77a 2.52 0.002 2,2,4-Trimethyl-1,3-pentanediol diisobutyrate 71 1442 ms 3.42 3.40 2.43 0.18 0.064 Total Esther and ether 1906.99b 1680.82ab 1385.33a 68.27 0.006 Isopropyl Alcohol 45 532 ms 119.01ab 163.82b 100.93a 9.65 0.039 1-Propanol 59 572 ms 39.39ab 59.98b 23.41a 3.96 0.002 2-Butanol 45 607 ms 21.64a 27.36a 30.26a 1.48 0.043 1-Butanol 56 707 ms 39.26b 40.08b 9.13a 3.13 <0.001 1-Penten-3-ol 57 730 ms 853.31 621.14 784.02 47.89 0.122 2-Pentanol 45 751 ms 124.97 209.61 202.82 18.56 0.088 1-Butanol, 3-methyl- 55 808 ms, lri 239.69a 1169.80b 4088.29c 253.84 <0.001 1-Butanol, 2-methyl- 57 812 ms 39.06a 238.09b 581.42c 42.81 <0.001 1-Pentanol 55 847 ms 576.25b 299.13a 189.49a 43.80 <0.001 2-Propanol, 2-methyl- 59 894 ms 22.58b 9.71a 17.36ab 1.92 0.016 2,3-Butanediol, [S-(R*,R*)]- 45 909 ms 69.08b 8.56a 2.13a 7.00 <0.001 3-Pentanol, 2,4-dimethyl- 73 954 ms 13.50 18.68 24.18 2.15 0.129
79
IV. Results and Discussion
Compound m/z LRI R Groups SEM P-value CO US-50 CV 1-Heptanol 70 1046 ms 109.18 96.80 71.31 8.50 0.202 1-Octen-3-ol 57 1051 ms, lri 3543.17 3818.07 3922.68 236.70 0.789 1-Heptanol, 2,4-diethyl- 69 1085 ms 112.27 71.78 77.41 9.03 0.108 2-Ethyl-1-hexanol 57 1094 ms 11.36ab 10.53a 15.90b 0.87 0.048 4-Ethylcyclohexanol 81 1104 ms 90.23a 129.55ab 141.39b 8.25 0.019 Benzyl alcohol 108 1124 ms, lri 131.16 145.59 153.53 7.36 0.444 1-Octanol 56 1127 ms 73.90ab 88.89b 49.90a 5.78 0.043 4-Methyl-5-decanol 55 1162 ms 25.30a 36.53a 74.05b 5.09 <0.001 p-Cresol 107 1178 ms 30.50 31.28 28.20 1.33 0.687 Phenylethyl Alcohol 92 1182 ms 13.89a 186.88a 883.92b 65.26 <0.001 1-Tetradecanol 68 1225 ms 28.08 31.26 33.29 1.36 0.281 1,4-Benzenediol, 2,5-bis(1,1-dimethylethyl)- 222 1485 ms 0.27a 0.41b 0.27a 0.02 <0.001 Total Alcohol 6548.61a 8599.43a 12199.24b 487.72 <0.001 Propanoic acid 74 827 ms 12.07 16.39 16.71 2.19 0.606 Propanoic acid, 2-methyl- 73 888 ms 74.38b 47.64ab 31.63a 5.69 0.005 Butanoic acid 60 918 ms 209.13c 74.58b 15.13a 14.47 <0.001 Butanoic acid, 3-methyl- 60 969 ms 427.98 329.99 366.87 33.67 0.459 Pentanoic acid 60 1083 ms 428.30c 274.79b 7.68a 28.77 <0.001 Octanoic acid 60 1224 ms 36.67c 20.14b 4.08a 2.72 <0.001 Total Carboxylic acid 1172.40c 950.08b 316.57a 58.15 <0.001 Fumaronitrile 78 646 ms 27.19b 17.32a 23.53ab 1.42 0.011 3-(1'-pyrrolidinyl)-2-butanone 98 906 ms 92.62 95.73 121.88 5.44 0.078 Pyrazine, 2,6-dimethyl- 108 978 ms, lri 347.01a 337.27a 478.72b 14.72 <0.001 1-(1'-pyrrolidinyl)-2-butanone 84 982 ms 90.39 97.20 117.94 5.32 0.110 Total Nitrogenous compounds 561.37a 550.57a 747.76b 20.62 0.000 Carbon disulfide 76 533 ms 157.74b 77.69a 195.02b 11.37 <0.001 Disulfide, dimethyl 94 781 ms, lri 1740.04b 206.48a 738.87a 141.24 <0.001
80 IV. Results and Discussion
Compound m/z LRI R Groups SEM P-value CO US-50 CV Dimethyl trisulphide 126 1035 ms 123.40b 10.27a 5.82a 10.58 <0.001 Sulphurous acid, decyl hexyl ester 85 1156 ms 110.15 122.77 104.36 11.50 0.835 Sulphurous acid, butyl dodecyl ester 85 1304 ms 31.82 38.24 37.81 1.86 0.254 Total Sulphur compounds 2213.62b 443.46a 1081.88a 161.36 0.000 Total Compounds 56662.84b 45848.47a 48407.25ab 1697.40 0.013 a-cMean values in the same row (corresponding to the same parameter) not followed by a common letter differ significantly (P <0.05; Tukey's Test). SEM: standard error of mean; m/z: Quantification ion; LRI: Lineal Retention Index calculated for DB-624 capillary column (JandW scientific: 30m×0.25mm id, 1.4 μm film thickness) installed on a gas chromatograph equipped with a mass selective detector; R: Reliability of identification; lri: linear retention index in agreement with literature (Domínguez et al., 2014; Lorenzo et al., 2012; Lorenzo, Bedia and Bañon, 2013; Lorenzo, 2014; Lorenzo and Domínguez, 2014; Lorenzo and Carballo, 2015; Pateiro et al., 2015; Purriños et al., 2011; Purriños et al., 2012, Purriños, Carballo and Lorenzo, 2013); ms: mass spectrum agreed with mass database (NIST14); s: mass spectrum and retention time identical with an authentic standard.
81
IV. Results and discussion
Regarding ketones, the CV group presented higher levels in four of the six odour active ketones found in this study, so the odour of this group of hams could be more floral and fruity compared with the others. On the other hand, alcohols with a low molecular weight confer a sweet and spirituous odour to ham, but as the molecular weight increases a fatty and irritating odour is perceived (Narváez-Rivas, Gallardo and León-Camacho, 2016). Samples from CV group showed higher values of 3-methyl butanol, compound associated to BF muscle (Sánchez-Peña et al., 2005), and 2-butanol than the other two groups. Additionally, it was observed fatty, balsamic and fruity notes reduction due to the lowest amounts of pentanol, octanol and butanol presented in these samples. It was not found significant differences in 1-octen-3-ol among the groups, a fact that was expected since this compound that contributes with a typical mushroom odour is derived from feeding system (Jurado et al., 2009). Among the esters reported in previous studies, only one was detected here. Ethyl ester butanoic acid was identified as a specific odour-active compound in Iberian (Carrapiso et al., 2010), Serrano (Flores et al., 1997) and Jinhua (Song, Cadwallader and Singh, 2008) hams. Finally, dimethyl disulphide and some carboxylic acids (butanoic, propanoic, pentanoic and 3-methyl butanoic acid) were previously reported like spoiled ham odorants (Carrapiso et al., 2010). In this context, CO group showed higher spoiled and rancid odour due to its higher amounts of butanoic, pentanoic, 3-methyl butanoic acid and dimethyl disulphide.
82 IV. Result and discussion
IV.3. EFFECTS OF THE APPLICATION OF HIGH-PRESSURE AS CORRECTIVE MEASURE TO DECREASE THE ADHESIVENESS
IV.3.1. BIOCHEMICAL EFFECTS
Fulladosa et al. (2009) found that applying pressures of 600 MPa on ham samples increased hardness and decreased pastiness. Bearing in mind that the degree of denaturation of the proteins contained in the muscle depends on the pressure, temperature, pH and processing time, hams with different degrees of proteolysis may arise, and therefore be more or less pasty. It is widely known that the application of high pressures together with high temperatures produces a greater effect at the ultrastructural level (Cheftel and Culioli, 1997). According to this, Coll-Brasas et al. (2019) demonstrated that combined treatments at 20 ºC were sufficient to correct the pastiness of ham samples with a PI lower than 36, but for samples with a higher PI, treatments at 35 ºC were required.
Considering the large volume of treatments and analyses carried out in addition to the format and limited amount of sample, it was not possible to obtain adhesiveness data in the present study. Despite this unexpected incident, noting that the previous evidence supported the purpose of the treatment as a pastiness corrective measure, it was decided to continue with the evaluation of the impact it has on the taste and smell of the final product.
Hereby, the first measure was moisture to check possible alterations. As result, statistical analysis did not show significant differences (P > 0.05) in moisture content among groups, presenting mean values of 58.12, 58.74, 58.64 and 58.68 g/100 g for CO, HPP-0, HPP-20 and HPP-35 groups, respectively (Fig. 19). Our moisture values were in the range of data (48.3-65.2 g/100 g) reported by other authors (Bermúdez et al., 2014; Prevolnik et al., 2011; Pugliese et al., 2015) for dry-cured ham, the same as happened in previous mentioned ultrasound treatments.
Figure 19. Effect of HPP treatments on moisture content of dry-cured ham. Mean values and standard deviations
IV.3.1. MICROSTRUCTURAL MODIFICATIONS
A comparison between the CO batch and HPP batch at 0 °C (HPP-0) was carried out to separate the effect of heating from that of the HPP and evaluate them independently. Representative 2- DE proteome images obtained from control and HPP-0 samples are shown in Fig. 19. The average number of protein spots detected was 116 and 123 in CO and HPP-0 samples,
83
IV. Results and discussion respectively. Each protein spot detected was matched and the gel spots with significant changes in abundance were marked and numbered on the image. In particular, eighteen spots with significantly differential abundance were found, resulting nine spots with significantly qualitative differences (spot no. 2, 3, 4, 12, 13, 14, 16, 17 and 18) and other nine spots with quantitative differences (spot no. 1, 5, 6, 7, 8, 9, 10, 11 and 15). However, it must be emphasized that the majority of these spots with qualitative changes were only present in HPP-0 treatment (8 of 9 spots). Consequently, it appears clear that proteomic profiles of CO and HPP-0 treated samples were highly differentiated. The preliminary study of the samples has shown that a higher number of spots detected on 2-DE gel were correlated to a higher PI. In this study, a higher number of spots was found in HPP samples, indicating more proteolytic degradation. This fact would suggest further proteolysis caused by the effect of pressure, matching with observations made in earlier studies (Ma and Ledward, 2004; Rakotondramavo et al., 2018). Overall, the curing step causes proteolysis whereas brining and tumbling mainly contribute to denaturation of the proteins. On the other hand, it has been reported that the HPP treatment leads to greater protein denaturation and oxidation causing aggregation, gelation or even increasing enzymatic activity. Although both proteomes must include a high level of protein breakdown, the HPP-0 treatment induced an increase in the proteolysis of dry- cured ham resulting in a higher number of spots. During the HPP process, the no covalent interactions in the tertiary (hydrophobic and ionic interactions) structure are weakened (Kaur et al., 2016). This change in protein structure could cause a partial unfolding of protein enabling hydrophobic interactions between proteins leading to their aggregation or causing higher proteolysis as the analysis suggested.
Figure 20. 2-DE gel images obtained from sliced dry-cured ham samples after a standard process (left) and an HPP treatment at 0 °C (right)
The eighteen differentially abundant spots were excised for further identification. Fourteen spots were identified with a high Mascot score (> 60) as indicated in Table 17. Among the identified spots, ten were actin, which as previously commented is an important fibrillar protein in meat, representing approximately 13% of the total muscle protein in two forms, G- and F-actin, being G-actin the monomer and F-actin the polymer (filament) (Appell et al., 2018). It is important to highlight that actin spots detected in 2-DE gel had different molecular weights ranging from 14.1 to 41.1 kDa. However, the actin isoform detected in this study has a theoretical molecular weight of around 42.0 KDa according to the Uniprot database. This difference between the theoretical and experimental mass of actin spots can be explained by their different degree of fragmentation after an HPP treatment.
84 IV. Result and discussion
Table 17. Identification of selected protein spots by MALDI-TOF/TOF Spot Protein Protein Mascot Sequence Number Theoretical Experimental no. a description b accession score b coverage of mass mass (2-DE (%) matched (UniProt, gel, KDa)d peptides KDa)c 1 Triosephosphate TPI1 87 29 6 26.7 41.1 isomerase 2 Alpha actin 1 ACTC1 93 9 3 42 41.1 3 Alpha actin 1 ACTC1 108 17 5 42 40 4 Myosin-7 MYH7 131 7 13 223.3 38.3 5 Alpha actin 1 ACTC1 340 24 9 42 38.4 6 Alpha actin 1 ACTC1 166 16 5 42 37.6 7 Hemoglobin HBB 91 15 2 16.2 37.7 subunit beta 9 Alpha actin 1 ACTC1 378 24 8 42 30 10 Alpha actin 1 ACTC1 119 30 11 42 30 11 Alpha actin 1 ACTC1 154 9 3 42 30 12 Alpha actin 1 ACTC1 90 23 8 42 29.8 14 Alpha actin 1 ACTC1 172 18 4 42 27.5 15 Triosephosphate TPI1 79 30 5 26.7 26 isomerase 17 Alpha actin 1 ACTC1 85 17 4 42 14.1 a The spot numbers are shown in Fig. 19. b Sus scrofa (pig) was employed as taxonomy filter in Mascot search. c Theoretical mass was provided by UniProt database. d Experimental mass was estimated on 2-DE gels using molecular weight standards.
The total RC value of actins was 2.51 indicating us that entire or fragmented actins are most abundant in HPP-0 samples (Table 18). On the other hand, the most abundant protein in the animal muscle (around 38%) is myosin. The myosin protein consists of two subunits with very different weights, called myosin light chain and heavy chain (Appell et al., 2018). In this comparative proteomic analysis, there was only a single myosin spot found in HPP-0 samples and identified as myosin heavy chain. In addition, the difference between the theoretical and experimental mass of myosin spot (223.3 kDa vs. 38.3 kDa) was very high and therefore it reasonable to suppose that spot number 4 is a myosin fragment. However, the presence of actin was more relevant than myosin based on the comparison of their RC (2.51 vs. 0.05). It has been reported that the actomyosin complex suffers a greater degradation in dry-cured ham during processing. Particularly, it has been described as an extensive degradation of myosin heavy chain while actin is remarkably more stable at this high ionic condition (Fabbro et al., 2016; Wang et al., 2017). Furthermore, a heating process causes the rupture of hydrogen bonds of proteins, in contrast to HPP-0 treatment, which affects the hydrophobic and electrostatic interactions (Duranton et al., 2012). For this reason, the proteomic analysis was carried out at 0 °C to avoid the temperature effect. In addition, the high ionic strength conditions during dry-cured ham process could change the myosin light chain conformation affecting the binding of myosin with actin through to their phosphorylation and dephosphorylation. In saline conditions, actin shows a high degree of phosphorylation improving its stability against μ-calpain (Wang et al., 2017). This suggests that salt curing of
85
IV. Results and discussion dry-cured ham produced an intense degradation of myosin that at the same time was unaffected by HPP. Regarding actin, the salty conditions should make this protein more stable, but the HPP-0 induces their fragmentation, confirmed by the presence of some actin fragments on the gel. According to Kęska and Stadnik (2017), myofibrillar proteins, specifically myosin-2, are precursors of peptides and amino acids which have a strong impact on the taste of dry-cured meat, while sarcoplasmic proteins had not incidence on taste-active components generation. On the other hand, two spots were identified as triosephosphate isomerase with a total RC of approximately 0.31. Triosephosphate isomerase is an enzyme that increases the glycolytic metabolism and produces NADH and ATP, consequently, it is strongly correlated with meat quality (Gagaoua, Monteils, Couvreur, and Picard, 2017; Kim and Dang, 2005; Schilling et al., 2017). In this proteomic study, spot number 1 identified as triosephosphate isomerase has an experimental mass similar to theoretical mass which would indicate that the protein is entire. On the contrary, the spot number 15 could suggest that there were protein aggregates even under reducing conditions since the theoretical mass was lower than experimental mass as occurred previously in other treatments (Di Luccia et al., 2015).
Table 18. Spot volumes with significant differences by the effect of a high-pressure treatment in sliced dry-cured ham. Fold change (FC) and relative change (RC) of the selected spots Spot Protein (gene name) Control HPP FC RC no. Mean ± SE Mean ± SE 1 Triosephosphate isomerase (TPI1) 124.48±38.76 249.53±45.25 +2 +0.25 2 Alpha actin 1 (ACTC1) – 337.46±152.42 +∞ +0.68 3 Alpha actin 1 (ACTC1) – 134.77±38.14 +∞ +0.27 4 Myosin-7 (MYH7) – 43.11±15 +∞ +0.09 5 Alpha actin 1 (ACTC1) 486.98±80.85 897.29±162.25 +1.84 +0.83 6 Alpha actin 1 (ACTC1) 340.11±52.4 191.86±47.54 −1.77 −0.3 7 Hemoglobin subunit beta (HBB) 109.81±30.37 35.04±10.43 −3.13 −0.15 8 Uncharacterized protein 179.16±11.03 227.48±18.14 +1.27 +0.1 9 Alpha actin 1 (ACTC1) 151.05±20.66 574.66±147.24 +3.8 +0.85 10 Alpha actin 1 (ACTC1) 132.3±27.02 473.8±110.71 +3.58 +0.69 11 Alpha actin 1 (ACTC1) 99.09±23 56.07±14.76 −1.77 −0.09 12 Alpha actin 1 (ACTC1) – 411.04±143.85 +∞ +0.83 13 Uncharacterized protein – 496.4±129.27 +∞ +1 14 Alpha actin 1 (ACTC1) – 175.91±82.73 +∞ +0.35 15 Triosephosphate isomerase (TPI1) 137.34±4.72 288.9±25.18 +2.1 +0.31 16 Uncharacterized protein – 142.94±46.31 +∞ +0.29 17 Alpha actin 1 (ACTC1) – 269.59±81.93 +∞ +0.54 18 Uncharacterized protein 153.73±16.46 – −∞ −0.31
IV.3.3. INSTRUMENTAL EVALUATION OF THE ORGANOLEPTIC PRECURSOR CHANGES
IV.3.3.1. Effects on free amino acid profile and taste implications
Table 19 shows the effect of different HPP-temperature treatments on the FAA content (expressed as mg/100 g DM) of dry-cured ham. Statistical analysis showed that total FAA
86 IV. Result and discussion content was significantly (P < 0.001) affected by treatments. HPP-35 group displayed the highest values (5313.16 vs. 4787.30 vs. 5072.48 vs. 6415.63 mg/100 g DM for CO, HPP-0, HPP- 20 and HPP-35 treatments, respectively).
Table 19. Effect of different HPP treatments on FAAs content (expressed as mg/100 g DM) of dry-cured ham Amino Acid/ Group SEM P-value Sensory attributes CO HPP-0 HPP-20 HPP-35 Aspartic acid 185.15b 119.45a 145.99a 240.20c 5.64 <0.001 Serine 201.65a 198.30a 200.45a 251.15b 5.65 <0.001 Glutamic acid 450.17a 382.41a 424.71a 588.97b 12.11 <0.001 Glycine 196.45a 202.46a 200.03a 238.69b 4.44 <0.001 Histidine 102.51b 82.62a 87.44ab 127.69c 2.88 <0.001 Taurine 93.22ab 83.48a 92.04ab 102.21b 2.30 0.045 Arginine 410.98b 295.84a 346.32ab 513.00c 11.99 <0.001 Threonine 221.57ab 223.76ab 216.03a 254.45b 5.28 0.037 Alanine 419.87a 471.30a 478.99ab 546.58b 10.75 <0.001 Proline 287.50 276.28 286.14 314.32 5.69 0.105 Cysteine 346.79b 61.44a 51.44a 553.79c 23.11 <0.001 Tyrosine 202.96c 116.37a 160.34b 121.24a 4.78 <0.001 Valine 393.22a 461.58b 471.45b 441.94ab 8.85 0.007 Methionine 203.84 205.41 216.23 223.19 4.30 0.330 Lysine 265.09a 256.41a 290.14a 448.34b 10.24 <0.001 Isoleucine 351.76 377.63 387.34 403.47 7.94 0.119 Leucine 588.53 629.06 654.06 664.31 12.86 0.146 Phenylalanine 391.88 343.50 363.34 382.06 7.05 0.086 Total Amino Acids 5313.16 4787.30 5072.48 6415.63 112.28 <0.001 Sweet 1 1310.60a 1372.10a 1379.22a 1587.65b 29.32 0.003 Bitter 2 1921.99 2017.17 2092.42 2083.20 38.73 0.362 Acid 3 737.84b 605.81a 667.62ab 956.87c 20.45 <0.001 Aged 4 653.20b 513.87a 601.86ab 833.88c 17.72 <0.001 a–c Mean values in the same row (corresponding to the same amino acid/sensory attribute) not followed by a common letter differ significantly (P < 0.05; Tukey's Test); SEM: standard error of the mean. 1Sweet flavour=Σ of alanine, glycine, threonine, serine and proline; 2Bitter flavour=Σ of leucine, valine, isoleucine, methionine and phenylalanine; 3Acid flavour=Σ of glutamic acid, aspartic acid and histidine; 4Aged flavour=Σ of lysine, tyrosine and aspartic acid.
No significant differences were observed among CO, HPP-0 and HPP-20 treatments. These values were in the range values 4000–7000 mg/100 g DM that was reported in previous studies (Bermúdez et al., 2014) on dry-cured ham FAAs composition. The higher total FAA content in HPP-35 samples was expected since it is well known that proteins are greatly influenced by temperature, so their structures could be degraded into smaller amino acids. In this regard, 13 of the 18 amino acids studied were significantly influenced by temperature- assisted HPP treatments. The samples submitted to HPP at 35 °C had the highest content in 12 amino acids (aspartic acid, serine, glutamine, glycine, histidine, taurine, arginine, threonine, alanine, cysteine, valine, and lysine). Tyrosine was the only amino acid that presented the highest level in untreated samples. Changes in individual AA content could promote changes in the final flavour of dry-cured ham (Hidalgo and Zamora, 2004; Jurado et al., 2007). Thereby, the higher content in specific amino acids showed in HPP-35 samples may influence the perception of sweet, acid and aged attributes in comparison to other treated and untreated
87
IV. Results and discussion
samples. In addition, previous studies showed that an increment of bitter taste in hams could be attributed to excessive proteolysis (Careri et al., 1993; Parolari, Virgili, and Schivazappa, 1994). However, the AAs responsible for the bitter taste were not affected by any treatment in the present study.
IV.3.3.2. Effects on the volatile profile and odour implications
Significant differences (P < 0.001) among treatments were found in the total content of volatile compounds. The highest values were observed in the HPP-35 batch (78,561.29 AU×103/g of dry-cured ham) while the lowest contents were obtained from the HPP-0 batch (28,487.02 AU×103/g of dry-cured ham) (Table 20).
Table 20. Levels of the main families of volatile compounds identified in untreated and HPP at 0° C, 20° C and 35° C treated dry-cured ham (expressed as quantified area units of the EIC (AU-EIC) x 103/g dry-cured ham) Compound Group SEM P-value CO HPP-0 HPP-20 HPP-35 Aliphatic 20538.44b 9197.34a 33631.08c 43688.86d 1469.50 <0.001 hydrocarbons Aromatic and cyclic 902.09c 486.35a 747.07b 1027.21c 27.31 <0.001 hydrocarbons Hydrocarbons 21440.53b 9683.69a 34385.34c 44716.06d 1489.52 <0.001 Aldehyde 22467.49c 9840.67a 12562.24a 17443.40b 607.51 <0.001 Ketone 2454.69a 2166.36a 3890.57b 5138.82c 124.64 <0.001 Esther and ether 1659.26a 1608.06a 1761.60a 2272.76b 43.34 <0.001 Alcohol 6465.70c 3900.03a 5295.68b 6826.08c 161.75 <0.001 Carboxylic acid 1027.64c 321.09a 680.18b 660.01b 31.48 <0.001 Nitrogenous compounds 585.65bc 524.52b 422.16a 648.02c 13.80 <0.001 Sulphur compounds 2178.20b 321.41a 206.63a 400.44a 88.24 <0.001 Chloro compounds 270.11b 121.20a 482.85c 455.68c 16.93 <0.001 Total Compounds 58549.27b 28487.02a 59641.13b 78561.29c 1986.98 <0.001 a-c Mean values in the same row (corresponding to the same parameter) not followed by a common letter differ significantly (P<0.05; Tukey´s Test). SEM: standard error of the mean.
In comparison to CO treatment, the samples after HPP-0 treatment showed significant declines in hydrocarbons, aldehydes, alcohols, carboxylic acids, sulphur compounds and chloro compounds content by 55%, 56%, 40%, 69%, 85% and 55%, respectively. Aldehydes, alcohols, carboxylic acids, nitrogenous and sulphur compounds content were reduced by 44%, 18%, 34%, 28% and 91%, respectively, in HPP-20 treated samples, while hydrocarbons, ketones and chloro compounds were incremented by 60% 58% and 79%, respectively, in comparison to CO. Furthermore, samples treated with HPP at 35 °C presented reduction in the aldehydes, carboxylic acid and sulphur compounds (22%, 36% and 82%, respectively) while hydrocarbons, ketones, ether and esters and chloro compounds were incremented by 109%, 109%, 37% and 69%, respectively, in comparison to CO. Since aldehydes, ketones, ester and ethers, and alcohols (to a limited extent) are the main families associated with the aroma of dry-cured ham (Carrapiso et al., 2010; García-González et al., 2008), the temperature-assisted HPP treatments may affect the quality of the final product. In this way, the HPP-35 treatment
88 IV. Result and discussion enhanced ester and ether contents, which are responsible for fruity odour notes. Meanwhile, all of HPP treatments caused a significant reduction of sulphur compounds, a fact that could modify the aroma by decreasing rotten egg and burnt notes. This data is in agreement with the results obtained by Martínez-Onandi et al. (2016b) in sliced Serrano dry-cured ham treated at 600 MPa and 21 °C for 2.5 min.
A total of 149 volatile compounds were identified and classified based on their origin according to Narváez-Rivas et al. (2012), Martín et al. (2006) and Fonseca et al. (2015). Of the 149 compounds, 92 were presumably originated from lipid oxidation, 21 were derived from proteolysis reactions, 21 were attributed to microbial activity and 15 had an unknown origin. Table 21 lists the compounds detected in the volatile fraction of the slices of dry-cured ham, as well as the effect of HPP treatments, the linear retention indexes, the ions used for quantification and the method used for identification. One hundred forty seven out of the 149 identified volatile compounds were significantly influenced by the HPP treatment. Regarding the origin of these compounds, the most probable origin was lipolysis, followed by proteolysis and microbial activity. The sum of secondary products of lipid oxidative decomposition was around 80% of the total volatile content in all treatments except for HPP-0, in which such compounds accounted for 67%. In contrast, the compounds derived from proteolysis represented 20% of the total volatile compounds in the HPP-0 group and 8–9% in the other treatments. These differences within families can be explained by the high temperatures (which promote lipolysis) and HPP that can induce protein denaturation (Guyon et al., 2018). Similar results were found by Martínez-Onandi et al. (2017) and Ramírez and Cava (2007) who reported values around 75% and 81.6% of total compounds were associated with lipid oxidation, respectively, and values of 20% and 12.7% of total compounds were attributed to proteolysis, respectively. Previous studies observed that the application of HPP at pressures below 300 MPa have minimum effect on lipid oxidation but higher pressures give an increase in the amount of aldehydes derived from lipolysis (Andrés et al., 2004; Fuentes et al., 2010). In contrast, Martínez-Onandi et al. (2016b) did not find any significant effect on linear aldehydes content in dry-cured ham treated at 600 MPa, and these authors concluded that HPP only influenced volatile compounds originated from microbial activity. Moreover, the majority of the most abundant volatile compounds were identified in either CO or HPP-35 samples (64 and 61 compounds, respectively).
Among the lipolysis-derived compounds, hexanal was the most abundant one, particularly in untreated samples. Conversely, the lowest value was observed in the HPP-0 batch. Interestingly, an increasing trend in hexanal content was observed as the temperature of treatment increased. This fact can be explained by the potential protective effect of the HPP against hexanal generation. This finding could be considered positive since high levels of this compound gives rancid notes to ham. In contrast, the aroma can turn grassier and more pleasant because of the hexanal reduction (Aparicio and Morales, 1998). On the contrary, previous studies on the effects of HPP on dry-cured hams showed that HPP increased the rancid odour perception due to an increment in aldehydes (Clariana et al., 2011; Fuentes et al., 2010). In agreement to Martínez-Onandi et al. (2017), nonanal, propanoic acid, butanoic acid, pentanoic acid, hexanoic acid and pentanal showed higher levels in untreated than in HPP samples. However, lower values of 2-pentanol were obtained from untreated samples.
89
IV. Results and discussion
Additionally, 1-Octen- 3-ol, a characteristic compound of dry-cured ham with a very low threshold in “Montanera hams” (Jurado et al., 2009), did not show significant differences between CO and HPP-35 samples, although the other two treatments (HPP-0 and HPP- 20) showed significantly lower values.
As expected, the main microbial activity-derived compounds detected in this study were esters whose formation are closely related to the microbial activity (Ramírez and Cava, 2007). Also, it is well known that temperature affects the ester compounds formation (Gorbatov and Lyaskovskaya, 1980). For this reason, it was no strange that CO and HPP-35 samples presented higher amounts of microbial activity-derived compounds than HPP-0 and HPP-20 samples, and, in the same way, the HPP-20 group presented higher values than HPP-0. Dimethyl disulphide was the main compound detected in CO samples, but it was greatly reduced by HPP treatments (1786.20 AU×103/g of dry-cured ham vs. 160.12 AU×103/g of dry-cured ham vs. 61.83 AU×103/g of dry-cured ham vs. 213.32 AU×103/g of dry-cured ham for CO, HPP-0, HPP-20 and HPP-35, respectively). Although the origin of dimethyl disulphide is usually related to the microbial activity, some previous studies established that amino acid catabolism can be another possible via (Ramírez and Cava, 2007; Sabio et al., 1998). Moreover, Muriel et al. (2004) found that dimethyl disulphide could result from the reaction between lipid oxidation products and cysteine. In the present study, a positive and significant (P < 0.05) correlation between cysteine and dimethyl disulphide formation cannot be discarded.
The compounds derived from proteolysis found in the present study that have been previously detected in dry-cured ham were 2-methyl propanal, 3-methyl butanal, 2-methyl butanal and 2- methyl-2-butenal (Andrés, Cava, and Ruiz, 2002; Sánchez-Peña et al., 2005; Timón et al., 2001). The highest values of 2-methyl propanal, 3-methyl butanal and 2-methyl- 2-butenal were observed in HPP-35 samples. Particularly for 2- methyl butanal, all HPP-treated samples (independently of assisted temperature) displayed higher values than CO samples, which may be due to the HPP effect on protein structures. Moreover, the statistical analysis showed a positive correlation (r=0.263, P < 0.01) between 2-methyl butanal and isoleucine, being the degradation of isoleucine the most probable origin of this compound (Ramírez and Cava, 2007).
Finally, fifteen compounds were classified as “unknown origin” which probable via/reaction was not found in the literature. Since their origin is not clear, it is not possible to include them into the three principal origins already commented. It is worth mentioning that the presence of p-Cresol could be associated with animal feed and further accumulation in the animal tissues (Sabio et al., 1998; Sánchez- Peña et al., 2005). The HPP-35 and CO samples showed higher contents of p-Cresol than HPP-0 and HPP-20 samples. Aromatic and cyclic hydrocarbons were also found: 1,3-dimethyl benzene, 1-ethyl-3-methyl cyclopentane and ethyl cyclopentane contents were reduced by HPP treatment.
90 IV. Result and discussion
Table 21. Effect of HPP treatments on volatile compound content (expressed as quantified area units of the EIC (AU-EIC)×103/g dry-cured ham) Group Compound m/z LRI R SEM P-value CO HPP-0 HPP-20 HPP-35 c a b b Pentane Ɏ 43 516 ms, lri, s 1166.82 341.36 568.52 722.43 39.322 <0.001 b a a a Propanal Φ 58 526 ms, lri, s 133.06 26.37 38.29 42.57 5.109 <0.001 ab b a c Acetone Φ 58 528 ms, lri 171.04 256.36 151.17 387.56 14.734 <0.001 a bc b c Isopropyl Alcohol ɎΦ 45 532 ms, lri 88.41 218.13 191.73 226.23 6.570 <0.001 a a b c 2,3-Hexanedione Φ 41 562 ms, lri 356.04 290.13 1252.05 2096.88 84.736 <0.001 a a b c n-Hexane Ɏ 57 562 ms, lri, s 822.88 706.36 2744.00 4705.52 185.161 <0.001 a b a c 1-Butene, 2,3-dimethyl- Ɏ 69 571 ms, lri 12.15 22.71 14.63 30.09 1.043 <0.001 a b bc c 1-Propanol ɎΦ 59 572 ms, lri 27.91 42.80 52.10 57.38 1.885 <0.001 c a ab b Butanal ɎΦ 72 584 ms, lri, s 24.28 8.00 11.96 15.52 0.808 <0.001 a ab b ab 2-Butanone Φ 72 596 ms, lri 182.67 255.04 281.36 247.02 11.320 0.012 a bc b c 2-Butanol ɎΦ 45 607 ms, lri 13.92 28.77 24.25 33.51 1.009 <0.001 c a a b Cyclopentanone, 3-methyl- Ɏ 56 667 ms, lri 45.68 9.53 16.76 28.13 1.683 <0.001 c a ab b Heptane Ɏ 71 675 ms, lri, s 1321.57 203.20 353.71 555.71 49.078 <0.001 c a ab b Furan, 2-ethyl- ɎΦ 81 703 ms, lri 40.15 7.25 12.78 16.35 1.492 <0.001 a a b b 1-Butanol ɎΦ 56 707 ms, lri 17.51 16.42 27.14 32.09 1.027 <0.001 b a ab c 2-Pentanone Φ 86 720 ms, lri 98.54 59.43 86.95 144.70 4.956 <0.001 c a a b Pentanal ɎΦ 57 728 ms, lri, s 1190.56 378.49 491.34 771.43 44.792 <0.001 c a b b 1-Penten-3-ol ɎΦ 57 730 ms, lri 1099.81 350.04 552.91 666.28 35.716 <0.001 a b b c 2-Pentanol ɎΦ 45 751 ms, lri 86.03 311.24 222.00 413.17 17.625 <0.001 b a a a Pentane, 2,3,4-trimethyl- Ɏ 71 756 ms, lri 181.85 115.66 98.42 108.51 6.903 <0.001 b a a a Pentane, 2,3,3-trimethyl- Ɏ 71 763 ms, lri 252.09 184.67 125.65 131.57 9.770 <0.001 b a a a Pentane, 3-ethyl- Ɏ 70 770 ms, lri 46.85 25.18 15.13 15.93 1.820 <0.001 b a c d 1-Pentene, 3-ethyl-2-methyl- Ɏ 83 774 ms, lri 32.62 14.34 55.02 85.19 2.815 <0.001 c b a a Hexane, 2,2,5-trimethyl- Ɏ 57 800 ms, lri 355.20 198.20 86.68 85.66 15.519 <0.001 c a ab b Octane Ɏ 85 822 ms, lri, s 3308.36 587.64 907.92 1277.65 124.815 <0.001 c a b b Propanoic acid ɎΦ 74 827 ms, lri 12.64 3.76 8.35 8.05 0.591 <0.001 c a a b 2-Octene, (E)- Ɏ 112 833 ms, lri 342.80 75.77 120.74 208.17 12.002 <0.001
91
IV. Results and discussion
Group Compound m/z LRI R SEM P-value CO HPP-0 HPP-20 HPP-35 c b a a Heptane, 3,4,5-trimethyl- Ɏ 85 842 ms, lri 76.88 49.25 9.68 9.50 3.621 <0.001 c a a b 3-Octene, (E)- Ɏ 112 845 ms, lri 170.74 39.14 55.81 99.32 6.553 <0.001 c a a b 1-Pentanol ɎΦ 55 847 ms, lri, s 500.17 136.34 220.83 385.19 19.128 <0.001 d a b c Hexanal ɎΦΨ 56 865 ms, lri 15270.28 3980.78 6595.32 9404.24 510.237 <0.001 b b a a Hexane, 2,2,5,5-tetramethyl- Ɏ 57 914 ms, lri 387.10 304.54 87.89 130.89 17.892 <0.001 c a b a Butanoic acid ɎΦ 60 918 ms, lri 191.58 50.50 113.16 69.28 7.191 <0.001 b a a b 4-Nonene Ɏ 70 926 ms, lri 198.55 128.93 152.16 230.92 7.011 <0.001 a a a b Heptane, 2-methyl-3-methylene- Ɏ 126 930 ms, lri 17.87 11.42 17.45 28.24 1.028 <0.001 c b a ab Nonane Ɏ 57 936 ms, lri, s 201.88 123.93 57.32 84.50 7.840 <0.001 b a a b 2-n-Butyl furan Ɏ 81 944 ms, lri 39.67 13.34 20.37 39.72 1.702 <0.001 a a b c 3-Heptanone Φ 57 960 ms, lri 42.37 47.19 70.34 117.12 3.546 <0.001 a a a b 2-Heptanone Ɏ 58 967 ms, lri 455.34 344.09 434.04 620.46 19.443 <0.001 c a ab b Heptanal ɎΦ 70 974 ms, lri, s 988.97 251.92 396.51 512.02 33.687 <0.001 ab a ab b 2-Nonen-4-one Φ 69 979 ms, lri 16.18 14.73 15.98 20.45 0.671 0.014 b a a ab 2-Octene, 4-ethyl- Ɏ 69 982 ms, lri 146.99 106.14 106.60 126.89 5.230 0.013 bc a ab c Octane, 3-methyl-6-methylene- Ɏ 70 985 ms, lri 303.36 198.09 243.42 365.97 13.905 <0.001 b ab a ab Octane, 4-ethyl- Ɏ 69 991 ms, lri 90.08 72.48 68.06 83.64 2.594 0.007 ab a a b 2-Hepten-4-one, 6-methyl- Ɏ 69 992 ms, lri 91.95 73.46 71.13 102.44 3.405 <0.001 b a a ab Pentane, 3,3-dimethyl- Ɏ 85 995 ms, lri 9.35 5.33 4.84 7.22 0.400 <0.001 a a a b Methional ɎΦ 104 999 ms, lri 201.58 211.98 252.49 387.58 15.839 <0.001 c b a b Nonane, 2,3-dimethyl- Ɏ 71 1003 ms, lri 87.74 62.48 40.21 63.90 3.371 <0.001 ab a ab b 1-Octene, 2,6-dimethyl- Ɏ 56 1010 ms, lri 104.76 77.37 89.61 119.04 4.298 0.004 ab a a b 3-Octene, 4-ethyl- Ɏ 69 1012 ms, lri 29.19 20.38 25.38 38.69 1.441 <0.001 ab a a b Nonane, 3-methylene- Ɏ 70 1022 ms, lri 236.74 188.34 180.06 284.91 10.133 <0.001 a a b c Heptane, 2,2,4,6,6-pentamethyl- Ɏ 57 1027 ms, lri 5140.73 1929.11 21626.35 27733.83 1183.950 <0.001 c c b a Decane Ɏ 57 1030 ms, lri, s 406.72 324.13 225.36 65.94 16.694 <0.001 ab a a b 3-Ethyl-3-hexene Ɏ 83 1042 ms, lri 62.24 47.97 56.07 78.99 2.493 <0.001
92 IV. Result and discussion
Group Compound m/z LRI R SEM P-value CO HPP-0 HPP-20 HPP-35 c a b bc 1-Heptanol ɎΦ 70 1046 ms, lri 91.50 36.91 62.38 71.79 3.218 <0.001 c a b c 1-Octen-3-ol ɎΦ 57 1051 ms, lri 3935.68 1915.73 2824.65 3607.07 117.522 <0.001 b ab a ab 5-Hepten-2-one, 6-methyl- Ɏ 69 1056 ms, lri 128.81 120.63 93.86 116.58 3.997 0.011 ab a b c 2-Octanone Ɏ 58 1059 ms, lri 41.33 38.50 51.94 72.99 2.061 <0.001 b a a a Octanal ɎΦ 56 1066 ms, lri, s 384.48 182.91 209.96 231.97 12.175 <0.001 a a b c Undecane, 3,6-dimethyl- Ɏ 57 1068 ms, lri 162.81 83.59 608.60 879.39 39.201 <0.001 b a a a Pentanoic acid ɎΦ 60 1083 ms, lri 394.31 212.80 257.50 210.40 13.552 <0.001 a a a b Undecane, 2,5-dimethyl- Ɏ 57 1085 ms, lri 163.08 142.14 186.12 258.59 9.209 <0.001 b b a b Decane, 2,3,5-trimethyl- Ɏ 57 1099 ms, lri 80.58 76.68 44.70 70.74 2.882 <0.001 a a ab b Undecane Ɏ 57 1113 ms, lri, s 1117.99 1034.94 1442.34 1864.94 73.182 <0.001 c a ab b 2-Octenal, (E)- ɎΦ 70 1123 ms, lri 52.79 9.63 19.13 22.76 1.993 <0.001 c a ab b 2,3-Dimethyl-3-heptene, (Z)- Ɏ 83 1123 ms, lri 56.77 15.27 23.21 27.74 1.972 <0.001 b a a a 1-Octanol ɎΦ 56 1127 ms, lri 63.72 43.46 48.67 48.86 1.806 <0.001 c a ab bc Decanal ɎΦ 81 1129 ms, lri, s 24.44 14.33 18.50 21.73 0.769 <0.001 b ab a b 2-Undecene, 9-methyl-, (Z)- Ɏ 70 1132 ms, lri 384.25 344.35 275.11 383.03 13.208 0.007 a ab a b 3-Nonanone Φ 113 1134 ms, lri 21.19 25.18 21.20 29.87 0.914 <0.001 a b c d 2-Nonanone Φ 58 1141 ms, lri 15.69 24.12 32.95 46.71 1.362 <0.001 bc ab a c 5-Undecene, 6-methyl- Ɏ 168 1144 ms, lri 11.40 9.13 7.32 12.51 0.477 <0.001 b a a a Nonanal ɎΦ 57 1148 ms, lri, s 538.49 307.02 303.28 327.20 14.872 <0.001 b ab a ab 4,4-Dipropylheptane Ɏ 85 1153 ms, lri 55.24 44.54 34.85 43.85 1.804 0.001 b ab a ab 5-Hexen-3-one Φ 57 1161 ms, lri 43.40 35.58 27.78 35.50 1.538 0.003 2-Undecene, 3-methyl-, (E)- Ɏ 70 1181 ms, lri 59.72 53.90 48.65 60.81 1.998 0.102 a a ab b Dodecane Ɏ 57 1188 ms, lri, s 701.16 663.91 932.47 1179.62 41.201 <0.001 b ab a ab 4-Nonene, 5-butyl- Ɏ 70 1197 ms, lri 24.54 22.39 18.01 20.65 0.857 0.043 b a a a 4-Nonenal, (E)- Ɏ 83 1201 ms, lri 31.24 18.32 18.06 23.63 1.001 <0.001 c a b b Octanoic acid ɎΦ 60 1224 ms, lri 30.74 9.66 21.75 18.36 1.197 <0.001 ab ab a b 1-Tetradecanol ɎΦ 68 1225 ms, lri 32.34 29.38 26.20 34.48 1.132 0.047
93
IV. Results and discussion
Group Compound m/z LRI R SEM P-value CO HPP-0 HPP-20 HPP-35 b a a ab Decane, 3-ethyl-3-methyl- Ɏ 57 1228 ms, lri 50.73 38.80 32.66 41.49 1.431 <0.001 c ab a bc 1-Tetradecene Ɏ 97 1236 ms, lri 30.34 23.99 19.00 25.23 0.845 <0.001 ab a bc c Tridecane Ɏ 71 1258 ms, lri, s 190.43 156.70 245.19 316.86 11.383 <0.001 b a a a 2-Decenal, (E)- Φ 70 1272 ms, lri 24.68 12.77 15.38 16.81 0.849 <0.001 b a a a 2,4-Decadienal, (E,E)- ɎΦ 81 1315 ms, lri 27.22 4.07 6.90 7.73 1.213 <0.001 b a a a 2-Undecenal ɎΦ 95 1339 ms, lri 5.96 1.11 1.42 1.92 0.266 <0.001 a bc b c Pentadecanal- Φ 82 1516 ms, lri, s 2.15 8.56 6.84 10.40 0.443 <0.001 b a b c Total lipolysis origin 45879.67 19311.22 47734.58 64466.40 1832.629 <0.001 b a a a Carbon disulphide Ɏ 76 533 ms, lri 225.07 119.20 119.13 148.43 8.542 <0.001 a ab ab b Propanal, 2-methyl- ɎΦ 72 557 ms, lri 161.98 225.54 190.25 241.26 9.171 0.008 c a a b Fumaronitrile Ɏ 78 646 ms, lri 29.18 11.61 10.27 21.33 0.948 <0.001 a a a b Butanal, 3-methyl- ɎΦ 58 659 ms, lri 1525.03 1973.95 1925.24 2925.94 86.149 <0.001 a b b b Butanal, 2-methyl- ɎΦ 57 671 ms, lri 758.92 1291.93 1195.03 1278.51 52.705 <0.001 a a a b 2-Butenal, 2-methyl- ɎΦ 84 801 ms, lri 76.70 63.38 53.95 106.33 3.759 <0.001 a c b b 1-Butanol, 3-methyl- ɎΦΨ 55 808 ms, lri 65.27 425.21 204.01 240.17 16.858 <0.001 a c b bc 1-Butanol, 2-methyl- ɎΦ 57 812 ms, lri 14.93 51.65 37.05 44.65 1.890 <0.001 c a b c Propanoic acid, 2-methyl- ɎΦ 73 888 ms, lri 51.23 18.99 34.63 60.85 2.289 <0.001 c a a b 2-Propanol, 2-methyl- ɎΦ 59 894 ms, lri 17.68 6.80 7.49 12.68 0.556 <0.001 b a a a 3-(1'-pyrrolidinyl)-2-butanone Ɏ 98 906 ms, lri 136.09 83.70 74.19 94.24 4.904 <0.001 a ab a b 3-Pentanol, 2,4-dimethyl- ɎΦ 73 954 ms, lri 7.84 8.75 7.43 10.29 0.236 <0.001 c a b bc Butanoic acid, 3-methyl- ɎΦ 60 969 ms, lri 349.62 139.30 250.02 327.58 14.309 <0.001 ab bc a c Pyrazine, 2,6-dimethyl- ɎΦ 108 978 ms, lri 290.52 344.23 241.18 395.39 10.530 <0.001 bc a ab c 1-(1'-pyrrolidinyl)-2-butanone Ɏ 84 982 ms, lri 129.87 84.97 98.05 138.36 5.375 <0.001 b a a a Dimethyl trisulphide ɎΦ 126 1035 ms, lri 179.23 13.88 7.19 11.39 8.662 <0.001 a a a b Benzaldehyde ɎΦ 106 1045 ms, lri 339.48 350.30 295.39 462.27 11.745 <0.001 ab a a b 1-Heptanol, 2,4-diethyl- ɎΦ 69 1085 ms, lri 90.78 73.41 69.19 108.27 3.947 <0.001 a b d c 2-Ethyl-1-hexanol ɎΦ 57 1094 ms, lri 6.65 58.12 128.81 86.52 5.083 <0.001
94 IV. Result and discussion
Group Compound m/z LRI R SEM P-value CO HPP-0 HPP-20 HPP-35 b a a a 5-Ethylcyclopent-1-enecarboxaldehyde 124 1099 ms, lri 27.85 10.17 13.43 14.93 0.934 <0.001 a ab ab b 2(3H)-Furanone, 5-ethyldihydro- ɎΦ 85 1158 ms, lri 174.77 197.82 178.62 215.24 5.130 0.014 b a a ab 4-Methyl-5-decanol ɎΦ 55 1162 ms, lri 21.99 12.83 15.13 17.55 0.750 <0.001 b b a b Sulphurous acid, butyl dodecyl ester ɎΦ 85 1304 ms, lri 27.37 28.21 18.48 27.32 0.772 <0.001 a a a b Total proteolysis origin 4708.05 5593.97 5174.15 6989.50 154.115 <0.001 a a a b Pentane, 2-methyl- ɎΦ 71 543 ms, lri 2.61 1.20 2.75 13.89 0.559 <0.001 bc b a c Acetic acid ethenyl ester Ɏ 86 588 ms, lri 25.79 21.53 14.90 28.64 0.898 <0.001 a b ab ab Ethyl Acetate Ɏ 61 598 ms, lri 148.15 238.69 213.97 215.97 10.382 0.013 b a b c Methane, oxybis[dichloro- Ɏ 83 611 ms, lri 270.11 121.20 318.55 455.68 16.324 <0.001 c a ab bc Propanoic acid, ethyl ester Ɏ 57 737 ms, lri 52.51 18.56 30.57 42.12 2.190 <0.001 b a a a Disulphide, dimethyl Φ 94 781 ms, lri 1786.20 160.12 61.83 213.32 77.445 <0.001 a a a b Butanoic acid, ethyl ester Ɏ 71 855 ms, lri 86.10 78.86 68.63 136.66 4.090 <0.001 c a ab bc Octane, 2-methyl- ɎΦ 71 899 ms, lri 16.79 10.29 11.93 15.87 0.627 <0.001 a ab a b Butanoic acid, 2-methyl-, ethyl ester Ɏ 102 908 ms, lri 49.05 65.69 56.95 85.20 2.925 <0.001 a a a b Butanoic acid, 3-methyl-, ethyl ester Ɏ 88 913 ms, lri 130.87 170.48 153.26 280.27 11.318 <0.001 c b a ab Oxalic acid, butyl propyl ester Ɏ 57 936 ms, lri 201.88 123.93 57.32 84.50 7.840 <0.001 a a b c Ethanol, 2-butoxy- Ɏ 57 985 ms, lri 431.27 353.25 797.03 947.37 29.190 <0.001 b ab a ab Carbonic acid, bis(2-ethylhexyl) ester Ɏ 112 1003 ms, lri 30.15 24.44 17.32 23.12 1.093 <0.001 a a a b Hexanoic acid, ethyl ester Ɏ 88 1050 ms, lri 167.48 205.07 205.07 274.88 7.699 <0.001 a a b c Tridecane, 6-methyl- ɎΦ 57 1079 ms, lri 323.72 207.70 636.89 884.36 35.976 <0.001 c a ab b 2-Piperidinecarboxylic acid, 1-acetyl-, ethyl ester Ɏ 84 1124 ms, lri 36.93 12.69 15.86 20.18 1.073 <0.001 Octanoic acid, ethyl ester Ɏ 88 1204 ms, lri 74.38 80.58 73.08 68.96 1.749 0.149 a a b c Dodecane, 2-methyl- ɎΦ 88 1233 ms, lri 22.03 23.92 40.66 53.41 2.015 <0.001 b b a b Tridecane, 3-methyl- ɎΦ 85 1304 ms, lri 27.62 28.46 18.60 27.30 0.823 <0.001 c b a ab Decanoic acid, ethyl ester Ɏ 88 1336 ms, lri 28.59 20.50 12.30 16.53 0.860 <0.001 a a a b 2,2,4-Trimethyl-1,3-pentanediol diisobutyrate Ɏ 71 1442 ms, lri 10.00 5.82 2.64 58.11 2.772 <0.001 c a b c Total microbial origin 3922.23 1972.97 2810.12 3946.33 97.439 <0.001
95
IV. Results and discussion
Group Compound m/z LRI R SEM P-value CO HPP-0 HPP-20 HPP-35 b a ab c Acetoin 45 787 ms, lri 478.36 299.84 367.90 625.50 19.901 <0.001 b a b c Cyclobutane, 1,1,2,3,3-pentamethyl- 70 813 ms, lri 326.38 172.20 402.58 569.07 20.318 <0.001 bc b a c Ethylbenzene 91 917 ms, lri 21.49 18.32 13.48 21.96 0.538 <0.001 c bc a ab Benzene, 1,3-dimethyl- 106 926 ms, lri 27.03 24.88 18.01 22.00 0.647 <0.001 ab a a b Cyclohexanone, 2-ethyl- 69 972 ms, lri 62.10 44.11 44.04 70.01 3.140 0.004 bc a ab c 4-Octanone, 5-hydroxy-2,7-dimethyl- 69 1042 ms, lri 13.29 9.05 10.38 14.64 0.447 <0.001 b a ab b 4-Ethylcyclohexanol 81 1104 ms, lri 120.90 86.88 106.36 118.21 3.718 0.006 b a a b Benzeneacetaldehyde 91 1119 ms, lri 712.67 514.37 501.69 680.17 18.764 <0.001 c a ab b Cyclopentane, 1-ethyl-3-methyl- 83 1123 ms, lri 56.77 15.27 23.21 27.74 1.972 <0.001 a b c d Benzyl alcohol 108 1124 ms, lri 125.21 291.08 425.52 552.39 17.344 <0.001 a a b c 1-Hexanone, 5-methyl-1-phenyl- 105 1137 ms, lri 11.53 11.66 34.01 75.99 2.754 <0.001 b a a a Cyclopentane, ethyl- 98 1148 ms, lri 261.83 134.68 142.63 156.46 7.681 <0.001 ab a a b p-Cresol 107 1178 ms, lri 29.90 23.63 27.27 33.88 0.908 <0.001 a b a a Phenylethyl Alcohol 92 1182 ms, lri 10.55 45.80 14.56 17.64 1.766 <0.001 c a b b Benzaldehyde, 3-ethyl- 134 1209 ms, lri 34.40 14.22 21.36 27.37 1.077 <0.001 b a b c Total unknown origin 2292.41 1705.98 2153.01 3013.04 56.635 <0.001 b a b c Total compounds 56802.37 28584.14 57871.86 78415.27 1973.358 <0.001 a–d Mean values in the same row (corresponding to the same compound) not followed by a common letter differ significantly (P < 0.05; Tukey's Test). Compound origin according to: ɎNarváez-Rivas et al. (2012); Φ Martín et al. (2006); Ψ Fonseca et al. (2015). SEM: standard error of the mean. m/z: Quantification ion; LRI: Lineal Retention Index calculated for DB-624 capillary column (JandW scientific: 30m×0.25mm id, 1.4 μm film thickness) installed on a gas chromatograph equipped with a mass selective detector; R: Reliability of identification; lri: linear retention index in agreement with literature (Domínguez et al., 2014; Lorenzo, 2014; Lorenzo, Bedia and Bañon, 2013; Lorenzo and Carballo, 2015; Lorenzo and Domínguez, 2014; Lorenzo et al., 2012; Pateiro et al., 2015; Purriños et al., 2011; Purriños, Carballo, and Lorenzo, 2013; Purriños et al., 2012); ms: mass spectrum agreed with mass database (NIST14); s: mass spectrum and retention time identical with an authentic standard.
96
V. CONCLUSIONS
Conclusions
1. In dry-cured ham, the adhesiveness increased with the proteolysis index. As the proteolysis got bigger, the total non-protein nitrogen content was increased but the total basic volatile nitrogen content and total free amino acid content were not significantly different. Taurine, arginine, cysteine and lysine showed higher concentrations in the dry-cured hams with low PI levels while the content of the rest of individual free amino acids was higher in dry-cured hams with high PI level, so higer proteolysis resulted in bitterer dry-cured hams. 2. A decrease in the total volatile compound content was observed as the extent of proteolysis was increased. Since most of the volatile compounds detected in dry-cured hams come from the oxidation of lipids and, keeping in mind that the processing conditions that favour the lipid oxidation inhibit the action of proteolytic enzymes, in this study the highest amounts of most of the volatile compounds was shown in the hams from the low proteolysis group (except for esters). 3. Proteolysis index is a reliable indicator of the extent of protein hydrolysis at proteomic scale. A total of five myofibrillar and sarcoplasmic proteins of biceps femoris muscle were identified as candidate markers for proteolysis and adhesiveness. However, two distinct isoforms of the myosin heavy chain (myosin-1 and myosin-4) and α-actin exhibited the strongest response to variable proteolysis as well as to adhesiveness, according to the measure of relative change. These proteins could also be potential candidate biomarkers for quality traits closely linked to proteolysis, such as pastiness. 4. The thermal treatment of sliced, vacuum packaged high proteolysis hams applied alone as much as assisted by ultrasonic treatment significantly decreased the adhesiveness of hams. However, both treatments significantly affected the total and individual free amino acid content, as well as an increment in all individual amino acid contents was observed after ultrasound application. Thus, the taste of the final product turned sweeter, bitterer and more aged, whereas a significant effect was observed on the volatile compounds, being outlined the reduction of aldehydes, causing loses in the typical fatty odour of dry-cured ham. 5. 2-DE coupled to MALDI-TOF/TOF MS revealed that ham samples subjected to power ultrasound exhibited significantly higher proteolysis than those conventionally heated. In sliced dry-cured hams there was more degraded myofibrillar protein actin after ultrasound application compared to conventional thermal treatment, suggesting that actin of dry- cured ham could be gradually suffering degradation by ultrasound treatment and downstream effects on quality attributes. In addition, FABP4/H, PRDX6, SOD, CBR1 and ACY1 were more abundant in ultrasound treated samples, being probably candidate for proteolysis-independent biomarkers of ultrasonic application. 6. Intense modifications were caused by the combination of high pressure and high temperatures (particularly at 35 °C) on free amino acid profile and volatile composition. Treatment at 35 °C caused increments in sweet, bitter and fatty taste, whereas odour became fattier. 7. Treatment temperatures between 0–20 °C could minimize the organoleptic changes from the high-pressure treatments. 8. The relative change obtained for actin (2.51) in samples which were treated by high pressures, together with the higher number of actin fragments found in these samples compared with the control group, indicated that high pressures promoted actin fragmentation.
99 V. Conclusions
100
VI. REFERENCES
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Wang, A., Kang, D., Zhang, W., Zhang, C., Zou, Y., and Zhou, G. (2018). Changes in calpain activity, protein degradation and microstructure of beef M. semitendinosus by the application of ultrasound. Food Chemistry, 245, 724-730. Wang, Z., Zhang, C., Li, Z., Shen, Q., and Zhang, D. (2017). Comparative analysis of muscle phosphoproteome induced by salt curing. Meat Science, 133, 19-25. Wu, H., Zhang, Y., Long, M., et al. (2014). Proteolysis and sensory properties of dry-cured bacon as affected by the partial substitution of sodium chloride with potassium chloride. Meat Science, 96, 1325-1331. Wu, H., Zhuang, H., Zhang, Y., et al. (2015). Influence of partial replacement of NaCl with KCl on profiles of volatile compounds in dry-cured bacon during processing. Food Chemistry, 172, 391-399. Yang, L. F. X. H. Q., and Lian-sheng, Y. E. Y. (2003). Preparation and Application of Starch Ester Octenylsuccinate [J]. Journal of South China University of Technology (Natural Science), 7. Zamora, R., Navarro, J. L., Aguilar, I., and Hidalgo, F. J. (2015). Lipid-derived aldehyde degradation under thermal conditions. Food Chemistry, 174, 89-96. Zhao, G. M., Tian, W., Liu, Y. X., Zhou, G. H., Xu, X. L., and Li, M. Y. (2008). Proteolysis in biceps femoris during Jinhua ham processing. Meat Science, 79(1), 39-45. Zhao, G. M., Zhou, G. H., Tian, W., Xu, X. L., Wang, Y. L., and Luo, X. (2005). Changes of alanyl aminopeptidase activity and free amino acid contents in biceps femoris during processing of Jinhua ham. Meat Science, 71(4), 612-619. Zhu, Z., Guan, Q., Koubaa, M., Barba, F. J., et al. (2017b). HPLC-DAD-ESI-MS2 analytical profile of extracts obtained from purple sweet potato after green ultrasound-assisted extraction. Food Chemistry, 215, 391-400. Zhu, Z., Wu, Q., Di, X., et al. (2017a). Multistage recovery process of seaweed pigments: Investigation of ultrasound assisted extraction and ultra-filtration performances. Food and Bioproducts Processing, 104, 40-47. Zou, Y., Xu, P., Wu, H., et al. (2018). Effects of different ultrasound power on physicochemical property and functional performance of chicken actomyosin. International Journal of Biological Macromolecules, 113, 640-647.
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VII. ANEXES
VII. Anexes
VII.1. PUBLICATIONS THAT INCLUDE THE RESULTS OF THIS DOCTORAL THESIS
119
Food Chemistry 244 (2018) 238–245
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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Comparative proteomic profiling of myofibrillar proteins in dry-cured ham MARK with different proteolysis indices and adhesiveness
M. López-Pedrousoa, C. Pérez-Santaescolásticab, D. Francob, E. Fulladosac, J. Carballod, ⁎ C. Zapataa, J.M. Lorenzob, a Department of Zoology, Genetics and Physical Anthropology, University of Santiago de Compostela, Santiago de Compostela 15872, Spain b Centro Tecnológico de la Carne de Galicia, Rúa Galicia N° 4, Parque Tecnológico de Galicia, San Cibrán das Viñas, 32900 Ourense, Spain c IRTA, XaRTA, Food Technology, Finca Camps i Armet, E-17121 Monells, Girona, Spain d Área de Tecnología de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain
ARTICLE INFO ABSTRACT
Keywords: Excessive proteolysis during dry-cured ham processing may lead to high adhesiveness and consumer dis- Defective ham texture satisfaction. The aim of this research is to identify biomarkers for proteolysis and adhesiveness. Two hundred Instrumental adhesiveness biceps femoris porcine muscle samples from Spanish dry-cured ham were firstly evaluated for various physico- Meat proteolysis chemical parameters, including their proteolysis indices and instrumental adhesiveness. Proteins of samples with Meat proteomics extreme proteolysis indices were separated by two-dimensional electrophoresis and identified by tandem mass Porcine proteome spectrometry (MALDI-TOF/TOF). We found that hams of higher proteolysis index had statistically significant Processed meat increased adhesiveness. Proteomic analysis revealed statistically significant qualitative and quantitative differ- ences between sample groups. Thus, protein fragments increased remarkably in samples with higher proteolysis index scores. In addition, higher proteolysis index hams showed increased degradation for a total of five non- redundant myofibrillar and sarcoplasmic proteins. However, myosin-1, α-actin and myosin-4 proteins were the biomarkers that underwent the most intense response to proteolysis and adhesiveness.
1. Introduction The intensity of proteolysis during dry-cured ham processing is often measured by the proteolysis index. It is defined as the percentage Dry-cured ham is a high-quality food product traditionally con- of non-protein nitrogen accounting for total nitrogen. The relationship sumed in Europe. A wide variety of physicochemical changes during the between proteolysis index and texture throughout the dry-cured ham elaboration process influence the final product characteristics, such as process has been previously studied under a variety of variables, in- flavor and texture (Bermúdez, Franco, Carballo, & Lorenzo, 2014). cluding pH, water and NaCl content, and lipid oxidation (García- Salting and ripening are the two main steps in the elaboration process Garrido, Quiles-Zafra, Tapiador, & Luque de Castro, 1999; García- of dry-cured ham. The curing processing requires salt as preserving Garrido, Quiles-Zafra, Tapiador, & Luque de Castro, 2000; Harkouss agent. The amount and type of salt have a significant influence on et al., 2015; Ruiz-Ramírez, Arnau, Serra, & Gou, 2006; Virgili, Parolari, flavor, texture, color and overall quality of the final product (Paredi Schivazappa, Bordini, & Borri, 1995). The proteolysis index of good et al., 2013; Toldrá, Flores, & Sanz, 1997). The proteins undergo an quality Spanish dry-cured ham is considered to be between 33 and 36%, intense proteolysis during the ripening process, which constitutes the whereas in Italian ham it is between 22 and 30% (Careri et al., 1993). most important enzymatic reaction regarding muscle proteins Myofibrillar and sarcoplasmic proteins are intensively degraded during (Bermúdez, Franco, Carballo, Sentandreu, & Lorenzo, 2014; Lorenzo, the ripening process contributing to dry-cured ham texture and ultimate Cittadini, Bermúdez, Munekata, & Domínguez, 2015). Salt content to- quality (Bermúdez et al., 2014b). However, myofibrillar proteins are a gether with many other factors, such as rearing conditions (e.g., major fraction of the total, accounting for 65–70% muscle proteins feeding, sex and slaughter age), pig line, features of raw product (initial (Lana & Zolla, 2016). Accordingly, proteolytic changes in this protein weight, fat level and pH), type of muscle and the ripening process, have fraction are important for the development of texture and sensorial a recognized impact on protein denaturation of dry-cured hams (Škrlep characteristics. In particular, myosin and actin are two main targets of et al., 2011; Théron et al., 2011). proteolysis (Mora, Sentandreu, & Toldrá, 2011; Théron et al., 2011).
⁎ Corresponding author. E-mail address: [email protected] (J.M. Lorenzo). http://dx.doi.org/10.1016/j.foodchem.2017.10.068 Received 23 June 2017; Received in revised form 27 September 2017; Accepted 10 October 2017 Available online 12 October 2017 0308-8146/ © 2017 Elsevier Ltd. All rights reserved. M. López-Pedrouso et al. Food Chemistry 244 (2018) 238–245
However, excessive proteolysis may generate the pastiness defect, 2.3. Chemical analysis characterized by excessive softness, mushy texture and unpleasant fla- vors (Škrlep et al., 2011). In this regard, Morales, Arnau, Serra, After instrumental adhesiveness determination, biceps femoris sam- Guerrero, and Gou (2008) showed that there is a close relationship ples were minced and subjected to chemical analysis in triplicate. Water between pastiness and adhesiveness (degree to which the surface of the content was analyzed by drying at 103 ± 2 °C until reaching a constant ham slice adheres to the palate when compressed by the tongue), as weight (AOAC, 1990); whereas the chloride content was analyzed ac- described by Guerrero, Gou, and Arnau (1999). Therefore, the de- cording to ISO 1841-2 (1996) using a potentiometric titrator 785 DMP termination of instrumental adhesiveness could be a good indicator of Titrino (Metrohm, Herisau, Switzerland) and results were expressed as pastiness level in dry-cured ham. percentage of NaCl. Proteomics has great potential to enhance our knowledge on the biochemical processes underlying the conversion of muscle into meat 2.4. Proteolysis index and identify biomarkers for meat quality traits (Lana & Zolla, 2016; Paredi, Raboni, Bendixen, de Almeida, & Mozzarelli, 2012; Paredi, Total nitrogen content was determined with Kjeldahl method (ISO Sentandreu, Mozzarelli, Fadda, Hollung, & de Almeida, 2013). In dry- R-937, 1978) using the Vapodest 50S analyzer (Gerhardt, Königswinter, cured ham, proteomic studies, generally based on one- or two-dimen- Germany). It involves a semi-micro rapid routine method using block- sional electrophoresis coupled to mass spectrometry, have tackled a digestion, copper catalyst and steam distillation into boric acid. A wide diversity of topics. For instance, variations in quality traits, evo- known quantity of the sample (1.0 ± 0.1 g) was taken in the Kjel- lution of proteolysis during processing, comparative proteomics pro- datherm digestion tube of the Vapodest and 20 mL of H2SO4 solution filing of biceps femoris and semimembranosus muscles and identification were added to the tube. Then, the tube was placed onto the Vapodest of antioxidant peptides (Di Luccia et al., 2005; Mora, Escudero, Fraser, and steam digestion was started for 4 min. The steam vapor was col- Aristoy, & Toldrá, 2014; Petrova, Tolstorebrov, Mora, Toldrá, & Eikevik, lected and titrated in a 250-mL volumetric flask. 2016; Škrlep et al., 2011; Théron et al., 2011). To the best of our For non-protein nitrogen, preparation of sample was performed as knowledge, however, proteome changes linked to differential adhe- described by Lorenzo, García Fontán, Franco, and Carballo (2008) siveness have not been previously reported. Sample (2.5 g) was homogenized in 25 mL of deionized water and In this study, we undertook a comparative proteomic profiling in centrifuged. Afterwards, 10 mL of 20% trichloroacetic acid (99.5% biceps femoris muscle from dry-cured hams with different proteolysis purity, Merck, Darmstadt, Germany) were added, stirred well and left to indices, to identify biomarkers for differential proteolytic activity and stabilize for 60 min at room temperature. After centrifugation, the su- adhesiveness, using two-dimensional electrophoresis and tandem mass pernatant was filtered and 15 mL of filtrate were used for determination spectrometry (MALDI-TOF/TOF MS). of nitrogen, as described above for total nitrogen (NT, ISO R-937, 1978). The proteolysis index was calculated as the ratio: 2. Materials and methods (non−× protein nitrogen/nitrogen total) 100,
2.1. Dry-cured ham samples according to Ruiz-Ramírez et al. (2006).
Two hundred raw hams from Large White × Landrace crosses 2.5. Protein extraction for proteomic analysis (average weight of 11.72 ± 1.06 kg), obtained from a commercial slaughterhouse, were elaborated according to the traditional system Total protein from biceps femoris muscle was extracted from 50 mg with some modifications regarding the temperature at specific steps, in of lyophilized dry-cured ham. Samples were homogenized with 1 mL of order to ensure hams with high proteolysis. At the end of the process, lysis buffer (7 M urea; 2 M thiourea; 4% CHAPS; 10 mM DTT, and 2% hams were cut and boned and the cushion part containing biceps femoris Pharmalyte™ pH 3–10; GE Healthcare, Uppsala, Sweden) and sonicated muscle was excised and sampled. Ten slices from each dry-cured ham (Sonifier 250; Branson Ultrasonics Corporation, Danbury, CT) in short were vacuum packed and stored at room temperature for no longer than pulses at 0 °C. Excess salts and other interfering substances were re- 4 weeks, for texture and chemical analysis. Dry-cured hams of low moved twice using the 2-D Clean-Up Kit (GE Healthcare) following proteolysis and high proteolysis were selected according to extreme manufacturer’s instructions. This method for selectively precipitating proteolysis index scores: low proteolysis samples with proteolysis index protein was carried out using 200 µL of sonicated sample and the re- lower than 33%, and high proteolysis samples with proteolysis index sulting pellet was dissolved in 210 µL of lysis buffer. The protein con- higher than 36%. Four biological replicates per treatment (i.e. low and centration was assessed using a commercial CB-X protein assay kit (G- high proteolysis hams) were used for proteomic analysis. Samples for Biosciences, St. Louis, MO) according to the manufacturer’s instructions proteomic analysis were lyophilised separately and subsequently frozen in a Chromate® microplate reader (Awareness Technology, Palm City, at −80 °C until the time of protein extraction. FL).
2.2. Instrumental texture 2.6. Two-dimensional electrophoresis (2-DE)
Textural analysis was performed using a texture analyzer (TA-XT The 2-DE was performed according to Franco et al. (2015a). Briefly, Plus; Stable Micro Systems, Godalming, UK) by carrying out a separa- 250 µg of protein in lysis buffer were mixed with rehydration buffer tion test using different load cells with a specific probe. Instrumental (7 M urea, 2 M thiourea, 4% CHAPS, 0.002% bromophenol blue), adhesiveness was measured in sliced ham samples (1 mm) by applying reaching 450 µL of total volume. Finally, 0.6% DTT and 1% IPG buffer probe tests and calculating the negative area of a force-time curve in (Bio-Rad Laboratories, Hercules, CA) were added. This protein extract tension tests with a single-cycle. The texturometer was equipped with a was loaded into immobilized pH gradient (IPG) strips (24 cm, pH 4–7 probe connected to a special device that enables horizontal probe dis- linear, Bio-Rad Laboratories). The isoelectric focusing (IEF) was carried placement. After separation of the slices, the probe returned to the in- out on a PROTEAN IEF cell system (Bio-Rad Laboratories). Low voltage itial position. The conditions for the measurement of adhesiveness of (50 V) was applied to rehydrate the strips and then an increasing vol- dry cured ham slices were: load cell = 5 N; speed = 0.5 mm/s and tage ramp at 70 kVh. After IEF, strips were soaked in equilibration distance = 100 mm. From the obtained graph of force vs. distance, the buffer (50 mM Tris pH 8.8, 6 M urea, 2% SDS, 30% glycerol) succes- adhesiveness was calculated. All the measurements were made in tri- sively supplemented with 1% DTT and 2.5% iodoacetamide for 15 min plicate, at room temperature. each. The second dimension separation was performed using an Ettan
239 M. López-Pedrouso et al. Food Chemistry 244 (2018) 238–245
DALTsix vertical gel system (GE Healthcare) with 12% SDS-PAGE gels search against UniProt/SwissProt database. Mascot search parameters at 18 mA/gel until the bromophenol blue dye front reached the end of were: precursor mass tolerance of 50 ppm, 0.6 Da MS/MS fragment the gels. The 2-DE gels were stained with SYPRO Ruby fluorescent stain tolerance, carbamidomethyl cysteine as fixed modification, oxidized (Lonza, Rockland, ME). methionine as variable modification and permitting one missed clea- vage. Proteins with at least two matched peptides and statistically fi 2.7. Image analysis of 2-DE gels signi cant (p-value < 0.05) Mascot scores were selected as positively identified. Gels were visualized and digitalized using the Gel Doc XR+ system (Bio-Rad Laboratories). The detection and quantification of 2-DE spot 2.9. Statistical analysis volumes were performed with PDQuest Advanced software v. 8.0.1 (Bio-Rad Laboratories) after background subtraction. Spot volume Statistical analysis for physicochemical parameters was performed normalization was performed using those validated across all replicate using the IBM SPSS Statistics V21.0 (SPSS, Chicago, IL) software gels. Observed values of molecular mass (Mr) were determined across package. protein spots from standard molecular mass markers ranging from 15 to Quantitative changes of 2-DE gel spot volumes in sample groups 200 kDa (Fermentas, Burlington, ON, Canada), whereas those of iso- were assessed using the measures “fold change” (FC) and “relative electric point (pI) were established according to their position on the change” (RC)(Franco et al., 2015a, 2015b). The measure fold change is
IEF-strips. given by FC =Vhigh/Vlow, where Vhigh and Vlow are the mean volumes in samples with high and low proteolysis index, respectively. Fold 2.8. Protein identification by mass spectrometry (MS) change values less than one were represented as their negative re- ciprocal. The relative change is provided by the relationship RC = DV/ − For MALDI TOF/TOF MS analysis, selected spots were excised from |DVmax|, where DV = Vhigh Vlow and DVmax is the maximum observed the gel and they were dehydrated with acetonitrile using a vacuum value of DV over spots. fi centrifuge. The gel piece was washed with Ambic buffer (50 mM am- Bootstrapping was used to obtain 95% con dence intervals for the monium bicarbonate in 50% methanol). The proteins were reduced means of spot volume across replicates as previously described (Franco with 10 mM DTT in 50 mM ammonium bicarbonate and alkylated with et al., 2015a, 2015b). For each set of (n = 4) volume estimates, 20,000 55 mM acetamide in 50 mM ammonium bicarbonate. Extracts were bootstrap samples of size n were obtained following a Monte Carlo al- fi repeatedly rinsed with Ambic buffer, dehydrated by addition of acet- gorithm. The 95% bootstrap con dence intervals were obtained by the onitrile and dried in a SpeedVac. Then the proteins were hydrolyzed bias-corrected percentile method from distribution of bootstrap mean fi with modified porcine trypsin (Promega, Madison, WI) at a final con- replications (Efron, 1982). Con dence intervals were adjusted for centration of 20 ng/μL of trypsin in 20 mM ammonium bicarbonate multiple hypothesis testing with the Bonferroni procedure. overnight at 37 °C. The total digest was incubated three times in 40 μL of 60% acetonitrile with 5% formic acid, concentrated in a SpeedVac 3. Results and discussion and stored at −20 °C until analysis. Dried samples were dissolved in 4 µL of 0.5% acetic acid. Equal volumes (0.5 µL) of peptide and matrix 3.1. Proteolysis index and instrumental adhesiveness of dry-cured hams solution, consisting of 3 mg of α-cyano-4-hydroxycinnamic acid dis- solved in 1 mL of 50% acetonitrile and 0.1% trifluoroacetic acid, were Mean ( ± SE) values of instrumental adhesiveness, moisture, salt deposited onto a 384 Opti-TOF MALDI plate (Applied Biosystems, content, non-protein nitrogen and total nitrogen in dry-cured hams with Foster City, CA) using the thin layer method (Vorm, extreme (< 33% or > 36%) proteolysis indices are shown in Table 1. Roepstorff, & Mann, 1994). Mass spectrometric data were obtained in Physicochemical parameter values both for the total of hams with ex- an automated analysis loop using 4800 MALDI-TOF/TOF analyzer treme proteolysis indices and for those samples used in the proteomic (Applied Biosystems). Mass spectra were acquired in positive-ion re- analysis are presented separately. It is noteworthy that physicochemical flector mode with an Nd:YAG laser operating at 355 nm and an average values in samples used for proteomic analysis were representative of the accumulation of 1000 laser shots. A minimum of three trypsin autolysis entire set of selected samples. Therefore, we will hereafter refer only to peaks were used for internal calibration, in order to decrease peptide physicochemical data of the entire set of selected samples. Mean mass errors for protein identification. All MS/MS spectra were per- ( ± SE) proteolysis indices in samples with low and high proteolysis formed by selecting the precursors with a relative resolution of 300 were 31.5 ± 0.2 and 38.5 ± 0.3, respectively (p < 0.001). Differ- (FWHM) and metastable suppression. Automated analysis of mass data ences in proteolysis index can be attributed to a large number of factors, was achieved using the 4000 Series Explorer Software v. 3.5 (Applied such as variable raw materials, salting procedures, ripening process, Biosystems). Peptide mass fingerprint and peptide fragmentation duration of the different steps involved in the elaboration, as well as spectra data of each sample were combined using GPS Explorer Soft- variations of temperature and relative humidity in dry-cured ham ware v. 3.6 and Mascot software v. 2.1 (Matrix Science, Boston, MA) to processing (García-Garrido et al., 1999; Pugliese et al., 2015; Škrlep
Table 1 Mean ( ± SE) values of physicochemical parameters in dry-cured hams with different proteolysis indices.
Parameters All samples Samples for proteomic analysis
Low proteolysis (n = 46) High proteolysis (n = 43) p-Value Low proteolysis (n = 4) High proteolysis (n =4) p-Value
Proteolysis index (%) 31.47 ± 0.19 38.47 ± 0.33 < 0.001 30.26 ± 0.68 37.96 ± 0.88 0.021 Instrumental adhesiveness (g) 82.36 ± 3.27 92.08 ± 2.11 0.030 66.75 ± 4.87 100.43 ± 2.86 0.021 Moisture (%) 59.02 ± 0.09 58.98 ± 0.14 0.941 59.10 ± 0.17 58.57 ± 0.16 0.058 Salt content (%) 4.82 ± 0.11 4.56 ± 0.13 0.135 4.67 ± 0.05 4.69 ± 0.10 1.000 Non-protein nitrogen (%) 1.55 ± 0.01 1.83 ± 0.01 < 0.001 1.50 ± 0.07 1.84 ± 0.04 0.021 Total nitrogen (%) 4.93 ± 0.02 4.75 ± 0.03 < 0.001 4.97 ± 0.19 4.84 ± 0.03 0.245
Low proteolysis (proteolysis index < 33%); High proteolysis (proteolysis index > 36%). p-Values were assessed by two-tailed Mann-Whitney U test.
240 M. López-Pedrouso et al. Food Chemistry 244 (2018) 238–245 et al., 2011; Zhao et al., 2008). In the present study, however, hams comparison of theoretical and observed molecular masses revealed that were elaborated under uniform conditions. It suggests that proteolysis an important number (55%) of identified spots contained protein can undergo large variations even under similar processing systems. fragments (Table 3). It is noteworthy, however, that most (86%) of Adhesiveness of sliced dry-cured ham was assessed, for the first these spots were actually unique spots present only in high proteolysis time, by mechanical procedures as an alternative to sensory analysis. samples (Table 2). Accordingly, the proteomic profile in dry-cured ham Our observations indicate that the instrumental adhesiveness was sig- samples of higher proteolysis index showed increased levels of protein nificantly (p < 0.05) higher in the high proteolysis batch than in the fragmentation. It also shows that proteolysis index scores can be good low proteolysis batch. In addition, we found a significant positive re- indicators of differential proteolysis over proteomes. The remaining lationship between proteolysis index and adhesiveness using the spots, with theoretical and empirical mass ratios below 1.5, were ex- Pearson product-moment correlation coefficient (r) and Spearman’s cluded from further analysis. It is not possible to assess whether they nonparametric coefficient (rs) of rank correlation (r = +236, actually contain either entire or slightly degraded proteins at the level p = 0.026, n = 89; rs = +0.242, p = 0.023). These results support the of resolution of 2-DE. The use of an internal standard in multiplexing conclusion that adhesiveness is dependent on the proteolysis index. methods such as two-dimensional difference gel electrophoresis (2-D Hams with defective texture can exhibit high moisture/protein ratios as DIGE) could reduce inter-gel variation, resulting in an increase of sta- a result of both increased moisture and decreased protein contents re- tistical power (Chevalier, 2010). However, 2-DE is able to identify the lative to ham with normal texture (García-Garrido et al., 1999). In strongest protein changes between sample groups, and therefore the addition, several authors (Bermúdez, Franco, Carballo, & Lorenzo, most useful biomarkers for proteolysis and adhesiveness. 2014a; Ruiz-Ramírez et al., 2006; Virgili et al., 1995) noticed that All fragments detected in our study corresponded to seven non-re- proteolytic activity in ham is governed by salt. However, García- dundant myofibrillar or sarcoplasmic muscle proteins: myosin-1 Garrido et al. (1999) showed hams with normal and defective texture (MYH1), myosin-4 (MYH4), α-4 glucan phosphorylase (F1RQQ8), α- containing salt contents from 6.2% to 8.1% by wet weight. In this study, actin (ACTS or ACTA1), heat shock 70 kDa protein 1-like (HS71L), there were no significant differences between sample groups for myosin-7 (MYH7) and vinculin (VINC). However, most fragments moisture and salt content. In contrast, non-protein nitrogen showed (86%) resulted from hydrolysis of myosin heavy chain and α-actin significant (p < 0.001) differences between treatments, since the myofibrillar proteins: nine MYH1 spots, four MYH4 spots, one MYH7 lowest mean values were observed in the low proteolysis batch (1.55 vs. spot and five ACTS spots (Table 3). It is noteworthy, however, that the 1.83%, for low and high proteolysis groups, respectively). This finding amount of protein fragments does not provide determinant information is in agreement with data reported by García-Garrido et al. (1999), who by itself to reliably evaluate the extent of differential proteolysis over observed that non-protein nitrogen levels were 30% higher in hams of proteins and sample groups. A complete characterization of differential defective texture than in normal pieces. Tyrosine crystals could be proteolysis not only requires determining the number of protein frag- considered markers of advanced and intense proteolysis. However, in ments, but also the quantification of their volumes. our study, we did not observe tyrosine crystals in any dry-cured ham samples (our hams were ripened for 14 months, not more). 3.4. Candidate biomarkers for differential proteolysis and adhesiveness
3.2. Comparison of proteomic profiles by 2-DE Quantitative differences in proteolysis intensity between low and high proteolysis ham batches were assessed by FC and RC statistics from High-quality 2-DE gels were obtained, despite dry-cured ham salt protein fragment volumes. Table 4 shows FC and RC values for each content. Representative 2-DE gel images of low and high proteolysis protein found to be differentially affected by proteolysis. There can be proteomes are shown in Fig. 1. The identification, matching and volume seen that both statistics provide very different information about the evaluation of 2-DE spots were obtained using PDQuest software. Satu- extent of proteolysis across proteins. It is worth noting that FC is a rated, faint and non-reproducible spots over replicates were excluded measure traditionally used to quantify differential protein abundance from further analysis. The total numbers of selected spots for proteomic between treatments. But it has the disadvantage that its range varies analysis were 92 and 123 spots in low and high proteolysis groups, from -∞ to +∞ and range boundaries are achieved with the presence respectively. We found that proteomic profiles of low and high pro- of unique spots independently of the existing differences in volume. In teolysis samples were remarkably differentiated (Table 2). In total, 58 contrast, RC always ranges from −1.0 to +1.0. It provides; therefore, a protein spots showed statistically significant differential abundance by more intuitive measure of the strength of change and maximum values the bootstrap re-sampling statistical method. It should be noted that of its range are not necessarily achieved with the mere occurrence of Bonferroni-corrected 95% bootstrap confidence intervals for means of unique spots (see Table 4). Accordingly, RC is a particularly appropriate spot volumes did not overlap in matched spots of different intensity or measure for the analysis of degraded proteome profiles exhibiting large did not overlap zero in unique spots. It is important to highlight that number of unique spots. In the present study, we found that RC-values only eight unique spots were observed in low proteolysis samples, of proteins ranged between −0.04 and +1.0 (Table 4). Only five whereas in high proteolysis samples there were 37 spots (p < 0.001, proteins (i.e. MYH1, ACTS, MYH4, HS71L and F1RQQ8) showed posi- Fisher’s exact test). This difference probably reflects an increased pro- tive RC-values, indicating that their fragments were over-represented in tein fragmentation in samples with high proteolysis. high-proteolysis hams. In contrast, MYH7 and VINC proteins underwent decreased proteolysis in high-proteolysis samples given that their RC- 3.3. Evaluation of protein fragmentation values were of negative sign. This result suggests that MYH7 and VINC proteins are not useful biomarkers of proteolysis intensity. Protein fragmentation was evaluated by the following procedure. MYH1, ACTS and MYH4 proteins showed the highest level of de- First, protein identification of differentially abundant spots between gradation in high proteolysis samples (RC-values > 0.40). Previous treatments was performed by MALDI-TOF/TOF MS. Second, 2-DE gel proteomic studies based on one-dimensional electrophoresis and 2-DE spots containing protein fragments were assessed by comparing the have systematically demonstrated that myosin heavy chain and α-actin theoretical molecular mass of each protein with the molecular mass are the main targets of proteolysis in the biceps femoris muscle, parti- observed on 2-DE gel. Protein fragments were eventually validated cularly at the end of ripening (Larrea, Hernando, Quiles, Lluch, & Pérez- when the ratio between theoretical and empirical masses was above Munuera, 2006; Tabilo, Flores, Fiszman, & Toldrá, 1999; Théron et al., 1.5. We found that most differentially abundant protein spots in low 2011; Toldrá, Rico, & Flores, 1993). In 12-month-old Parma and S. and high proteolysis ham samples (40 out of 58 spots) were successfully Daniele dry-cured ham, most isoforms of myosin and actin were found identified (p < 0.05) by MALDI-TOF/TOF MS (Table 3). The to be completely hydrolyzed (Di Luccia et al., 2005). We found that
241 M. López-Pedrouso et al. Food Chemistry 244 (2018) 238–245
Fig. 1. 2-DE gel images showing the proteome profile of A) Low proteolysis index dry-cured ham with low (A) and high (B) proteolysis index. Protein spots with statistically significant qualitative (pre- 4 pI 7 sence/absence) and quantitative (changes in intensity) dif- ferences are marked and numbered. All these spots were Mr (kDa) 1 excised for further analysis by MALDI-TOF/TOF MS. 2 3 70
60
45 24 29 40 23 34 30 35 37 39 41 30
46 47 58 49 54
B) High proteolysis index 4 pI 7
Mr (kDa) 2 1 70 13 14 4 12 5 6 7 60 8 11 15 9 10 16 20 17 18 45 19
24 28 21 40 22 25 26 27 34 33 43 30 31 37 32 41 36 38 39 42 40 30 57 44 45 48 49 55 56 47 50 51 52 53 58
MYH1 (RC = +1) was a more sensitive biomarker for proteolysis than NaCl concentration, higher water content and increased proteolytic ACTS (RC = +0.60). This difference can be attributed to the fact that activity. Taken together, the available evidence suggests that MYH1 myosin is more sensitive to denaturation by salt content (Graiver, and MYH4 can be suitable biomarkers for proteolysis under different Pinotti, Califano, & Zaritzky, 2006). However, we found that two spe- scenarios. cific isoforms of myosin heavy chain (MYH1 and MYH4) were in- Of the five fragmented proteins over-represented in high proteolysis tensively degraded in response to proteolysis. It suggests that these two hams, two were sarcoplasmic proteins: HS71L and F1RQQ8. They are myosin heavy chain isoforms might exhibit differential susceptibility to proteins with a considerably lower relative representation in the pro- degradation by proteolytic enzymes during dry-cured ham processing. teome of biceps femoris muscle, which explains their low RC values In this regard, Théron et al. (2011) reported differential MYH1 or (< 0.10). The HS71L protein is a molecular chaperone that appears to MYH4 fragmentation in biceps femoris and semimembranosus muscles play a critical role in multiple cellular functions, including activation of with different proteolytic activity, due to differences in salt and proteolysis of misfolded proteins, controlling the targeting of proteins moisture content in the course of dry-cured ham processing. Specifi- for subsequent degradation and protection of the proteome in response cally, fragments of these two myosin heavy chains isoforms were to stress (Archibald et al., 2010; Radons, 2016; The UniProt overrepresented in biceps femoris muscle, an internal muscle with lower Consortium, 2017). On the other hand, the F1RQQ8 protein is a
242 M. López-Pedrouso et al. Food Chemistry 244 (2018) 238–245
Table 2 Spot volumes with statistically significant (p-value < 0.05) differential abundance in dry-cured hams of low and high proteolysis level.
Spot No. Low proteolysis High proteolysis
Mean ( ± SE) Volume ̂ ̂ 95% bootstrap CI (CL, CU) Mean ( ± SE) Volume ̂ ̂ 95% bootstrap CI (CL, CU) p(θθB ⩽ ) p(θθB ⩽ )
1 684 ± 31 0.57 617, 746 280 ± 75 0.53 79, 409 2 741 ± 150 0.53 353, 962 1531 ± 128 0.52 1259, 1742 3 392 ± 81 0.55 247, 554 −−− 4 −−− 1360 ± 215 0.54 815, 1712 5 −−− 307 ± 18 0.75 281, 333 6 −−− 271 ± 25 0.73 236, 306 7 −−− 366 ± 113 0.58 121, 566 8 −−− 2010 ± 419 0.60 1241, 2904 9 −−− 2186 ± 473 0.56 1320, 3073 10 −−− 2360 ± 500 0.53 1348, 3212 11 −−− 1174 ± 342 0.56 647, 2156 12 −−− 688 ± 95 0.49 520, 881 13 −−− 667 ± 219 0.54 53, 1014 14 −−− 1302 ± 257 0.58 976, 1830 15 −−− 661 ± 58 0.55 509, 764 16 −−− 508 ± 43 0.56 422, 589 17 −−− 655 ± 185 0.64 377, 1074 18 −−− 619 ± 194 0.60 229, 1003 19 −−− 582 ± 193 0.56 237, 974 20 −−− 163 ± 13 0.75 145, 182 21 −−− 468 ± 116 0.53 259, 695 22 −−− 798 ± 176 0.49 437, 999 23 234 ± 16 0.75 211, 257 −−− 24 725 ± 183 0.49 341, 993 1801 ± 212 0.68 1419, 2259 25 −−− 1459 ± 56 0.76 1379, 1537 26 −−− 1980 ± 327 0.75 1518, 2443 27 −−− 477 ± 112 0.51 248, 602 28 −−− 3396 ± 855 0.62 2016, 5152 29 283 ± 122 0.52 67, 510 −−− 30 235 ± 65 0.67 84, 310 489 ± 65 0.67 409, 639 31 −−− 324 ± 95 0.51 99, 541 32 −−− 507 ± 160 0.61 185, 826 33 −−− 477 ± 112 0.51 248,602 34 1079 ± 177 0.75 829, 1329 443 ± 178 0.62 318, 652 35 524 ± 99 0.77 394, 674 −−− 36 −−− 387 ± 16 0.61 359, 422 37 255 ± 6 0.76 246, 263 333 ± 40 0.64 284, 426 38 −−− 142 ± 66 0.67 37, 289 39 252 ± 29 0.54 172, 302 455 ± 98 0.58 338, 658 40 −−− 266 ± 47 0.53 158, 358 41 1756 ± 408 0.56 957, 2485 3274 ± 249 0.56 2990, 3783 42 965 ± 267 0.55 649, 1511 2041 ± 254 0.56 1577, 2555 43 −−− 544 ± 82 0.52 372, 667 44 −−− 1103 ± 113 0.74 943, 1264 45 1145 ± 197 0.56 814, 1556 −−− 46 465 ± 43 0.76 405, 525 −−− 47 475 ± 86 0.73 354, 597 1469 ± 302 0.56 722, 1963 48 −−− 608 ± 31 0.63 567, 679 49 779 ± 34 0.62 706, 843 1517 ± 312 0.58 1112, 2441 50 −−− 1370 ± 46 0.59 1277, 1462 51 −−− 622 ± 33 0.71 0.569, 0.697 52 1089 ± 344 0.66 543, 1862 −−− 53 −−− 2544 ± 665 0.62 1485, 4037 54 1622 ± 462 0.55 654, 2496 −−− 55 −−− 313 ± 116 0.58 46, 537 56 −−− 661 ± 292 0.61 28, 1180 57 683 ± 67 0.74 589, 777 352 ± 62 0.75 264, 440 58 643 ± 90 0.63 634, 849 399 ± 121 0.56 156, 623
Gel position of spots is shown in Fig. 1. Mean ( ± SE) volumes were obtained from four biological replicates. Bootstrap confidence intervals (CIs) were obtained by the bias-corrected percentile method from 20,000 bootstrap mean replications; Bonferroni method was applied to obtain si- multaneous CIs over comparisons; CL and CL are the lower and upper bounds, respectively. ̂ ̂ ̂ ̂ The bootstrap distribution was median biased if p(θθB ⩽ ) ≠ 0.50, where θB and θ are the bootstrap and sample mean estimates, respectively. phosphorylase that catalyzes and regulates the breakdown of glycogen femoris and semimembranosus muscles (Théron et al., 2011). Specifi- to glucose-1-phosphate for the generation of ATP during glycogenolysis cally, the biceps femoris muscle showed more F1RQQ8 fragments than (Archibald et al., 2010; Gautron et al., 1987; The UniProt Consortium, the semimembranosus muscle during the ripening of dry-cured ham, due 2017). Fragments of F1RQQ8 resulting from proteolytic activity were to its higher proteolytic activity (Théron et al., 2011). It follows also detected in post-mortem longissimus dorsi porcine muscle FIRQQ8 is a good biomarker of proteolysis in agreement with our ob- (Lametsch, Roepstorff, & Bendixen, 2002), as well as in dry-cured biceps servations.
243 M. López-Pedrouso et al. Food Chemistry 244 (2018) 238–245
Table 3 Protein identification by MALDI-TOF/TOF MS of differentially (P-value < 0.05) represented 2-DE spots in dry-cured hams with low and high proteolysis index.
Spot No. Protein Abbrev. Accession number Mascot Sequence coverage Number of matched pI Mr (Uniprot) score (%) peptides th/obs th/obs (kDa)
1 Vinculin VINC P26234 60 19 17 5.6/6.2 124.4/76.1 Fragment 2 Serum albumin ALBU P08835 144 21 13 6.1/6.1 71.6/72.9 3 Serum albumin ALBU P08835 125 21 14 6.1/6.3 71.6/73.2 4 Serum albumin ALBU P08835 601 42 19 6.1/6.5 71.6/70.7 5 Serum albumin ALBU P08835 56 10 7 6.1/6.1 71.6/66.3 9 Myosin-1 MYH1 Q9TV61 503 17 36 5.6/5.6 224.4/59.6 Fragment 10 Myosin-1 MYH1 Q9TV61 373 15 31 5.6/5.6 224.4/62.6 Fragment 11 Myosin-1 MYH1 Q9TV61 493 16 35 5.6/5.7 224.4/62.8 Fragment 12 Myosin-1 MYH1 Q9TV61 582 16 30 5.6/4.7 224.4/53.3 Fragment 13 Myosin-1 MYH1 Q9TV61 331 8 15 5.6/4.8 224.4/52.9 Fragment 14 Myosin-1 MYH1 Q9TV61 467 15 28 5.6/4.9 224.4/52.8 Fragment 15 Myosin-1 F1SS62 Q9TV61 287 24 34 5.5/5.1 171.0/61.2 Fragment 16 Myosin-4 MYH4 Q9TV62 249 11 19 5.6/5.1 224.0/60.8 Fragment 17 Myosin-1 MYH1 Q9TV61 249 15 25 5.6/5.2 224.4/59.4 Fragment 20 α-1,4-Glucan phosphorylase F1RQQ8 F1RQQ8 102 13 10 6.7/6.5 97.7/55.4 Fragment 21 α-Actin, skeletal muscle ACTS P68137 180 28 9 5.2/5.9 42.4/45.5 22 Heat shock 70 kDa protein 1-like HS71L A5A8V7 66 6 4 5.6/6.7 70.7/45.1 Fragment 23 Myosin-7 MYH7 P79293 380 13 21 5.6/4.4 223.0/44.2 Fragment 24 α-Actin, skeletal muscle ACTS P68137 96 14 4 5.2/4.9 42.4/40.1 25 Myosin-4 MYH4 Q9TV62 241 12 21 5.6/4.9 224.0/43.4 Fragment 26 Myosin-4 MYH4 Q9TV62 701 15 30 5.6/5.1 224.0/43.8 Fragment 28 α-Actin, skeletal muscle ACTS P68137 255 34 10 5.2/5.6 42.4/40.1 29 α-Actin, skeletal muscle ACTS P68137 69 19 5 5.2/4.7 42.4/39.1 30 Desmin DESM P02540 87 10 4 5.2/4.4 53.6/38.0 31 α-Actin, skeletal muscle ACTS P68137 94 13 4 5.2/4.8 42.4/42.6 32 Myosin-4 MYH4 Q9TV62 424 11 22 5.6/4.9 224.0/39.3 Fragment 34 F-Actin-capping protein subunit CAZA2 Q29221 269 47 11 5.6/5.8 33.1/39.1 alpha-2 36 F-Actin-capping protein subunit CAZA2 Q29221 67 9 2 5.6/6.1 33.1/35.7 alpha-2 40 β-Enolase ENOB Q1KYT0 92 23 7 8.1/6.5 47.4/35.1 44 F-Actin-capping protein subunit CAPZB A0PFK7 395 46 13 5.5/4.9 31.6/31.0 beta 45 α-Actin, skeletal muscle ACTS P68137 149 17 5 5.2/5.3 42.4/32.6 46 α-Actin, skeletal muscle ACTS P68137 159 30 8 5.2/4.5 42.4/25.5 Fragment 47 α-Actin, skeletal muscle ACTS P68137 117 12 4 5.2/5.3 42.4/25.4 Fragment 48 Myosin-1 MYH1 Q9TV61 415 15 32 5.6/5.5 224.4/62.5 Fragment 49 α-Actin, skeletal muscle ACTS P68137 180 17 5 5.2/5.6 42.4/25.4 Fragment 50 Peroxiredoxin-6 PRDX6 Q9TSX9 665 58 15 5.7/5.7 25.0/25.5 51 α-Actin, skeletal muscle ACTS P68137 126 14 4 5.2/5.3 42.4/24.0 Fragment 53 α-Actin, skeletal muscle ACTS P68137 180 14 4 5.2/5.5 42.4/24.2 Fragment 55 Multiprotein bridging factor 1 A6N8P5 A6N8P5 70 49 10 10.0/6.1 16.4/24.0 56 Triosephosphate isomerase TPIS Q29371 85 33 8 7.0/6.6 26.9/24.0
All identified proteins were matched to Sus scrofa (pig) proteins. The Mascot baseline statistically significant (p-value < 0.05) score was 56. Sequence coverage (%): percentage of coverage of the entire amino acid sequence by matched peptides. Number of matched peptides: total number of identified spectra matched for the protein.
Theoretical (Th) isoelectric point (pI) and molecular mass (Mr) were obtained from UniProtKB/Swiss-Prot databases.
Observed (Ob) pI and Mr were obtained from the spot position on the gel.
Protein fragments: Mr (Th)/Mr (Obs) ratio higher than 1.5.
Table 4 In the present study, we found that the instrumental adhesiveness is Fold change (FC) and relative change (RC)ofdifferentially (p < 0.05) represented pro- dependent on the proteolytic activity in dry-cured ham. Therefore, the ff tein fragments in dry-cured ham with di erent proteolysis indices. identified biomarkers also apply for the meat quality trait of adhe-
Spot No. Protein (abbrev.) fragment FC RC siveness. These biomarkers provide non-invasive tools alternative to sensory analysis or mechanical measures, to assess variations in adhe- 9–15, 17, 48 Myosin-1 (MYH1) +∞ +1.00 siveness. The identified proteins can also be potential biomarkers for α 46, 47, 49, 51, 53 -Actin, skeletal muscle (ACTS) +13.23 +0.60 other proteolysis-related ham quality traits. It is particularly true in the 16, 25, 26, 32 Myosin-4 (MYH4) +∞ +0.43 22 Heat shock 70 kDa protein 1-like (HS71L) +∞ +0.08 case of pastiness, considering that pastiness variations are closely re- 20 α-1,4-Glucan phosphorylase (F1RQQ8) +∞ +0.02 lated with the extent of proteolysis and adhesiveness (Morales et al., 23 Myosin-7 (MYH7) −∞ −0.02 2008; Škrlep et al., 2011). 1 Vinculin (VINC) −2.44 −0.04 4. Conclusions FC and RC values of MYH1, ACTS and MYH4 were computed from all fragments of the same protein on 2-DE gels. Comparison of dry-cured ham proteomic profiles with extreme proteolysis index scores allowed us to identify novel candidate bio- markers for differential proteolytic activity underlying quality traits. First of all, we found that the proteolysis index is a reliable indicator of
244 M. López-Pedrouso et al. Food Chemistry 244 (2018) 238–245 the extent of protein hydrolysis at proteomic scale. In addition, hams Harkouss, R., Astruc, T., Lebert, A., Gatellier, P., Loison, O., Safa, H., ... Mirade, P. S. (2015). Quantitative study of the relationships among proteolysis, lipid oxidation, with higher proteolysis indices showed increased instrumental adhe- structure and texture throughout the dry-cured ham process. Food Chemistry, 166, siveness. A total of five myofibrillar and sarcoplasmic proteins of biceps 522–530. femoris muscle were identified as candidate markers for proteolysis and ISO (1978). Determination of nitrogen content. ISO 937:1978 Standard. International standards meat and meat products. Ginebra. Suiza: International Organization for adhesiveness. However, two distinct isoforms of the myosin heavy Standardization. chain (myosin-1 and myosin-4) and α-actin exhibited the strongest re- ISO 1841-2 (1996). Meat and meat products. 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(2011). – response to pre-slaughter stress. Journal of Proteomics, 122,73 85. Proteomic analysis of semimembranosus and biceps femoris muscles from Bayonne dry- Franco, D., Mato, A., Salgado, F., López-Pedrouso, M., Carrera, M., Bravo, S., ... Zapata, C. cured ham. Meat Science, 88(1), 82–90. fi (2015b). Quanti cation of proteome changes in bovine muscle from two-dimensional Toldrá, F., Flores, M., & Sanz, Y. (1997). Dry-cured ham flavour: enzymatic generation – electrophoresis data. Data in Brief, 4, 100 104. and process influence. Food Chemistry, 59(4), 523–530. García-Garrido, J. A., Quiles-Zafra, R., Tapiador, J., & Luque de Castro, M. (1999). Toldrá, F., Rico, E., & Flores, J. (1993). Cathepsins L, D, H and L activities in the pro- Sensory and analytical properties of Spanish dry-cured ham of normal and defective cessing of dry-cured ham. Journal of the Science of Food and Agriculture, 62(2), – texture. Food Chemistry, 67(4), 423 427. 157–161. García-Garrido, J. A., Quiles-Zafra, R., Tapiador, J., & Luque de Castro, M. D. (2000). Virgili, R., Parolari, G., Schivazappa, C., Bordini, C. S., & Borri, M. (1995). Sensory and Activity of cathepsin B, D, H and L in Spanish dry-cured ham of normal and defective texture quality of dry-cured ham as affected by endogenous cathepsin b activity and – texture. Meat Science, 56,1 6. muscle composition. Journal of Food Science, 60(6), 1183–1186. Gautron, S., Daegelen, D., Mennecier, F., Dubocq, D., Kahn, A., & Dreyfus, J.-C. (1987). Vorm, O., Roepstorff, P., & Mann, M. (1994). Improved resolution and very high sensi- ’ fi Molecular mechanisms of McArdle s disease (muscle glycogen phosphorylase de - tivity in MALDI TOF of matrix surfaces made by fast evaporation. Analytical – ciency). Journal of Clinical Investigation, 79, 275 281. Chemistry, 66(19), 3281–3287. ff Graiver, N., Pinotti, A., Califano, A., & Zaritzky, N. (2006). Di usion of sodium chloride in Zhao, G. M., Tian, W., Liu, Y. X., Zhou, G. H., Xu, X. L., & Li, M. Y. (2008). Proteolysis in – pork tissue. Journal of Food Engineering, 77, 910 918. biceps femoris during Jinhua ham processing. Meat Science, 79(1), 39–45. Guerrero, L., Gou, P., & Arnau, J. (1999). Influence of meat pH on mechanical and sensory textural properties of dry-cured ham. Meat Science, 52, 267–273.
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Food Research International
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Effect of proteolysis index level on instrumental adhesiveness, free amino T acids content and volatile compounds profile of dry-cured ham
C. Pérez-Santaescolásticaa, J. Carballob, E. Fulladosac, José V. Garcia-Perezd, J. Beneditod, ⁎ J.M. Lorenzoa, a Centro Tecnológico de la Carne, Rúa Galicia N°4, Parque Tecnológico de Galicia, San Cibrán das Viñas, 32900, Ourense, Spain b Área de Tecnología de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain c IRTA, XARTA, Food Technology Program, Finca Camps i Armet s/n, 17121 Monells, Girona, Spain d UPV, Department of Food Technology, Universitat Politècnica de València, Camí de Vera s/n, E-46022 València, Spain
ARTICLE INFO ABSTRACT
Keywords: Defective textures in dry-cured ham are a common problem that causes important economic losses in the ham Ham texture industry. An increase of proteolysis during the dry-cured ham processing may lead to high adhesiveness and Pastiness consumer rejection of the product. Therefore, the influence of proteolysis index (PI) on instrumental adhe- Proteolysis siveness, free amino acids and volatile profile of dry-cured ham was assessed. Two hundred Spanish dry-cured Texture defects ham units were firstly classified according to their PI: low PI (< 32%), medium PI (32–36%) and high PI Nitrogen fraction (> 36%). Instrumental adhesiveness was affected by PI, showing the lowest values in the batch with low PI. Aroma Significant differences (P < 0.05) among groups were found in six amino acids: serine, taurine, cysteine, me- thionine, isoleucine and leucine. The content of leucine, serine, methionine, and isoleucine significantly (P < 0.05) increased as the proteolysis index rose. However, taurine and cysteine content showed an opposite behaviour, reaching the highest values in the dry-cured hams with low PI. Significant differences (P < 0.001) in the total content of volatile compounds among ham groups were observed, with the highest concentration in the batch with low PI, and decreasing the concentration as the PI increased. Regarding the different chemical families of volatiles, the hydrocarbons (the main family), alcohols, aldehydes, ketones and acids were more abundant in the hams showing the lowest PI. Esters did not show significant differences among the three batches of hams studied. The present study demonstrated that, apart from the effect on the adhesiveness, an excessive proteolysis seems to be associated with negative effects on the taste and aroma of the dry-cured ham.
1. Introduction is affected by many processing parameters such as water content, temperature, salt content, anatomic location and fresh ham pH Texture is an important quality criterion for the certification of the (Bermúdez, Franco, Carballo, Sentandreu, & Lorenzo, 2014; Ruiz- Traditional Spanish dry-cured ham “Jamón Serrano” as a Guaranteed Ramírez, Arnau, Serra, & Gou, 2005; Ruiz-Ramírez, Arnau, Serra, & Traditional Speciality (Fundación Jamón Serrano, 1998). Dry-cured Gou, 2006). The intensity of proteolysis during dry-cured ham proces- hams are usually classified into four texture types: very pasty, pasty, sing is often measured by the proteolysis index, that is defined as the soft and normal, each identified by various properties (Harkouss et al., non-protein nitrogen content expressed as percentage of the total ni- 2015). Texture problems, such as pastiness, crusting and softness, fre- trogen. In this regard, a relationship between proteolytic index (PI) and quently hinders slicing and provides a mouth-coating sensation, un- texture throughout the dry-cured ham manufacture process has been derlining the important role of texture for both retailer and consumer reported by several authors (Garcıa-Garrido,́ Quiles-Zafra, Tapiador, & acceptability. Pastiness is a texture defect that appears in dry-cured De Castro, 1999; Harkouss et al., 2015; Ruiz-Ramírez et al., 2006). In ham when there is an excessive breakdown of the protein structure of addition, Harkouss et al. (2015) showed that adhesiveness can be es- the muscle due to the action of a series of autochthonous enzymes and timated as a function of PI values, water and salt content. therefore related to an excessive proteolysis. Proteolysis, one of the main biochemical reactions during the dry- Several authors reported that proteolysis activity in dry-cured ham cured ham processing, is considered to be the major contributor to
⁎ Corresponding author. E-mail address: [email protected] (J.M. Lorenzo). https://doi.org/10.1016/j.foodres.2018.03.001 Received 16 December 2017; Received in revised form 26 February 2018; Accepted 1 March 2018 Available online 02 March 2018 0963-9969/ © 2018 Elsevier Ltd. All rights reserved. C. Pérez-Santaescolástica et al. Food Research International 107 (2018) 559–566 texture changes (Jurado, García, Timón, & Carrapiso, 2007). It has been adhesiveness was measured in sliced ham samples (1 mm) by applying shown that textural defects are closely related to anomalous proteolysis probe tests and calculating the negative area of a force-time curve in (Virgili, Parolari, Schivazappa, Bordini, & Borri, 1995). In addition, li- tension tests with a single-cycle. The texturometer was equipped with a polysis and proteolysis are the main biochemical reactions involved in probe connected to a special device that enables horizontal probe dis- the generation of a wide range of volatile compounds (Bermúdez, placement. After the separation of the slices, the probe returned to the Franco, Carballo, & Lorenzo, 2015; Fulladosa et al., 2010). In this re- initial position. The conditions for the measurement of adhesiveness of gard, the volatile compounds of dry-cured ham give an indication of the dry cured ham slices were reported by López-Pedrouso et al. (2018). chemical and metabolic process that occurs during the ripening process. From the obtained graph force vs. distance, the adhesiveness was cal- Several groups of volatile compounds have been reported in dry-cured culated. All the measurements were made in triplicate, at room tem- ham, such as hydrocarbons, aldehydes, ketones, alcohols, esters, lac- perature. tones, terpenes, nitrogen compounds, sulfur compounds, carboxylic acids and chloride compounds. These volatiles can be grouped ac- 2.3. Chemical analysis cording to their possible origin, into volatiles from lipid autooxidation fi (aldehydes, hydrocarbons, alcohols and ketones), microbial esteri ca- After instrumental adhesiveness determination, the biceps femoris tion (e.g. propyl acetate and ethyl propanoate), carbohydrate fermen- samples were minced and subjected to chemical analysis in triplicate. tation (e.g. 1,3-butanediol and phenyl acetaldehyde), amino acid cat- Water content was analysed by drying at 103 ± 2 °C until reaching a abolism (e.g. 2-methylbutanal, 2-methyl-1-butanol and 2,3-butanediol) constant weight (AOAC, 1990), whereas the chloride content was and other origins (Lorenzo, Franco, & Carballo, 2014; Lorenzo, Montes, analysed according to the ISO 1841–2(ISO, 1996) standard using a Purriños, & Franco, 2012; Narváez-Rivas, Gallardo, & León-Camacho, potentiometric titrator 785 DMP Titrino (Metrohm, Herisau, Switzer- 2012; Purriños, Carballo, & Lorenzo, 2013), although several of these land), and results were expressed as percentage of NaCl. volatiles may have more than one origin (Lorenzo, Bedia, & Bañón, 2013). In order to deepen the knowledge of the correlation between proteolysis and the different sensory quality parameters of ham, the 2.4. Nitrogen fraction analysis objective of this study was to assess the effect of the proteolysis index on the free amino acid and volatile profiles and the adhesiveness of Total nitrogen content (NT) was determined according to the Spanish dry-cured ham. Kjeldahl method (ISO, 1978) using the Vapodest 50S analyzer (Ger- hardt, Königswinter, Germany). It concerns a semi-micro rapid routine 2. Materials and methods method using block-digestion, copper catalyst and steam distillation into boric acid. A known quantity of the sample (1 ± 0.1 g) was ana- 2.1. Samples lysed. The content of non-protein nitrogen was assessed as described by Two hundred raw hams with a pH value < 5.5, which were more Lorenzo, García Fontán, Franco, and Carballo (2008). Two and half g of prone to develop defective textures, were obtained from a commercial sample were homogenized in 25 mL of deionized water and centrifuged. slaughterhouse. Hams were coming from pigs belonging to crosses of Afterwards, 10 mL of 20% trichloroacetic acid (99.5% purity, Merck, Large white and Landrace breeds (medium fat content). All animals Darmstadt, Germany) were added, and the mix was stirred well and let (castrated male) were reared in the conditions. The pigs were allowed to stabilize for 60 min at room temperature. Next, it was centrifuged at fi ad libitum access to water and feed. The basal diet contained: barley 1734g during 10 min. After centrifugation, the supernatant was ltered, fi (81.08%), lard (4.0%), soya (12.05%), methionine (0.08%), lysine and 15 mL of ltrate were used for determination of the nitrogen con- (0.30%), threonine (0.11%), calcium carbonate (0.96%), dicalcium tent, following the same method used for the total nitrogen (NT) de- phosphate (0.66%), salt (0.33%) and minerals and vitamins (0.4%). All termination (ISO, 1978). The proteolytic index (PI) was calculated as hams (n = 200) were weighted (11.9 kg ± 1.1 kg) and salted ac- the ratio (non-protein nitrogen/total nitrogen) × 100 according to cording to the traditional system. Hams were manually rubbed with the Ruiz-Ramírez et al. (2006). Total volatile basic nitrogen (TVB-N) content was assessed ac- following mixture: 0.15 g of KNO3, 0.15 g of NaNO2, 1.0 g of dextrose, 0.5 g of sodium ascorbate and 10 g of NaCl per kilogram of raw ham. cording to the Commission Regulation (EC) No 2074/2005 The hams were next pile salted at 3 ± 2 °C and 85 ± 5% RH during 4 (Commission Regulation, 2011). A 10 g sample of muscle was homo- (n = 50), 6 (n = 50), 8 (n = 50) or 11 days (n = 50) according to their genized with 90 mL of perchloric acid, and the resulting suspension was corresponding raw weight. After salting, hams were washed with cold centrifuged at 10000g for 10 min using an Allegra X-22 centrifuge water and post-salted at 3 ± 2 °C and 85 ± 5% RH during 45 days. (Beckman Coulter, California, EEUU). Fifty milliliter of the supernatant Drying of hams were performed at 12 ± 2 °C and 70 ± 5% RH until were analysed for the nitrogen content following the Kjeldahl method reaching a weight loss of 29%, later they were vacuum packaged and using a Vapodest 50S analyzer (Gerhardt, Königswinter, Germany). The kept at 30 °C during 30 days to induce proteolysis. After this time, hams TVB-N values were expressed as mg nitrogen/100 g of dry matter. ff continued the drying process at 12 ± 2 °C and 65 ± 5% RH until Finally, dry-cured hams were categorized in di erent proteolysis reaching a weight loss of 34%, later they were vacuum packaged again index level groups according to their proteolysis index: low proteolysis and kept at 30 °C during 30 days more. After this period, hams were level (IP < 32%) (LP), medium proteolysis level (32% < IP > 36%) dried again until the end of the drying process (weight loss of 36%). At (MP) and high proteolysis level (IP > 36%) (HP). the end of the process, hams were cut and boned and the cushion part containing the biceps femoris muscle was excised and sampled. Ten 2.5. Free amino acid analysis slices from each dry-cured ham were vacuum packed and stored at room temperature (20 °C) for no longer than 4 weeks, for texture and The amino acids were extracted following the procedure described chemical analysis. by Lorenzo, Cittadini, Bermúdez, Munekata, and Domínguez (2015). Amino acids were derivatizated with 6-aminoquinolyl-Nhydrox- 2.2. Instrumental adhesiveness ysuccinimidyl carbamate (Waters AccQ-Fluor reagent kit) and analysed by RP-HPLC using a Waters 2695 Separations Module with a Waters Textural analysis was performed using a texture analyzer (Stable 2475 Multi Fluorescence Detector, equipped with a Waters AccQ-Tag Micro Systems, TA-XT Plus, London, UK) by carrying out a separation amino acid analysis column. The results were expressed as mg of free test using different load cells with a specific probe. Instrumental amino acid/100 g of dry matter.
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2.6. Volatile compound analysis contents from 6.2 to 8.1% in wet weight in agreement with the results of the present study (salt contents ranging from 4.48 to 4.96%). Sta- The extraction of the volatile compounds was performed using tistical analysis did not show significant differences on salt content solid-phase microextraction (SPME). A SPME device (Supelco, among the three PI levels studied, presenting mean values of 11.63 g/ Bellefonte, USA) containing a fused silica fibre (10 mm length) coated 100 g dry matter. with a 50/30 layer of divinylbenzene/carboxen/polydimethylsiloxane No works related to instrumental adhesiveness of dry-cured ham was used. Headspace SPME extraction (from 1 g of sample) and chro- slices were found in literature. Results from this study showed that matographic analyses were carried out under the conditions described significant (P < 0.001) differences between PI levels groups were by Domínguez, Gómez, Fonseca, and Lorenzo (2014). Volatile com- found. Thereby, the higher the PI, the higher the adhesiveness (71.43, pounds were identified by comparing their mass spectra with those 77.20 and 90.15 g, for LP, MP and HP groups, respectively). According contained in the NIST14 (National Institute of Standards and Tech- to Garcıa-Garridó et al. (1999), hams with a defective texture exhibited nology, Gaithersburg) library, and/or by comparing their mass spectra high moisture/protein ratios as a result of both increased moisture and and retention time with authentic standards (Supelco, Bellefonte, PA, decreased protein contents related to hams with a normal texture. USA), and/or by calculation of retention index relative to a series of The PI is defined as the percentage of non-protein nitrogen ac- standard alkanes (C5–C14) (for calculating Kovats indexes, Supelco counting for total nitrogen and it is often used to describe the intensity 44,585-U, Bellefonte, PA, USA) and matching them with data reported of proteolysis during dry-cured ham processing. In Spanish dry-cured in literature. The results are expressed as area units (AU) × 106/g of dry ham, the PI reflecting a good quality could be considered between 33 matter. and 36, whereas in Italian dry-cured ham is between 22 and 30 (Careri et al., 1993). Result in the present study are in agreement with data ı́ 2.7. Statistical analysis reported by other authors (Garc a-Garrido et al., 1999; Pugliese et al., 2015; Zhao et al., 2008) who observed values between 17.23 and 35.2 ff The effect of proteolysis index group/level was examined using a in dry-cured hams. These di erences in PI among hams could be due to ff one-way ANOVA, where this parameter was set as factor. The values di erences in raw materials, salting procedure, ripening process, were given in terms of mean values and standard error of the means duration of steps, and temperature and relative humidity used in the (SEM). When a significant effect (P < 0.05) was detected, means were processing of dry-cured hams. In addition, Ruiz-Ramírez et al. (2006) compared using the Tukey's test. Correlations between variables were observed that the anatomic location of the muscle, the fresh ham pH, ff determined by correlation analyses using the Pearson's linear correla- and the amount of added NaCl a ected the proteolysis index at the end tion coefficient. All analyses were conducted using the IBM SPSS of the dry-cured ham process. Statistics 24.0 program (IBM Corporation, 2016) software package. Another noted relation is that stablished between the content of the nitrogen compounds and the proteolysis reactions, because proteolytic processes break down the proteins giving rise to smaller peptides and 3. Results and discussion free amino acids (Armenteros, Aristoy, & Toldrá, 2009). In addition, Petrova, Tolstorebrov, Mora, Toldrá, and Eikevik (2016) noticed that 3.1. Instrumental adhesiveness, chemical parameters and nitrogen fractions the PI during dry-cured ham processing is directly related to the en- zymatic activity. In this regard, in the present study the non-protein Table 1 shows the instrumental adhesiveness, chemical composi- nitrogen content also showed significant (P < 0.001) differences tion, nitrogen fractions and proteolysis index of dry-cured hams for the among ham groups, since the lowest values were observed in the LP ff di erent proteolysis levels (low, medium and high). Several authors batch (3.76 vs. 4.02 vs. 4.42 g/100 g of dry matter, for LP, MP and HP (Bermúdez et al., 2014; Ruiz-Ramírez et al., 2006; Virgili et al., 1995) groups, respectively). This is an expected result since the hams have noticed that proteolytic activity in ham is highly correlated to salt been classified according to their IP. Low activity values of proteolytic content. The negative relationship between salt content and proteolysis enzymes would result in low protein degradation and in a smaller index has been extensively reported (Flores et al., 2006; Armenteros, amount of non-protein nitrogen in samples (Petrova et al., 2016). This Aristoy, Barat, & Toldrá, 2009; dos Santos et al., 2015). In this regard, finding is in agreement with data reported by Garcıa-Garridó et al. in the present study, it was found that there is a negative correlation (1999) who observed that the non-protein nitrogen levels were 30% − (r = 0.218, P < 0.01, data not shown) between the proteolysis index higher in hams of defective texture than in normal pieces. In addition, ı́ and the salt concentration. However, Garc a-Garrido et al. (1999) Martín, Córdoba, Antequera, Timón, and Ventanas (1998) noticed that showed hams of both normal and defective texture may contain salt the high temperatures during the drying stage stimulate the formation of non‑nitrogenous compounds as the enzymatic activity increases. Table 1 Finally, the basic volatile nitrogen content was not affected by Effect of proteolysis index on instrumental adhesiveness, chemical parameters and ni- trogen fractions of dry-cured ham. proteolysis index, showing this nitrogen fraction mean values of 389.88 mg/100 g of dry matter (Table 1). These values were higher Parameters Groups SEM P-value than those reported by other authors in dry-cured ham (values ranging from 50 to 240 mg/100 g of dry matter) (Martín et al., 1998; Ventanas LP MP HP et al., 1992), and also higher than data reported by Lorenzo et al. Instrumental adhesiveness (g) 71.43a 77.20a 90.15b 1.580 0.005 (2008) in dry-cured lacón (85.6–109.7 mg/100 g of dry matter). Moisture (%) 58.98 58.83 58.86 0.071 0.065 Salt (% dry matter) 11.88 11.86 11.16 0.135 0.067 3.2. Free amino acids TN (% dry matter) 11.85 11.76 11.70 0.027 0.062 NPN (% dry matter) 3.76a 4.02b 4.42c 0.025 < 0.001 ff TBVN (mg/100 g dry matter) 385.79 389.21 394.65 2.612 0.112 The e ect of proteolysis index on free amino acid content (expressed Proteolysis index (%) 31.10a 34.50b 38.59c 0.249 < 0.001 as mg/100 g dry matter) of dry-cured ham is shown in Table 2.No significant differences in the total amount of free amino acids among a–c Mean values in the same row (corresponding to the same parameter) not followed by a the three different groups (mean values of 5370 mg/100 g of dry ff fi common letter di er signi cantly (P < 0.05; Tukey's Test). matter) were observed. The total free amino acid content observed in SEM: standard error of mean. Groups: LP = low proteolysis (PI < 32%); MP = medium proteolysis the present study was higher than those in previous studies on dry- (32% < PI > 36%) and HP = high proteolysis (PI > 36%). cured ham (about 4000 mg/100 g dry matter; Córdoba et al., 1994; TN: Total Nitrogen; NPN: Non-protein nitrogen; TBVN: Total basic volatile nitrogen. Martín, Antequera, Ventanas, Benítez-Donoso, & Córdoba, 2001; Ruiz
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Table 2 (Table 2). According to Bermúdez et al. (2014), the free amino acid Effect of proteolysis index on free amino acids content (expressed as mg/100 g dry content variations depend on the ratio between free amino acid for- matter) in dry-cured ham. mation and degradation. In addition, during ripening process the en-
Amino acids Groups SEM P-value zymes continue the protein degradation producing mainly small pep- tides and free amino acids (Toldrá, 2006). Some of these free amino LP MP HP acids contribute directly to taste (Jurado et al., 2007), whereas other ones participate indirectly in flavor development because they are Aspartic acid 183.62 181.84 182.64 2.53 0.961 Serine 176.95a 193.86b 203.05b 2.92 0.001 precursors of many odourants (Hidalgo & Zamora, 2004) important for Glutamic acid 451.20 447.19 462.25 5.70 0.538 dry-cured meat products. Glycine 198.66 194.48 195.69 2.54 0.788 These differences in the individual free amino acid content among Histidine 98.08 102.24 100.76 1.44 0.497 the three ham groups studied could induce differences in flavor. In this Taurine 97.45b 91.18ab 85.97a 1.27 0.001 regard, Henriksen and Stahnke (1997) noticed that specific amino acid Arginine 397.50 386.43 379.00 5.62 0.398 ff Threonine 209.87 220.07 223.57 2.83 0.117 groups might have an impact exceeding the individual e ects on sen- Alanine 414.96 406.56 416.48 5.11 0.706 sorial properties. In this sense, the concentration of alanine, serine, Proline 275.58 279.87 290.94 3.43 0.163 proline, threonine and glycine is related with sweet taste; bitter taste is b a a Cysteine 443.09 286.77 269.44 9.93 < 0.001 mainly associated with aromatic amino acids such as leucine, valine, Tyrosine 189.49 194.15 197.59 2.78 0.485 Valine 383.68 389.34 400.21 4.42 0.291 isoleucine, methionine, while phenylalanine, histidine, glutamic and Methionine 194.50a 206.57ab 216.58b 2.61 0.002 aspartic acids impart an acid taste, and a characteristic aged flavor have Lysine 266.70 251.38 248.40 3.70 0.094 been linked to lysine, tyrosine and aspartic acid (Table 2). According to a ab b Isoleucine 338.43 349.81 371.49 4.20 0.004 the free amino acid profile and to the differences observed in the pre- Leucine 566.83a 586.15ab 623.74b 6.89 0.002 sent study, our results seem to indicate that only bitter taste could be Phenylalanine 374.93 392.15 400.76 4.62 0.061 fi ff Total free amino acids 5399.04 5333.27 5406.31 62.77 0.878 signi cantly (P < 0.05) a ected by PI, presenting the highest values in Flavors dry-cured hams with high PI. This result is in agreement with data re- Sweet1 1235.86 1267.92 1299.09 12.348 0.096 ported by other authors (Careri et al., 1993; Parolari, Virgili, & 2 a ab b Bitter 1860.39 1924.03 2003.98 21.417 0.018 Schivazappa, 1994) who noticed that an excess of proteolysis is un- Acid3 718.79 729.23 737.25 7.775 0.601 Aged4 632.46 621.49 623.19 5.852 0.703 desirable because it may give a bitter or metallic aftertaste in dry-cured hams. – a bMean values in the same row (corresponding to the same parameter) not followed by a common letter differ significantly (P < 0.05; Tukey's Test). 3.3. Volatile compounds SEM: standard error of mean. Groups: LP = low proteolysis (PI < 32%); MP = medium proteolysis Table 3 shows the effect of proteolysis index on the volatile com- (32% < PI > 36%) and HP = high proteolysis (PI > 36%). fi 1 Sweet flavor = ∑ of alanine, glycine, threonine, serine and proline. pounds pro le of dry-cured ham. An increase in the relative abundance 2 Bitter flavor = ∑ of leucine, valine, isoleucine, methionine and phenylalanine of total volatiles in headspace of ham might suppose a more intense 3 Acid flavor = ∑ of glutamic acid, aspartic acid and histidine odour or flavor, or not, or it might have a negative or positive effect; 4 Aged flavor = ∑ of lysine, tyrosine and aspartic acid. this will depend on the type of volatile compounds that are formed. Thirty-nine volatile compounds were identified and quantified and they et al., 1999). However, other studies showed higher total concentra- were classified into the following chemical families: hydrocarbons (14), tions in dry-cured ham (about 12,500 g/100 g dry matter, Jurado et al., alcohols (5), aldehydes (4), esters (2) ketones (4) acid (1), sulfur 2007; Zhao et al., 2005). compounds (1) and other compounds (2) according to Lorenzo and In general, the free amino acid profile observed in the present study Carballo (2015). Most of the volatile compounds come from chemical or basically coincides with those reported in different types of dry cured enzymatic oxidation of unsaturated fatty acids and further interactions ham (Bermúdez et al., 2014; Jurado et al., 2007; Martín et al., 2001; with proteins, peptides and free amino acids. Other volatile compounds Virgili, Saccani, Gabba, Tanzi, & Bordini, 2007; Zhao et al., 2005). The result from Strecker degradation of free amino acids and Maillard re- individual free amino acids showed higher values in dry-cured hams actions (Toldrá & Flores, 1998). Statistical analysis showed significant with high PI, except for taurine, arginine, cysteine and lysine that differences (P < 0.001) in the total volatiles content between groups, presented higher concentrations in dry-cured ham with low PI than in with the highest concentration observed in the batch with low PI, and medium and high PI groups. As discussed previously, an excess of decreasing as the proteolysis index increased (1575.24 vs. 133,781 vs. proteolysis causes a texture defect which translates into a non-accep- 997.49 AU × 106/g of dry matter for LP, MP and HP batches, respec- tance by the consumers; this excess of proteolysis also entails an in- tively). crease in the concentration of nitrogen compounds of low molecular As shown in Table 3, the main family of volatile compounds were weight, such as peptides and free amino acids (Toldrá, 1998). The the hydrocarbons. These compounds derived from the oxidative de- factors associated with animals (genotype, age, sex of animals, pre and composition of lipids, which may be catalyzed by hemocompounds post-mortem treatments) and the processing conditions and technolo- such as hemoglobin and myoglobin (Ramírez & Cava, 2007). In addi- gical processes used (pH, humidity, water activity, time, temperature, tion, Martín, Córdoba, Aranda, Córdoba, and Asensio (2006) suggested salt concentration, etc.) have great important in the activity of the that methyl hydrocarbons could be synthesized by molds as a product enzymes that cause proteolysis reactions (Sanz & Toldrá, 2002). of secondary degradation of triglycerides. It was observed a higher On the other hand, six of the 18 free amino acids quantified in this content of hydrocarbons in the batch with lower PI compared to the study showed significant differences among PI levels (P < 0.05): other ones (759 vs. 605 vs. 416 AU × 106/g of dry matter for LP, MP serine, taurine, cysteine, methionine, isoleucine and leucine (Table 2). and HP groups, respectively). These outcomes could be due to the Leucine was the major amino acid in all groups, showing a significant greater lipid oxidation in the low PI ham group compared to the other increase (P < 0.01) when proteolysis index increased (566.83, 586.15 two groups. However, at the sensory level, these differences do not have and 623.75 mg/100 g of dry mater for LP, MP and HP batches, re- a great impact on the quality of the final product since the hydro- spectively). A similar trend was observed for serine, methionine and carbons are compounds that have little contribution to aroma because isoleucine, showing the highest levels in dry-cured hams with high PI. of their high odour threshold values (Wu et al., 2015). Among hydro- However, taurine and cysteine content presented an opposite beha- carbons, undecane was the most abundant in the three ham categories viour, reaching the highest values in dry-cured hams with low PI studied and this compound could be used to discriminate dry-cured
562 C. Pérez-Santaescolástica et al. Food Research International 107 (2018) 559–566
Table 3 Effect of proteolysis index on volatile compounds content (expressed as Area Units (AU) × 106/g dry matter) of dry-cured ham.
Volatile compounds LRI R Groups SEM P-value
LP MP HP
Octane 800 ms,lri,s 57.49c 36.59b 26.07a 1.759 < 0.001 Decane 1000 ms,lri,s 61.95c 47.44b 30.13a 1.753 < 0.001 Undecane 1100 ms,lri,s 143.42c 123.26b 71.39a 4.014 < 0.001 6-Tridecene 1223 ms 10.66b 10.11b 6.05a 0.442 < 0.001 Dodecane 1200 ms,lri,s 84.90b 80.25b 46.84a 2.366 < 0.001 Tridecane 1300 ms,lri,s 27.44b 26.79b 17.07a 0.729 < 0.001 Total lineal hydrocarbons 377.69c 323.96b 194.36a 9.309 < 0.001 Pentane, 2,3,4-trimethyl- 666 ms 13.92ab 15.54b 11.50a 0.53 0.006 Pentane, 2,3,3-trimethyl- 675 ms 33.08b 18.68a 24.44a 1.239 < 0.001 Heptane, 3-methylene- 743 ms 30.73b 22.33a 19.37a 0.905 < 0.001 Heptane, 3-ethyl- 866 ms,lri 24.88c 15.49b 9.48a 0.761 < 0.001 2,3-Dimethyl-3-heptene, (Z)- 898 ms 8.69b 6.49a 5.83a 0.254 < 0.001 Octane, 3-ethyl- 996 ms 23.39b 19.23a 16.15a 0.611 < 0.001 Nonane, 3-methyl- 999 ms 16.97c 12.85b 8.94a 0.422 < 0.001 Cyclohexane, 1,2-diethyl-1-methyl- 1041 ms 13.85c 11.27b 5.69a 0.449 < 0.001 Cyclopentane, pentyl- 1082 ms 66.26c 50.50b 33.98a 2.151 < 0.001 5-Undecene, 9-methyl-, (Z)- 1169 ms 78.70c 64.56b 34.22a 2.05 < 0.001 Undecane, 3-methyl- 1215 ms 31.98c 27.08b 19.68a 0.844 < 0.001 Undecane, 3-methylene- 1233 ms 12.58b 13.04b 8.69a 0.405 < 0.001 5-Undecene, 3-methyl-, (E)- 1235 ms 12.46c 9.92b 5.77a 0.527 < 0.001 10-Methylnonadecane 1293 ms 2.92b 2.68b 1.92a 0.117 < 0.001 Total branched hydrocarbons 347.95c 283.77b 213.44a 9.29 < 0.001 Total hydrocarbons 759.93c 605.28b 416.99a 21.711 < 0.001 2-Pentanone 620 ms,lri 10.82b 7.94a 10.66b 0.307 < 0.001 2-Butanone, 3-hydroxy- 711 ms,lri 25.60b 21.52a 19.56a 0.531 < 0.001 3-Heptanone 940 ms 4.24c 2.97b 2.20a 0.147 < 0.001 2-Heptanone 950 ms,lri 11.08 11.13 9.93 0.264 0.089 Total ketones 48.85b 43.22a 42.06a 0.654 < 0.001 Ethylalcohol 307 ms 256.05b 255.00b 223.95a 5.257 0.018 1-Butanol, 3-methyl- 737 ms 23.73c 17.61b 12.99a 0.92 < 0.001 1-Hexanol 932 ms,lri 20.43b 17.67b 11.70a 0.812 < 0.001 1-Octen-3-ol 1062 ms,lri 60.28c 47.67b 30.22a 2.208 < 0.001 Benzyl Alcohol 1157 ms,lri 24.78c 21.97b 17.53a 0.444 < 0.001 Total Alcohols 364.49b 357.68b 299.65a 6.092 < 0.001 Butanal, 3-methyl- 537 ms,lri 82.17b 82.72b 68.65a 1.985 0.005 Hexanal 814 ms,lri,s 104.42c 79.42b 43.87a 3.592 < 0.001 Heptanal 959 ms,lri,s 21.64c 17.02b 11.60a 0.554 < 0.001 Benzeneacetaldehyde 1154 ms 22.85c 17.34b 14.55a 0.572 < 0.001 Total Aldehydes 232.10c 195.75b 140.52a 5.969 < 0.001 Acetic acid, ethylester 437 ms 35.57 31.22 34.97 0.909 0.128 Decanoic acid, ethylester 1442 ms 4.92c 4.13b 3.23a 0.123 < 0.001 Total Esters 40.92 35.45 38.11 0.937 0.072 Acetic acid 571 ms 55.16b 40.46a 35.72a 1.519 < 0.001 Total Acids 55.16b 40.46a 35.72a 1.519 < 0.001 Disulfide, dimethyl 702 ms,lri 4.86a 6.06b 4.30a 0.179 < 0.001 Total Sulfur Compounds 4.86a 6.06b 4.30a 0.179 < 0.001 Pyrazine, 2,6-dimethyl- 964 ms,lri 15.44b 13.74a 14.21ab 0.259 0.029 Ethanol, 2-butoxy- 974 ms 41.42b 31.17a 25.95a 1.187 < 0.001 Total Other Compounds 56.86b 44.91a 40.16a 1.328 < 0.001 Total Compounds 1575.24c 1337.81b 997.49a 37.224 < 0.001
– a cMean values in the same row (corresponding to the same parameter) not followed by a common letter differ significantly (P < 0.05; Tukey's Test). SEM: standard error of mean; LRI: Lineal Retention Index calculated for DB-624 capillary column (J&W scientific: 30 m × 0.25 mm id, 1.4 μm film thickness) installed on a gas chromatograph equipped with a mass selective detector; R: Reliability of identification; lri: linear retention index in agreement with literature (Domínguez et al., 2014; Flores, Nieto, Ferrer, & Flores, 2005; Pateiro, Franco, Carril, & Lorenzo, 2015); ms: mass spectrum agreed with mass database (NIST14); s: mass spectrum and retention time identical with an authentic standard. Groups: LP = low proteolysis (PI < 32%); MP = medium proteolysis (32% < PI > 36%) and HP = high proteolysis (PI > 36%). hams according to their PI. three ham groups, ethyl alcohol was the most abundant and represented Regarding alcohols, significant differences were observed in the about 72% of the total alcohols. On the other hand, high 1-octen-3-ol total content (P < 0.001) among groups, as well as in all of individual content was also found in the three groups (60.28 vs. 47.67 vs. compounds. In all cases, the highest values corresponded to the hams 30.22 AU × 106/g of dry matter for LP, MP and HP groups, respec- with lower PI (Table 3). Alcohols follow the same mechanism of gen- tively). Mainly in hams with low PI, this alcohol has low odour eration as acids; straight-chain aliphatic alcohols can be generated by threshold and is associated with mushroom-like, earth, dust, fatty, the oxidation of lipids, whereas branched alcohols are most likely de- sharp and rancid odours (García-González, Tena, Aparicio-Ruiz, & rived from the Strecker degradation of amino acids through the re- Morales, 2008; Théron et al., 2010). In addition, it was found a positive duction of their respective aldehydes (Pérez-Palacios, Ruiz, Martín, correlation between 1-octen-3-ol and cysteine (r = 0.766; P < 0.01). Grau, & Antequera, 2010). Alcohols, because of their low odour Aldehydes are known as the major contributors to the unique flavor threshold, contribute to the aroma of ham, with fatty, woody and of dry-cured ham due to their rapid formation during lipid oxidation herbaceous notes (García & Timón, 2001). Among the alcohols, in the and their low odour thresholds (Ramírez & Cava, 2007). Linear
563 C. Pérez-Santaescolástica et al. Food Research International 107 (2018) 559–566 aldehydes come mainly from an oxidative degradation of the un- is originated from carbohydrate fermentation by microorganisms saturated fatty acids: oleic, linoleic, linolenic and arachidonic (Chan & (Kandler, 1983) and from the Maillard reaction according to others Coxon, 1987; Sabio, Vidal-Aragon, Bernalte, & Gata, 1998). On the (Martín et al., 2006). other hand, the major formation pathway of the branched chain alde- Most of the volatile compounds detected in the present study come hydes seems to be the oxidative deamination-decarboxylation, probably from the oxidation of lipids. Usually the processing conditions that fa- via Strecker-degradation (Narváez-Rivas et al., 2012). Statistical ana- vour the lipid oxidation (e.g. increased salt content) inhibit the action lysis showed that the total aldehyde content was significantly affected of proteolytic enzymes. This is probably the reason by which hams (P < 0.001) by PI, reaching the highest values in dry-cured hams with having the low PI showed the highest amounts of most of the volatile low PI (232.10 vs. 195.75 vs. 140.52 AU × 106/g of dry matter for LP, compounds determined. Apart from the volatiles formed directly from MP and HP groups, respectively). Within aldehydes, hexanal was the the lipid oxidation, oxidized lipids formed during ripening could react most abundant, showing significant differences (P < 0.001) among with the free amino acids converting them into Strecker aldehydes, α- batches (104.42 vs. 79.42 vs. 43.87 AU × 106/g of dry matter for LP, keto acids and amines. The lipid oxidation products (free radicals and MP and HP groups, respectively). Hexanal at low concentrations has a reactive carbonyls) can also influence the subsequent reactions suffered pleasant and grassy aroma (Aparicio & Morales, 1998), which turns by these compounds: the formation of Strecker aldehydes and other fatty at medium concentration and extremely rancid and tallowy at aldehydes from α-keto acids, the formation of Strecker aldehydes and high concentrations (Morales, Rios, & Aparicio, 1997). At the con- olefins from amines, the formation of shorter aldehydes from Strecker centrations determined in the analysed hams, hexanal contributes to aldehydes, and the addition reactions suffered by the olefins produced grassy odour in hams with high PI, and, perhaps, to a fatty perception in from the amines (Hidalgo & Zamora, 2016). This could be the most the case of hams with low PI. It was found a positive correlation be- probable origin of the butanal, 3-methyl (from leucine) and the ben- tween cysteine and hexanal (r = 0.599; P < 0.01), heptanal zeneacetaldehyde (from phenylalanine) detected in the present study; (r = 0.516; P < 0.01) and benzeneacetaldehyde (r = 0.561; the 1-butanol, 3-methyl also probably comes from reduction of the P < 0.01). butanal, 3-methyl having this same origin. The formation of these The low odour thresholds of ketones indicate that they have a great Strecker aldehydes from Maillard reactions is unlikely in hams, given impact on ham aroma. In dry-cured ham, their origin can be diverse. the very low amounts of reducing sugars present in such food matrix. Ramírez and Cava (2007) found that the majority of ketones originated On the other hand, these compounds could also be formed by reactions from lipid oxidation, whereas a few others, such as 3-hydroxybutan-2- between protein carbonyls and amino acids (Estévez, Ventanas, & one, are formed through Maillard reactions; the methyl ketones are Heinonen, 2011), but this origin in the present study is also unlikely, generated by microorganism esterification. Hams with low PI presented given that hams with a more intense proteolysis were those that pre- higher total ketones content than those in the two other groups (48.85 sented the lowest values of these two compounds. vs. 43.22 vs. 42.06 AU × 106/g of dry matter for LP, MP and HP groups, Due to the non-polar character of most of the volatiles determined, respectively). Although, the concentration of 2-heptanone did not show the special structure of the hams with abundant fat infiltrated in muscle significant differences among groups, this compound contributes to tissue could favour the retention of these compounds. Fat solubilizes ham aroma with spicy/blue cheese/acorn sensory notes due to low and traps these compounds avoiding its loss in the prevailing en- odour thresholds. Esters did not show significant differences among the vironmental conditions during maturation. three batches studied (40.92 vs. 35.45 vs. 38.11 AU × 106/g of dry matter for LP, MP and HP groups, respectively). Esters are formed 4. Conclusions through the enzymatic esterification of fatty acids and alcohols during curing, mostly by the action of microorganisms such as lactic acid Proteolysis significantly increased the adhesiveness of dry-cured bacteria and Micrococcaceae (Purrinos, Bermúdez, Franco, Carballo, & ham. The basic volatile nitrogen content and total free amino acid Lorenzo, 2011). Esters have low olfaction threshold values; however, content was not significantly affected by the proteolysis index. taking into account that the analysed samples presented very low va- Individual free amino acids content was higher in dry-cured hams with lues of these compounds, it can be considered that they do not con- high PI level, except for taurine, arginine, cysteine and lysine that tribute to the aroma of dry-cured ham. showed higher concentrations in the dry-cured hams with low PI levels. Sulfur compounds mainly originate from the catabolism of amino The bitter amino acids were significantly (P < 0.05) affected by PI, acids that contain sulfur (Ramírez & Cava, 2007; Sabio et al., 1998) showing the highest values in high PI level. Total content of volatile from ribonucleotides (Dumont & Adda, 1972), or they are generated by compound were significantly different among PI level groups, showing the microbial population (Martín et al., 2006). Only a sulfur compound, a decrease with the increase of the proteolysis index. Regarding the dimethyl disulfide, was found at very low concentrations in the three different chemical families of volatiles, the hydrocarbons (the main batches studied, and its presence could come from the degradation of family), alcohols, aldehydes, ketones and acids were more abundant in sulfur amino acids through a microbial deamination (Belitz & Grosch, the hams showing the lowest PI. However, esters did not show sig- 1999). However, a significant correlation between dimethyl disulfide nificant differences among the three batches of hams studied. Most of and cysteine, taurine and methionine was not found. the volatile compounds detected in the present study come from the Finally, acetic acid was the only acid identified in the headspace of oxidation of lipids. Usually the processing conditions that favour the the dry-cured ham samples, showing the highest content in hams with lipid oxidation inhibit the action of proteolytic enzymes. This is prob- low PI (55.16 vs. 40.46 vs. 35.72 AU × 106/g of dry matter for LP, MP ably the reason by which hams having the low PI showed the highest and HP groups, respectively). This outcome is in agreement with data amounts of most of the volatile compounds determined. Apart from the reported by Pérez-Juan, Flores, and Toldrá (2006) who observed that effect on the adhesiveness, an excessive proteolysis seems to be asso- acetic acid was the most abundant acid detected in dry-cured ham. The ciated with negative effects on the taste and aroma of the dry-cured main straight-chain carboxylic acids are derived from the hydrolysis of ham. triglycerides and phospholipids and mainly from the oxidation of un- saturated fatty acids (Pugliese et al., 2015). In addition, some branched Acknowledgements acids could be also originated from the oxidation of their respective Strecker aldehydes, for example, 2-methyl butanal would come from This research was supported by Grant RTA 2013-00030-CO3-03 the degradation of isoleucine amino acid and 2-methyl butanoic acid from INIA (Spain). Acknowledgements to INIA for granting Cristina would be formed from later oxidation (Ramírez & Cava, 2007). The Pérez Santaescolástica with a predoctoral scholarship. José M. Lorenzo origin of acetic acid in ham is not clear. According to some authors, this is member of the MARCARNE network, funded by CYTED (ref.
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Food Research International
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Application of temperature and ultrasound as corrective measures to decrease the adhesiveness in dry-cured ham. Influence on free amino acid T and volatile compound profile
Pérez-Santaescolástica C.a, Carballo J.b, Fulladosa E.c, Garcia-Perez José V.d, Benedito J.d, ⁎ Lorenzo J.M.a, a Centro Tecnológico de la Carne, Rúa Galicia No 4, Parque Tecnológico de Galicia, San Cibrán das Viñas, 32900 Ourense, Spain b Área de Tecnología de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain c IRTA, XARTA. Food Technology Program, Finca Camps i Armet, s/n 17121, Monells, Girona, Spain d UPV, Department of Food Technology, Universitat Politècnica de València, Camí de Vera s/n, E-46022, Valencia, Spain
ARTICLE INFO ABSTRACT
Keywords: The impact of low temperature treatment and its combination with ultrasound has been evaluated in order to Adhesiveness correct texture defects in dry-cured hams. A total of 26 dry-cured hams, classified as high proteolysis index Dry-cured ham (PI > 36%), were used. From these hams, ten slices from each ham sample were cut, vacuum packed and Free amino acid content submitted to three different treatments: control (without treatment), conventional thermal treatments (CV) and Heat treatment thermal treatment assisted by power ultrasound (US). The impact of these treatments on instrumental adhe- Proteolysis siveness, free amino acid and volatile compounds profile were assessed. Statistical analysis showed that both US Ultrasound treatment fi Volatile compounds and CV treatments, signi cantly (P < .001) decreased the instrumental adhesiveness of dry-cured hams from 85.27 g for CO to 40.59 and 38.68 g for US and CV groups, respectively. The total free amino acid content was significantly (P < .001) affected by both treatments, presenting higher values the samples from the US group (6691.5 vs. 6067.5 vs. 5278.2 mg/100 g dry matter for US, CV and CO groups, respectively). No significant differences were observed between US and CV treatments. All the individual free amino acids were influenced by ultrasound and temperature treatments, showing the highest content in sliced dry-cured ham submitted to ultrasounds at 50 °C, except for isoleucine which presented the highest level in samples from CV group. Similarly, significant differences (P < .05) were also detected in the total volatile compound content between CO and US groups, with a higher concentration in the CO batch (56,662.84 AU × 103/g of dry-cured ham) than in the US treatment (45,848.47 AU × 103/g of dry-cured ham), being the values in the CV treatment intermediate (48,497.25 AU × 103/g of dry-cured ham). Aldehydes, ethers and esters, carboxylic acids and sulphur compounds were more abundant in the CO group, while CV group showed higher concentrations of ketones, alcohols and nitrogen compounds.
1. Introduction However, negative impact on texture quality due to the reduction of salt in dry-cured meat products has been widely reported (Armenteros, In terms of economic value, dry-cured ham is the most important Aristoy, Barat, & Toldrá, 2009; Flores et al., 2006; Lorenzo, Fonseca, meat product in the Spanish market. Nevertheless, its production ex- et al., 2015). In this regard, excessive proteolysis during dry-cured ham perienced a gradual reduction during the last years (Ministerio de processing may lead to a high instrumental adhesiveness, a high pas- Agricultura y Pesca, 2017). This may be a consequence of consumer's tiness perception and thus a decrease of consumers' acceptability increasing concern for health. Dry-cured products have been reported (López-Pedrouso et al., 2018). In addition, other factors such as prop- to be one of the main sources of dietary salt in Spain, and it is known erties of fresh pieces (pH, fat level, weight), ripening process and type that sodium is highly related to cardiovascular diseases (WHO, 2012). of muscle have been related to proteolysis index of dry-cured ham Consequently, the reduction of salt in dry-cured ham could improve the (Škrlep et al., 2011). López-Pedrouso et al. (2018) noticed that the value of this product by addressing consumer's requirements. determination of instrumental adhesiveness could be a good indicator
⁎ Corresponding author. E-mail address: [email protected] (J.M. Lorenzo). https://doi.org/10.1016/j.foodres.2018.08.006 Received 11 June 2018; Received in revised form 17 July 2018; Accepted 2 August 2018 Available online 04 August 2018 0963-9969/ © 2018 Elsevier Ltd. All rights reserved. C. Pérez-Santaescolástica et al. Food Research International 114 (2018) 140–150 of pastiness level in dry-cured ham. These authors also observed that defined as the time needed to reach in the centre of the slice a tem- hams with higher proteolysis indices displayed increased instrumental perature 5 °C below that in the heating medium, measured using a adhesiveness. thermocouple. Thus, average ultrasonic treatment time was of 7.5 min. On the other hand, consumer preference highly depends on the Finally, samples were kept in a water bath (50 °C) to complete 5 h of sensory properties of slices, which are mainly determined by aroma, treatment. This heating temperature and time were chosen to avoid the taste and texture (Narváez-Rivas, Gallardo, & León-Camacho, 2012). In appearance of cooking flavours in the ham, as found in preliminary this regard, aroma of dry-cured ham is due to the presence of many experiments. Thermal treatments were applied in an ultrasonic bath volatile compounds generated by chemical and enzymatic mechanisms (600 W, 25 kHz, model GAT600W, ATU, Spain) using water as heating during the ripening process (Bermúdez, Franco, Carballo, & Lorenzo, fluid. 2015). A great number of volatile compounds has been found in dry- b) Conventional thermal treatments (CV) where samples were kept cured ham, including hydrocarbons, ketones, acids, terpenes, ketones, in a water bath for 5 h at 50 °C. alcohols, nitrogen and sulphur compounds, and others. However, only a limited number of volatile compounds contribute to the overall ham 2.2. Instrumental adhesiveness flavor (mainly aldehydes and ketones) (Carrapiso, Ventanas, & García, 2002). Textural analysis was performed using a texture analyzer (Stable Mild thermal treatments (around 30 °C) during a long time (between Micro Systems, TA-XT Plus, London, UK) by carrying out a separation 7 and 10 days) have been used to correct the softness and pastiness of test using different load cells with a specific probe. Instrumental ad- dry-cured ham (Gou, Morales, Serra, Guàrdia, & Arnau, 2008; Morales, hesiveness was measured in sliced ham samples (1 mm) by applying Arnau, Serra, Guerrero, & Gou, 2008). However, these treatments are probe tests and calculating the negative area of a force-time curve in not useful for the meat industries because they require a long proces- tension tests with a single cycle. The texturometer was equipped with a sing time which could affect to sensorial characteristics (mainly aroma probe connected to a special device that enables horizontal probe dis- and color) of dry-cured hams. Thus, in order to avoid these defects and placement. After the separation of the slices, the probe returned to the improve the final quality of dry-cured ham, new corrective measures initial position. The conditions for the instrumental measurement of that produce a more homogeneous increase of temperature of the ham adhesiveness of dry cured ham slices were reported by López-Pedrouso need to be explored. In this regard, the application of ultrasounds (US) et al. (2018). From the graph force vs. distance obtained, the adhe- treatment could be a suitable alternative to conventional thermal siveness was calculated. All the measurements were made in triplicate treatment (Önür et al., 2018). In addition, US can induce chemical, and carried out at room temperature. biological and mechanical changes in meat and meat products due to cavitations in liquid systems (Kang et al., 2016) and its effect of dry- 2.3. Moisture content cured hams has not been previously investigated. Low-intensity US waves are used to obtain information about the Moisture content was quantified according to the ISO recommended propagation medium, while high-intensity waves, or high-power US, standards 1442:1997 (ISO, 1997). are used to make permanent changes in the medium (Robles-Ozuna & Ochoa-Martínez, 2012). High-intensity US application is based in the 2.4. Free Amino acid analysis elastic deformation of ferroelectric materials caused by the mutual at- traction of polarized molecules into an electric field (Raichel, 2006). In The free amino acids were extracted following the procedure de- addition, Sajas and Gorbatow (1978) considered that ultrasonic in- scribed by Lorenzo, Cittadini, Bermúdez, Munekata, and Domínguez tensity is closely related to the appearance and magnitude of US effects. (2015b). Amino acids were derivatizated with 6-aminoquinolyl-Nhy- In a previous study, Contreras, Benedito, Bon, and Garcia-Perez (2018) droxysuccinimidyl carbamate (Waters AccQ-Fluor reagent kit) and noticed that heating caused an increase in hardness and elasticity of analyzed by RP-HPLC using a Waters 2695 Separations Module with a dry-cured ham, whereas the application of US did not modify the tex- Waters 2475 Multi Fluorescence Detector, equipped with a Waters ture parameters. However, to date the application of US as a corrective AccQ-Tag amino acid analysis column. The results were expressed as measure for adhesiveness of dry-cured meat products has not been mg of free amino acid/100 g of dry matter. explored. Previous studies noticed that the structure and the function of 2.5. Volatile compound analysis protein can be modified by the application of US. Thus, the objective of this study was to evaluate the high-power US combined with moderate The extraction of the volatile compounds was performed using thermal treatments as a non-invasive intervention strategy to decrease solid-phase microextraction (SPME). A SPME device (Supelco, the adhesiveness of sliced dry-cured ham, as well as the assessment of Bellefonte, USA) containing a fused silica fibre (10 mm length) coated the effects of these treatments on the free amino acid and volatile with a 50/30 layer of divinylbenzene/ carboxen/polydimethylsiloxane compound contents of ham samples. was used. Chromatographic analyses were carried out under the con- ditions described by Domínguez, Gómez, Fonseca, and Lorenzo (2014) 2. Materials and methods with modifications, and a gas chromatograph 7890B (Agilent Tech- nologies, Santa Clara, CA, USA) equipped with a mass selective detector 2.1. Samples 5977B (Agilent Technologies) was used. For extraction, 1 g of each sample was weighed in a 20 mL vial, after being ground using a com- For this study, a total of 26 dry-cured hams, classified as having a mercial grinder. The conditioning, extraction and injection of the high proteolysis index (PI > 36%) were used. Hams were manufactured samples were carried out with an autosampler PAL-RTC 120. Volatile according the process reported by Fulladosa et al. (2018). At the end of compounds were identified by comparing their mass spectra with those the process, hams were cut and boned and the cushion part containing contained in the NIST14 (National Institute of Standards and Tech- the Biceps femoris muscle was excised and sampled. Ten slices from each nology, Gaithersburg) library, and/or by comparing their mass spectra ham sample were vacuum packed and submitted to three different and retention time with authentic standards (pentane, octane, decane, treatments: control (without treatment), conventional thermal treat- undecane, dodecane, tridecane, propanal, butanal, pentanal, hexanal, ments (CV) and thermal treatment assisted by power ultrasound (US). heptanal, octanal, decanal, nonanal and pentadecanal) (Supelco, Bel- a) Thermal treatments assisted by power ultrasound (US), where lefonte, PA, USA), and/or by calculation of retention index relative to a ultrasound was only applied during the heating stage, which was series of standard alkanes (C5–C14) (for calculating Kovats indexes,
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ff Fig. 1. E ect of temperature treatment alone (CV) or US assisted (US) on in- Fig. 2. Effect of temperature treatment alone (CV) or US assisted (US) on strumental adhesiveness of dry-cured ham. Plotted values are means and moisture content of dry-cured ham. Plotted values are means and standard standard deviations of the results from twenty-six samples of each group. deviations of the results from twenty-six samples of each group.
Supelco 44,585-U, Bellefonte, PA, USA) and matching them with data difference between control and US starch corn samples, but they found fi reported in literature. The results are expressed as quanti ed area units a lower hardness, elasticity and brittleness in US treated samples. 3 (AU) × 10 /g of sample. Taking into account that texture is one the most important sensory attributes of dry-cured ham, which affect its acceptability by consumer, 2.6. Statistical analysis the application of both treatments, US and CV, could be used to reduce the instrumental adhesiveness of dry-cured ham slices by immersing the The effect of treatment was examined using a one-way ANOVA, packaged samples in a water bath during a short period of time. where this parameter was set as factor. The values were given in terms of mean values and standard error of the means (SEM). When a sig- 3.2. Effect of treatments on moisture content nificant effect (P < .05) was detected, means were compared using the Tukey's test. All analyses were conducted using the IBM SPSS Statistics The effect of temperature treatment alone or US assisted on 24.0 program (IBM Corporation, Somers, NY, USA) software package. moisture content is presented in Fig. 2. Statistical analysis did not show Correlations between variables (P < .05) were determined using the significant differences on moisture content among groups, presenting Pearson's linear correlation coefficient. mean values of 59.01, 58.68 and 58.57 g/100 g; P > .05, for CO, US and CV groups, respectively. Our moisture values were in the range of 3. Results and discussion data (48.3–65.2 g/100 g) reported by other authors (Bermúdez, Franco, Carballo, and Lorenzo, 2014; Prevolnik et al., 2011; Pugliese et al., 3.1. Effect of treatments on instrumental adhesiveness 2015) for dry-cured ham.
The effect of temperature treatment alone or US assisted on in- 3.3. Effect of treatments on free amino acid content strumental adhesiveness of dry-cured ham is shown in Fig. 1. Statistical analysis showed that both, US and CV treatments, significantly Table 1 shows the effect of temperature treatment alone or US as- (P < .001) decreased the instrumental adhesiveness of dry-cured hams sisted on the free amino acids of dry-cured ham. Statistical analysis from 85.27 g for CO to 40.59 and 38.68 g for US and CV groups, re- displayed that total free amino acid content was significantly spectively. However, there was not significant differences between US (P < .001) affected by both treatments, presenting the higher values and CV treatments. The decrease of instrumental adhesiveness in dry- the samples from the US group (6691.5 vs. 6067.5 vs. 5278.2 mg/100 g cured ham slices may be due to the fact that the intramolecular hy- dry matter for US, CV and CO groups, respectively). No significant drogen connections can break due to the mechanical vibration and the differences were observed between US and CV treatments. These values effects of thermal and ultrasonic cavitation causing loosening of the are within the range of free amino acid contents (from 4000 to molecular structure and reduction of molecular nodes (Luo, Huang, & 12,500 mg/100 g dry matter) described by other authors (Bermúdez, Yang, 2003). In addition, denaturation and structural changes of pro- Franco, Carballo, Sentandreu, and Lorenzo, 2014; Jurado, García, teins due to thermal treatment could also decrease the instrumental Timón, & Carrapiso, 2007; Martín, Antequera, Ventanas, Benítez- adhesiveness of dry-cured ham slices (Tornberg, 2005). Finally, some Donoso, & Córdoba, 2001) in dry-cured ham. The higher total free changes such as the aggregation of the globular heads of myosin amino acid content in samples submitted to ultrasound at 50 °C could (Morales et al., 2008), cell membrane destruction (Rowe, 1989) and the be due to the release of some free amino acids from cell tissues that transversal and longitudinal shrinkage of meat fibers (Tornberg, 2005) were destroyed by the ultrasounds. could take place during the thermal treatment. All the individual free amino acids were influenced by ultrasound The findings in the present work are in agreement with data re- and temperature treatments, showing the highest content in sliced dry- ported by Morales et al. (2008) who showed that the thermal treatment cured ham submitted to ultrasounds at 50 °C, except for isoleucine at 30 °C for 168 h on both sliced and whole dry-cured ham decreased which presented the highest level in samples from CV group. According softness, adhesiveness and pastiness in BF muscle, without increasing to Jambrak, Mason, Lelas, Paniwnyk, and Herceg (2014), the ultra- hardness in SM muscle or affecting their physicochemical parameters sound treatment can modify the protein structure due to partial clea- (moisture, activity water and proteolysis index). In addition, Gou et al. vage of intermolecular hydrophobic interactions, rather than peptide or (2008) observed a decrease of soft textures in whole dry-cured ham disulphide bonds increased the release of free amino acids. It could be pieces without affecting the sensory properties after a treatment of seen that leucine, glutamic acid and alanine were the most abundant 10 days ageing process at 30 °C. Regarding US application, our out- free amino acid in the three studied groups and the sum of these three comes are in agreement with data reported by Contreras et al. (2018) amino acids reached around 27% of the total free amino acids. who did not find any significant difference in hardness and elasticity of On the other hand, the flavour of dry-cured ham could be linked to dry-cured ham slices between ultrasonically assisted heated and con- the amount of the individual free amino acid. In this regard, sweet taste ventionally heated samples. However, our results are in disagreement is associated with the level of alanine, serine, proline, threonine and with those reported by Hu et al. (2014) who did not show significant glycine; bitter taste is related to aromatic amino acids such as leucine,
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Table 1 used as a method to improve the food preservation (Knorr et al., 2011) Effect of treatments on free amino acids content (expressed as mg/100 g dry together with the hypothesis that spoilage could originate higher con- matter) in dry-cured ham. Values are means of the results from twenty-six centrations of volatile compounds in the headspace (Carrapiso, Martín, samples of each group. Jurado, & García, 2010), could explain the less content of total volatile Tratamiento SEM P-value compounds in the US group. Regarding the different chemical families, except for hydrocarbons, the sum of the volatile compounds of each CO US CV family showed significant differences among groups. Moreover, the fi fl Aspartic acid 164.65a 212.10b 149.32a 5.122 < 0.001 levels of 94 individually volatile compounds were signi cantly in u- Serine 191.48a 243.71b 204.82a 5.820 < 0.001 enced by the treatment (24 hydrocarbons, 15 ketones, 15 alcohols, 21 Glutamic acid 430.61a 544.77b 463.93a 12.375 < 0.001 aldehydes, 10 ester and ethers, 4carboxilic acids, 3 sulfur compounds a c b Glycine 187.99 245.58 216.85 5.917 < 0.001 and 2 nitrogenous compounds). a b a Histidine 99.02 133.55 113.51 3.641 < 0.001 As shown in Table 2, hydrocarbons were the most numerous che- Taurine 80.95a 102.75b 100.04b 2.592 < 0.001 ff Arginine 364.86a 518.93b 361.99a 14.676 < 0.001 mical family with up to 56 di erent compounds, 24 of them have al- Threonine 218.46a 281.96c 250.30b 6.642 < 0.001 ready been identified in other previous studies in hams (Bermúdez Alanine 398.16a 544.41c 461.75b 12.949 < 0.001 et al., 2015; Narváez-Rivas et al., 2012; Pérez-Santaescolástica et al., a c b Proline 287.99 372.34 330.99 8.804 < 0.001 2018). Hydrocarbons represented a percentage of 30% of the total area Cisteine 287.14a 437.18b 417.09b 17.045 < 0.001 Tyrosine 181.33a 228.49b 219.62b 6.942 < 0.001 of the volatile compounds in control samples, whereas, in both US and Valine 385.79a 484.95b 428.48a 10.053 < 0.001 CV groups, this chemical family was the most abundant (accounting for Metionine 213.90a 259.31b 250.63b 6.074 < 0.001 43% and 37%, for US and CV batches, respectively). The aliphatic hy- a b a Lysine 247.69 351.95 276.72 9.506 < 0.001 drocarbon, that was found in higher concentration was 2,2,4,6,6-pen- a b b Isoleucine 364.94 411.06 421.89 8.196 < 0.001 tamethyl heptane, followed by octane, and then, with similar values, Leucine 608.59a 750.85b 700.38b 15.831 < 0.001 Phenilalanine 391.01a 495.85b 459.91b 11.808 < 0.001 pentane, hexane, undecane and dodecane. It is well known that sig- Total Aas 5278.18a 6691.53b 6067.45b 148.807 < 0.001 nificant differences in the hydrocarbons content does not originate Sweet1 1328.43a 1705.69c 1499.88b 33.752 < 0.001 important odour changes due to their low threshold values (Carrapiso 2 a b b Bitter 2014.89 2289.93 2256.99 36.002 < 0.001 et al., 2002). Acid3 699.95a 904.94b 765.60a 16.902 < 0.001 Aged4 601.69a 767.19b 645.23a 14.888 < 0.001 Meanwhile, the main family of volatile compounds in CO group were the aldehydes (approximately 41% of the total area of volatile a-bMean values in the same row (corresponding to the same parameter) not compounds). In this regard, Garcia et al. (1991) identified linear alde- followed by a common letter differ significantly (P < .05; Tukey's Test). hydes as a secondary product of lipid oxidative decomposition and at- SEM: standard error of mean. tributed the origin of branched aldehydes to non-enzymatic Strecker Treatments: CO = control (without treatment), CV = conventional thermal degradation of valine, leucine and isoleucine. In our work an important treatments and US = thermal treatment assisted by power ultrasound. reduction of total aldehydes content in US group was observed, as well 1 fl ∑ 2 Sweet avor = of alanine, glycine, threonine, serine and proline; Bitter as a higher decrease in CV batch (23,509.08 vs. 10,307.72 vs. flavor = ∑ of leucine, valine, isoleucine, methionine and phenylalanine; 3Acid 2381.68 AU × 103/g of dry-cured ham for CO, US and CV groups, re- flavor = ∑ of glutamic acid, aspartic acid and histidine; 4Aged flavor = ∑ of lysine, tyrosine and aspartic acid. spectively). According with previous studies in ham (Andres, Cava, Ventanas, Muriel, & Ruiz, 2007; Garcia et al., 1991; García-González, phenylalanine, methionine, valine and isoleucine; whereas acid taste is Tena, Aparicio-Ruiz, & Morales, 2008; Jurado, Carrapiso, Ventanasa, & linked to histidine, glutamic and aspartic acids, and aged flavour is García, 2009; Sánchez-Peña, Luna, García-González, & Aparicio, 2005), associated with the content of lysine, tyrosine and aspartic acid hexanal was the predominant linear aldehyde in CO and US groups, fi with the highest content presented in CO samples (12,264.83 vs. (Table 1). According to this classi cation, both treatments (ultrasound 3 and temperature) significantly increased the bitter taste of dry-cured 5747.78 vs. 185.78 AU × 10 /g of dry-cured ham for CO, US and CV ham. On the other hand, the use of temperature did not significantly groups, respectively). Hexanal is considered the main volatile com- modify the acid and aged taste, whereas these two tastes were sig- pound derived from oxidation of n-6 fatty acids such as linoleic and nificantly increased by using ultrasounds. The temperature significantly arachidonic acids, which contributes to the green, greasy and fatty fl increased the sweet taste of hams and this taste was significantly further distinctive avour in matured hams (García-González et al., 2008). In increased by the ultrasound treatment at 50 °C. These variations in free contrast, CV batch presented propanal as the main aldehyde, whose amino acid content could be affected the acceptance of dry-cured ham concentration was higher than in the other two groups. On the other for the consumers. hand, 3-methyl butanal was the most abundant branched aldehyde determined in all cases but presenting significant differences (P < .001) among the groups. CO samples showed the highest con- 3.4. Effect of treatments on volatile compound profile centration of this compound, while CV group registered the lowest one. In this way, Pérez-Santaescolástica et al. (2018) found that high-pro- The effect of temperature treatment alone or US assisted on the teolytic hams presented lower amounts of hexanal and 3-methyl bu- volatile fraction of dry-cured ham can be observed in Table 2. A total of tanal than low-proteolytic hams. Lower amounts of these aldehydes in 155 volatile compounds were found in headspace of the dry-cured ham. both treatment groups than in control was expected since high tem- These volatile compounds were classified as part of some of the main peratures promote protein degradation and enhance proteolytic reac- chemical families according to Narváez-Rivas et al. (2012) and tions. According to Ramirez and Cava (2007), who proposed the de- Purriños, Franco, Bermudez, Carballo, and Lorenzo (2011): 56 hydro- gradation of isoleucine amino acid as the most probably origin of 2- carbons, 23 aldehydes, 21 ketones, 16 esters and ethers, 24 alcohols, 6 methyl butanal, a negative correlation between these compounds was carboxylic acids, 4 nitrogenous compounds and 5 sulphur compounds. found (r = −0.547; P < .01), as well as significant (P < .001) dif- Significant differences (P < .05) were detected in the total volatile ference among the groups, obtaining higher levels in CV group than in compound content between CO and US groups, with a higher con- the others ones. centration in the CO batch (56,662.84 AU × 103/g of dry-cured ham) Likewise, the total alcohol content showed higher levels in CV than in the US treatment (45,848.47 AU × 103/g of dry-cured ham), samples than in the other two groups (6548.61 vs. 8599.43 vs. being the values in the CV treatment intermediate 12,199.24 AU × 103/g of dry-cured ham for CO, US and CV groups, (48,497.25 AU × 103/g of dry-cured ham). The fact that US had been respectively). This high content of total alcohols found in CV group is a
143 C. Pérez-Santaescolástica et al. Food Research International 114 (2018) 140–150
Table 2 Effect of treatments on volatile compounds content (expressed as quantifier area units (AU) x 103/g dry cured ham. Values are means of the results from twenty-six samples of each group.
Compound m/z LRI R Treatment SEM P-value
CO US CV
Pentane 43 500 ms, lri, s 883.71a 688.22a 1471.54b 94.956 0.005 Pentane, 2-methyl- 71 543 ms, lri 2.57a 3.29ab 4.50b 0.289 0.023 1-Butene, 2,3-dimethyl- 57 571 ms 19.51a 10.68a 30.18b 1.734 < 0.001 n-Hexane 69 600 ms, lri, s 810.40b 529.80a 1541.71c 61.771 < 0.001 Heptane 71 700 ms, lri, s 802.78 514.56 879.78 68.817 0.103 Pentane, 2,3,4-trimethyl- 71 756 ms, lri 232.76a 365.58ab 437.24b 26.540 0.003 Pentane, 2,3,3-trimethyl- 71 763 ms, lri 319.34a 508.02b 620.06b 34.305 < 0.001 Pentane, 3-ethyl- 70 770 ms, lri 51.97a 77.48ab 85.39b 5.219 0.015 1-Pentene, 3-ethyl-2-methyl- 83 774 ms 32.98 37.73 45.65 2.220 0.069 Hexane, 2,2,5-trimethyl- 57 799 ms 374.97a 655.05ab 705.58b 51.550 0.010 Octane 85 800 ms, lri, s 1942.31 1335.15 1731.67 154.326 0.257 2-Octene, (E)- 112 833 ms, lri 201.22 122.73 157.6 14.935 0.078 Heptane, 3,4,5-trimethyl- 85 842 ms 67.19a 110.46b 120.25b 7.106 0.002 3-Octene, (E)- 112 845 ms, lri 84.68 59.41 70.66 6.160 0.217 Octane, 2-methyl- 71 899 ms 12.42 15.12 13.79 1.002 0.530 Hexane, 2,2,5,5-tetramethyl- 57 914 ms, lri 301.96 409.36 394.91 26.669 0.168 4-Nonene 70 926 ms 130.55 148.11 173.08 7.236 0.057 Nonane 126 900 ms, lri, s 131.63a 167.86ab 193.45b 9.614 0.024 Heptane, 2-methyl-3-methylene- 57 930 ms 12.74a 14.51ab 17.80b 0.743 0.020 2-Octene, 4-ethyl- 69 982 ms 121.06 109.24 139.94 7.447 0.322 Octane, 3-methyl-6-methylene- 70 985 ms 204.18a 223.88ab 286.28b 12.678 0.028 Octane, 4-ethyl- 69 991 ms 72.43a 83.39ab 99.48b 4.114 0.026 Heptane, 3,3,4-trimethyl- 69 994 ms 6.01a 11.98b 3.49a 0.730 < 0.001 Pentane, 3,3-dimethyl- 85 995 ms 6.14 5.74 7.14 0.432 0.483 Decane 57 1000 ms, lri, s 392.40 484.05 448.96 35.082 0.536 Nonane, 2,3-dimethyl- 71 1003 ms 62.32 61.17 73.08 3.761 0.440 1-Octene, 2,6-dimethyl- 56 1010 ms 72.47 78.95 89.54 4.118 0.252 3-Octene, 4-ethyl- 69 1012 ms 23.62 22.29 26.35 1.302 0.519 Nonane, 3-methylene- 70 1022 ms 165.31 193.91 219.60 9.675 0.068 Heptane, 2,2,4,6,6-pentamethyl- 57 1027 ms, lri 3130.36ab 6386.68b 2772.86a 571.676 0.023 3-Ethyl-3-hexene 83 1042 ms 46.18a 68.29a 99.93b 5.404 < 0.001 Undecane, 3,6-dimethyl- 57 1068 ms 247.95ab 333.34b 119.46a 31.537 0.042 Tridecane, 6-methyl- 57 1079 ms, lri 241.55 296.61 296.67 18.192 0.326 Undecane, 2,5-dimethyl- 57 1085 ms 159.26 140.65 150.96 11.186 0.788 Decane, 2,3,5-trimethyl- 57 1099 ms 102.23b 56.83a 81.27ab 7.435 0.032 Undecane 57 1100 ms, lri, s 930.86 1346.47 1216.44 83.082 0.085 2,3-Dimethyl-3-heptene, (Z)- 83 1123 ms, lri 56.04b 25.71a 10.65a 4.093 < 0.001 2-Undecene, 9-methyl-, (Z)- 70 1132 ms 368.85 345.35 367.91 22.501 0.900 5-Undecene, 6-methyl- 168 1144 ms 11.24 8.17 9.33 0.741 0.202 4,4-Dipropylheptane 85 1153 ms 51.23 43.30 50.12 3.096 0.548 2-Undecene, 3-methyl-, (E)- 70 1181 ms 60.96 55.41 61.11 3.488 0.774 4-Nonene, 5-butyl- 70 1197 ms 24.26 23.38 20.87 1.532 0.678 Dodecane 57 1200 ms, lri, s 664.51 948.13 849.77 53.501 0.066 Decane, 3-ethyl-3-methyl- 57 1228 ms 50.22 42.58 46.32 2.933 0.551 Dodecane, 2-methyl- 57 1233 ms 23.00a 38.36b 30.39ab 2.057 0.005 1-Tetradecene 97 1236 ms, lri 31.84 30.42 28.93 2.097 0.857 Tridecane 71 1300 ms, lri, s 228.76 318.27 217.88 21.114 0.131 Tridecane, 3-methyl- 85 1304 ms 31.82 38.27 37.84 1.868 0.252 Total Aliphatic hydrocarbons 15,578.28 19,062.05 17,144.10 1014.413 0.356 Furan, 2-ethyl- 81 703 ms, lri 38.75ab 14.06a 60.00b 4.756 0.001 Toluene 92 804 ms 122.47a 131.23a 178.32b 5.716 < 0.001 Cyclobutane, 1,1,2,3,3-pentamethyl- 70 813 ms 247.78 268.52 288.93 13.907 0.490 Ethylbenzene 91 917 ms, lri 17.64 18.84 17.70 0.814 0.811 Benzene, 1,3-dimethyl- 106 926 ms 19.44 21.44 21.39 0.603 0.267 2-n-Butyl furan 81 944 ms, lri 35.70 32.04 42.78 2.845 0.383 Cyclopentane, 1-ethyl-3-methyl- 83 1123 ms 56.04b 25.71a 10.65a 4.093 < 0.001 Cyclopentane, ethyl- 98 1148 ms, lri 300.84c 173.68b 38.57a 20.284 < 0.001 Total Aromatic and cyclic hycrocarbons 808.45 743.01 769.51 26.041 0.565 Total Hydrocarbons 16,867.18 19,912.67 17,932.30 1045.388 0.479 Propanal 58 526 ms, lri, s 139.01a 102.85a 751.47b 43.600 < 0.001 Propanal, 2-methyl- 72 557 ms, lri 213.22b 173.69b 7.43a 16.502 < 0.001 Butanal 72 584 ms, lri, s 23.16c 10.81b 1.45a 1.688 < 0.001 Butanal, 3-methyl- 58 659 ms, lri 1968.06c 1240.06b 68.91a 142.214 < 0.001 Butanal, 2-methyl- 57 671 ms, lri 1139.71b 929.14b 43.06a 84.003 < 0.001 Pentanal 57 728 ms, lri, s 951.76 640.68 697.89 65.639 0.090 2-Butenal, 2-methyl- 84 801 ms 104.37b 55.38a 27.29a 7.598 < 0.001 Hexanal 56 865 ms, lri, s 12,264.83c 5747.78b 185.13a 889.713 < 0.001 Heptanal 70 974 ms, lri, s 853.54c 401.98b 25.49a 68.206 < 0.001 Methional 104 999 ms, lri 134.75b 134.52b 7.04a 12.331 < 0.001 Benzaldehyde 106 1045 ms, lri 352.12c 200.47b 67.03a 22.052 < 0.001 Octanal 56 1066 ms, lri, s 370.02c 249.58b 98.19a 23.992 < 0.001 (continued on next page)
144 C. Pérez-Santaescolástica et al. Food Research International 114 (2018) 140–150
Table 2 (continued)
Compound m/z LRI R Treatment SEM P-value
CO US CV
5-Ethylcyclopent-1-enecarboxaldehyde 124 1099 ms 32.99b 17.82a 10.03a 2.308 < 0.001 Benzeneacetaldehyde 91 1119 ms, lri 796.26c 356.03b 37.78a 52.710 < 0.001 2-Octenal, (E)- 70 1123 ms, lri 44.78b 17.22a 10.22a 3.112 < 0.001 Decanal 81 1129 ms, lri, s 24.68 23.26 23.18 1.663 0.912 Nonanal 57 1148 ms, lri, s 614.70c 380.07b 133.97a 38.155 < 0.001 4-Nonenal, (E)- 83 1201 ms 33.21b 23.96ab 23.29a 1.657 0.013 Benzaldehyde, 3-ethyl- 134 1209 ms 33.46b 27.15b 8.76a 2.527 < 0.001 2-Decenal, (E)- 70 1272 ms, lri 28.90b 19.66ab 13.75a 1.793 0.001 2,4-Decadienal, (E,E)- 81 1315 ms, lri 23.10b 8.08a 1.22a 2.199 < 0.001 2-Undecenal 95 1339 ms, lri 6.56b 2.44a 2.76a 0.624 0.004 Pentadecanal- 82 1516 ms, lri, s 3.90a 9.02b 4.73a 0.682 0.003 Total Aldehyde 23,509.08c 10,307.72b 2381.68a 1562.858 < 0.001 Acetone 58 528 ms 246.04a 438.13b 958.64c 50.416 < 0.001 2,3-Hexanedione 41 562 ms 391.05b 226.53a 696.97c 30.694 < 0.001 2-Butanone 72 596 ms 177.17a 264.28b 504.65c 22.630 < 0.001 Cyclopentanone, 3-methyl- 56 667 ms 30.74ab 18.76a 34.05b 2.459 0.043 2-Pentanone 86 720 ms, lri 101.75a 78.17a 305.68b 25.871 0.001 Acetoin 45 787 ms, lri 484.13a 501.60a 2031.51b 153.676 < 0.001 3-Heptanone 57 960 ms, lri 43.80 37.03 37.54 1.883 0.225 2-Heptanone 58 967 ms, lri 427.95a 664.14ab 980.43b 62.048 0.001 Cyclohexanone, 2-ethyl- 69 972 ms 39.00a 42.78a 65.73b 3.247 0.002 2-Nonen-4-one 69 979 ms 13.48 14.36 17.24 0.940 0.272 2-Hepten-4-one, 6-methyl- 69 992 ms 72.65a 80.61ab 99.82b 3.864 0.015 4-Octanone, 5-hydroxy-2,7-dimethyl- 69 1042 ms 9.29a 18.03ab 21.64b 1.615 0.003 1-Octen-3-one 70 1046 ms, lri 109.18 96.80 71.31 8.502 0.202 5-Hepten-2-one, 6-methyl- 69 1056 ms, lri 104.35ab 93.37a 134.10b 5.814 0.026 2-Octanone 58 1059 ms, lri 38.35a 95.71a 163.52b 12.653 < 0.001 3-Nonanone 113 1134 ms 23.48 21.34 23.80 1.588 0.818 1-Hexanone, 5-methyl-1-phenyl- 105 1137 ms 15.19a 28.98b 24.08b 1.564 < 0.001 2-Nonanone 58 1141 ms, lri 16.85a 71.11b 56.62b 6.375 < 0.001 2(3H)-Furanone, 5-ethyldihydro- 85 1158 ms, lri 187.86 226.67 199.86 8.500 0.156 5-Hexen-3-one 57 1161 ms 48.92 38.56 53.49 3.652 0.298 2,6-Bis(1,1-dimethylethyl)-4-(1-oxopropyl)phenol 233 1448 ms 11.04b 0.00a 0.00a 1.497 0.001 Total Ketone 2322.78a 3046.03b 6772.32c 265.182 < 0.001 Acetic acid ethenyl ester 86 588 ms 25.62a 17.51a 50.61b 3.166 < 0.001 Ethyl Acetate 61 598 ms 107.45 162.28 142.48 13.452 0.210 Methane, oxybis[dichloro- 83 611 ms 224.46 251.18 231.85 14.170 0.734 Propanoic acid, ethyl ester 57 737 ms 46.38b 15.79a 19.06a 3.404 < 0.001 Butanoic acid, ethyl ester 71 855 ms 77.53c 53.05b 22.14a 4.569 < 0.001 Butanoic acid, 2-methyl-, ethyl ester 102 908 ms 46.49 49.14 39.04 3.892 0.624 Butanoic acid, 3-methyl-, ethyl ester 88 913 ms 121.86ab 138.61b 67.83a 10.093 0.024 Oxalic acid, butyl propyl ester 57 936 ms 131.63a 167.86ab 193.45b 9.614 0.024 Ethanol, 2-butoxy- 57 985 ms, lri 394.15b 296.66ab 218.86a 22.783 0.004 Carbonic acid, bis(2-ethylhexyl) ester 112 1003 ms 25.20 25.06 28.09 1.605 0.736 Hexanoic acid, ethyl ester 88 1050 ms 184.39b 150.70b 79.11a 11.285 < 0.001 2-Piperidinecarboxylic acid, 1-acetyl-, ethyl ester 84 1124 ms 30.54b 18.80a 15.15a 1.887 0.001 Carbonic acid, tridecyl vinyl ester 57 1168 ms 210.11a 163.66a 189.81a 15.263 0.447 Octanoic acid, ethyl ester 88 1204 ms 75.26b 77.21b 42.04a 4.187 0.001 Decanoic acid, ethyl ester 88 1336 ms 33.57b 27.32b 12.77a 2.519 0.002 2,2,4-Trimethyl-1,3-pentanediol diisobutyrate 71 1442 ms 3.42a 3.40a 2.43a 0.182 0.064 Total Esther and ether 1906.99b 1680.82ab 1385.33a 68.273 0.006 Isopropyl Alcohol 45 532 ms 119.01ab 163.82b 100.93a 9.654 0.039 1-Propanol 59 572 ms 39.39ab 59.98b 23.41a 3.963 0.002 2-Butanol 45 607 ms, lri 21.64 27.36 30.26 1.483 0.043 1-Butanol 56 707 ms, lri 39.26b 40.08b 9.13a 3.127 < 0.001 1-Penten-3-ol 57 730 ms 853.31 621.14 784.02 47.894 0.122 2-Pentanol 45 751 ms 124.97 209.61 202.82 18.563 0.088 1-Butanol, 3-methyl- 55 808 ms, lri 239.69a 1169.80b 3556.89c 253.843 < 0.001 1-Butanol, 2-methyl- 57 812 ms 39.06a 238.09b 581.42c 42.813 < 0.001 1-Pentanol 55 847 ms, lri 576.25b 299.13a 189.49a 43.802 < 0.001 2-Propanol, 2-methyl- 59 894 ms 22.58b 9.71a 17.36ab 1.924 0.016 2,3-Butanediol, [S-(R*,R*)]- 45 909 ms 69.08b 8.56a 2.13a 7.003 < 0.001 3-Pentanol, 2,4-dimethyl- 73 954 ms 13.50 18.68 24.18 2.149 0.129 1-Heptanol 70 1046 ms 109.18 96.80 71.31 8.502 0.202 1-Octen-3-ol 57 1051 ms, lri 3543.17 3818.07 3922.68 236.699 0.789 1-Heptanol, 2,4-diethyl- 69 1085 ms 112.27 71.78 77.41 9.031 0.108 2-Ethyl-1-hexanol 57 1094 ms 11.36ab 10.53a 15.90b 0.875 0.048 4-Ethylcyclohexanol 81 1104 ms 90.23a 129.55ab 141.39b 8.253 0.019 Benzyl alcohol 108 1124 ms, lri 131.16 145.59 153.53 7.361 0.444 1-Octanol 56 1127 ms, lri 73.90ab 88.89b 49.90a 5.781 0.043 4-Methyl-5-decanol 55 1162 ms 25.30a 36.53a 74.05b 5.088 < 0.001 p-Cresol 107 1178 ms 30.50 31.28 28.20 1.333 0.687 Phenylethyl Alcohol 92 1182 ms 13.89a 186.88a 883.92b 65.261 < 0.001 (continued on next page)
145 C. Pérez-Santaescolástica et al. Food Research International 114 (2018) 140–150
Table 2 (continued)
Compound m/z LRI R Treatment SEM P-value
CO US CV
1-Tetradecanol 68 1225 ms 28.08 31.26 33.29 1.363 0.281 1,4-Benzenediol, 2,5-bis(1,1-dimethylethyl)- 222 1485 ms 0.27a 0.41b 0.27a 0.017 < 0.001 Total Alcohol 6548.61a 8599.43a 12,199.24b 487.720 < 0.001 Propanoic acid 74 827 ms, lri 12.07 16.39 16.71 2.193 0.606 Propanoic acid, 2-methyl- 73 888 ms, lri 74.38b 47.64ab 31.63a 5.693 0.005 Butanoic acid 60 918 ms, lri 209.13c 74.58b 15.13a 14.471 < 0.001 Butanoic acid, 3-methyl- 60 969 ms, lri 427.98 329.99 366.87 33.667 0.459 Pentanoic acid 60 1083 ms, lri 428.30c 274.79b 7.68a 28.766 < 0.001 Octanoic acid 60 1224 ms 36.67c 20.14b 4.08a 2.717 < 0.001 Total Carboxylic acid 1172.40c 950.08b 316.57a 58.148 < 0.001 Fumaronitrile 78 646 ms 27.19b 17.32a 23.53ab 1.418 0.011 3-(1′-pyrrolidinyl)-2-butanone 98 906 ms 92.62 95.73 121.88 5.438 0.078 Pyrazine, 2,6-dimethyl- 108 978 ms, lri 347.01a 337.27a 478.72b 14.720 < 0.001 1-(1′-pyrrolidinyl)-2-butanone 84 982 ms 90.39 97.20 117.94 5.324 0.110 Total Nitrogenous compounds 561.37a 550.57a 747.76b 20.616 < 0.001 Carbon disulfide 76 533 ms 157.74b 77.69a 195.02b 11.366 < 0.001 Disulfide, dimethyl 94 781 ms, lri 1740.04b 206.48a 738.87a 141.238 < 0.001 Dimethyl trisulfide 126 1035 ms, lri 123.40b 10.27a 5.82a 10.579 < 0.001 Sulfurous acid, decyl hexyl ester 85 1156 ms 110.15 122.77 104.36 11.499 0.835 Sulfurous acid, butyl dodecyl ester 85 1304 ms 31.82 38.24 37.81 1.862 0.254 Total Sulfur compounds 2213.62b 443.46a 1081.88a 161.357 < 0.001 Total Compounds 56,662.84b 45,848.47a 48,407.25ab 1697.399 0.013 a-cMean values in the same row (corresponding to the same parameter) not followed by a common letter differ significantly (P < .05; Tukey's Test). SEM: standard error of mean; m/z: Quantification ion; LRI: Lineal Retention Index calculated for DB-624 capillary column (J&W scientific: 30 m × 0.25 mm id, 1.4 μm film thickness) installed on a gas chromatograph equipped with a mass selective detector; R: Reliability of identification; lri: linear retention index in agreement with literature (Domínguez et al., 2014; Lorenzo, Montes, Purriños, & Franco, 2012; Lorenzo, Bedia, & Bañon, 2013; Lorenzo, 2014; Lorenzo & Domínguez, 2014; Lorenzo & Carballo, 2015; Pateiro, Franco, Carril, & Lorenzo, 2015; Pérez-Santaescolástica et al., 2018; Purriños, Franco, Bermúdez, Temperan, Carballo, and Lorenzo, 2011; Purriños, Franco, Carballo, & Lorenzo, 2012, Purriños, Carballo, & Lorenzo, 2013); ms: mass spectrum agreed with mass database (NIST14); s: mass spectrum and retention time identical with an authentic standard. Treatments: CO = control (without treatment), CV = conventional thermal treatments and US = thermal treatment assisted by power ultrasound. consequence of the higher amounts of three specific individual alcohols: was not observed in the present study, since the CV samples showed the 2-methyl butanol, 3-methyl butanol and phenylethyl alcohol. The in- lowest total content of esters (1906.99 vs. 1680.82 crement of 2-methyl butanol and 3-methyl butanol in CV group could vs.1385.33 AU × 103/g of dry-cured ham for CO, US and CV groups, be explained for the decrease observed in the 2-methyl butanal and 3- respectively). This fact may be explained because the high temperature methyl butanal since that branches alcohols may be originated, among produced losses by volatilisation. others reasons, from the reduction of branched aldehydes (Martín, Regarding carboxylic acids, total content was 20% less in US group Córdoba, Aranda, Córdoba, & Asensio, 2006). Otherwise, the major and 70% in CV treatment than in CO group. The highest differences alcohol detected in similar levels in all the groups was 1-octen-3-ol were found between pentanoic acid and butanoic acid contents. (3543.17 vs. 3818 vs. 3922.68 AU × 103/g of dry-cured ham for CO, US On the other hand, 2,6-dimethyl pyrazine was found as the main and CV groups, respectively). nitrogenous compound. Pyrazines are usual compounds in meat and In addition to aldehydes, Carrapiso et al. (2002) identified ketones meat products cooked at high temperatures (Mussinan & Walradt, as important compounds to odour contribute in dry-cured ham. In our 1974), and their formation is a result of the reaction between diketones study, statistical analysis showed that the total ketones content was and amino compounds at high temperatures (Shibamoto & Bernhard, significantly (P < .001) affected by the treatment, observing the 1976). According to this, CV samples showed higher significant values greatest level in CV group, and being the 2-heptanone and the acetoin (P˂0.001) than the other batches, whereas US batch did not show any the most abundant ones with higher amount in CV samples than in CO difference compared with CO group. It is possible that the structural and US groups (427.95 vs. 664.14 vs. 980.43 and 484.130 vs. 501.60 vs. changes that were originated by US application can prevent reactions 231.51 AU × 103/g of dry-cured ham for CO, US and CV groups, re- between diketones and amino compounds. spectively). In agreement with previous studies (Ramirez & Cava, 2007; Finally, the temperature application also originated an important Sabio, Vidal-Aragón, Bernalte, & Gata, 1998), other 2-ketones were also decrease in the sulfur compounds, being the dimethyl disulfide the most found, such as 2-butanone, 2-pentanone, 2-octanone and 2-nonanone. affected compound (1740.04 vs. 206.48 vs. 738.87 AU × 103/g of dry- All these compounds presented the highest values in the samples from cured ham for CO, US and CV groups, respectively). The sulfur amino CV treatment. acids showed a negative and significant (P < .01) correlation with Esters and ethers, carboxylic acids, nitrogenous compounds and dimethyl disulfide (r = −0.557, r = −0.614 and r = −0.512, for sulfur compounds were the chemical families that presented minor le- taurine, cysteine and methionine, respectively) and dimethyl trisulfide vels of volatile compounds. Esters are compounds distributed in the (r = −0.550, r = −0.599 and r = −0.493, for taurine, cysteine and essential oils with a high flavouring effects, derived from the reaction of methionine, respectively), suggesting that these compounds could be an alcohol or phenol with acids (Reineccius, 1991). Some studies re- originated by the amino acids catabolism (Sabio et al., 1998). ported low values of esters in volatile dry-cured ham profiles (Martín et al., 2006), whereas other studies carried out in cooked pork meat showed a greater content of these compounds (Gorbatov & 3.5. Effect of treatment on sensory attributes Lyaskovskaya, 1980). According to this, it could be assumed that temperature affects the ester compound formation. However, this effect It is worth noting that not all the volatile compounds contribute in the same way to the final odour because only a small percentage of
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Fig. 3. Comparative sensory descriptors among treatments. Sensory descriptions are given in agreement with: García-González et al. (2008), Carrapiso et al. (2010); Carrapiso et al. (2002) and Narváez-Rivas et al. (2012). Selected sensory descriptors related to each volatile compound were grouped in three intervals for a better comprehension: A (0-15,000 AU × 103/g of dry-cured ham), B (0-2000 AU × 103/g of dry-cured ham) and C (0–400 AU × 103/g of dry-cured ham. them are odour active and the sensory characteristics can change de- described as odour descriptors, octane, heptane, hexane, ethyl benzene pending on their concentrations and on the synergies with other com- and 2-ethyl furan, whose contribution is related with sweet notes. As pounds of the matrix (Aparicio & Morales, 1998). Over the years, some mentioned above, this chemical family has not very odorant impact, authors have investigated the relationship between volatile compounds because of its high threshold. Considering their low threshold, alde- and the odour characteristics (Carrapiso et al., 2010; García-González hydes are the most intensive compounds followed by ketones and es- et al., 2008; Narváez-Rivas et al., 2012). In this context, Fig. 3 shows ters, and to a lesser extent by alcohols. Hexanal and 3-methyl butanol the most odour compounds in dry-cured ham identifying and com- are the most odour-active compounds identified in hams (Carrapiso paring their contents in the different treatments. Due to different et al., 2002) and were the main volatile compounds showed in CO amounts, selected sensory descriptors related to each volatile com- samples, contributing principally with the characteristic greasy odour pound were grouped in three intervals for a better comprehension: A of ham and to a lesser extent with fruity notes. Significant lower levels (0–15,000 AU × 103/g of dry-cured ham), B (0–2000 AU × 103/g of of hexanal were found in treated groups, observing the lowest content dry-cured ham) and C (0–400 AU × 103/g of dry-cured ham). in CV group. Lower contents in CV batch also detected for nonanal, In case of the hydrocarbons, only five compounds were previously octanal, heptanal, 2-methyl butanal, 3-methyl butanal, 2,4-decadienal,
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Fig. 3. (continued)
4-nonenal, 2-octenal 2-methyl propanal, methional and benzaldehyde. differences in 1-octen-3-ol among the groups, a fact that was expected According to this, the application of high temperature without ultra- since this compound that contributes with a typical mushroom odour is sound could promote an important reduction, specially, on fatty and derived from feeding system (Jurado et al., 2009). Among the esters grassy notes. Regarding ketones, the CV group presented higher levels reported in previous studies, only one was detected here. Ethyl ester in four of the six odour active ketones found in this study, so the odour butanoic acid was identified as a specific odour-active compound in of this group of hams could be more floral and fruity compared with the Iberian (Carrapiso et al., 2010), Serrano (Flores, Grimm, Toldrá, & others. On the other hand, alcohols with a low molecular weight confer Spanier, 1997) and Jinhua (Song, Cadwallader, & Singh, 2008) hams. a sweet and spirituous odour to ham, but as the molecular weight in- Finally, dimethyl disulfide and some carboxylic acids (butanoic, creases a fatty and irritating odour is perceived (Narváez-Rivas, propanoic, pentanoic and 3-methyl butanoic acid) were previously re- Gallardo, & León-Camacho, 2016). Samples from CV group showed ported like spoiled ham odorants (Carrapiso et al., 2010). In this con- higher values of 3-methyl butanol, compound associated to biceps fe- text, CO group showed higher spoiled and rancid odour due to its moris muscle (Sánchez-Peña et al., 2005), and 2-butanol than the other higher amounts of butanoic, pentanoic, 3-methyl butanoic acid and two groups. Additionally, it was observed fatty, balsamic and fruity dimethyl disulfide (see Fig. 3b and c). notes reduction due to the lowest amounts of pentanol, octanol and butanol presented in these samples. It was not found significant
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4. Conclusions of jumbo squid (Dosidicus gigas) meat by ultrasonic treatment using response surface methodology. Food Chemistry, 160, 219–225. ISO (1997). Determination of moisture content, ISO 1442:1997 standard. In International The thermal treatment (5 h at 50 °C) of sliced, vacuum packaged standards meat and meat products. Geneva, Switzerland: International Organization for high proteolysis hams applied both alone and assisted by ultrasonic Standardization. treatment during the first 7.5 min of thermal treatment significantly Jambrak, A. R., Mason, T. J., Lelas, V., Paniwnyk, L., & Herceg, Z. (2014). Effect of ul- decreased the adhesiveness of hams. However, both treatments sig- trasound treatment on particle size and molecular weight of whey proteins. Journal of Food Engineering, 121,15–23. nificantly affected the total and individual free amino acid content. Jurado, Á., Carrapiso, A. I., Ventanasa, J., & García, C. (2009). Changes in SPME-ex- These treatments had also a significant effect on the total volatile tracted volatile compounds from Iberian ham during ripening. Grasas y Aceites, 60(3), – compounds and on the contents of the different families of volatiles. 262 270. Jurado, Á., García, C., Timón, M. L., & Carrapiso, A. I. (2007). Effect of ripening time and Taking into account the specific taste of some free amino acids and also rearing system on amino acid-related flavour compounds of Iberian ham. Meat the particular aroma notes of the different volatile compounds, and Science, 75(4), 585–594. despite the limitations of the present work (no quantification or nor- Kang, D. C., Zou, Y. H., Cheng, Y. P., Xing, L. J., Zhou, G. H., & Zhang, W. G. (2016). Effects of power ultrasound on oxidation and structure of beef proteins during curing malization was done for the extraction of volatile molecules and sen- processing. Ultrasonics Sonochemistry, 33,47–53. sorial analyses were not carried out), an effect of these two treatments Knorr, D., Froehling, A., Jaeger, H., Reineke, K., Schlueter, O., & Schoessler, K. (2011). on the taste and odor of ham could be expected. Emerging Technologies in Food Processing. Annual Review of Food Science and Technology, 2(1), 203–235. López-Pedrouso, M., Pérez-Santaescolástica, C., Franco, D., Fulladosa, E., Carballo, J., Acknowledgements Zapata, C., & Lorenzo, J. M. (2018). Comparative proteomic profiling of myofibrillar proteins in dry-cured ham with different proteolysis indices and adhesiveness. Food Chemistry, 244, 238–245. This research was supported by Grant RTA 2013-00030-CO3-03 Lorenzo, J. M. (2014). Influence of the type fiber coating and extraction time on foal dry- from INIA (Spain). Acknowledgements to INIA for granting Cristina cured loin volatile compounds extracted by solid-phase microextraction (SPME). Pérez Santaescolástica with a predoctoral scholarship (grant number Meat Science, 96, 179–186. fl CPD2015-0212). José M. Lorenzo is member of the MARCARNE net- Lorenzo, J. M., Bedia, M., & Bañon, S. (2013). Relationship between avour deterioration and the volatile compound profile of semi-ripened sausage. Meat Science, 93, work, funded by CYTED (ref. 116RT0503). 614–620. Lorenzo, J. M., & Carballo, J. (2015). Changes in physico-chemical properties and volatile References compounds throughout the manufacturing process of dry-cured foal loin. Meat Science, 99,44–51. Lorenzo, J. M., Cittadini, A., Bermúdez, R., Munekata, P. E., & Domínguez, R. (2015). fl Andres, A. I., Cava, R., Ventanas, S., Muriel, E., & Ruiz, J. (2007). Effect of salt content In uence of partial replacement of NaCl with KCl, CaCl2 and MgCl2 on proteolysis, and processing conditions on volatile compounds formation throughout the ripening lipolysis and sensory properties during the manufacture of dry-cured lacón. Food of Iberian ham. European Food Research and Technology, 225(5–6), 677–684. Control, 55,90–96. Aparicio, R., & Morales, M. T. (1998). Characterization of olive ripeness by green aroma Lorenzo, J. M., & Domínguez, R. (2014). Cooking losses, lipid oxidation and formation of compounds of virgin olive oil. Journal of Agricultural and Food Chemistry, 46(3), volatile compounds in foal meat as affected by cooking procedure. Flavour and 1116–1122. Fragance Journal, 29, 240–248. Armenteros, M., Aristoy, M. C., Barat, J. M., & Toldrá, F. (2009). Biochemical changes in Lorenzo, J. M., Fonseca, S., Gómez, M., & Domínguez, R. (2015). Influence of the salting dry-cured loins salted with partial replacements of NaCl by KCl. Food Chemistry, time on physico-chemical parameters, lipolysis and proteolysis of dry-cured foal 117(4), 627–633. “cecina”. LWT- Food Science and Technology, 60, 332–338. Bermúdez, R., Franco, D., Carballo, J., & Lorenzo, J. M. (2014). Physicochemical changes Lorenzo, J. 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Study of volatile alcohols foal meat. LWT- Food Science and Technology, 58, 439–445. and esters from the subcutaneous fat during ripening of Iberian dry-cured ham. A tool Flores, M., Barat, J. M., Aristoy, M. C., Peris, M. M., Grau, R., & Toldrá, F. (2006). for predicting the dry-curing time. Grasas y Aceites, 67(4), 166. Accelerated processing of dry-cured ham. Part 2. Influence of brine thawing/salting Önür, İ., Misra, N. N., Barba, F. J., Putnik, P., Lorenzo, J. M., Gökmen, V., & Alpas, H. operation on proteolysis and sensory acceptability. Meat Science, 72(4), 766–772. (2018). Effects of ultrasound and high pressure on physicochemical properties and Flores, M., Grimm, C. C., Toldrá, F., & Spanier, A. M. (1997). Correlations of Sensory and HMF formation in Turkish honey types. Journal of Food Engineering, 219, 129–136. Volatile Compounds of Spanish “Serrano” Dry-Cured Ham as a Function of Two Pateiro, M., Franco, D., Carril, J. A., & Lorenzo, J. M. (2015). 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Journal of Proteomics 193 (2019) 123–130
Contents lists available at ScienceDirect
Journal of Proteomics
journal homepage: www.elsevier.com/locate/jprot
Proteomic footprint of ultrasound intensification on sliced dry-cured ham T subjected to mild thermal conditions López-Pedrouso M.a, Pérez-Santaescolástica C.b, Franco D.b, Carballo J.c, Garcia-Perez José V.d, ⁎ Benedito J.d, Zapata C.a, Lorenzo J.M.b, a Department of Zoology, Genetics and Physical Anthropology, University of Santiago de Compostela, Santiago de Compostela 15872, Spain b Centro Tecnológico de la Carne de Galicia, Rúa Galicia N° 4, Parque Tecnológico de Galicia, San Cibrán das Viñas, 32900 Ourense, Spain c Área de Tecnología de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain d Universitat Politècnica de València, Department of Food Technology, Camí de Vera s/n, 46022 València, Spain
ARTICLE INFO ABSTRACT
Keywords: Ultrasound can intensify the heating process used to correct texture defects in dry-cured hams. The effect of Ham proteolysis biomarkers ultrasound-assisted heating on the proteome of sliced dry-cured ham was evaluated. Dry-cured hams with high Actin proteolysis index (PI > 36) were sliced, vacuum packed and subjected to conventional (CV) and ultrasound- Emerging technologies assisted (US) thermal treatments. Comparative proteome profiling between sample groups was assessed bytwo- Ultrasound biomarkers dimensional electrophoresis (2-DE) coupled to tandem mass spectrometry. It was found that protein fragmen- tation increased markedly after US thermal treatment. Specifically, fragments of the major myofibrillar protein, actin, were abundantly over-represented following US heating. In addition, five unfragmented sarcoplasmic proteins (i.e. fatty acid-binding protein, peroxiredoxin-6, superoxide dismutase, carbonyl reductase and ami- noacylase) showed increased abundance in the US sample group. These results suggest candidate biomarkers to monitor proteolysis intensity and proteolysis-independent effects linked to cured ham quality by ultrasound application. Significance: The present proteome profiling study of treated dry-cured ham demonstrates the impact ofultra- sound action on proteins. Moreover, negative organoleptic effects can be appearing with ultrasound treatment due to proteolysis increase. Therefore, the proteolysis monitoring could help to control these effects. In this regards, our results suggest that actin can be a candidate biomarker to monitor proteolysis intensity.
1. Introduction The main sensory attributes of dry-cured ham for the consumer acceptability are appearance, odor, texture and flavor. Gradual pro- Dry-cured ham is a high-quality product traditionally produced by teolysis of sarcoplasmic and myofibrillar muscle proteins during the Mediterranean countries but its consumption is currently increasing ripening process affects the sensorial features of dry-cured ham[5]. The worldwide. The traditional manufacturing process involving the steps proteolytic activity of endogenous enzymes releases small peptides and of salting, drying and ripening [1,2] requires further processing im- aliphatic acids [5,6]. Proteolytic activity is mainly influenced by tem- provements to satisfy the new consumer requirements (sliced, low salt perature, relative air humidity and salt content [7]. In addition, in- content, vacuum packaged, no pastiness, etc.). Part of the production of tensive proteolysis may lead to lower quality texture attributes such as dry-cured ham is currently commercialized as vacuum-packed slices. pastiness or adhesiveness and consumer rejection [8–10]. In this regard, Packaged ham slices increase the number of parameters influencing the mild thermal treatments cause an increase in the hardness and elasticity final product quality but it offers clear advantages: longer shelf-life of dry-cured ham, which could be considered as correction actions to because the growth of microorganisms is reduced, prevents moisture reduce the intensity of ham pastiness defects [11]. losses and lipid oxidation, and maintains organoleptic properties for Novel emerging technologies such as ultrasound could be used to longer time [3]. In this sense, there are growing efforts to control the intensify traditional processes although its use could have a marked deboning, slicing and packaging processes leading to safer products impact on sensorial and textural characteristics of dry-cured ham [11]. with a higher sensorial quality [4]. It is well known that ultrasound can modify the diffusivity of both
⁎ Corresponding author. E-mail address: [email protected] (J.M. Lorenzo). https://doi.org/10.1016/j.jprot.2018.10.002 Received 6 June 2018; Received in revised form 2 October 2018; Accepted 8 October 2018 Available online 10 October 2018 1874-3919/ © 2018 Elsevier B.V. All rights reserved. M. López-Pedrouso et al. Journal of Proteomics 193 (2019) 123–130 sodium chloride and water, during the salting stage of dry-cured ham, After this time, hams continued the drying process at 12 ± 2 °C and thus improving its textural features. Hence ultrasound can be used to 65 ± 5% RH until reaching a weight loss of 34%, later they were va- intensify mass transfer processes taking place between meat and brine cuum packaged again and kept at 30 °C during 30 days more. After this [12,13]. Ultrasounds are high frequency elastic waves that propagate period, hams were dried again until the end of the drying process through a medium. When the energy of the wave reaches a threshold, (weight loss of 36%). From these hams, we selected eight dry-cured the cavitation phenomenon appears. Cavitation refers to the formation, hams with high proteolysis index values (PI > 36), which were used in growth, and implosion of tiny bubbles in a liquid when ultrasound this study. Hams were cut and boned and the cushion part containing travels through it. This bubble implosion gives rise to high temperature biceps femoris muscle was excised and sampled. Ten slices from each (hot spots) and pressure, which involves thermal, mechanical and ham were vacuum packed and stored at room temperature (22 °C) for chemical changes in the food matrix [14]. Regarding meat products, no longer than 4 weeks. Ham slices were randomly divided into two ultrasound has influence on tenderization due to proteolytic degrada- groups and subjected separately to conventional (CV) and ultrasound- tion. It has been reported that protein stability and proteolytic enzymes assisted (US) thermal treatments: are related to disruption of myofibril structure leading to higher ten- derness. Hence structural changes of proteins and their post-transla- a) Thermal treatments assisted by power ultrasound (US), where ul- tional modifications such as phosphorylation have been related to dif- trasound was only applied during the heating stage, which was ferential myofibrillar degradation [15,16]. It has been demonstrated defined as the time needed to reach in the centre of theslicea that application of ultrasound caused acceleration of proteolysis which temperature 5 °C below that in the heating medium, measured using increases tenderness followed by a fragmentation of proteins [17]. a thermocouple. Thus, average ultrasonic treatment time was of Depending on the ultrasonic intensity applied, the muscle fibers are 7.5 min. Finally, samples were kept in a water bath (50 °C) to more or less weakened because of the degradation of sarcoplasmic and complete 5 h of treatment. This heating temperature and time were myofibrillar proteins, causing the meat tissue tenderization [18]. Ul- chosen to avoid the appearance of cooking flavours in the ham, as trasound systems are being developed to characterize dry-cured ham as found in preliminary experiments. Thermal treatments were applied analytical tool in a non-invasive way [19]. Ultrasound can be used to in an ultrasonic bath (600 W, 25 kHz, model GAT600W, ATU, Spain) intensify the heating process carried out to correct texture defects using water as heating fluid. (pastiness or adhesiveness) in dry-cured hams [11]. In this regard, b) Conventional thermal treatments (CV) where samples were kept in a Pérez-Santaescolastica et al. [20] showed that both, conventional water bath for 5 h at 50 °C. thermal treatments (CV) and ultrasound-assisted (US) thermal treat- ment significantly (P-value < .001) decreased the instrumental adhe- For proteomic analyses, four independent biological samples for siveness of sliced dry-cured ham from 85.27 g for control group to each treatment were lyophilized and frozen at −80 °C. 40.59 and 38.68 g for US and CV groups, respectively. However, to our knowledge, there is no literature about the effect of US thermal treat- 2.2. Protein extraction for proteomic analysis ment on the protein degradation of vacuum-packaged sliced dry-cured ham. Other emerging technologies to produce cured meat products with Total protein was extracted from 50 mg of lyophilized ham slices long shelf life such as high pressure are being developed and protein according to the following protocol. One technical replicate was carried profile has been characterized to monitor this process [21,22]. out for each biological replicate. Samples were mixed with 1 mL of lysis Proteomics has become an important tool in meat quality research buffer (7 M urea; 2 M thiourea; 4% CHAPS; 10 mM DTT,and2% to understand the factors that lead to the quality defect and identify Pharmalyte™ pH 3–10, GE Healthcare, Uppsala, Sweden) and sonicated specific biomarkers [23]. It has been successfully applied to understand (Sonifier 250, Branson, Danbury, CC, USA) at 0 °C. Samples weresub- the biochemical processes of meat quality attributes such as tenderness sequently cleaned twice with Clean-Up kit (GE Healthcare) following [24], color [25], water-holding capacity [26], pastiness [10] and ad- the manual instructions. The resulting protein was dissolved in 200 μL hesiveness [8]. In particular, proteomic changes linked to variable of lysis buffer and it was quantified by using the CB-X protein assaykit proteolysis intensity during dry-cured ham processing have been pre- (G-Biosciences, St. Louis, MO, USA) in a Chromate® microplate reader viously described [8,10,27]. Thus, the aim of this study was to assess (Awareness Technology, Palm City, FL, USA). the impact of US heating treatments on myofibrillar and sarcoplasmic proteins of dry-cured ham using a combination of 2-DE and mass 2.3. Two-dimensional electrophoresis (2-DE) and image analysis spectrometry (MS) technologies. The identification of potential bio- markers associated with ultrasound application can be used to control The first dimension was performed with immobilized pH gradient and predict the effects of this emerging technology on the final quality (IPG) strips (24 cm, pH 4–7 linear, Bio-Rad Laboratories, Hercules, CA, of dry-cured ham. USA). The protein solution loaded onto a strip consisted of 250 μg of protein in lysis buffer mixed with rehydration buffer (7 M urea,2M 2. Materials and methods thiourea, 4% CHAPS, 0.002% bromophenol blue) reaching a 450 μL of total volume, together with 0.6% DTT and 1% IPG buffer pH 4–7 (Bio- 2.1. Dry-cured samples Rad Laboratories). The isoelectric focusing (IEF) was performed using a PROTEAN IEF cell system (Bio-Rad Laboratories). The IEF protocol was: Hams were coming from pigs belonging to crosses of Large White Step-1 (rehydration), 50 V for 12 h; Step-2, 250 V for 30 min; Step-3, and Landrace breeds (medium fat content). All animals (castrated male) 500 V for 1 h; Step-4, 1000 V for 1 h; Step-5, 4000 V for 2 h; Step-6, were reared in the same conditions. The hams were salted according to 8000 V for 2 h; and Step-7, 10,000 V until to reach 70,000 V. In the the traditional system (manually rubbed with the following mixture: equilibration step, the strips were washed in buffer I (50 mM Tris 0.15 g of KNO3, 0.15 g of NaNO2, 1.0 g of dextrose, 0.5 g of sodium pH 8.8, 6 M urea, 2% SDS, 30% glycerol, 1% DTT) for 15 min and buffer ascorbate and 10 g of NaCl per kilogram of raw ham). The hams were II (50 mM Tris pH 8.8, 6 M urea, 2% SDS, 30% glycerol, 2.5% iodoa- next pile salted at 3 ± 2 °C and 85 ± 5% RH during 4, 6, 8 or 11 days cetamide) for another 15 min. Subsequently, the separation of proteins according to their corresponding raw weight. After salting, hams were in second dimension was performed in 12% SDS-PAGE (24 × 20 cm) washed with cold water and post-salted at 3 ± 2 °C and 85 ± 5% RH using an Ettan DALTsix vertical gel system (GE Healthcare). The second during 45 days. Drying of hams were performed at 12 ± 2 °C and dimension was carried out at 5 mA for 1 h, followed by 16 mA until 70 ± 5% RH until reaching a weight loss of 29%, later they were va- bromophenol blue tracking dye reached the botton of the gel. Finally, cuum packaged and kept at 30 °C during 30 days to induce proteolysis. the resulting gels were stained with SYPRO Ruby fluorescent stain
124 M. López-Pedrouso et al. Journal of Proteomics 193 (2019) 123–130
Fig. 1. 2-DE protein profiles of sliced dry-cured ham after conventional (A) and ultrasound-assisted (B) thermal treatments. The numbered 2-DE proteinspots corresponding to differentially abundant proteins were analyzed by MALDI TOF/TOF MS.
(Lonza, Rockland, ME, USA). Acquisition of gel images was carried out corrected percentile method as previously described [24]. The 95% using Gel Doc XR+ system (Bio-Rad Laboratories). The analysis of 2-DE bootstrap confidence interval corrected by Bonferroni's method was gel images was performed using PDQuest Advanced software v. 8.0.1 constructed from 20,000 bootstrap samples of size N = 4. Descriptive (Bio-Rad Laboratories). Molecular mass (Mr) and isoelectric point (pI) statistics and other statistical analysis were performed with IBM SPSS values for each spot on 2-DE gels were obtained using as reference Statistic V21.0 (SPSS, Chicago, IL, USA) software package. standard molecular mass markers ranging from 15 to 200 kDa The strength of differential protein abundance between CV andUS (Fermentas, Ontario, Canada) and linear IPG strips, respectively. thermal treatments was assessed with fold change (FC) and relative change (RC) measures [28]. The measure FC is defined as FC = Vus/Vcv, where V and V are the spot volumes averaged over replicates in US 2.4. Tryptic digestion of protein spots and mass spectrometry analysis us cv and CV thermal treatments, respectively. Values of FC < 1.0 were converted into their reciprocals with a negative sign. Therefore, FC has Protein spots of interest were excised from 2-DE gels and digested a range from −∞ to +∞. RC-values can be obtained by the eq. with trypsin as previously described in Franco et al. [28]. Peptide ex- RC = DV/│DV │, where DV = V − V and DV is the maximum tracts were concentrated in a SpeedVac (Thermo Fisher Scientific, max us cv max observed value of DV across spots in the study. The measure RC ranges Waltham, MA, USA) and stored at −20 °C until analysis. Dried samples from −1.0 to +1. Negative and positive RC-values indicate proteins of each spot were dissolved in 4 μL of 0.5% acetic acid and subsequently over-represented in CV and US samples, respectively. The UPGMA analyzed by MALDI-TOF/TOF MS. Peptide solution (0.5 μL) was mixed (Unweighted Pair Group Method with Arithmetic mean) clustering with an equal volume of matrix solution (3 mg of α-cyano-4-hydro- method was used to produce a dendrogram from distance matrix of xycinnamic acid dissolved in 1 mL of 50% acetonitrile and 0.1% tri- pairwise values of RC over proteins with the XLSTAT v.2014.5.01 sta- fluoroacetic acid), and the resulting mixture was deposited onto a384 tistical software. Opti-TOF MALDI plate (Applied Biosystems, Foster City, CA, USA) using the thin-layer method [29]. All mass spectra were obtained using 4800 MALDI-TOF/TOF analyzer mass spectrometer (Applied Biosystems) 3. Results and discussion operating in positive-ion reflector mode with an Nd:YAG 355 nmwa- velength laser and a minimum of three trypsin autolysis peaks as in- 3.1. 2-DE profiles of dry-cured ham after CV and US thermal treatments ternal standard for spectrum calibration. Precursor ions were selected using a relative mass window of 300 (FWHM, full-width half mass) and Representative 2-DE gel images of the proteome of dry-cured ham metastable suppression. Raw mass data were acquired with 4000 Series after CV and US thermal treatments are shown in Fig. 1. A total of 79 Explorer software v. 3.5 (Applied Biosystems). The combination of MS and 112 matched, well-resolved and reproducible 2-DE spots were de- and MS/MS spectra was analyzed by GPS Explorer software v. 3.6 tected across biological replicates in CV and US sample groups, re- (Applied Biosystems9) and Mascot software v. 2.1 (Matrix Science, spectively. The Bonferroni-corrected 95% bootstrap confidence inter- Boston, MA, USA) using the UniProt/SwissProt database. Search set- vals for means of spot volumes across replicates revealed that 40 tings were: oxidation of methionine and carbamidomethylation of cy- protein spots showed significant (P-value < .05) differences between steine as variable and fixed modifications, respectively; one missed sample groups (Table 1). Most spots (i.e. 33 out of 40 spots) were un- cleavage; precursor mass tolerance of 50 ppm; and fragment ion mass ique spots present in only one of the sample groups. Specifically, most tolerance of 0.6 Da. Positive protein identifications required statistically percentage (94%) of the unique spots was detected in the US sample significant Mascot scores at the significance level of α = 0.05 andthree group (P < .05, Fisher's exact test). It can be concluded, therefore, that matched peptides as minimum. the proteome of dry-cured hams subjected to CV and US thermal treatments was markedly different at the qualitative level. As itwas 2.5. Statistical analysis previously pointed out, the commonly used FC measure does not pro- vide useful information on quantitative differences among unique Statistical quantitative and qualitative differences in 2-DE spot vo- protein spots because of it gives −∞ or +∞ values [8,28]. On the lumes between CV and US thermal treatments were assessed by boot- other hand, only seven non-unique protein spots (i.e. spots 4, 5 19, 20, strap re-sampling methods. Non-parametric bootstrap confidence in- 26, 36 and 37) showed statistically significant quantitative differences terval was obtained for the mean volume of each spot by the bias- in volume, with FC expressed in absolute value ranging from 1.8 to 2.9.
125 M. López-Pedrouso et al. Journal of Proteomics 193 (2019) 123–130
Table 1 It cannot be excluded that other spots with ratios between the theore- Differentially abundant (P < .05) 2-DE spot volumes in dry-cured ham sub- tical and observed Mr close to 1.5 (e.g. spots 19, 20 and 21) contained jected to conventional (CV) and ultrasound-assisted (US) thermal treatments. partially degraded actins. This result can be understood taking into Spot no. Average volume ± SE Fold change (FC) account that most abundant muscle proteins (60–70%) belong to the myofibrillar fraction [30]. More specifically, the myofibrillar proteins CV US actin and myosin account for ca. 29 and 13% of muscle proteins, re- spectively, after rigor mortis but before degradation changes post-mortem 1 – 530 ± 86 +∞ 2 – 1687 ± 299 +∞ [31]. Therefore, the major proportion of actin in muscle contributes to 3 – 790 ± 221 +∞ explain higher release, extraction and degradation of actin from rup- 4 1825 ± 390 5295 ± 645 +2.9 tured myofibrils following US. Accordingly, Kang et al.[32] found 5 10,802 ± 1220 5500 ± 830 −2.0 higher fragmentation of myofibril proteins by ultrasound application in 6 – 656 ± 178 +∞ 7 – 2602 ± 267 +∞ beef loin during curing. Ultrasonic cavitation during salting of pork 8 – 396 ± 93 +∞ meat also enhanced the extraction of proteins such as actin, produced 9 – 2581 ± 1274 +∞ by the dissociation of actomyosin [13]. On the other hand, chicken 10 – 613 ± 231 +∞ meat treated by ultrasound (150 W) was shown to generate electro- 11 – 2710 ± 367 +∞ phoretic patterns with a significant increased intensity of actin bands as 12 – 1005 ± 204 +∞ 13 – 1763 ± 100 +∞ compared to control samples [33]. 14 – 2647 ± 411 +∞ It is noteworthy that no fragment of the myofibrillar protein myosin 15 2386 ± 186 – −∞ was detected on 2-DE gels even though it is a highly represented protein 16 – 1643 ± 455 +∞ in the muscle. Experimental observations obtained in brine-cooked ham 17 – 1436 ± 232 +∞ 18 – 405 ± 146 +∞ assisted by ultrasound have demonstrated that denaturation tempera- 19 8081 ± 881 3619 ± 288 −2.2 ture of myosin is lower than actin using differential scanning calori- 20 6439 ± 808 2787 ± 307 −2.3 metry [34]. In addition, Kang et al. [32] reported that oxidized myosin 21 – 1080 ± 420 +∞ by the ultrasound application was responsible for its protein aggrega- 22 – 505 ± 208 +∞ tion. Overall, oxidation and polymerization processes may change the 23 1153 ± 727 – −∞ 24 – 2460 ± 582 +∞ structure and conformation of the myosin increasing susceptibility to 25 – 1391 ± 312 +∞ proteolysis. Therefore, US treatment could lead to a higher denatura- 26 964 ± 174 2538 ± 259 +2.6 tion of myosin caused by molecular frictions and a possible acoustic 27 – 1775 ± 769 +∞ cavitation in salty meat. This cavitation could produce hot spots and 28 – 1979 ± 78 +∞ 29 – 872 ± 512 +∞ pressure fluctuations facilitating the weakening of the myosin structure 30 – 882 ± 304 +∞ and contributing to its destabilization. However, there were no statis- 31 – 1178 ± 324 +∞ tically significant differences of myosin degradation between CVand 32 – 1206 ± 231 +∞ US thermal treatments in a range of Mr from 10 to 70 kDa on 2-DE gels 33 – 571 ± 190 +∞ (Fig. 1b). Therefore, it is not possible to determine whether myosin 34 – 853 ± 349 +∞ 35 – 624 ± 203 +∞ degradation was due to US or CV thermal treatment. 36 6324 ± 1070 11,068 ± 712 +1.8 It is well known that the proteolysis during the ripening process 37 376 ± 102 1064 ± 175 +2.8 plays a critical role on the sensorial and textural features of dry-cured 38 – 582 ± 241 +∞ ham. Post-ripening treatments such as deboning, slicing and packaging 39 – 1769 ± 1000 +∞ 40 – 1023 ± 553 +∞ also contribute to protein breakdown [2]. Our observations showed that the proteolysis increased markedly in sliced dry-cured hams subjected Location of numbered spots on 2-DE gels is shown in Fig. 1. to power ultrasound. Several authors have previously reported that Average ( ± SE, Standard error) volumes were assessed from four independent proteolysis is intensified by ultrasound treatment [18,32,35]. In the biological replicates using the PDQuest software. present study, proteolysis can be facilitated by the fact that dry-cured Statistically significant (P < .05) differences between pairs of matched spots of ham was cut into thin slices. In addition, US thermal treatment can CV and US thermal treatments were assessed using 95% bootstrap confidence produce intensification of heat transfer by convention due to thevi- intervals with the Bonferroni correction from spot volumes averaged over re- brations [36]. The sonication phenomenon triggers mechanical vibra- plicates. tion, agitation, shear forces, turbulence, acoustic cavitation and free radicals which could change protein structure conducing to increased 3.2. Effect of CV and US thermal treatments on myofibrillar proteins proteolysis. Degradation of myofibrillar proteins by power ultrasound alters the All spots with significant differential abundance were selected for structural integrity of the myofibrils producing changes in textural protein identification by tandem mass spectrometry (MALDI-TO/TOF properties [37]. In the present study, we found that the degradation of MS). The list of proteins that were unambiguously identified is shown in actin increased significantly with US thermal treatment. Interestingly, Table 2. Only seven non-redundant myofibrillar and sarcoplasmic differentially degraded actins were detected on 2-DE gels. Therefore, proteins were identified: actin (ACTS); aminoacylase (ACY1); peroxir- the actin can be a useful candidate biomarker to monitor the level of edoxin-6 (PRDX6), carbonyl reductase (CBR1), superoxide dismutase proteolysis of dry-cured ham subjected to different power ultrasound (SOD), cyclin-G1 (CCNG1) and two fatty acid-binding protein isoforms and treatment times and their effects on quality attributes. (FABP4/H). It is noteworthy that most spots (76%) contained the myofibrillar protein actin. Protein fragmentation for each individual 3.3. Search of biomarkers to control ultrasound process spot was assessed through the difference between the observed Mr on 2- DE gel and its theoretical value obtained from UniprotKB/Swiss-Prot A total of five unfragmented sarcoplasmic proteins showed sig- databases. A ratio between the theoretical and observed M above 1.5 r nificant (P < .05) differential abundance following US thermal treat- was used as validation criterion of protein fragments on 2-DE gels as ment: ACY1, PRDX6, CBR1, SOD and FABP4/H (Tables 1 and 2). It is previously [8]. Using this criterion, we found higher protein fragmen- noteworthy that these differences could affect dry-cured ham quality in tation after US thermal treatment (P < .05, Fisher's exact test). terms of sensorial and textural characteristics, considering that flavor Most fragments (90%) detected on 2-DE gels corresponded to actins. and aroma of dry-cured ham is related to non-volatile compound
126 M. López-Pedrouso et al. Journal of Proteomics 193 (2019) 123–130
Table 2 List of protein identifications by tandem mass spectrometry (MALDI-TOF/TOF MS).
Spot no. Protein (abbrev.) Accesion no. Mascot score Sequence Number of pI Mr Protein (Uniprot) coverage (%) matched peptides fragment Th/Obs Th/Obs (kDa)
4 Actin, alpha P68137 142 16 5 5.2/5.3 42.4/45.3 − skeletal muscle (ACTS) 5 Actin, alpha P68137 48 11 3 5.2/5.5 42.4/45.1 − skeletal muscle (ACTS) 7 Actin, alpha P68137 88 31 9 5.2/5.9 42.4/42.9 − skeletal muscle (ACTS) 8 Aminoacylase-1 P37111 63 25 10 5.6/6.1 42.2/43.3 − (ACY1) 9 Actin, alpha P68137 111 30 9 5.2/6.2 42.2/43.4 − skeletal muscle (ACTS) 10 Actin, alpha P68137 46 22 6 5.2/6.2 42.2/40.4 − skeletal muscle (ACTS) 11 Actin, alpha P68137 165 19 6 5.2/5.7 42.2/40.6 − skeletal muscle (ACTS) 12 Actin, alpha P68137 52 30 9 5.2/5.9 42.2/41.1 − skeletal muscle (ACTS) 13 Actin, alpha P68137 50 24 7 5.2/5.5 42.2/36.0 − skeletal muscle (ACTS) 14 Actin, alpha P68137 123 24 7 5.2/5.7 42.2/37.3 − skeletal muscle (ACTS) 15 Actin, alpha P68137 71 39 10 5.2/5.9 42.2/36.2 − skeletal muscle (ACTS) 19 Actin, alpha P68137 76 27 10 5.2/4.8 42.2/31.7 − skeletal muscle (ACTS) 20 Actin, alpha P68137 87 20 6 5.2/4.9 42.2/31.8 − skeletal muscle (ACTS) 21 Actin, alpha P68137 50 8 3 5.2/5.1 42.2/30.4 − skeletal muscle (ACTS) 22 Actin, alpha P68137 45 24 7 5.2/5.1 42.2/28.4 + skeletal muscle (ACTS) 23 Actin, alpha P68137 58 33 9 5.2/5.3 42.2/28.4 + skeletal muscle (ACTS) 25 Actin, alpha P68137 89 23 6 5.2/5.8 42.2/29.0 + skeletal muscle (ACTS) 26 Peroxiredoxin-6 Q9TSX9 303 47 10 5.7/6.0 25.1/28.0 − (PRDX6) 27 Actin, alpha P68137 70 20 7 5.2/4.7 42.2/23.6 + skeletal muscle (ACTS) 28 Actin, alpha P68137 53 28 8 5.2/5.1 42.2/27.0 + skeletal muscle (ACTS) 29 Actin, alpha P68137 109 14 4 5.2/5.0 42.2/24.0 + skeletal muscle (ACTS) 30 Actin, alpha P68137 87 34 10 5.2/5.3 42.2/26.5 + skeletal muscle (ACTS) 31 Actin, alpha P68137 107 11 4 5.2/5.4 42.2/27.3 + skeletal muscle (ACTS) 33 Carbonyl Q28960 59 32 10 7.6/5.6 32.0/24.4 − reductase (CBR1) 35 Actin, alpha P68137 49 24 7 5.2/6.4 42.2/22.0 + skeletal muscle (ACTS) (continued on next page)
127 M. López-Pedrouso et al. Journal of Proteomics 193 (2019) 123–130
Table 2 (continued)
Spot no. Protein (abbrev.) Accesion no. Mascot score Sequence Number of pI Mr Protein (Uniprot) coverage (%) matched peptides fragment Th/Obs Th/Obs (kDa)
37 Superoxide P04178 102 41 4 6.0/6.6 16.0/16.0 − dismutase (SOD) 38 Cyclin-G1 Q52QT8 49 27 9 9.1/6.4 34.6/14.5 + (CNG1) 39 Fatty acid- O97788 59 40 6 6.3/5.9 14.8/12.3 − binding protein, adypocites (FABP4) 40 Fatty acid- O02772 89 44 6 6.1/6.1 14.8/13.4 – binding protein, heart (FABPH)
All identifications were matched to Sus scrofa (pig) protein databases. Proteins were positively identified (P < .05) according with Mascot scores. Sequence coverage (%) is the proportion of the overall amino acid sequence covered by matched peptides. Number of peptides matched is the total number of identified spectra matched for the protein.
Theoretical (Th) pI and Mr values for each protein spot were obtained from UniProtKB/Swiss-Prot databases.
Observed (Obs) pI and Mr values for each protein spot were assessed using lineal IPG strips and molecular mass markers, respectively. Protein fragments verify that the ratio between the theoretical and observed Mr is above 1.5.
bovine muscle because of its relationship with μ-calpain activity and proteolysis [40]. SOD also showed a relevant level of extraction after ultrasound treatment (RC = +0.25). It is an oxidative enzyme that leads to hydrogen peroxide and inactivate superoxide radicals, which are intermediate products of oxidative rancidity in dry-cured meat [41]. Accordingly, a previous study has demonstrated that SOD has anti- oxidant activity in dry-cured ham [42]. The proteins CBR1 (RC = +0.20) and ACY1 (RC = +0.14) were only present in dry-cured hams samples subjected to ultrasound treatment, but their effects on dry-cured ham quality are yet little known. The increased extraction susceptibility of FABP4/H and the rest of sarcoplasmic proteins found to be differentially represented in dry- cured ham subjected to US thermal treatment suggests that they can be candidate biomarkers to assess ultrasonic application. The effects of US heating are probably enhanced in sliced dry-cured ham. Further re- search is required to assess whether these potential biomarkers can be applied to non-sliced dry-cured ham. Fig. 2. The extent of change of differentially abundant sarcoplasmic proteins in CV and US samples as measured by the relative change (RC) coefficient. 4. Conclusions
(protein) profile [38]. The extent of change for each protein was 2-DE coupled to MALDI-TOF/TOF MS revealed that ultrasound ap- quantified by the RC measure (Fig. 2). We found that all RC-values were plication during mild thermal treatment had a considerable impact on of positive sign, which indicates that proteins were over-represented in the proteome of sliced dry-cured ham. Ham samples subjected to power the US sample group. In addition, change intensity was noticeably ultrasound exhibited significantly higher proteolysis than those con- different among proteins, with RC-values ranging from +0.14 (ACY1) ventionally heated. In slice dry-cured hams there was more degraded to +1.0 (FABP4/H). The UPGMA dendrogram based on the RC distance myofibrillar protein actin after ultrasound application compared to matrix showed that the value of RC was notably higher for FABP4/H conventional thermal treatment. This finding could suggest that actin of isoforms than for the other proteins (Fig. 3a). The difference was found dry-cured ham is gradually suffering degradation by ultrasound treat- to be statistically significant using 95% bootstrap confidence intervals ment and downstream effects on quality attributes. In addition, five (Fig. 3b). FABP is a protein that exhibits a diversity of isoforms related unfragmented sarcoplasmic proteins (FABP4/H, PRDX6, SOD, CBR1 to the transfer of fatty acids into cells and their combustion in the and ACY1) were more abundant in samples following power ultra- mitochondria [39]. sound. These sarcoplasmic proteins can be candidate proteolysis-in- The protein PRDX6 also showed a high value of RC (RC = +0.56). dependent biomarkers of ultrasonic application. Future research is ne- It is a member of the thiol-specific antioxidant protein family that cessary for in-depth understanding the changes in the proteome and protects cells against oxidative injury, prevents lipid oxidation and quality attributes of dry-cured ham associated with ultrasound-assisted participates in the regulation of phospholipid turnover. It has been heating. reported that the level of PRDX6 is correlated to meat tenderness in
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[33] Y. Zou, P. Xu, P. Li, K. Zhang, M. Zhang, Z. Sun, J. Cao, Effects of different ultra- volatile compounds throughout the manufacture of Celta dry-cured ham, Food Sci. sound power on physicochemical property and functional performance of chicken Technol. Int. 21 (8) (2015) 581–592. actomyosin, Int. J. Biol. Macromol. 113 (2018) 640–647. [39] D. Nechtelberger, V. Pires, J. Söolknet, G. Brem, M. Mueller, S. Mueller, [34] C.K. McDonnell, P. Allen, C. Morin, J.G. Lyng, The effect of ultrasonic salting on Intramuscular fat content and genetic variants at fatty acid-binding protein loci in protein and water–protein interactions in meat, Food Chem. 147 (2014) 245–251. Austrian pigs, J. Anim. Sci. 79 (11) (2001) 2798–2804. [35] A. Wang, D. Kang, W. Zhang, C. Zhang, Y. Zou, G. Zhou, Changes in calpain activity, [40] X. Jia, E. Veiseth-Kent, H. Grove, P. Kuziora, L. Aass, K. Hildrum, K. Hollung, protein degradation and microstructure of beef M. semitendinosus by the application Peroxiredoxin-6–A potential protein marker for meat tenderness in bovine long- of ultrasound, Food Chem. 245 (2018) 724–730. issimus thoracis muscle, J. Anim. Sci. 87 (2009) 2391–2399. [36] M. Legay, N. Gondrexon, S. Le Person, P. Boldo, A. Bontemps, Enhancement of heat [41] C.E. dos Santos Cruxen, G.D. Funck, G. da Silva Dannenberg, L. Haubert, J. de Lima transfer by ultrasound: review and recent advances, Int. J. Chem. Eng. (2011). Marques, I.S. Kroning, Â.M. Fiorentini, Characterization of Staphylococcus xylosus [37] Y. Zou, W. Zhang, D. Kang, G. Zhou, Improvement of tenderness and water holding LQ3 and its application in dried cured sausage, LWT-Food Sci. Technol. 86 (2017) capacity of spiced beef by the application of ultrasound during cooking, Int. J. Food 538–543. Sci. Technol. 53 (3) (2018) 828–836. [42] Y.Y. Hu, L.J. Xing, G.H. Zhou, W.G. Zhang, Antioxidant activity of crude peptides [38] R. Bermúdez, D. Franco, J. Carballo, J.M. Lorenzo, Influence of type of muscle on extracted from dry-cured Jinhua ham, J. Food Nutr. Res. 4 (6) (2016) 377–387.
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Food Research International
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Influence of high-pressure processing at different temperatures onfree T amino acid and volatile compound profiles of dry-cured ham Pérez-Santaescolástica C.a, Carballo J.b, Fulladosa E.c, Munekata P.E.S.a, ⁎ Bastianello Campagnol P.C.d, Gómez B.a, Lorenzo J.M.a, a Centro Tecnológico de la Carne, Rúa Galicia N°4, Parque Tecnológico de Galicia, San Cibrán das Viñas, 32900 Ourense, Spain b de Tecnología de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, Área 32004, Ourense, Spain c IRTA, XARTA, Food Technology program, Finca Camps i Armet, s/n, 17121, Monells, Girona, Spain d Universidade Federal de Santa Maria, CEP 97105-900 Santa Maria, Rio Grande do Sul, Brazil
ARTICLE INFO ABSTRACT
Keywords: The effect of high pressure processing (HPP) (600 MPa during 6 min) at different temperatures (0, 20and35°C) High hydrostatic pressure in dry-cured ham has been studied in order to optimize the technique and reduce its impact on chemical cha- Volatile compound racteristics, which are widely related with sensorial parameters. Vacuum-packed slices from 120 dry-cured hams Free amino acids were used. These slices were submitted to four different treatments: without application of pressure or tempe- Dry-cured ham rature (CO), high pressure treatment at 0 °C (HPP-0), high pressure treatment at 20 °C (HPP-20), and high- pressure treatment at 35 °C (HPP-35). The effect of the treatments on free amino acids and volatile compounds profile was evaluated. The HPP-35 treatment significantly (P < 0.001) increased the total free amino acid content (6415.63 mg/100g dry matter) when compared to the contents of the CO, HPP-0 and HPP-20 treatments (5313.16, 4787.30 and 5072.48 mg/100g dry matter, respectively). Significant differences were also found among treatments in the content of 13 individual free amino acids, and HPP-35 samples presented the highest values in 12 of them. Similarly, the total volatile compound content was influenced by temperature-assisted HPP treatments. The HPP-35 treated samples showed the highest content (78,415.27 AU × 103/g dry-cured ham) and the HPP-0 treated samples the lowest content (28,584.14 AU × 103/g dry-cured ham). No significant differences were observed between CO and HPP-20 treatments. The fractions of volatile compounds derived from lipolysis, proteolysis and microbial activity were significantly modified by the different treatments. HPP-0 samples pre- sented lower values of alcohol and hydrocarbon contents, whereas HPP-35 samples showed higher ketone and ester contents.
1. Introduction (Liu, Selomulyo, & Zhou, 2008; Rivalain, Roquain, & Demazeau, 2010). Since some protein conformations are sensitive to pressure, the appli- The use of high pressure processing (HPP) in food technology began cation of high pressure treatments can induce modifications on enzyme in the 90s. Since then, most of the studies have focused on reducing the activity (Buckow, Truong, & Versteeg, 2010; Chéret, Hernández- microbial load of food and obtaining safer products with a longer shelf Andrés, Delbarre-Ladrat, De Lamballerie, & Verrez-Bagnis, 2006), life. According to Duranton, Simonin, Guyon, Jung, and de Lamballerie which can result in texture changes, mainly by increasing hardness and (2014), HPP has other potential applications. For instance, HPP has elasticity of the product (Duranton et al., 2012; Yoshioka & Yamada, been studied as an auxiliary method in the food elaboration processes in 2002). It is worth mentioning that Tao, Sun, Hogan, and Kelly (2014) recent years. In this line of thought, Duranton, Simonin, Chéret, concluded that moderate pressure does not cause significant changes in Guillou, and de Lamballerie (2012) used HPP on salting stage of dry- the flavor of products when high hydrostatic pressures were usedfor cured ham processing to improve salt diffusion and to reduce the sterilization. amount of salt in the formulation. However, some studies found that, The most important attributes affecting consumer's purchase pre- under certain conditions, HPP can cause physico-chemical, sensory, and ference are related to odour and taste. The aroma is originated by even functional alterations, particularly on proteins, lipids and starches chemical and enzymatic reactions during the processing of dry-cure
⁎ Corresponding author. E-mail address: [email protected] (J.M. Lorenzo). https://doi.org/10.1016/j.foodres.2018.12.039 Received 30 September 2018; Received in revised form 6 December 2018; Accepted 22 December 2018 Available online 26 December 2018 0963-9969/ © 2018 Elsevier Ltd. All rights reserved. C. Pérez-Santaescolástica et al. Food Research International 116 (2019) 49–56 hams (Bermúdez, Franco, Carballo, & Lorenzo, 2015). Concerning the 0 °C (HPP-0), the second at 20 °C (HPP-20) and the third at 35 °C (HPP- effect of HPP on the enzymes that originate flavor compounds, con- 35). In order to evaluate the effects of HPP treatments, a fourth group of trasting results have been reported in scientific literature. In this way, samples was not treated and was used as a control (CO) batch. both Clariana et al. (2011) and Clariana, Guerrero, Sárraga, and Garcia- Regueiro (2012) observed an increase in the superoxide dismutase ac- 2.3. Free amino acid analysis tivity, but no effect was shown on catalase and glutathione peroxidase activity after HPP treatments at 400 MPa. Conversely, superoxide dis- The free amino acids were extracted following the procedure de- mutase and glutathione peroxidase activities were reduced without any scribed by Lorenzo, Cittadini, Bermúdez, Munekata, and Domínguez effect on catalase activity after treatment at 900 MPa. Nevertheless, (2015). Amino acids were derivatized with 6-aminoquinolyl-Nhydrox- HPP at 600 MPa showed no effect on the activity of none of the anti- ysuccinimidyl carbamate (Waters AccQ-Fluor reagent kit) and analysed oxidant enzymes. by RP-HPLC techniques using a Waters 2695 Separations Module Moreover, the compositional characteristics of the product could equipped with a Waters AccQ-Tag amino acid analysis column and with influence the volatile compound profile. For example, highin- a Waters 2475 Multi Fluorescence Detector. The results were expressed tramuscular fat content in ham can increase the concentration of as mg of free amino acid/100 g of dry matter. compounds such as acetic acid, methylbenzene or phenol, whereas low intramuscular fat content can lead to greater contents of 2-propanol and dimethyl sulfide, among others (Martínez-Onandi, Rivas-Cañedo, 2.4. Volatile compound analysis Núñez, & Picón, 2016). On the other hand, the chemical and enzymatic reactions during the process involve the modification of protein struc- For the volatile compound extraction, a solid-phase micro extraction tures in order to develop a particular ham taste. Due to the fact that (SPME) device (Supelco, Bellefonte, PA, USA) containing a fused-silica HPP treatment could promote changes in cellular structures (dos fibre (10 mm length) coated with a 50/30 mm thickness of DVB/CAR/ Santos, Cristianini, & Sato, 2018) and temperature could have an im- PDMS (divinylbenzene/carboxen/polydimethylsiloxane) was used. For pact in the development of reactions, it is emerging the necessity to find the volatile compound determination, a gas chromatograph 7890B out the consequences in the final flavor after the application ofthis (Agilent Technologies, Santa Clara, CA, USA) equipped with a DB-624 technique. capillary column (30 m, 0.25 mm i.d., 1.4 μm film thickness; J&W In this way, interesting results were obtained by using HPP tech- Scientific, Folsom, CA, USA) coupled to a mass selective detector 5977B nique during pre and post rigor stage of dry-cured ham to improve (Agilent Technologies) was used. texture (Fulladosa, Serra, Gou, & Arnau, 2009) and in vacuum-pack- The extraction of the volatile compounds (SPME) was performed aged products to enhance shelf life (Fuentes, Ventanas, Morcuende, following the procedure described by Domínguez, Gómez, Fonseca, and Estévez, & Ventanas, 2010). The impact of HPP on sensory properties of Lorenzo (2014) with some modifications. One g of each sample (after the packaged dry-cured ham was previously studied regarding to the being ground using a commercial grinder) was weighed in a 20 mL vial. pressure effect, but there are no studies about the combined effectof The vials were subsequently screw-capped with a laminated Teflon- temperature and HPP processing in volatile and free amino acid com- rubber disc. The fibre was previously conditioned by heating in aFibre position of dry-cured ham. Due to the multitude of current applications Conditioning Station at 270 °C for 30 min. The conditioning, extraction of the HPP as well as their potential uses in the future, it is interesting to and injection of the samples were carried out with an autosampler PAL- study the impact of the HPP on chemical changes as a first step to RTC 120. The extractions were carried out at 37 °C for 30 min, after understand the effects on sensory attributes. Therefore, the objective of equilibration of the samples for 15 min at the temperature used for this study was to evaluate the effect of HPP treatment assisted with extraction, which ensured a homogeneous temperature for both sample three different temperatures on free amino acid content and volatile and headspace. Once sampling was finished, the fibre was transferred to compound of dry-cured ham. the injection port of the gas chromatograph–mass spectrometer (GC–MS) system. The SPME fibre was desorbed and maintained in the 2. Materials and methods injection port at 260 °C during 8 min. The samples were injected in splitless mode. Helium was used as a carrier gas with a flow of 1.2 mL/ 2.1. Samples min (9.59 psi). The temperature program was firstly isothermal for 10 min at 40 °C, then raised to 200 °C at 5 °C/min and next to 250 °C at One hundred and twenty raw hams with pH < 5.5, which are more 20 °C/min, and finally held for 5 min; total run time was 49.5 min.In- prone to develop defective texture properties, from animals belonging jector and detector temperatures were both set at 260 °C. The mass to crosses of Large White and Landrace breeds (medium fat content) spectra were obtained using a mass selective detector working in were obtained from a commercial slaughterhouse. All hams were electronic impact at 70 eV, with a multiplier voltage of 850 V and col- weighted (11.9 kg ± 1.1 kg) and manufactured according to the tra- lecting data at 6.34 scans/s over the range m/z 40–550. Compounds ditional system. Dry-cured hams, the aitch bone, the butt and the femur were identified by comparing their mass spectra with those contained bone were excised and the cushion part, containing Biceps femoris (BF) in the NIST14 (National Institute of Standards and Technology, Gai- muscle, was obtained and trimmed. thersburg) library, and/or by comparing their mass spectra and reten- After that, the 120 hams were divided into treatments (30 hams per tion time with authentic standards (Supelco, Bellefonte, PA, USA), and/ treatment). From each ham unit, three 1.5 mm-thick slices were va- or by calculation of retention index relative to a series of standard al- cuum packed in individual plastic bags of polyamide/polyethylene kanes (C5–C14) (for calculating Kovats indexes, Supelco 44,585-U, 3 2 Bellefonte, PA, USA) and matching them with data reported in litera- (oxygen permeability of 50 cm /m /24 h at 23 °C and water perme- 3 ability of 2.6 g/m2/24 h at 23 °C and 85% RH, Sacoliva® S.L., Spain) ture. The results were expressed as quantified area units (AU) ×10 /g and stored in a chamber at 4 °C ± 2 °C until the treatment application. of sample.
2.2. HPP treatments 2.5. Statistical analysis
The treatment of the packaged slices was applied using a NC The effect of treatments was examined using a one-way ANOVA. Hyperbaric WAVE 6000/120 equipment (NC Hyperbaric, Burgos, When a significant effect (P < 0.05) was detected, means were com- Spain). Three different treatments were performed at 600 MPa during pared using Tukey's test. Analyses were conducted using the IBM SPSS 6 min, each one accompanied by a different temperature: the first at Statistics 19.0 (IBM Corporation, Somers, NY, USA) software package.
50 C. Pérez-Santaescolástica et al. Food Research International 116 (2019) 49–56
Table 1 Effect of different HPP treatments on free amino acids content (expressed as mg/100 g dry matter) of dry-cured ham. Values are means ofthirtyhamsforeach treatment.
Treatment SEM p-value
CO HPP-0 HPP-20 HPP-35
Aspartic acid 185.15b 119.45a 145.99a 240.20c 5.643 < 0.001 Serine 201.65a 198.30a 200.45a 251.15b 5.647 0.001 Glutamine 450.17a 382.41a 424.71a 588.97b 12.112 < 0.001 Glycine 196.45a 202.46a 200.03a 238.69b 4.444 0.001 Histidine 102.51b 82.62a 87.44ab 127.69c 2.879 < 0.001 Taurine 93.22ab 83.48a 92.04ab 102.21b 2.305 0.045 Arginine 410.98b 295.84a 346.32ab 513.00c 11.986 < 0.001 Threonine 221.57ab 223.76ab 216.03a 254.45b 5.278 0.037 Alanine 419.87a 471.30a 478.99ab 546.58b 10.754 < 0.001 Proline 287.50 276.28 286.14 314.32 5.691 0.105 Cysteine 346.79b 61.44a 51.44a 553.79c 23.108 < 0.001 Tyrosine 202.96c 116.37a 160.34b 121.24a 4.777 < 0.001 Valine 393.22a 461.58b 471.45b 441.94ab 8.847 0.007 Methionine 203.84 205.41 216.23 223.19 4.303 0.330 Lysine 265.09a 256.41a 290.14a 448.34b 10.239 < 0.001 Isoleucine 351.76 377.63 387.34 403.47 7.937 0.119 Leucine 588.53 629.06 654.06 664.31 12.864 0.146 Phenylalanine 391.88 343.50 363.34 382.06 7.045 0.086 TOTAL 5313.16a 4787.30a 5072.48a 6415.63b 112.28 < 0.001 Sweet1 1310.60a 1372.10a 1379.22a 1587.65b 29.320 0.003 Bitter2 1921.99 2017.17 2092.42 2083.20 38.731 0.362 Acid3 737.84b 605.81a 667.62ab 956.87c 20.453 < 0.001 Aged4 653.20b 513.87a 601.86ab 833.88c 17.725 < 0.001 a–cMean values in the same row (corresponding to the same amino acid/sensory attribute) not followed by a common letter differ significantly (P < 0.05; Tukey's Test). SEM: standard error of mean. Treatments: CO = control (without treatment); HPP-0 = High pressure treatment at 0 °C; HPP-20= High pressure treatment at 20 °C; HPP-35= High pressure treatment at 35 °C. 1 Sweet flavor = ∑ of alanine, glycine, threonine, serine and proline. 2 Bitter flavor = ∑ of leucine, valine, isoleucine, methionine and phenylalanine. 3 Acid flavor = ∑ of glutamic acid, aspartic acid and histidine. 4 Aged flavor = ∑ of lysine, tyrosine and aspartic acid.
3. Results and discussion threonine and glycine content), acid (calculating as sum of phenylala- nine, histidine, glutamic and aspartic acid content) and aged (calcu- 3.1. Free amino acids lating as sum of lysine, tyrosine and aspartic acid content) attributes in comparison to other treated and untreated samples (Table 1). In addi- Table 1 shows the effect of different HPP-temperature treatments on tion, previous studies showed that an increment of bitter taste in hams the free amino acid content (expressed as mg/100 g dry matter) of dry- could be attributed to excessive proteolysis (Careri et al., 1993; cured ham. Statistical analysis showed that total free amino acid con- Parolari, Virgili, & Schivazappa, 1994). However, the amino acids re- tent was significantly (P < 0.001) affected by treatments. HPP-35 sponsible for the bitter taste were not affected by any treatment in the group displayed the highest values (5313.16 vs. 4787.30 vs. 5072.48 present study. vs. 6415.63 mg/100 g dry matter for CO, HPP-0, HPP-20 and HPP-35 tretaments, respectively). No significant differences were observed 3.2. Volatile compounds among CO, HPP-0 and HPP-20 treatments. These values were in the range values 4000–7000 mg/100 g dry matter that was reported in Significant differences (P < 0.001) among treatments were found previous studies (Bermúdez, Franco, Carballo, Sentandreu, & Lorenzo, in the total content of volatile compounds. The highest values were 2014; Pérez-Santaescolástica et al., 2018a; Pérez-Santaescolástica et al., observed in the HPP-35 batch (78,415.27 AU × 103/g of dry-cured 2018b) about dry-cured ham volatile composition. The higher total free ham) while the lowest contents were obtained from the HPP-0 batch amino acid content in HPP-35 samples was expected since it is well (28,584.14 AU × 103/g of dry-cured ham) (Table 2). In comparison to known that proteins are greatly influenced by temperature, so their HPP-0 treatment, the samples showed significant declines in hydro- structures could be degraded into smaller amino acids. In this regard, carbons, aldehydes, alcohols, carboxylic acids, sulphur compounds and 13 of the 18 amino acids studied were significantly influenced by chloro compounds content by 55%, 56%, 40%, 69%, 85% and 65%, temperature-assisted HPP treatments. The samples submitted to HPP at respectively. Aldehydes, alcohols, carboxylic acids, nitrogenous and 35 °C had the highest content in 12 amino acids (aspartic acid, serine, sulphur compounds content were reduced by 44%, 18%, 34%, 28% and glutamine, glycine, histidine, taurine, arginine, threonine, alanine, cy- 91%, respectively, in HPP-20 treated samples, while hydrocarbons, steine, valine, and lysine). Tyrosine was the only amino acid that pre- ketones and chloro compounds were incremented by 60% 58% and sented the highest level in untreated samples. 79%, respectively, in comparison to CO. Furthermore, samples treated Changes in individual amino acid content could promote changes in with HPP at 35 °C presented reduction in the aldehydes, carboxylic acid the final flavor of dry-cured ham(Hidalgo & Zamora, 2004; Jurado, and sulphur compounds (22%, 36% and 82%, respectively) while hy- García, Timón, & Carrapiso, 2007). Thereby, the higher content in drocarbons, ketones, ether and esters and chloro compounds were in- specific amino acids showed in HPP-35 samples may influence the cremented by 109%, 109%, 37% and 69%, respectively, in comparison perception of sweet (calculating as sum of alanine, serine, proline, to CO. It is well known that aldehydes, ketones, ester and ethers, and
51 C. Pérez-Santaescolástica et al. Food Research International 116 (2019) 49–56
Table 2 Levels (expressed as quantified area units (AU)3 x10 /g dry cured ham) of the mainly families of volatile compounds identified in untreated and HPP at 0 ° C, 20°C and 35° C treated dry cured ham. Values are means of thirty hams for each treatment.
Compound Treatment SEM p-value
CO HPP-0 HPP-20 HPP-35
Aliphatic hydrocarbons 20,538.44b 9197.34a 33,631.08c 43,688.86d 1469.501 < 0.001 Aromatic and cyclic hydrocarbons 902.09c 486.35a 747.07b 1027.21c 27.306 < 0.001 Hydrocarbons 21,440.53b 9683.69a 34,385.34c 44,716.06d 1489.524 < 0.001 Aldehyde 22,467.49c 9840.67a 12,562.24a 17,443.40b 607.511 < 0.001 Ketone 2454.69a 2166.36a 3890.57b 5138.82c 124.636 < 0.001 Esther and ether 1659.26a 1608.06a 1761.60a 2272.76b 43.336 < 0.001 Alcohol 6465.70c 3900.03a 5295.68b 6826.08c 161.750 < 0.001 Carboxylic acid 1027.64c 321.09a 680.18b 660.01b 31.479 < 0.001 Nitrogenous compounds 585.65bc 524.52b 422.16a 648.02c 13.799 < 0.001 Sulphur compounds 2178.20b 321.41a 206.63a 400.44a 88.244 < 0.001 Chloro compounds 270.11b 121.20a 482.85c 455.68c 16.934 < 0.001 Total Compounds 58,549.27b 28,487.02a 59,641.13b 78,561.29c 1986.982 < 0.001 a–dMean values in the same row (corresponding to the same family) not followed by a common letter differ significantly (P < 0.05; Tukey's Test). SEM: standard error of mean. Treatments: CO = control (without treatment); HPP-0 = High pressure treatment at 0 °C; HPP-20 = High pressure treatment at 20 °C; HPP-35 = High pressure treatment at 35 °C. alcohols (to a limited extent) are the main families associated with the influenced volatile compounds originated from microbial activity. aroma of dry-cured ham (Carrapiso, Martín, Jurado, & García, 2010; Moreover, the majority of the most abundant volatile compounds were García-González, Tena, Aparicio-Ruiz, & Morales, 2008). Therefore the obtained in either CO or HPP-35 samples (64 and 61 compounds, re- temperature-assisted HPP treatments may affect the quality of the final spectively). product. In this way, the HPP-35 treatment enhanced ester and ether Among the lipolysis-derived compounds, hexanal was the most contents, which are responsible for fruity odour notes. Meanwhile, all abundant, particularly in untreated samples. Conversely, the lowest of HPP treatments caused a significant reduction of sulphur compounds, value was observed in the HPP-0 batch. Interestingly, an increasing a fact that could modify the aroma by incrementing rotten egg and trend in hexanal content was observed as the temperature of treatment burnt notes. In addition, our data are in agreement with the results increased. This fact can be explained by the potential protective effect obtained by Martínez-Onandi, Rivas-Cañedo, Picón, and Núñez (2016) of the HPP against hexanal generation. This finding could be considered in sliced Serrano dry-cured ham treated at 600 MPa and 21 °C for positive since high levels of this compound gives rancid notes to ham. In 2.5 min. contrast, the aroma can turn grassier and more pleasant because of the A total of 149 volatile compounds were identified and classified hexanal reduction (Aparicio & Morales, 1998). On the contrary, pre- based on their origin according to Narváez-Rivas, Gallardo, and León- vious studies about the effects of HPP on dry-cured hams showed that Camacho (2012), Martín et al. (2006) and Fonseca, Gómez, Domínguez, HPP increased the rancid odour perception due to an increment in al- and Lorenzo (2015). Of the 149 compounds, 92 were presumably ori- dehydes (Clariana et al., 2011; Fuentes et al., 2010). In agreement to ginated from lipid oxidation, 21 were derived from proteolysis reac- Martínez-Onandi et al. (2017), nonanal, propinoic acid, butanoic acid, tions, 21 were attributed to microbial activity and 15 had an unknown pentanoic acid, hexanoic acid and pentanal showed higher levels in origin. Table 3 lists the compounds detected in the volatile fraction of untreated than in HPP samples. However, lower values of 2-pentanol the slices of dry-cured ham, as well as the effect of HPP treatments, the were obtained from untreated samples (Table 3). Additionally, 1-Octen- linear retention indexes, the ions used for quantification and the 3-ol, a characteristic compound of dry-cured ham with a very low method used for identification. threshold in “Montanera hams” (Jurado, Carrapiso, Ventanasa, & 147 out of the 149 identified volatile compounds were significantly García, 2009), did not show significant differences between CO and influenced by the HPP treatment. Regarding the origin of these com- HPP-35 samples, although the other two treatments (HPP-0 and HPP- pounds, the most probable origin was lipolysis, followed by proteolysis 20) showed significant lower values. and microbial activity. The sum of secondary products of lipid oxidative As expected, the main microbial activity-derived compounds de- decomposition was around 80% of the total volatile content in all tected in this study were esters whose formation are closely related to treatments with the exception of HPP-0, in which such compounds microbial activity (Ramírez & Cava, 2007). Also, it is well known that accounted for 67%. In contrast, the compounds derived from proteo- temperature affects the ester compounds formation (Gorbatov & lysis represented 20% of the total volatile compounds in the HPP-0 Lyaskovskaya, 1980). For this reason, it was no strange that CO and group and 8–9% in the other treatments. These differences within fa- HPP-35 samples presented higher amounts of microbial activity-derived milies can be explained by the high temperatures (which promote li- compounds than HPP-0 and HPP-20 samples, and, in the same way, the polysis) and HPP that can induce protein denaturation (Guyon, Le HPP-20 group presented higher values than HPP-0. Dimethyl disulfide Vessel, Meynier, & de Lamballerie, 2018). Similar results were found by was the main compound detected in CO samples, but it was greatly Martínez-Onandi et al. (2017) and Ramírez and Cava (2007) who re- reduced by HPP treatments (1786.20 AU × 103/g of dry-cured ham vs ported values around 75% and 81.6% of total compounds were asso- 160.12 AU × 103/g of dry-cured ham vs 61.83 AU × 103/g of dry- ciated with lipid oxidation, respectively, and values of 20% and 12.7% cured ham vs 213.32 AU × 103/g of dry-cured ham for CO, HPP-0, of total compounds were attributed to proteolysis, respectively. Pre- HPP-20 and HPP-35, respectively). Although the origin of dimethyl vious studies observed that the application of HPP at pressures below disulfide is usually related to the microbial activity, some previous 300 MPa have minimum effect on lipid oxidation but higher pressures studies established that amino acid catabolism can be another possible give an increase in the amount of aldehydes derived from lipolysis via (Ramírez & Cava, 2007; Sabio, Vidal-Aragon, Bernalte, & Gata, (Andrés, Møller, Adamsen, & Skibsted, 2004; Fuentes et al., 2010). In 1998). Moreover, Muriel, Antequera, Petrón, Andrés, and Ruiz (2004) contrast, Martínez-Onandi, Rivas-Cañedo, Núñez, and Picón (2016) did found that dimethyl disulfide could result from the reaction between not find any significant effect on linear aldehydes content in dry-cured lipid oxidation products and cysteine. In the present study, a positive ham treated at 600 MPa, and these authors concluded that HPP only and significant (P < 0.05) correlation between cysteine and dimethyl
52 C. Pérez-Santaescolástica et al. Food Research International 116 (2019) 49–56
Table 3 Effect of treatments on volatile compound content (expressed as quantified area units (AU)×103/g dry cured ham). Values are means of thirty hams for each treatment.
Compound m/z LRI R Treatment SEM p-value
CO HPP-0 HPP-20 HPP-35
Pentane Ɏ 43 516 ms, lri, s 1166.82c 341.36a 568.52b 722.43b 39.322 < 0.001 Φ Propanal 58 526 ms, lri, s 133.06b 26.37a 38.29a 42.57a 5.109 < 0.001 Φ Acetone 58 528 ms, lri 171.04ab 256.36b 151.17a 387.56c 14.734 < 0.001 Φ Isopropyl Alcohol Ɏ 45 532 ms, lri 88.41a 218.13bc 191.73b 226.23c 6.570 < 0.001 Φ 2,3-Hexanedione 41 562 ms, lri 356.04a 290.13a 1252.05b 2096.88c 84.736 < 0.001 n-Hexane Ɏ 57 562 ms, lri, s 822.88a 706.36a 2744.00b 4705.52c 185.161 < 0.001 1-Butene, 2,3-dimethyl- Ɏ 69 571 ms, lri 12.15a 22.71b 14.63a 30.09c 1.043 < 0.001 Φ 1-Propanol Ɏ 59 572 ms, lri 27.91a 42.80b 52.10bc 57.38c 1.885 < 0.001 Φ Butanal Ɏ 72 584 ms, lri, s 24.28c 8.00a 11.96ab 15.52b 0.808 < 0.001 Φ 2-Butanone 72 596 ms, lri 182.67a 255.04ab 281.36b 247.02ab 11.320 0.012 Φ 2-Butanol Ɏ 45 607 ms, lri 13.92a 28.77bc 24.25b 33.51c 1.009 < 0.001 Cyclopentanone, 3-methyl- Ɏ 56 667 ms, lri 45.68c 9.53a 16.76a 28.13b 1.683 < 0.001 Heptane Ɏ 71 675 ms, lri, s 1321.57c 203.20a 353.71ab 555.71b 49.078 < 0.001 Φ Furan, 2-ethyl- Ɏ 81 703 ms, lri 40.15c 7.25a 12.78ab 16.35b 1.492 < 0.001 Φ 1-Butanol Ɏ 56 707 ms, lri 17.51a 16.42a 27.14b 32.09b 1.027 < 0.001 Φ 2-Pentanone 86 720 ms, lri 98.54b 59.43a 86.95ab 144.70c 4.956 < 0.001 Φ Pentanal Ɏ 57 728 ms, lri, s 1190.56c 378.49a 491.34a 771.43b 44.792 < 0.001 Φ 1-Penten-3-ol Ɏ 57 730 ms, lri 1099.81c 350.04a 552.91b 666.28b 35.716 < 0.001 Φ 2-Pentanol Ɏ 45 751 ms, lri 86.03a 311.24b 222.00b 413.17c 17.625 < 0.001 Pentane, 2,3,4-trimethyl- Ɏ 71 756 ms, lri 181.85b 115.66a 98.42a 108.51a 6.903 < 0.001 Pentane, 2,3,3-trimethyl- Ɏ 71 763 ms, lri 252.09b 184.67a 125.65a 131.57a 9.770 < 0.001 Pentane, 3-ethyl- Ɏ 70 770 ms, lri 46.85b 25.18a 15.13a 15.93a 1.820 < 0.001 1-Pentene, 3-ethyl-2-methyl- Ɏ 83 774 ms, lri 32.62b 14.34a 55.02c 85.19d 2.815 < 0.001 Hexane, 2,2,5-trimethyl- Ɏ 57 800 ms, lri 355.20c 198.20b 86.68a 85.66a 15.519 < 0.001 Octane Ɏ 85 822 ms, lri, s 3308.36c 587.64a 907.92ab 1277.65b 124.815 < 0.001 Φ Propanoic acid Ɏ 74 827 ms, lri 12.64c 3.76a 8.35b 8.05b 0.591 < 0.001 2-Octene, (E)- Ɏ 112 833 ms, lri 342.80c 75.77a 120.74a 208.17b 12.002 < 0.001 Heptane, 3,4,5-trimethyl- Ɏ 85 842 ms, lri 76.88c 49.25b 9.68a 9.50a 3.621 < 0.001 3-Octene, (E)- Ɏ 112 845 ms, lri 170.74c 39.14a 55.81a 99.32b 6.553 < 0.001 Φ 1-Pentanol Ɏ 55 847 ms, lri, s 500.17c 136.34a 220.83a 385.19b 19.128 < 0.001 Φ Hexanal Ɏ Ψ 56 865 ms, lri 15,270.28d 3980.78a 6595.32b 9404.24c 510.237 < 0.001 Hexane, 2,2,5,5-tetramethyl- Ɏ 57 914 ms, lri 387.10b 304.54b 87.89a 130.89a 17.892 < 0.001 Φ Butanoic acid Ɏ 60 918 ms, lri 191.58c 50.50a 113.16b 69.28a 7.191 < 0.001 4-Nonene Ɏ 70 926 ms, lri 198.55b 128.93a 152.16a 230.92b 7.011 < 0.001 Heptane, 2-methyl-3-methylene- Ɏ 126 930 ms, lri 17.87a 11.42a 17.45a 28.24b 1.028 < 0.001 Nonane Ɏ 57 936 ms, lri, s 201.88c 123.93b 57.32a 84.50ab 7.840 < 0.001 2-n-Butyl furan Ɏ 81 944 ms, lri 39.67b 13.34a 20.37a 39.72b 1.702 < 0.001 Φ 3-Heptanone 57 960 ms, lri 42.37a 47.19a 70.34b 117.12c 3.546 < 0.001 2-Heptanone Ɏ 58 967 ms, lri 455.34a 344.09a 434.04a 620.46b 19.443 < 0.001 Φ Heptanal Ɏ 70 974 ms, lri, s 988.97c 251.92a 396.51ab 512.02b 33.687 < 0.001 Φ 2-Nonen-4-one 69 979 ms, lri 16.18ab 14.73a 15.98ab 20.45b 0.671 0.014 2-Octene, 4-ethyl- Ɏ 69 982 ms, lri 146.99b 106.14a 106.60a 126.89ab 5.230 0.013 Octane, 3-methyl-6-methylene- Ɏ 70 985 ms, lri 303.36bc 198.09a 243.42ab 365.97c 13.905 < 0.001 Octane, 4-ethyl- Ɏ 69 991 ms, lri 90.08b 72.48ab 68.06a 83.64ab 2.594 0.007 2-Hepten-4-one, 6-methyl- Ɏ 69 992 ms, lri 91.95ab 73.46a 71.13a 102.44b 3.405 0.001 Pentane, 3,3-dimethyl- Ɏ 85 995 ms, lri 9.35b 5.33a 4.84a 7.22ab 0.400 < 0.001 Φ Methional Ɏ 104 999 ms, lri 201.58a 211.98a 252.49a 387.58b 15.839 < 0.001 Nonane, 2,3-dimethyl- Ɏ 71 1003 ms, lri 87.74c 62.48b 40.21a 63.90b 3.371 < 0.001 1-Octene, 2,6-dimethyl- Ɏ 56 1010 ms, lri 104.76ab 77.37a 89.61ab 119.04b 4.298 0.004 3-Octene, 4-ethyl- Ɏ 69 1012 ms, lri 29.19ab 20.38a 25.38a 38.69b 1.441 < 0.001 Nonane, 3-methylene- Ɏ 70 1022 ms, lri 236.74ab 188.34a 180.06a 284.91b 10.133 < 0.001 Heptane, 2,2,4,6,6-pentamethyl- Ɏ 57 1027 ms, lri 5140.73a 1929.11a 21,626.35b 27,733.83c 1183.950 < 0.001 Decane Ɏ 57 1030 ms, lri, s 406.72c 324.13c 225.36b 65.94a 16.694 < 0.001 3-Ethyl-3-hexene Ɏ 83 1042 ms, lri 62.24ab 47.97a 56.07a 78.99b 2.493 < 0.001 Φ 1-Heptanol Ɏ 70 1046 ms, lri 91.50c 36.91a 62.38b 71.79bc 3.218 < 0.001 Φ 1-Octen-3-ol Ɏ 57 1051 ms, lri 3935.68c 1915.73a 2824.65b 3607.07c 117.522 < 0.001 5-Hepten-2-one, 6-methyl- Ɏ 69 1056 ms, lri 128.81b 120.63ab 93.86a 116.58ab 3.997 0.011 2-Octanone Ɏ 58 1059 ms, lri 41.33ab 38.50a 51.94b 72.99c 2.061 < 0.001 Φ Octanal Ɏ 56 1066 ms, lri, s 384.48b 182.91a 209.96a 231.97a 12.175 < 0.001 Undecane, 3,6-dimethyl- Ɏ 57 1068 ms, lri 162.81a 83.59a 608.60b 879.39c 39.201 < 0.001 Φ Pentanoic acid Ɏ 60 1083 ms, lri 394.31b 212.80a 257.50a 210.40a 13.552 < 0.001 Undecane, 2,5-dimethyl- Ɏ 57 1085 ms, lri 163.08a 142.14a 186.12a 258.59b 9.209 < 0.001 Decane, 2,3,5-trimethyl- Ɏ 57 1099 ms, lri 80.58b 76.68b 44.70a 70.74b 2.882 < 0.001 Undecane Ɏ 57 1113 ms, lri, s 1117.99a 1034.94a 1442.34ab 1864.94b 73.182 < 0.001 Φ 2-Octenal, (E)- Ɏ 70 1123 ms, lri 52.79c 9.63a 19.13ab 22.76b 1.993 < 0.001 2,3-Dimethyl-3-heptene, (Z)- Ɏ 83 1123 ms, lri 56.77c 15.27a 23.21ab 27.74b 1.972 < 0.001 Φ 1-Octanol Ɏ 56 1127 ms, lri 63.72b 43.46a 48.67a 48.86a 1.806 < 0.001 Φ Decanal Ɏ 81 1129 ms, lri, s 24.44c 14.33a 18.50ab 21.73bc 0.769 < 0.001 2-Undecene, 9-methyl-, (Z)- Ɏ 70 1132 ms, lri 384.25b 344.35ab 275.11a 383.03b 13.208 0.007 Φ 3-Nonanone 113 1134 ms, lri 21.19a 25.18ab 21.20a 29.87b 0.914 0.001 Φ 2-Nonanone 58 1141 ms, lri 15.69a 24.12b 32.95c 46.71e 1.362 < 0.001 (continued on next page)
53 C. Pérez-Santaescolástica et al. Food Research International 116 (2019) 49–56
Table 3 (continued)
Compound m/z LRI R Treatment SEM p-value
CO HPP-0 HPP-20 HPP-35
5-Undecene, 6-methyl- Ɏ 168 1144 ms, lri 11.40bc 9.13ab 7.32a 12.51c 0.477 < 0.001 Φ Nonanal Ɏ 57 1148 ms, lri, s 538.49b 307.02a 303.28a 327.20a 14.872 < 0.001 4,4-Dipropylheptane Ɏ 85 1153 ms, lri 55.24b 44.54ab 34.85a 43.85ab 1.804 0.001 Φ 5-Hexen-3-one 57 1161 ms, lri 43.40b 35.58ab 27.78a 35.50ab 1.538 0.003 2-Undecene, 3-methyl-, (E)- Ɏ 70 1181 ms, lri 59.72 53.90 48.65 60.81 1.998 0.102 Dodecane Ɏ 57 1188 ms, lri, s 701.16a 663.91a 932.47ab 1179.62b 41.201 < 0.001 4-Nonene, 5-butyl- Ɏ 70 1197 ms, lri 24.54b 22.39ab 18.01a 20.65ab 0.857 0.043 4-Nonenal, (E)- Ɏ 83 1201 ms, lri 31.24b 18.32a 18.06a 23.63a 1.001 < 0.001 Φ Octanoic acid Ɏ 60 1224 ms, lri 30.74c 9.66a 21.75b 18.36b 1.197 < 0.001 Φ 1-Tetradecanol Ɏ 68 1225 ms, lri 32.34ab 29.38ab 26.20a 34.48b 1.132 0.047 Decane, 3-ethyl-3-methyl- Ɏ 57 1228 ms, lri 50.73b 38.80a 32.66a 41.49ab 1.431 < 0.001 1-Tetradecene Ɏ 97 1236 ms, lri 30.34c 23.99ab 19.00a 25.23bc 0.845 < 0.001 Tridecane Ɏ 71 1258 ms, lri, s 190.43ab 156.70a 245.19bc 316.86c 11.383 < 0.001 Φ 2-Decenal, (E)- 70 1272 ms, lri 24.68b 12.77a 15.38a 16.81a 0.849 < 0.001 Φ 2,4-Decadienal, (E,E)- Ɏ 81 1315 ms, lri 27.22b 4.07a 6.90a 7.73a 1.213 < 0.001 Φ 2-Undecenal Ɏ 95 1339 ms, lri 5.96b 1.11a 1.42a 1.92a 0.266 < 0.001 Φ Pentadecanal- 82 1516 ms, lri, s 2.15a 8.56bc 6.84b 10.40c 0.443 < 0.001 Total lipolysis origin 45,879.67b 19,311.22a 47,734.58b 64,466.40c 1832.629 < 0.001 Carbon disulfide Ɏ 76 533 ms, lri 225.07b 119.20a 119.13a 148.43a 8.542 < 0.001 Φ Propanal, 2-methyl- Ɏ 72 557 ms, lri 161.98a 225.54ab 190.25ab 241.26b 9.171 0.008 Fumaronitrile Ɏ 78 646 ms, lri 29.18c 11.61a 10.27a 21.33b 0.948 < 0.001 Φ Butanal, 3-methyl- Ɏ 58 659 ms, lri 1525.03a 1973.95a 1925.24a 2925.94b 86.149 < 0.001 Φ Butanal, 2-methyl- Ɏ 57 671 ms, lri 758.92a 1291.93b 1195.03b 1278.51b 52.705 < 0.001 Φ 2-Butenal, 2-methyl- Ɏ 84 801 ms, lri 76.70a 63.38a 53.95a 106.33b 3.759 < 0.001 Φ 1-Butanol, 3-methyl- Ɏ Ψ 55 808 ms, lri 65.27a 425.21c 204.01b 240.17b 16.858 < 0.001 Φ 1-Butanol, 2-methyl- Ɏ 57 812 ms, lri 14.93a 51.65c 37.05b 44.65bc 1.890 < 0.001 Φ Propanoic acid, 2-methyl- Ɏ 73 888 ms, lri 51.23c 18.99a 34.63b 60.85c 2.289 < 0.001 Φ 2-Propanol, 2-methyl- Ɏ 59 894 ms, lri 17.68c 6.80a 7.49a 12.68b 0.556 < 0.001 3-(1′-pyrrolidinyl)-2-butanone Ɏ 98 906 ms, lri 136.09b 83.70a 74.19a 94.24a 4.904 < 0.001 Φ 3-Pentanol, 2,4-dimethyl- Ɏ 73 954 ms, lri 7.84a 8.75ab 7.43a 10.29b 0.236 < 0.001 Φ Butanoic acid, 3-methyl- Ɏ 60 969 ms, lri 349.62c 139.30a 250.02b 327.58bc 14.309 < 0.001 Φ Pyrazine, 2,6-dimethyl- Ɏ 108 978 ms, lri 290.52ab 344.23bc 241.18a 395.39c 10.530 < 0.001 1-(1′-pyrrolidinyl)-2-butanone Ɏ 84 982 ms, lri 129.87bc 84.97a 98.05ab 138.36c 5.375 0.001 Φ Dimethyl trisulfide Ɏ 126 1035 ms, lri 179.23b 13.88a 7.19a 11.39a 8.662 < 0.001 Φ Benzaldehyde Ɏ 106 1045 ms, lri 339.48a 350.30a 295.39a 462.27b 11.745 < 0.001 Φ 1-Heptanol, 2,4-diethyl- Ɏ 69 1085 ms, lri 90.78ab 73.41a 69.19a 108.27b 3.947 0.001 Φ 2-Ethyl-1-hexanol Ɏ 57 1094 ms, lri 6.65a 58.12b 128.81d 86.52c 5.083 < 0.001 5-Ethylcyclopent-1-enecarboxaldehyde 124 1099 ms, lri 27.85b 10.17a 13.43a 14.93a 0.934 < 0.001 Φ 2(3H)-Furanone, 5-ethyldihydro- Ɏ 85 1158 ms, lri 174.77a 197.82ab 178.62ab 215.24b 5.130 0.014 Φ 4-Methyl-5-decanol Ɏ 55 1162 ms, lri 21.99b 12.83a 15.13a 17.55ab 0.750 < 0.001 Φ Sulfurous acid, butyl dodecyl ester Ɏ 85 1304 ms, lri 27.37b 28.21b 18.48a 27.32b 0.772 < 0.001 Total proteolysis origin 4708.05a 5593.97a 5174.15a 6989.50b 154.115 < 0.001 Φ Pentane, 2-methyl- Ɏ 71 543 ms, lri 2.61a 1.20a 2.75a 13.89b 0.559 < 0.001 Acetic acid ethenyl ester Ɏ 86 588 ms, lri 25.79bc 21.53b 14.90a 28.64c 0.898 < 0.001 Ethyl Acetate Ɏ 61 598 ms, lri 148.15a 238.69b 213.97ab 215.97ab 10.382 0.013 Methane, oxybis[dichloro- Ɏ 83 611 ms, lri 270.11b 121.20a 318.55b 455.68c 16.324 < 0.001 Propanoic acid, ethyl ester Ɏ 57 737 ms, lri 52.51c 18.56a 30.57ab 42.12bc 2.190 < 0.001 Φ Disulfide, dimethyl 94 781 ms, lri 1786.20b 160.12a 61.83a 213.32a 77.445 < 0.001 Butanoic acid, ethyl ester Ɏ 71 855 ms, lri 86.10a 78.86a 68.63a 136.66b 4.090 < 0.001 Φ Octane, 2-methyl- Ɏ 71 899 ms, lri 16.79c 10.29a 11.93ab 15.87bc 0.627 < 0.001 Butanoic acid, 2-methyl-, ethyl ester Ɏ 102 908 ms, lri 49.05a 65.69ab 56.95a 85.20b 2.925 < 0.001 Butanoic acid, 3-methyl-, ethyl ester Ɏ 88 913 ms, lri 130.87a 170.48a 153.26a 280.27b 11.318 < 0.001 Oxalic acid, butyl propyl ester Ɏ 57 936 ms, lri 201.88c 123.93b 57.32a 84.50ab 7.840 < 0.001 Ethanol, 2-butoxy- Ɏ 57 985 ms, lri 431.27a 353.25a 797.03b 947.37c 29.190 < 0.001 Carbonic acid, bis(2-ethylhexyl) ester Ɏ 112 1003 ms, lri 30.15b 24.44ab 17.32a 23.12ab 1.093 < 0.001 Hexanoic acid, ethyl ester Ɏ 88 1050 ms, lri 167.48a 205.07a 205.07a 274.88b 7.699 < 0.001 Φ Tridecane, 6-methyl- Ɏ 57 1079 ms, lri 323.72a 207.70a 636.89b 884.36c 35.976 < 0.001 2-Piperidinecarboxylic acid, 1-acetyl-, ethyl ester Ɏ 84 1124 ms, lri 36.93c 12.69a 15.86ab 20.18b 1.073 < 0.001 Octanoic acid, ethyl ester Ɏ 88 1204 ms, lri 74.38 80.58 73.08 68.96 1.749 0.149 Φ Dodecane, 2-methyl- Ɏ 88 1233 ms, lri 22.03a 23.92a 40.66b 53.41c 2.015 < 0.001 Φ Tridecane, 3-methyl- Ɏ 85 1304 ms, lri 27.62b 28.46b 18.60a 27.30b 0.823 < 0.001 Decanoic acid, ethyl ester Ɏ 88 1336 ms, lri 28.59c 20.50b 12.30a 16.53ab 0.860 < 0.001 2,2,4-Trimethyl-1,3-pentanediol diisobutyrate Ɏ 71 1442 ms, lri 10.00a 5.82a 2.64a 58.11b 2.772 < 0.001 Total microbial origin 3922.23c 1972.97a 2810.12b 3946.33c 97.439 < 0.001 Acetoin 45 787 ms, lri 478.36b 299.84a 367.90ab 625.50c 19.901 < 0.001 Cyclobutane, 1,1,2,3,3-pentamethyl- 70 813 ms, lri 326.38b 172.20a 402.58b 569.07c 20.318 < 0.001 Ethylbenzene 91 917 ms, lri 21.49bc 18.32b 13.48a 21.96c 0.538 < 0.001 Benzene, 1,3-dimethyl- 106 926 ms, lri 27.03c 24.88bc 18.01a 22.00ab 0.647 < 0.001 Cyclohexanone, 2-ethyl- 69 972 ms, lri 62.10ab 44.11a 44.04a 70.01b 3.140 0.004 4-Octanone, 5-hydroxy-2,7-dimethyl- 69 1042 ms, lri 13.29bc 9.05a 10.38ab 14.64c 0.447 < 0.001 4-Ethylcyclohexanol 81 1104 ms, lri 120.90b 86.88a 106.36ab 118.21b 3.718 0.006 Benzeneacetaldehyde 91 1119 ms, lri 712.67b 514.37a 501.69a 680.17b 18.764 < 0.001 Cyclopentane, 1-ethyl-3-methyl- 83 1123 ms, lri 56.77c 15.27a 23.21ab 27.74b 1.972 < 0.001 (continued on next page)
54 C. Pérez-Santaescolástica et al. Food Research International 116 (2019) 49–56
Table 3 (continued)
Compound m/z LRI R Treatment SEM p-value
CO HPP-0 HPP-20 HPP-35
Benzyl alcohol 108 1124 ms, lri 125.21a 291.08b 425.52c 552.39d 17.344 < 0.001 1-Hexanone, 5-methyl-1-phenyl- 105 1137 ms, lri 11.53a 11.66a 34.01b 75.99c 2.754 < 0.001 Cyclopentane, ethyl- 98 1148 ms, lri 261.83b 134.68a 142.63a 156.46a 7.681 < 0.001 p-Cresol 107 1178 ms, lri 29.90ab 23.63a 27.27a 33.88b 0.908 0.001 Phenylethyl Alcohol 92 1182 ms, lri 10.55a 45.80b 14.56a 17.64a 1.766 < 0.001 Benzaldehyde, 3-ethyl- 134 1209 ms, lri 34.40c 14.22a 21.36b 27.37b 1.077 < 0.001 Total unknoun origin 2292.41b 1705.98a 2153.01b 3013.04c 56.635 < 0.001 Total compounds 56,802.37b 28,584.14a 57,871.86b 78,415.27c 1973.358 < 0.001 a–dMean values in the same row (corresponding to the same compound) not followed by a common letter differ significantly (P < 0.05; Tukey's Test). Φ Compound origin according to: ɎNarváez-Rivas et al. (2012) Martín et al. (2006)ΨFonseca et al. (2015). SEM: standard error of mean. m/z: Quantification ion; LRI: Lineal Retention Index calculated for DB-624 capillary column (J&W scientific: 30 m × 0.25 mm id, 1.4 μm film thickness) installedona gas chromatograph equipped with a mass selective detector; R: Reliability of identification; lri: linear retention index in agreement with literature (Domínguez et al., 2014; Lorenzo, 2014; Lorenzo, Bedia, & Bañon, 2013; Lorenzo & Carballo, 2015; Lorenzo & Domínguez, 2014; Lorenzo, Montes, Purriños, & Franco, 2012; Pateiro, Franco, Carril, & Lorenzo, 2015; Pérez-Santaescolástica et al., 2018a; Pérez-Santaescolástica et al., 2018b; Purriños et al., 2011; Purriños, Carballo, & Lorenzo, 2013; Purriños, Franco, Bermudez, Carballo, & Lorenzo, 2011; Purriños, Franco, Carballo, & Lorenzo, 2012); ms: mass spectrum agreed with mass database (NIST14); s: mass spectrum and retention time identical with an authentic standard. Treatments: CO = control (without treatment); HPP-0 = High pressure treatment at 0 °C; HPP-20 = High pressure treatment at 20 °C; HPP-35 = High pressure treatment at 35 °C. disulfide (r = 0.200) was observed and therefore this via for the di- from INIA (Spain). Acknowledgements to INIA for granting Cristina methyl disulfide formation can not be discarded. Pérez Santaescolástica with a predoctoral scholarship. José M. Lorenzo The compounds derived from proteolysis found in the present study is member of the MARCARNE network, funded by CYTED (ref. that have been previously detected in dry-cured ham were 2-methyl 116RT0503). Paulo E. Munekata acknowledges postdoctoral fellowship propanal, 3-methyl butanal, 2-methyl butanal and 2-methyl-2-butenal support from Ministry of Economy and Competitiveness (MINECO, (Andrés, Cava, & Ruiz, 2002; Sánchez-Peña, Luna, García-González, & Spain) “Juan de la Cierva” program (FJCI-2016-29486). Aparicio, 2005; Timón, Ventanas, Carrapiso, Jurado, & García, 2001). The highest values of 2-methyl propanal, 3-methyl butanal and 2-me- References thyl-2-butenal were observed in HPP-35 samples. Particularly for 2- methyl butanal, all HPP-treated samples (independently of assisted Andrés, A. I., Cava, R., & Ruiz, J. (2002). Monitoring volatile compounds during dry- temperature) displayed higher values than CO samples, which may be cured ham ripening by solid-phase microextraction coupled to a new direct-extrac- tion device. Journal of Chromatography A, 963(1–2), 83–88. due to the HPP effect on protein structures. Moreover, the statistical Andrés, A. I., Møller, J. K., Adamsen, C. E., & Skibsted, L. H. (2004). High pressure analysis showed a positive correlation (r = 0.263, P < 0.01) between treatment of dry-cured Iberian ham. Effect on radical formation, lipid oxidation and 2-methyl butanal and isoleucine. The degradation of isoleucine is the colour. European Food Research and Technology, 219(3), 205–210. Aparicio, R., & Morales, M. T. (1998). Characterization of olive ripeness by green aroma most probable origin of this compound, as reported by previous studies compounds of virgin olive oil. Journal of Agricultural and Food Chemistry, 46(3), (Ramírez & Cava, 2007). 1116–1122. Finally, fifteen compounds were classified as “unknown origin” Bermúdez, R., Franco, D., Carballo, J., & Lorenzo, J. M. (2015). Influence of type of whose probable via/reaction was not found in literature. Since their muscle on volatile compounds throughout the manufacture of Celta dry-cured ham. Food Science and Technology International, 21(8), 581–592. origin is not clear, it is not possible to include them into the three Bermúdez, R., Franco, D., Carballo, J., Sentandreu, M. A., & Lorenzo, J. M. (2014). principal treatments already commented. It is worth mentioning that Influence of muscle type on the evolution of free amino acids and sarcoplasmic and the presence of p-Cresol could be associated with animal feed and myofibrillar proteins through the manufacturing process of Celta dry-cured ham. Food Research International, 56, 226–235. further accumulation in the animal tissues (Sabio et al., 1998; Sánchez- Buckow, R., Truong, B. Q., & Versteeg, C. (2010). Bovine cathepsin D activity under high Peña et al., 2005). The HPP-35 and CO samples showed higher contents pressure. Food Chemistry, 120(2), 474–481. of p-Cresol than HPP-0 and HPP-20 samples. Aromatic and ciclyc hi- Careri, M., Mangia, A., Barbieri, G., BOUONI, L., VIRGILI, R., & PAROLARI, G. (1993). Sensory property relationships to chemical data of Italian-type dry-cured ham. drocarbons were also found: 1,3-dimethyl benzene, 1-ethyl-3-methyl Journal of Food Science, 58(5), 968–972. cyclopentane and ethyl cyclopentane contents were reduced by HPP Carrapiso, A. I., Martín, L., Jurado, Á., & García, C. (2010). Characterisation of the most treatment. odour-active compounds of bone tainted dry-cured Iberian ham. Meat Science, 85(1), 54–58. Chéret, R., Hernández-Andrés, A., Delbarre-Ladrat, C., De Lamballerie, M., & Verrez- 4. Conclusion Bagnis, V. (2006). Proteins and proteolytic activity changes during refrigerated sto- rage in sea bass (Dicentrarchus labrax L.) muscle after high-pressure treatment. European Food Research and Technology, 222(5–6), 527. HPP is a promising technology to process food, specially products Clariana, M., Guerrero, L., Sárraga, C., Díaz, I., Valero, Á., & García-Regueiro, J. A. affected by higher temperatures. From the results obtained in thepre- (2011). Influence of high pressure application on the nutritional, sensory andmi- crobiological characteristics of sliced skin vacuum packed dry-cured ham. Effects sent study, it can be concluded that HPP can be applied to dry-cured along the storage period. Innovative Food Science & Emerging Technologies, 12(4), ham but in the range 0–20 °C in order to minimize the impact of such 456–465. treatments on free amino acid and volatile compounds. This re- Clariana, M., Guerrero, L., Sárraga, C., & Garcia-Regueiro, J. A. (2012). Effects of high pressure application (400 and 900 MPa) and refrigerated storage time on the oxi- commendation is supported by the intense modifications caused HPP dative stability of sliced skin vacuum packed dry-cured ham. Meat Science, 90(2), and high temperature (particularly at 35 °C) on free amino acid profile 323–329. and volatile composition, which could reduce product quality. Domínguez, R., Gómez, M., Fonseca, S., & Lorenzo, J. M. (2014). Effect of different cooking methods on lipid oxidation and formation of volatile compounds in foal meat. Meat Science, 97(2), 223–230. Acknowledgements Duranton, F., Simonin, H., Chéret, R., Guillou, S., & de Lamballerie, M. (2012). Effect of high pressure and salt on pork meat quality and microstructure. Journal of Food Science, 77(8), E188–E194. This research was supported by Grant RTA 2013-00030-CO3-03
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Food Research International
journal homepage: www.elsevier.com/locate/foodres
Molecular insight into taste and aroma of sliced dry-cured ham induced by protein degradation undergone high-pressure conditions T
López-Pedrouso M.a, Pérez-Santaescolástica C.b, Franco D.a, Carballo J.c, Zapata C.b, ⁎ Lorenzo J.M.a, a Department of Zoology, Genetics and Physical Anthropology, University of Santiago de Compostela, Santiago de Compostela 15872, Spain b Centro Tecnológico de la Carne de Galicia, Rúa Galicia N° 4, Parque Tecnológico de Galicia, San Cibrán das Viñas 32900, Ourense, Spain c Área de Tecnología de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain
ARTICLE INFO ABSTRACT
Keywords: High pressure processing (HPP) is currently being developed to increase the shelf-life of sliced dry-cured ham in Myofibrillar proteins convenience package without detrimental effects on texture and sensorial characteristics. This study is focused Strecker metabolites on protein degradation under pressure conditions and its contribution to taste and aroma. Samples of sliced dry- Linear and branched aldehydes cured ham undergone HPP (600 Pa, 0–35 °C) were analyzed from different approaches including proteomic and Proteolysis and lipid oxidation chemical analysis (amino acids and volatile compounds). Proteomic analysis revealed that high-pressure conditions caused a higher level of proteolysis, displaying that actin (ACTC1) was differentially degraded, unlike myosin. Furthermore, main Strecker metabolites-isoleucine and leucine-were more abundant at lower temperatures as opposed to 2-methyl butanal and 3-methyl butanal under HPP. Moreover, this study confirmed that HPP affected positively linear aldehydes (pentanal, hexanal, heptanal and nonanal) because of produce a decrease of them, which could improve flavor and taste of dry-cured ham.
1. Introduction the breakdown of myofibrillar structures (Čandek-Potokar & Škrlep, 2012; Mora, Sentandreu, & Toldrá, 2011). For these reasons HPP is The traditional manufacturing process of dry-cured ham includes being boosted within food industry, but concomitant effects on odor the following steps: salting, post-salting, ripening and drying for a total (rancidity) and texture attributes have been revealed particularly on period between 12 and 24 months (Bermúdez, Franco, Carballo, & sliced dry-cured hams (Lorido, Estévez, Ventanas, & Ventanas, 2015; Lorenzo, 2014). All these steps have a strong impact on the meat quality Pérez-Santaescolástica et al., 2019). It has been reported that HPP due to complex chemical reactions (Harkouss et al., 2015; López- modified the protein structure causing alterations in meat color and Pedrouso et al., 2018; López-Pedrouso et al., 2019; Lorenzo et al., 2015; texture parameters such as pastiness and hardness (Garcıa-Esteban,́ Petrova, Tolstorebrov, Mora, Toldrá, & Eikevik, 2016). Nowadays, Ansorena, & Astiasarán, 2004). Moreover, volatile compounds have consumers demand the use of convenience package to increase the shelf also been affected by HPP and storage conditions of sliced vacuum- life of the products as well as to maintain microbiological safety. To packaged dry-cured ham (Clariana et al., 2011; Fuentes, Utrera, overcome the problem of possible food contaminations without the loss Estévez, Ventanas, & Ventanas, 2014). of quality, innovative and nonthermal techniques are being developed Flavor and aroma are influenced by proteolysis and lipolysis phe- such as high-pressure processing (HPP). However, the introduction of nomena, which release free fatty acids, peptides and free amino acids. novel technology is another important factor, which has a major in- These complex processes are attributed to enzymatic and chemical re- fluence on sensory features. Along with the whole process, the volatile actions such as Maillard reactions and Strecker degradations (Estévez, compounds profile (aldehydes, ketones, alcohols, hydrocarbons and Ventanas, & Heinonen, 2011; Petrova et al., 2016). In the muscle, lipids esters) which defines aroma could be changed due to protein de- and proteins are transformed into flavor precursors depending on pro- gradation (Martínez-Arellano, Flores, & Toldrá, 2016; Pérez- cess conditions (Degnes, Kvitvang, Haslene-Hox, & Aasen, 2017; Toldrá Santaescolástica et al., 2018b). In addition, other key characteristic & Flores, 1998). Specifically, in dry-cured ham process, the Maillard associated with proteolysis is the tenderization, which is mainly due to reaction is a chemical reaction between a reducing sugar and an amino
⁎ Corresponding author. E-mail address: [email protected] (J.M. Lorenzo). https://doi.org/10.1016/j.foodres.2019.01.037 Received 10 December 2018; Received in revised form 14 January 2019; Accepted 15 January 2019 Available online 17 January 2019 0963-9969/ © 2019 Elsevier Ltd. All rights reserved. M. López-Pedrouso et al. Food Research International 122 (2019) 635–642 compound favored by long drying period and low water activity up to 800 spots (Lametsch & Bendixen, 2001; Morzel et al., 2004). This (Flores, 2018). On the other hand, the Strecker degradation of amino would provide us enough quantity of data to carry out this experiment acids lead to their deamination and decarboxylation turning out alde- between two conditions. Furthermore, the combination of pressure and hydes and ketones. These amino acids are intermediates from Maillard temperature produce protein denaturation particularly acute in com- reaction which means that both reactions are closely related (Van bination of high pressure and temperature (Guyon, Meynier, & De Boekel, 2006). Therefore, the aroma and flavor are strongly affected by Lamballerie, 2016). In order to avoid interaction between these two processing techniques, which could modify the kinetics of both reac- variables, the selected temperature for proteomic analysis was 0 °C with tions. These sensorial attributes could also be studied in detail using the the aim of identify the potential biomarkers related exclusively to high identification and quantification of their volatiles (Bermúdez, Franco, pressure distinguishing them from biomarkers associated with high Carballo, Sentandreu, & Lorenzo, 2014; Pérez-Santaescolástica et al., temperature. 2018a). Consequently, the combination of non-volatile and volatile For each biological replica, a lyophilized sample of dry-cured ham compounds defines a specific taste and smell, which is highly appre- (50 mg) was mixed with 1 mL of lysis buffer (7 M urea; 2 M thiourea; ciated. Indeed, juiciness and flavor intensity of dry-cured ham are the 4% CHAPS; 10 mM DTT, and 2% Pharmalyte™ pH 3–10, GE Healthcare, most important positive characteristics assessed by consumers (Ruiz, Uppsala, Sweden) and sonicated (Sonifier 250, Branson, Danbury, CC, Garcıa, Muriel, Andrés, & Ventanas, 2002). USA) under refrigerated conditions (0 °C). Protein purification was To monitor the quality, safety and nutritional requirements during performed twice with Clean-Up Kit (GE Healthcare) and subsequently production and storage, foodomics is a very useful tool for the taste, the pellet was dissolved in 200 μL of lysis buffer. Protein content in flavor and texture characterization. A proteomic approach using bidi- samples was estimated by CB-X protein assay kit (G-Biosciences, St. mensional electrophoresis (2-DE) coupled with mass spectrometry (MS) Louis, MO, USA) using a Chromate® microplate reader (Awareness could also provide biomarkers to optimize food processing (Picard & Technology, Palm City, FL, USA). The isoelectric focusing (IEF) was Gagaoua, 2017; Picard, Gagaoua, & Hollung, 2017). carried out in 24 cm pH 4–7 IPG strips (Bio-Rad Laboratories, Hercules, In fact, it was employed in thermal, microwaves, high pressure, CA, USA). In the first step, the strips were rehydrated for 12 h in 450 μL pulsed electric field and ultrasound treatments (López-Pedrouso et al., of a solution 0.6% DTT and 1% IPG buffer (Bio-Rad Laboratories) which 2018; Piras, Roncada, Rodrigues, Bonizzi, & Soggiu, 2016). In addition, contained 250 μg of protein in lysis buffer and rehydration buffer (7 M this approach has been used to study cured and cooked pork ham urea, 2 M thiourea, 4% CHAPS, 0.002% bromophenol blue). Afterward, products (Pioselli, Paredi, & Mozzarelli, 2011; Škrlep et al., 2011; the voltage was sequentially raising until reaching 70,000 Vh, and then Théron et al., 2011). From a proteomic point of view, the proteolysis of the strips were equilibrated with buffer I (50 mM Tris pH 8.8, 6 M urea, dry-cured ham produced during their manufacturing process has also 2% SDS, 30% glycerol, 1% DTT) for 15 min and buffer II (50 mM Tris been studied (Fabbro et al., 2016; López-Pedrouso et al., 2018; Pérez- pH 8.8, 6 M urea, 2% SDS, 30% glycerol, 2.5% iodoacetoamide) for Santaescolástica et al., 2018b), but only few researches have focused on another 15 min. Finally, the second dimension was carried out in 12% proteins of dry-cured ham submitted to HPP. SDS-PAGE using an Ettan DALTsix vertical gel system (GE Healthcare). Therefore, the aim of this study was to evaluate the protein al- The resulting 2-DE gels were stained with SYPRO Ruby fluorescent stain terations of sliced dry-cured ham undergone high-pressure processing. (Lonza, Rockland, ME, USA) and the images of gels were obtained by The contribution of protein degradation to the release of volatile Gel Doc XR+ system (Bio-Rad Laboratories). Image analysis of gels was compounds and their metabolites should be studied to optimize high- carried using PDQuest Advanced software v. 8.0.1 (Bio-Rad pressure treatment on a final meat product. Laboratories) and experimental values of isoelectric point (pI) and mass
(Mr) were estimated. 2. Material and methods 2.3.2. Protein identification by mass spectrometry 2.1. Samples The excision and in-gel tryptic digestion of protein gel spots were carried out according to Franco et al. (2015). The digested spots with Sixteen dry-cured hams, manufactured according to the traditional trypsin were ionized using α-cyano-4-hydroxycinnamic acid as matrix system, were sliced (1.5 mm-thick) and vacuum packed in individual and deposited onto a 384 Opti-TOF MALDI plate (Applied Biosystems, plastic bags of polyamide/polyethylene (oxygen permeability of Foster City, CA, USA). Mass spectrometry analysis was performed on a 50 cm3/m2/24 h at 23 °C and water permeability of 2.6 g/m2/24 h at MALDI-TOF/TOF tandem mass spectrometer 4800 MALDI-TOF/TOF 23 °C and 85% RH, Sacoliva® S.L., Spain) and stored in a chamber at (Applied Biosystems, Foster City, CA, USA) using 4000 Series Explorer 4 °C ± 2 °C until the treatment application. software v. 3.5 (Applied Biosystems, Foster City, CA, USA). The search was carried out using Mascot software v. 2.1 (Matrix Science, Boston, 2.2. HPP treatments MA, USA) to identify proteins from peptide mass fingerprint data em- ploying UniProt/SwissProt database. The treatment of the packaged slices was applied using a NC Hyperbaric WAVE 6000/120 equipment (NC Hyperbaric, Burgos, 2.4. Free leucine and isoleucine: extraction, identification and Spain). Three different treatments were performed at 600 MPa during quantification 6 min, each one accompanied by a different temperature: 0 °C, 20 °C and 35 °C. In order to evaluate the effect of HPP treatments, a fourth The extraction of free leucine and isoleucine was carried out ac- group of samples were not treated and used as a control batch. For cording to the procedure described by Pérez-Palacios, Ruiz, Barat, proteomic analysis, only samples from control and HPP (0 °C) groups Aristoy, and Antequera (2010) with some modifications. Briefly, five were analyzed. Samples were stored at room temperature (20 °C) for no grams of sliced-dry cured ham were homogenized with 25 mL of hy- longer than 4 weeks. drochloric acid 0.1 N, in an Ika Ultra-Turrax for 8 min under re- frigerated conditions by submerging the extract in ice. Afterwards, the 2.3. Proteomic analysis solution was centrifuged at 5240g for 20 min at 4 °C and the super- natant layer was filtered through glass wool prior to further analyses. 2.3.1. Protein extraction and two-dimensional electrophoresis (2-DE) Two hundred ml of this extract was deproteinized by adding 800 mL of A comparison between control (without HPP treatment) and HPP acetonitrile and centrifuged for 3 min at 5240g. (0 °C) batch was carried out using four biological replicas in each case. The derivatization, identification and quantification were carried It is well known that 2-DE maps of porcine muscles were used to detect out according to Franco and Lorenzo (2014). The derivatization and
636 M. López-Pedrouso et al. Food Research International 122 (2019) 635–642
Fig. 1. 2-DE gel images obtained from sliced dry-cured ham samples after a standard process (left) and a HPP treatment at 0 °C (right). Spots with significant differences (P < .05) by effect of pressure treatment are indicated and numbered. These spots were analyzed by MALDI-TOF/TOF MS. chromatographic analysis conditions were as follows: 10 μL sample was comparing their mass spectra and retention time with authentic stan- buffered to pH 8.8 (AccQFluor borate buffer) to yield a total volume of dards (Supelco, Bellefonte, PA, USA) and c) calculating the retention
100 μL. The derivatization reaction was initiated by the addition of index relative to a series of standard alkanes (C5-C14) (for calculating 20 μL AccQ-Fluor reagent (3 mg/mL in acetonitrile). The chromato- Kovats indexes, Supelco 44,585-U, Bellefonte, PA, USA). Results were graphic separation was carried out in a Waters AccQ-Tag column expressed as area units (AU) × 103/g of dry matter. (3.9 mm × 150 mm with a 4 μm of particle size) with a flow rate of 1.0 mL/min at 37 °C, using a HPLC (Alliance model 2695, Waters, 2.6. Statistical analysis Milford, MA, USA) equipped with a 2475 scanning fluorescence de- tector. The detection of leucine and isoleucine was accomplished by For statistical evaluation of data, the IBM SPSS Statistics 23.0 pro- fl uorescence with excitation at 250 nm and emission at 395 nm. Both gram (IBM Corporation, Somers, NY, USA) was used. To select spots for fi amino acids were identi ed by retention time using an amino acid mass spectrometry analysis, spot volumes were analyzed by Mann- ™ standard. The Empower 2 advanced software was employed to control Whitney test. The fold change (FC) and relative change (RC) were used system operation and results management. to assess the changes of the spot volume after high-pressure treatment
compared to controls. The fold change (FC) is given by FC=VHP/Vcontrol 2.5. Aldehydes: extraction, identification and quantification where VHP and Vcontrol were the average spot volume in control group and group after a high-pressure treatment, respectively. If the FC values The extraction of aldehydes compounds was performed according to were less than one, they were represented as their negative reciprocal. │ │ methodology proposed by Lorenzo, Gómez, Purriños, and Fonseca The equation used to calculate RC was RC = DV/ DVmax where (2016). A solid-phase microextraction (Supelco, Bellefonte, PA, USA) DV = VHP-Vcontrol and DVmax was the maximum observed value of DV. ff composed by fused-silica fiber (10 mm length) coated with a 50/30 μm To assess the e ect of HPP on leucine, isoleucine and branched and thickness of DVB/CAR/PDMS (divinylbenzene/carboxen/poly- linear aldehydes, the four groups of samples were analyzed (control vs. dimethylsilox-ane) was used for aldehydes extraction. One gram of three HPP treatments) using an analysis of variance (ANOVA) of one ff fi sliced dry-cured ham was weighted into a 40 mL vial and then the vial way. When the HPP e ect was signi cant (P < .05), the least squares fi was screw-capped with a laminated teflon-rubber disk. The fiber means were separated using Duncan's t-test at the 95% of con dence (SPME) was introduced into the sample vial through the septum and level. exposed to headspace. Before determination, the fiber was conditioned by heating it into a gas chromatograph injection port at 270 °C for 3. Results and discussion 60 min, according to specifications manufacturer. Aldehyde extraction was carried out in an oven (35 °C for 30 min) to ensure a homogeneous 3.1. Proteomic analysis by 2-DE and mass spectrometry temperature for the sample and headspace. Prior to extraction samples were maintained for 15 min at the extraction temperature. Once ex- A comparison between a control batch and HPP batch (600 MPa, traction was finished, the fiber was withdrawn into the needle and 6 min) at 0 °C was carried out to differentiate the heating caused by HPP transferred to the injection port of the gas chromatograph effect. In the Fig. 1, representative 2-DE proteome images obtained (6890 N)–mass spectrometer detector (5973 N) system (Agilent Tech- from control and HPP samples are shown. The average number of nologies Spain, S.L., Madrid, Spain). A DB-624 capillary column (J&W protein spots detected was 116 and 123 in control and HPP samples, scientific: 30 m, 0.25 mm id, 1.4 μm film thickness) was used to alde- respectively. Each protein spot detected was matched using PDQuest hydes separation. The SPME fiber was desorbed and maintained in the software for the biological replicates. In addition, those gel spots with injection port at 260 °C for 5 min. Sample injection was in split-less significant changes in abundance were marked and numbered on the mode, using Helium as a carrier gas with a linear velocity of 40 cm/s. A image (Fig. 1). In particular, eighteen spots with significantly differ- gradient of temperature was as follow: initially isothermal at 40 °C for ential abundance were found, resulting nine spots with significantly 10 min, then raised to 200 °C at a rate of 5 °C/min, further raised to qualitative differences (spot no. 2, 3, 4, 12, 13, 14, 16, 17 and 18) and 250 °C at a rate of 20 °C/min, and finally held at 250 °C for 5 min: total other nine spots with quantitative differences (spot no. 1, 5, 6, 7, 8, 9, runtime 49.5 min. Injector and detector temperatures were both set at 10, 11 and 15). However, it must be emphasized that the majority of 260 °C. The mass spectra were obtained using a mass selective detector these spots with qualitative changes were only present in HPP treat- working in electronic impact at 70 eV, with a multiplier voltage of ment (8 spots of 9 spots). Consequently, it appears clear to assume that 1953 V and collecting data at a rate of 6.34 scans/s over the range m/z proteomic profiles of control and HPP treated samples were highly 40–300. Aldehydes were identified using three methods: a) comparing differentiated. Previous studies of sliced dry-cured ham have shown their mass spectra with those contained in the NIST14 library, b) that a higher number of spots detected on 2-DE gel were correlated to a
637 M. López-Pedrouso et al. Food Research International 122 (2019) 635–642 higher proteolysis index (López-Pedrouso et al., 2018). In the present It has been reported that the actomyosin complex suffers a greater work, a higher number of spots were found in HPP samples, indicating degradation dry-cured ham during processing. Particularly, it has been more proteolytic degradation. This fact would suggest further proteo- described as an extensive degradation of myosin heavy chain while lysis caused by the effect of pressure matching those observed in earlier actin is remarkably more stable at this high ionic condition (Fabbro studies (Ma & Ledward, 2004; Rakotondramavo, Rabesona, Brou, de et al., 2016; Wang, Zhang, Li, Shen, & Zhang, 2017). Furthermore, a Lamballerie, & Pottier, 2018). Overall, the curing step causes proteo- heating process causes the rupture of hydrogen bonds of proteins, in lysis whereas brining and tumbling mainly contribute to denaturation contrast to HPP treatment, which affects the hydrophobic and electro- of the proteins. On the other hand, it has been reported that the HPP static interactions (Duranton, Simonin, Chéret, Guillou, & de treatment lead to greater protein denaturation and oxidation causing Lamballerie, 2012). For this reason, the proteomic analysis was carried aggregation, gelation or even increasing enzymatic activity. Although out at 0 °C to avoid the temperature effect. In addition, the high ionic both proteomes must include a high level of proteins breakdown, the strength conditions during dry-cured ham process could change the HPP treatment induced an increase in the proteolysis of dry-cured ham myosin light chain conformation affecting the binding of myosin with resulting in a higher number of spots. During the HPP process, the non- actin through to their phosphorylation and dephosphorylation. In saline covalent interactions in the tertiary (hydrophobic and ionic interac- conditions, actin shows a high degree of phosphorylation improving its tions) structure are weakened (Kaur et al., 2016). This change in pro- stability against μ-calpain (Wang et al., 2017). This suggests that salt tein structure could causes a partial unfolding of protein enabling to curing of dry-cured ham produced an intense degradation of myosin enable hydrophobic interactions between proteins leading to their ag- that at the same time was unaffected by HPP. Regarding actin, the salty gregation or could give rise to a higher proteolysis as the analysis conditions should make this protein more stable, but the HPP induces suggested. their fragmentation, confirmed by the presence of some actin fragments The eighteen differentially abundant spots were excised for further on the gel. According to Kęska and Stadnik (2017), myofibrillar pro- identification using MALDI TOF/TOF MS. Fourteen spots were identi- teins, specifically myosin-2, are precursors of peptides and amino acids, fied with a high Mascot score (> 60) as indicated in Table 1. Among the which have a strong impact on the taste of dry-cured meat, while sar- identified spots, ten were actin, which is an important fibrillar protein coplasmic proteins had not incidence on taste-active components gen- in meat, representing approximately 13% of the total muscle protein in eration. two forms G, and F-actin, being G-actin a monomer of F-actin (Appell, On the other hand, two spots were identified as triosephosphate Hurst, & Finley, 2018). It is important to highlight that actin spots isomerase with a total RC of approximately 0.31. Triosephosphate detected in 2-DE gel had different molecular weights ranging from 41.1 isomerase is an enzyme, which increases the glycolytic metabolism and to 14.1 KDa (Table 1). However, the actin isoform detected in this study produces NADH and ATP, consequently it is strongly correlated with has a theoretical molecular weight of around 42.0 KDa according to meat quality (Gagaoua, Monteils, Couvreur, & Picard, 2017; Kim & Uniprot database. This difference between theoretical and experimental Dang, 2005; Schilling et al., 2017). In this proteomic study, spot mass of actin spots can be explained by their different degree of frag- number 1 identified as triosephosphate isomerase has an experimental mentation after a HPP treatment. The total RC value of actins was 2.51 mass similar to theoretical mass which would indicate that the protein indicating us entire or fragmented actins are most abundant in HPP is entire. On the contrary, the spot number 15 could suggest that there samples (Table 2). On the other hand, the most abundant protein in the were protein aggregates even under reducing conditions due to the animal muscle (around 38%) is myosin. The myosin protein consists of theoretical mass was lower than experimental mass as in other treat- two subunits with very different weights, called myosin light chain and ments occurred previously (Di Luccia et al., 2015; López-Pedrouso heavy chain (Appell et al., 2018). In this comparative proteomic ana- et al., 2018). lysis, there was only a single myosin spot found in HPP samples and fi ff identi ed as myosin heavy chain. In addition, the di erence between 3.2. Levels of leucine and isoleucine affected by HPP treatment theoretical and experimental mass of myosin spot (223.3 kDa vs. 38.3 kDa) was very high and therefore it reasonable to suppose that As hydrolysis of proteins by enzyme action and chemical reaction spot number 4 is a myosin fragment. However, the presence of actin release free amino acids, their quantification can be used as an indirect was more relevant than myosin based on the comparison of their RC way to quantify proteolysis. Among free amino acids, leucine and iso- (2.51 vs. 0.05) (Table 2). leucine have a great importance because of their high concentration
Table 1 Identification of selected protein spots by MALDI-TOF/TOF.
Spot no.a Protein descriptionb Protein Mascot Sequence coverage Number of matched Theoretical mass Experimental mass (2-DE gel, accessionb score (%) peptides (UniProt, KDa)c KDa)d
1 Triosephosphate isomerase TPI1 87 29 6 26.7 41.1 2 Alpha actin 1 ACTC1 93 9 3 42.0 41.1 3 Alpha actin 1 ACTC1 108 17 5 42.0 40.0 4 Myosin-7 MYH7 131 7 13 223.3 38.3 5 Alpha actin 1 ACTC1 340 24 9 42.0 38.4 6 Alpha actin 1 ACTC1 166 16 5 42.0 37.6 7 Hemoglobin subunit beta HBB 91 15 2 16.2 37.7 9 Alpha actin 1 ACTC1 378 24 8 42.0 30.0 10 Alpha actin 1 ACTC1 119 30 11 42.0 30.0 11 Alpha actin 1 ACTC1 154 9 3 42.0 30.0 12 Alpha actin 1 ACTC1 90 23 8 42.0 29.8 14 Alpha actin 1 ACTC1 172 18 4 42.0 27.5 15 Triosephosphate isomerase TPI1 79 30 5 26.7 26.0 17 Alpha actin ACTC1 85 17 4 42.0 14.1
a The spot numbers are shown in Fig. 1. b Sus scrofa (pig) was employed as taxonomy filter in Mascot search. c Theoretical mass was provided by UniProt database. d Experimental mass was estimated on 2-DE gels using molecular weight standards.
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Table 2 Spot volumes with significant differences by effect of a high-pressure treatment in sliced dry-cured ham. Fold change (FC) and relative change (RC) of the selected spots.
Spot no. Protein (gene name) Control HPP FC RC
Mean ± SE Mean ± SE
1 Triosephosphate isomerase (TPI1) 124.48 ± 38.76 249.53 ± 45.25 + 2.00 + 0.25 2 Alpha actin 1 (ACTC1) – 337.46 ± 152.42 + ∞ + 0.68 3 Alpha actin 1 (ACTC1) – 134.77 ± 38.14 + ∞ + 0.27 4 Myosin-7 (MYH7) – 43.11 ± 15.00 + ∞ + 0.09 5 Alpha actin 1 (ACTC1) 486.98 ± 80.85 897.29 ± 162.25 + 1.84 + 0.83 6 Alpha actin 1 (ACTC1) 340.11 ± 52.40 191.86 ± 47.54 − 1.77 − 0.30 7 Hemoglobin subunit beta (HBB) 109.81 ± 30.37 35.04 ± 10.43 − 3.13 − 0.15 8 Uncharacterized protein 179.16 ± 11.03 227.48 ± 18.14 + 1.27 + 0.10 9 Alpha actin 1 (ACTC1) 151.05 ± 20.66 574.66 ± 147.24 + 3.80 + 0.85 10 Alpha actin 1 (ACTC1) 132.30 ± 27.02 473.80 ± 110.71 + 3.58 + 0.69 11 Alpha actin 1 (ACTC1) 99.09 ± 23.00 56.07 ± 14.76 − 1.77 −0.09 12 Alpha actin 1 (ACTC1) – 411.04 ± 143.85 + ∞ + 0.83 13 Uncharacterized protein – 496.40 ± 129.27 + ∞ + 1.00 14 Alpha actin 1 (ACTC1) – 175.91 ± 82.73 + ∞ + 0.35 15 Triosephosphate isomerase (TPI1) 137.34 ± 4.72 288.90 ± 25.18 + 2.10 + 0.31 16 Uncharacterized protein – 142.94 ± 46.31 + ∞ + 0.29 17 Alpha actin 1 (ACTC1) – 269.59 ± 81.93 + ∞ + 0.54 18 Uncharacterized protein 153.73 ± 16.46 – −∞ −0.31
Average ( ± SE, Standard error) volumes were assessed from 2-DE gels using the PDQuest software.
Santaescolástica et al., 2018a). There was a significant difference (P < .05) between control sam- ples and samples after HPP treatment. As shown in the Fig. 2, it can be observed that there was significant (P < .05) differences between samples after HPP treatment at low temperatures (0 °C and 20 °C) and samples at 35 °C. In particular, the relative leucine and isoleucine content increased under HPP at low temperatures (0 °C and 20 °C) in comparison with control samples unlike 35 °C with the lowest values for both amino acids. Therefore, these findings suggest that the effect of HPP treatment on relative isoleucine and leucine content is highly de- pendent on temperature. According to Guyon et al. (2016), the high- pressure process greatly contributes more to the hydrolysis of proteins into amino acids such as leucine and isoleucine. This suggests that all HPP treatments should increase both concentrations in dry-cured ham Fig. 2. Relative leucine and isoleucine content in dry-cured ham under HPP due to protein degradation. On the other hand, Kim, Kemp, and treatment at different temperatures. Mean ± standard deviation expressed in Samuelsson (2016) have described that leucine and isoleucine are mg FAA/ mg total FAA per 100 g dry matter. Differences in letter: a to c per metabolites involved in chemical reactions forming volatiles com- fi ff each FAA represent signi cant di erences (P < .05) among treatments. pounds as aldehydes. This means an indirect way of influence in taste and aroma as mentioned above. It is possible to hypothesize that the and are classified as lipophilic amino acids. Both are related to meat higher temperatures facilitate the Strecker reaction between leucine taste and flavor directly or indirectly via for being metabolites of and isoleucine and α‑carbonyl compounds and this fact could explain Strecker aldehydes. As shown in the Fig. 2, relative leucine content was the lower content of both amino acids after HPP treatment at 35 °C. higher than isoleucine in all treatments. The average relative content of leucine ranged from 102.62 to 130.50 mg/100 g dry cured ham (d.b.) whereas isoleucine varied from 62.18 to 77.43 mg/100 g dry cured ham 3.3. Aldehyde volatile compounds affected by HPP treatment (d.b.). Indeed, leucine was the most abundant free amino acid in dry- cured ham according to Pérez-Santaescolástica et al. (2018a) and both Wide varieties of volatile compounds were detected using SPME are considered as predominant free amino acids like in the other types followed by GC–MS (Pérez-Santaescolástica et al., 2018b). Volatile of hams such as Jinhua, Xuanwei, Parma and Bama (Zhang et al., compounds are classified in chemical families, but aldehydes are a key 2018). Both amino acids have a specific tastes and high concentrations group in dry-cured ham for their abundance and important contribution consequently, it can be supposed that both directly influence the de- to taste and aroma. Their sensorial notes are fruity, toasted, oily, fatty velopment of aroma and taste. It is known that leucine together with and rancid, commonly associated with cured meats (Sánchez-Peña, glutamate, lysine, alanine and lactate are the highest contributor of Luna, García-González, & Aparicio, 2005). As shown in Table 3, the taste in dry-cured hams (Zhang et al., 2018). Specifically, leucine and HPP treatment significantly (P < .001) affected the total aldehydes isoleucine were strongly associated with bitter taste of dry-cured meat concentration resulting in the highest values in control samples products (Kęska & Stadnik, 2017). However, it has also indicated that (23,430.18 AU × 103/g of dry-cured ham). Again, the temperature the spatial arrangement of amino acids can be changed with the food fixed in each HPP treatment resulted in significant (P < .05) differ- process, resulting different tastes from the same amino acid (Solms, ences in total aldehydes between samples at low temperature (average 1969). On the other hand, it has recently reported that high levels of value of 12,331.38 AU × 103/g of dry-cured ham) and at 35 °C leucine and isoleucine were correlated with proteolysis index con- (19,687.56 AU × 103/g of dry-cured ham). Among the aldehydes de- tributing to a high adhesiveness in dry-cured hams (Pérez- termined with statistical significance, the most abundant were two branched aldehydes as 2-methyl-butanal and 3-methyl-butanal and four
639 M. López-Pedrouso et al. Food Research International 122 (2019) 635–642
Table 3 Effect of HPP at different temperatures on main branched and linear aldehydes of dry-cured ham expressed as AU × 103/g of dry-cured ham (mean ± standard deviation).
m/z LRI R Treatment P-value
CO HPP-0 HPP-20 HPP-35
2-Methyl butanal 57 671 ms, lri 863.96 ± 326.20 a 879.99 ± 427.70 a 1168.02 ± 483.10 ab 1334.05 ± 423.90 b P = .001 3-Methyl butanal 58 659 ms, lri 1736.34 ± 521.48 a 1547.91 ± 340.04 a 1878.31 ± 791.48 a 2945.25 ± 802.78 b P < .001 Pentanal 57 728 ms, lri, s 1215.3 ± 547.46 b 541.18 ± 203.79 a 594.13 ± 192.92 a 810.22 ± 323.57 a P < .001 Hexanal 56 865 ms, lri 15,717.34 ± 4194.15 c 6040.25 ± 1821.88 a 7535.70 ± 2212. a 03 11,088.96 ± 3395.96 b P < .001 Heptanal 70 974 ms, lri, s 1025.45 ± 324.52 c 384.07 ± 105.33 a 450.05 ± 163. ab 91 597.94 ± 157.35 b P < .001 Nonanal 57 1148 ms, lri, s 553.39 ± 170. 73 b 346.82 ± 74.69 a 338.68 ± 91.06 a 397.50 ± 121.93 a P < .001 Total aldheydes 23,430.18 ± 4949.22 c 11,065.57 ± 2337.37 a 13,597.19 ± 3035.38 a 19,687.56 ± 4283.05 b P < .001 a–c Mean values in the same row (corresponding to the same compound) not followed by a common letter differ significantly (P < .05). m/z: Quantification ion; LRI: Lineal Retention Index calculated for DB-624 capillary column (J&W scientific: 30 m × 0.25 mm id, 1.4 μm film thickness) installed on a gas chromatograph equipped with a mass selective detector; R: Reliability of identification; lri: linear retention index in agreement with literature (Domínguez, Gómez, Fonseca, & Lorenzo, 2014; Lorenzo, 2014; Lorenzo, Bedia, & Bañon, 2013; Lorenzo & Carballo, 2015; Lorenzo & Domínguez, 2014; Lorenzo, Montes, Purriños, & Franco, 2012; Pateiro, Franco, Carril, & Lorenzo, 2015; Pérez-Santaescolástica et al., 2018a; Pérez-Santaescolástica et al., 2018b; Purriños et al., 2011; Purriños, Carballo, & Lorenzo, 2013; Purriños, Franco, Carballo, & Lorenzo, 2012); ms: mass spectrum agreed with mass database (NIST14); s: mass spectrum and retention time identical with an authentic standard. Treatments: CO = control (without treatment); HPP-0 = High pressure treatment at 0 °C; HPP-20 = High pressure treatment at 20 °C; HPP-35 = High pressure treatment at 35 °C. linear aldehydes as pentanal, hexanal, heptanal and nonanal. content, as noted above. Furthermore, it is necessary to highlight that it All linear aldehydes showed a significant reduction (P < .05) due has been reported that the ability of 2-methyl-butanal to bind peptide to the HPP effect which means that its contribution in overall aroma extracts of dry-cured ham was too weak to be detected (Martínez- was decreased. The reductions ranged from 37.43% to 62.54% for Arellano et al., 2016). It seems that Strecker aldehydes are less reactive nonanal and heptanal, respectively. The most likely explanation for than linear aldehydes and consequently more stable. Based on these these results may be the fact that aldehydes are considered highly re- reasons, it can be concluded that Strecker aldehydes can be associated active molecules especially α- and β-unsaturated (alkenals, alkadienals, with greater proteolysis degree of dry-cured ham after HPP treatment. and hydroxyalkenals) which could react with proteins (Guyon et al., 2016). The results suggest that linear aldehydes could be reacting to a 4. Conclusions great extent in HPP conditions; therefore, their concentration decreases in comparison with samples untreated. In the case of the most abundant High pressure conditions caused a significant impact on proteome of aldehyde, the hexanal concentration diminished > 2.5 times by high- sliced dry-cured ham. The effect of HPP treatment was clearly de- pressure effect. This result is consistent with Fuentes et al. (2014) that monstrated on myofibrillar proteins such as actin which was likely indicated a decrease of hexanal level in dry-cured ham slices treated given rise to isoleucine and leucine. In addition, 2-methyl-butanal and with high pressure (600 MP). In addition, hexanal plays a key role in 3-methyl-butanal coming from both Strecker metabolites increased dry-cured products and at high concentration lead to the development their concentration due to HPP effect. Therefore, protein degradation of an unpleasant rancid odor (Carrapiso, Martín, Jurado, & García, by HPP effect can trigger a series of chain reactions depending on 2010; Lorenzo, Carballo, & Franco, 2013; Shahidi & Pegg, 1994). In- conditions (pressure and temperature) which cause a strong impact on deed, like other linear aldehydes, hexanal come from lipid oxidation by volatile compound profile. A further research needs to be done to es- breakdown products of unsaturated lipids (Petričević, Radovčić, Lukić, tablish whether all changes in the volatile profile induced by high- Listeš, & Medić, 2018). Specifically, hexanal could be formed from the pressure process will be positive for the improvement of dry-cured ham breakdown of linoleic, gamma-linolenic and arachidonic acids (Shahidi, sensorial attributes. 2001), hence is widely used as oxidation level indicator. It may, therefore, be concluded that HPP prevents the lipid oxidation, parti- Acknowledgements cularly at low temperatures. On the other hand, branched aldehydes, which are 2-methyl-butanal This research was supported by Grant RTA 2013-00030-CO3-03 and 3-methyl-butanal, are related to proteolysis and amino acids de- from INIA (Spain). Acknowledgements to INIA for granting Cristina gradation (Toldra, 1998). From a sensorial point of view, both alde- Pérez Santaescolástica with a predoctoral scholarship. José M. Lorenzo hydes are potent odorants associated with the nutty, cheesy and salty is member of the MARCARNE network, funded by CYTED (ref. sensory notes (Sánchez-Peña et al., 2005). Both aldehydes come from 116RT0503). isoleucine and leucine, via Strecker degradation, which is considered as a step within Maillard reaction. This reaction involves oxidative dea- References mination followed by decarboxylation resulting aldehydes with one carbon atom less the corresponding amino acid (Estévez et al., 2011; Appell, M., Hurst, W. J., & Finley, J. W. (2018). Amino acids and proteins. Principles of Resconi, Escudero, & Campo, 2013). The results show that HPP treat- food chemistry (pp. 117–164). Cham: Springer. Bermúdez, R., Franco, D., Carballo, J., & Lorenzo, J. M. (2014). Physicochemical changes ment at 35 °C had an impact on both aldehydes, because of their con- during manufacture and final sensory characteristics of dry-cured Celta ham. Effect of centration significantly increased (P < .05) with increments of 35.30% muscle type. Food Control, 43, 263–269. and 41.05% for 2-methyl-butanal and 3-methyl-butanal, respectively. Bermúdez, R., Franco, D., Carballo, J., Sentandreu, M.Á., & Lorenzo, J. M. (2014). fi Influence of muscle type on the evolution of free amino acids and sarcoplasmic and However, these ndings do not support previous research of Martínez- myofibrillar proteins through the manufacturing process of Celta dry-cured ham. Onandi, Rivas-Cañedo, Picon, and Nuñez (2016) who found that HPP Food Research International, 56, 226–235. treatment (600 MPa, 6 min) produced a reduction in the amount of 2- Čandek-Potokar, M., & Škrlep, M. (2012). Factors in pig production that impact the – methyl-butanal. Although these results must be interpreted with cau- quality of dry-cured ham: A review. Animal, 6(2), 327 338. Carrapiso, A. I., Martín, L., Jurado, Á., & García, C. (2010). Characterisation of the most tion because they are influenced by water activity and intramuscular fat odour-active compounds of bone tainted dry-cured Iberian ham. Meat Science, 85(1),
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Contents lists available at ScienceDirect
Trends in Food Science & Technology
journal homepage: www.elsevier.com/locate/tifs
Review Application of non-invasive technologies in dry-cured ham: An overview T Cristina Pérez-Santaescolásticaa, Ilse Fraeyeb, Francisco J. Barbac, Belen Gómeza, ∗ Igor Tomasevicd, Alberto Romeroe, Andrés Morenof, Fidel Toldrág, Jose M. Lorenzoa, a Centro Tecnológico de la Carne de Galicia, rúa Galicia n° 4, Parque Tecnológico de Galicia, San Cibrao das Viñas, 32900, Ourense, Spain b KU Leuven Technology Campus Ghent, Leuven Food Science and Nutrition Research Centre (LFoRCe), Research Group for Technology and Quality of Animal Products, Gebroeders De Smetstraat 1, BE-9000, Gent, Belgium c Nutrition and Food Science Area, Preventive Medicine and Public Health, Food Sciences, Toxicology and Forensic Medicine Department, Faculty of Pharmacy, Universitat de València, Avda. Vicent Andrés Estellés, s/n, 46100, Burjassot, València, Spain d Department of Animal Source Food Technology, University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080, Belgrade, Serbia e Departamento de Ingeniería Química, Universidad de Sevilla, Facultad de Física, Sevilla, 41012, Spain f University of Castilla-La Mancha, Faculty of Chemical Sciences and Technologies (San Alberto Magno Building), Department of Organic Chemistry, Av. Camilo José Cela, 10, Ciudad Real, 13071, Spain g Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Avenue Agustín Escardino 7, 46980, Paterna, Valencia, Spain
ARTICLE INFO ABSTRACT
Keywords: Background: Dry-cured ham is one of the most valued food products by Mediterranean consumers. In this sense, Dry-cured ham the appropriate development of its different production stages is essential to ensure the quality requirements. For Non-invasive technologies this reason, non-invasive technologies have gained popularity and have been reported as useful not only to High pressure ensure the food safety of different products, but also to monitor fundamental stages in the production process, Ultrasound such as the salting stage, to analyze the content of different compounds without sample losses, and to correct Infrared spectroscopy possible defects in the final product. Magnetic resonance Pulse electric fields Scope and approach: This work has been focused on summarizing the studies that describe and have successfully Time domain reflectometry microwave applied these techniques, as well as on mentioning other technologies with potential use in dry-cured ham Data mining manufacture which have not been studied enough. Finally, the potential next steps to improve and optimize the Laser backscattering imaging process, as well as the suitability of creating new products with added value based on the new quality standards, have also been evaluated. Key findings and conclusions: Innovative non-invasive technologies such as high pressure (HP), ultrasound (US), pulsed electric fields (PEF), microwaves, irradiation, etc. can be used as promising tools to effectively control salting and curing stages as well as for checking defects of the final product and/or ensuring food safety. HP and US are useful tools for the determination of salt and fat content, and for monitoring the salting process. Moreover, HP enhances salty taste perception, which makes it a useful tool to reduce salt addition. Both, HP and US, can correct texture defects. In addition, NIRS allows predicting the state of the meat to remove those pieces that could result in defective products. Moreover, RAMAN or MRI are able to detect anomalous textures at the end of the process. Microwaves could be useful for the online estimation of salt, water and fat contents easily with portable equipment. Finally, data mining, that allows to make predictions based on an immense data file, is the most promising discovery in recent years for detecting defects or classifying products according to sensory attributes.
1. Introduction lengthy dry-ageing stages (Bermúdez, Franco, Franco, Carballo, & Lorenzo, 2012). Throughout the process, many lipid and protein con- Dry-cured ham is a highly consumed product around the world, version reactions take place in the raw meat, giving rise to the parti- whose organoleptic characteristics are strongly valued by consumers. cular flavour of dry-cured ham (Bermúdez, Franco, Carballo, & Lorenzo, Its production process consists of salting the entire back leg of the pig in 2015)(Fig. 1). In this context, in order to get a tasty and safe product, a order to stabilize the raw meat by reducing water availability, followed good control of all process stages is absolutely necessary. For example, by a dry-curing process which includes post-salting, pre-drying and manufacturing temperature greatly affects lipolysis reactions (Ripollés,
∗ Corresponding author. E-mail address: [email protected] (J.M. Lorenzo). https://doi.org/10.1016/j.tifs.2019.02.011 Received 29 November 2018; Received in revised form 29 January 2019; Accepted 6 February 2019 Available online 08 February 2019 0924-2244/ © 2019 Elsevier Ltd. All rights reserved. C. Pérez-Santaescolástica, et al. Trends in Food Science & Technology 86 (2019) 360–374
Fig. 1. Dry cured ham elaboration process, characteristics of the product and possible alterations.
Campagnol, Armenteros, Aristoy, & Toldrá, 2011), and salt content and 2015a; 2015b), many studies in dry-cured ham have focused on re- water activity (aw) play an important role in proteolysis reactions and duction of NaCl content and its substitution by other salts (Armenteros, microbial spoilage (Bermúdez, Franco, Carballo, Sentandreu, & Aristoy, Barat, & Toldrá, 2011). However, NaCl reduction implies a Lorenzo, 2014a, 2014b; Purriños, García Fontán, Carballo, & Lorenzo, longer post-salting time to reduce water activity levels, assuring mi- 2013). In addition, salt is involved in textural functions, organoleptic crobial stability (Harkouss, Chevarin, Daudin, Sicard, & Mirade, 2018). characteristics and water holding capacity (WHC) (Taormina, 2010). On the other hand, evaluation of physicochemical characteristics along The salting stage is one of the longest steps in dry-cured ham produc- the entire process is mostly done through traditional methods. These tion, due to the high resistance of cell membranes to mass transfer methods are often lengthy, arduous and, above all, in many cases cause (Janositz, Noack, & Knorr, 2011), extending the process time. While the product losses (Pérez-Palacios, Caballero, Caro, Rodríguez, & use of salt is fundamental from a technological point of view, the high Antequera, 2014). amount that is incorporated turns dry-cured ham into a high salt con- To solve the current drawbacks in dry-cured ham production, and to tent product. It is well known that high salt intake is related with hy- be able to optimize analytical procedures, non-invasive technologies pertension, stroke risk, different types of cancers and osteoporosis have been the object of several studies. In this way, the monitoring and owing to a high level of calcium excretion (Karppanen & Mervaala, characterization of ham during the salting stage facilitate the salt re-
2006). As a result, during the last years, new consumer requirements duction (Fulladosa, Muñoz, Serra, Arnau, & Gou, 2015a). Likewise, aw are being established, increasing the need to improve processing tech- monitoring allows to assess the final product safety (Santos-Garcés, niques and to create new products with added value based on the new Gou, Garcia-Gil, Arnau, & Fulladosa, 2010). Furthermore, non-invasive quality standards. technologies such as near infrared or microwave spectroscopy can be Regarding lifestyle changes, one of the manufacturing strategies is used to determine textural characteristics of the product (Damez & the development of ready-to-eat (RTE) foods. In this regard, slices of Clerjon, 2013; Fulladosa et al., 2018; García-Rey, García-Olmo, De dry-cured ham are commercialized in RTE-vacuum packages (Jin et al., Pedro, Quiles-Zafra, & de Castro, 2005; Ortiz, Sarabia, García-Rey, & de 2012). Despite the great ease for consumers, this product format in- Castro, 2006; Rubio-Celorio, Fulladosa, Garcia-Gil, & Bertram, 2016). volves more handling, increasing the microbial contamination risks (Jin The objective of the present review is to summarise and update on the et al., 2012; Sánchez-Molinero, García-Regueiro, & Arnau, 2010). Fur- use of non-invasive technologies, and their potential applications, in the thermore, since products with low sodium content are increasingly at- processing of dry-cured ham. tractive to consumers and demanded for healthy reasons (Lorenzo et al.,
361 .PrzSnasoátc,e al. et Pérez-Santaescolástica, C.
Table 1 Summary of non-invasive technologies used in dry-cured hams sorted by their fundament and their potential applications.
Technology Working principle Alternatives Product application Usefulness
Irradiation techniques Technology based on the application of sources, like alpha, beta, gamma rays or X-rays X-Ray simple To sterilize Processing that have enough energy to ionize the matter, extracting electrons which are linked to the X-Ray multi energy detector Analytical atom. Gamma irradiation Electron Bean Irradiated Computed Tomography (CT) To monitor process To model and to estimate components Magnetic Resonance Imaging Absorption spectroscopy based in the absorption of energy (radio frequencies) by a Proton-based magnetic resonance imaging (1H-MRI) To monitor process Analytical (MRI) magnetically active nucleus, which is oriented within a magnetic field, changing its Sodium-based magnetic resonance imaging (23Na- To predict salt and fat content orientation as a result of that energy. MRI) by one or two dimensional analysis To predict muscle volumes To identify feeding backgrounds Infrared spectroscopy Absorption of infrared radiation by molecules in vibration, which begin to vibrate in a Near infrared imaging (NIRS) To predict sensory attributes Analytical certain way thanks to the energy supplied by infrared light. Near infrared hyperspectral imaging (hyperspectral and components eNIRS)
362 Visible-Near infrared imaging (Visible eNIRS) To model and to monitor RAMAN process High Pressure (HP) Application of instantaneous and uniform high pressure High Pressure (HP) To reduce microbial Processing population To correct sensory attributes Ultrasound It is based on the difference of speed of an ultrasonic wave when crossing different Through-transmission ultrasound To sterilize Processing materials. Pulse-echo mode To monitor process Analytical Air-coupled ultrasound To predict water, fat and salt contents Scanning acoustic microscopy To correct texture defects Pulse electric fields (PEF) Application of high voltage pulses for short periods of time to a food, which is placed in Pulse electric fields (PEF) To accelerate salting Processing an electrolytic solution, between two electrodes. Electromagnetic waves Based on the electrical properties of a flow of photons when go through a medium Microwave spectrometry To predict components Analytical Time Domain Reflectometry Trends inFoodScience&Technology86(2019)360–374 Magnetic Induction Laser Backscattering Imaging Based on the obtaining of absorption, transmission and reflectance by the passage of light Laser Backscattering Imaging To predict sensory attributes Analytical at different wavelengths through the product and components To monitor process To detect defects Data mining Based on exploring and analyzing a high range of data sets Data mining To predict components Analytical To classify in function of sensorial traits C. Pérez-Santaescolástica, et al. Trends in Food Science & Technology 86 (2019) 360–374
2. New technologies ham samples (De Prados et al., 2015; Fulladosa et al., 2015a, 2015b). In addition, it uses multi energy sensor for predicting salt contents There are some non-destructive techniques that have been eval- (Fulladosa, Gou, & Muñoz, 2016) or making textural measures uated widely with regard to their implementation in the dry-cured ham (Fulladosa et al., 2018). Unfortunately, composition identifications are manufacture, while very little research has been performed on others. bound to error due to the fact that X-Ray technology is influenced by The techniques that are discussed in this review are summarised in the density of the object. In this sense, dense objects produce more Table 1 along with their respective working principles, current subtypes attenuation, but also variations in thickness can result in different at- and the main potential applications. tenuations (Fulladosa et al., 2015b). Moreover, increments in attenua- − tion has been observed in the presence of salt, since Na+ and Cl ions 2.1. Irradiation technologies are denser than the inorganic substances present in the medium (Fulladosa, Santos-Garcés, Picouet, & Gou, 2010; Håseth et al., 2008). Irradiation technologies are based on attenuation variations which These attenuation increments were mainly observed at low energy ra- are caused by radiation passing through different materials influenced diations (Kalender, 2000, p. 220). For that, Fulladosa et al. (2015b) by energy (Farkas, 2006). Even though high efficacy on microorganism concluded that the salt content could only be predicted by using 50 kV. control was shown (Jin et al., 2012; Kong et al., 2017), consumers as- However, variations in part of the obtained data were shown, which sociate irradiated products with quality reductions (Lee, Love, & Ahn, could be caused by the fact that the water content influences sample 2004). However, irradiated food is permitted in a wide range of densities to a large extent. The same energy was tested as effective countries (Jin et al., 2012) because it is declared completely reliable method to predict fat contents (Table 2), and it is possible to improve and, in meat products, this technique has been considered as one of the the resulting predictions by adding the information obtained using most effective preservation procedures (Alfaia et al., 2007; Kong et al., 70 kV. In this regard, De Prados et al. (2015) considered the best po- 2017). The purpose of its application is based on the absorbed radiation sitively correlated conditions at 90 kV and 4 mA (R2 = 0.57) and at dose (Farkas, 2006). In this regard, while doses below 1 kGy are used to 70 kV and 8 mA (R2 = 0.53). Lower attenuations were shown at 80, 110 delay ripening, inhibit germination and kill insects, doses between 1 and 140 kV in samples in which proteolysis induction times were ex- and 10 kGy are applied for spoilage reduction and microbial control in tended (Fulladosa et al., 2018). The reason for that could be the protein fresh products. degradation and the associated induction of changes in cell structure, Among the irradiation techniques, gamma irradiation, electron originating from modifications in the X-Ray scattering (Fulladosa et al., beam generators and X-Ray accelerators are the most commonly in- 2017, 2018; Hoban et al., 2016). vestigated. The interest of researchers for these techniques began a long On the other hand, computed tomography (CT) also employs X-Ray time ago. The first evidence was in 1895 when a German Physics for irradiations. The scanning parameters (voltage, energy, slice thick- Professor, called Wilhelm Conrad, discovered X-Rays. Initially, the in- ness, etc.) affect the quality of images, for example noise or low re- terest in this technique related mainly to medical purposes. After that, solution can be reduced by the use of a higher radiation dose (Goldman, the number of studies focusing on the application of these promising 2007). CT equipment loses part of the X-Ray energy when the body technologies increased and later on, the first implementations in food tissues interact with them, depending on differences in the tissue den- manufacture were established: grain disinfestation in 1980 in the Soviet sities and was thus mainly used to predict pigs body composition and to Union, poultry meat decontamination in 1991 in France, fish products evaluate the content and distribution of salt in meat (Frøystein, sterilization in Belgium and France, fermented sausage sanitisation in Sørheim, Berg, & Dalen, 1989; Sørheim & Berg, 1987; Vestergaard, Thailand, space foods sterilization in the USA and frozen meals in South Risum, & Adler-Nissen, 2005). The first studies in dry-cured ham were Africa (Farkas, 2006). performed by Sørheim and Berg (1987) who demonstrated the viability In spite of the great potential and the wide range of research about of CT for studying the salting process, and Frøystein et al. (1989) who the use of gamma and energy electron beams irradiation, very few reported the effect of freezing on salt penetration. As in previous cases, studies focused on their application to dry-cured ham. Only two pub- because of the high costs and the low technical development, the lications were found in the literature (Table 2). Jin et al. (2012) eval- technique began to be studied in depth only a few years ago uated the effects on the physicochemical and sensory characteristics of (Vestergaard et al., 2005). Finally, CT was declared useful in curing absorbed doses of gamma irradiations between 2.5 and 10 kGy, since processes, mainly to monitor the salting and post-salting steps, con- the effectiveness of 10 kGy and lower doses were previously probed for sidering that the addition of NaCl, due to its high density, increases X- microbial reductions (FSIS, 1999). The study confirmed its potential use Ray attenuation, facilitating salt monitoring (Fulladosa et al., 2010; and reported that doses below 5 kGy could maintain the sensorial Håseth et al., 2008). The first published calibration model for pre- properties intact, while higher doses result in colour changes, lipid dicting salt contents in ham was developed by Håseth, Egelandsdal, oxidation and the increase of off-flavours. Bjerke, and Sørheim (2007). The obtained prediction error at 130 kV Regarding electron beam irradiation, although there is little in- was 0.8% NaCl; this precision was improved one year later by opti- formation available, it is important to note that Kong et al. (2017) mizing the tube voltage (Håseth et al., 2008) concluding that the most demonstrated that this irradiation maintains sensory properties better useful voltage to predict salt content, according to later studies than gamma irradiation. It is well known that lipid oxidation is the (Fulladosa et al., 2010), was 80 kV, or even better, a combination of 80 major consequence of irradiation and its effects on nutritional value and and 120 kV. In addition, it was shown that a voltage of 120 kV is also sensory characteristics are meat colour fading and off-odour production the best option for the prediction of water content. Furthermore, it was (Alfaia et al., 2007). In this regard, Kong et al. (2017) carried out an observed that both fat and water content had a great influence on the evaluation of both technologies (irradiated by electron beam and model precisions (Fulladosa et al., 2010). Thus, the models could be gamma rays) focusing on their effects on volatile compounds and, in unfeasible when there are areas with fat contents above 30% on dry contrast to previous evidence in meat products, no differences were matter of dry-cured ham or when the measurement is made at the end shown in aldehyde and ketone contents between control and both ir- of the process (Fulladosa et al., 2010; Santos-Garcés et al., 2010). radiated hams. Therefore, the main odour modifications were not CT imaging applications have been extended, through the genera- considered to be originated from lipid oxidation. tion of models for salt and water contents in different stages along the On the other hand, while electron accelerating equipment produces process, for characterizing and optimizing the dry-cured ham process high energy electron beams, X-Ray technology turns electron energy (Santos-Garcés, Muñoz, Gou, Sala, & Fulladosa, 2011; Håseth, Sørheim, into electromagnetic X-Rays (Farkas, 2006). This latest technology uses Høy, & Egelandsdal, 2012). Additionally, it has been demonstrated that single or dual absorptiometry to analyze the composition of dry-cured CT scanning can be used for detecting unsuitable practices in the dry-
363 C. Pérez-Santaescolástica, et al. Trends in Food Science & Technology 86 (2019) 360–374
Table 2 Gamma, electron beam and X-Ray irradiation-based technologies and their applications across the dry-cured ham process sorted by method parameters.
Technology Measure condition Purpose of the research References
Gamma irradiation 0, 2.5, 5.0, 7.5, and 10 kGy To determine the effects on physicochemical and sensory Jin et al. (2012) properties Gamma irradiation 3 and 6 kGy To eliminate pathogens and extend shelf life Kong et al. (2017) Electron beam irradiation 3 and 6 kGy To eliminate pathogens and extend shelf life Kong et al. (2017)
Voltage Intensity Sample thickness
X-Ray 90 kV 4 mA Whole piece To predict salt content in bone-in hams after salting Fulladosa et al. (2015a) 70 kV 8 mA 76.3 ± 3.5 mm To predict salt and fat content Fulladosa et al. (2015b) 50 kV 15 mA Whole piece To predict fat content for industrial classification purposes De Prados et al. (2015) X-Ray multi energy 50 keV 1.5 mA Variable (between 1.2 and To determine changes of the energy spectra, energy bands Fulladosa et al. (2016) detector 80 keV 1.7 mA 9 mm) and ratios due to salt addition 140 kV 1 mA 20 mm To characterize and classification commercial samples Fulladosa et al. (2018) 110 kV 1.5 mA according to their proteolysis index or defective texture 80 kV 2.8 mA level Computed tomography 130 kV 10 mm To monitor salt penetration Vestegaard et al. (2005) (CT) 80 kV 106 mA 5 mm To determine salt content Håseth et al. (2007) 110 kV 2 mm To determine effects of CT scanner parameters on CT values variations 130 kV 10 mm To determine optimal tube voltage settings and modeling Håseth et al. (2008) conditions for NaCl prediction at different production stages 80 kV 250 mA 10 mm To predict water and salt transfers. Fulladosa et al. (2010) 120 kV To predict water and salt transfers. Santos-Garcés et al. (2010) 140 kV To estimate the effect of freezing and thawing treatment, Picouet, Gou, Fulladosa, Santos- before salting, on the apparent NaCl diffusion Garcés, and Arnau (2013) 140 kV 145 mA 10 mm To estimate lean content for further drying processing Picouet, Teran, Gispert, and i Furnols (2010) 3 GHz Unknown To predict water and salt transfers, water activity and Harkouss et al. (2018) proteolysis during the early stages of the process cured ham manufacture process by determining the relationship be- Rodriguez, Cernadas, & Ruiz, 2003) or on the type of muscle tween the salt distribution and the physical characteristics of the pro- (Antequera, Caro, Rodríguez, & Pérez, 2007; Caro, Durán, Rodríguez, duct (Vestergaard et al., 2005). Moreover, Harkouss et al. (2018) in- Antequera, & Palacios, 2003). The use of MRI to determine the content dicated that CT models can be used to monitor the post-salting stage in and distribution of intramuscular fat is very important to improve the dry-cured hams with reduced salt content in order to evaluate the time dry-cured ham process, since penetration of salt and water loss are very required to obtain the same characteristics in the final product. influenced by the fat content (Ripollés et al., 2011). As shown in Table 3, water content and salt diffusion are the most studied properties among the MRI applications. Fantazzini et al. (2005) observed a good 2.2. Magnetic resonance imaging correlation between the experimental salt-to-moisture ratio (S/M) and the ratio obtained by MRI, allowing the non-invasive control of mi- Magnetic resonance imaging (MRI) is based on the knowledge that crobial populations during the process. Texture analysis based on MRI fi certain atomic nuclei absorb energy when subjected to a magnetic eld confirmed the advantage of applying MRI technology in dry-cured hams and stimulated by radio waves with appropriate frequency. The atomic in order to differentiate between pigs fed diets based on acorn and grass nuclei release the absorbed energy (resonance signal) when the mag- and diets based on concentrates of oleic acid (Pérez-Palacios et al., fi netic eld ceases. This energy is received and analyzed and, in this 2010b, 2011). As a result of these studies, it was noted that the biggest sense, MRI allows to observe the composition inside bodies and this is difference in texture characteristics was shown in semimembranosus why medical science frequently uses it for diagnoses. In the case of food (SM) muscles due to their higher sensibility to the ripening process. In science, the use of this technique is not so extended even though some this regard, Antequera et al. (2007) used MRI combined with an Active reviews on its possibilities published in the 1990s. There was some Contour to observe and identify changes in muscle volumes during ri- research about food morphology characterization to identify defects pening. These results showed high correlations between the informa- ’ (Ciampa, Dell Abate, Masetti, Valentini, & Sequi, 2010) and some re- tion from joint MRI and Active Contour and, on the other hand, weight lated to optimised treatments in food processing (Bouhrara et al., and water content, obtaining better results for biceps femoris (BF) 2011). In the case of meat products, the application of MRI was eval- muscle than SM, since this muscle is an internal muscle and its ex- uated as a method to analyze the lipid distribution (Beauvallet & Renou, traction is more difficult, generating more variability in the data. 1992), to characterize muscle structure (Bonny et al., 2001) and to Additionally, aw, soluble solids and salt contents were characterized quantify muscle and subcutaneous and intermuscular fat contents by MRI to monitor the process not only in SM and BF muscles, but also (Monziols et al., 2006). Furthermore, MRI is used to check the water in semitendinosus (ST) and rectus femoris (RF), finding a similar corre- distribution along meat processing steps such as a freeze-thawing lation as the one observed in BF (Manzocco et al., 2013). Besides that, (Guiheneuf, Parker, Tessier, & Hall, 1997) or drying (Ruiz-Cabrera, Pérez-Palacios et al. (2011) focused on the prediction of sensory Gou, Foucat, Renou, & Daudin, 2004), and to quantify changes in the properties by fat and texture features characterization through MRI and moisture and structure of cooked chicken meat (Shaarani, Nott, & Hall, they reported that fatty acids could participate in the hardness of the fat 2006). Furthermore, this technology, in combination with computer and lean, flavour intensity, brightness and juiciness, while texture vision recognition techniques, could be useful for the evaluation of features can define the marbling, the intensity of the odour and flavour fi quality characteristics in pork products, for instance, for a classi cation and the redness resulting at the end of the curing process. On the other based on feed (Pérez-Palacios, Ruiz, Tejeda, & Antequera, 2009), on hand, the use of 23Na isotope for MRI analysis was investigated since it intramuscular fat content, on sensory properties (Antequera, Muriel,
364 C. Pérez-Santaescolástica, et al. Trends in Food Science & Technology 86 (2019) 360–374
Table 3 Magnetic resonance imaging technology applications across the dry-cured ham process sorted by method parameters.
Technology Measure conditiona Purpose of the research Reference
FOV ET TR Section
Non specified-Magnetic resonance 120 × 85 mm 20 ms 500 ms 2.0 mm To predict lipid content, sensory Pérez-Palacios et al. (2010a) imaging (MRI) traits and pigs fed. Pérez-Palacios et al. (2010b) To discriminate different feeding Pérez-Palacios et al. (2011) backgrounds Unknown Unknown 2.0 mm To characterize fat content and Caro et al. (2003) distribution Proton-based magnetic resonance 390 × 390 mm 22 ms 567 ms 4.0 mm To estimate salt content in Manzocco et al. (2013) imaging (1H-MRI) 134 ms 7520 ms different stages 120 × 85 mm 20 ms 500 ms 2.0 mm To determine BF and SM volume Antequera et al. (2007) during the ripening process 180 × 180 mm 10, 14, 20, 50, 80, 130, 3000 ms 100 mm To monitoring changes during Fantazzini, Gombia, Schembri, 210, 350, 550, 750 ms processing, Simoncini, and Virgili (2009) To predict salt-to-moisture ratio Fantazzini et al. (2005) Sodium-based magnetic resonance 64 × 64 mm 2.7 ms 250 ms 20 mm To quantify salt diffusion Vestergaard et al. (2005) imaging (23Na-MRI)
a FOV= Field Of View; ET = Echo Time; TR = Repetition Time. is able to measure the molecular nuclei directly instead of through at- fundamental ways: they extend, increasing the interatomic distance tenuation differences. Measurements can be performed by either one- along the axis between two atoms (which occurs at higher frequencies or two-dimensional profile maps. While two-dimensional analyses give or shorter wavelengths), or they bend (at lower frequencies or greater information about the sodium position, namely information of sample wavelength) by changing the bond angle between two atoms (Alomar & structural features, one-dimensional profiles provide information about Fuchslocher, 1998). Absorption is selective and depends on the mole- the water proton mobility, thus the concentration can be calculated as a cular groups involved. Therefore, it is estimated by the difference be- distance function (Vestergaard et al., 2005). They have been applied in tween incident light and reflected or transmitted light. some foods to quantify salt contents: paste from fermented soy, vinegar Some researchers have reviewed its applications in meat technology cucumbers, plums, crabs and meat (Nagata, Chuda, Yan, Suzuki, & (Prieto, Roehe, Lavín, Batten, & Andrés, 2009) and many others have Kawasaki, 2000). However, only one publication was found on dry- used NIR for studying quality parameters (Berzaghi, Dalle Zotte, cured ham, whose conclusions reported that both application types Jansson, & Andrighetto, 2005; García-Rey et al., 2005), sensory attri- were viable to investigate salt contents, although they have some lim- butes (Liu, He, Wang, & Sun, 2011) and for predicting aW, moisture, fat, itations. The main limitation is related to the salt contents of the protein and NaCl contents (Collell, Gou, Picouet, Arnau, & samples. Good results were obtained in 23Na-MRI from two-dimensions Comaposada, 2010). Unfortunately, in the case of dry-cured hams, the when samples had more than 2.5 g NaCl/100 g and in 23Na-MRI from available literature about the potential use of NIR is scarce. There are one dimension at concentrations above of 0.9 g NaCl/100 g. only few studies, as reported in Table 4, focused on predicting the (Vestergaard et al., 2005). In conclusion, 1H-MRI is considered a good content of diverse compounds, monitoring processes and classifying method for dynamical monitoring of moisture losses (Antequera et al., according to different characteristics. 2007), while 23Na-MRI is capable to observe the dynamic tracking of Regarding the classification studies, García-Rey et al. (2005) salt content into the ham (Vestergaard et al., 2005). showed the viability of NIR to classify dry-cured hams based on dif- ferent levels of pastiness and anomalous colours. To illustrate this point, 2.3. Infrared spectroscopy using the content predictions obtained by the NIRS analysis, the main result consisted of an overall accuracy of 88.5% for the pastiness clas- fi Infrared spectroscopy (IS) techniques are based on the absorption of si cation, and 79.7% for colour defects. However, the authors observed infrared light due to the changes in the vibrational states of organic in the Principal Component Analysis (PCA) a high number of factors to molecules. Due to the opacity of certain samples, their use is limited explain the variance for pastiness predictions, which were attributed to fl (Rinnan, van den Berg, & Engelsen, 2009), although the viability of IS the numerous elements that in uence the NIR measurements. For the fi to quantify and to characterize different components in meat and fish quanti cation of compounds, Collell et al. (2010; 2011) developed ff products has been evaluated (Ziadi, Maldague, Saucier, Duchesne, & models to predict the aW content in di erent meat products, obtaining Gosselin, 2012). Moreover, the ability of IS based techniques for the better results in sausage samples than in dry-cured hams, probably due ff identification of different meat species, allowing the detection of food to the di erences in the homogeneity between samples. In the same fraud, has been reported (Damez & Clerjon, 2013; Kamruzzaman, study, the NaCl content was also predicted, but the obtained Root Mean Barbin, ElMasry, Sun, & Allen, 2012). Nevertheless, it should be noted Squared Error Cross Validation (RMSECV) was not very successful. that the different existing techniques have a variety of bandwidths and Additionally, the use of wavelength in the visible range (visible-NIRS) optical parameters, such as transmission, reflection or scattering, was also examined. García-Rey et al. (2005) concluded that it is a po- among others, which may need to be adjusted to get better results tential method for predicting sensory attributes such as texture and (Damez & Clerjon, 2013). colour, and Ortiz et al. (2006) also assayed its suitability in dry-cured fi Among the different existing techniques, Near Infrared reflectance hams classi cation according to other sensory parameters, such as spectroscopy (NIRS) is the measurement of the wavelength and in- pastiness, crusting or marbling. tensity of near infrared light absorption (700 nm - 2500 nm). When the On the other hand, Geesink et al. (2003) indicated that use of NIR light hits a sample, part of the photons can be transmitted through it, allows to classify raw meat according to the WHC (superior or inferior) and the rest is absorbed by some covalent bonds that act as oscillating and, consequently, this fact allows the early detection of meat that springs, coupled with the exact frequency or wavelength of the light could result in anomalous products at the end of the process (García- radiation (Cajarville, Repetto, Curbelo, Soto, & Cozzolino, 2003). By Rey et al., 2005), reducing associated economic losses. absorbing energy, the bonds of the molecules vibrate in two The combination of classical computer vision and spectroscopy
365 C. Pérez-Santaescolástica, et al. Trends in Food Science & Technology 86 (2019) 360–374
Table 4 Infrared spectroscopy based technologies and their applications across the dry-cured ham process sorted by method parameters.
Technology Processing condition Purpose of the research Reference
Near infrared spectroscopy (NIRS) Intervals: 2 nm To classify dry cured hams as a function of their texture García-Rey et al. (2005) Wavelength range: and colour evaluation 400–2200 nm 32 scans To predict moisture, water activity and NaCl content Collell, Gou, Arnau, and Comaposada − Resolution:8cm 1 and to determine the optimum number of spectra per (2011) sample. Near infrared hyperspectral imaging 0.7 × 0.8 mm2 To construct models for predicting NaCl and moisture Garrido-Novell, Garrido-Varo, Pérez-Marín, (hyperspectral eNIRS) Intervals: 3.15 nm contents Guerrero-Ginel, and Kim (2015) Wavelength range: 900–1700 nm Resolution: 20 nm To analyze water, fat and salt contents Gou et al. (2013) Wavelength range: To predict moisture and salt content at different stages Liu et al. (2013) 400–1000 nm in the salting process Visible-Near infrared spectroscopy Intervals: 2 nm To predict sensory quality Ortiz et al. (2006) (Visible-NIRS) Wavelength range: 400–2500 nm gives rise to the hyperspectral technique, which combines the strengths that its application potential to this particular product is still unknown. of both providing spectral and spatial information. Although this technique was used initially for geological purposes, there are some 2.4. High pressure studies about its application to evaluate the quality of different meat products (Barbin, ElMasry, Sun, & Allen, 2012; ElMasry, Iqbal, Sun, The use of high-pressure technology is one of the most investigated Allen, & Ward, 2011; Måge, Wold, Bjerke, & Segtnan, 2013). techniques across time. In 1899, Bert Hite designed a prototype HP ElMasry et al. (2011) applied hyperspectral imaging to investigate system, which was used to pasteurize some foods, but in this time its the effect of different processes in cooked hams from turkeys, while viability in food industry was limited due to the high cost involved. It Talens et al. (2013) tested the feasibility of this technology to predict was not until 1993 that the HP technology was introduced in food compounds, like fat, water and protein content, and to classify cooked manufacture with stabilizing purposes (Rivalain, Roquain, & hams from pig meats based on different qualities using the information Demazeau, 2010). The mechanism of action of HP units resides in a from fat, water and protein predictions. However, only a few publica- chamber in which the food, previously packed, is positioned. After tions have been found related to dry-cured hams. As shown in Table 4, closing the vessel, the chamber is filled with a medium capable of three studies focused on moisture and salt predictions. Liu, Qu, Sun, Pu, transmitting the pressure. and Zeng (2013) obtained a good coefficient of determination for both The traditional main purpose of HP treatments has been the mi- water and salt content, although Gou et al. (2013) showed better values crobial reduction in order to extend shelf life (Duranton, Simonin, reducing the wavelength interval. In addition to water and salt content, Guyon, Jung, & de Lamballerie, 2015). In this sense, Bajovic, Bolumar, Gou et al. (2013) also predicted the fat content, obtaining a Root Mean and Heinz (2012) observed that pressures around 10 or 50 MPa had an Squared Error (RMSE) of 1.36%. effect on microbial growth, even though higher pressures are needed for In the range of middle IS, it is worth highlighting the Raman the inactivation of microorganisms. spectroscopy, a high-resolution photonic technique that provides in a On the other hand, it has been observed that in dry-cured loins there few seconds chemical and structural information of almost any organic is an increase in autolytic activity when a pressure of 300 MPa was or inorganic material or compound, allowing its identification. To ob- applied, however at 500 MPa the activity of aminopeptidases was de- tain molecular information, the characteristic vibrational energy levels creased (Campus, Flores, Martinez, & Toldrá, 2008). HP application of the atoms of the bond, its conformation and its environment are implies modifications in the final products and the food could undergo analyzed. These levels have characteristic resonance frequencies, which physical, chemical, organoleptic, technical and functional alterations or are a function of the mass of the molecules and the strength of their all at once (Liu, Selomulyo, & Zhou, 2008; Rivalain et al., 2010). bonds. Unlike other IS technologies, which require a change in the di- However, these changes are not necessarily negative. In this regard, pole moment of the molecule, Raman spectroscopy requires a change in dry-cured products showed an increase in saltiness perception caused polarizability, which allows obtaining complementary spectral in- by HP (Clariana, Guerrero, Sárraga, & Garcia-Regueiro, 2012, 2011; formation on homonuclear molecules. Moreover, its main advantage is Fulladosa, Sala, Gou, Garriga, & Arnau, 2012; Rubio, Martinez, Garcia- the minimal influence of water in the samples (Damez & Clerjon, 2013). Cachan, Rovira, & Jaime, 2007), which is interesting with regard to Raman spectroscopy proved that it was possible to determine the un- reduced salt product elaborations. saturation degree of animal fats (Olsen, Rukke, Flåtten, & Isaksson, In relation to dry-cured ham, Table 5 shows the studies carried out 2007) in pork meat. In this sense, in pork loins, it was shown that in last years. Likewise, the initial purpose of HP use has also been mi- Raman spectroscopy was able not only to predict the texture, tender- crobial inactivation (Clariana et al., 2011; Garriga, Grebol, Aymerich, ness (Beattie, Bell, Farmer, Moss, & Patterson, 2004), juiciness and Monfort, & Hugas, 2004; Rubio-Celorio et al., 2016). Since dry-cured chewiness of pork loins (Wang, Lonergan, & Yu, 2012), but also to ham is a highly salted product, and so the water activity level is low, evaluate the effect of thermal treatments and salt addition (Herrero, thus the main spoilage presented corresponds to Gram-positive bacteria Carmona, López-López, & Jiménez-Colmenero, 2008). Additionally, and yeast, which are mainly located at the surface of the product. It has there is a recent advance in this technology; Sowoidnich, Schmidt, been proved that HP treatments at 600 MPa keep the microbial load at Kronfeldt, and Schwägele (2012) developed a portable Raman device low levels during storage with a remarkable effect on psychrotrophs, allowing a quick identification of pork meat spoilage. Therefore, Raman resulting in an extension of the shelf life up to 4 months (Garriga et al., could be considered useful in dry-cured ham processing, due to evi- 2004). Furthermore, moulds require pressures between 200 and dence suggesting its potential application for detecting microbial po- 300 MPa for their inactivation, while their spores need 400 MPa pulation, characterizing textural attributes and even for monitoring the (Aymerich et al., 2008). There was a study focused on purine and drying process. However, there are no studies in dry-cured ham yet so pyrimidine content, which are compounds declared as indicators of
366 C. Pérez-Santaescolástica, et al. Trends in Food Science & Technology 86 (2019) 360–374
Table 5 High pressure technology applications across the dry-cured ham process sorted by method parameters.
Measure Condition Sample type Purpose of the research Reference
Pressure Time Temperature
200 MPa 5 min 20 °C Sliced vacuum-packed To reduce intrinsic water population Rubio-Celorio et al. 400 MPa dry-cured ham (2016) 600 MPa 600 MPa 6 min Variable Sliced, skin vacuum- To reduce intrinsic microbial population Clariana et al. 900 MPa 5 min packed dry-cured ham (2011) 600 MPa 6 min 31 °C Sliced, skin vacuum- To inactivate microbial population Garriga et al. (2004) packed dry-cured ham 400 MPa 10 min Variable Frozen vacuum packed To evaluate the effects on physicochemical parameters and on antioxidant and proteolytic Serra et al. (2007a) 600 MPa ham enzyme activities at different stages of ham elaboration and on the final product Serra et al. (2007b) characteristics 600 MPa 6 min 12 °C Sliced vacuum-packed To examine lipid and protein oxidation and sensory properties Fuentes et al. (2010) dry-cured ham 300 MPa 5 min 12 °C Sliced vacuum-packed To examine structural and molecular changes affecting sodium and water dynamics Picouet et al. (2012) 600 MPa dry-cured ham 900 MPa 500 MPa 3 min 3 °C Sliced, skin vacuum- To evaluate textural changes Garcia-Gil et al. packed dry-cured ham (2014) 400 MPa 10 min 12 °C Sliced vacuum-packed To investigate changes in the volatile fraction influenced by packaging material. Rivas-Cañedo et al. dry-cured ham (2009) 600 MPa 6 min 21 °C Sliced, skin vacuum- To evaluate the influence on the volatile fraction Martinez-Onandi packed dry-cured ham (2017) Martinez-Onandi (2018) microbial spoilage, in which no differences were observed between pressure application. However, modifications in protein links, protein untreated samples and samples treated at 600 and 900 MPa, maybe due solubility losses and decreases in cathepsin B, D and L activity have to the water and salt contents, since a low aw preserves the cells from been observed in HP-treatments above 400 MPa (Campus et al., 2008). pressure effects (Clariana et al., 2011). Moreover, diverse studies The provided effect of HP on both enzyme activity and protein dena- showed that the HP application at 100 MPa causes lysosomal mem- turation, which are widely associated with the final texture, have branes destruction and reversible protein denaturation, and the protein caused an increasing interest in its potential use in meat tenderization denaturation at pressures beyond 200 MPa becomes irreversible (dos (Ichinoseki, Nishiumi, & Suzuki, 2006; Jung, de Lamballerie-Anton, & Santos, Cristianini, & Sato, 2018; Qi, Ren, Xiao, & Tomasula, 2015) Ghoul, 2000). In spite of previous evidence in meat showing sig- facilitating proteolytic reactions. These structural protein modifications nificantly higher shear force values in HP-treated salted pork meat also cause higher water losses. Therefore, higher pressures result in (Duranton, Simonin, Chéret, Guillou, & de Lamballerie, 2012) and in higher water losses (Fulladosa, Serra, Gou, & Arnau, 2009; Garcia-Gil cooked beef meat (Jung et al., 2000), the textural effect on dry-cured et al., 2014; Picouet et al., 2012; Serra et al., 2007a). In this regard, hams has not been thoroughly studied. To date, only a few studies have Rubio-Celorio et al. (2016) observed a 0.43% increase in water loss for been published about it, reporting higher hardness values in pressurized every pressure increment of 100 MPa, while Picouet et al. (2012), ob- samples (Fuentes, Ventanas, Morcuende, Estévez, & Ventanas, 2010; served even higher losses up to 0.82%. In addition to this, the pressure Fulladosa et al., 2009; Rubio-Celorio et al., 2016). Fuentes et al. (2010) could induce physicochemical changes, such as reductions in water reported greater hardness and chewiness in pressurized samples, and holding capacity with treatments between 300 and 600 MPa (Fulladosa they observed that the treated samples were less juicy and doughy than et al., 2009; Picouet et al., 2012). Previously, Serra et al. (2007a) the controls. showed a 24% increase in salting weight losses in samples treated be- On the other hand, colour parameters can also be affected by the fore the salting process at 600 MPa compared to samples treated at application of pressure, as increments in reflectance and lightness (L*) 400 MPa and both HP-treated samples showed lower NaCl content than and lower redness (a*) values have been reported (Andrés, Møller, control samples. This fact is attributed to an increase in protein dena- Adamsen, & Skibsted, 2004; Fulladosa et al., 2009; Hughes, Oiseth, turation caused by pressurization, in turn increasing salting weight Purslow, & Warner, 2014; Rubio-Celorio et al., 2016), modifications losses (Serra et al., 2007a). Due to this lower NaCl content, significantly which could be explained by the fact that HP promotes changes in higher levels of aw were observed in pressured samples from BF muscle, protein structural conformations, which could have an undesirable whereas no differences were found in SM muscle, may be due to the impact on the organoleptic characteristics of final products (Serra et al., large variability in the composition values of this muscle (Virgili & 2007b). The increase in salty taste is another organoleptic change ob- Schivazappa, 2002). served after pressure treatments. Picouet et al. (2012) explained this Moreover, there are some studies about the effects of pressure observation by the increment of the amount of sodium ions released treatments on enzymatic activity. These studies concluded that most of due to differences in salt and protein interactions after the application enzyme activities is generally unaffected at 200 MPa and below of pressure. (Masson, Tonello, & Balny, 2001), but inactivation begins to occur as Since the final aroma is influenced by all the reactions occurring the pressure increases, with differences depending on the enzyme during the dry-curing process, such as proteolysis and lipid oxidation (Rivalain et al., 2010). In this sense, it has been shown that pressures of reactions, several researchers have focused on investigating the volatile 600 MPa reduce glutathione peroxidase (GSHPx), superoxide dismutase profile changes due to pressure application to determine the organo- (SOD) and catalase (CAT) activity. Moreover, at 400 MPa, the in- leptic impact (Clariana et al., 2011; Lorido, Estévez, Ventanas, & activation percentage of CAT increases. Based on this evidence, Serra Ventanas, 2015). In this sense, since pressure promotes lipolysis reac- et al. (2007a) concluded that the ageing conditions and the dry-curing tions (Fuentes et al., 2010, 2014), mainly at pressures around 600 MPa process would have a greater effect on the enzymatic activity than the (Andrés et al., 2004; Fuentes et al., 2010), the amount of some lipolysis
367 C. Pérez-Santaescolástica, et al. Trends in Food Science & Technology 86 (2019) 360–374 derived volatiles increases in HP-treated samples. Martínez-Onandi level of intensity. For this reason, it is useful for characterization pur- (2018; 2017; 2016a; 2016b) evaluated the changes in the volatile poses within food processing (Chandrapala, Oliver, Kentish, & profile of dry-cured ham slices treated at 600 MPa and its evolution Ashokkumar, 2012; Koch et al., 2011a, 2011b). On the contrary, high- after 5 months of refrigerated storage. They observed higher levels of intensity ultrasound could induce physical, chemical and/or mechan- 12 volatile compounds in the treated samples, whereas, after the sto- ical modifications (Jayasooriya, Bhandari, Torley, & D'arcy, 2004) and rage period, only 9 of the total volatile compounds were enhanced. On this is why its traditional use in food manufacture has been for gen- the other hand, at 400 MPa, Rivas-Cañedo, Fernández-García, and erating emulsions (Dolatowski, Stadnik, & Stasiak, 2007). Nuñez (2009) also observed increased amounts in volatile compounds, As far as characterization uses are concerned, salt, fat and water whereas, at treatments between 200 and 600 MPa, Andrés et al. (2004), content have been the most studied constituents in dry-cured hams. In reported an important increase in hexanal which is considered the main this sense, salt analyses are performed in both pre and post-salting compound derived from lipid oxidation and it contributes to the fatty process stages (De Prados et al., 2015, 2016). The importance of post- distinctive matured ham flavour. Its amount was even more pro- salting research consists of the process optimization since US allows the nounced at 800 MPa. Similarly, Fuentes et al. (2010) noticed higher classification according to different levels of salt content with the aim of levels in 5 aldehydes after treatment at 600 MPa, which caused an in- the development of the following stages (De Prados, Garcia-Perez, & crement of the rancid odour perception while differences in rancid Benedito, 2017). flavour were not shown. However, enhanced saltiness, bitterness and Since ultrasonic velocity depends on the aggregation states of the cured flavour were obtained in the HP-treated samples. matter, the use of this technology to measure fat content is strongly Likewise, HP treatments could reduce the levels of some volatile affected by the temperature of the sample. In this sense, fat is solid at compounds due to enzyme inactivation or matrix structure modifica- low temperatures so that ultrasound velocity is enhanced and the dif- tions (Garcia-Gil et al., 2014; Picouet et al., 2012). Thus, Martínez- ferentiation between fatty and lean tissues is clearly possible. Onandi, Rivas-Cañedo, Picon, and Nuñez (2018) established reductions Conversely, at high temperatures, fat becomes liquid and the velocity of in 23 volatile compounds, as performed by Rivas-Cañedo et al. (2009) ultrasonic waves is reduced, approaching that in lean tissue. In this and, Martínez-Onandi, Rivas-Cañedo, Picon, and Nuñez (2016b) in 6 regard, De Prados et al. (2015) concluded that temperatures around volatile compounds and by Martínez-Onandi et al. (2017) in 31 volatile 2 °C are suitable for fat determinations due to the remarkable difference compounds. in both velocities. In contrast, Fulladosa et al. (2015b) showed that at In summary, the effects of HP treatments depend on the pressure 2 °C the velocity in fatty tissues is similar to that in the protein matrix level, although the influence of factors such as the measuring conditions and they suggested to calculate the velocity variation between 2 and of the HP treatment, packaging material or storage cannot be ignored 15 °C, showing great fat and protein identification at 15 °C. In spite of (Martínez-Onandi et al., 2017). this result, they concluded that the US feasibility to create models for fat and protein contents is not very disheartening, since they showed other factors, like water content, which influence US to a greater extent 2.5. Ultrasound than lipid and protein matrices. However, it has been proved that ul- trasonic models provide a better percentage of hams correctly classified Ultrasound technology (US) is based on pressure fluctuations caused into different fat levels compared to other non-invasive technologies by sound waves that increase temperature and pressure leading to ca- like X-Ray (88.5% vs 65.4%, respectively) (De Prados et al., 2015). In vitation phenomena which could accelerate mass transfer (Jambrak addition, US implementation in the ham manufacture is cheaper and et al., 2010) and favors extraction processes (Zhu et al., 2017a; 2017b). easier than, for instance, MNR or X-Ray techniques (Corona et al., As shown in Table 6, the interest about the potential uses of US has 2013). Despite of the commented advantages, some production system increased significantly in the last years. However, it is difficult to adaptations are required for its effective incorporation (Corona et al., compare the different results obtained due to the high variability 2013). among studies, including the many parameters that could influence the Until now, commented results are obtained using the through- outcomes (Berlan & Mason, 1992). Regarding the intensity applied, US transmission mode, namely a method in which two transducers are is classified into low-intensity, (frequencies above 100 kHz and in- − connected directly at both sides of the sample (De Prados et al., 2017). tensities below 1 W cm 2) and high-intensity ultrasound (frequencies − That is the reason why it can be very difficult to introduce this mode of from 18 to 100 kHz and intensities above 1 W cm 2)(McClements, application in certain stages of the dry-cured ham process. However, 1995). The first one can be considered as non-invasive due to the lower
Table 6 Ultrasound technology applications across the dry-cured ham process sorted by method parameters.
Technology Measure condition Purpose of the research Reference
Intensity Temperature (°C)
Ultrasound (US) 1 MHz 2 °C To predict the fat content in dry-cured hams for De Prados et al. (2015) Through-transmission industrial classification To monitor dry salting De Prados et al. (2016) 2 °C and 15 °C To predict salt and fat contents Fulladosa et al. (2015b) 0, 2, 4, 6, 8, 10, 12, 14, 20, 22 To characterize the melting properties of Niñoles, Mulet, Ventanas, and and 24 °C subcutaneous fat Benedito (2010) 50 W 40, 45 and 50 °C To shorten the hot air mild thermal treatment Contreras et al. (2018) applied to correct the texture 4.2, 11 and 19 W Below 21 °C To accelerate the curing stage McDonnell et al. (2014a,b) cm-2 600 W 50 °C To correct texture defects Pérez-Santaescolastica et al. (2018) Ultrasound (US) 1 MHz 2 °C To monitor dry salting and to predict the final De Prados et al. (2017) Pulse-echo mode salt content Air-coupled ultrasound 0.75 MHz 6 °C To characterize Corona et al. (2013b) Scanning acoustic 10 MHz 6 °C To characterize microscopy
368 C. Pérez-Santaescolástica, et al. Trends in Food Science & Technology 86 (2019) 360–374 the pulse-echo mode allows to simplify its implementation in the pro- Different factors are involved in the effectiveness of the process, and cess, considering that only one transducer is positioned in the sample, these factors could be technological, such as the intensity, time or working as emitter and transducer at the same time (Awad, Moharram, temperature of the application, or derived from the matrix in which Shaltout, Asker, & Youssef, 2012). Traditionally, this mode has been they are applied, e.g. conductivity, pH or fat content (McDonnell, Allen, applied for the detection of defects in metallic materials, although its Chardonnereau, Arimi, & Lyng, 2014b). use for the characterization of components in food has been recently PEF can be applied to enhance mass transfer, will modify the investigated (De Prados et al., 2017). By measuring the Time of Flight characteristics of a product in a different way depending on its own variation (ΔTOF) of the pulse-echo mode it is possible to monitor the nature. In this context, it is important to take into account that meat is a salting process in hams with a mean thickness of 15.7 cm (De Prados complex set of connective tissue, adipose, vascular and nervous tissues, et al., 2017), showing a high ΔTOF reduction at few hours after the and longitudinal, multinucleated muscle cells. In general, PEF proces- beginning of the salting (De Prados et al., 2016). Additionally, the sing affects the muscle cell membranes influencing the interaction be- prediction model for salt diffusion could be improved by the addition of tween fatty acids and cell membrane phospholipids with prooxidants in fat and water gain information (De Prados et al., 2017). meat (Faridnia et al., 2015). Such interactions can originate undesirable In addition, it was shown that the US applications around 64 or compounds able to decompose into secondary products which can − 51 W cm 2 could accelerate the transport of water and salt due to the cause off-flavors and odors in meat and reduce its sensorial and nutri- changes caused by cavitation phenomena (Cárcel, Benedito, Bon, & tional quality. Mulet, 2007). Nevertheless, in recent studies, Siró et al. (2009) defined It was shown that PEF application allows the reduction in the curing − 2–4Wcm 2 as the intensity in which the diffusion of salt is strongly time since mass transfer is improved (Chauhan & Unni, 2015). Pre- enhanced, while McDonnell, Lyng, and Allen (2014a) obtained the viously, Toepfl and Heinz (2007) observed improvements in salt dif- − highest increment with the application of for 25 min at 19 W cm 2. fusion after applying PEF in dry-cured products. This fact could be Additionally, McDonnell et al. (2014a) also established treatments for explained by myofibril fragmentation, caused by the treatment, which − 10 or 25 min at intensities of 19 W cm 2 as a good method to increase would break the structure of the muscle (O'Dowd, Arimi, Noci, Cronin, the water gain. Despite of previous studies in meat which have not & Lyng, 2013). Consequently, this results in increases in weight loss. shown any effect on texture after US application, McDonnell et al. Moreover, the weight loss is influenced by the frequency of the treat- (2014a) found lower cohesiveness and gumminess values in treated ment, as shown by McDonnell et al. (2014b), who observed a higher hams. Moreover, hardness was found to increase upon treatments with water content in samples treated at 100 Hz than in the ones treated at high intensity US, which could be due to the induced heating the 200 Hz, whereas the latter frequency resulted in higher WHC values treated samples (Contreras, Benedito, Bon, & Garcia-Perez, 2018). compared to the first one. In the same study, it was also proved that Pérez-Santaescolastica et al. (2018) found a decrease in adhesiveness treatments at 100 Hz combined with 300 pulses enhanced the NaCl values, although the treated samples presented modifications in the content and the samples tended to be harder and chewier. This study volatile compounds profile and enhanced the sweet, acid, bitter and was the only one found in literature about the potential PEF applica- aged tastes compared to the untreated samples. tions in dry-cured hams. Finally, there are alternative techniques based on ultrasonic waves Likewise, electrical impedance scanning (EIS) has also been poorly that have not been studied yet even though could be useful for im- studied. This technique is based on the application of a potential signal proving the dry-cured ham process. For instance, air-coupled ultra- to an electrode and the measurement of its current response at different sound has been used for foreign body detection (Pallav, Hutchins, & frequencies, obtaining an impedance spectrum. The first research in Gan, 2009), as well as to identify modifications in physicochemical which the EIS treatment was applied in hams as a classification method properties of liquids (Meyer, Hindle, Sandoz, Gan, & Hutchins, 2006) based on different ranges of pH and fat in raw meat (Oliver et al., and to find anomalies in starchy solid consistency (Gan, Hutchins, & 2001). The early identification of potential pale, soft and exudative Billson, 2002). Corona et al. (2013) suggested that the use of air-cou- (PSE) hams could help to prevent textural and flavour anomalies in the pled ultrasound could lead to an important reduction in the time and final product, thus improving the process. As a result, 88.46% of hams manipulations throughout the process. In addition, they commented with pH above 6.1 were well-classified, while hams with pH under 5.95 that scanning acoustic microscopy (SAM), which is a potential tech- were classified with an accuracy of 92.31%. nique to analyze the distribution of different tissues, should be in- On the other hand, electromagnetic waves have been widely used in vestigated. meat products for investigating quality attributes, like sensory and nutritional characteristics, chemical and physicochemical properties, 3. Other emerging technologies and safety (Damez & Clerjon, 2013). An electromagnetic technique worth highlighting is microwave spectroscopy, whose main advantages In the early 90's, food manufacture started to use electricity to include energy efficiency and food safety (Han, Cai, Cheng, & Sun, process foods, particularly in milk pasteurization, although other po- 2018). Nevertheless, nowadays its use in meat manufacture is not en- tential uses were also investigated (Chauhan & Unni, 2015). In this ough investigated and no studies were found in relation to dry-cured context, in 1960, Doevenspeck patented the pulse electric fields (PEF) hams. The available publications related to meat address structural and technique and in 1967, Sale and Hamilton probed its effectiveness in compositional measures (Nelson & Trabelsi, 2012; 2008) and foreign reducing the bacterial population and inactivating enzymes (Chauhan & body detection Damez & Clerjon, 2013. Another example of microwave Unni, 2015). However, the PEF treatment in dry-cured hams has been spectroscopy technology is the namely Time Domain Reflectometry poorly studied (Table 7). (TDR), which is based on the determination of the propagation speed of This technology is based on the application of pulses during short an electromagnetic wave after being reflected. It is characterized for a times, usually 1–100 μs every 0.001–1 ms, which can expand the cell faster response, being easier to use and providing a portable equipment membrane pores or, even generate new ones (Chauhan & Unni, 2015). (Miura, Yagihara, & Mashimo, 2003). This new technology has been These pores increase the permeability of the membrane causing cell considered capable of water and salt estimations in dry-cured hams, content losses or the entry of external substances, with both situations with a RMSEV of 1.67% and 0.22% respectively, and for fat content resulting in cell death (Rodrigo, Sampedro, Silva, Palop, & Martínez, estimation with lower precision (RMSEV = 2.81%) (Fulladosa et al., 2010). Depending on the applied intensity the process can be reversible 2013). Moreover, Rubio, Fulladosa, Claret, Guàrdia, and García-Gil or irreversible. In addition to microbial inactivation, the process can (2013) observed its potential for classifying slices based on different result in the modification of some food properties, such us texture or pastiness levels, with 93% probability of correct classification. water holding capacity (WHC) (Gudmundsson & Hafsteinsson, 2001). Meanwhile, Magnetic induction (MI) is the process by which
369 C. Pérez-Santaescolástica, et al. Trends in Food Science & Technology 86 (2019) 360–374
Table 7 Other non-invasive technologies with potential use in the dry-cured ham process sorted by measure conditions.
Technology Measure conditions Purpose of the research Reference
PULSE ELECTRIC FIELDS (PEF) VOLTAGE 25 kV To accelerate salting McDonnell et al. PULSE WIDTH 4–32 μs (2014a,b) FREQUENCY 1000 Hz ELECTRICAL IMPEDANCE SPECTROSCOPY ELECTRICAL IMPEDANCE From 8 kHz to 1 MHz To classify based on quality Oliver et al. (2001) (EIS) characteristics ELECTRICAL IMPEDANCE From 8 kHz to 1 MHz To predict salt content and texture Guerrero et al. (2004) TIME DOMAIN REFLECTOMETRY (TDR) FREQUENCY 20 MHz-5 GHz To predict salt, water and fat contents Fulladosa et al. (2013)
TIME-BASE RESOLUTION 10 ps To predict salt content, texture and aw FREQUENCY up to 5 × 109 Hz To classify according to the pastiness Rubio et al. (2013) TIME-BASE RESOLUTION 0–2.56 ns level MAGNETIC INDUCTION (MI) INTENSITY 1 μT To predict salt content in whole hams Schivazappa et al. (2017) INSPECTION VOLUME 45 cm width, 27 cm height, and after salting 60 cm length LASER BACKSCATTERING IMAGING (LBI) RESOLUTION IMAGEN 1280 × 960 pixels with To determine composition and textural Fulladosa ey al. (2017) WAVELENGTHS RANGE 400–750 nm. characteristics magnetic fields generate electric fields in a conductive material. The use according to the post-salting time and muscle type. of MI has been investigated recently in food technology, having the great advantage that electrodes are not needed for its application (Schivazappa et al., 2017). There are several studies in which the 4. Conclusions and future trends spectra of agricultural products have been analyzed using multi-fre- quency MI (Barai, Watson, Griffiths, & Patz, 2012) and single-frequency The most important aspect in the dry-cured ham process is to MI (Euring, Russ, & Wilke, 2011). Regarding hams, Schivazappa et al. maintain a rigorous control of the salting and curing stages, since all the (2017) obtained a higher signal in salted hams than in fresh ones, and reactions that give rise to the typical sensory properties of the product they developed a model for salt prediction in which the RMSEC was take place at these stages. The control of the salt distribution in the 0.18%. There is little difference between this RMSEC and the one ob- muscle is essential for an appropriate development of the process. This tained by using other technologies previously cited. This fact demon- has been and continues to be the most widespread study object among strated the feasibility of MI to predict the salt content in dry-cured the research carried out with new technologies. However, it is not the products although more studies should be carried out to extend its use only application for which emerging non-invasive technologies can be in the food industry. useful, since detecting defects of the final product or ensuring food Another satisfactory technology for process monitoring is Laser safety are examples of applications that can help to optimize and im- backscattering imaging (LBI), since it is an inexpensive technology in prove the process. which the sample microstructure affects the light scattering, offering As has been shown, there is a wide variety of technologies that are information about the changes that can take place in the physical potentially applicable in ham, although further research is needed for a properties and in the component distributions (Fulladosa et al., 2017). number of them to determine their application potential. Regarding In general, this technology was successfully developed for quality techniques based on irradiation, they can ensure commercial sterility. evaluation of different products (Adebayo, Hashim, Abdan, & Hanafi, However, some of them, such as gamma radiation, promote lipid oxi- 2016; Mollazade, Omid, Tab, & Mohtasebi, 2012; 2013; Qing, Ji, & dations causing colour changes and off-flavors, and they are not widely Zude, 2007; 2008), for detecting damages in bananas (Hashim et al., accepted by consumers; therefore, their use is limited. Among the most 2013), for detecting citrus decomposition (Lorente et al., 2013), and for studied techniques are HP and US, which have demonstrated their monitoring drying processes in fruits and vegetables (Romano, Nagle, potential use as texture defect correctives, but for US implementation, Argyropoulos, & Müller, 2011; Udomkun, Nagle, Mahayothee, & despite being a cheaper technique, production line adjustments are Müller, 2014). In meat products, one research was found in which the needed. However, HP was shown to be practical in order to reduce the freshness of pork was analyzed (Li et al., 2016). In hams, there is a salt content since the salty taste is enhanced with HP treatments. On the recent publication by Fulladosa et al. (2017), whose results confirmed other hand, NIRS can be used to classify based on WHC at the beginning that the proteolysis caused by drying and the water content variations of the process, allowing for the early detection and disposal of meat that influence the response of the light backscattering and then, the esti- could result in defective products in the following process stages, while mation of texture properties is not possible using only LBI technique. RAMAN or MRI are able to detect anomalous textures at the end of the However, they suggest that the combination with others technologies process. could be feasible for these purposes. A less studied technology is that based on microwaves, which is Finally, one of the techniques that have aroused most interest in characterized by its energy efficiency. It could be useful to online es- recent years is data mining. This new technology consists in exploring timate salt, water and fat contents in an easy way due to the availability and analyzing many data sets in order to predict and describe new data of portable equipment. Like PEF and EIS, it could be a good choice for (Witten, Frank, Hall, & Pal, 2016). Some studies have applied it on food further research since their multiple uses have been proved in other (Wu, Sun, & He, 2012; Holmes, Fletcher, & Reutemann, 2012. Liu et al. foods. Finally, data mining, which allows to make predictions based on (2013)) used data mining to predict water activity and NaCl content in an immense data file archive, is the most promising discovery in recent pork meat, but it was in 2010 when it was first applied in hams and years for detecting defects or for classifying products according to considered useful for classifying them as a function of the pig feeding sensory attributes. (Pérez-Palacios, Antequera, Molano, Rodríguez, & Palacios, 2010a; To sum up, there is still much to explore in the use of these tech- 2011). After that, Pérez-Palacios et al. (2014) investigated the ability of nologies in dry-cured ham and there are many alternatives and poten- data mining to estimate quality traits. Finally, Caballero et al. (2016) tial applications across the dry-cured ham manufacture which could used the Waikato Environment for Knowledge Analysis (WEKA) free provide great advantages. software and proved its feasibility for classifying dry-cured hams
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