Doctoral Thesis University of Castilla-La Mancha 2017

Adrián Rabadán Guerra

UNIVERSIDAD DE CASTILLA-LA MANCHA

ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS Y DE MONTES

DEPARTAMENTO DE PRODUCCIÓN VEGETAL Y TECNOLOGÍA AGRARIA

NUTS AS RAW MATERIAL FOR OIL EXTRACTION IN THE FOOD INDUSTRY: CHARACTERIZATION, PRODUCT AND PROCESS INNOVATION AND ECONOMICS

Memoria presentada para aspirar al grado de Doctor por la Universidad de Castilla-La Mancha

ADRIÁN RABADÁN GUERRA

ALBACETE, 2017

Agradecimientos

Llega el momento de dar las gracias a todas las personas que de una forma u otra han contribuido y ayudado en la realización de este trabajo. Mirando hacia atrás te das cuenta de a cuanta gente tienes que agradecer el que esta tesis sea hoy una realidad.

Fundamental para el correcto desarrollo de una tesis es la adecuada elección de los directores. En este sentido he tenido la enorme suerte de contar con José Emilio Pardo, Ricardo Gómez y Manuel Álvarez, que más allá de una inmejorable guía en todo lo relacionado en la parte más técnica de tesis, han sabido, además, aportarme el apoyo personal que se necesita cuando te embarcas en un proyecto como éste. A los tres, de verdad, muchas gracias.

Tengo que agradecer además su ayuda a todos mis compañeros del grupo de investigación de “Calidad, Seguridad e Higiene de Productos Agroalimentarios”, especialmente a Andrés Alvarruiz, Arturo Pardo, Eulogio López y Sergio Díaz, por todo el apoyo técnico, científico y personal.

Más allá de mi propio grupo de investigación, tengo que hacer mención especial a otros profesores e investigadores que de una forma u otra han participado en este proyecto. Así, tengo que agradecer a Miguel Olmeda los consejos y el apoyo antes y durante la realización de mi tesis, y a Ángela Triguero por, a pesar de la falta de tiempo, aceptar a dirigirme el TFM que ahora se ha convertido en parte de esta tesis. Tengo que dar las gracias también a Lourdes Gallardo y Beatriz Gandul, del Instituto de la Grasa, a Concepción de Miguel de la Universidad de Extremadura y a Joana Santos y Beatriz Oliveira de la Universidad de Oporto por toda la ayuda en los análisis y en la interpretación de resultados que ha permitido desarrollar esta tesis.

La idea original de embarcarme en este proyecto podría no haber llegado a buen fin de no haberse desarrollado en un marco tan motivador como el que me proporcionó mi trabajo de becario en el Vicerrectorado de Investigación y Política Científica de la UCLM. Por ello, tengo que dar las gracias a Paco Sáez, Leonor Prieto, Mercedes Acebal, Antonio Alfaro, Santiaga Gutiérrez, Cecilia Fernández y Julián Garde, por ayudarme en todo en este primer contacto que esta beca supuso con la investigación, y por animarme a realizar la tesis. No puedo olvidarme de Sisco, ni de mis compañeros

Agradecimientos becarios allí, Guadalupe, Santa y a “los Joses”, siempre dispuestos a ayudar y a comentar los problemas variados que iban aconteciendo con un café en una mesa del Vice.

También agradecer a todo el personal del Departamento de Producción Vegetal y Tecnología Agraria, de la Escuela Técnica Superior de Ingenieros Agrónomos y de Montes (ETSIAM) y de la Escuela Internacional de Doctorado por los medios y las facilidades con todas las gestiones. También merece especial mención la propia UCLM, que en el marco de su Plan Propio de Investigación me ha proporcionado la financiación necesaria para desarrollar esta tesis.

No me puedo olvidar tampoco de todos los que contribuyeron a hacer de mi estancia en la Universidad de Adelaida una experiencia inolvidable. A Michelle Wirthensohn, primero por darme la posibilidad de trabajar con ella, y segundo, por su cercanía y ayuda en absolutamente todo, tuviera o no que ver con el doctorado. Como parte de esa experiencia, tengo que nombrar también a Jana Kolesik, a Shashi, y en general a todo el personal del South Australian Research & Development Institute por la excepcional acogida.

El desarrollo de una tesis ocasiona sin duda momentos de duda o hartazgo, que deben ser superados para llegar a buen puerto. Para ello, he tenido la suerte de contar con los ánimos de muchos amigos a los que ahora tengo la oportunidad de agradecer ese apoyo. Así, tengo que agradecer a Irene por aguantarme al teléfono hablando de cuartiles y editores, a los belmonteños (imposible nombrarlos a todos) por arrastrarme lejos del ordenador cuando era necesario, y a los albaceteños, Álvaro, Juan Ángel, Javier, Ana, Cristina y los Carlos, por hacer lo propio cuando estaba en la capital.

Y por último, a mi familia, a toda ella en general, y de forma muy particular a mis padres y mi hermana, porque sin ellos, esta tesis no hubiera sido posible.

Gracias.

Abstract/Resumen

Abstract/Resumen

Abstract

The health-promoting properties of nuts encourage their study and the development of industrial processes to obtain high quality oils and other by products derived from the extraction process such as partially defatted flours. Although many papers have focused on these products, there are still many lacks remaining, related to the characterization of cultivars, and their influence on the oils and defatted flours characteristics, the changes originated by the extraction process which leads to the elaboration of high quality products from the sensory and physicochemical point of view, the product innovation to improve oil characteristics and the analysis of economic factors related to nuts and nut oils.

Thus, as a first step, the characterization of cultivars from almond, pistachio and walnut was developed attending to physical, chemical and sensory analysis of kernels, to evaluate the differences that raw material may originate in the oils characteristics. In addition, the effect of the crop year was also evaluated.

In almond, pistachio and walnut kernels, physical parameters (size, weight and colour) proved to be useful for cultivar discrimination. Regarding sensory analysis, consumers showed preferences in overall visual acceptance and taste for some almond (Vairo, Belona and Penta) and pistachio cultivars (Larnaka, Aegina and Sirora) when affective tests were performed. When the oils from the different cultivars were evaluated, differences were found in physical parameters like viscosity, showing strong correlations with the oil fatty acid profile and the concentration of specific triglycerides.

The oils obtained from the studied cultivars showed significant differences in their chemical composition. Oil yield was similar in all the selected almond cultivars, but significant differences were found in the oxidative stability or in their fatty acid profile, mainly in the content of linoleic, stearic and palmitoleic acids. Regarding pistachio oil, three different groups of cultivars were defined according to the physicochemical composition and oil yield. Cultivars in group 1 showed a higher content of linoleic fatty acid (higher than 26.4%), while cultivars in group 3 type showed a stronger presence of oleic acid (higher than 67.8%), resulting in oils that

Abstract/Resumen were more stable to oxidation. Cultivars with intermediate characteristics were included in group 2. Among walnut cultivars, significant differences were found in the fatty acid profile and concentration of minor components of interest as polyphenols. Linoleic fatty acid compound more than 47.5% of total fatty acids in all cultivars, accounting up to 62.13% and 60.02% in cvs. Pedro and Mollar de Nerpio, respectively. As expected, the oxidative stability index of walnut oil was lower than the reported in other plant oils due to the high presence of polyunsaturated fatty acids.

After oil extraction, the remaining pressing cake may be transformed into partially defatted flour, with a high proportion of protein, fibre and essential minerals. This flour also showed differentiated nutritional characteristics depending on the cultivar of almond, pistachio or walnut considered.

In the analysis of the variability reported by the genotype (G), the crop year (CY) and the interaction GxCY, the genotype remained as the main factor determining oil chemometrics. However, significant variability of the crop year and the interaction GxCY was found for most of the parameters. In comparison to almond and pistachio oils, a greater effect of the crop year has been reported for most of the fatty acids in walnut oil, including stearic, oleic and linolenic. Moreover, the crop year affected the concentration of some minor components of crucial nutritional interest in nuts oils as total polyphenols and phytosterols.

Regarding oil extraction process, innovation is crucial for the development of high quality products. For this reason, the analysis of the influence of oil extraction parameters on product quality has been addressed. Within extraction methods, pressing by using two different presses (hydraulic and screw) has been considered. The extraction of vegetable oils by means of screw presses originates an increase in oil temperature due to the friction of the raw material. Oil temperature may reach values around 60˚C in almond and pistachio when oil is extracted at low temperatures, being somewhat lower in walnut oil. Results show that extraction temperature influenced greatly the physicochemical parameters of regulated quality of the oils.

Abstract/Resumen

When the oil obtained from the screw and the hydraulic press are compared, the use of higher temperatures on the first one resulted in changes in physicochemical and sensory properties of the oils. In the crew press, oil extraction with lower rotational speed resulted in higher yields and the oil was more valued by consumers. In the hydraulic press extraction, no differences were found regarding yield and physicochemical characteristics in oils by changing extraction parameters. The lack of influence of extraction parameters in physicochemical characteristics of oil suggest changing the focus to the influence of these parameters in yields and sensory evaluation.

To improve sensory attributes, a roasting step can be included in the nut oil production process before extraction. Pistachio roasting before extraction increased consumer preference, but at the same time it caused changes in pistachio oil physical parameters, oil oxidative stability and oil pigment concentration. Severe roasting conditions increased the oxidative stability of oils due to the antioxidant activity of Maillard reaction products. Different roasting intensities result in pistachio oils that can be classified into two groups. The first group of oils, obtained from natural pistachio or low temperature roasted pistachios showed yellow colour, while a second group of oils with green colour, were obtained after more severe roasting. Regarding pigments, carotenoids are transferred from pistachio to oil in greater proportion than chlorophylls in all samples. Therefore, the ratio between the chlorophyll and the carotenoid fraction is reduced considerably in oils. Higher roasting temperatures favoured the solubilisation of pigments in the oil, mainly in the fraction of chlorophyll derivatives.

Roasting has also an effect on the flours that appear as a by-product after oil extraction. The study of the effect of roasting on the antioxidant properties of defatted walnut flours concluded that all walnut flours had about 42% of protein and a significant amount of dietary fibre (17%), not being affected by the roasting process. Nonetheless, the fat content increased around 50% in walnuts flours subjected to longer and higher roasting temperatures. The lipid fraction that remains in the flour showed a good nutritional quality with a high vitamin E content (mainly γ-tocopherol)

Abstract/Resumen and fatty acid profile rich in linoleic and linolenic acids. The high phenolic content also provides great antioxidant capacity to the flours.

Regarding oil storage, the nuts oils are prone to rancidity due to the high proportion of unsaturated fatty acids. Thus, studies about storage conditions are necessary in order to evaluate the shelf life of these oils. Almond, pistachio and walnut oils were stored for a 16 months period under different conditions. Storage at room temperature originated a fast increase in the peroxide values, especially in the case of pistachio oil exposed to daylight. The oxidative stability decreased during the storage period for all three nut oils, regardless of the storage conditions. Pistachio oil remained the most stable oil at the end of the storage period, followed by almond oil. In addition, the percentage of polyunsaturated fatty acids decreased slightly throughout the storage.

Another innovation in nuts oils may be the addition of natural antioxidants, like garlic. The direct addition of garlic showed slight reductions on oils stability. However, differences appear depending on the garlic cultivar and the way of garlic addition. The addition of the whole clove, cause lower reductions on oil stability than the addition of crushed garlic, but only on roasted oils. Within the considered garlic cultivars, the “Purple garlic from Las Pedroñeras” (European Protected Geographical Indication) showed the better results for further study.

Economic factors related to nut and nut oil production are also considered. The effect of food safety legislation on pistachio trade is analysed by using a gravity model evaluating the effect of importer aflatoxin legislation on main exporting countries. In this sense, results show that stricter requirements regarding food safety are positive for exporting countries, regardless of their level of economic development.

Moreover, the production cost of two proposed production lines for the industrial production of pistachio have been calculated. According to production costs, the breakeven value that makes screw press extraction line sustainable is 70.4 €/litre, while for the hydraulic press extraction line is 91.0 €/litre, due mainly to a lower extraction yield and the larger extraction time required.

Abstract/Resumen

Resumen

Las propiedades beneficiosas que los frutos secos tienen para la salud hacen aconsejable su estudio y el desarrollo de procesos industriales destinados a obtener a partir de ellos aceites de elevada calidad y otros productos derivados de esta extracción, como harinas parcialmente desengrasadas. Aunque muchos estudios han analizado estos productos, existen ciertas carencias en algunas áreas relacionadas con la caracterización de variedades y su influencia en los aceites y las harinas obtenidas, los cambios que el proceso de extracción utilizado tiene en las características sensoriales y fisicoquímicas de los productos obtenidos, la innovación de producto para mejorar las características del aceite y el análisis de los factores económicos relacionados con los propios frutos secos y los aceites obtenidos a partir de ellos.

Por ello, como primer paso, se han caracterizado diferentes variedades de almendra, pistacho y nuez atendiendo a parámetros físicos, químicos y sensoriales, con el objetivo de evaluar la influencia que la materia prima puede tener en las características de los aceites obtenidos. Además, se ha analizado la influencia de la campaña en la composición química de los aceites y las harinas obtenidas.

Los parámetros físicos han demostrado ser útiles para la discriminación de diferentes variedades de almendra, pistacho y nuez. En el análisis sensorial, y utilizando pruebas afectivas, los consumidores mostraron preferencias de sabor y atractivo visual por algunas variedades de almendra (Vairo, Belona y Penta) y de pistacho (Larnaka, Aegina y Sirora). Respecto a los parámetros físicos de los aceites obtenidos, se encontraron diferencias significativas en parámetros como el color o la viscosidad. Además, la viscosidad de los aceites obtenidos mostró fuertes correlaciones con parámetros químicos de gran interés como el perfil de ácidos grasos y el perfil de triglicéridos.

Los aceites obtenidos de las diferentes variedades estudiadas mostraron también diferencias en su composición química. En las variedades de almendra, el rendimiento de aceite fue similar, encontrándose sin embargo diferencias significativas en la estabilidad oxidativa y en el perfil de ácidos grasos,

Abstract/Resumen fundamentalmente en las concentraciones de linoleico, esteárico y palmitoleico. En relación al aceite de pistacho, se identificaron tres grupos de variedades en función de su composición fisicoquímica y el rendimiento de aceite. Las variedades del grupo 1 mostraron un mayor contenido de ácido linoleico (superior al 26.4%), mientras las variedades del grupo 3 mostraron una mayor proporción de ácido oleico (mayor del 67.8%), dando así lugar a aceites más estables frente a la oxidación. Las variedades con características intermedias fueron las incluidas en el grupo 2. Entre las variedades de nuez, se encontraron diferencias significativas en el perfil de ácidos grasos y en la concentración de componentes minoritarios de interés como la concentración de polifenoles. El ácido linoleico supuso más del 47.5% del total de ácidos grasos en todas las variedades de nuez, suponiendo más del 62.13% y del 60.02% en las variedades Pedro y Mollar de Nerpio, respectivamente. Como se esperaba, la estabilidad oxidativa de los aceites de nuez fue inferior a la de los otros frutos secos debido a su elevado contenido en ácidos grasos poliinsaturados.

Tras la extracción de aceite, la torta de prensado que queda como subproducto puede ser triturada para transformarla en una harina parcialmente desengrasada. Esta harina tiene un gran interés nutricional, debido a su elevada proporción de proteínas, de fibra y de minerales esenciales. El perfil nutricional de las harinas obtenidas a partir de las diferentes variedades de almendra, pistacho y nuez analizadas, también ha mostrado diferencias nutricionales significativas entre las mismas.

En el análisis de la variabilidad aportada por el genotipo (G), la campaña (C) y la interacción GxC en las características de los aceites, el genotipo fue identificado como el factor que más influencia tenía en las características químicas de los mismos. Sin embargo, la variabilidad aportada por la campaña y la resultante de la interacción entre el genotipo y el año también resultaron ser significativas para la mayoría de los parámetros. Frente a los aceites de almendra y pistacho, en los aceites de nuez se ha observado una mayor influencia de la campaña, afectando en mayor medida a ácidos grasos relevantes como el esteárico, el oleico y el linoleico. Además, la campaña ha demostrado ser la principal causante de variabilidad en la concentración de

Abstract/Resumen componentes minoritarios de interés en los aceites de frutos secos, como el contenido total de polifenoles y de fitosteroles.

En relación al proceso de extracción de aceite, la innovación se presenta como un elemento crucial para obtener productos de elevada calidad. Por esta razón, se ha evaluado la influencia que los parámetros de extracción de aceite tienen sobre la calidad del producto obtenido. De entre los posibles métodos de extracción de aceite de frutos secos que existen, se han evaluado dos métodos de presión utilizando prensas diferentes (prensa de tornillo y prensa hidráulica). La extracción de aceites vegetales utilizando la prensa de tornillo origina un aumento en la temperatura del aceite obtenido debido a la fricción de la propia materia prima en el interior del tornillo. Por ello, en la extracción de almendra y de pistacho, el aceite alcanza temperaturas de en torno a 60˚C, siendo algo menor en el caso de la nuez. Los resultados obtenidos, demuestran además, que la temperatura de extracción afecta de forma notable a los parámetros fisicoquímicos de calidad regulada de los aceites obtenidos.

Cuando los aceites obtenidos con la prensa de tornillo y la hidráulica se comparan, se observa como el uso de las elevadas temperaturas en la extracción con la primera origina diferencias en los parámetros fisicoquímicos y en las propiedades sensoriales de los aceites obtenidos. En la prensa de tornillo, la extracción de aceite a velocidades de rotación más bajas da lugar a mayores rendimientos y a aceites mejor valorados por los consumidores. En cambio, en la extracción con la prensa hidráulica, no se encontraron diferencias en el rendimiento y en las características fisicoquímicas de los aceites obtenidos en función de los parámetros de funcionamiento. La falta de influencia de los parámetros de extracción en los parámetros fisicoquímicos de los aceites obtenidos con las dos prensas, aconseja cambiar el objeto de estudio para centrarse en su influencia en los rendimientos y en la valoración sensorial.

El tostado del fruto seco como paso previo a la extracción de aceite sirve para mejorar los atributos sensoriales de los aceites obtenidos. Así, el tostado del pistacho previo a la extracción, aumenta la preferencia de los consumidores; causando al mismo tiempo cambios en los parámetros físicos, en la estabilidad oxidativa y en la

Abstract/Resumen concentración de pigmentos de los aceites. Condiciones severas de tostado aumentan la estabilidad oxidativa de los aceites debido a la actividad antioxidante de los productos que resultan de la reacción de Maillard. Los resultados apuntan a que diferentes intensidades de tostado dan lugar a aceites diferentes, que pueden ser clasificados en dos grupos. El primero estaría compuesto por aquellos aceites amarillos, obtenidos a partir del pistacho natural o de pistachos ligeramente tostados, mientras un segundo grupo estaría compuesto por aceites de color verde, obtenidos tras tostados más intensos. En relación a los pigmentos, los carotenoides se transfieren del pistacho al aceite en mayor proporción que las clorofilas en todas las muestras. Así, el ratio entre la fracción de las clorofilas y la de los carotenoides se reduce considerablemente en los aceites. Los resultados demuestran, sin embargo, que las temperaturas de tostado más elevadas favorecen la solubilización de los pigmentos en el aceite, principalmente de aquellos de la facción de la clorofila y sus derivados.

El tostado previo del fruto también afecta a las harinas que aparecen como subproducto tras la extracción de aceite. El estudio de los efectos del tostado en las características de la harina de nuez desengrasada concluyó que todas las harinas obtenidas tenían un contenido en proteínas de alrededor del 42% y una cantidad significativa de fibra (17%), independientemente de las condiciones de tostado. Sin embargo, el contenido de lípidos aumentaba aproximadamente un 50% en aquellas harinas que habían sido sometidas a unas temperaturas de tostado más elevadas durante más tiempo. La fracción lipídica de las harinas mostró una buena calidad nutricional, con un contenido elevado de vitamina E (fundamentalmente γ-tocoferol) y un perfil de ácidos grasos con contenidos elevados de linoleico y linolénico. Además, el elevado contenido fenólico identificado en las harinas proporciona a las mismas gran capacidad antioxidante.

Uno de los posibles limitantes para la difusión del consumo de aceites de frutos es su correcto almacenamiento, ya que por su elevada proporción de ácidos grasos insaturados resultan especialmente sensibles al enranciamiento. Así, el estudio de las condiciones de almacenamiento más adecuadas es también un tema importante a considerar. Para ello los aceites de almendra, pistacho y nuez se almacenaron

Abstract/Resumen durante 16 meses bajo diferentes condiciones. El almacenamiento a temperatura ambiente ocasionó un rápido incremento de los valores de peróxidos, especialmente en el caso del aceite de pistacho expuesto a luz solar. La estabilidad oxidativa de los tres aceites disminuyó durante el almacenamiento, independientemente de las condiciones. El aceite de pistacho mostró una mayor estabilidad al final del almacenamiento, seguido por el aceite de almendra. En los tres aceites se observó una reducción significativa del porcentaje de ácidos grasos poliinsaturados tras el almacenamiento.

Una posible innovación en los aceites de frutos secos es la adición de antioxidantes naturales, como el ajo, con el objetivo de aumentar su estabilidad. La adición directa de ajo causó ligeras reducciones en la estabilidad oxidativa de los aceites de almendra. Sin embargo, se observaron diferencias en función de la variedad de ajo utilizada y la forma de adición del ajo. La adición del diente entero causó una reducción menor de la estabilidad que la adición del ajo triturado, pero sólo en los aceites obtenidos a partir de las almendras tostadas. De entre las variedades de ajo utilizadas, el “Ajo Morado de Las Pedroñeras” (Indicación Geográfica Protegida, IGP) mostró los mejores resultados para ser utilizado en futuros estudios.

Los factores económicos relacionados con los frutos secos y los aceites de frutos secos también deben ser tenidos en cuenta a la hora de valorar la creación de una industria de este tipo. El efecto de la legislación sobre seguridad alimentaria en el comercio internacional de pistacho fue analizado utilizando un modelo de gravedad para evaluar el efecto de la legislación sobre aflatoxinas sobre las exportaciones de los principales países productores. Los resultados indican que una legislación más restrictiva en los países importadores tiene un efecto positivo para los países exportadores, independientemente de su nivel de desarrollo económico.

Además, se han evaluado los costes de producción de dos líneas diferentes de producción de aceite de pistacho a escala industrial. De acuerdo a los costes de producción, el precio que haría sostenible la producción de aceite de pistacho con la línea de producción que incluye la prensa de tornillo sería de 70.4 €/litro, mientras para la extracción con prensa hidráulica sería de 91.0 €/litro. Las diferencias en el

Abstract/Resumen coste de producción estarían debidas sobre todo a los menores rendimientos y al mayor tiempo de extracción necesario con la prensa hidráulica.

i

Index/Índice

ii

Index/Índice

...... 1 1.2. Caracterización de los frutos secos ...... 5 1.3. Innovación de proceso y de producto ...... 11 1.4. Factores económicos...... 16 1.5. Objetivos ...... 20 1.6. Bibliografía ...... 21 ...... 29 2.1. Physical and sensory analysis of nuts cultivars ...... 31 Physical and sensory analysis of almond and pistachio kernels ...... 33

Physical analysis of pistachio kernels and pistachio oils ...... 51

Evaluation of physical parameters of walnut kernels and walnut products ...... 69 2.2. Physicochemical analysis of oils and partially defatted flours ...... 91 Suitability of Spanish almond cultivars for the industrial production of almond oil and defatted flour ...... 93

Chemometric characterization of pistachio oils and defatted flours regarding cultivar and geographic origin ...... 115 2.3. Influence of crop year ...... 143 Influence of genotype and crop year in the chemometrics of almond and pistachio oils ...... 145

Effect of genotype and crop year in the nutritional value of walnut virgin oil and defatted flour ...... 169

...... 193 3.1. Process innovation ...... 195 Influence of the temperature in the extraction of nuts oils by means of screw press 197

Optimization of pistachio oil extraction regarding processing parameters of screw and hydraulic presses ...... 219

Storage stability and composition changes of nuts oils under refrigeration and room temperature conditions ...... 243 3.1. Product innovation ...... 261 Changes in physical parameters, chlorophyll and carotenoids in pistachio oil with previous roasting ...... 263

Effect of roasting conditions on the composition and antioxidant properties of defatted walnut flour ...... 281

iii

Index/Índice

Effect of almond roasting and addition of different garlic cultivars on oil stability .... 305

...... 319

Influence of food safety on pistachio trade ...... 321

Analysis of production costs for the industrial production of pistachio oil by using two different production lines ...... 357

...... 379

...... 389

iv

1

Introducción

2

Introducción

1.1. Actualidad de los frutos secos

Durante los últimos diez años, se ha observado un incremento del interés de los consumidores por el consumo de frutos secos, que ha propiciado un importante incremento en la producción de los mismos. Si se analizan los datos de producción de frutos secos a nivel mundial, es fácil observar el crecimiento importarte de las producciones. La producción de nuez a nivel mundial ha pasado de las 1.6 x 106 toneladas de 2004 a las más de 3.5 x 106 toneladas de 2014. En el caso de la almendra, en estos mismos años, la producción ha pasado de las 1.6 x 106 toneladas de 2004 a 2.7 x 106 toneladas de 2014. La producción de pistacho ha sido tradicionalmente inferior comparada con la de los frutos secos anteriores, sin embargo, la progresión ha sido similar, pasando de producciones mundiales de 0.44 x 106 toneladas en 2004 a las más de 0.85 x 106 toneladas de 2014 (FAO, 2016).

El cultivo de frutos secos está limitado geográficamente a zonas secas y con temperaturas elevadas, en las que, en comparación con otros cultivos, estos árboles son capaces de proporcionar los rendimientos más altos y los frutos secos de mayor calidad. Así, podemos identificar tres grandes zonas productoras a nivel mundial: la cuenca mediterránea, oriente medio y Asia central y Estados Unidos (EEUU) (Figura 1). La producción de nuez se concentra mayoritariamente en China, EEUU, e Irán con producciones también significativas en Turquía. La producción de almendra ha estado tradicionalmente liderada por EEUU, seguida de lejos por España; pero en los últimos años la producción de Australia ha aumentado de forma notable, superando la producción de nuestro país en algunas campañas. Por otro lado, la producción de pistacho está completamente dominada por las producciones de Irán y EEUU, que han producido, de media, el 70% de la producción global de pistacho en el periodo 1993-2013 (FAO, 2016).

Analizando los países productores nos damos cuenta de que pueden ser fácilmente clasificados en dos grupos muy diferenciados. El primero estaría compuesto por países en desarrollo y subdesarrollados de oriente medio y Asia central, con producciones caracterizadas por un escaso uso de tecnología, y cuyas ventajas estarían fundamentadas en unos bajos costes de producción (basados en bajos costes

3

Introducción de explotación y de mano de obra). El segundo grupo, compuesto por países desarrollados como EEUU o Australia, obtendría sus ventajas de un elevado uso de tecnología, de inputs y de innovación en sus producciones.

Figura 1. Porcentaje de producción de almendra, nuez y pistacho sobre el total de la producción mundial en 2014. Elaboración propia a partir de FAO (2016).

Esta realidad deja a España en una posición intermedia entre ambos grupos, que puede tener efectos negativos. En España, los frutos secos han sido un cultivo tradicional, que se ha desarrollado mayoritariamente en tierras marginales, donde era imposible implantar otro tipo de cultivos, teniendo así un papel importante en la conservación de los suelos en zonas áridas y de pendiente. Para muchas familias, los frutos secos han sido un complemento a su renta, frente a otra actividad agraria principal, como el cereal, el olivar o el viñedo. Debido a todas estas condiciones, la producción nacional de frutos secos, y en mayor medida la producción de Castilla-La Mancha, es una producción poco especializada, poco intensiva y poco competitiva,

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Introducción que podría tener problemas de viabilidad ante un aumento de la producción de frutos secos a nivel mundial.

Es necesario también apuntar que los frutos secos han experimentado en los últimos años un aumento de precios muy importante, debido fundamentalmente a la gran demanda que apenas puede ser cubierta con la producción actual. Sin embargo, en algunos frutos secos como la almendra, las organizaciones agrarias ya están apuntando a la creación de burbujas debidas, precisamente, a este aumento desmedido de los precios. Ante un escenario de mercados colapsados o de precios bajos, la producción nacional y regional, tendría muchos problemas para ser rentable.

Ante esta realidad se hace necesario impulsar el desarrollo de la producción nacional de frutos secos hacia un escenario más competitivo. Por ello, el uso de la tecnología y la innovación en la producción y en la posterior transformación y comercialización de los frutos secos se concibe como un elemento fundamental a potenciar. Ante este escenario, nuestra propuesta es el uso de los frutos secos (almendra, nuez y pistacho) en procesos industriales dedicados a la extracción del aceite contenido en su interior, analizando también las características y posibles usos de las harinas parcialmente desengrasadas que se obtienen en el citado proceso.

1.2. Caracterización de los frutos secos

El interés por el estudio de los frutos secos y sus productos derivados, está fundamentado en los beneficios que su consumo tiene para la salud humana. Entre sus beneficios destaca la reducción que su consumo ocasiona en los niveles de colesterol LDL y el aumento de los niveles de HDL, la mejora del tránsito intestinal, la prevención de la anemia, la reducción de la tensión arterial y la prevención del cáncer, ya que su consumo estimula los mecanismos inmunológicos, presentando además un elevado contenido en compuestos antioxidantes, que protegen de la acción de los radicales libres (Abbey et al., 1994; Awad y Fink, 2000; Amaral et al., 2005; Martínez et al., 2006; Gentile et al., 2007; Yang et al., 2009; Kodad et al., 2011; Robbins et al., 2011). Además, a pesar de su elevado contenido en lípidos, el consumo

5

Introducción de frutos secos no está relacionado con un aumento de peso (Ibarrola-Jurado et al., 2013).

Por todo lo anterior, los frutos secos y su estudio han venido llamando la atención de los investigadores durante los últimos años, aumentado de forma muy notable el número de artículos científicos publicados sobre ellos (figura 2). El interés es tal, que desde el año 2000 el número de publicaciones relacionadas con la almendra, la nuez y el pistacho no ha dejado de aumentar año a año, pasando de las 294 publicaciones que incluían estos términos en el año 2000 a las 1.125 del año 2016.

Figura 2. Evolución del número de trabajos científicos sobre frutos secos disponibles en la base de datos de Scopus (https://www.scopus.com). Términos incluidos en el buscador: Pistachio, Walnut y Almond.

Una de las características principales de los frutos secos es su elevada proporción de lípidos (Tabla 1). En la almendra, los lípidos suponen un 40 - 67 % del peso del fruto (Yada et al., 2011), siendo similar en el pistacho, con un 50 - 62 % (Catalán et al., 2017). En la nuez esta proporción es superior, suponiendo un intervalo de 60 - 72 % del peso total del fruto (Amaral et al., 2003; Kodad et al., 2016). Entre los lípidos, destaca la gran proporción de ácidos grasos insaturados, destacando fundamentalmente el elevado contenido de ácido oleico, superior al 50 % en la almendra y el pistacho (Roncero et al., 2016; Catalán et al., 2017), y los porcentajes elevados de linoleico en el perfil de ácidos grasos de la nuez (Crews et al., 2005; Martínez et al., 2006). Además de los ácidos grasos, los frutos secos presentan un elevado contenido en proteínas, que además han sido identificadas como proteínas

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Introducción de elevada calidad debido a su elevada digestibilidad y a la elevada concentración de aminoácidos esenciales que presentan (Sze-Tao y Sathe, 2000). Los frutos secos tienen además una interesante cantidad de minerales, muchos de ellos esenciales (Roncero et al., 2016), entre los que destacan algunos como el magnesio, el calcio o el potasio, que unidos previenen la desmineralización de los huesos, reduciendo además la presión arterial y el riesgo de enfermedades cardiovasculares (Segura et al., 2006).

Tabla 1. Contenido en aceite y perfil de ácidos grasos de la almendra, la nuez y el pistacho. Ácidos grasos (% sobre el total) Contenido en Frutos secos Ácido Ácido Ácido Ácido aceite (%) oleico linoleico linolénico Palmítico Almendra 40,0 - 67,0 57,5 – 78,7 12,0 – 33,9 0,05 – 0,09 5,2 – 7,7 Nuez 60,0 - 72,0 14,2 – 25,0 50,0 – 72,1 10,0 – 12,5 7,4 – 8,0 Pistacho 50,0 - 62,0 51,6 – 76,5 8,1 – 29,8 0,27 – 0,45 8,5 – 13,0 Amaral et al. (2003); Crews et al. (2005); Martínez et al. (2006); Yada et al. (2011); Cuesta et al. (2017); Kodad et al. (2016); Roncero et al. (2016); Catalán et al. (2017).

Más allá del estudio de componentes de gran interés, como los ácidos grasos, el estudio de los frutos secos ha prestado especial atención a los componentes minoritarios, debido a la elevada influencia que éstos tienen en la calidad nutricional de los mismos. Los aceites de frutos secos se consideran así una fuente de vitamina E, debido a su elevado contenido en tocoferoles. La concentración de tocoferoles se sitúa entre 194 a 297 mg/kg para la nuez (Amaral et al., 2005), 350 a 550 mg/kg en el aceite de almendra (Kodad et al., 2014) y en torno a 350 mg/kg en el aceite de pistacho (Ling et al., 2016). Además de tocoferoles, existen otros compuestos minoritarios de gran interés, como los fitosteroles, que aparecen en concentraciones muy elevadas en el aceite de pistacho, de hasta 271,9 mg por cada 100 g de aceite. En el aceite de almendra y el aceite de nuez, esta concentración es menor con 161,8 mg/100 g de aceite de almendra (Kornsteiner-Krenn et al., 2013) y 140 mg/100g como término medio en el aceite de nuez (Crews et al., 2005).

Todas las ventajas que tiene para la salud el consumo de frutos secos se deben a la presencia de los diferentes componentes comentados anteriormente, que varían en

7

Introducción función del fruto seco considerado; pero también en función de otros factores que es necesario evaluar, como la variedad concreta analizada. En los pistachos de diferentes variedades, diversos estudios han encontrado diferencias en el perfil de ácidos grasos, triglicéridos, fitosteroles, aminoácidos y minerales (Dyszel y Pettit, 1990; Küçüköner y Yurt, 2003; Arena et al., 2007; Bellomo y Fallico, 2007; Mahmoodabadi, 2012). En la almendra se han encontrado diferencias en parámetros como el contenido total de aceite, el perfil de ácidos grasos, los triglicéridos o la concentración de tocoferoles, entre otros (Kodad et al., 2011; Yada et al., 2011; Kodad et al., 2014; Roncero et al., 2016). De igual forma, en la nuez, las variedades estudiadas han demostrado producir frutos con diferencias en su perfil de ácidos grasos, tocoferoles, triglicéridos, esteroles o contenido proteico (Amaral et al., 2003; Amaral et al., 2005; Crews et al., 2005; Bada et al., 2010; Kodad et al., 2016).

Más allá de la variedad, las condiciones climáticas y las prácticas culturales que se realizan en campo son también factores fundamentales a analizar, ya que su influencia en las características de los frutos secos obtenidos ha demostrado ser crucial (García-López et al., 1996; Sánchez-Bel et al., 2008; Yada et al., 2011; Kodad et al., 2014; Carbonell-Barrachina et al., 2015; Zhu et al., 2015). De esta forma, los frutos secos cultivados en lugares diferentes y, por tanto, sometidos a diferentes condiciones climáticas y diferentes prácticas culturales, han mostrado presentar diferencias significativas en algunos parámetros fisicoquímicos.

En el caso del pistacho, los frutos secos cultivados en lugares como Túnez (Chahed et al., 2008; Ghrab et al., 2010), Turquía (Küçüköner y Yurt, 2003; Seferoglu et al., 2006; Harmankaya et al., 2014;) o Irán (Kamangar y Farsam, 1977; Mahmoodabadi, 2012) presentan diferencias en su composición. Resultados similares se han obtenido para la nuez (Amaral et al., 2005; Crews et al., 2005) y la almendra (García-López et al., 1996; Yada et al., 2011; Maestri et al., 2015; Roncero et al., 2016;). Cuando las diferentes variedades son cultivadas en distintos lugares, la influencia de las condiciones ecológicas y las prácticas culturales pueden causar diferencias en los frutos secos que no deben ser atribuidas a la variedad. Así, por ejemplo, se ha demostrado el efecto directo que el riego tiene sobre el contenido total de

8

Introducción tocoferoles en frutos secos como la almendra o la nuez (Amaral et al., 2005; Crews et al., 2005; Bada et al., 2010; Kodad et al., 2016).

La mayoría de los trabajos de investigación realizados han evaluado frutos secos de diferentes variedades producidos, además, en lugares diferentes, siendo pocos los estudios que han analizado diferentes variedades cultivadas en el mismo lugar, y por tanto, sometidas a las mismas condiciones climáticas y a las mismas prácticas culturales (Bellomo y Fallico, 2007; Tsantili et al., 2010; Tsantili et al., 2011; Maestri et al., 2015). Teniendo en cuenta todo lo anterior, parece necesario analizar las características fisicoquímicas y sensoriales de las diferentes variedades de almendra, pistacho y nuez disponibles en el mercado, cultivadas bajo las mismas condiciones. Sólo de esa manera se puede identificar el verdadero alcance de la variedad en la aparición de diferencias.

Con esta idea, el primer bloque de capítulos de esta tesis está centrado en el análisis de las características fisicoquímicas y sensoriales de diferentes variedades de almendra, nuez y pistacho, cultivadas bajo las mismas condiciones. Las variedades consideradas son aquellas que de forma mayoritaria se han utilizado en la producción de frutos secos en España. Se debe tener en cuenta que el cultivo de las variedades incluidas en este estudio se ha realizado en el marco del clima mediterráneo- continentalizado propio de la meseta sur, en la región de Castilla-La Mancha.

Concretamente, se han analizado las características físicas y sensoriales de las almendras, las nueces y los pistachos obtenidos de diferentes variedades. A pesar de la importancia que los parámetros físicos y sensoriales de los frutos secos, y los productos obtenidos a partir de ellos, tienen para los consumidores y para la industria alimentaria, estos parámetros no han sido extensamente analizados en la bibliografía (Tsantili et al., 2010). Parámetros como el tamaño, el color o la dureza del fruto seco, determinan las preferencias de los consumidores o los posibles usos que la industria puede hacer de ellos (Kader et al., 1982; Tsantili et al., 2010). Entre los parámetros físicos del aceite, además del color, la viscosidad aparece con un parámetro clave a considerar, debido a la utilidad que presenta para determinar la calidad del aceite y

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Introducción a su importancia en el diseño industrial de procesos que incluyan la producción o utilización de estos aceites.

Con todo ello, parece necesario analizar las características físicas y sensoriales de diferentes variedades de frutos secos, analizando si estos parámetros pueden ser utilizados para discriminar entre las diferentes variedades consideradas. Además, cabe la posibilidad de utilizar los parámetros físicos analizados y las valoraciones de los consumidores para analizar la formación de preferencias de los mismos.

Más allá de los parámetros físicos y sensoriales, desde el punto de vista nutricional, el estudio de la composición química de los aceites y harinas obtenidos a partir de las diferentes variedades consideradas es crucial para identificar su calidad. Precisamente, este análisis de los componentes nutricionales de las diferentes variedades es el objetivo de varios capítulos integrados dentro del bloque dedicado a la caracterización.

Así, se han analizado las diferencias que existen en el aceite y las harinas obtenidas a partir de diez variedades de almendra diferentes, analizando además la utilidad de cada una de las variedades para la producción de aceite, utilizando tres parámetros considerados fundamentales para la industria agroalimentaria (Maestri et al., 2015): el rendimiento de aceite, el índice de yodo de los aceites obtenidos y la estabilidad oxidativa de los mismos.

De forma similar, se han analizado hasta veinte variedades diferentes de pistacho, analizando las diferencias nutricionales que existen en los aceites y las harinas obtenidas a partir de cada una de ellas. Además, agrupando las variedades por origen, se ha determinado si se pueden establecer diferencias entre ellas en función del país en el que aparecieron o fueron documentadas por primera vez.

La influencia de las condiciones climáticas, anteriormente descritas como fundamentales, se puede analizar teniendo en cuenta los efectos de la campaña en las características fisicoquímicas de los frutos secos obtenidos cada año (Amaral et al., 2005; Maestri et al., 2015). Para ello es necesario contar con datos obtenidos a partir de frutos secos que hayan sido recogidos de los mismos árboles durante al

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Introducción menos dos años. De esta forma se puede identificar si las condiciones climáticas, fundamentalmente la temperatura en el caso de que los árboles cuenten con un sistema de riego, afecta a las características de los frutos secos o productos obtenidos a partir de ellos.

Para analizar la influencia de la campaña en las características de los aceites y las harinas obtenidos, se ha realizado un seguimiento de las diferentes variedades de almendra, pistacho y nuez durante dos años consecutivos, concretamente durante las campañas 2015 y 2016. Los datos obtenidos se han utilizado para el desarrollo de la segunda parte de este primer bloque, dedicado a la caracterización de frutos secos, aceites y harinas. Concretamente, se ha analizado de forma conjunta el efecto de la campaña en los aceites y harinas de almendra, pistacho y nuez, analizando así qué parte de la variabilidad observada en los datos se debe a la propia variedad, qué parte de debe a la campaña y qué parte se debe a la interacción entre ambos factores.

Con todo ello, el trabajo realizado en el marco de este primer bloque temático tendría como objetivo la caracterización de los frutos secos y de los aceites y de las harinas obtenidas a partir de ellos. Supone así una interesante base de datos con parámetros de interés para agricultores, personal implicado en programas de cruzamiento de variedades y para responsables de la industria alimentaria. Además, permite identificar para cada uno de los parámetros fisicoquímicos considerados qué porcentaje de la variabilidad observada se debe a la influencia de la variedad y qué parte puede, por el contrario, ser atribuida a la campaña, sirviendo como punto de partida para el desarrollo de un producto de la mayor calidad nutricional y lo más homogéneo posible, siendo éstas algunas de las demandas fundamentales de los mercados actuales.

1.3. Innovación de proceso y de producto

La producción de aceites y harinas a partir de frutos secos, se presenta como una línea a explorar, por su capacidad para dinamizar y hacer más competitivas las producciones nacionales, aumentando el valor añadido de estos cultivos y posibilitando la creación de una industria transformadora sostenible, dedicada a la

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Introducción producción de alimentos funcionales con grandes perspectivas de crecimiento en los próximos años. El desarrollo de esta industria de bajo a medio contenido tecnológico, y su mantenimiento en el tiempo, precisa de un importante esfuerzo de innovación con el objetivo de fomentar la diferenciación de los productos obtenidos y mejorar la eficiencia de los procesos de producción. Por ello, la innovación de proceso y de producto es el tema del segundo bloque de capítulos de esta tesis.

La extracción de aceites de frutos secos se puede hacer utilizando diferentes métodos, que presentan diferentes rendimientos y sirven para producir aceites de diferentes calidades (Figura 3). La extracción con disolventes es el método que permite obtener los mayores rendimientos, dando también lugar a los aceites con menor calidad, debido a la aparición de sabores y olores poco deseables, causando además la inactivación o desaparición de vitaminas y otras sustancias bioactivas de interés (Satil et al., 2003; Miraliakbari y Shahidi, 2008; Abdolshahi et al., 2015). En los últimos años, el uso de fluidos supercríticos ha emergido como alternativa al uso de disolventes (Palazoglu y Balaban, 1998; Jokić et al., 2012). Concretamente, ha sido el uso de CO2 supercrítico el que ha permitido obtener los aceites de mayor calidad, debido a que con este método la extracción de aceite puede realizase a bajas temperaturas (Abbasi et al., 2008; Chan y Ismail, 2009). Utilizando fluidos supercríticos, la calidad del aceite obtenido y el rendimiento de aceite evolucionan en direcciones contrarias, ya que la reducción de temperatura, presión y caudal de

CO2, suponen una disminución de los rendimientos obtenidos (Jokić et al., 2012). Sin embargo, son los elevados costes de producción los que en mayor medida condicionan el uso de este método, limitando su utilización a la obtención de productos que presenten elevados precios (Rosa y Meireles, 2005).

El prensado en frío permite en cambio obtener aceites de elevada calidad a precios asequibles. Mediante prensado se pueden obtener aceites que pueden ser consumidos directamente, sin necesidad de refinado, conservando así todos los beneficios asociados al consumo directo de frutos secos (Álvarez-Ortí et al., 2012). El prensado en frío se realiza fundamentalmente con dos tipos de prensas: prensas de tornillo y prensas hidráulicas. Aunque la extracción utilizando la prensa de tornillo se ha clasificado habitualmente como una extracción en frío, para el adecuado

12

Introducción funcionamiento de la prensa es necesario aplicar temperaturas elevadas en la boquilla donde se produce la rotura del fruto y la salida del aceite. La aplicación de temperaturas elevadas, puede afectar a la calidad de los aceites obtenidos. Por ello, se ha analizado cómo la temperatura en diferentes puntos de la prensa de tornillo afecta a la calidad del aceite de almendra, nuez y pistacho, evaluando el verdadero alcance de esta variable sobre los parámetros fisicoquímicos de los aceites obtenidos.

Figura 3. Diagramas del sistema de extracción con fluidos supercríticos y de los sistemas de presión. Catalán et al. (2017).

Como ya se ha comentado anteriormente, además del prensado utilizando la prensa de tornillo, el prensado en frío para la obtención de aceite de frutos secos también puede realizarse utilizando prensas hidráulicas. La extracción utilizando este tipo de prensas permite obtener aceite a temperatura ambiente (Sena-Moreno et al., 2015), por lo que podría permitir obtener aceites de mayor calidad. La optimización de la extracción de aceite de pistacho con una prensa hidráulica y una prensa de tornillo en función de rendimientos, parámetros fisicoquímicos, análisis sensoriales, así como

13

Introducción la comparación de ambos sistemas de prensado, es también uno de los temas abordados en el marco de esta tesis.

El uso de una prensa u otra no sólo condiciona el rendimiento o la calidad del aceite obtenido, afecta también a los procesos previos a los que se puede someter el fruto seco del que se va a extraer el aceite. Uno de los pasos previos que se puede considerar en la producción de aceite de frutos secos, con el objetivo de diferenciar el producto, es el tostado del fruto seco. Sin embargo, esta operación sólo es recomendable cuando se utiliza la prensa hidráulica, debido a que el propio funcionamiento de la prensa de tornillo ya otorga a los aceites obtenidos propiedades similares a las que se consiguen con este tostado previo (Sena-Moreno et al., 2015).

La exposición de los frutos secos a elevadas temperaturas, bien sea debido a procesos de secado o tostado, puede aumentar la estabilidad oxidativa de los aceites obtenidos, debido a la aparición de productos de la reacción de Maillard que actúan como antioxidantes (Veldsink et al., 1999; Choe y Min, 2006). Además, estos productos resultantes de la reacción de Maillard, han demostrado otorgar al producto olores y colores agradables para los consumidores (Kashani y Valadon, 1984; Aceña et al., 2010), por lo que el tostado de frutos como la almendra o el pistacho pueden servir para aumentar la preferencia de los consumidores por los aceites obtenidos a partir de ellos. Además, el tostado de los frutos secos puede inactivar las enzimas responsables del enranciado (Durmaz y Gökmen, 2011; Pumilia et al., 2014), aumentando la calidad y la vida útil de los frutos secos. Sin embargo, también puede causar cambios en la composición fisicoquímica de los aceites y las harinas resultantes de la extracción, ocasionando la rotura de las paredes celulares, la desnaturalización de las proteínas, la esterilización o desactivación de las enzimas termosensibles de interés o cambios en pigmentos como carotenoides y clorofilas (Figura 4) (Bellomo et al., 2009; Pumilia et al., 2014; Savoire et al., 2013; Vaidya y Eun, 2013).

Para analizar los efectos del tostado previo del fruto en las características de los aceites y las harinas obtenidos a partir de ellos, en este segundo bloque de estudios centrado en la innovación de proceso y de producto, se ha analizado el efecto de las

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Introducción condiciones de tostado del pistacho (tiempo y temperatura) en la concentración de pigmentos y las características físicas del aceite obtenido con la prensa hidráulica. De forma similar, también se ha evaluado cómo la temperatura y el tiempo de tostado de la nuez afectan a la composición y al poder antioxidante de las harinas parcialmente desengrasadas.

Figura 4. Clorofila y sus derivados. Pumilia et al. (2014).

Para la correcta comercialización de estos aceites resulta fundamental identificar su vida útil y las condiciones más adecuadas para su conservación. La estabilidad oxidativa de los aceites vegetales está directamente influida por las condiciones de almacenamiento (temperatura, luz y disponibilidad de oxígeno), la composición de ácidos grasos y la presencia de componentes minoritarios en cada aceite concreto, incluyendo, ácidos grasos libres, mono y diacilgliceroles, fosfolípidos, clorofilas, tocoferoles, fitosteroles, compuestos fenólicos o carotenoides (Choe y Min, 2006).

Las diferencias identificadas en la composición de los aceites de almendra, pistacho y nuez hacen que se espere una diferente evolución de estos aceites durante su conservación. Por ello, es necesario analizar las condiciones de conservación más adecuadas para cada uno de los aceites de frutos secos estudiados. Con este objetivo, esta tesis incluye el estudio de la evolución en el tiempo de diferentes parámetros

15

Introducción fisicoquímicos de los aceites de almendra, pistacho y nuez, evaluando los efectos que diferentes condiciones de conservación tienen sobre ellos. Las condiciones de conservación planteadas combinan condiciones de luz y oscuridad con diferentes temperaturas de almacenamiento.

Una de las líneas de investigación que podría tener mayor recorrido, es el estudio de los procesos y sustancias que pueden ser utilizados para aumentar la vida útil de estos aceites, garantizando que lleguen al consumidor en condiciones óptimas y se mantengan así durante el mayor tiempo posible. Un posible aditivo de interés que podría ser añadido a los aceites de frutos secos, es el ajo. El ajo posee características que lo convierten en un producto interesante para ser añadido a diferentes productos, ya que ha demostrado poseer propiedades antifúngicas, antibacterianas, antivirales, antimicrobianas, anticancerígenas, antimutagénicas, antiasmáticas y antioxidantes (Corzo-Martínez et al., 2007; Iciek et al., 2009). Además, posee la ventaja de ser aceptado fácilmente por los consumidores, que prefieren el uso de productos naturales a aditivos alimentarios sintéticos (Pokorný, 1991). Más allá de sus beneficios para la salud, el ajo puede aportar a estos aceites nuevas características organolépticas, ofreciendo así nuevas posibilidades en el ámbito culinario. Diferentes experimentos in vitro han demostrado diferencias en la actividad antioxidante de diferentes variedades de ajo (Chen et al., 2013; Denre et al., 2013; Bhyari et al., 2014). Por ello, se ha analizado la influencia en el aceite de añadir cuatro tipos diferentes de ajo: ajo blanco, ajo violeta, ajo IGP “Ajo Morado de las Pedroñeras” y ajo negro (ajo morado fermentado). Además, se han considerado diferentes formas de añadir el ajo al aceite.

1.4. Factores económicos

Uno de los factores que más importancia tiene en la producción, comercio y consumo del pistacho es el riesgo sanitario que puede presentar para la salud su consumo de este producto. Esto se debe a que el pistacho ha sido identificado como el producto crudo que mayor riesgo presenta de causar contaminación por aflatoxinas en humanos (Pittet, 1998).

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Introducción

Las aflatoxinas son micotoxinas producidas por algunas de especies de Aspergillus (fundamentalmente Aspergillus flavus y Aspergillus parasiticus). La legislación recoge fundamentalmente cuatro tipos de aflatoxinas (B1, B2, G1 y G2), estando todas ellas catalogadas desde hace mucho tiempo como carcinogénicas por el Centro Internacional de Investigaciones sobre el Cáncer (IARC, 2002). De entre ellas, la aflatoxina B1 ha sido identificada como el carcinogénico de hígado más potente conocido (Wu y Guclu, 2012). Debido al elevado riesgo de contaminación por aflatoxinas que tiene el pistacho, diferentes organizaciones internacionales, como la Organización de las Naciones Unidas para la Alimentación y la Agricultura (FAO) o la Organización Mundial de la Salud (OMS), recomiendan implementar una estricta regulación sobre la concentración máxima de aflatoxinas permitida en este fruto seco, garantizando así la seguridad de los consumidores. Atendiendo a estas recomendaciones y con el objetivo de prevenir estos riesgos, la mayoría de países han creado regulaciones donde se incluyen las concentraciones máximas de aflatoxinas que pueden tener los productos producidos e importados, incluyendo regulaciones específicas para frutos secos y los pistachos (Tabla 2).

En el ámbito del comercio internacional de productos agroalimentarios, esta legislación restrictiva puede causar problemas a los países que exportan productos vulnerables a este tipo de contaminaciones, debido a los costes que supone la necesidad de realizar un control más intensivo (Otsuki et al., 2001). El rechazo de cargamentos en las fronteras aumenta los costes, causa alarma social y daña la reputación del exportador. Sin embargo, estos estándares más restrictivos pueden beneficiar a aquellos países que producen alimentos de elevada calidad, motivándolos a cumplir esta normativa si el precio ofrecido por producir de acuerdo a estas normas es superior. Estos límites más restrictivos podrían incluso beneficiar a los países exportadores que producen alimentos de menor calidad, presionándolos para que adopten tecnologías y métodos de control de la calidad, dando lugar a un escenario en el que todas las partes, incluidos los consumidores, obtendrían beneficios (Wu, 2008).

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Tabla 2. Límites máximos de concentración de aflatoxinas en pistachos para diferentes países. Standard para aflatoxinas totales en Standard para aflatoxinas totales País 1995 (FAO, 1997) (ng/g) en 2003 (FAO, 2004) (ng/g) EEUU 15 15 Irán Sin regulación 15 Unión Europea Sin regulación 4 (Cambia a 10 en 2009) Bélgica Sin regulación 4 (Cambia a 10 en 2009) Canadá 15 15 Alemania 4 4 (Cambia a 10 en 2009) Hong Kong 15 15 Japón 20* 201 Arabia Saudí Sin regulación Sin regulación China Sin regulación Sin regulación Egipto Sin regulación Sin regulación Países Bajos 101 4 (Cambia a 10 en 2009) Rusia 5 5 1Japón y los Países Bajos solo tienen un estándar para la aflatoxina B1 por lo que la concentración máxima para esa aflatoxina específica se ha duplicado. Bui-Klimke et al. (2014).

Sería esperable que un mayor nivel de desarrollo del país productor, pudiera garantizar ventajas a la hora de producir un producto más seguro (Jongwanich, 2009; Bao y Qiu, 2012; Chen et al., 2008; Drogué y DeMaria, 2012; Li y Beghin, 2014; Melo et al., 2014), que en este caso sería un pistacho con una menor concentración de aflatoxinas. Ésta es a priori una de las ventajas que podría tener la producción nacional de pistacho frente a la producción de países en desarrollo como Irán o Turquía.

Por todo lo anterior, el estudio sobre la influencia de la legislación sobre seguridad alimentaria en el comercio internacional de pistacho es también objeto de estudio de esta tesis. Para ello se han analizado todas las importaciones de pistacho desde EEUU e Irán en el periodo 1996-2014, analizando la influencia de la legislación sobre aflatoxinas de los países importadores sobre las exportaciones de estos dos países productores. Para ello, se han propuesto cuatro indicadores diferentes sobre la regulación de aflatoxinas. Además, se ha analizado la influencia de la innovación en la producción y en la gestión en las exportaciones desde estos dos países.

La segunda parte de este tercer bloque de factores económicos, evalúa la viabilidad de la producción de aceite de pistacho a escala industrial. El pistacho es uno de los

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Introducción frutos secos que mayor precio alcanza en el mercado, por lo que la producción de aceite de pistacho está considerada como un proceso costoso. En los estudios actualmente publicados sobre aceites de frutos secos, no se ha prestado apenas atención a los costes de producción, existiendo sólo algunos ejemplos de estudios sobre la viabilidad económica de producción de otros aceites, como el aceite de palma (Owolarafe et al., 2002) o el aceite de pepita de uva (Fiori, 2010).

La producción de aceite de frutos secos, y de pistacho en particular, es un proceso relativamente sencillo; pero que necesita de un minucioso control en cada una de las etapas de producción. Una correcta elección de la materia prima (Álvarez-Ortí et al., 2012), un adecuado control de las condiciones de secado o tostado (Sena-Moreno et al., 2015), o la correcta elección del método y los parámetros de extracción son fundamentales para la obtención de aceites de calidad.

Por todo ello, la parte final de esta tesis, utiliza los conocimientos previos adquiridos sobre las condiciones más adecuadas de extracción para cada una de las prensas (prensa de tornillo y prensa hidráulica), para evaluar las características del aceite de pistacho producido utilizando dos líneas de producción diferentes, que muy bien podrían representar dos líneas reales de producción de aceite en una industria. Tras analizar las características de los aceites obtenidos, se han analizado los costes de producción de aceite de pistacho con cada una de las líneas planteadas, identificando así el precio mínimo que debería tener el aceite de pistacho para cubrir los costes que tiene su producción.

Por todo ello, esta tesis recoge un estudio amplio y transversal de los frutos secos y los aceites y harinas obtenidos a partir de ellos, incluyendo factores agronómicos, nutricionales, industriales y económicos. El objetivo es sentar las bases para el desarrollo de una industria de producción de productos semielaborados a partir de frutos secos basada en la innovación, que haga viable la producción de frutos secos nacional y regional ante cualquier escenario venidero.

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1.5. Objetivos

El objetivo fundamental de esta tesis doctoral es crear las bases para el desarrollo de una industria dedicada a la producción de aceites de frutos secos y harinas desengrasadas de almendra, nuez y pistacho basada en la innovación, que otorgue valor añadido a la producción de frutos secos nacional y regional. Para ello, se identifican los siguientes objetivos secundarios:

. Analizar los parámetros físicos de la materia prima (fruto seco) y de los aceites.

. Caracterizar los aceites y las harinas obtenidos a partir de las diferentes variedades de cada uno de los frutos secos considerados.

. Analizar la variabilidad que aporta la campaña a los aceites en comparación con la aportada por el genotipo.

. Analizar la influencia de la temperatura en la extracción de aceites de frutos secos con la prensa de tornillo.

. Optimizar el proceso de extracción industrial de aceite de pistacho con prensa hidráulica y prensa de tornillo.

. Analizar la influencia del tostado previo del pistacho en la concentración de pigmentos y en características físicas del aceite obtenido.

. Analizar la influencia del tostado previo de la nuez en las propiedades antioxidantes de la harina desgrasada obtenida tras la extracción de aceite.

. Analizar cómo influyen las condiciones de conservación en la calidad de los aceites.

. Analizar cómo influye el tostado previo de la almendra y la adición de ajo en la conservación del aceite de almendra.

. Analizar la influencia de la legislación sobre seguridad alimentaria en las exportaciones de pistacho.

. Analizar los costes de producción industrial de aceite de pistacho.

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1.6. Bibliografía

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Introducción pistachio kernels (Pistacia vera L.) during roasting. Food Research International 65, 193-198.

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2.1. Physical and sensory analysis of nuts cultivars

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Physical and sensory analysis of almond and pistachio kernels

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Physical and sensory analysis of almond and pistachio kernels

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Influence of raw material in oil characteristics

Physical and sensory analysis of almond and pistachio kernels

ABSTRACT Keywords: The differences on the characteristics of kernels form different almond and Pistacia vera Prunus dulcis pistachio genotypes have been widely analysed attending to chemical Consumer characteristics with little attention been paid to physical parameters. Sensory analysis Regarding sensory analysis, most of studies have relied on train judges to Colour differentiate almond and pistachio genotypes, while the preferences of consumer type panellist have not been properly considered. In this study, the analysis of physical parameters size, weight, colour and hardness using technical procedures and consumers preferences have been performed in ten different almond genotypes and ten pistachio genotypes grown on the same plot. Results show that physical parameters are useful for cultivar discrimination even when nuts are grown on the same plot. Consumers show significant preferences in overall visual acceptance and taste for some almond (Vairo, Belona and Penta) and pistachio cultivars (Larnaka, Aegina and Sirora). In almonds, physical parameters width and colour parameter b* inside the kernel are correlated with higher consumer valuations for overall visual acceptance and taste, respectively. Correlations between physical parameters and consumer valuations can be the first step in the study of formation of consumer preferences. Breeders and the food industry should take into account consumer preferences for some cultivars.

1. Introduction

The quality of nuts has been primarily based on their nutritional composition, mainly fatty acid profile and bioactive compounds (Chen et al., 2006; Yada et al., 2011; Roncero et al., 2016a; Catalán et al., 2017). Almonds and pistachios show a similar fatty acid profile with dominance of unsaturated fatty acids, mainly oleic and linoleic (Roncero et al., 2016a, b; Catalán et al., 2017;), and a wide range of health promoting components as fibre, tocopherols, minerals and phytonutrients (Kamil and Chen,

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Physical and sensory analysis of almond and pistachio kernels

2012). A wide range of studies have analysed the chemical differences of different cultivars (Roncero et al., 2016b; Catalán et al., 2017; Rabadán et al., 2017a), while less attention has been paid to physical differences among cultivars (Contador et al., 2015; Rabadán et al., 2017c), that should also be considered as quality related parameters.

Physical parameters in nuts have effects on consumer preference and industrial processes (Tsantili et al., 2010; Rabadán et al., 2017c). They can be evaluated by using technical procedures or sensory analysis (Aktas et al., 2007; Tsantili et al., 2010). By using technical procedures, significant differences have been reported in the kernel size, colour, crispness and firmness in kernels from different pistachio cultivars (Kader et al., 1982; Tsantili et al., 2010). Differences in colour related to the cultivar have been also found in pistachio by-products (Rabadán et al., 2017c). In almonds, the mechanical properties of the shell and kernel have been widely studied using technical procedures (Aydin, 2003; Ledbetter and Palmquist, 2006; Aktas et al., 2007).

Regarding sensory analysis of almonds and pistachios, most of the studies have analysed the effect of processing parameters, mainly roasting time and temperature, roasting system and salting on sensory characteristics (Vázquez-Araújo et al., 2009; Shakerardekani et al., 2011; Penci et al., 2013; Milczarek et al., 2014; Rabadán et al., 2017b). In pistachios, the aroma after roasting has been identified as determinant for consumer acceptance (Aceña et al., 2010) while the storage at different temperatures, although increased the roasting flavour, did not increased significantly overall flavour intensity (Kader et al., 1982). In almonds, the roasting decrease sweetness and increased bitterness and grittiness (Gou et al., 2000).

As a result, sensory characteristics of almonds and pistachios have been mainly described in the roasted kernel (Guerrero et al., 1997; Varela et al., 2006; Varela et al., 2008; Vázquez-Araújo et al., 2009; Shakerardekani et al., 2011; Penci et al., 2013; Mokhtarian et al., 2017;) with little attention been paid to the fresh almonds (Contador et al., 2015) and pistachios (Kader et al., 1982). Nut flavour, texture, colour, and appearance are modified with roasting resulting in a completely different

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Influence of raw material in oil characteristics

product (Nikzadeh and Sedaghat, 2008; Shakerardekani et al., 2011). Then the results of the sensory analysis of roasted almonds and pistachios should not be directly assumed as proportional to those obtained on the fresh nuts, as the appearance and evolution of main components of sensory odour and flavour aromas with roasting also differ depending on the genotype (Vázquez-Araújo et al., 2009).

Sensory studies have been focused on the descriptive characterization and the sensory evaluation of nuts by trained and semi-trained judges (Kader et al., 1982; Nikzadeh and Sedaghat, 2008; Vázquez-Araújo et al., 2009; Contador et al., 2015; Lynch et al., 2016) paying little attention to consumer preferences. In almonds, trained judges have reported differences in main physical parameters of kernels from different cultivars, including flavour intensity, colour, roughness, crispness, crunchiness and hardness (Contador et al., 2015). In pistachios, trained judges did not find significant differences in the flavour of four cultivars grown in the same orchard (Kader et al., 1982), however, a posterior study performed by minimum trained panellist, that considered a larger number of cultivars, reported significant differences (Tsantili et al., 2010). In the same study, differences attending to the visual acceptance of kernels were not reported.

The objective of this paper is to analyse the usefulness of physical parameters to discriminate almond and pistachio kernels obtained from different genotypes. Differences on the consumer acceptance of kernels from these different cultivars are also reported. The correlation of the results of physical analysis and consumer valuation can provide useful information about the formation of consumer preferences.

2. Materials and methods

2.1. Plant material

Almonds and pistachios were collected in 2016 in the south of Spain from experimental orchards. Almonds were collected at the Instituto Técnico Agronómico Provincial of Albacete. Ten different cultivars were collected. On the other side, ten

37

Physical and sensory analysis of almond and pistachio kernels different pistachio cultivars were collected at an experimental orchard in the Centro de Mejora Agraria el Chaparrillo of Ciudad Real.

2.2. Almond and pistachio characterization

2.2.1. Physical properties

Kernel length, width and weight were recorded on sub-samples of 10 kernels for each almond and pistachio cultivar.

The colour of almond and pistachio kernels was measured in the kernel peel and inside the kernel. The colour of the kernels was measured by reflection in three zones of ten different kernels for each cultivar using a Minolta CR-200 colorimeter (Minolta Camera Co., Ltd., Osaka, Japan). As almonds show more homogeneous colour, colour was measured in random zones while in pistachio the colour in the darkest zone (pink-purple to black area) was measured in the kernel membrane. The tristimulus values obtained were used to calculate the CIELAB chromatic coordinates a* (red– green component) and b* (yellow–blue component) (CIE, 1986).

To evaluate the texture of almonds and pistachios, 10 kernels of each variety were analyzed. The cutting force was measured using a TA-XT Plus texture analyser (Stable Micro Systems, Godalming, UK). The kernels were cut perpendicularly to their major axis with a Warner Bratzler blade with rectangular slot blade at a constant velocity of 2 mm s-1.

2.2.2. Sensory analysis

Affective test was used to evaluate the acceptance of consumers. Testing was carried out in the sensory laboratory of the Higher Technical School of Agricultural and Forestry Engineering (Albacete, Spain). In affective test, almonds and pistachio kernels were randomly labelled and 102 consumer-type panellists were asked to evaluate overall visual acceptance and taste of nuts from every single cultivar. Each hedonic description was assigned a nine-point scale (−4: dislike extremely, 0: neither like nor dislike, 4: like extremely).

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Influence of raw material in oil characteristics

2.3. Statistical analysis

Physical parameters and sensory attributes were processed using variance analysis (ANOVA). Differences between means were compared using a Duncan test with a 95% significant level (p < 0.05). Correlation analyses were performed employing Pearson´s test. A multivariate statistical analysis with variables that had previously shown significant differences between cultivars was performed using principal component analysis (PCA). All statistical analysis were carried out using the SPSS programme, release 23.0 for Windows.

3. Results and discussion

3.1. Physical parameters

3.1.1. Almond

All physical parameters of almond kernels considered showed significant differences between cultivars (Duncan test, p < 0.01) (Table 1). Figure 1 shows the principal component analysis of almond cultivar kernels. Some cultivars show similar values for the analysed parameters. This is the case of Ferraduel, Antoñeta, Vairo, Guara and Penta. These five cultivars show average size and weight with similar and average values for the colours outside and inside the kernel.

As expected, strong correlations exist among the parameters length, width, weight and hardness on almond kernels (table 2). Those kernels that are longer are also wider, heavier and, as a result, harder. Belona and Ferragnes have the longest kernels and Marcona and Ayles the widest. Attending to length and width, cultivars Ferragnes, Tardona, Marcona and Penta can be completely discriminated. As the weight of almond kernels is more intensively correlated with the width than with the length, Marcona and Ayles are also the cultivars with the heaviest kernels. Between these four largest cultivars (Belona, Ferragnes, Marcona and Ayles) there is not significant differences in the cutting force needed to cut the kernel (hardness). As reported by Contador et al (Contador et al., 2015), Marcona produce one of the hardest cultivars, although in our study the values of Belona are even higher.

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Physical and sensory analysis of almond and pistachio kernels

Figure 1. Principal component analysis of physical parameters in kernels from different almond cultivars. Abbreviations: L: kernel length; S: hardness; We: kernel weight; Wi: kernel width; ICa*: internal colour a* parameter; ICb*: internal colour b* parameter; ECa*: external colour a* parameter; ECb*: external colour b* parameter

Table 1. Physical parameters of almond kernels from different cultivars

Length Width Height Hardness Cultivar ECa ECb ICa ICb (mm) (mm) (g) (N) Antoñeta 22.60d 14.80b 1.20de 17.61ab 35.25a -0.23c 12.12bcde 61.15abc Ayles 24.70b 16.60a 1.57ab 14.81c 29.26c -0.23c 12.06bcde 64.16abc Belona 24.40bc 16.40a 1.45c 18.68a 35.69a -0.15c 11.55de 72.47a Ferraduel 24.5bc 14.40b 1.31d 18.47ab 34.20a -0.20c 11.59de 53.44cd Ferragnes 25.80a 14.60b 1.48bc 18.46ab 34.91a 0.28a 11.74cde 60.60bc Guara 23.60bcd 14.50b 1.26de 18.17ab 35.59a -0.02bc 12.66abc 54.07cd Marcona 23.90bc 16.60a 1.63a 14.62c 31.31bc -0.07bc 11.10e 66.74ab Penta 23.40cd 12.80c 1.05f 17.88ab 35.30a 0.22ab 13.53a 55.37bcd Tardona 19.40e 11.60d 0.72g 18.21ab 35.29a 0.03abc 12.33bcd 47.90e Vairo 24.60b 15.00b 1.17e 17.33b 33.93ab -0.11c 12.89ab 52.67cd P *** *** *** *** *** *** *** *** ECa: external colour, parameter a*; ECb: external colour, parameter b*; ICa: internal colour, parameter a*; ICb: internal colour, parameter b*. Numbers are means. Means in a column not sharing the same letter are significant different by Duncan test (p < 0.05). ** Significant at p < 0.05. *** Significant at p < 0.01.

Colour also shows significant differences between almond kernels, both in the peel and in the inside of the kernel. On the peel, a* and b* parameters are strongly correlated (0.677, p < 0.01). Belona is the cultivar that shows the highest values for

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Influence of raw material in oil characteristics

the parameters a* and b*, while Ayles and Marcona show the lowest values for the colour parameters. The kernels of some of the cultivars can be differentiated attending to their external colour.

Table 2. Correlations between almond kernel physical parameters Length Width Weight ECa ECb ICa ICb Hardness Length 1 .605** .734** -.150 -.227* .041 -.125 .128 Width 1 .874** -.386** -.339** -.222* -.314** .362** Weight 1 -.397** -.362** -.086 -.329** .344** CEa 1 .677** .109 .185 -.092 CEb 1 .160 .245* .005 CIa 1 .084 -.076 CIb 1 -.218* Hardness 1 ECa: external colour, parameter a*; ECb: external colour, parameter b*; ICa: internal colour, parameter a*; ICb: internal colour, parameter b*. * Significant at p < 0.05. ** Significant at p < 0.01.

The parameter b* of kernel peel is correlated with the same parameter for the intern colour of the kernel (0.245, p < 0.05). This means that the yellower the kernel peel is, the yellower the kernel is also on the inside. Penta, Vairo and Guara are the cultivars with the higher values for b* on the inside, while Marcona shows the lower values. Values for a* parameter inside the kernel are close to zero in all cultivars, with Penta showing the highest average values (0.28) and Antoñeta and Ayles (-0.23) showing the lowest values.

The colour parameter b* inside the almond kernel is negatively correlated with the strength (-0.218, p < 0.05). This means that the yellower kernels are inside, the less hard they are. However, the strength required to cut the kernel is also heavily correlated the width (0.344, p < 0.01) and the weight (0.362, p < 0.01) of the kernel.

3.1.2. Pistachio

For pistachio kernels, all parameters showed significant differences with the exception of the colour parameter b* measured in the interior of the kernel (table 3). As it can be observed in figure 2, main differences in pistachio cultivars are related to the size, weight and strength (PC1), but also to colour parameters (CP2). In 41

Physical and sensory analysis of almond and pistachio kernels accordance to previous studies, Kerman cultivar produces the largest kernels (Tsantili et al., 2010). Only length, width or weight are enough to differentiate Kerman kernels from the rest of the cultivars even when pistachios are grown in the same orchard and management and environmental effects are controlled. In pistachios, the correlation of kernel weight with size is higher with the length (0.825, p<0.01) than with the width (0.739, p<0.01).

Figure 2. Principal component analysis of physical parameters in kernels from different pistachio cultivars. Abbreviations: L: kernel length; S: hardness; We: kernel weight; Wi: kernel width; ICa*: internal colour a* parameter; ECa*: external colour a* parameter; ECb*: external colour b* parameter

The peel colour is also useful for cultivar discrimination. The higher values of a* and the lower values of b* correspond to the kernels with the stronger pink to purple colours. This is the case of Napoletana and Sirora kernels. On the other hand, the cultivars with the less purple colours on their kernel membrane are Kerman and Sfax.

On the inside, pistachio kernels show bright green to yellow colour. Cultivars with the lower values for a* measured inside the kernel are those with greener colours. This is the case of Larnaka and Ouleimy. Cultivars Mateur and Sfax have the kernels with the less green colour inside. Cultivar Avidon shows b* values especially higher, indicating yellower colours inside the kernel.

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Influence of raw material in oil characteristics

Table 3. Physical parameters of pistachio kernels from different cultivars Length Width Height Hardness Cultivar ECa ECb ICa ICb (mm) (mm) (g) (N) Aegina 16.80bcd 7.80e 0.60c 66.31ab 14.67cde -0.30a 9.35 33.51de Alpina 16.20de 7.90e 0.54d 61.65b 14.03cde -0.28a 9.41 31.73e Avidon 16.40cd 7.90e 0.60c 77.25a 15.34cd -1.26ab 15.12 28.84e Kerman 19.00a 10.10a 0.80a 65.58ab 19.20ab -2.13ab 6.93 46.52ab Larnaka 17.10bc 8.80bcd 0.63c 70.73ab 15.04cde -7.72c 9.58 45.79abc Mateur 16.90bcd 8.50cde 0.63bc 75.06ab 17.10bc 1.63a 8.54 42.17bcd Napoletana 16.70bcd 7.80e 0.52d 44.39c 6.26f -1.20ab 7.50 53.84a Ouleimy 17.40b 9.40b 0.69b 62.66ab 12.04de -5.39bc 9.32 44.42abc Sfax 15.50e 8.20de 0.50d 74.72ab 20.91a 0.41a 5.24 41.81bcd Sirora 17.30b 9.00bc 0.65bc 71.50ab 11.65e -0.13a 4.42 36.29cde P *** *** *** *** *** *** NS *** ECa: external colour, parameter a*; ECb: external colour, parameter b*; ICa: internal colour, parameter a*; ICb: internal colour, parameter b*. Numbers are means. Means in a column not sharing the same letter are significant different by Duncan test (p < 0.05). ** Significant at p < 0.05. *** Significant at p < 0.01.

As reported by Kader et al. (Kader et al., 1982), Kerman kernels show high resistance to cutting, but considering a larger number of cultivars no significant differences were found with Napoletana and Ouleimy. In fact, Napoletana is the hardest cultivar and, at the same time, the cultivar with the lower values for external colour parameters a* and b*, indicating greener colours. As had also been observed in the almond kernels, hardness of pistachio kernel is negatively correlated with the measure of parameter b* in the inside of the nut (-0.227, p < 0.05) (table 4). As every cultivar was pick up at the most appropriate harvest date, these differences in colour and hardness cannot be considered the result of different maturity stages. In pistachios, none of the size parameters considered showed correlation with kernel hardness.

3.2. Consumer preference

3.2.1. Almond

Regarding aspect, significant differences appear on the valuation that consumers do on visual acceptance and taste of almond kernels (Duncan Test, p < 0.01) (table 5). All cultivars receive positive valuations for kernel visual acceptance and taste (average ratings above 0).

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Physical and sensory analysis of almond and pistachio kernels

Table 4. Correlations between pistachio kernel physical parameters Length Width Weight ECa ECb ICa ICb Hardness Length 1 .614** .825** -.130 -.103 -.113 .011 .118 Width 1 .739** .069 .173 -.075 -.148 .118 Weight 1 .033 .085 -.072 .018 .013 CEa 1 .555** .180 -.044 -.091 CEb 1 .144 -.020 -.098 CIa 1 -.655** .008 CIb 1 -.227* Hardness 1 ECa: external colour, parameter a*; EC: external colour, parameter b*; ICa: internal colour, parameter a*; ICb: internal colour, parameter b*. * Significant at p < 0.05. ** Significant at p < 0.01.

Regarding visual acceptance, Belona and Vairo are the cultivars that obtain the highest valuations, although significant differences were not found with other cultivars (Marcona, Antoñeta and Ferragnes). Belona and Vairo show some physical similarities that must be analysed to understand the characteristics that may influence consumer preference. Both produce kernels slightly wider than the average, while their values of weight and external colour parameters a* and b* are average.

Attending to the taste, the cultivars that consumers valued more positively are Penta and Vairo (although no significant differences were found with Tardona). Penta and Vairo kernels have some common physical characteristics, as they both have higher internal values for b* (yellower) and medium hardness. Although the Spanish cultivar Marcona is widely known for its high sensory quality, Marcona kernels received low scores in taste. This result is however in agreement with results obtained in previous studies performed on fresh Marcona almonds (Contador et al., 2015). Consumer panellist gave the lowest scores to Ayles and Ferraduel kernels.

3.2.2. Pistachio

Significant differences were found in the consumer valuations of physical aspect and taste of different pistachio cultivars, however the differences where higher regarding the physical parameters than regarding the kernel taste (table 5). The results are opposite to those obtained by a study developed using minimal trained judges

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Influence of raw material in oil characteristics

(Tsantili et al., 2010), which found more significant differences in overall flavour than in visual acceptance. However, as the cultivars analysed are different, and due to the high variability reported in the physicochemical characteristics of pistachio cultivars (Rabadán et al., 2017b; Rabadán et al., 2017c), different results can be expected depending on the considered cultivars.

Table 5. Sensory attributes of almond and pistachio kernels. The evaluation guidelines considered a continuous scale, ranging from -4 (lowest score) to 4 (highest score). Almond Pistachio Visual Visual Cultivar Taste Cultivar Taste acceptance acceptance Antoñeta 2.03ab ± 1.10 1.30bcd ± 1.62 Aegina 2.47ab ± 1.01 1.63ab ± 1.25 Ayles 1.47bcd ± 1.36 0.30e ± 2.04 Alpina 1.67c ± 1.37 1.70ab ± 1.06 Belona 2.43a ± 1.22 1.47bc ± 1.55 Avidon 0.87d ± 1.36 0.70c ± 1.68 Guara 1.47bcd ± 1.28 0.87cde ± 2.01 Kerman 1.97abc ± 1.52 1.03abc ± 1.30 Ferraduel 1.00d ± 1.58 0.37de ± 1.77 Larnaka 2.60a ± 1.40 1.53abc ± 1.36 Ferragnes 1.93abc ± 1.36 0.93cde ± 1.57 Mateur 1.77bc ± 0.97 1.33abc ± 1.56 Marcona 2.10ab ± 1.49 0.80cde ± 2.12 Napoletana 0.43de ± 1.72 0.79c ± 1.45 Penta 1.20cd ± 1.54 2.40a ± 1.16 Ouleimy 0.57de ± 1.77 1.00bc ± 1.76 Tardona 0.80d ± 1.88 1.67abc ± 1.47 Sfax -0.10e ± 1.52 1.50abc ± 1.46 Vayro 2.33a ± 1.15 2.03ab ± 1.06 Sirora 1.77bc ± 0.94 1.87a ± 1.33 P *** *** P *** ** Numbers are means of total valuations. Means in a column not sharing the same letter are significant different by Duncan test (p < 0.05). ** Significant at p < 0.05. *** Significant at p < 0.01.

Consumers valued positively the visual acceptance and taste of all pistachio cultivars with the exception of the visual acceptance of Sfax kernels. The negative valuation of Sfax kernels can be the result of the lower values of colour parameter b* in the kernel peel combined with their smaller size. Cultivars Aegina and Larnaka received the higher valuation regarding physical aspect, although no significant differences were found with Kerman kernels. If the physical similarities of Aegina and Larnaka are considered, it can be observed how they both have medium size and weight, medium values for a* parameter and medium to low values of b* parameter on the kernel peel.

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Physical and sensory analysis of almond and pistachio kernels

Regarding taste, cultivars Alpina and Sirora received the highest valuations from consumers, but significant differences with most of the cultivars were not identified. These two cultivars that received the highest scores showed high values for a* parameter inside the kernel (less green colour) and low hardness.

3.3. Matching physical traits with consumer preference

3.3.1. Almond

Attending to the external aspect, consumers valued more positively the cultivars with wider kernels that are also the hardest (table 6). However, it should not be considered that consumers prefer harder almonds, as this is only the result of the high correlation between width and hardness (0.820, p<0.01).

Table 6. Correlation of physical parameters and sensory evaluation by consumer type panellist of almond and pistachio kernels from different cultivars Almond Length Width Weight ECa ECb ICa ICb Hardness Visual acceptance .509 .696* .540 -.156 -.041 -.181 -.284 .667* Taste -.344 -.512 -.635* .355 .535 .422 .687* -.277 Pistachio Length Width Weight ECa ECb ICa ICb Hardness Visual acceptance .453 .189 .421 .189 .099 -.191 .070 -.206 Taste -.186 -.051 -.151 .298 .205 .167 -.495 -.337 ECa: external colour, parameter a*; ECb: external colour, parameter b*; ICa: internal colour, parameter a*; ICb: internal colour, parameter b*. * Significant at p < 0.05. ** Significant at p < 0.01.

If we analyse the correlations of taste punctuations and physical parameters evaluated, it can be observed how consumers give higher punctuations in taste to those cultivars than produce the less heavy kernels. The relation of breakdown of the kernel almond and the higher acceptability of the consumers reported in a previous study (Varela et al., 2008) was not found in the correlation of hardness and taste (- 0.277). On the other side, cultivars with kernels that are more yellow on the inside (higher values of b*) also get more positive valuations in taste.

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Influence of raw material in oil characteristics

3.3.2. Pistachio

Although significant correlations are not shown in pistachio, some tendencies can be observed (table 6). Regarding the external appearance, the highest values are obtained by the cultivars with the longest and heavier kernels. The less homogenous colour of pistachio kernels hampesr the measurement of the kernel colour, especially the kernel membrane colour. Consumer visual preference cannot be identified with a particular colour in the kernel membrane.

The taste is partially correlated with the interior colour parameter b*. This is the more appreciated cultivars are those that show less yellowish colour on the inside. However, no significant correlations appear relating colour or hardness with consumer preference.

4. Conclusions

Physical parameters on almond and pistachio cultivars, related to kernel size, weight, colour and hardness can be used for cultivar discrimination. Among the almond cultivars considered, consumers valued more positively kernels from cultivars Vairo, Belona and Penta, while the pistachio cultivars that received the more positive valuations were Larnaka, Aegina and Sirora. Significant differences in the overall visual acceptance and the taste of kernels from different cultivars reveal the importance of considering consumer preferences in cultivar selection and breeding beyond traditional factors as crop yield or industrial preferences.

In almonds the visual acceptance of consumers is higher for the wider almonds, while the taste is correlated with the weight (lighter almonds) and the higher values of b* parameter inside the kernel (yellower colours). In pistachios although some tendencies can be observed, influence of physical parameters on consumer preferences have not been detected.

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Physical and sensory analysis of almond and pistachio kernels

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Physical analysis of pistachio kernels and pistachio oils

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Physical analysis of pistachio kernels and pistachio oils

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Influence of raw material in oil characteristics

Physical analysis of pistachio kernels and pistachio oils

ABSTRACT Keywords: Pistachio kernel characteristics are influenced by cultivar on a greater way Pistacia vera Kernel size than other tree nuts. Previous studies have focused on the analysis of Colour chemical traits of pistachio cultivars, with little attention been paid to Viscosity differences in their physical parameters. To solve this disregard, differences in the physical traits of twenty different cultivars from nine different countries are evaluated in this study. To identify the cultivar effect, all pistachio varieties were grown in the same plot to remove environmental and land management effects on kernel traits. Regarding kernel size, significant differences were found in the kernel length and width, but not in the relation length/width. Significant differences in the colour of pistachio by-products, pistachio oil and flour, were found. Colour parameter b* in oils and L* in flours allowed the differentiation of 25% and 40% of considered cultivars, respectively. The differences in the fatty acid profile of cultivars seems to affect oil viscosity, allowing the calculation of the content in oleic and linoleic acids with the viscosity data. Viscosity values ranged from 59.71 mPa·s in Kerman to 63.93 mPa·s in Avdat. By grouping the considered cultivars depending on their origin countries, significant differences were found in the kernel length and oil viscosity but not in the colour parameters (L*, a*, b*). This study certifies the usefulness of some physical parameters to confirm authenticity of kernels and pistachio by products depending on the cultivar and its origin.

1. Introduction

The identification of differences between pistachio cultivars has focused in the chemical parameters, mainly fatty acids, tocopherols and phytosterols (Arena et al., 2007; Tsantili et al., 2010; Catalán et al., 2016) paying little attention to physical parameters. However, physical characteristics of pistachio kernels and pistachio by-

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Physical analysis of pistachio kernels and pistachio oils products are important properties in the pistachio market. In particular, size and colour have been reported as some on the most important characteristics in the kernel market (Seferoglu et al., 2006). Moreover, if pistachios are used to obtain secondary products, such as oils, viscosity appears as a main parameter to consider as it is related to oil quality and the design of any engineering process related to its industrial production.

Consumers prefer greener and larger pistachios (Balta, 2002; Tsantili et al., 2010), that also have been reported to produce the best quality oils (Álvarez-Ortí et al., 2012). Although a more intense taste has been attributed to smaller kernels (Kallsen et al., 2009). Cultivars that produce the largest pistachios (as Iranian Kerman) are widely preferred for direct consumption. The correlation of length and width of kernels has been related to the cultivar origin (Caruso et al., 1998), although subsequent studies have revealed the limited scope of established correlations (Tsantili et al., 2010).

Colour is an important parameter in pistachio quality. Major pigments in pistachios are chlorophylls and carotenoids. Chlorophylls (a and b) provide pistachios with their characteristic bright green colour while the predominant carotenoid, lutein, gives them a yellow colour (Giuffida et al., 2006). Beyond its active role in pistachio coloration, these pigments show antioxidant properties that increase the nutritional benefits of pistachio consumption (Hsu et al., 2013). Raw pistachios have a chlorophyll content of about 9.72 mg per kg of wet weight and a lutein content of 8.12 mg /kg of wet weight (Pumilia et al., 2014). Studies have analysed the differences in the colour of pistachio kernels depending mainly on their origin (Zakynthinos and Rouskas, 1994; Agar et al., 1998; Bellomo and Fallico, 2007) or the influence of roasting on the colour of kernels or by-products (Pumilia et al., 2014; Ling et al., 2015; Ling et al., 2016). Beyond direct measures in kernels, the variations that appear on the colour of pistachio products because of the cultivar effect should be quantified, as their influence in the product quality is crucial.

Pistachio oil has become an interesting product, with potential for future development (Catalán et al., 2016). To maximize its commercial opportunities, quality

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Influence of raw material in oil characteristics

should be controlled from origin, and the cultivar effect must be considered. Within oil parameters, viscosity is related to oil processing and oil quality. Research has proved that oil viscosity is related to the temperature of the oil (Ginner et al., 1996; Sadat and Khan, 2007; Kemar et al., 2013), the degree of unsaturation (Kim et al., 2010; Oliveira et al., 2016) and the triglyceride composition of the vegetable oil (Geller and Goodrum, 2000; Sadat and Khan, 2007). The study of the correlation between rheology and the chemical composition of vegetable oils has been verified for popular edible vegetable oils used for frying (Kim et al., 2010) paying little attention to direct consumption oils such as pistachio oil. Conte et al. (2011), suggested the existence of differences in the viscosity of oils obtained from three different pistachio cultivars, but a deeper analysis with a significant number of cultivars should be performed.

The selection of the cultivar and the location have been reported to have major influence on the characteristics of pistachio kernels (Seferoglu et al., 2006; Tsantili et al., 2010). Few studies have analysed the differences attributed to the cultivar effect by using pistachio cultivars that have been grown in the same plot (Tsantili et al., 2010; Tsantili et al., 2011). When cultivars are grown in the same plot, the influence of ecological conditions (Silver et al., 1984; Seferoglu et al., 2006) and management practises (Sánchez et al., 2008; Carbonell-Barrachina et al., 2015) are removed, allowing the identification of the true cultivar effect on pistachio characteristics.

The objective of this study is to analyse the physical differences that appear in pistachios because of the cultivar effect by controlling the effects of the environment and the land management. The kernel size, the colour of the pistachio oil and pistachio flour and the oil viscosity were studied attending to its utility for cultivar and origin differentiation.

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Physical analysis of pistachio kernels and pistachio oils

2. Materials and Methods

2.1. Plant material

Pistachios were collected at an experimental orchard in the Centro de Mejora Agraria el Chaparrillo of Ciudad Real in the south of Spain in 2015. Twenty different cultivars (Aegina, Ajamy, Albina, Ashoury, Avdat, Avidon, Batoury, Boundoky, Bronte, Iraq, Joley, Kastel, Kerman, Larnaka, Lathwardy, Mateur, Napoletana, Ouleimy, Sfax and Sirora) with nine different origins (Iran, Iraq, Syria, Israel, Cyprus, Greece, Italy, Tunisia and Australia) were evaluated. One kilogram of pistachios was collected from every one of the three trees analyzed within each cultivar. Pistachios were picked at the most appropriate harvest date for each cultivar.

2.2. Analysis

Oil extraction was carried out using a hydraulic press (MECAMAQ Model DEVF 80, Vila-Sana, Lleida, Spain). The remaining pressing cake was ground and sieved to obtain a pistachio flour with particle size lower than 1 mm. Pistachio flour is then a by-product of pistachio oil extraction, although it can be also considered as the main production objective (Rabadan et al., 2017).

The colour of the oil samples was measured using a spectrophotometer UV/Vis Jasco V-530 (Jasco Analytical, Madrid, Spain). Oil was placed in quartz cuvettes (1cm path length) for their analysis. N-hexane was used as blank reference. The colour of the flour was measured by reflection in five random zones with a Minolta CR-200 colorimeter (Minolta Camera Co., Ltd., Osaka, Japan) in each flour. The illuminant used was D65. The tristimulus values obtained were used to calculate the CIELAB chromatic coordinates: L∗ (brightness), a∗ (red–green component), b∗ (yellow–blue component) as recommended by the Commission Internationale de l’Eclairage (CIE, 1986). Chroma values (C*) were calculated using the expression:

퐶∗ = (푎∗2 + 푏∗2)1/2

In order to determine fatty acids composition (%), the methyl-esters were prepared by vigorous shaking of a solution of oil in hexane (0.2 g in 3 ml) with 0.4 ml of 2 N

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Influence of raw material in oil characteristics

methanolic potassium hydroxide solution, and analysed by GC with a Hewlett- Packard (HP 6890) chromatograph equipped with a FID Detector. A fused silica column (50 m length x 0.25 mm i.d.), coated with SGL-1000 phase (0.25 µm thickness; Sugerlabor), was used. Helium was employed as a carrier gas with a flow through the column of 1 ml min-1. The temperatures of the injector and detector were set at 250°C with an oven temperature of 210°C. An injection volume of 1 µL was used (Regulation EEC 2568/91, corresponding to AOCS method Ch 2–91).

Viscosity was measured by a rotary viscometer test method (Visco Basic Plus, Fungilab S.A.) using the method described by Xu et al., (2007).

2.3. Statistical analysis

Significant differences among varieties were determined by ANOVA and Duncan test with a 95% significance level (P < 0.05) using the SPSS programme, release 23.0 for Windows. To select the most convenient method for cultivar differentiation using colour parameters, the variable that created the most groups using the Duncan test was selected.

3. Results and discussion

3.1. Kernel size

Differences can be found in the size of pistachios obtained from different cultivars, even when controlling the effect of the environment and the land management. The pistachios kernel length and width reported in our study are in general smaller than previously observed in those studies that considered the whole nut (with shell) (Tsantili et al., 2010). Attending to the length and width of kernels, three different groups can be identified (p<0.01) (figure 1). Cultivars Kastel (Israel), Sirora (Australia) and Kerman (Iran) produced the larger pistachios, while Boundoky pistachios (Syria) were the smallest. However, most of the cultivars are included in an intermediate group that showed average size.

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Physical analysis of pistachio kernels and pistachio oils

Attending to the kernel form, Italian, Greek and Tunisian varieties are considered elongated (length/width > 1.80), while Iranian, Turkish and Syrian are considered ovoid (length/width > 1.50 - 1.80) (Caruso et al., 1998). Our Greek (Aegina) and Italian cultivars (Napoletana and Bronte) met this rule, but only one of the two Tunisian cultivars did. The cultivars from the middle-East region presented a wide variability of kernel forms, with eight cultivars within the proposed range l/w=1.50-1.80 (Ajamy, Ashoury, Avidon, Batoury, Boundoky, Kastel, Kerman and Lathwardy) and five cultivars above it (Albina, Avdat, Iraq, Joley and Ouleimy).

Our results support Tsantili et al., (2010) findings about the differences in size of pistachio kernels. However, for our larger number of cultivars, we only found statistical differences between cultivars in the kernel length (p > 0.01) and width (p > 0.05) but not in relation length/width.

Figure 1. Length and width of pistachio kernels from twenty pistachio cultivars grown in the same plot.

3.2. Colour parameters

Tsantili et al., (2010), reported the importance of pistachio colour for cultivar differentiation. They studied the differences that appear in the colour of kernels from eight pistachio cultivars that had been grown in the same plot finding significant differences in chroma (intensity of colour). Our results support those and show that

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Influence of raw material in oil characteristics

the differences in colour remain if pistachio by products (oil and defatted flour) are considered for our larger number of varieties (Table 1).

Table 1. Physical parameters proposed for cultivar differentiation of pistachio oil and pistachio flour. Cultivar Flour Oil Cultivar origin L* a* b* L* a* b* Viscosity Aegina Greece 52.51 f -2.09 cd 22.11 g 94.65 hi -7.76 abc 71.49 h 62.79 def Ajamy Syria 53.10 e -3.22 gh 24.25 d 95.59 bcd -7.96 abcd 69.51 j 61.72 g Albina Iran 44.05 l -1.13 a 19.09 k 95.40 cde -8.31 d 72.73 f 62.89 cde Ashoury Syria 56.71 bc -0.93 a 21.25 j 95.22 ef -7.94 abcd 68.1 k 63.36 b Avdat Israel 49.65 i -1.66 b 21.62 hi 95.90 ab -8.20 bcd 68.36 k 63.96 a Avidon Israel 50.54 h -3.55 h 21.35 ij 95.30 de -7.82 abc 71.78 gh 60.72 i Batoury Syria 52.49 f -2.25 cd 21.86 gh 94.96 fgh -7.95 abcd 83.29 b 63.13 bcd Boundoky Syria 43.63 m -3.23 gh 21.45 ij 95.29 de -8.20 bcd 73.26 e 63.43 b Bronte Italy 49.82 i -3.26 gh 21.83 gh 95.22 ef -7.77 abc 71.30 h 63.33 b Iraq Iraq 52.46 f -3.29 gh 22.46 f 95.75 b -7.99 abcd 71.62 h 61.30 h Joley Iran 51.08 g -3.54 h 24.21 d 96.17 a -7.62 a 62.25 l 61.22 h Kastel Israel 51.31 g -2.38 de 26.16 a 95.20 ef -8.00 abcd 72.18 g 59.88 j Kerman Iran 56.81 b -4.66 i 25.69 b 93.80 j -7.57 a 87.04 a 59.71 j Larnaka Cyprus 45.96 k -2.33 cde 21.99 g 93.41 k -7.64 a 75.22 d 63.90 a Lathwardy Syria 68.70 a -4.48 i 24.44 cd 95.09 efg -7.73 ab 80.59 c 63.28 b Mateur Tunisia 56.50 c -2.27 cd 22.61 f 95.83 b -8.22 cd 70.55 i 62.68 df Napoletana Italy 52.45 f -2.68 ef 21.30 ij 95.08 efg -8.03 abcd 75.35 d 62.50 f Ouleimy Syria 56.85 b -3.02 fg 22.71 f 95.65 bc -7.90 abcd 70.41 i 63.24 bc Sfax Tunisia 47.82 j -3.27 gh 23.79 e 94.79 ghi -7.88 abcd 73.615 e 60.41 i Sirora Australia 54.45 d -1.93 bc 24.63 c 94.60 i -7.89 abcd 80.39 c 62.57 df nº groups 13 9 11 11 4 12 10 nº cultivars completely 8 0 4 2 0 5 1 differentiated P *** *** *** *** ** *** *** Numbers are means. Means in a column not sharing the same letter are significant different by Duncan test (p < 0.05). Viscosity is measured in mPa·s. ** Significant at p < 0.05 and *** Significant at p < 0.01

The pistachio flours have a light greenish yellow colour. Varieties Boundoky and Albina showed the darkest flours, with L* values below 43.63, while highest L* values were reported in the Syrian cultivars Lathwardy, Ouleimy and Ashoury. Cultivars Kerman and Lathwardy showed the greener flours, and Kastel and Kerman the highest values for the b* parameter (yellower flours).

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Physical analysis of pistachio kernels and pistachio oils

Colour intensity in pistachio oil is higher than the reported in the flours. The brightness in pistachio oil is higher than the reported in the flours. Varieties Joley and Avdat showed the the brightest oils, while the Cypriot Larnaka produced the darkest oils. Cultivars Albina and Mateur produced the greener oils, which have been reported as more valued by consumers (Álvarez-Ortí et al., 2012). On the other hand, Kerman is the cultivar that produced the less green oils. Attending to the b* parameter, the highest values (this is more yellow oils) were reported in cultivars Kerman and Batoury. Our results showed oils with more intense colours than reported for Pumilia et al., (2014) for one cultivar. Brightness values (L*) are similar.

Chroma values in defatted flours ranged from 26.27 in Kastel to 19.12 in Albina. In the oils, these values were significantly higher and ranged from 87.37 in Kerman to 62.71 in Joley. Depending on the preferred intensity in the colour of pistachio products, some cultivars could be considered over the rest.

Although significant differences between cultivars were found for all the CIELAB chromatic coordinates, some parameters could be identified as more convenient for cultivar differentiation.

To differentiate cultivars depending on the colour of the oil, the parameter b* (p < 0.01) would be the most convenient, although significant differences were also found for L* (p < 0.01, 11 groups) and a* (p < 0.05, 4 groups) (Table 1). Attending to the colour parameter b*, 12 statistically different groups are defined (p < 0.05). By using this parameter, up to five of the 20 cultivars considered can be completely differentiated and 12 more are reduced to only two possibilities.

In the case of defatted pistachio flour, parameter L* (p < 0.01) is considered the most appropriate one. Significant differences were also found for b* (p < 0.01, 11 groups) and a* (p < 0.01, 9 groups). The Duncan test on the data for the L* parameter created 13 statistically different groups. Eight groups are formed by only one cultivar (total differentiation), and three more are composed by two cultivars.

CIELAB chromatic coordinates showed correlations between oil and flour parameters (Table 2). These correlations showed that the greener the oil, the less green the flour

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Influence of raw material in oil characteristics

(r = -0.556, p < 0.05). This correlation may appear due to the transference of chlorophylls from the kernel to oil during extraction, as these pigments are oil- soluble. A negative correlation exists in pistachio oil between the L* and the b* (r = - 0.680, p < 0.01). This was the stronger correlation reported, meaning that when the oil is more yellow, it tends to be darker. A similar correlation is observed if the a* parameter is considered, as greener oils also tend to by lighter (r = -0.511, p < 0.05). Further analysis of the mechanisms that explain the reported correlations should be needed, as they are not the mainly on the study, which focus mainly in the observed cultivar variability.

Table 2. Correlation matrix showing the Pearson coefficients found among the flour and the oil parameters considered for cultivar colour analysis. Flour Oil L a b L a b L 1 -.349 .453* .044 .326 .293 Flour a 1 -.525* .100 -.556* -.296 b 1 -.174 .489* .260 L 1 -.511* -.680** Oil a 1 .237 b 1 * Significant at p < 0.05 and ** Significant at p < 0.01

3.3. Oil viscosity

Viscosity values showed significant differences among pistachio cultivars (Table 1), identifying oil viscosity as a useful variable for cultivar differentiation. Oils obtained from the Avdat and Larnaka cultivars showed the highest viscosity values (above 63.90 mPa·s), while Kastel and Kerman showed the smallest values. Attending to viscosity, 10 different groups are created to differentiate the twenty cultivars considered (Duncan test, p <0.05), however only the variety Ajamy would be completely differentiated.

Conte et al. (2011) studied the differences in the viscosity of oils from three pistachio cultivars (Bianca, Larnaka and Kerman). They concluded that the dynamic properties of pistachio oils are dependent upon the aggregate sizes of oil components. Our results show that those differences in the size of pistachio oil components are useful

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Physical analysis of pistachio kernels and pistachio oils for cultivar differentiation. As expected, a strong correlation of pistachio oils viscosities with the percentage of oleic and linoleic acids was found (Kim et al., 2010) (Figure 2). Considering the need to design fast, cheap and accurate techniques to determine viscosity in vegetable oils to confirm authenticity, detect adulteration and design processing (Sadat and Khan, 2007; Kemar et al., 2013), empirical equations for predicting the viscosities values are proposed.

The strong dependence between the oil viscosity and the fatty acids profile (Geller, 2000; Kim et al., 2010; Conte et al., 2011; Oliveira et al., 2016) allows the calculation of the viscosity by using the percentage of oleic (q1) or linoleic (q2) fatty acids. The equations q1 and q2 are able to explain up to 92% of the total variance observed in viscosity values (Figure 2). Agreeing with the previous results obtained for other plant oils (Kim et al., 2010), the viscosity of pistachio oil in the different cultivars was however not related to the proportion of saturated (R2= 0.01) or unsaturated fatty acids (R2= 0.00). This could be attributed to the low concentration of saturated fatty acids of pistachio oil, as oleic and linoleic acids compound at least the 86.05% of the total fatty acid profile in the pistachio cultivars considered.

Figure 2. Interrelationship between the oleic and linoleic concentration and cultivars oil viscosity.

푉𝑖푠푐표푠𝑖푡푦 = 43.10 + 0.284푥 (q1) where x = oleic content (%)

푉𝑖푠푐표푠𝑖푡푦 = 67.88 − 0.291푦 (q2) where y = linoleic content (%)

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Influence of raw material in oil characteristics

3.4. Usefulness of physical parameters for cultivar origin differentiation

Among the physical parameters considered, only the length of pistachio kernels and the viscosity of pistachio oils showed significant differences when cultivars are grouped depending on their country of origin. Any of the colour parameters considered (L*, a*, b*) showed significant differences if cultivars origins are considered (Table 3).

Table 3. Differences in the physical parameters of cultivars grouped by geographical origin. Kernel Kernel Origin Relation Flour Flour Flour Oil Oil Oil Viscosity length width country L/W L* a* b* L* a* b* (mPa·s) (mm) (mm) Greece 17.00 ab 9.33 1.82 52.51 -2.09 22.10 94.65 -7.76 71.49 62.57 abcd (n=1x3) Syria 17.22 ab 9.28 1.86 55.25 -2.85 22.66 95.30 -7.95 74.19 63.02 ab (n=6x3) Iran 16.33 bc 9.00 1.82 50.65 -3.11 23.00 95.12 -7.83 74.01 61.27 d (n=3x3) Israel 16.56 bc 9.11 1.82 50.50 -2.52 23.04 95.47 -8.01 70.77 61.52 bcd (n=3x3) Italy 16.83 ab 8.83 1.91 51.14 -2.97 21.67 95.15 -7.90 73.33 62.91 abc (n=3x3) Iraq 15.00 c 9.00 1.68 52.46 -3.29 22.46 95.75 -7.99 71.62 61.30 cd (n=1x3) Cyprus 16.00 bc 8.33 1.92 45.96 -2.33 21.99 93.41 -7.64 75.22 63.90 a (n=1x3) Tunisia 17.17 ab 9.00 1.93 52.16 -2.77 23.20 95.31 -8.04 72.08 61.54 bcd (n=2x3) Australia 18.33 a 10.67 1.72 54.45 -1.93 24.63 94.60 7.89 80.39 62.57 abcd (n=1x3) P ** NS NS NS NS NS NS NS NS *** n = number of cultivars considered from the country x number of replicates for each cultivar. Numbers are means of cultivars from the same origin country. Different letters in columns indicate significant differences by Duncan test (P< 0.05). NS, not significant. ** Significant at P < 0.05. *** Significant at P < 0.001.

Kernel length is a useful parameter for origin differentiation (p<0.05). By origin countries, Iranian cultivars were reported to produce the shortest nuts, while Italian were the longest (Caruso et al., 1998). In our study, the longest cultivar is Sirora from Australia, while the Iraqi cultivar Iraq produced the shortest kernels. These two cultivars have been neglected in most of previous studies. Within the rest, statistical

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Physical analysis of pistachio kernels and pistachio oils differences where not found. For kernel width and the relation length/width, significant differences were not reported. This low variability in kernel physical parameters could be the result of considering kernels from cultivars that have been all grown in the same plot. Then, the differences reported in previous studies (Caruso et al., 1998), could be partially attributed to the environment and land management and not to the cultivar itself.

Pistachio oil viscosity was also identified as a useful parameter for origin differentiation. Cultivars from Iran (Kerman, Albina and Joley) showed the lowest viscosity values that could be attributed to the higher proportion of linoleic fatty acid (Kim et al., 2010; Oliveira et al., 2016). On the other hand, cultivars from Cyprus (Larnaka) and Syria (Ajamy, Ashoury, Batoury, Boundoky, Lathwardy and Ouleimy) showed the highest values (means over 63.02 mPa·s). Viscosity could be proposed as a useful parameter to detect adulteration of varietal pistachio oils.

4. Conclusion

Differences in length and width of pistachio kernels were found when the effects of the environment and the land management were controlled. However, differences in the relation length/width were not reported. Regarding pistachio by products, colour can be identified as an useful variable for cultivar differentiation of pistachio oil and flour. The b* parameter in pistachio oils and the parameter L* in pistachio defatted flours are considered the most adequate parameters for cultivar differentiation. Varieties also produce oils with different values for viscosity and as a result, this value could be used for cultivar authentication. Beyond these differences, viscosity in pistachio oils is directly correlated with the percentage of oleic and linoleic fatty acids in pistachio cultivars (R=0.92). By grouping cultivars depending on their origin, differences were found in the kernel length and in the oil viscosity, but not in the kernel width nor in the colour parameters. Utility of physical parameters for pistachio cultivar differentiation and cultivar origin differentiation is certified.

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Tsantili, E., Takidelli, C., Christopoulos, M. V., Lambrinea, E., Rouskas, D. Roussos, P.A., 2010. Physical, compositional and sensory differences in nuts among pistachio (Pistacia vera L.) varieties. Scientia Horticulturae 125, 562–568.

Yeganeh, R., 2013. Colour optimisation of ground pistachio during roasting. Quality Assurance and Safety of Crops & Foods 5, 357-363.

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Evaluation of physical parameters of walnut kernels and walnut products

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Evaluation of physical parameters of walnut kernels and walnut products

ABSTRACT Keywords: The differences in walnut cultivars attending to their chemical composition Juglans regia Walnut oil have been widely reported. These chemical differences result in differences Walnut flour in the physical parameters of walnut oils and walnut defatted flours (DF) Viscosity CIELCH obtained from cold pressing. In this study, the physical parameters of Crop year walnut kernels, walnut oils and DF obtained from nine different cultivars

are analyzed. Additionally, the effect of the genotype, the crop year and the interaction between both in the colour parameters of walnut oil and DF is studied. Results show that oil colour is mainly determined by the cultivar, while the colour of DF is determined for the genotype, the crop year and the interaction of both. L* parameter is the most useful for cultivar discrimination if the two crop years are considered. Within physical parameters of walnut products, oil viscosity can be considered a parameter of oil quality as it shows strong correlations with the oil fatty acid profile and the concentration of specific triglycerides (LLL, OLO and POO). Physical parameters have been reported to be crucial for consumers and for the food industry and therefore should be considered as quality parameters to be considered in walnut by-products.

1. Introduction

The walnut has been classified as a strategic species for human nutrition and has been included in the FAO’s list of priority plants (Kasmi et al., 2013). The nutritional benefits of walnut are associated to its high content of unsaturated fatty acids profile, presence of bioactive components, high quality proteins and its significant content of essential minerals (Amaral et al., 2005; Crews et al., 2005; Cuesta et al., 2017).

The use of walnut kernels to obtain new products that can be consumed directly or added to food to improve its characteristics has been widely studied. The high

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Evaluation of physical parameters of walnut kernels and walnut products content of lipids in walnut, that ranges between 52 to 74% of kernel weigh depending on the cultivar (Amaral et al., 2003; Cuesta et al., 2017; Kodad et al., 2016; Martínez et al., 2006), encourages the use of walnuts for oil production. Walnut oil shows high concentration of polyunsaturated fatty acids, mainly linoleic (Amaral et al., 2003), and important concentrations of antioxidants as vitamin E (Amaral et al., 2005; Misawa, 2009). Although different methods can be used for oil extraction (Roncero et al., 2016), cold pressing has been identified as the most convenient method as it allows the production of the high quality oils at affordable prices (Rabadán et al., 2017a). The main by-product resulting from oil extraction is a partially defatted walnut flour. This flour is expected to contain some of the beneficial properties reported in the whole nut, with special importance of the protein fraction due to its balanced content of essential amino acids (Cuesta et al., 2017).

The influence of genotype, origin and land management practices on the chemical composition of walnuts have been widely studied. The fatty acid profile, tocopherols, triglycerides, sterols and protein content, among other parameters, are effectively affected by the walnut genotype and walnut origin (Amaral et al., 2003;Amaral et al., 2005; Bada et al., 2010; Crews et al., 2005; Kodad et al., 2016). Beyond the effect of genotype and origin, land management practices, as irrigation (Verardo et al., 2009) and fertilization (Verardo et al., 2013), affect the fatty acid profile and the presence of minor components. Most of the research has focused on the characterization and the chemical analysis of walnut genotypes, while little attention has been focused on the physical parameters of walnuts and walnuts by-products. Although nutritional value is directly correlated with the chemical characteristics of the oil and flour, physical parameters are more easily accessible to consumers, cheap and faster to measure and have main importance in the food industry. Beyond that, physical parameters are correlated with crucial chemical parameters in walnut products (Geller and Goodrum, 2000; Kim et al., 2010; Oliveira et al., 2016).

Within oil parameters, viscosity is related to oil processing and oil quality. Research has proved that oil viscosity is related to the temperature of the oil (Kumar et al., 2013), the degree of unsaturation (Kim et al., 2010; Oliveira et al., 2016) and the triglyceride composition of the vegetable oil (Geller and Goodrum, 2000). Variability

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in the reported viscosity values of walnut oil are expected to be high due to the important differences that appear in the fatty acid profile of walnut cultivars (Cuesta et al., 2017; Yerlikaya et al., 2012). Moreover, the high presence of double bonds in the fatty acids that compound walnut oil makes it more sensitive to rapid changes in viscosity with temperature (Kim et al., 2010). Gharibzahedi et al. (2014), suggested the existence of differences in the viscosity of oils obtained from three different walnut cultivars collected in Iran, but a deeper analysis, with a significant number of cultivars, should be performed.

One physical parameter that usually do not received enough attention is colour (Moyano et al., 2010). Colour has been identified as a parameter influencing consumer preference in olive oils (Gámbaro et al., 2014; Recchia et al., 2012) so its influence in the quality of walnut oil should be considered. The green to yellowish colour of plant oils is the result of various pigments, mainly carotenoids and chlorophylls (Misawa, 2009). Carotenoids have been widely studied in walnut oil due to their protective effects against cardiovascular diseases, certain cancers and ageing diseases (Misawa, 2009). In walnuts, carotenoids were reported to range between 0.08 and 0.49 mg/kg depending on the cultivar analyzed (Özrenk et al., 2012). Beyond the cultivar itself, the content of carotenoids in plants are also affected by environmental conditions (Strzałka et al., 2003) and land management practices (Leskovar et al., 2009).

The oil colour is mainly attributed to the existence of the pigments that are extracted along with the oil during extraction (Gharibzahedi et al., 2014). The remaining flour that appear as by-product of oil extraction retain the pigments that are not lipid- soluble, and those that are retained in the lipid fraction that remains in the defatted flour. Martínez et al. (2008) studied the effect of seed moisture and the extraction temperature on the colour of walnut oils obtained by screw pressing. They concluded that the values of b* in oils increased with the seed moisture content resulting in yellower oils. However, oils obtained with supercritical CO2 extraction were even darker than those obtained with the screw press possibly due to a higher content of

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Evaluation of physical parameters of walnut kernels and walnut products carotenoids. The extraction pressure used in the supercritical oil extraction also caused increases in the carotenoid content.

In this study, the physical parameters of walnut kernels, walnut oils and walnut defatted flours obtained using a hydraulic press from nine different walnut cultivars are analyzed. The usefulness of physical parameters for cultivar discrimination is evaluated. Additionally, the effect of the genotype and the crop year in the colour parameters of walnut oil and walnut defatted flour is analyzed.

2. Materials and Methods

2.1. Plant material

Walnuts were collected in the Instituto Técnico Agronómico Provincial of Albacete in the southeast of Spain in 2015 and 2016. Nine different walnut cultivars were considered (Franquette, Hartley, Hugget, Mayette, Mollar de Nerpio, Parisienne, Payne, Pedro and Serr). One kilogram of walnuts was collected from every one of the three trees analysed within each cultivar. Walnuts were picked at the most appropriate harvest date for each cultivar.

2.2. Oil extraction

Walnuts were cracked and shelled manually in controlled conditions for immediate drying at room temperature. Oil extraction was carried out using a hydraulic press (MECAMAQ Model DEVF 80, Vila-Sana, Lleida, Spain) at pressure of 150 bar. One kilogram of ground walnuts was placed each time on the press. After pressing, oil was centrifuged to remove remaining solids. Oils and flours were stored in dark glass bottles at 5˚C to avoid degradation until analysis (Rabadán et al., 2017a; Rabadán et al., 2017b).

2.3. Analysis

Kernel length, width and weight were recorded on sub-samples of 10 kernels for each walnut cultivar.

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The colour of the oil samples was measured using a spectrophotometer UV/Vis Jasco V-530 (Jasco Analytical, Madrid, Spain). Oil was placed in quartz cuvettes (1cm path length) for their analysis. N-hexane was used as blank reference. The colour of the flour was measured by reflection in five random zones with a Minolta CR-200 colourimeter (Minolta Camera Co., Ltd., Osaka, Japan) in each flour. The illuminant used was D65. The tristimulus values obtained were used to calculate the CIELAB chromatic coordinates: L∗ (brightness), a∗ (red–green component), b∗ (yellow–blue component) as recommended by the Commission Internationale de l’Eclairage (CIE, 1986). The recorded values, were converted into h˚ and C* according to the equations 1 and 2, respectively. Plotting the hue coordinates, a colour wheel subtends 360˚, with red-purple traditional placed at the far right (angle of 0˚); yellow, bluish-green, and blue follow counter clockwise at 90˚, 180˚ and 270˚, respectively (McGuire, 1992).

푏∗ ℎ° = 푡푎푛−1 ( ) 푤ℎ푒푛 푎∗ > 0 푎푛푑 푏∗ > 0 푎∗

푏∗ ℎ° = 180° + 푡푎푛−1 ( ) 푤ℎ푒푛 푎∗ < 0 (1) 푎∗

푏∗ ℎ° = 360° + 푡푎푛−1 ( ) 푤ℎ푒푛 푎∗ > 0 푎푛푑 푏∗ < 0 푎∗

퐶∗ = (푎∗2 + 푏∗2)1/2 (2)

To evaluate the texture of walnuts, 10 kernels of each variety were analyzed. The cutting force was measured using a TA-XT Plus texture analyzer (Stable Micro Systems, Godalming, UK). The kernels were cut perpendicularly to their major axis with a Warner Bratzler blade with rectangular slot blade at a constant velocity of 2 mm s-1.

In order to determine fatty acids composition (%), the methyl-esters were prepared by vigorous shaking of a solution of oil in hexane (0.2 g in 3 ml) with 0.4 ml of 2 N methanolic potassium hydroxide solution, and analysed by GC with a Hewlett- Packard chromatograph (HP 6890). A fused silica column (50 m length x 0.25 mm i.d.), coated with SGL-1000 phase (0.25 µm thickness; Sugerlabor), was used. Helium was

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Evaluation of physical parameters of walnut kernels and walnut products employed as a carrier gas with a flow through the column of 1 ml min-1. The temperatures of the injector and detector were set at 250°C with an oven temperature of 210°C. An injection volume of 1 µL was used (Regulation EEC 2568/91, corresponding to AOCS method Ch 2–91).

Triglycerides have been determined using HPLC-IR after weighting 0.5g of oil in 10mL of acetone, mobile phase 50:50 acetone: acetonitrile at a flow rate 1 mL/min and column ACE18 (250 mm length, x 4.6 mm i.d x 5 µm).

Viscosity was measured by a rotary viscometer test method (Visco Basic Plus, Fungilab S.A.) using the method described by Xu et al. (2007).

2.3. Statistical analysis

Determinations in this study are means of triplicate measurements from three independent samples for each walnut genotype. Statistical differences were estimated from ANOVA test at the 5% level of significance and Duncan test (p < 0.05). Correlation analyses were performed employing Pearson´s test. In order to analyse the comparative responses of walnut genotypes in both 2015 and 2016 crop years and the possible interaction, data were also analysed by two way ANOVA. All statistical analysis were carried out using the SPSS programme, release 23.0 for Windows.

3. Results and discussion

3.1. Walnut kernel size, form and hardness

Table 1 shows the data of main physical parameters of walnut kernels. Significant differences were reported for weight, width, length and the relation length/width. The weight of the kernel from cv. Pedro and Mollar de Nerpio were 2.79 and 2.66g, respectively. These values almost double the weight of others as Parisienne. However, the weight of the reported cultivars is within the lower values reported by cultivars in Iran (Ebrahimi et al., 2015) and also lower than the reported for kernels from Turkey (Akça et al., 2014). Attending to relation L/W two groups can be

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identified attending to cultivars with kernels with values close or far from zero (round to elliptic). In previous studies, the shell strength had been measured, but not the kernel hardness (Ebrahimi et al., 2015; Khadivi-Khub et al., 2015). Our results show that the hardness of walnut kernels does not show statistically significant values among cultivars.

3.2. Oil density and viscosity

Density and viscosity are parameters of crucial interest in the food industry. Our results show that density is similar in oils obtained from different cultivars, while the viscosity of oils depends on the cultivar used for oil extraction (table 2). The density of walnut oils ranged between 0.83 to 0.89 mg/cm3, slightly lower than the reported in previous studies (Iqbal et al., 2016). These differences could be attributed to different oil extraction methods as extraction methods affects the level of impurities in oils (Rabadán et al., 2017b). Regarding viscosity, cv. Scharch-Franquette and Parisienne are those with the higher values, with 46.78 and 46.41 mPa·s, respectively. Differences appear with the viscosity values obtained in previous studies (Gharibzahedi et al., 2014). These differences could be attributed to differences in the measurement method, the temperature of the oil (Kim et al., 2010) or the walnut cultivars analysed (Cuesta et al., 2017). Viscosity values obtained for walnut oils were lower than those obtained for pistachio oil, this could be the result of the higher content of oleic in pistachio oil (Rabadán et al., 2017c).

Table 1. Data of kernel size, form and hardness of walnut kernels from different cultivars. Cultivar Weight (g) Width (mm) Length (mm) Length/Width Hardness (N) Scharch- 1.86cd ± 0.15 23.1cd ± 1.0 22.9d ± 0.1 1.00cd ± 0.07 36.86 ± 10.62 Franquette Harthey 2.17bc ± 0.02 25.0b ± 1.0 29.0ab ± 0.1 1.16a ± 0.01 24.26 ± 6.33 Nugget 2.39ab ± 0.20 30.3a ± 0.6 27.7b ± 0.1 0.91e ± 0.05 39.11 ± 13.54 Mayette 1.56d ± 0.25 21.7de ± 1.5 25.3c ± 0.1 1.17a ± 0.08 27.87 ± 5.21 Nerpio 2.66a ± 0.25 30.2a ± 0.6 28.7ab ± 0.1 0.95de ± 0.02 43.92 ± 14.80 Payne 1.46d ± 0.41 21.5de ± 0.5 23.0d ± 0.0 1.07bc ± 0.02 33.34 ± 5.66 Parisienne 1.42d ± 0.25 20.7e ± 0.6 22.6d ± 0.1 1.10ab ± 0.00 32.97 ± 10.09 Pedro 2.79a ± 0.26 29.7a ± 1.5 30.0a ± 0.1 1.01cd ± 0.05 32.67 ± 8.85 Serr 2.47ab ± 0.24 24.5bc ± 0.5 28.1b ± 0.1 1.14ab ± 0.02 36.22 ± 18.17 P *** *** *** *** ns

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Mean values (±standard deviation) were the averages of ten independent measurements. Different letters on the column indicate significant differences among genotypes (p<0.05). ns, not significant; **p<0.05; ***p<0.01.

As expected, a strong correlation of walnut oils viscosities with the oil fatty acid profile was found (Kim et al., 2010) (Figure 1). This reported dependence (Conte et al., 2011; Geller and Goodrum, 2000; Kim et al., 2010; Oliveira et al., 2016), allows the calculation of viscosity by using the percentage of monounsaturated (MUFA) or polyunsaturated (PUFA) fatty acids or vice versa. The equations proposed in figure 1 are able to explain up to 83 and 87% of the total variance observed in walnut oil viscosity values. Agreeing with the previous results obtained for other plant oils (Kim et al., 2010), the viscosity of walnut oil in the different cultivars was however not related to the proportion of saturated (R2= 0.08). Similar results had been previously reported for pistachio oil (Rabadán et al., 2017c). This could be attributed to the low concentration of saturated fatty acids of nut oils, in which oleic and linoleic compound almost completely the fatty acid profile.

Table 2. Density (mg/cm3) and viscosity (mPa·s) values of walnut oils obtained from different cultivars. Cultivar Density Viscosity Scharch- Franquette 0.87 ± 0.02 46.78a ± 0.28 Hartley 0.86 ± 0.01 43.85f ± 0.08 Nugget 0.85 ± 0.03 44.97d ± 0.25 Mayette 0.85 ± 0.02 45.60c ± 0.15 Nerpio 0.88 ± 0.02 44.09ef ± 0.18 Parisienne 0.84 ± 0.03 46.41b ± 0.32 Payne 0.83 ± 0.01 44.10ef ± 0.07 Pedro 0.89 ± 0.02 44.45e ± 0.21 Serr 0.85 ± 0.04 45.44c ± 0.13 P ns *** Mean values (±standard deviation) were the averages of three independent measurements. Different letters on the column indicate significant differences among genotypes (p<0.05). ns, not significant; **p<0.05; ***p<0.01.

A strong correlation between viscosity and the triglyceride profile was also reported (Figure 2). Triglycerides whose content showed the strongest correlation with viscosity values were LLL, OLO and POO. As previously reported for fatty acids, triglycerides composed for polyunsaturated fatty acids were found in higher

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concentration in oils with lower viscosity values. These results agree with the conclusions of previous studies about the influence of triglyceride composition on the viscosity of vegetable oils (Geller and Goodrum, 2000).

Figure 1. Medium values obtained for each cultivar considered for oil viscosity and content of polyunsaturated (PUFA) and monounsaturated (MUFA) fatty acids.

Figure 2. Medium values obtained for each cultivar considered for oil viscosity and content of triglycerides LLL, OLO and POO. Where: L, linoleic acid; O, oleic acid; P, palmitic acid.

Considering the need to design fast, cheap and accurate techniques to determine viscosity in vegetable oils to confirm authenticity, detect adulteration and design industrial processes (Kumar et al., 2013), empirical equations for predicting the viscosities values could be used.

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3.3. Colour parameters

Table 3 shows the results of the study of colour in the walnut kernel, the walnut flour obtained after walnut gridding and the colour of the walnut flour after oil extraction (defatted walnut flour). The colour of the walnut kernel is different from the colour of the walnut flour and the two of them are different from the colour of the flour obtained after oil extraction, especially regarding C* values.

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The colour of walnut peel shows significant differences among cultivars for the three parameters considered in CIELCH (L*, h˚, C*). Previously the colour of walnut kernels had been mainly analysed by using colour scales finding differences among cultivars (Khadivi-Khub et al., 2015). Lightness values (L*) are higher in cv. Parisienne, Scharch- Franquette and Nugget, meaning kernels with the darkest brown peels. Cultivars with the lower values for lightness also show the lower values for hue (h˚) and chroma (C*) as significant correlations between parameters have been reported (Table 4). Cultivars Mollar de Nerpio and Serr show the lowest values in the colour coordinate h˚, with values closer to the 0˚ axis (red). This seems to be an intrinsic characteristic of these cultivars that could be used for cultivar discrimination.

Table 4. Correlation values obtained employing Pearson´s test. N L* N h˚ N C* F L* F h˚ F C* DF L* DF h˚ DF C* N L* 1 .786** .524** .656** .604** .188 .172 .520** -.107 N h˚ 1 .667** .488** .578** .227 -.025 .587** -.229 N C* 1 .525** .515** .094 .077 .456** -.104 F L* 1 .748** .500** .273 .449* .072 F h˚ 1 .195 .164 .601** .142 F C* 1 -.067 .238 -.284 DF L* 1 .282 .621** DF h˚ 1 -.059 DF C* 1 Abbreviations: N, whole walnut kernel; F, walnut flour; DF, Defatted walnut flour. *p<0.05; **p<0.01.

The lightness of the colour (L*) is higher in the defatted walnut flour than in the walnut flour, while C* values show opposite evolution. The extraction of the oil also means the extraction of most of the lipid-soluble compounds (Gharibzahedi et al., 2014), which causes the reduction of the total concentration of pigments in the remaining flour. This means that by extracting the oil of the walnut flour we obtain defatted flours with less intense colours that move from yellow-brown to greyish colours. The colour of the walnut flour and the defatted flour is correlated with the colour of the whole walnuts (Table 4). Values for h˚ in walnut flour and the defatted walnut flour are correlated with colour parameters reported in the whole walnut.

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3.4. Influence of crop year in colour parameters

Walnut oils obtained by cold pressing are pale yellow in colour. Although at first sight all seem similar, by using CIELCH coordinates we found significant differences in the oils obtained from different cultivars (Table 5). Main differences appear in the lightness and the chroma values. Cultivars Scharch-Franquette and Parisienne show the highest values for lightness, while colour saturation (C*) is higher in cv. Mayette and Mollar de Nerpio. These higher values of C* indicate oils with more vivid yellow colours, while lower values indicate whitish oils. Lightness and chroma values were higher in most of the oils obtained from the 2015 harvest, while hue values were similar in the two crop years. The content of total carotenoids in the cv. Parisienne was the lower between the cultivars considered by Misawa (2009), this could be the cause of the lower values for C*, meaning less intense yellow oil.

Table 5. Colour parameters lightness (L*), hue (h) and chroma (C) in walnut oil from different cultivars in 2015 and 2016.

Cultivar L* h˚ C* 2015 2016 Mean 2015 2016 Mean 2015 2016 Mean Scharch-Franquette 97.08a 92.25bc 94.67 94.48c 94.41c 94.45 18.31g 16.52d 17.42 Hartley 94.51c 91.52d 93.02 94.63ab 95.23b 94.93 20.54e 17.99c 19.27 Nugget 95.63b 92.36ab 94.00 94.57c 95.67a 95.12 19.07f 19.28b 19.18 Mayette 90.64e 89.64f 90.14 93.43d 93.43e 93.43 28.27a 28.27a 28.27 Nerpio 95.36b 91.28e 93.32 94.35bc 95.11b 94.73 26.63b 18.32c 22.48 Parisienne 95.81b 92.56a 94.19 95.15a 95.78a 95.46 17.76g 16.41d 17.08 Payne 94.18c 87.34g 90.76 94.17bc 93.94d 94.06 22.67c 19.27b 20.97 Pedro 95.51b 92.10c 93.81 94.60ab 95.22b 94.91 20.98de 18.14c 19.56 Serr 92.53d 85.93h 89.23 93.85cd 95.22b 94.54 21.47d 18.61c 20.04 P *** *** *** *** *** *** Mean values (±standard deviation) were the averages of three independent measurements. Different letters on the column indicate significant differences among genotypes (p<0.05). ns, not significant; **p<0.05; ***p<0.01.

Using CIELAB coordinates, Gharibzahedi et al. (2014) reported values for oils of 1.2 for a* and 11.5 for b*. Previously, in a study about the colour of the oil obtained from a mixed range of walnut cultivars, different results were obtained with -1.9 and 6.2 for a* and b*, respectively (Sze-Tao and Sathe, 2000). In our study the CIELAB coordinates a* and b*, were used to calculate CIELCH coordinates. Our previous

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Evaluation of physical parameters of walnut kernels and walnut products values for a* were similar to some of the reported values (Sze-Tao and Sathe, 2000), while values for b* were slightly higher. Although similar colours are reported in all the studies, differences in the extraction methods and the methods for colour measurement could explain the observed differences. Our walnut oils obtained by hydraulic pressing show more vivid yellow colours than some of the reported previously, due to higher b* values that also result in higher C* values.

In the case of the defatted walnut flour, significant differences were reported for the lightness of flours (p<0.001) (table 6). However, hue values showed no discriminant power for the flours obtained from the 2015 harvest. Lightness is again the most useful parameter for cultivar discrimination, with cv. Parisienne, Payne and Serr obtaining mean values over 80 for the two crop years considered. On the other hand cv. Pedro and Mayette showed the lower values for lightness indicating darker flours.

Table 6. Colour parameters lightness (L*), hue (h˚) and chroma (C*) in walnut defatted flour (DF) from different cultivars in 2015 and 2016. L* h˚ C* Cultivar 2015 2016 Mean 2015 2016 Mean 2015 2016 Mean Scharch- 78.97a 80.46a 79.72 84.26 84.60ab 84.43 17.47bcd 16.80a 17.14 Franquette Hartley 79.37a 74.22b 76.80 85.56 84.53b 85.04 18.35b 16.84a 17.59 Nugget 79.86a 79.44a 79.65 85.16 84.68ab 84.92 17.54bc 17.16a 17.35 Mayette 66.75b 79.24a 72.99 87.00 84.68ab 85.84 24.88a 16.61a 20.74 Nerpio 79.68a 75.12b 77.40 85.85 82.43b 84.14 17.73bc 16.98a 17.35 Parisienne 82.04a 78.87a 80.45 85.55 84.60ab 85.07 15.85de 17.70a 16.78 Payne 80.35a 80.13a 80.24 85.18 85.58a 85.38 16.57cde 17.22a 16.90 Pedro 65.03b 67.26c 66.15 84.41 84.74ab 84.58 15.82e 15.41b 15.61 Serr 82.47a 77.53ab 80.00 86.03 84.59ab 85.31 17.04bcde 17.12a 17.08 P *** *** ns *** *** ** Mean values (±standard deviation) were the averages of three independent measurements. Different letters on the column indicate significant differences among genotypes (p<0.05). ns, not significant; **p<0.05; ***p<0.01.

Results of the two-way ANOVA are shown in Table 7. The source of variability in the colour parameters of walnut oils and walnut defatted flours is different. Although the genotype, the crop year and the interaction between both are all significant in the determination of colour in walnut oil, the greatest source of variability in the three parameters is the crop year. Regarding flours, lightness is mainly determined for the

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Influence of raw material in oil characteristics genotype while the hue is determined mainly for the interaction between the genotype and the crop year. No effect of the year is reported in these parameters. However, the chroma values of walnut defatted flours is strongly determined by the crop year.

Table 7. Analysis of variance for colour parameters of walnut oil and walnut defatted flour Mean Source of variation df F-test p-value square Oil L* Genotype 8 23.75*** 324.51 <0.001 Year 1 219.29*** 2996.56 <0.001 YearxGenotype 8 5.07*** 69.21 <0.001 Error 54 Oil h˚ Genotype 8 2.20*** 29.34 <0.001 Year 1 3.81*** 50.81 <0.001 YearxGenotype 8 0.43*** 5.79 <0.001 Error 54 Oil C* Genotype 8 67.48*** 572.63 <0.001 Year 1 87.41*** 741.72 <0.001 YearxGenotype 8 9.40*** 79.80 <0.001 Error 54 DF L* Genotype 8 168.62*** 22.24 <0.001 Year 1 1.07 ns 0.14 0.709 YearxGenotype 8 57.31*** 7.56 <0.001 Error 54 7.58 DF h˚ Genotype 8 3.59*** 5.47 <0.001 Year 1 2.18 ns 3.31 0.074 YearxGenotype 8 5.66*** 8.62 <0.001 Error 54 0.66 DF C* Genotype 8 15.02*** 23.13 <0.001 Year 1 19.30*** 29.73 <0.001 YearxGenotype 8 16.27*** 25.06 <0.001 Error 54 0.65 Abbreviations: DF, defatted walnut flour. ns, not significant; **p<0.05; ***p<0.01.

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4. Conclusions

Different walnuts cultivars can be used to produce oils and flours with different physical characteristics. Physical parameters of walnut kernels, walnut oil and walnut defatted flour are useful for cultivar discrimination. Beyond the traditional use of scales for the analysis of physical parameters as walnut colour or form, our results prove that analytical methods are useful for that purpose. Viscosity in walnut oils is highly correlated with the level of MUFA and PUFFA and also with the content of specific triglycerides. This provides useful information to develop cheap and fast methods to obtain information about chemical characteristics by using information about physical parameters. Regarding colours, walnut oil colour depends on the walnut cultivar used for oil extraction while the colour of the defatted flour depends also on the crop year and the interaction between the cultivar and the crop year. Results encourage the use of physical parameters to develop cheap and fast methods of analysis that can provide information about the chemical characteristics of walnut products and at the same time detect product adulteration.

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Conte, P., Mineo, V., Bubici, S., De Pasquale, C., Aboud, F., MacCotta, A., Planeta, D., Alonzo, G., 2011. Dynamics of pistachio oils by proton nuclear magnetic resonance relaxation dispersion. Analytical and Bioanalytical Chemistry 400, 1443-1450.

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Ebrahimi, A., Khadivi-Khub, A., Nosrati, Z., Karimi, R., 2015. Identification of superior walnut (Juglans regia) genotypes with late leafing and high kernel quality in Iran. Scientia Horticulturae 193, 195-201.

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Gharibzahedi, S.M.T., Mousavi, S.M., Hamedi, M., Khodaiyan, F., 2014. Determination and characterization of kernel biochemical composition and functional compounds of Persian walnut oil. Journal of Food Science and Technology 51, 34-42.

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Martínez, M.L., Mattea, M.A., Maestri, D.M., 2008. Pressing and supercritical carbon dioxide extraction of walnut oil. Journal of Food Engineering 88, 399-404.

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Özrenk, K., Javidipour, I., Yarilgac, T., Balta, F., Gündoǧdu, M., 2012. Fatty acids, tocopherols, selenium and total carotene of pistachios (P. vera L.) from Diyarbakir (Southestern Turkey) and walnuts (J. regia L.) from Erzincan (Eastern Turkey). Food Science and Technology International 18, 55-62.

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Rabadán, A., Pardo, J.E., Gómez, R., Alvarruiz, A., Álvarez-Ortí, M., 2017c. Usefulness of physical parameters for pistachio cultivar differentiation. Scientia Horticulturae 222, 7-11.

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2.2. Physicochemical analysis of oils and partially defatted flours

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Suitability of Spanish almond cultivars for the industrial production of almond oil and

defatted flour

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Suitability of Spanish almond cultivars for the industrial production of almond oil and defatted flour

ABSTRACT Keywords: The combined evaluation of almond oils and flours obtained by cold Almond flour Almond oil pressing provides a comprehensive approach about the nutritional and Cultivars industrial interest of Spanish almond cultivars for these production Fatty acids Oxidative purposes. Almonds of ten different cultivars were collected from the same stability plot to remove the environmental and agricultural management effects on almond chemical traits. Results show that oil yield was similar for all the selected cultivars, however, obtained oils showed significant differences in their fatty acid profile, including essential fatty acids of main nutritional interest. According to the triglyceride profile, significant differences were found for main tri-unsaturated triglycerides (mainly, OOO and OLL) but not for OLO. The concentration of minor components with health promoting properties also was different in the oil obtained from different cultivars. For the selection of the optimal cultivar for oil production, three parameters are proposed as main parameters to consider: iodine value, oxidative stability and oil yield. According to these parameters, the varieties Guara, Ferragnes and Belona would be the most appropriate cultivars for almond oil production. Regarding almond flours obtained, large differences were found in the content of available carbohydrates (276.4 – 156.1 g/kg) and proteins (581.3 – 379.4 g/kg) resulting in flours with potential different uses. The total mineral content in flours and the presence of specific minerals (as Fe and Zn) also showed significant differences among cultivars.

1. Introduction

The almond (Prunus dulcis) is the most important nut in terms of commercial production. Almond tree cultivation is focused mainly in three regions: California, the Mediterranean basin and central Asia - Middle East. Cultivation distribution is limited

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Evaluation of almond cultivars due to the specific characteristics of hot and dry conditions where the tree produces the highest yields of high quality almonds. Almond production has increased drastically in the last years, from 1,251 billion tons in 2003 to 2,917 billion tons in 2013. The United States and Spain are the larger almond producers, producing, respectively, 43% and 7% of the total almond produced in the last decade (FAO, 2016).

In almond seeds, lipids appear stored in oil droplets (Gallier et al., 2012) and they account from 40 to 67 g/100 g of dry almond weight (Yada et al., 2011). Substantial quantities of triacylglycerol have been reported in almond oil (Martín-Carratalá et al., 1999; Cherif et al., 2004). The proportion of proteins (14-26%) and carbohydrates (2- 8%) in the dry almond seed are lower than lipid content although high variability has been described (Roncero et al., 2016a).

Extensive research has been developed regarding the fatty acid profile of almond oils. Almond oil is mainly composed of mono and di-unsaturated fatty acids (Roncero et al., 2016b). Differences in almond oil fatty acid profile attending to the almond origin have been widely described (García-López et al., 1996; Kodad and Socias I Company, 2008; Yada et al., 2011; Maestri et al., 2015). In order of importance, the main fatty acids that appear in almond oil are oleic (50-80%), linoleic (11-37%), palmitic (5-16%) and stearic (1-4%) acids (Askin et al., 2007). Linolenic acid appears in concentrations lower than 0.1% (Maestri et al., 2015) although percentages higher than 11% have been reported in some cultivars (Askin et al., 2007). Almond breeding programs have shown promising results of superior genotypes that could be selected for almond oil production although further work will be needed (Kodad and Socias I Company, 2008; Zhu et al., 2016).

Minor components, as sterols or tocopherols are decisive for almond oil quality (Maestri et al, 2015). Almond oil sterols are almost entirely composed by β-sitosterol (95% of total sterols) and minor concentrations of campesterol and stigmasterol. Previous research found that tocopherol concentration in the Spanish genetic bank varies from 350 to 550 mg/kg oil, with major presence of α-tocopherol (Kodad et al., 2014). Differences in the content of tocopherol homologues in almonds grown in

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Spain and Morocco were found, but total tocopherol content remained (Kodad et al., 2011). The active role of tocopherols in the oil protection against lipid oxidation make them an important quality parameter in almond oil.

Beyond cultivar effect, other factors such as soil and climate have proven to have influence on almond oil (García-López et al., 1996; Yada et al., 2013; Kodad et al., 2014). The effects of irrigation on almond oil content have also be analyzed, but studies lead to different results, with some finding a significant influence of irrigation supplement on almond oil composition (Schirra and Aggabio, 1989; Sánchez-Bel et al., 2008), while others report opposite results (Egea et al., 2009).

Cultivar selection and the growing region have been reported to have both a significant effect on almond quality and nutritional value (Kodad et al., 2011). Our proposal is the selection of the optimal almond cultivar for production of almond oil and flour considering some of the main cultivars grown in a continental Mediterranean climate in Spain attending to nutritional and industry production parameters.

2. Materials and Methods

2.1. Plant material

Almond seeds were collected at an experimental orchard in the Instituto Técnico Agronómico Provincial of Albacete in the southeast Spain in 2015. Ten different cultivars (Antoñeta, Ayles, Belona, Ferraduel, Ferragnes, Guara, Marcona, Penta, Tardona and Vairo) were analysed. One kilogram of almonds was collected from every one of the three trees analyzed within each cultivar. Almonds were picked at the most appropriate harvest date for each cultivar. By considering almond seeds that have all being grown at the same plot, result deviations that could appear on almond oil and flour production due to differences in local environmental conditions (Kodad et al., 2011) or land management, such as irrigation (Sánchez-Bel et al., 2008; Kodad et al., 2011), are controlled. Thus, the observed differences are related to the cultivar effect.

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Although almond tree cultivation is mainly concentrated in areas with Mediterranean or Mediterranean type climates, differences in water deficit (Zhu et al., 2015) and temperatures (Kodad et al., 2006) should always be considered, as they have major influence in almond production and characteristics. Analysis on the most adequate almond cultivar for oil and flour production must always be linked to the characteristics of a geographic location.

2.2. Oil extraction

Almonds were cracked and shelled manually in controlled conditions for immediate drying. Moisture was calculated after drying in a desiccation oven for 12h at 100˚C. Three samples for each genotype were processed.

Oil extraction was carried out using a hydraulic press (MECAMAQ Modelo DEVF 80, Vila-Sana, Lleida, Spain) at pressures of 50, 100, 150 and 200 bar with increasing pressure every 5 minutes (Rabadán et al., 2017a). One kilogram of ground almonds was placed each time on the hydraulic press. After pressing, oil was centrifuged to remove remaining solids. Oil was stored in dark glass bottles at 5˚C to avoid degradation until analysis (Rabadán et al., 2017b).

The remaining pressing cake was ground and sieved to obtain a flour with particle size lower than 1 mm.

2.3. Oil and flour analysis

Oxidative stability was evaluated by the Rancimat method (Gutiérrez, 1989). Stability was expressed as the oxidation induction time (hours), measured with the Rancimat 743 apparatus (Metrohm Co., Basel, Switzerland). An oil sample of 3.5 g was used, warmed to 100°C under an air flow of 10 l h-1.

In order to determine fatty acids composition (%), the methyl-esters were prepared by vigorous shaking of a solution of oil in hexane (0.2 g in 3 ml) with 0.4 ml of 2 N methanolic potassium hydroxide solution, and analysed by GC with a Hewlett- Packard (HP 6890) chromatograph equipped with a FID Detector. A fused silica column (50 m length x 0.25 mm i.d.), coated with SGL-1000 phase (0.25 µm thickness;

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Sugerlabor), was used. Helium was employed as a carrier gas with a flow through the column of 1 ml min-1. The temperatures of the injector and detector were set at 250°C with an oven temperature of 210°C. An injection volume of 1µL was used (Regulation EEC 2568/91, corresponding to AOCS method Ch 2–91).

Sterols were determined with a Hewlett-Packard (HP 6890) gas chromatograph with a capillary column (25 m length x 0.25 mm i.d.) coated with SGL-5 (0.25 µm thickness; Sugerlabor). Working conditions were as follows: carrier gas, helium; flow through the column, 1.2 ml min-1; injector temperature, 280°C; detector temperature, 290°C; oven temperature, 260°C; injection volume 1 µl (Regulation EEC 2568/91, corresponding to AOCS method Ch 6–91).

The concentration of total polyphenols (ppm) was estimated using the method proposed by Bail et al. (2008) based on the determination of total phenols of oil using the the Folin–Ciocalteau colorimetric method. The absorption of the solution was measured on a spectrophotometer Hewlett-Packard 8450 A UV/Vis.

The tocopherol content (mg/Kg) was analysed by HPLC (model 360, Kontron, Eching, Germany) in accordance with the IUPA2432 method (IUPAC, 1987). 1.5 g oil was dissolved in the mobile phase (10 ml) of 0.5% isopropanol in n-hexane. A normal phase column Lichrosphere Si60 (250 mm length, 4.6 mm i.d. and 5 µm particle size) was used with an injection volume of 20 µl and a flow rate of 1.0 ml/min.

Triglycerides have been determined using HPLC-IR after weighting 0.5g of oil in 10mL of acetone, mobile phase 50:50 acetone: acetonitrile at a flow rate 1 mL/min and column ACE18 (250 mm length, x 4.6 mm i.d x 5 µm).

Analytical tests were performed in triplicate.

Maestri et al., (2015) identified oil production, fatty acid composition and the parameters related to oil oxidative stability as main criteria to consider in the selection of the optimal almond cultivar for almond oil production. Based on these criteria, the identification of the most adequate cultivar for almond oil extraction was developed by considering these three parameters equally important. Oil production

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Evaluation of almond cultivars is considered using the oil yield obtained with the hydraulic press extraction; fatty acid composition was taken into account by calculating the iodine value (I2V) and parameters related to oil oxidative stability with the results of oxidative stability obtained with the Rancimat method (Gutiérrez, 1989). We consider that the evaluation of these three main variables provides a valid approximation of cultivar potential for almond oil production.

Iodine values are a simple method to obtain information about the amount of unsaturation in fatty acids in oil. I2V was calculated from fatty acid percentages by using the formula (Torres and Maestry, 2006) that considers the three main unsaturated fatty acids that can be found on almond oil:

I2V = (% palmitoleic acid ∗ 1.001) + (% oleic acid ∗ 0.899) + (% linoleic acid ∗ 1.814)

After grinding, main nutritional components of extracted flour were also determined. The method used to determine the water content in the flour consisted on measuring the loss of weight after oven drying at 105ºC for 72h at least (Lau, 1982). Protein content was calculated by multiplying the total nitrogen content, obtained by the Kjeldahl method (FOSS, 2003) by a conversion factor of 6.25. To determine ash content, flours were ashed at 550ºC to constant weight (MAPA, 1998). Crude fat (Ethyl ether extract) was estimated gravimetrically by filter bag technique after petroleum ether extraction of the dried sample in an extraction system Ankom XT10 (ANKOM, 2009). To determine the content of crude fibre, the Weende technique adapted to the filter bag technique was applied. This method determines the organic residue remaining after digestion with solutions of sulfuric acid and sodium hydroxide, using an Ankom 220 fibre analyser (ANKOM, 2008). Total carbohydrate content was calculated by subtracting the sum of the crude protein, total fat, water and ash from the total weight of the flour (Sullivan, 1993). Available carbohydrate content (nitrogen-free) was calculated by subtracting the crude fibre from the total carbohydrate content (González et al., 1987).

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Contents of mineral elements in flours were obtained by inductively coupled plasma mass spectrometry (ICP-MS) using Jobin Yvon JY 50-P equipment (Instruments S.A., Jobin Yvon, Longjuemeau, France).

3. Results and discussion

3.1. Oil yield, fatty acid composition and minor components.

The differences in the oil content of different almond cultivars do not influence oil yield (table 1). Extraction yields with the hydraulic press were between 32.2-34.9 g of oil per 100g of almonds. Although higher extraction yields were obtained in Belona, Marcona and Tardona, significant differences with other cultivars were not found. Press characteristics and established extraction time (20 minutes) limit a more effective extraction. The slight increase in the moisture content, up to 4%-5%, has been identify with higher oil extraction for some seeds (Subroto et al., 2015) but, within the reduced moisture contents observed in our samples (3.94 - 5.93%), no significant variations in the obtained yield was observed in our study.

Fatty acid composition shows significant differences among cultivars (table 1). The wide range of fatty acid composition reported for some studies as Askin et al., (2007), can also be identified in the cultivars analysed in our study. The main six fatty acids found on almond oil are useful for almond cultivar discrimination (P < 0.01). As reported before, oleic is the dominant fatty acid in almond oil (Askin et al., 2007; Maestri et al., 2015; Zhu et al., 2015; Roncero et al., 2016a), with percentages that in our study range from 72.99% of total fatty acids in Guara to 65.37% in Ayles. The percentage of oleic obtained in our study for Guara is higher that the reported by Maestri et al., (2015) in a crop in Argentine, but similar to the one reported by Kodad et al., (2014). Linoleic content also shows high variability within the considered cultivars (23.54% - 16.90%), becoming a parameter of main nutritional interest as it is the main essential fatty acid found in almond oil. On the other side, palmitic and stearic fatty acids, are the most common saturated fatty acids in almond oil, with percentages in our samples below 7.64 and 3.21%, respectively.

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The nutritional value of almond oil is highly influenced by the high presence of unsaturated fatty acids, and although all cultivars show a healthy fatty acid profile, some differences can be observed. For instance, unsaturated fatty acids compound up to 91.54% of total fatty acids in Tardona or 91.41% in Belona. To compare the degree of unsaturation the iodine values of the considered cultivars are presented in table 1. Iodine values in Penta and Ayles were the highest, so more unsaturated oils are obtained from these cultivars. On the other hand, Guara (96.81) and Marcona (96.49) showed the more saturated oils.

The content of polyphenols, tocopherols and sterols showed significant differences, becoming useful parameters for cultivar almond oil discrimination (figure 1). Regarding the polyphenols content, the cultivars Guara, Ferraduel and Vairo can be discriminated from the rest (figure 1a). Beyond its active health promoting properties (Vanamala, 2017), the antioxidant properties of polyphenols (Tomaino et al., 2010) are related to higher stability of almond oils. Oil stability is considered an important parameter for food industry as it affects the self-life of the product. The oil obtained from the different cultivars also showed differences in the sterol content. The cultivar Ferraduel produced the oil with the higher concentration of sterols (2563 mg/kg), while the oil from the cultivar Tardona, showed the lowest (1819 mg/kg).

While the information about polyphenols and sterols is more limited, the differences in tocopherols in almond cultivars have been widely studied (Kodad et al., 2006; Kodad et al., 2011; Kodad et al., 2014; Zhu et al., 2015, Maestri et al., 2015). Tocopherols content (figure 1c) showed high variations, with concentrations of 503.1 mg/kg in Guara and 412.9 mg/kg in Vairo and values lower than 250 mg/kg in Tardona and Ayles. The high variability in the tocopherol content of the Spanish almond genetic bank was already reported by Kodad et al. (2014). In our study, the total tocopherol content of Marcona agrees with the range described by Kodad et al. (2014) for Spain, while the same cultivar in Argentine was reported to have a larger tocopherol content (Maestri et al., 2015). The tocopherols in Ferragnes and Ferraduel are slightly under the range reported in Kodad et al., (2011) for North-eastern Spain. The effect of the location and other factors such as rainfall or irrigation supplementation have been proposed as factors that have effects in the tocopherols

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Evaluation of almond cultivars in almonds (Kodad et al., 2011; Zhu et al., 2015), discouraging the comparison of the tocopherol content of the same cultivar in different plots.

Figure 1. Concentration of polyphenols (a), total sterols (b) and tocopherols (c) in almond oils. Different letters on the bars indicate significant differences (p<0.05) among cultivars (Duncan test).

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3.2. Triglyceride composition

Triglyceride composition of almond oils has been identified as a useful variable to differentiate almond origin (Martín-Carratalá, 1999; Cherif et al., 2004), but selected Spanish and French cultivars could to be too close to find those differences (Martín- Carratalá, 1999). Previous research found differences in triglyceride composition of almond cultivars with very different origins (Cherif et al., 2004), but our results prove that these differences also appear when comparing a larger number of cultivars even if they have a closer origin (table 2).

The healthy fatty acid profile of almond oil was also reflected in the triglycerides profile. Most common triglycerides on almond oil are triunsaturated (from three unsaturated fatty acids), mainly OOO, OLO, OLL and LLL. Diunsaturated triglycerides (SOO and POO) compound the rest of triglyceride profile, while disaturated triglycerides (PPL and PPO) are only testimonial in some cultivars. The highest proportion of triunsaturated fatty acids, leading to healthier oils, appear in Belona and Marcona (768.0 and 758.3 g/kg respectively). Disaturated triglycerides, although scarce, appear in higher concentrations in Guara and Penta (10.20 and 9.90 g/kg, respectively) making these oils less convenient for human consumption. Within reported triglycerides all but OLO and PPL, showed significant differences.

3.3. Principal component analysis

By considering all the parameters of interest, a principal component analysis (PCA) was carried out (Figure 2). The first principal component explained almost 61% of total variance and allowed the separation of Guara and Ferragnes (with higher oleic acid and oxidative stability) and Ayles and Penta, which produce less stable oils due to their higher linoleic acid content. The second principal component increased the variability explained in a 31.4% attending mainly to the content of tocopherols and the cultivar oil yield. Belona, Marcona and Tardona showed higher oil yields while Antoñeta, Ferraduel and Vairo present a higher tocopherol content with lower oil yields. Attending to the parameter selected as production objective (i.e. higher oil yield, better physicochemical quality or longer storage time, among others) different oils can be obtained by using different almond cultivars.

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Figure 2. Principal component analysis of parameters in oils obtained from ten different almond cultivars.

Antoñeta 100 Vairo Ayles 90

Tardona 80 Belona

70

Penta Ferraduel

Marcona Ferragnes

Guara

Iodine value Oil yield Oxidative stability

Figure 3. Suitability of almond cultivars for oil extraction. Parameters considered: Iodine values, oil yield obtained with the hydraulic press and oil oxidative stability. Value 100 is assigned to the highest value for each parameter.

Selection of optimal almond cultivar for almond oil production relies on producer market strategy. Maestri et al. (2015) identifies oil production, fatty acid composition and the parameters related to oil oxidative stability as main criteria to consider for almond oil production. Figure 3 results if value 100 is assigned to the highest value of cultivars for these three parameters and data are weighted. Oil yield and iodine value show lower differences among cultivars being the oxidative stability the parameter that varies the most. Iodine value and oxidative stability present a strong and negative correlation (figure 3) so intermediated values or the selection of one

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Evaluation of almond cultivars parameter, at the expense of the other, should be made when selecting the optimal cultivar for almond production. Attending to the results, cultivars Guara, Ferragnes and Belona are, in that order, the cultivars that reached the higher medium values if these three parameters are considered equally important.

3.4. Flour composition and minerals

Defatted almond flours appear as a result of almond oil production, their nutritional composition makes them a valuable product. Some differences in macronutrient composition and mineral content had been found in almonds from places as California, Spain or Tunisia (Yada et al., 2011), but the analysis of almond flours obtained from almond cultivars grown at the same plot is scarce. Results prove that the use of different cultivars for almond oil production also results in defatted almond flours that show differences both in their macronutrients composition and in their mineral content (table 3).

The protein contents obtained were within the rage of results obtained in other studies about Spanish almond (Saura-Calixto et al., 1983; Yada et al., 2011), but important differences appear among cultivars. High protein content in flour was observed in some almond cultivars as Ayles (581.3 g/kg) and Marcona (559.3 g/kg). This high concentration could be interesting due to the positive influence of the protein content to the structure of dough when baking, enabling a higher proportional replacement of wheat flour by almond flour (Pineli et al., 2015). That replacement should be lower when using flour from cultivars as Penta and Tardona that present protein concentrations below 400 g/kg. Almond defatted flours that have the lower protein content (Penta and Tardona) present the higher concentrations of available carbohydrates.

Available carbohydrates also show significant differences between cultivars, with values ranging from 276.4 g/kg in Tardona to 149.7 g/kg in Marcona. As expected, crude fat content in flour was highly correlated with higher oil content in the almond seed (r=0.874, P < 0.01). The fibre content in defatted almond flour was lower than values obtained in wheat flour (130g/kg) (USDA, 2016). This lower fibre content is related to lower capacity for hold oil of doughs (Sharma and Gujral, 2014).

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The energy value of obtained flours was similar for all the reported cultivars. However, it can be observed how the cultivar Ayles showed an energy value slightly lower than the cultivars Antoñeta, Belona, Guara, Penta, Tardona and Vairo. The energy value of almond flour is higher in all analysed cultivars that in traditional flours as wheat flour (Pineli et al., 2015) due to the higher fat content, even after oil extraction.

Regarding the mineral content in flours, differences appear in the total mineral content and in the concentration of specific minerals. Mineral content is higher in Ferragnes (70.0 g/kg) and Tardona (67.5 g/kg) and lower in Guara (53.0 g/kg) and Antoñeta (52.8 g/kg). Of the 14 minerals identified as principal minerals for the human body (Latham, 1997) up to nine appear in almond flour in relevant concentrations. Most common minerals in obtained almond flours are phosphor, magnesium, calcium, sulphur and potassium with significant differences among cultivars. High concentrations of other vital minerals such as zinc (up to 55.88 mg/kg in Marcona) and iron (up to 38.19 mg/kg in Guara) and are of interest. Compared to wheat flour, almond flours present a content several times higher of Ca, Fe, Na, K, Zn, Cu and Mg (Pineli et al., 2015; USDA, 2016) and similar contents of Se (USDA, 2016).

4. Conclusions

If production of almond oil or almond flour becomes the main destiny of almond production, the selection of the adequate almond cultivar is crucial. Important differences in the oil and flour obtained from almond cultivars grown in a continental Mediterranean climate in Spain have been identified. Significant differences have been obtained in almond oils with regard to the fatty acid profile, oxidative stability, minor components content and the triglyceride composition. Within the main oil parameters considered for almond cultivar selection, the oxidative stability shows high differences among cultivars with fewer variations of oil yield and iodine value. As expected, iodine value and oxidative stability present a strong negative correlation so one should be prioritise over the other. Cultivar almond flours also show significant differences both in the macronutrient composition, affected mainly by the fat content of the kernel, and in the content of specific minerals (especially P, Ca, K, S, B

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Influence of raw material in oil characteristics and Zn). Effects of the cultivar selection in the obtained oil and flour encourages the selection of the cultivar depending on the production objective of almond oil and flour producers.

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Chemometric characterization of pistachio oils and defatted flours regarding cultivar and

geographic origin

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Chemometric characterization of pistachio oils and defatted flours regarding cultivar and geographic origin

ABSTRACT Keywords: The present study analyses pistachio oil and defatted flour obtained from Pistacia vera Food analysis twenty cultivars originating in eight different countries. To identify the Pistachio flour cultivar effect, all pistachio varieties were grown in the same plot to control Minerals Phytosterols environmental and agricultural management effects on kernel chemical Origin traits. Regarding oil, three different groups were defined according to the fatty acid profile, K270, K232, oxidative stability, phytosterols (stigmasterol and betasitosterol) and oil yield. Cultivars in group 1 showed a higher content of linoleic fatty acid (higher than 26.4%), while cultivars in group 3 type showed a stronger presence of oleic acid (higher than 67.8%), resulting in oils that were more stable to oxidation. Cultivars with intermediate characteristics were included in group 2. Defatted flours showed significant differences in nutritional quality parameters, such as protein content (34.6 - 46.1%) or concentration of essential minerals, such as Fe (17.3 - 45.1 mg/kg) or Mg (0.13 - 0.18 g/100g). Beyond the cultivar effect, the geographical origin of the cultivar was identified for the first time as a source of variability in pistachio product traits. The content of Fe in pistachio flours and the content of stigmasterol in pistachio oils are proposed as useful parameters for discrimination of cultivars attending to their origin.

1. Introduction

The pistachio (Pistacia vera L.) is a native tree from the Middle East (Whitehose, 1957) that has been largely spread around the Mediterranean basin and other areas with Mediterranean type climates, such as California and Australia. This expansion has resulted in the development of different cultivars adapted to the environmental conditions of their growing area or the preferences of the local markets. All these

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Evaluation of pistachio cultivars cultivars compose a huge genetic resource of plant material that should be studied in order to identify their characteristics and make the best use of it.

The consumption of pistachio by-products has interesting commercial opportunities due to their health benefits. Pistachio consumption has been shown to be associated with a significant increase in high-density lipoprotein (HDL) (Sheridan et al., 2007) with a reduction in low-density lipoprotein (LDL) (Sari et al., 2010). It also reduces the level of triglycerides in blood (Dreher, 2012; Sauder et al., 2015) and the risk of coronary heart disease (Dreher, 2012; Braschi and Naismith, 2008). Moreover, pistachio consumption does not lead to weight gain and may even improve the risk factors associated with metabolic syndrome (Wang et al., 2012).

Pistachios beneficial effects have mainly been related with the presence of five components: unsaturated fatty acids, phytosterols, dietary fibre, proteins and magnesium (Dreher, 2012). Within nutritional benefits of pistachio consumption, the study of the lipid fraction is crucial. In pistachio kernels, lipids account for 50 to 62 g/100 g (Catalán et al., 2017). Pistachio fatty acid profile is mainly compound by oleic (51 - 81%), linolenic (8 - 31 %) and palmitic (7 – 15%) fatty acids (Dyszel and Petit, 1990; Satil et al., 2003; Tsantili et al., 2010; Catalán et al., 2017). Among interesting minor components in nut oils, previous studies reported high contents of tocopherols and phytosterols, reported as decisive for nut oil quality (Roncero et al., 2016, Maestri et al., 2015; Stuetz et al., 2017). Specifically, pistachio oil shows the higher concentration of total phytosterols compared to any other nut oil, up to 271.9 mg per 100 g oil (Kornsteiner-Krenn et al., 2013; Arena et al. 2007), with high concentrations of β-sitosterol (up to 90%) and minor concentrations of campesterol and stigmasterol (Catalán et al., 2017). However, the tocopherol content in pistachios is lower than in other nuts as almonds, with average concentrations around 350 mg/kg oil (Ling et al., 2016a). The analysis of how these and other components vary depending on the cultivar and the origin of the cultivar would provide useful information.

The analysis of chemical traits to identify the origin of food products has been largely used for products such as wine (Etiévant et al., 1988; Aires-De-Sousa, 1996) and more

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Influence of raw material in oil characteristics recently in other products such as olive oil (Tapp et al., 2003) or coffee (Anderson and Smith, 2002). Several studies have found differences in pistachio seeds according to their origin country by analysing the fatty acids (Dyszel and Petit, 1990), triglycerides (Dyszel and Petit, 1990), ultimate elements (Anderson and Smith, 2005), phytosterols (Arena et al., 2007) or major coloured compounds (Bellomo and Fallico, 2007). However, the origin country effect comprises the differences that appear depending on several secondary variables (mainly cultivar, environment and land management), so the independent effect of each one of these variables should be singly studied.

Few studies have analysed the differences attributed to the cultivar effect by using pistachio cultivars that have been grown in the same plot (Bellomo and Fallico, 2007; Tsantili et al., 2011; Rabadán et al., 2017a). When cultivars are grown in the same plot, the influence of ecological conditions (Silver et al., 1984) and management practises (Sánchez-Bel et al 2008; Carbonell-Barrachina et al., 2015) are controlled, allowing the identification of the true cultivar effect. By analysing pistachios from different varieties grown in the same plot, it can be known which proportion of the differences related to the widely studied “origin effect” are the result of the cultivar effect.

The study of native pistachio cultivars of such countries as Tunisia (Chahed et al., 2008; Ghrab et al., 2010), Turkey (Seferoglu et al., 2006; Küçüköner and Yurt, 2003; Harmankaya et al., 2014) or Iran (Kamangar and Farsam, 1977; Mahmoodabadi et al., 2012) has shown significant differences in chemical components, such as fatty acids, glycolipids, minerals or amino acids. The reported variability in pistachio cultivars within the same country has discouraged the study of common chemical traits in the native cultivars from each country. Studies developed on other nuts, such as hazelnut, show that the geographical origin of the cultivar has no influence in the fatty acids, tocopherols and sterols (Parcerisa et al., 1998). To our knowledge, no study concerning pistachio has specifically addressed this issue.

Most studies have evaluated the differences that appear in pistachio kernels depending on the origin country. By controlling the differences that can appear as a result of the environment and land management, this study analyses the differences

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Evaluation of pistachio cultivars that appear in pistachio oil and flour due to the cultivar and the geographical origin of the cultivar.

2. Materials and Methods

2.1. Plant material

Pistachios were collected at an experimental orchard in the Centro de Mejora Agraria el Chaparrillo of Ciudad Real in the south of Spain. All varieties were grafted on Pistacia terebinthus L. and Peters and C. especial were used as pollinators. Within each genotype considered, kernels from three different trees were considered, and within each tree, kernels were collected from different parts of the tree. This sampling plan was designed to reduce the effect of the biological variability to a minimum.

The shell of the pistachios was removed in controlled conditions for immediate drying of the seeds. Pistachios were dried at room temperature until they reached a moisture content lower than 6%, avoiding the use of high temperatures that could affect kernel characteristics (Álvarez-Ortí et al., 2012; Sena-Moreno et al., 2015).

Twenty cultivars with different origins (Iran, Iraq, Syria, Israel, Cyprus, Greece, Italy and Tunisia) were evaluated. The differences in the oils and flours with respect to the geographical origin is made by grouping cultivars with the same origin. Our goal is to identify if the cultivars from one specific country have chemical differences that make oils and flours different from others with different origin. Even if they are grown on the same plot, the historical development of those cultivars in a specific environment (country) may lead to some common characteristics that made them different to others historically developed in a different country.

2.2. Oil extraction

Before oil extraction, pistachios were ground using a blender (GM200-RETSCH). Oil extraction was performed using a hydraulic press (MECAMAQ Model DEVF 80, Vila- Sana, Lleida, Spain) at pressures of 50, 100, 150 and 200 bar with increasing pressure

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Influence of raw material in oil characteristics each 5 minutes (20 minutes per sample). One kilogram of ground pistachios was placed each time on the hydraulic press. After pressing, the oil was centrifuged to remove remaining solids. Oil was stored in dark glass bottles at 5˚C to avoid degradation until analysis (Rabadán et al., 2017b).

The remaining pressing cake was ground using the blender so that pistachio flour passed a 1 mm mesh sieve.

2.3. Oil and flour analysis

2.3.1. Oil analysis

2.3.1.1. Oxidative stability

Oxidative stability was evaluated by the Rancimat method (Gutiérrez, 1989). Stability was expressed as the oxidation induction time (hours), measured with the Rancimat 743 apparatus (Metrohm Co., Basel, Switzerland). An oil sample of 3.5 g was used, warmed to 100°C under an air flow of 10 l h-1.

2.3.1.2. Fatty acids

To determine fatty acid composition (%), the methyl esters were prepared by vigorous shaking of a solution of oil in hexane (0.2 g in 3 ml) with 0.4 ml of 2 N methanolic potassium hydroxide solution, and analysed by GC with a Hewlett- Packard (HP 6890) chromatograph equipped with an FID Detector. A fused silica column (50 m length x 0.25 mm i.d.), coated with SGL-1000 phase (0.25 µm thickness; Sugerlabor), was used. Helium was employed as a carrier gas with a flow rate through the column of 1 ml min-1. The temperatures of the injector and detector were set at 250°C with an oven temperature of 210°C. An injection volume of 1 µl was used (Regulation EEC 2568/91, corresponding to AOCS method Ch 2–91).

2.3.1.3. Iodine values

Iodine values are a simple method to obtain information about the amount of unsaturation in oils. I2V was calculated from fatty acid percentages by using the formula (Torres and Maestry, 2006):

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I2V = (% palmitoleic acid ∗ 1.001) + (% oleic acid ∗ 0.899) + (% linoleic acid ∗ 1.814)

2.3.1.4. Sterols

Sterols (%) were determined with a Hewlett-Packard (HP 6890) gas chromatograph with a capillary column (25 m length x 0.25 mm i.d.) coated with SGL-5 (0.25 µm thickness; Sugerlabor). Working conditions were as follows: carrier gas, helium; flow through the column, 1.2 ml min-1; injector temperature, 280°C; detector temperature, 290°C; oven temperature, 260°C; injection volume 1 µl (Regulation EEC 2568/91, corresponding to AOCS method Ch 6–91).

2.3.1.5 Tocopherols

The tocopherol content (mg/kg) was analysed in accordance with the IUPAC 2432 method (IUPAC, 1987). Oil (1.5 g) was dissolved in the mobile phase (10 ml) of 0.5% isopropanol in n-hexane. A normal phase column (Lichrosphere Si60, 250 mm length, 4.6 mm i.d. and 5 µm particle size) was used with an injection volume of 20 µl and a flow rate of 1.0 ml/min.

2.3.1.6. Triglycerides

Triglycerides were determined using HPLC-IR after weighing 0.5 g of oil in 10 ml of acetone, mobile phase 50:50 acetone/acetonitrile at a flow rate 1 ml/min and an ACE18 column (5 µm x 4.6 mm x 250 mm).

2.3.2. Flour analysis

2.3.2.1. Main nutritional components

After grinding, main nutritional components of extracted flour were determined. Protein content was calculated by multiplying the total nitrogen content, obtained by the Kjeldahl method (FOSS, 2003), by a conversion factor of 6.25. To determine ash content, flours were ashed at 550°C to constant weight (MAPA, 1998). Crude fat (ether extract) was estimated gravimetrically by the filter bag technique after petroleum ether extraction of the dried sample in an Ankom XT10 extraction system

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(ANKOM, 2009). To determine the content of crude fibre, the Weende technique adapted to the filter bag technique was applied. This method determines the organic residue remaining after digestion with solutions of sulfuric acid and sodium hydroxide, using an Ankom 220 fibre analyser (ANKOM, 2008). Total carbohydrate content was calculated by subtracting the sum of the crude protein, total fat, water and ash from the total weight of the flour (Sullivan, 1993). Available carbohydrate content (nitrogen-free) was calculated by subtracting the crude fibre from the total carbohydrate content (González et al., 1987).

2.3.2.2. Minerals

Contents of mineral elements in flours were obtained by inductively coupled plasma mass spectrometry (ICP-MS), capable of detecting metals and some non-metals at low concentrations. The operating conditions were radiofrequency applied power of 1.4 kW, sample uptake rate of 300 μL/min, sample depth 7.5 mm, peak hopping scanning mode, dwell time 0.05 s, nebulizer gas flow-rate of 0.86 L/min, plasma gas flow-rate of 18.0 L/min, auxiliary gas flow-rate of 1.8 L/min, and sheath gas flow-rate of 0.13 L/min (Ling et al., 2016a).

2.3.3. Statistical analysis

Differences between means of triplicate measurements were tested for statistical significance using ANOVA and separated with Duncan's test (P < 0.05). Variables considered for the Principal Component Analysis were factors related to oil quality as

K232, K270, fatty acid profile (including oleic, linoleic, linolenic, palmitoleic, stearic and arachidic fatty acids), the proportion of stigmasterol and β-sitosterol and others that have main interest for the food industry as the oil yield or the oxidative stability values. Hierarchical clustering was used to discriminate three groups of pistachio cultivars. All statistical analysis were carried out using the SPSS programme, release 23.0 for Windows.

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3. Results and discussion

3.1. Pistachio oils

3.1.1. Fatty acids

The fatty acid profile of pistachio has been widely described as beneficial for human health due to the high proportion of unsaturated fatty acids, primarily oleic and linoleic, as shown in Table 1. Overall, the fatty acid composition reported in the present study is in agreement with many others (Arena et al., 2007; Tsantili et al., 2010; Chahed et al., 2008; Okay, 2002; Shakerardekani et al. 2015; Ling et al., 2016a; Catalán et al., 2017). Within the evaluated cultivars, Sfax, Kastel and Kerman showed a higher percentage of linoleic acid. In previous studies, Kerman has been reported to have the higher linoleic content (Tsantili et al., 2010), but our results suggest that Sfax and Kastel present a linoleic content that can be even higher. However, Kerman remained as the cultivar with the highest linolenic content. High content of linolenic in Kerman kernels had already been reported by Martínez et al., (2016). Sfax, Kastel and Kerman also showed the highest I2V values, so greater nutritional value could be attributed to them. On the other hand, Avdat, Larnaka and Lathwardy showed lower unsaturated fatty acid profiles that result in lower nutritional quality due to the larger content of palmitoleic fatty acid reported. Although not presented in Table 1, myristic, arachidonic and behenic acids were detected in all analysed cultivars at low concentrations while margaric acid was found only in Avidon, Batoury and Larnaka varieties.

The fatty acid profile has been widely used to differentiate the origin of pistachio seeds from Turkey and Iran (Aslan et al., 2002) and Turkey, Iran, Italy and Greece (Arena et al., 2007). Palmitic acid had been reported as useful for origin discrimination (Aslan et al., 2002; Arena et al., 2007); however, our results suggest that palmitic acid is not useful to differentiate the origin of pistachio cultivars when trees are grown in the same plot and environmental and management factors are corrected. The remaining fatty acids (palmitoleic, stearic, oleic, linoleic and linolenic) were significantly affected by variety, with the exception of gondoic acid. These results for our higher number of cultivars support the findings of Tsantili et al., (2010).

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Table 1. Fatty acid composition (%) of pistachio cultivars and iodine value (I2V) Palmitic Palmitoleic Stearic Oleic Linoleic Linolenic Gondoic Cultivar I2V C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:1 Aegina 9.44 0.86 ef 1.83 bcd 69.70 cde 17.00 def 0.32 efg 0.36 93.73 Ajamy 9.65 0.97 bc 1.47 fg 67.33 fg 19.32 cd 0.39 bc 0.37 96.21 Albina 9.97 0.97 bc 1.72 cde 68.85 def 17.36 def 0.32 efg 0.33 94.36 Ashoury 8.98 0.84 ef 1.61 ef 71.05 bc 16.34 ef 0.31 fg 0.39 94.36 Avdat 9.24 0.85 ef 2.05 a 72.94 a 13.76 g 0.27 g 0.36 91.38 Avidon 9.77 0.81f 1.33 gh 63.88 h 23.02 b 0.37 cde 0.39 100.00 Batoury 9.31 0.85 ef 1.90 abc 69.60 cde 17.12 def 0.35 cdef 0.39 94.62 Boundoky 9.24 0.84 ef 1.87 abc 70.24 bcd 16.58 ef 0.33 def 0.37 94.06 Bronte 8.99 0.92 bcde 1.50 fg 70.68 bcd 16.70 ef 0.32 efg 0.40 94.76 Iraq 9.06 0.93 bcde 1.24 h 66.22 g 21.35 bc 0.33 def 0.40 99.19 Joley 9.51 0.95 bcd 1.27 h 64.42 h 22.58 b 0.39 bc 0.38 99.82 Kastel 9.87 0.87 def 1.25 h 58.73 i 28.14 a 0.38 cd 0.39 104.72 Kerman 10.84 1.13 a 1.15 h 59.21 i 26.44 a 0.44 a 0.33 102.31 Larnaka 9.35 0.87 def 2.02 ab 71.65 ab 14.93 fg 0.30 fg 0.34 92.37 Lathwardy 9.71 0.87 def 1.85 abcd 71.19 abc 15.13 fg 0.32 efg 0.36 92.32 Mateur 9.56 0.90 cdef 1.88 abc 69.90 bcd 16.60 ef 0.32 efg 0.35 93.85 Napoletana 9.26 0.85 ef 1.75 cde 69.55 cde 17.41 def 0.33 def 0.39 94.96 Ouleimy 9.72 1.01b 1.62 ef 69.59 cde 16.89 def 0.33 def 0.35 94.21 Sfax 10.20 0.92 cde 1.23 h 57.85 i 28.53 a 0.43 ab 0.38 104.68 Sirora 10.11 0.96 bc 1.66 def 67.82 efg 18.24 de 0.37 cde 0.36 95.02 P NS *** *** *** *** *** NS Values are means of three replicates. Means in a column not sharing the same letter are significantly different by Duncan test (P< 0.05). NS, not significant. *** Significant at P < 0.001.

3.1.2. Polyphenols, tocopherols and sterols.

Minor components are a major parameter to evaluate oil quality (Maestri et al., 2015). Polyphenols have antioxidant properties (Tomaino, 2010) and, beyond their nutritional value, the active role of tocopherols in the protection of oils against lipid oxidation makes them an important quality parameter. Pistachios have been reported as the nut with the highest sterol content (Dreher, 2012) providing them with intrinsic cholesterol-lowering properties (Segura et al. 2006).

Total tocopherol content also showed significant differences, with cultivars Ashoury and Avdat (559.20 mg/kg and 496.10 mg/kg, respectively) doubling the concentration found in Bronte (244.40 mg/kg) (table 2). Cultivar Kerman, showed a total tocopherol

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content of 457.93 mg/kg, higher than the data reported for the same cultivar previously (Ling et al., 2016a). These differences could be attributed to different environmental conditions, as temperature has a direct influence in the tocopherol content of some nuts as almonds (Kodad et al., 2006). The different content of tocopherols in pistachio oils could be useful to identify the best cultivar according to their antioxidant capacity.

Table 2. Content of minor components in pistachio oils obtained from different cultivars Sterols (% total) Total Total Cultivars Tocopherols Sterols D7 Campesterol Stigmasterol Betasitosterol (mg/Kg) (mg/Kg) Stigmasterol Aegina 415.85 bcde 3554 bcdefg 4.05 abc 1.25 efgh 93.40 abc 0.45 cd Ajamy 352.50 def 3860 ab 4.20 abc 2.10 ab 92.20 d 0.50 bcd Albina 370.70 cdef 3304 efgh 3.70 cd 1.00 gh 94.10 ab 0.50 bcd Ashoury 406.60 bcde 3689 bcde 4.10 abc 1.60 cde 92.80 bcd 0.50 bcd Avdat 496.10 ab 3410 cdefgh 4.00 abcd 1.00 gh 93.40 bcd 0.70 ab Avidon 433.70 bcd 3036 h 3.45 d 0.80 h 94.35 a 0.50 bcd Batoury 416.65 bcde 3219 gh 3.85 abcd 1.15 efgh 93.55 abc 0.65 abc Boundoky 388.70 bcdef 3803 abc 4.10 abc 1.20 efgh 93.00 bcd 0.70 ab Bronte 244.40 g 3732 bcd 4.00 abcd 1.80 bcd 92.50 d 0.80 a Iraq 559.20 a 3664 bcdef 4.40 a 1.20 efgh 93.10 bcd 0.60 abcd Joley 283.10 fg 3639 bcdef 4.20 abc 1.50 def 92.90 cd 0.60 abcd Kastel 476.20 abc 3503 bcdefg 3.90 abcd 1.40 defg 93.50 abc 0.50 bcd Kerman 457.93 abcd 3565 bcdefg 4.30 ab 1.20 efgh 93.30 abcd 0.50 bcd Larnaka 344.03 def 3445 cdefg 3.73 bcd 2.50 a 92.20 d 0.53 bcd Lathwardy 406.50 bcde 3281 fgh 4.10 abc 1.10 fgh 92.70 cd 0.70 ab Mateur 397.85 bcde 3378 defgh 4.05 abc 1.15 efgh 93.50 abc 0.50 bcd Napoletana 304.65 efg 3609 bcdefg 3.95 abcd 1.60 cde 93.00 bcd 0.65 abc Ouleimy 351.95 def 3414 cdefgh 4.15 abc 0.80 h 93.70 abc 0.60 abcd Sfax 392.00 bcdef 4147 a 4.15 abc 0.80 h 93.75 abc 0.50 bcd Sirora 417.45 bcde 3442 cdefg 4.10 abc 2.00 bc 92.15 d 0.40 d P *** *** ** *** ** ** Values are means of three replicates. Means in a column not sharing the same letter are significantly different by Duncan test (P< 0.05). ** Significant at P < 0.05. *** Significant at P < 0.001.

The discriminatory power of sterols has been previously reported for several cultivars of different origin (Arena et al., 2007), but there is a lack of evidence about the influence of cultivar when the country effect is controlled. Pistachio oil sterols are

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Influence of raw material in oil characteristics almost completely composed of β-sitosterol (94.35% in Avidon to 92.25% in Sirora), which shows significant differences between cultivars (P < 0.05). Stigmasterol showed a higher discriminatory power among sterols (P < 0.001) ranging from 2.50% in Larnaka to 0.80% in Avidon, Sfax and Ouleimy. Our results show significant differences both in the total concentration of sterols and in the percentage of specific sterols. Due to the effects of phytosterols in the reduction of total plasma cholesterol and low-density lipoprotein cholesterol (Martínez et al., 2006) additional beneficial effects can be expected from cultivars Sfax and Ajamy.

3.1.3. Principal component analysis

The result of the principal component analysis is reported in Figure 1. The two linear combinations explain, overall, 72.3% of the variance. CP1 comprises the information about the fatty acid profile, K270, K232 and oxidative stability, while CP2 shows the differences regarding the sterols (stigmasterol and betasitosterol) and the oil yield obtained in oil extraction.

Figure 1. Principal component analysis of parameters in oils obtained from twenty different pistachio cultivars

Three different groups of varieties can be identified attending to the characteristics of the pistachio oil obtained using hierarchical clustering. The main differences

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between the three groups are related to their fatty acid profile, with the oil from cultivars in group 1 been more unsaturated and the oil from group 3 cultivars been less unsaturated and more stable to oxidation. The correlation between the degree of saturation of oils and their oxidative stability has been widely described for different plant oils, including nut oils (Rabadán et al., 2017c). Group 2 is compound by cultivars with intermedium characteristics. Within the concentration of different sterols and obtained oil yield (CP2), differences within the named groups could not be identified.

3.1.4. Triglycerides

The triglyceride profile of the analysed pistachio cultivars is shown in Table 3.

Table 3. Triglyceride composition (%) of oils obtained from different pistachio cultivars Cultivar LLL OLL PLL OLO PPL OOO POO PPO PPP SOO Aegina 3.75 gh 13.05 cdef 2.82 def 24.12 abcd 0.81 cde 29.17 cdef 13.30 cde 1.64 0.38 abcd 2.20 bc Ajamy 5.39 cd 14.30 cd 3.83 ab 23.15 cde 0.92 bcde 27.35 ef 12.34 efg 1.53 0.35 cd 1.73 de Albina 4.03 fgh 13.11 cdef 3.26 bcd 23.13 cde 1.00 abcd 28.13 def 13.40 cde 1.85 0.47 abc 2.10 cd Ashoury 3.56 gh 12.13 efg 2.72 defgh 24.24 abcd 0.75 cde 31.14 bcde 12.91 def 1.46 0.42 abcd 2.04 cd Avdat 2.65 i 10.00 hi 2.04 h 22.92 de 0.63 e 33.69 ab 14.98 a 1.53 0.56 a 2.60 ab Avidon 5.97 c 17.27 b 4.00 b 24.98 ab 0.94 bcde 23.12 g 11.13 gh 1.32 0.33 cd 1.18 fg Batoury 3.43 ghi 11.44 fgh 2.54 efgh 23.56 bcde 0.78 cde 32.09 abcd 13.69 abcde 1.57 0.27 de 2.33 cd Boundoky 3.27 hi 10.52 ghi 2.45 fgh 22.94 de 0.80 cde 32.96 abc 14.41 abc 1.68 0.39 abcd 2.25 bc Bronte 2.65 i 9.21 i 2.08 gh 22.22 e 0.75 cde 35.73 a 14.86 ab 1.66 0.36 bcd 2.84 a Iraq 4.18 fgh 12.99 def 2.80 defg 24.13 abcd 0.67 de 29.61 cdef 13.18 cde 1.60 0.39 abcd 2.21 bc Joley 4.02 fgh 12.59 def 2.86 def 23.64 bcde 0.72 cde 29.75 cdef 13.37 cde 1.61 0.39 abcd 2.35 bc Kastel 3.67 gh 11.77 fg 2.64 defgh 23.94 bcde 0.72 cde 30.93 bcde 13.54 bcde 1.62 0.43 abcd 2.28 bc Kerman 5.15 cde 14.79 c 3.35 abc 25.69 a 0.79 cde 30.66 bcde 11.73 fg 1.34 0.27 de 1.43 ef Larnaka 7.51 b 19.37 a 5.08 a 24.70 abc 1.17 ab 19.06 h 9.96 hi 1.32 0.00 f 0.95 gh Lathwardy 7.75 b 19.76 a 5.27 a 24.58 abcd 1.26 a 18.56 h 9.68 i 1.40 0.00 f 0.72 h Mateur 3.67 gh 12.17 efg 2.89 def 23.50 bcde 0.79 cde 29.92 bcdef 13.77 abcd 1.78 0.44 abcd 2.33 bc Napoletana 4.76 def 13.19 cdef 3.11 def 23.13 cde 0.81 cde 29.19 cdef 13.14 cde 1.57 0.43 abcd 2.12 bc Ouleimy 4.15 fgh 12.69 def 3.03 def 23.15 cde 0.88 bcde 29.22 cdef 13.83 abcd 1.80 0.39 abcd 1.97 cd Sfax 9.13 a 20.54 a 5.62 a 23.64 bcde 1.18 ab 17.46 h 9.40 i 1.36 0.15 ef 0.91 gh Sirora 4.30 efg 13.85 cde 3.03 def 23.49 bcde 1.03 abc 26.35 fg 13.29 cde 1.89 0.55 ab 2.03 cd P *** *** *** ** ** *** *** NS *** *** Values are means of three replicates. Means in a column not sharing the same letter are significantly different by Duncan test (P< 0.05). Triglyceride composition in which L represents linoleic acid; P, palmitic acid; O, oleic acid; and S, stearic acid. NS, not significant. ** Significant at P < 0.05. *** Significant at P < 0.001.

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Most common triglycerides of pistachio oil are triunsaturated, mainly OOO, OLO and OLL, in agreement with previous studies (Ballistreri et al., 2010). OOO is the most common triglyceride ranging from 35.73% in Bronte to 17.46% in Sfax. The differences found among varieties in the concentration of OOO and OLL are significant at the 1% level, while the OLO differences are significant at the 5% level. Diunsaturated triglycerides (SOO and POO) composed the rest of the triglyceride profile, while disaturated triglycerides (PPL and PPO) and trisaturated triglycerides (PPP) were found only in some cultivars. PPL was the only triglyceride without discriminatory power among pistachio varieties.

3.2. Pistachio defatted flour

3.2.5. Components of pistachio defatted flour

After oil extraction, the pistachio flour still holds a percentage of lipids. The percentage of oil extracted from the total oil content of pistachio seeds ranged from 59.0% in the Ajamy cultivar to 64.84% in Avdat. As expected, a strong correlation between the total fat content of the pistachio seed and the crude fat that remains in the flour after oil extraction appeared (r = 0.997, P < 0.01). With similar extraction efficiency, the total lipid content of the seed is determinant for the crude fat content of the flour. The crude fat that remained in pistachio flour showed significant differences between cultivars (P < 0.05) due to the different oil content of pistachio seeds. Crude fat content was higher in Kastel (24.30%) and Avidon (23.69%), while in Alpina and Batoury crude fat represented less than 19% of total flour weight.

Proteins are the main component of pistachio defatted flours (Figure 2) agreeing with the results of Ling et al., (2016b) for raw pistachio press-cake flour. The highest proportion appeared in Napoletana, in which flour proteins represented 46.13% of flour weight. On the other hand, Sfax and Kastel proteins only represented 34.57% and 35.25%, respectively. In previous studies, Turkish pistachio proteins represented only 22-27% of seed weight as the lipid concentration exceed 50% (Küçüköner and Yurt, 2003; Seferoglu et al., 2006; Harmankaya and Özcan, 2014). The protein part of some nuts has received major attention lately due to the high presence of highly

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Evaluation of pistachio cultivars digestible proteins and the balanced presence of essential amino acids (Sze-Tao and Sathe, 2000).

100 90 80 70 60 50 40 30 20 10 0

Proteins (%) Ashes (%) Crude fibre (%) Crude fat (%) Available carbohydrates (%)

Figure 2. Proximate composition of defatted flours of pistachio cultivars. Components as percentage of total flour dry weight

Available carbohydrates compose about a third of the flour components and were higher in Sfax (34.79%) and lower in Ashoury (28.10%). This proportion of carbohydrates agrees with previous studies about proximate composition of pistachio defatted flours (Ling et al., 2016b). The differences in the protein and in the available carbohydrate concentrations between cultivars were both significant at the 1% level. Fibre is one of the components that supports the nutritional benefits attributed to pistachios (Dreher et al., 2012). The fibre content in defatted flour is higher than in the seeds, although high variability exists (Kamangar and Farsam, 1977; Seferoglu et al., 2006). Our results showed significant differences in the crude fibre content of flour (p < 0.001), which ranged from 6.56% in the Kastel cultivar to 4.33% in Alpina flour.

The ash content in pistachio seeds has been reported to be approximately 2.60% of seed weight in Turkish cultivars (Küçüköner and Yurt, 2003; Harmankaya et al., 2014) and 3.0% in Kerman cultivar (Martínez et al., 2016). In our defatted pistachio flours, the mineral concentration was higher, ranging between 4.95 - 4.21%. This content is similar to the reported by Rabadán et al., (2017d) in defatted flours obtained using different pressing systems. The ash content showed significant differences for some

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Influence of raw material in oil characteristics cultivars if pistachio seeds are considered (Küçüköner and Yurt, 2003; Seferoglu et al., 2006). In our flours, however, significant differences were not detected. Our results prove that when the effects of the environment and the management practises are corrected, the ash content remained homogeneous among cultivars. Different results have been obtained for the mineral content of almond flours (Rabadán et al., 2017c). However, although in pistachio flour the total mineral content was consistent, the content of each specific mineral was different for each variety as reported below.

3.2.6. Minerals in pistachio flour

The distribution of minerals in the plant parts is a distorted reflection of the trace mineral compositions of the soil and environment in which the plant grows (Esechie, 1992). However, the high selectivity for mineral bioaccumulation in plants can result in similar trace minerals in plants of the same species, even if they are grown in different environments. Our results show the differences that appear in pistachio varieties attending only to the genotype, as environmental and management differences were controlled.

Pistachio flour appears as an adequate source of some macro (P, Mg, Ca, K) and microelements (B, Fe, Mn, Rb, Sr). Compared to wheat flour, defatted pistachio flour shows a higher content of Ca, Fe and K (Pineli et al., 2015). The high concentration of Mg in pistachio defatted flour (Küçüköner and Yurt, 2003; Harmankaya et al., 2014) has been reported as one of its most important nutritional values (Dreher, 2012). A high intake of magnesium, together with calcium and potassium, prevents bone demineralization, lowers blood pressure and reduces the risk of cardiovascular diseases (Segura et al., 2006) making defatted pistachio flour an interesting product from a health perspective.

The concentration of minerals in pistachio seeds has been used for cultivar discrimination (Küçüköner and Yurt, 2003; Harmankaya et al., 2014) and, at a lower level, for origin discrimination (Anderson and Smith, 2005). Anderson and Smith (2005) found small discriminating power of macro elements after analysing kernels from three different geographic growing areas (California, Iran and Turkey).

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Microelements presented higher discriminating power, but definitive origin differentiation was not observed even when pistachios had been grown in completely different environments. By analysing pistachios grown in the same plot, we obtained similar results.

Regarding the concentration of specific macro elements (Table 4), significant differences were found in the concentration of P and Mg (P < 0.05) and Ca (P < 0.001), while significant differences were not found for K. The Kerman cultivar showed the highest concentration of K (up to 1.49 g/100 g), while the concentration of Mg was highest in Joley (0.18 g/100 g) and P was higher in Albina (0.77 g/100 g). Within the considered microelements, Fe is the most common, ranging from 45.12 mg/kg in Bronte to 17.25 mg/kg in Iraq. These concentrations of minerals agree in general with previous studies (Küçüköner and Yurt, 2003, Arena et al., 2007; Ling et al. 2016a). All considered microelements showed significant discriminatory power (P < 0.001) but, as reported for macro elements, singly any element would be useful for total cultivar differentiation. Depending on the preferred content of specific minerals in pistachio flour, one cultivar could be selected among the analysed.

3.3. Effect of cultivar geographical origin

The differences of the cultivars depending on their geographical origin country are presented in table 5. The 20 cultivars analysed are native to eight different countries (Greece, Syria, Iran, Israel, Italy, Iraq, Cyprus and Tunisia).

The number of cultivars considered for each country was not the same. Up to seven cultivars were considered for Syria, three were considered for Iran and Israel, two for Tunisia and Italy and only one cultivar was considered for Greece, Iraq and Cyprus. The first limitation that should be assumed is that we are only considering pistachio cultivars with commercial interest. Therefore, the number of cultivars considered for each country reflects, partially, the number of cultivars with commercial interest originating in that country. For some countries, the consideration of a single cultivar is considered enough to comprise the national effect. This is, for example, the case of cultivar Larnaka for Cypriot pistachio cultivars, Iraq for Iraqi cultivars or Aegina for Greek native cultivars.

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According to the fatty acid profile, significant differences between countries could be observed according to the presence of stearic, oleic, linoleic and linolenic acids (P < 0.05). Differences in the percentages of palmitic acid were not significant. The Cyprus cultivar (Larnaka) presented the highest percentage of stearic acid, whilst the Iraq cultivar (Iraq) showed the lowest percentage. The oleic acid content was also in higher percentage in the oil of the variety native from Cyprus (71.65%). The presence of oleic was lower in the native cultivars from Tunisia (Mateur and Sfax), Israel (Kastel, Avdat and Avidon) and Iran (Kerman, Albina and Joley). The linoleic acid content was highest in cultivars native from Tunisia, Israel and Iraq.

The sterol composition of pistachio has been identified as a discriminatory parameter for pistachio origin (Arena et al., 2007). Studies have found differences if the pistachios are cultivated in their native countries, but to the best of our knowledge, those differences have not been reported when cultivars are grown in the same plot. In this study, significant differences were found in the concentration of stigmasterol (P < 0.001) and betasitosterol (P < 0.05) but not for campesterol. Stigmasterol concentration was higher in cultivars native from Cyprus (Larnaka) and Italy while at a lower concentration was found in cultivars native from Tunisia and Israel. The beta- sitosterol content was higher in cultivars from Greece, Israel, Iran and Tunisia and lower in cultivars from Cyprus. This was the result of the negative correlation between stigmasterol and beta-sitosterol (r = -0.660, P < 0.01).

The triglycerides showed significant differences (P < 0.05) for OOO, OLL, POO and LLL. The OOO percentage was higher in pistachio cultivars from Italy (Bronte and Napoletana) and lower in Cypriot cultivar Larnaka. The OOO and OLL percentages were negatively correlated. The concentration of LLL was higher in cultivars from Cyprus and Tunisia (Mateur and Sfax) and lower in Italian cultivars.

Regarding the presence of specific minerals in pistachio flours, significant differences were also detected. Differences had been reported when pistachios were grown in their native countries, but we can confirm that those differences could be partially attributed to the cultivar origin itself, as they also appear in our study. The mineral concentration of flours presented differences in Fe (P < 0.001) and Mg, B, Mn, Rb and

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Sr (P < 0.05). The concentration of Fe was higher in Italian cultivars (Bronte and Napoletana) and lower in cultivars from Iraq and Cyprus. The concentrations of Mg and Mn were higher in Iranian cultivars (Kerman, Albina and Joley).

Table 5. Differences in the oil and flour from cultivars depending on their geographical origin country Greece Syria Iran Israel Italy Iraq Cyprus Tunisia P n=1x3 n=7x3 n=3x3 n=3x3 n=2x3 n=1x3 n=1x3 n=2x3 Fatty acids Stearic 1.83 ab 1.71 abc 1.38 cd 1.54 bcd 1.63 bc 1.24 d 2.02 a 1.55 bcd ** Palmitic 9.44 9.53 10.11 9.63 9.13 9.06 9.35 9.88 NS Oleic 69.70 abc 69.54 abc 64.16 c 65.18 bc 70.11 ab 66.22 abc 71.65 a 63.88 c ** Linoleic 17.00 ab 17.08 ab 22.13 a 21.64 a 17.05 ab 21.35 a 14.93 b 22.57 a ** Linolenic 0.32 ab 0.34 ab 0.38 a 0.34 ab 0.33 ab 0.33 ab 0.30 b 0.38 a ** Sterols Campesterol 4.07 4.09 4.07 3.79 3.97 4.40 3.73 4.12 NS Stigmasterol 1.27 bc 1.42 bc 1.23 bc 1.07 c 1.70 b 1.20 bc 2.50 a 0.98 c *** Betasitosterol 93.40 a 92.92 ab 93.43 a 93.62 a 92.58 ab 93.10 ab 92.23 b 93.63 a ** Tryglicerides OOO 29.17 ab 28.24 ab 29.51 ab 29.25 ab 32.46 a 29.61 ab 19.06 c 23.69 bc ** OLO 24.12 23.59 24.15 23.95 22.67 24.13 24.70 23.57 NS OLL 13.05 bc 13.53 bc 13.50 bc 13.01 bc 11.20 c 12.99 bc 19.37 a 16.36 ab ** POO 13.30 ab 12.88 ab 12.83 ab 13.22 ab 14.00 a 13.18 ab 9.96 c 11.58 bc ** LLL 3.75 c 4.55 bc 4.40 bc 4.10 c 3.71 c 4.18 c 7.51 a 6.40 ab ** Minerals P 0.64 0.59 0.68 0.60 0.60 0.64 0.56 0.60 NS Mg 0.15 ab 0.15 ab 0.17 a 0.15 ab 0.16 ab 0.16 ab 0.14 b 0.15 b ** Ca 0.13 0.12 0.13 0.11 0.12 0.14 0.10 0.12 NS K 1.25 1.31 1.43 1.30 1.38 1.23 1.28 1.19 NS B 17.46 a 17.33 a 17.37 a 17.13 a 18.96 a 15.69 a 11.19 b 16.23 a ** Fe 36.05 ab 35.96 ab 36.78 ab 36.43 ab 40.17 a 17.25 d 29.28 c 32.31 bc *** Mn 19.41 ab 18.38 b 21.41 a 19.34 ab 19.41 ab 18.21 b 18.30 b 17.14 b ** Rb 6.97 b 8.79 ab 10.76 a 8.67 ab 7.12 b 10.68 a 7.84 b 9.23 ab ** Sr 24.33 ab 17.44 bc 22.85 ab 13.58 c 15.42 bc 26.61 a 16.10 bc 20.23 abc ** n = number of cultivars considered from the country x number of replicates for each cultivar. Values are means of cultivars from the same origin country. Origins considered: Greece: Aegina; Syria: Ajamy, Ashoury, Batoury, Boundoky, Lathwardy, Ouleimy, Sirora; Iran: Albina, Joley, Kerman; Israel: Avdat, Avidon, Kastel; Italy: Bronte, Napoletana; Iraq: Iraq; Cyprus: Larnaka; Tunisia: Mateur, Sfax. Means in a row not sharing the same letter are significantly different by Duncan test (P< 0.05). NS, not significant. ** Significant at P < 0.05. *** Significant at P < 0.001.

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4. Conclusions

A wide variability in the chemical characteristics of pistachio cultivars is reported. However, pistachio cultivar oils can be grouped according to chemical parameters, mainly due to similarities in their fatty acid profile, into three different groups. This study also shows that even when native cultivars from different countries are cultivated in the same plot, several differences related to their origin remain. The concentration of stigmasterol in pistachio oil and the presence of Fe in pistachio flour appear as the most effective discriminatory variables to identify the origin of pistachio cultivars, although singly they would not be sufficient to differentiate cultivar origin. Results support that geographical origin of the cultivar has a larger influence in pistachio than in other nuts, such as hazelnuts (Parcerisa et al., 1998), where significant differences were not reported.

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2.3. Influence of crop year

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Influence of genotype and crop year in the chemometrics of almond and pistachio

oils

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Influence of genotype and crop year in the chemometrics of almond and pistachio oils

ABSTRACT Keywords: Almond and pistachio oils can be considered as interesting products to Prunus dulcis Pistacia vera produce and commercialize due to their health promoting properties. Cultivars However, these properties are not consistent because of the differences Nut oil Oil composition that appear in oils as a result of the genotype and the crop year. The analysis of these variations and their origin is decisive to ensure the commercial future prospects of these nut oils. Although significant variability has been reported in almond and pistachio oils as a result of the crop year and the interaction between the crop year and the genotype, the genotype itself remains as the main factor determining oil chemometrics. Oils fatty acid profile has been mainly determined by the genotype, with the exception of palmitic fatty acid in pistachio oil. However, the crop year affects the concentration of some minor components of crucial nutritional interest as total polyphenols and phytosterols. Regarding reported differences in oil, some almond and pistachio genotypes should be prioritised for oil extraction. Breeding programmes focused on the improvement of specific characteristics of almond and pistachio oils should focus on chemical parameters mainly determined by the genotype.

1. Introduction

The increase in the demand and price of almonds and pistachios has led to an escalation in the cultivation of these nuts, with increases in the global production of 8.8 and 6.1%, respectively, in the last six years (FAO, 2017). The production of these two nuts sometimes compete for the space, as their cultivation is restricted to areas with hot and dry conditions where trees produce the highest yields of high quality kernels. As a result, the distribution of almond and pistachio trees is mainly focused on the Mediterranean basin, the Middle East, U.S. and Australia (FAO, 2017). Traditional genotypes available worldwide, in addition to cultivars from recent

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Available almond and pistachio genotypes have different physical and chemical characteristics that need to be considered (Roncero et al., 2016a, b; Catalán et al., 2017; Rabadán et al., 2017c;). In pistachio kernels from different cultivars, several studies have found differences in size (Rabadán et al., 2017c), fatty acids (Dyszel and Pettit, 1990; Arena et al., 2007), triglycerides (Dyszel and Pettit, 1990), phytosterols (Arena et al., 2007), amino acids (Mahmoodabadi, 2012), minerals (Küçüköner and Yurt, 2003; Anderson and Smith, 2005) or major coloured compounds (Bellomo and Fallico, 2007). Similar results have been reported for almonds (Martín-Carratalá et al., 1999; Kodad et al., 2006; Kodad et al., 2011; Yada et al., 2011; Maestri et al., 2015;). Most of the studies have analysed kernels from different cultivars produced in different countries, with few studies considering trees grown in the same plot (Bellomo and Fallico, 2007; Tsantili et al., 2010; Tsantili et al., 2011; Maestri et al., 2015;). When cultivars are not grown in the same plot, the influence of ecological conditions (García-López et al., 1996; Kodad et al., 2014; Yada et al., 2011) and management practises (Sánchez-Bel et al., 2008; Carbonell-Barrachina et al., 2015; Zhu et al., 2015) may cause differences that should not be attributed to the genotype.

The global demand of almonds and pistachios has been associated to the health benefits of nut consumption. Research has reported that nut consumption decreases the risk of coronary heart disease (Braschi and Naismith, 2008; Dreher, 2012), reduces the level of triglycerides in blood (Dreher, 2012; Sauder et al., 2015) and increases the high-density lipoprotein (HDL) (Sheridan et al., 2007), while reducing the low-density lipoprotein (LDL) (Sari et al., 2010). It also has been associated with cancer prevention due to the stimulation of immunological mechanisms and the protection against free radicals (Gentile et al., 2007; Kodad et al., 2011). Moreover, it does not lead to weight gain and may even improve the risk factors associated with the metabolic syndrome (Dreher, 2012; Wang et al., 2012).

In pistachios, beneficial effects are mainly related to their fatty acid profile (rich in unsaturated fatty acids) and high concentration of phytosterols, dietary fibre,

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Influence of raw material in oil characteristics proteins and magnesium (Dreher, 2012). In almonds, the fatty acid profile and the tocopherol concentration have been the main nutritional components analysed, with important research developed in breeding programmes to increase the quality of genotypes attending to these parameters (Kodad et al., 2006; Zhu et al., 2015). Within nutritional benefits of almond and pistachio consumption, the study of the lipid fraction is crucial. In almond kernels, lipids account from 40 to 67 g/100 g of almond weight (Yada et al., 2011) and, in pistachios, about 50 to 62 g/100 g (Catalán et al., 2017). Almond and pistachio fatty acid profile is composed mainly of oleic acid that constitutes more than 50% of the total fatty acids in most of cultivars (Roncero et al., 2016b; Catalán et al., 2017;). Linoleic acid, is the second fatty acid of importance in both nuts, with percentages that reach 12-34% in almonds and around 10 to 30% in pistachios (Catalán et al., 2017; Roncero et al., 2016b). As expected, substantial quantities of triacylglycerols have been reported in both nuts (Martín- Carratalá et al., 1999; Cherif et al., 2004; Chahed et al., 2008).

The high content of lipids and their health promoting properties makes interesting the production of almond and pistachio oils for human consumption. Among interesting minor components in nut oils, previous studies reported high contents of tocopherols and phytosterols, reported as decisive for nut oil quality (Maestri et al., 2015; Roncero et al., 2016a). Specifically, pistachio oil shows the higher concentration of total phytosterols compared to any other nut oil, up to 271.9 mg per 100 g oil. In almond oil, this concentration is lower, around 161.8 mg per 100 g oil (Kornsteiner- Krenn et al., 2013). However, the phytosterols composition in almond and pistachio oils is similar, with high concentrations of β-sitosterol (up to 95% in almond and more than 90% in pistachios) and minor concentrations of campesterol and stigmasterol (Maestri et al., 2015; Catalán et al., 2017). Tocopherol content has been reported highly variable depending on the almond and pistachio cultivar. In almond oils, tocopherol content ranges between 350 to 550 mg/kg oil (Kodad et al., 2014), while in pistachios this content is lower, around 350 mg/kg oil (Ling et al., 2016). Unlike the phytosterols composition, tocopherol composition is different in almonds and pistachios, α-tocopherol is the main tocopherol in almonds while γ-tocopherol is the most common in pistachios (Stuetz et al., 2017).

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The huge variability in parameters of major significance as fatty acid profile, tocopherols, phytosterols or triglycerides attending to the genotype or the crop year can constrain the production of almond and pistachio oil at an industrial scale due to the reduced certainty about the chemical characteristics of obtained oil. The wide range of almond and pistachio genotypes available for kernel production should be individually evaluated attending to oil production parameters, as oil yield or specific nutritional properties (Rabadán et al., 2017a; Rabadán et al., 2017b). The present study was aimed to evaluate genotype and crop year effects on oil chemical parameters from several almond and pistachio genotypes.

2. Materials and methods

2.1. Plant material

Almonds and pistachios were collected in the Instituto Técnico Agronómico Provincial of Albacete (Southeastern Spain), during two consecutive crop years. Ten almond cultivars (Antoñeta, Ayles, Belona, Ferraduel, Ferragnes, Guara, Marcona, Penta, Tardona and Vairo) and ten pistachio cultivars (Aegina, Alpina, Avidon, Kerman, Larnaka, Mateur, Napoletana, Ouleimy, Sfax and Sirora) were considered. One kilogram of kernels was collected from every one of the three trees analysed within each cultivar. Nuts were picked at the most appropriate harvest date for each cultivar.

Although almond and pistachio production is mainly concentrated in areas with Mediterranean or Mediterranean type climates, differences in water deficit (Zhu et al., 2015) and temperatures (Kodad et al., 2006) should always be considered, as they have major influence in nut characteristics. As a result, the characterization of genotypes must always be linked to the characteristics of a geographic location. Table 1 shows climatic conditions at the evaluated growing site. For the considered years, the precipitation was below the historical average in 2015 and on the average in 2016. Supplemental irrigation was added both years in order to meet crop water requirements. Temperatures were within the historical records.

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Table 1. Average monthly temperatures (ºC), rainfall (mm) and reference evapotranspiration (mm) measured at the station located La Gineta (Albacete, Spain) during 2015 and 2016 crop years.

2015 crop year 2016 crop year Month Tm (ºC) Rainfall (mm) ETo (mm) Tm (ºC) Rainfall (mm) ETo (mm) January 3.7 7.7 45.0 7.2 15.8 38.6 February 4.9 32.3 50.4 6.9 18.9 54.3 March 9.4 48.8 86.4 7.6 39.0 83.5 April 12.0 17.9 105.6 11.2 40.6 99.7 May 18.1 30.4 171.8 14.4 61.9 134.5 June 21.1 37.6 182.9 21.5 0.6 197.8 July 27.0 0.0 227.2 25.2 0.6 224.1 August 24.2 7.9 183.7 24.4 5.9 192.0 September 18.4 73.2 120.2 20.7 0.4 141.4 October 14.7 12.7 76.2 15.7 44.3 77.9 November 9.1 37.3 49.0 8.4 57.4 38.6 December 7.0 2.6 31.9 5.8 58.4 18.9 14.1 308.4 1330.3 14.1 343.8 1301.3

2.2. Oil extraction

Almonds and pistachios were cracked and shelled manually in controlled conditions for immediate drying at room temperature. Oil extraction was carried out using a hydraulic press (MECAMAQ Model DEVF 80, Vila-Sana, Lleida, Spain) at pressure of 150 bar. One kilogram of ground nuts was placed each time on the press. After pressing, oil was centrifuged to remove remaining solids. Oil was stored in dark glass bottles at 5˚C to avoid degradation until analysis (Rabadán et al., 2017a; Rabadán et al., 2017b).

2.3. Analytical methods

Conjugated trienes (K270) and conjugated dienes (K232) coefficients were calculated from absorbance of a 1% solution of oil in cyclohexane at 270 and 232 nm, respectively, with a UV/VIS spectrophotometer Jasco V-530 (Jasco Analitica Spain, Madrid, Spain), and a path length of 1 cm (EEC, 1991). Free acidity, given as % of oleic acid, was determined by titration of a solution of oil dissolved in ethanol/ether (1:1) with 0.1 M potassium hydroxide ethanolic solution (EEC, 1991).

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In order to determine fatty acids composition (%), the methyl-esters were prepared by vigorous shaking of a solution of oil in hexane (0.2 g in 3 ml) with 0.4 ml of 2 N methanolic potassium hydroxide solution, and analyzed by GC with a Hewlett- Packard (HP 6890) chromatograph equipped with a FID Detector. A fused silica column (50 m length x 0.25 mm i.d.), coated with SGL-1000 phase (0.25 µm thickness; Sugerlabor), was used. Helium was employed as a carrier gas with a flow through the column of 1 ml min-1. The temperatures of the injector and detector were set at 250°C with an oven temperature of 210°C. An injection volume of 1µL was used (Regulation EEC 2568/91, corresponding to AOCS method Ch 2–91).

Oxidative stability was evaluated by the Rancimat method (Gutiérrez, 1989). Stability was expressed as the oxidation induction time (hours), measured with the Rancimat 743 apparatus (Metrohm Co., Basel, Switzerland). An oil sample of 3.0 g was used, warmed to 100°C under an air flow of 10 l h-1.

The concentration of total polyphenols (ppm) was estimated using the method proposed by Bail et al. (2008) based on the determination of total phenols of oil using the the Folin–Ciocalteau colorimetric method. The absorption of the solution was measured on a spectrophotometer Hewlett-Packard 8450 A UV/Vis.

The tocopherol content (mg kg-1) was analysed by HPLC (model 360, Kontron, Eching, Germany) in accordance with the IUPA2432 method (IUPAC, 1947). 1.5 g oil was dissolved in the mobile phase (10 ml) of 0.5% isopropanol in n-hexane. A normal phase column Lichrosphere Si60 (250 mm length, 4.6 mm i.d. and 5 µm particle size) was used with an injection volume of 20 µl and a flow rate of 1 mL min-1.

Sterols were determined with a Hewlett-Packard (HP 6890) gas chromatograph with a capillary column (25 m length x 0.25 mm i.d.) coated with SGL-5 (0.25 µm thickness; Sugerlabor). The sample preparation and working conditions used were the described by Madawalaa et al. (2012). Apparent β-sitosterol was calculated as the sum of β-sitosterol, Δ5,23-stigmastadienol, clerosterol, sitostanol, and Δ5,24- stigmastadienol.

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Triglycerides have been determined using HPLC-IR after weighing 0.5g of oil in 10mL of acetone, mobile phase 50:50 acetone: acetonitrile at a flow rate 1 mL min-1 and column ACE18 (250 mm length, x 4.6 mm i.d x 5 µm) by using the method proposed by Holčapek et al. (2005).

2.4. Statistical analyses

Determinations in this study are means of triplicate measurements from three independent samples for each almond and pistachio genotype. Statistical differences were estimated from ANOVA test at the 5% level of significance and Duncan test (p < 0.05). In order to analyse the comparative responses of almond and pistachio genotypes in both 2015 and 2016 crop years and the possible interaction, data were also analysed by two way ANOVA. Principal component analysis (PCA) was developed for almond and pistachio genotypes for the crop years considered including variables that are of major nutritional interest that had previously showed significant differences. All statistical analysis were carried out using the SPSS programme, release 23.0 for Windows.

3. Results and discussion

As reported in several studies, the almond oil chemical composition depends mainly on the genotype (Kodad et al., 2014; Kodad et al., 2011; Kodad et al., 2006; Maestri et al., 2015; Martín-Carratalá et al., 1999). Except for CT values, the chemical parameters of almond oils studied here show significant differences among genotypes, in both crop years analysed (Table 2). Values obtained for all oils were below the maximum values established for non-refined oils in the Codex Alimentarious (FAO, 2001).

In agreement with previous studies, oleic was the main fatty acid found in almond oils (Kodad et al., 2014; Yada et al., 2011). Attending to the fatty acid profile analysed, significant differences were found in all the fatty acids analysed. The oleic content was higher in cultivar Guara for the two crop years considered (72.82 and 73.52%), although no significant differences appear with Ferragnes in 2015 (72.53%) and

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Ferragnes and Vairo in 2016 (71.97 and 73.45%). On the other hand, Penta and Ayles are the cultivars with the higher content of linoleic acid (23.63-24.60% and 23.20- 23.52%, respectively) for the two crop years considered. As previously reported (Askin et al., 2007; Maestri et al., 2015), a strong negative correlation between oleic and linoleic fatty acids was observed (r = -0.980 p < 0.001). At the same time, the fatty acid profile is one of the factors that influence oxidative stability. In our study, OSI values were positively correlated with oleic acid (r = 0.494 p < 0.05) and negatively with linoleic acid (r = -0.544 p < 0.05). OSI values ranged from 16.3 h in Ferraduel oil in 2016 to 26.5 h in Guara in 2015. The OSI values of our study are higher than the values reported in other studies (Maestri et al., 2015; Moayedi et al., 2011), but differences can be attributed to dissimilarities in the conditions of analysis using the Rancimat method.

Among minor components, almond oil is considered a rich source of tocopherols, which are antioxidants of main nutritional interest (Chen et al., 2006). In our study, Guara is the cultivar with the higher concentration of total tocopherols in the two crop years considered (501 and 490 mg/kg, respectively) while Tardona is the cultivar with the lower concentration (182 and 163 mg/kg). Previously Kodad et al. (2014) had reported a high variability of tocopherols in Spanish almond genotypes, with concentrations of α-tocopherol between 313–616 mg/kg.

Although the amount of polyphenols in oils is low due to reduced solubility (Torres et al., 2009; Maestri et al., 2015), the total polyphenols content varied from 18.53 to 32.40 ppm in the first CY, and from 8.53 to 21.57 ppm in the second CY (table 2). The total concentration of phytosterols and the proportion of each specific phytosterols also showed significant differences among genotypes. β-Sitosterol in Belona and Ayles reached values higher than 95.2% of total phytosterols.

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Triacylglycerol composition of almond oils was also a useful parameter to distinguish almond genotypes (table 2). Triglycerides had been previously reported as useful to classify almond attending to their origin (Martín-Carratalá et al., 1999). Main triglyceride found in almond oil was OOO that ranged from 23.97-31.81% in 2015 and 25.79-36.59% in 2016. This result agrees with previous studies developed in Spain in almonds grown at different sites, reporting the effectiveness of triacylglycerol composition for almond genotype discrimination independently from environmental effects (Prats-Moya et al., 1999).

In pistachio oils, the three parameters of regulated quality considered (CD, CT and AV) meet Codex Alimentarious (FAO, 2001) requirements and show significant differences among genotypes. Values for the two crop years considered are similar.

Overall, the fatty acid composition reported in the present study is in agreement with previous studies (Arena et al., 2007; Chahed et al., 2008; Tsantili et al., 2010; Catalán et al., 2017;). The fatty acid profile of different pistachio genotypes shows significant differences for all fatty acids analysed (Duncan test, p < 0.05) (table 3). Higher nutritional value could be attributed to genotypes with the higher percentage of linoleic acid. That is the case of Sfax genotype, which had a linoleic acid percentage of 29.57% in 2015 and 31.41% in 2016. Previously, Kerman had been identified as the cultivar with the higher linoleic content in a study that did not consider Sfax (Tsantili et al., 2010). Cultivars with the higher concentration of linoleic acid (Sfax and Kerman) are those with the less stable oils as OSI values are strongly correlated with linoleic fatty acid content (r = -0.961 p < 0.001).

Phytosterols are one of the most important minor components in pistachios (Dreher, 2012). The discriminatory power of phytosterols has been previously reported for pistachio genotypes with different origin (Arena et al., 2007), but our results prove the usefulness of the total concentration of these compounds for genotype discrimination when cultivars are grown in the same plot (table 3). Among genotypes considered, Sfax showed the greatest concentration of phytosterols (4042 and 4197 mg/kg fat in 2015 and 2016, respectively). On the contrary, the lowest phytosterols concentrations were found in Alpina and Aegina genotypes from the 2016 harvest.

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The differences found on the fatty acid profile can also be identified attending to the triglyceride profile. Significant differences (Duncan test, p < 0.05) were found for all triglycerides analysed. Triglycerides have been reported to be useful for discrimination of pistachio oils from different geographic origins (Ballistreri et al., 2010), reporting significant differences in most common triglycerides such as OOO or OLO. In our results the greatest difference appears in the content of OOO. The concentration of this triglyceride ranges between 16.84 % - 31.02 % (crop year 2015), and between 16.61 % - 35.10% (crop year 2016) for Sfax (lower values) and Larnaka (higher values) genotypes. As triglycerides have more possible combinations than fatty acids, they have been reported to be more discriminating (Holčapek et al., 2003; Ballistreri et al., 2010).

Analysis from two-way ANOVA (table 4) shows that the main variability source for chemical composition and characteristics of almond and pistachio oils differs depending on the parameter considered.

The fatty acid profile of almond and pistachio oils is mainly affected by the genotype with the exception of palmitic acid in pistachio oil. In pistachio oils, these short chained fatty acids seem to be more strongly affected by the crop year, although a significant effect of the genotype and the interaction G x CY was also found. Results for almond oil agree with the conclusions of Kodad et al. (2014) although this study also reported a small effect of the CY in the variability of oleic and linoleic acids that is not found in our results. These slight differences could be attributed to larger differences in the annual weather conditions or the different genotypes considered. However, all studies agree in the stronger effect of the genotype in the fatty acid profile of almond oil compared to crop year (Kodad et al., 2014; Maestri et al., 2015).

Regarding the triglyceride profile of almond oil, the crop year has been identified as main variability source. Opposite results have been obtained for pistachio oils in which genotype is the main source of variability for all triglycerides except for OLO. However, the high proportion of OLO in the pistachio triglyceride profile makes the influence of crop year determinant.

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Table 4. Variability expressed as percentage of the total sum of squares for chemical composition and characteristics from almond and pistachio oils.

Pistachio Almond Parameter Genotype (G) Crop year (CY) G x CY Genotype (G) Crop year (CY) G x CY OC 75.15** 20.61** 4.23* 74.82** 20.09** 5.09* C16:0 19.37** 77.2** 3.43** 85.85** 6.17 7.99 C18:0 51.52** 40.31** 8.17** 85.95** 1.83** 12.22** C18:1 87.12** 8.53** 4.35** 79.55** 2.52 17.92** C18:2 91.31** 2.43** 6.27** 81.87** 0.08 18.05** Polyp 6.81** 86.38** 6.81** 2.57** 94.47** 2.96** OSI 57.19** 36.87** 5.94** 17.49** 76.00** 6.50** Ste 39.70** 51.52** 8.78** 27.95** 65.91** 6.15** Camp 13.97** 84.20** 1.83** 93.39** 1.00 5.61** β-Sit 34.27** 45.81** 19.92** 81.00** 16.19** 2.81** Toc 49.66** 24.33** 26.01** 63.40** 29.56** 7.03** OLL 69.18** 25.10** 5.72** 13.50** 82.26** 4.24** OLO 12.19 59.67** 28.15** 10.74** 87.99** 1.28 SLL+PLO 80.99** 14.15** 4.86** 35.83** 59.74** 4.43** OOO 82.46** 13.28** 4.26** 19.97** 75.72** 4.31** POO 80.30** 4.75* 14.96** 31.93** 59.97** 8.11** Abbreviations: OC, oil content; C16:0, palmitic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; OSI, oxidative stability index; Polyp, total polyphenols; Ste, total sterols; β-Sit, aparent β-Sitosterol; Camp, campesterol; Toc, tocopherol content. Triacylglycerols: L, linoleic acid; P, palmitic acid; O, oleic acid; and S, stearic acid. *p<0.05; **p<0.01.

Attending to minor components, the variability in the polyphenol content depends mainly on the CY in both oils. Although, the tocopherol content has been reported to be directly affected by temperature (Kodad et al., 2006), in our study, the variability in the tocopherol content in oils was greatly influenced by the genotype. In this regard, similar results had been obtained for the content of α-tocopherol content in almond cultivars grown in Argentina (Maestri et al., 2015) and local Spanish almond cultivars (Kodad et al., 2014). These results about tocopherols support the effectiveness of breeding programmes in the development of almond cultivars with high tocopherol content (Zhu et al., 2016). On the other hand, the total phytosterols content is determined to a larger extent for the CY. However, in the composition of specific phytosterols, different results were obtained. In pistachio oils, campesterol and β-Sitosterol were greatly affected by the CY, while the same components in almond oil showed larger variations as a result of the genotype effect.

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Influence of crop year in walnut oils and flours

Differences regarding the genotype and the CY in almond oils can be observed in figure 1. CP1 comprises mainly the information of oleic, linoleic, OSI and polyphenols while CP2 includes the information about palmitoleic and tocopherols. It can be observed that major differences in the crop years considered appear due to the concentration of polyphenols and OSI values. Oils obtained in 2016 show larger concentration of palmitoleic, while oils from 2015 show higher content of polyphenols and a higher OSI values. Some genotypes show greater differences regarding the CY than others, this is the case of Guara and Tardona. Vairo is the genotype with the lower variability related to the crop year.

Figure 1. Principal Component Analysis of almond genotypes in 2015 and 2016. Abbreviations: C16:1, palmitoleic acid; C18:1, oleic acid; C18:2, linoleic acid; OSI, oxidative stability index; Polyp, total polyphenols; Toc, tocopherol content.

Figure 2 shows the differences that appear in pistachio genotypes for the two crop years considered. CP1 comprises the information about palmitic, stearic, oleic, total polyphenols and OSI values, while CP2 is associated with the information about linoleic and total sterols. The concentration of total polyphenols and the percentage of palmitic are the variables that greatly affect crop year variability. Oils obtained from genotypes in 2015 showed a higher content of polyphenols and phytosterols

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Influence of raw material in oil characteristics than oils obtained in 2016. On the other hand, oils obtained in 2016 showed a larger presence of palmitic acid. As reported before, and regardless of the crop year, Sfax and Kerman are the genotypes that show the largest concentration of linoleic acid and therefore, the lower concentration of oleic acid and the smallest values for OSI. Differences reported in genotypes attending to the crop year is not homogeneous. Cultivars Avidon and Sfax show smaller differences attending to the crop year than the rest of the genotypes considered.

Figure 2. Principal Component Analysis of pistachio genotypes in 2015 and 2016. Abbreviations: C16:0, palmitic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; OSI, oxidative stability index; Polyp, total polyphenols; Ste, total sterols; Toc, tocopherol content.

4. Conclusion

A wide range of variability was found in the study in oils from different almond and pistachio genotypes collected in two consecutive crop years. Regarding the study of the main variability source in chemical parameters, similar results were found for almonds and pistachios. In both, oil content and fatty acid profile (oleic, linoleic and stearic fatty acids) were mainly determined by the genotype. However, the

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Influence of crop year in walnut oils and flours concentration of palmitic acid in pistachio oils was strongly determined by the CY. The variability in the concentration of some compounds of nutritional interest such as polyphenols and phytosterols was mainly determined by the CY while the tocopherols content was strongly influenced by the genotype. The analysis of the source of the reported variability provides crucial information for the development of a food industry based on the production of almond and pistachio oils. Moreover, according to the provided information, breeding programmes should focus on the study of parameters that are more directly affected by the genotype.

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Effect of genotype and crop year in the nutritional value of walnut virgin oil and

defatted flour

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Influence of crop year in walnut oils and flours

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Influence of raw material in oil characteristics

Effect of genotype and crop year in the nutritional value of walnut virgin oil and defatted flour

ABSTRACT Keywords: The present study analyses the health-promoting compounds of walnut oil Juglans regia Human health and walnut defatted flour obtained using and hydraulic press. To identify Tocopherols the cultivar effect, all walnut varieties were harvested in the same plot for Proteins Essential two years to control environmental and agricultural management effects minerals on kernel chemical traits. Beyond the variability reported in the products obtained from the different cultivars analysed, the crop year shows a crucial effect in the nutritional value of walnut products. Specifically, the variability caused for the crop year in the presence of oleic fatty acid, total phytosterols, polyphenols, tocopherols, protein concentration and presence of essential minerals is larger than the reported for the cultivar. As a result, focus must change in the study of cultivar effect on walnut products to a wider analysis of specific variables as temperature due to walnut development, as they are expected to be crucial in determining walnut nutritional value.

1. Introduction

Walnut is the most widespread tree nut in the world. In the last decade, the production of walnuts worldwide has increased from 1.6 x 106 tons in 2004 up to 3.5 x 106 tons in 2014 (FAO, 2016). Native from Central Asia, walnut cultivation has currently reached places as far as Ukraine, Mexico, Romaine, France or Egypt. However, the main production countries are still China, US, Iran and Turkey (FAO, 2016). The cultivation of walnut tree in these different places, has encouraged the development of new walnut genotypes, adapted to the environmental conditions of their growing area and the preferences of the local markets. All these cultivars

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Influence of crop year in walnut oils and flours compose a huge genetic resource of plant material that should be studied in order to know their specific characteristics and make the best use of them.

The interest of walnut production and consumption is associated to its health- promoting properties. Walnuts consumption improves the flood lipoprotein profile, shows anticancer and antitherogenic effect, shows high antioxidant capacity and contributes to the regulation of immunological activity and inflammatory response (Awad and Fink, 2000; Yang et al., 2009). Moreover, walnut consumption is not associated to weight gain (Ibarrola-Jurado et al., 2013). Some of the main benefits of walnuts are associated to its high concentration of polyunsaturated fatty acid acids that show a protective effect with respect to coronary heart disease events (Amaral et al., 2003). Walnut is the nut with the highest content of linoleic reported (Robbins et al., 2011), with contents between 50.2 to 73.5% of total fatty acids depending on the genotype considered (Crews et al., 2005; Martínez et al., 2006). Moreover, walnut contains a wide range of phytochemicals as phenolic acids or flavonoids with antioxidant properties (Trandafir et al., 2016).

Walnuts are considered a rich source of vitamin E, with concentrations of tocopherols between 194 to 297mg/kg (Amaral et al., 2005). Main tocopherols found in walnut oil are α-tocopherol and δ-tocopherol (Crews et al., 2005; Martínez et al., 2006), while high concentrations of γ-tocopherol have been found in partially defatted flours. These lipid-soluble compounds are involved in crucial functions, acting as antioxidants but also as membrane stabilisers (Amaral et al., 2005). Walnuts also have significant concentration of phytosterols, linked to the reduction of total plasma cholesterol and low-density lipoprotein cholesterol (Abbey et al., 1994; Martínez et al., 2006). Sterols in walnut oils are found in concentrations about 140mg/100g, although high variability has been found attending to walnut origin (Crews et al., 2005). β-sitosterol is the most common (81-92%) (Crews et al., 2005; Martínez et al., 2006), with minor concentrations of Δ5-avenasterol and campesterol also reported.

Within the proximate profile of walnut, the content of lipids, proteins and minerals are crucial in walnut nutritional value. Fat content compound over 60% of nut weight (Amaral et al., 2003; Kodad et al., 2016), while proteins range between 12 to 15% of

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Influence of raw material in oil characteristics total nut weight (Amaral et al., 2003). The protein part of walnut has recently focus major attention due to the high presence of highly digestible proteins and the balanced presence of essential amino acids (Sze-Tao and Sathe, 2000). Total ash content in walnuts is usually around 2%, similar values than in other nuts as almonds (Roncero et al., 2016). High concentration of essential minerals as Ca, K, P, Fe, Zn or Cu have been reported in walnut kernels (Gharibzahedi et al., 2012, 2014). These minerals play crucial roles as catalysts and antioxidants (Gharibzahedi et al., 2012) becoming determinant in the study of nutritional value of walnuts.

Walnuts can be consumed directly or be used for oil extraction. The use of the hydraulic press allows the production of high quality oils at reasonable prices (Rabadán et al., 2017a; Rabadán et al., 2017c). By using cold pressing extraction, all components previously identified as interesting for human consumption are expected to remain in the virgin walnut oil and in the partially defatted walnut flour that appears as by-product (Cuesta et al., 2017).

The walnut genotype, origin and the land management practices have a major impact in the chemical composition of walnuts. The fatty acid profile, tocopherols, triglycerides, sterols and protein and mineral content, among other parameters, are effectively affected by the walnut genotype and walnut origin (Amaral et al., 2003; Amaral et al., 2005; Crews et al., 2005; Bada et al., 2010; Gharibzahedi et al., 2014; Kodad et al., 2016). Beyond the effect of genotype and origin, land management practices affect the fatty acid profile and the presence of minor components. Studies report differences in the content of linoleic acid due to irrigation (Verardo et al., 2009), and together, nitrogen fertilization and irrigation were able to enrich walnuts in phenolic compounds (Verardo et al., 2013). The environment has been one of the factors that has been identified as responsible of changes in the total tocopherol content in some nuts, including walnuts (Amaral et al., 2005; Crews et al., 2005; Bada et al., 2010; Kodad et al., 2016).

By analysing walnuts that have all been grown and harvested in the same plot, the effect of origin and land management practices and origin on walnut chemometrics can be controlled. The aim of this two year study is to analyse the main health-

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Influence of crop year in walnut oils and flours promoting compounds of walnut oil and walnut defatted flour obtained by cold pressing and identity if the source of the reported variability is the genotype itself, and therefore can be easily controlled, or if that variability is the result of the crop year, including factors that are not easy to control, as temperature.

2. Materials and methods

2.1. Plant material

Walnuts were collected in the Instituto Técnico Agronómico Provincial of Albacete in the southeast of Spain in 2015 and 2016. Ten different walnut cultivars were considered (Franquette, Hartley, Hugget, Mayette, Mollar de Nerpio, Parisienne, Payne, Pedro and Serr). One kilogram of kernels was collected from every one of the three trees analysed within each cultivar. Nuts were picked at the most appropriate harvest date for each cultivar.

The influence of environment and land management practices in the chemical characteristics of walnuts have been already reported (Verardo et al., 2009; Verardo et al., 2013). As a result, the characterization of walnut genotypes must always be linked to the characteristics of a geographic location. Climatic conditions at the experimental plot were walnuts were collected are showed in table 1. For the considered years, the precipitation was below the historical average in 2015 and on the average in 2016. Supplemental irrigation was added both years in order to meet walnut trees water requirements. Temperatures were within the historical records.

2.2. Oil extraction

Walnuts were cracked and shelled manually in controlled conditions for immediate drying at room temperature. Oil extraction was carried out using a hydraulic press (MECAMAQ Model DEVF 80, Vila-Sana, Lleida, Spain) at pressure of 150 bar. One kilogram of ground walnuts was placed each time on the press. After pressing, oil was centrifuged to remove remaining solids. Oil was stored in dark glass bottles at 5˚C to avoid degradation until analysis (Rabadán et al., 2017a; Rabadán et al., 2017c).

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Influence of raw material in oil characteristics

Table 1. Average monthly temperatures (ºC), rainfall (mm) and reference evapotranspiration (mm) measured at the station located La Gineta (Albacete, Spain) during 2015 and 2016 crop years.

2015 crop year 2016 crop year Tm Rainfall ETo Tm Rainfall ETo Month (ºC) (mm) (mm) (ºC) (mm) (mm) January 3.7 7.7 45.0 7.2 15.8 38.6 February 4.9 32.3 50.4 6.9 18.9 54.3 March 9.4 48.8 86.4 7.6 39.0 83.5 April 12.0 17.9 105.6 11.2 40.6 99.7 May 18.1 30.4 171.8 14.4 61.9 134.5 June 21.1 37.6 182.9 21.5 0.6 197.8 July 27.0 0.0 227.2 25.2 0.6 224.1 August 24.2 7.9 183.7 24.4 5.9 192.0 September 18.4 73.2 120.2 20.7 0.4 141.4 October 14.7 12.7 76.2 15.7 44.3 77.9 November 9.1 37.3 49.0 8.4 57.4 38.6 December 7.0 2.6 31.9 5.8 58.4 18.9 14.1 308.4 1330.3 14.1 343.8 1301.3

2.3. Analytical methods

In order to determine fatty acids composition (%), the methyl-esters were prepared by vigorous shaking of a solution of oil in hexane (0.2 g in 3 ml) with 0.4 ml of 2 N methanolic potassium hydroxide solution, and analyzed by GC with a Hewlett- Packard chromatograph (HP 6890). A fused silica column (50 m length x 0.25 mm i.d.), coated with SGL-1000 phase (0.25 µm thickness; Sugerlabor), was used. Helium was employed as a carrier gas with a flow through the column of 1 ml min-1. The temperatures of the injector and detector were set at 250°C with an oven temperature of 210°C. An injection volume of 1µL was used (Regulation EEC 2568/91, corresponding to AOCS method).

Oxidative stability was evaluated by the Rancimat method (Gutiérrez, 1989). Stability was expressed as the oxidation induction time (hours), measured with the Rancimat 743 apparatus (Metrohm Co., Basel, Switzerland). An oil sample of 3.0 g was used, warmed to 100°C under an air flow of 10 l h-1.

The concentration of total polyphenols (ppm) was estimated using the method proposed by Bail et al. ( 2008) based on the determination of total phenols of oil using

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Influence of crop year in walnut oils and flours the the Folin–Ciocalteau colorimetric method. The absorption of the solution was measured on a spectrophotometer Hewlett-Packard 8450 A UV/Vis.

The tocopherol content (mg/Kg) was analysed by HPLC (model 360, Kontron, Eching, Germany) in accordance with the IUPA2432 method (IUPAC, 1947). 1.5 g oil was dissolved in the mobile phase (10 ml) of 0.5% isopropanol in n-hexane. A normal phase column Lichrosphere Si60 (250 mm length, 4.6 mm i.d. and 5 µm particle size) was used with an injection volume of 20 µl and a flow rate of 1.0 ml/min.

Sterols were determined with a Hewlett-Packard (HP 6890) gas chromatograph with a capillary column (25 m length x 0.25 mm i.d.) coated with SGL-5 (0.25 µm thickness; Sugerlabor). Working conditions were as follows: carrier gas, helium; flow through the column, 1.2 ml min-1; injector temperature, 280°C; detector temperature, 290°C; oven temperature, 260°C; injection volume 1 µl (Regulation EEC 2568/91, corresponding to AOCS method Ch 6–91). Apparent β-sitosterol was calculated as the sum of β-sitosterol, Δ5,23-stigmastadienol, clerosterol, sitostanol, and Δ5,24- stigmastadienol.

Water content in flour was measured by the loss of weight after oven drying at 105ºC for 72h at least (Lau, 1982). Protein content was calculated by multiplying the total nitrogen content, obtained by the Kjeldahl method (FOSS, 2003), by a conversion factor of 5.18 (Greenfield, 2003). To determine ash content, flours were ashed at 550ºC to constant weight. Crude fat (ethyl ether extract) was estimated gravimetrically by filter bag technique after petroleum ether extraction of the dried sample in an extraction system Ankom XT10 (ANKOM, 2009). To determine the content of crude fibre, the Weende technique adapted to the filter bag technique was applied. This method determines the organic residue remaining after digestion with solutions of sulfuric acid and sodium hydroxide, using an Ankom 220 fibre analyser (ANKOM, 2008). Total carbohydrate content was calculated by subtracting the sum of the crude protein, total fat, water and ash from the total weight of the flour (Sullivan, 1993). Available carbohydrate content (nitrogen-free) was calculated by subtracting the crude fibre from the total carbohydrate content (González et al., 1987).

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Contents of mineral elements in flours were obtained by inductively coupled plasma mass spectrometry (ICP-MS), using Jobin Yvon JY 50-P equipment (Instruments S.A., Jobin Yvon, Longjuemeau, France).

2.4. Statistical analyses

Determinations in this study are means of triplicate measurements from three independent samples for each walnut genotype involved in the two year study. Statistical differences were estimated from ANOVA test at the 5% level of significance and Duncan test (p < 0.05). Correlation analyses were performed employing Pearson´s test. In order to analyze the comparative responses of walnut genotypes in both 2015 and 2016 crop years and the possible interaction, data were also analyzed by two way ANOVA.

3. Results and discussion

3.1. Walnut oils

3.1.1. Fatty acid profile

Data about fatty acid profile of walnut genotypes in 2015 and 2016 is shown in table 2. As previously reported, walnut oil is mainly composed by polyunsaturated and monounsaturated fatty acids (Amaral et al., 2003; Crews et al., 2005; Martínez et al., 2006; Bada et al., 2010). This profile makes walnut oil one of the most interesting oils from health perspective due to the benefits of unsaturated fatty acids in the reduction of the risk of cardiovascular diseases (Crews et al., 2005).

Significant differences among cultivars were found for all individual fatty acids and the index PUFA/MUFA. Palmitic and stearic were the main saturated fatty acids found, agreeing with previous studies (Amaral et al., 2003; Crews et al., 2005; Bada et al., 2010). Linoleic fatty acid compound more than 47.5% of total fatty acids in all cultivars involved in the two year study, accounting up to 62.13% and 60.02% in cv. Pedro and Mollar de Nerpio in 2015. In 2015 the concentration of linoleic in walnut oils was higher in most of the cultivars. Some cultivars as Scharch-Franquette showed

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Influence of crop year in walnut oils and flours a small concentration of linoleic acid compared to the rest (50.39 and 47.49% in 2015 and 2016, respectively) agreeing with the results obtained for Franquette cultivar in Argentina (Martínez et al., 2006), although higher concentrations for the same cultivar had been reported in Portugal (Amaral et al., 2003). The strong variability in the concentration of linoleic attending to both the genotype and the crop yeat (CY) can be the cause of these different results.

The percentage of oleic acid is higher in all cultivars in 2016 compared to 2015 values with the exceptions of cv. Parisienne and Serr. The values for the correlation between PUFA and MUFA were higher for Pedro, Hartley, Nerpio and Payne in 2015. Higher values for the PUFA/MUFA index indicate more polyunsaturated oils with stronger health promoting characteristics, but also oils more sensitive to oxidation processes (Rabadán et al., 2017b).

3.1.2. Minor compounds and oxidative stability

One of the main nutritionals benefits of walnut consumption is associated to the elevated concentration of tocopherols (Amaral et al., 2005). These compounds have a strong antioxidant effect, successfully correlated to a lower risk of cancer and cardiovascular diseases. In our study, Hartley in CY2015 and Pedro in CY2016 showed the highest value reported (326 mg/kg), while values lower than 200 mg/kg were found in Scharch-Franquette oils in 2015. Values in our study are similar to other literature values (Amaral et al., 2005), but smaller to the reported for some unspecified cultivars in the north of Spain (Bada et al., 2010). Regarding the two crop years considered, the tocopherol content found in walnut oils showed significant differences among cultivars in 2015 but not in 2016 (Table 3). Values are similar to those reported for Moroccan seedlings where high variability regarding the crop year was also observed (Kodad et al., 2016).

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Table 2. Fatty acid profile of walnut oils in the two year study. PUFA/ Genotype C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 MUFA CY2015 Scharch- 7.82b ± 0.16bc ± 3.40a ± 27.47a ± 50.39f ± 10.25d ± 2.20e ± Franquette 0.10 0.01 0.10 0.55 0.40 0.25 0.07 7.32cd ± 0.13d ± 3.14bc ± 17.33d ± 58.77bc ± 12.92ab ± 4.11b ± Hartley 0.10 0.01 0.05 0.35 0.80 0.08 0.12 7.06e ± 0.15c ± 3.00cd ± 20.11c ± 58.96bc ± 10.50d ± 3.43c ± Nugget 0.13 0.01 0.09 0.56 1.00 0.50 0.17 7.96ab ± 0.17b ± 3.17b ± 22.54b ± 54.16d ± 11.67c ± 2.90d ± Mayette 0.14 0.01 0.11 1.16 0.55 0.45 0.15 7.87ab ± 0.19a ± 2.58f ± 17.33d ± 60.02b ± 11.43c ± 4.08b ± Nerpio 0.13 0.01 0.08 0.70 0.21 0.21 0.17 7.16de ± 0.15c ± 2.81e ± 27.46a ± 52.49e ± 9.49e ± 2.25e ± Parisienne 0.14 0.01 0.10 1.50 0.45 0.20 0.13 8.11a ± 0.19a ± 2.49fg ± 17.59d ± 58.20c ± 12.55c ± 3.98b ± Payne 0.20 0.01 0.08 0.52 0.86 0.25 0.09 7.21cde ± 0.15c ± 2.38g ± 14.56e ± 62.13a ± 13.09a ± 5.12a ± Pedro 0.14 0.01 0.08 0.45 0.70 0.10 0.10 7.44c ± 0.16bc ± 2.88de ± 23.46b ± 54.24d ± 11.40c ± 2.78d ± Serr 0.13 0.01 0.06 0.61 1.10 0.30 0.11 CY2016 Scharch- 7.16bc ± 0.15bcd ± 4.04a ± 32.32a ± 47.49e ± 8.21e ± 1.72g ± Franquette 0.14 0.01 0.06 0.50 0.50 0.20 0.02 7.02cd ± 0.13d ± 3.16e ± 21.49e ± 57.36a ± 10.35b ± 3.13c ± Hartley 0.21 0.01 0.11 0.50 1.15 0.35 0.11 6.87d ± 0.14cd ± 3.48c ± 24.82d ± 55.51b ± 8.70de ± 2.57d ± Nugget 0.08 0.01 0.11 0.95 0.40 0.10 0.09 7.29b ± 0.15bcd ± 3.85b ± 28.88b ± 50.52d ± 8.70de ± 2.04f ± Mayette 0.19 0.01 0.12 0.45 0.50 0.20 0.02 7.80a ± 0.15bc ± 2.65f ± 20.07f ± 58.16a ± 10.61b ± 3.40a ± Nerpio 0.10 0.01 0.08 0.35 1.15 0.30 0.01 7.16bc ± 0.15bcd ± 3.44c ± 26.55c ± 52.73c ± 9.49c ± 2.33e ± Parisienne 0.16 0.01 0.08 0.55 0.70 0.50 0.09 7.92a ± 0.18a ± 3.25de ± 26.25c ± 52.81c ± 9.29c ± 2.35e ± Payne 0.14 0.01 0.05 0.55 0.91 0.20 0.08 7.25bc ± 0.14cd ± 3.34cd ± 21.39e ± 58.25a ± 9.11cd ± 3.13c ± Pedro 0.11 0.01 0.10 0.36 0.25 0.10 0.06 7.42b ± 0.16b ± 2.56f ± 20.96ef ± 57.15a ± 11.57a ± 3.25b ± Serr 0.13 0.01 0.06 0.45 0.65 0.30 0.03 Abbreviations and units: CY, crop year; C16:0, palmitic acid; C16:1, palmitoleic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3, linolenic acid; PUFA, total polyunsaturated fatty acids. MUFA, total monounsaturated fatty acids. Fatty acids are expressed as % of total fatty acids. Mean values (±standard deviation) were the averages of three independent measurements. Different letters on the column indicate significant differences among genotypes, Duncan test (p<0.05).

Significant concentration of phytosterols were also reported in walnut oils, showing the potential of these oils to reduce the risk of coronary heart disease (Awad and Fink, 2000). The sterol total content ranged between 94.8 and 130.4 mg/100g in

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Payne (2016) and Pedro (2015), respectively. These results are in agreement with those already described for walnuts (Amaral et al., 2003), but are lower than those found in other nut oils as almond (Rabadán et al., 2017b) and pistachio oil (Rabadán et al., 2017a). A previous study had reported a range of 103.7 - 161.4mg/100g for phytosterols in Spanish walnuts (Crews et al., 2005). Our values agree with the lowest values of the proposed range. The total content of sterols showed no significant differences between cultivars for any of the two crop years considered.

The proportion of specific sterols in walnut oil is significantly affected by the cultivar. Similar results had been previously obtained by Martínez et al. (2006) in a three cultivar study involving two years and Amaral et al. (2003) in a one year study involving six cultivars. The main sterol found in walnut oil is apparent β-Sitosterol (sum of β-sitosterol and the minor percentages of Δ5,23-stigmastadienol, clerosterol, sitostanol, and Δ5,24-stigmastadienol). Values for campesterol, Δ7-Stigmastenol and stigmasterol were within the values obtained in other studies of Spanish walnut oils (Bada et al., 2010).

Oxidative stability of walnut kernels is related to the fatty acid composition (Verardo et al., 2013). As expected, our results show that cultivars with higher concentrations of linoleic and linolenic showed lower OSI values. On the contrary, higher values of OSI are positively correlated with stearic fatty acid concentration (r=0.789, p <0.01). For some oils, as olive oil, a correlation between polyphenols and OSI values have been widely reported (Carrasco-Pancorbo et al., 2005). However, in our results no positive correlation between the polyphenols content and the OSI values was found. This can be the result of the low concentration of polyphenols found in walnut oil compared to olive oil. In walnut oil, OSI values seem to be determined by fatty acid profile and the concentration of tocopherols (r=0.439, p <0.01).

3.2. Walnut flours

3.2.1. Proximate composition

The proximal analysis of defatted walnut flour shows significant differences for walnut cultivars for the two years considered (table 4) agreeing in general with

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Influence of raw material in oil characteristics previous studies (Amaral et al., 2003; Labuckas et al., 2014; Kodad et al., 2016). After oil extraction, the main components remaining in the walnut flour by-product obtained are carbohydrates, with percentages that range between 40.35 - 50.78%. As reported by Labuckas et al. (2014) after oil extraction proteins compound about a third of total components in walnut flour. Specifically, walnut proteins have large effect on the nutritional quality of walnut flours as they are highly digestible and show a balanced presence of essential amino acids (Sze-Tao and Sathe, 2000). Cvs. Serr and Mayette from the 2016 harvest are those that reported the highest protein content with values of 39.61% and 38.26%, respectively. Significant quantities of ashes are also reported. The reduction in the energy value of partially defatted flour compared to the whole nut is important as fat contributes largely than other components to increase energy value. Whole walnuts have energy value contents around 686 – 710 kcal (Amaral et al., 2003) while in defatted walnut flours these values reduce to 421 – 453 kcal.

3.2.2. Essential minerals

Relevant concentrations of essential minerals have been found on walnut flours. Specifically, five different macro minerals and five micro minerals appear in significant concentrations (Table 5). All essential minerals show significant differences among cultivars and important differences regarding the crop year.

In agreement with previous studies (Gharibzahedi et al., 2014), potassium (0.62 - 0.92 g/100g) and phosphorous (0.55 -0.84 g/100g) are the main minerals found in walnuts. Among the essential minerals reported, calcium (0.15 - 0.24 g/100g), iron (22.01 – 43.55 mg/Kg) and zinc (11.78 – 47.54 mg/ Kg) are within the most important minerals in human nutrition (Latham, 1997). Some cultivars, as Pedro, show high concentration of zinc for both harvested years, however high variability appear regarding the crop year.

As the oil fraction has been partially removed, the result is a walnut flour with a higher content of macro minerals and most of micro minerals compared to previous studies that considered the whole kernel (Gharibzahedi et al., 2012, 2014). The presence of minerals such as K, Ca, Mg, P, Fe, Zn, Mn and Cu improves the nutritional

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Influence of crop year in walnut oils and flours value of walnut flour as these minerals are an essential part of enzymes, and important play roles as catalysts and antioxidants (Gharibzahedi et al., 2012). Magnesium (0.24 - 0.33 g/100g), together with calcium and potassium, prevents bone demineralization, lowers blood pressure and reduces the risk of cardiovascular diseases (Segura et al., 2006). As examples, sulphur (0.19 - 0.38 g/100g) is needed in the amino acids that compound proteins (Latham, 1997), iron is essential in blood formation (Gharibzahedi et al., 2014) and copper (19.73 – 28.89 mg/Kg) is involved in normal carbohydrate and lipid metabolism (Kabas et al., 2007). To our knowledge, significant concentrations of nickel (0.79 – 3.91 mg/Kg) have been reported for the first time in walnut flours (Gharibzahedi et al., 2012, 2014).

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183

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3.4. Origin of variability

The two-way ANOVA showed that most parameters were significantly affected by the genotype, the crop year and the interaction between both (GxCY) (Table 6). The influence of crop year in chemical parameters in walnuts is higher than the reported in other nuts as almonds (Kodad et al., 2014).

Our results show that the main source of variability in the fatty acid profile of walnuts is the crop year. Previous studies obtained different results. In Moroccan walnuts, palmitic acid was significantly affected by the genotype and the interaction GxCY, while the study of Martínez et al. (2006) identified the genotype as main source of variability for palmitic acid. Our study found intermediate results, showing a similar effect of genotype and the crop year in the reported variability of palmitic fatty acid. The variability in the content of stearic and linolenic in our study was caused mainly for the crop year in agreement with Kodad et al. (2016). Differences between studies were expected due to the different cultivars analysed, however, some similar tendencies can be observed.

As reported previously by Amaral et al. (2005), tocopherol content in walnuts is strongly influenced by crop year. The contribution of the crop year to the total variability observed in tocopherol content in our study was 88.18%. The effect of temperature in the tocopherol content reported for other nuts as almonds (Kodad et al., 2006), can be the reason of this result. The differences reported in previous studies about the content of total tocopherols in walnuts from different countries could be then the result of different weather conditions (Crews et al., 2005). The strong effect of crop year in the variability of fatty acids and tocopherol content results in a stronger variability of OSI values regarding the crop year due to the reported correlations.

Other group of phytochemicals mainly affected by the crop year are sterols (75.28% of reported variability). Few studies have analysed how sterol concentration is affected by the genotype and the crop year at the same time. Only Martínez et al. (2006) studied how crop year affects the proportion of campesterol in walnut oil.

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Their results suggested a major effect of genotype in the presence of campesterol. Although our results show a significant effect of genotype in the presence of campesterol, is the interaction GxCY the one that showed the stronger influence.

Table 6. Variability expressed as percentage of the total sum of squares for chemical composition and characteristics of walnut oils and walnut flours. Genotype Crop year Parameter G x CY (G) (CY) Walnut oil C16:0 46.65** 46.24** 7.11** C16:1 43.93** 51.21** 4.87* C18:0 22.13** 70.69** 7.18** C18:1 30.37** 63.51** 6.12** C18:2 52.43** 41.38** 6.18** C18:3 8.56** 85.97** 5.47** PUFA/MUFA 25.70** 66.99** 7.31** Total Sterols 17.44* 75.38** 7.18 Campesterol 43.01** 0.39 56.60** Stigmasterol 54.50** 35.26** 10.24** Aparent β-Sitosterol 19.85** 42.60** 37.55** Δ7-Stigmastenol 33.94** 31.89** 34.17** Polyphenols 1.84** 96.75** 1.42** Tocopherols 6.68** 88.18** 5.14** OSI 5.78** 92.01** 2.21** Walnut partially defatted flour Proteins 18.72** 80.65** 0.63* Ashes 30.84** 40.17** 28.99** Crude fibre 70.53** 21.07* 8.40 Crude fat 95.53** 2.59 1.88* Carbohydrates 22.3** 77.04** 0.66 Energy value 90.51** 6.58** 2.91** Ca 73.09** 4.89 22.02** K 7.39** 84.4** 8.21** Mg 10.9** 74.81** 14.28** P 14.88** 50.89** 34.23** S 2.05** 97.1** 0.85** Zn 29.57** 20.79** 49.65** Cu 11.33** 77.68** 10.99** Fe 4.65** 87.27** 8.08** Mn 22.25** 47.52** 30.24** Mo 0.81** 98.37** 0.81** Ni 12.64** 72.54** 14.82** Abbreviations: C16:0, palmitic acid; C16:1, palmitoleic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3, linolenic acid; PUFA, total polyunsaturated fatty acids; MUFA, total monounsaturated fatty acids; OSI, oxidative stability index. *p<0.05; **p<0.01.

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Regarding partially defatted walnut flour composition, crude fat content is mainly determined by the genotype compared to crop year. The same results were reported by Kodad et al. (2016) considering the total oil content of Moroccan walnuts. Crude fibre is also determined by the genotype. However, although genotype, crop year and GxCY affect significantly the protein content, the crop year itself explained 80.65% of observed variability. This result for proteins agrees with a previous study developed in the whole walnut (Kodad et al., 2016) and encourages the study of the effect of crop year on the protein content due to the key role of proteins in walnut nutritional quality.

Essential minerals are crucial for walnut flour nutritional quality. The reported variability for most of them is attributed to crop year. Only calcium concentration is greatly affected by the cultivar while the interaction GxCY is important for the concentration of phosphorus, zinc and manganese. As any fertilizer was added in the two years study, the reported differences should be explained through environmental factors as soil pH, mineral availability and the effects of water and temperature on walnut tree mineral intake.

As the amount of water available for all genotypes was the same and the land management practices were similar in the two years studied, temperature is proposed as main factor affecting chemical parameters in walnuts oils and flours. However, attending to the weather data provided in table 1, not the annual medium temperature, but the temperature in the different stages of walnut development may be the crucial factor for further analysis.

4. Conclusion

Walnut cultivars show different nutritional qualities that result in oils and defatted flours with different characteristics that may be of great benefit to health and nutrition in the human diet. Beyond the variability reported in the different cultivars analysed, the crop year shows a crucial effect of walnut health-promoting compounds. Specifically, the variability caused for the crop year in the presence of oleic fatty acid, total phytosterols, polyphenols, tocopherols, proteins and essential

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Influence of raw material in oil characteristics minerals is larger than the reported for the cultivar. As a result, focus must be change in the study of the cultivar effect on walnut products and change to a wider analysis of the effects of crop year. Moreover, further study of the effects of the specific factors related to the crop year that affect the content of crucial components for the nutritional quality of walnut oil and defatted flour must be performed.

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Sullivan, D.M., 1993. Proximate and mineral analysis, in: Methods of Analysis for Nutrition Labeling. AOAC International, Arlington (VA), USA.

Sze-Tao, K.W.C., Sathe, S.K., 2000. Walnuts (Juglans regia L): Proximate composition, protein solubility, protein amino acid composition and protein in vitro digestibility. Journal of the Science of Food and Agriculture 80, 1393-1401.

Trandafir, I., Cosmulescu, S., Botu, M., Nour, V., 2016. Antioxidant activity, and phenolic and mineral contents of the walnut kernel (Juglans regia L.) as a function of the pellicle color. Fruits 71, 177-184.

Verardo, V., Bendini, A., Cerretani, L., Malaguti, D., Cozzolino, E., Caboni, M.F., 2009. Capillary gas chromatography analysis of lipid composition and evaluation of phenolic compounds by micellar electrokinetic chromatography in italian walnut (juglans regia l.): Irrigation and fertilization influence. Journal of Food Quality 32, 262-281.

Verardo, V., Riciputi, Y., Sorrenti, G., Ornaghi, P., Marangoni, B., Caboni, M.F., 2013. Effect of nitrogen fertilisation rates on the content of fatty acids, sterols, tocopherols and phenolic compounds, and on the oxidative stability of walnuts. LWT - Food Science and Technology 50, 732-738.

Yang, J., Liu, R.H., Halim, L., 2009. Antioxidant and antiproliferative activities of common edible nut seeds. LWT - Food Science and Technology 42, 1-8.

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3.1. Process innovation

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Influence of the temperature in the extraction of nuts oils by means of screw

press

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Influence of the temperature in the extraction of nuts oils by means of screw press

ABSTRACT Keywords: The extraction of vegetable oils by means of screw presses is considered as a Nut oils Temperature cold extraction. However, this type of machinery needs a pre-heating of the Regulated nozzle in which the separation of the oil takes place so that it is produced in a quality Cold pressed satisfactory way. In this study, the extraction of almond, pistachio and walnut

oil has been evaluated in a screw press, analysing the effect of the temperature applied on the barrel and the crew speed selected on oil temperature. To evaluate the influence of the temperature on the oil extraction, five points of the press were selected and the temperature was monitored during the extraction process using thermocouples. At low processing temperatures, an increase in oil temperature was observed due to the friction of the raw material, which reached values around 60˚C in almond and pistachio, being somewhat smaller in walnut oil. At higher temperatures (between 100 to 200°C), the oil temperature was not increased above 84°C by the cooling produced by the continuous supply of the fresh raw material. By increasing the screw seed, the continuous supply of fresh raw material in a faster way contributes to the cooling of the system, avoiding an excessive increase of the temperature. Results show that temperature of oil influenced greatly the physicochemical parameters of regulated quality of the oils. On the other hand, elevated temperatures may originate the appearance of Maillard reaction compounds with antioxidant capacity that may contribute to increase the oxidative stability of the oils.

1. Introduction

Benefits associated to nut consumption have been widely recognised due to the high amount of health-promoting components identified on them. Research has reported that nut consumption reduces the low-density lipoprotein (LDL) (Sari et al., 2010) and increases the high-density lipoprotein (HDL) (Sheridan et al., 2007), reducing the level of triglycerides in blood (Dreher, 2012; Sauder et al., 2015) and the risk of coronary

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Influence of temperature in oil extracted with the crew press heart disease (Braschi and Naismith, 2008; Dreher, 2012). It also has been associated with cancer prevention due to the stimulation of immunological mechanisms and the protection against free radicals (Gentile et al., 2007; Kodad et al., 2011). Moreover, nut consumption does not lead to weight gain and would even improve the risk factors associated with the metabolic syndrome (Dreher, 2012; Wang et al., 2012).

All these benefits are possible because of nuts proximate composition and their high content of bioactive components. Fat content is crucial to evaluate the nutritional value of nuts. In almond kernels, lipids account from 40 to 67 g/100 g of almond weight (Yada et al., 2011) and in pistachios, about 50 to 62 g/100 g (Catalán et al., 2017). In walnuts the concentration is even larger, up to 60 to 72 g/100 g of total nut weight (Amaral et al., 2003; Kodad et al., 2016). Fatty acid profile in nuts is mainly composed for unsaturated fatty acids that represent more than 85% of total fatty acids. In almond and pistachio, fatty acid profile is compounded mainly by oleic acid that constitutes more than 50% of the total fatty acids in most cultivars (Roncero et al., 2016b; Catalán et al., 2017). In walnuts, linoleic acid is dominant, with percentages around 50.2 to 73.5% of total fatty acids depending on the cultivar considered (Crews et al., 2005; Martínez et al., 2006). Beyond the fatty acid profile, nut oils show high contents of tocopherols and phytosterols, reported as decisive for nut oil quality due to their antioxidant properties and their benefits for human health (Maestri et al., 2015; Roncero et al., 2016a).

The high content of lipids, together with the high nutritional value of lipid profile and bioactive compounds than remain in oil after extraction (Amaral et al., 2005; Catalán et al., 2017), makes advisable the production of nuts oils by using methods that do not affect these beneficial components.

Nut oil extraction can be performed by using different methods that result in oils with different characteristics. Higher oil yields can be obtained by using solvents as hexane or diethyl ether for oil extraction (Bellomo et al. 2009; Conte et al. 2011). However, this method shows serious constraints, as later refining is needed if oil is destined to human consumption, making impossible the production of “virgin” oils (Roncero et al., 2016). Moreover, the use of solvents can cause the inactivation or disappearance

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Product and process innovation of vitamins and other relevant bioactive substances in oils (Satil et al., 2003; Miraliakbari and Shahidi, 2008; Abdolshahi et al., 2015). Extraction using supercritical fluids is the newest alternative for nut oil production (Palazoglu and Balaban, 1998;

Jokić et al., 2012). By using supercritical fluids, mainly CO2, high quality oils can be obtained due to the use of low temperatures for oil extraction (Abbasi et al., 2008; Chan and Ismail, 2009). However, by using supercritical fluids, the quality of oil and the oil yield, evolve in contrary directions, as a reduction of temperature, pressure and CO2 flow, results in lower oil yields (Jokić et al., 2012). Although the supercritical fluid extraction is a technology with huge potential of development, currently it involves high cost of investment, constraining its use to high valuable products (Rosa and Meireles, 2005).

Pressing systems provide high quality oils at affordable prices (Khoddami et al., 2014; Rabadán et al., 2017c). The presses that have been widely used for nut oil extraction are the screw press and the hydraulic press. Oil extraction with hydraulic press demands high labour, however, oils obtained show better maintenance of their physicochemical and sensory properties due to the extraction is performed at room temperature (Álvarez-Ortí et al., 2012). On the other side, although screw press is considered a cold extraction system, the adequate functioning of the press requires an increase of temperature on the barrel to ensure a correct extraction (Álvarez-Ortí et al., 2012). This increase of temperature can have main influence on the quality of obtained oils (Sena-Moreno et al., 2015), requiring a further study of how press extraction conditions can affect the quality of obtained nuts oils.

In this study, the effect of extraction temperature using a screw press on the quality parameters of almond, walnut and pistachio oils is evaluated. With that purpose, the influence of extraction conditions of a screw press (temperature applied on the barrel and rotational speed of the screw) on the temperature of the obtained oils have been studied.

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2. Materials and methods

2.1. Raw material

Almonds, walnuts and pistachio were collected at the Instituto Técnico Agronómico Provincial de Albacete in south east Spain. The shells were removed in controlled conditions for immediate drying. Nuts were dried at room temperature for three days.

2.2. Oil extraction and temperature measurement

Oil extraction was performed using a screw press model Komet Oil Press CA59G (IBG Monforts Oekotec GmbH & Co. KG, Mönchengladbach, Germany). This press includes a heating ring able to reach up to 200˚C and a variable speed drive. Twelve different extraction conditions were evaluated regarding different temperature and speed conditions. The influence of four different temperatures (50°C, 100°C, 150°C, 200°C) and three rotational speed conditions (17 rpm, 49 rpm and 96 rpm) were tested.

Temperature was monitored in different places of the screw press by using thermocouples (Figure 1). Locations selected for temperature monitoring were: the beginning of the screw which is the way of raw material entrance (T1), the end of the screw that is the oil extraction point (T2), the nozzle, where pellet is ejected (T3), the extracted oil (T4) and the oil collector beaker (T5).

Figure 1. Location of points selected for temperature measurement in the screw press. Locations: T1, beginning of the screw, raw material entrance point; T2, end of the screw, oil extraction point; T3, nozzle, pellet ejection point; T4, extracted oil; T5, oil collector beaker.

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2.3. Oil analysis

Free acidity, given as % of oleic acid, was determined by titration of a solution of oil dissolved in ethanol/ether (1:1) with 0.1 mol/L potassium hydroxide ethanolic solution (EEC, 1991). K270 and K232 extinction coefficients were calculated from absorbance of a 10 μl/ml solution of oil in cyclohexane at 270 and 232 nm, respectively, with a UV/VIS spectrophotometer Jasco V-530 (Jasco Analitica Spain, Madrid, Spain), and a path length of 1 cm (EEC, 1991).

Oxidative stability was evaluated by the rancimat method (Gutiérrez, 1989). Stability was expressed as the oxidation induction time (hours), measured with the Rancimat 743 apparatus (Metrohm Co., Basel, Switzerland). An oil sample of 3.5 g was used, warmed to 100 °C under an air flow of 10 l h−1.3.

In order to determine fatty acids composition (%), the methyl-esters were prepared by vigorous shaking of a solution of oil in hexane (0.2 g in 3 ml) with 0.4 ml of 2 N methanolic potassium hydroxide solution, and analyzed by GC with a Hewlett- Packard chromatograph (HP 6890). A fused silica column (50 m length x 0.25 mm i.d.), coated with SGL-1000 phase (0.25 µm thickness; Sugerlabor), was used. Helium was employed as a carrier gas with a flow through the column of 1 ml min-1. The temperatures of the injector and detector were set at 250°C with an oven temperature of 210°C. An injection volume of 1µL was used (Regulation EEC 2568/91, corresponding to AOCS method Ch 2–91).

The concentration of total polyphenols (ppm) was estimated using the method proposed by Bail et al. ( 2008) based on the determination of total phenols of oil using the the Folin–Ciocalteau colorimetric method. The absorption of the solution was measured on a spectrophotometer Hewlett-Packard 8450 A UV/Vis.

The tocopherol content (mg/Kg) was analysed by HPLC (model 360, Kontron, Eching, Germany) in accordance with the IUPA2432 method (IUPAC, 1947). 1.5 g oil was dissolved in the mobile phase (10 ml) of 0.5% isopropanol in n-hexane. A normal phase column Lichrosphere Si60 (250 mm length, 4.6 mm i.d. and 5 µm particle size) was used with an injection volume of 20 µl and a flow rate of 1.0 ml/min.

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Sterols were determined with a Hewlett-Packard (HP 6890) gas chromatograph with a capillary column (25 m length x 0.25 mm i.d.) coated with SGL-5 (0.25 µm thickness; Sugerlabor). Working conditions were as follows: carrier gas, helium; flow through the column, 1.2 ml min-1; injector temperature, 280°C; detector temperature, 290°C; oven temperature, 260°C; injection volume 1 µl (Regulation EEC 2568/91, corresponding to AOCS method Ch 6–91). Apparent β-sitosterol was calculated as the sum of β-sitosterol, Δ5,23-stigmastadienol, clerosterol, sitostanol, and Δ5,24- stigmastadienol.

2.4. Statistical analyses

Determinations in this study are means of triplicate measurements from three independent samples for each nut and extraction conditions. Statistical differences were estimated from ANOVA test at the 5% level of significance and Duncan test (p < 0.05). All statistical analysis were carried out using the SPSS programme, release 23.0 for Windows.

3. Results and discussion

3.1. Temperature monitoring

To evaluate the effects of the temperature selected in the heater ring in the temperature of the selected points in the press, the temperature at those different points was monitored though all the oil extraction process. The temperature recorded in the different locations changed depending on the heating ring temperature used. In figure 2, it can be observed how temperature increases progressively until the temperature selected in the ring (50˚C). When low functioning temperatures are selected, the raw material entrance increases the oil temperature beyond the selected temperature in the heating ring due to the friction produced inside the press. However, when high temperatures are used (150˚C) (Figure 3), the continuous entrance of raw material contributes to cool down the whole system, preventing the oil to reach temperatures higher than 70˚C.

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Figure 2. Temperature evolution in the points selected for measurement in the screw press for almond oil extraction. Ring heater temperature selected: 50ºC. Rotational speed selected: 49rpm.

Figure 3. Temperature evolution in the points selected for measurement in the screw press for almond oil extraction. Ring heater temperature selected: 150ºC. Rotational speed selected: 49rpm.

As it can be observed, the temperature selected on the heating ring influences the temperature of oils, but it is not completely decisive. The variation of temperature from 50˚C to 150˚C in the heating ring only causes a slight increase in oil temperature that is not proportional to the increase in the temperature applied on the barrel.

The simultaneous influence of selected temperature applied on the barrel and the rotational speed of the screw on the temperature of extracted oil was also measured

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(Table 1). As expected, higher temperatures applied on the barrel result in oils with higher temperatures, however the influence of rotational speed is determinant. In this regard, the use of lower rotational speeds, increased the temperature of obtained oil, especially when higher temperatures of extraction were used. On the other hand, when using higher rotational speeds, the faster provision of fresh raw material (nuts) into the screw contributed to the system refrigeration, reducing the temperature of extracted oil. A similar evolution of the temperatures can be observed for almond, walnut and pistachio oils.

Table 1. Average temperatures in the oils extracted from almond, walnut and pistachio using different temperatures in the heating ring and different screw rotational speeds.

Heating ring Tª 17 rpm 49 rpm 96 rpm Almond 50 ºC 48.2 52.5 57.28 100 ºC 59.21 62.13 55.98 150 ºC 68.04 64.35 61.52 200 ºC 80.5 78.4 73.1 Walnut 50 ºC 42.25 44.57 48.61 100 ºC 52.99 53.08 60.77 150 ºC 72.8 66.63 62.54 200 ºC 83.9 74.1 70.9 Pistachio 50 ºC 39.59 41.18 36.64 100 ºC 45.38 41.65 42.7 150 ºC 61.79 53.35 46.99 200 ºC 75.6 65.67 53.16 Colour scale: greener colours indicate oils with lower temperatures while reddish indicate higher temperatures. Data show average temperatures of oil measured each second though all the oil extraction process at the described conditions.

When low temperatures in the ring are used for oil extraction, the influence of screw rotational speed in oil temperature is not clear. This is due to the bad functioning of the screw press at low temperatures, causing problems to obtain the oil. For a correct functioning of the press, is advisable to use temperatures of at least 100˚C. For this very same reason, the use of higher rotational speeds would be more appropriate at

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Product and process innovation those high temperatures, as they contribute to the obtainment of oil with lower temperatures.

3.2. Chemical parameters

3.2.1. Oil quality

Free acidity, peroxide value and extinction coefficients (K232 and K270) were evaluated to estimate oils quality. Higher drying temperatures (over 70˚C) have been reported to increase the peroxide values and K270 when using screw and hydraulic presses for pistachio oil extraction (Sena-Moreno et al., 2015). In our study, the temperature of extracted oil using the screw press reached temperatures over 40˚C, even when low temperatures are used for extraction, so differences in oil quality were expected.

In almond oil, higher temperatures result in significant increases in the peroxides content, while in walnut oil it caused increases in the K270 values. No significant impact of temperature in the other parameters of nut oils were observed depending on the temperature selected in the barrel. It should be considered that even when using high temperatures on the barrel, the increases of temperature in oils are substantially lower, this can be the reason of the absence of statistical differences on some quality related parameters.

However, rotational speed has a larger impact on oil quality parameters that the temperature applied on the barrel. Figure 4, shows the data about pistachio oil quality parameters when using different speeds for pistachio extraction. Extinction parameters, acidity, and peroxide values show lower values when oil is extracted at 96 rpm. When extraction is performed at higher speeds, the extraction time is shorter, oils are extracted at lower temperatures (Table 1) and therefore they show higher quality.

Oxidative stability is other parameter that provides information about oil quality, since it refers to the susceptivility of the oil to lipid oxidation. Results show slight increases in the oxidative stability values of oils when they are extracted at higher temperatures, but also when using higher rotational speeds for extraction (Table 2).

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The increse of oxidative stability values in oils with temperature can be attibuted to the formation of Maillard reaction compounds with antioxidant activity, increasing the oxidative stability values of these oils (Veldsink et al., 1999). However, for this very same reason, a reduction in the oxidative stability of oils when using higher speeds in the screw press was expected, as higher speed implies lower temperatures in the oil, due to the cool down effect of faster raw material entrance. However, the extraction at low speeds, also implies a longer extraction process that can cause the degradation of antioxidants components such as tocopherols (Durmaz and Gökmen, 2011). The further degradation of antioxidants can be the cause of the slight decrease of oxidative stability with lower extraction speeds.

Figure 4. Differences in the quality parameters of pistachio oil attending to rotational speed for oil extraction (17 rpm, 49 rpm and 96 rpm). a, K270 values; b, K232 values; c, free acidity values; d, peroxide index.

Within the reported values, pistachio oil extracted at 200˚C is the one with the higher oxidative stability, up to 32.90h. Values reported for pistachio oil are higher than the previously found when using a hydraulic press for pistachio oil extraction (Rabadán et al., 2017a; Rabadán et al., 2017c).

The effect of temperature and rotational speed on the fatty acid profile and sterols of nuts oil is shown in tables 3 and 4, respectively.

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Table 2. Oxidative stability values in the oils extracted using different temperatures in the heat ring and different rotational speeds. Nut oil Temperature (˚C) Rotational speed (rpm) 50 100 150 200 17 49 96 19.45 ± 20.83 ± 20.13 ± 20.33 ± 19.93 ± 19.53 ± 20.56 ± Almond 1.62 0.93 0.35 1.25 1.10 1.54 0.50 6.30 ± 6.10 ± 6.60 ± 6.50 ± 6.34 ± 6.20 ± 6.43 ± Walnut 0.07 0.00 0.17 0.27 0.11 0.61 0.35 30.73 ± 29.53 ± 32.33 ± 32.90 ± 30.82 ± 30.52 ± 31.38 ± Pistachio 1.28 0.72 0.21 1.45 1.71 1.28 2.39 Mean values (±standard deviation) were the averages of three independent measurements. Different letters on the row indicate significant differences among genotypes, Duncan test (p<0.05).

Table 3. Differences in fatty acid profile and sterols in the nuts oils (almond, walnut and pistachio) obtained using different temperatures in the screw press. Fatty acids Sterols Aparent Total Δ7- Tª Palmitic Estearic Oleic Linoleic Linolenic Campest. β- sterols Stigmast. sitosterol Almond 6.55 ± 2.20 ± 69.85 ± 20.45 ± 0.10 ± 2097 ± 2.65 ± 95.50 ± 0.50a ± 50 0.21 0.14 1.34 1.06 0.00 58 0.07 0.00 0.00 6.40 ± 2.10 ± 70.60 ± 19.77 ± 0.10 ± 2200 ± 2.77 ± 95.50 ± 0.40b ± 100 0.00 0.00 0.53 0.15 0.00 31 0.06 0.10 0.00 6.37 ± 2.10 ± 71.10 ± 19.53 ± 0.10 ± 2264 ± 2.67 ± 95.50 ± 0.43b ± 150 0.06 0.00 0.17 0.12 0.00 48 0.06 0.10 0.06 6.37 ± 2.10 ± 71.13 ± 19.47 ± 0.10 ± 2143 ± 2.70 ± 95.43 ± 0.40b ± 200 0.06 0.00 0.15 0.12 0.00 7 0.00 0.06 0.00 Walnut 7.80 ± 2.70 ± 19.05a ± 57.80 ± 12.10 ± 1444 ± 4.40a ± 94.75 ± 0.25a ± 50 0.00 0.00 0.07 0.07 0.00 4 0.00 0.07 0.07 7.83 ± 2.70 ± 18.27b ± 58.27 ± 12.30 ± 1359 ± 4.30ab ± 94.90 ± 0.10d ± 100 0.06 0.00 0.35 0.35 0.10 11 0.00 0.00 0.00 7.77 ± 2.70 ± 18.70ab ± 57.90 ± 12.33 ± 1490 ± 4.27b ± 94.87 ± 0.17b ± 150 0.06 0.00 0.20 0.20 0.15 51 0.06 0.12 0.06 7.83 ± 2.73 ± 18.20b ± 58.23 ± 12.43 ± 1449 ± 4.27b ± 94.87 ± 0.20ab ± 200 0.06 0.06 0.40 0.40 0.06 41 0.06 0.15 0.00 Pistachio 10.60 ± 1.10 ± 54.60 ± 31.50 ± 0.50 ± 3431 ± 4.17 ± 94.03b ± 0.40 ± 50 0.00 0.00 0.20 0.20 0.00 283 0.06 0.15 0.00 10.60 ± 1.00 ± 54.37 ± 31.77 ± 0.50 ± 3361 ± 4.17 ± 94.23a ± 0.37 ± 100 0.00 0.00 0.15 0.12 0.00 112 0.06 0.06 0.06 10.60 ± 1.00 ± 54.50 ± 31.67 ± 0.50 ± 3327 ± 4.17 ± 94.27a ± 0.37 ± 150 0.00 0.00 0.17 0.12 0.00 99 0.06 0.06 0.06 10.57 ± 1.00 ± 54.60 ± 31.63 ± 0.50 ± 3417 ± 4.20 ± 94.37a ± 0.30 ± 200 0.06 0.00 0.20 0.15 0.00 148 0.00 0.06 0.06 Mean values (±standard deviation) were the averages of three independent measurements. Different letters on the column for each nut indicate significant differences among extraction conditions, Duncan test (p<0.05). Fatty acids are expressed as % of total fatty acids. Specific sterols are expressed as % of total sterols. Units: Total sterols (mg/100g fat).

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Table 4. Differences in fatty acid profile and sterols in the nuts oils (almond, walnut and pistachio) obtained using different rotational speeds in the screw press. Fatty acids Sterols Rotational Aparent Total Δ7- speed Palmitic Estearic Oleic Linoleic Linolenic Campest. β- sterols estigmast. (rpm) sitosterol Almond 6.37 ± 2.10 ± 71.10 ± 19.53 ± 0.10 ± 2202 ± 2.73 ± 95.50 ± 0.40 ± 17 0.06 0.00 0.17 0.12 0.00 48 0.06 0.00 0.00 6.53 ± 2.20 ± 69.82 ± 20.37 ± 0.10 ± 2200 ± 2.63 ± 95.47 ± 0.45 ± 49 0.19 0.11 1.17 0.90 0.00 135 0.10 0.12 0.05 6.40 ± 2.10 ± 70.88 ± 19.70 ± 0.10 ± 2163 ± 2.70 ± 95.40 ± 0.46 ± 96 0.00 0.00 0.11 0.10 0.00 47 0.00 0.07 0.05 Walnut 7.81 ± 2.70 ± 18.69 ± 58.14 ± 12.11 ± 1376 ± 4.33 ± 94.82 ± 0.19 ± 17 0.06 0.00 0.42 0.42 0.25 78 0.10 0.12 0.06 7.75 ± 2.75 ± 18.65 ± 57.68 ± 12.58 ± 1465 ± 4.30 ± 94.85 ± 0.18 ± 49 0.10 0.10 0.33 0.33 0.56 76 0.00 0.13 0.05 7.80 ± 2.73 ± 18.33 ± 58.17 ± 12.37 ± 1432 ± 4.30 ± 94.87 ± 0.17 ± 96 0.00 0.06 0.38 0.38 0.15 74 0.00 0.06 0.06 Pistachio 10.58 ± 1.02 ± 54.50 ± 31.66 ± 0.50 ± 3403 ± 4.20a ± 94.22 ± 0.34 ± 17 0.04 0.04 0.24 0.18 0.00 109 0.00 0.15 0.05 10.60 ± 1.02 ± 54.42 ± 31.70 ± 0.50 ± 3314 ± 4.20a ± 94.20 ± 0.38 ± 49 0.00 0.04 0.11 0.12 0.00 134 0.00 0.19 0.04 10.60 ± 1.02 ± 54.54 ± 31.62 ± 0.50 ± 3398 ± 4.14b ± 94.26 ± 0.38 ± 96 0.00 0.04 0.22 0.22 0.00 192 0.05 0.05 0.04 Mean values (±standard deviation) were the averages of three independent measurements. Different letters on the column for each nut indicate significant differences among extraction conditions, Duncan test (p<0.05). Fatty acids are expressed as % of total fatty acids. Specific sterols are expressed as % of total sterols. Units: Total sterols (mg/100g fat).

The fatty acid profile of nuts oils is characterized by the high content of monounsaturated and polyunsaturated fatty acids whose consume is associated to the reduction of the risk of cardiovascular diseases (Crews et al., 2005). In agreement with previous studies, almond and pistachio oil fatty profiles are composed mainly by oleic and linoleic fatty acids (Roncero et al., 2016b; Catalán et al., 2017), while in walnut oil the linoleic content is clearly higher (57.68 – 58.27%) (Amaral et al., 2003; Cuesta et al., 2017). Higher content of polyunsaturated fatty acids indicates oils with stronger health promoting characteristics, but also oils more sensitive to oxidation processes (Rabadán et al., 2017b). Differences in the content of polyunsaturated fatty acids between pistachio and almond oil and walnut oil result in important differences in the oxidative stability values shown in table 2.

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The different extraction conditions with the screw press did not affect the fatty acid profile of nuts oils. Previously it had been proved that different cold extraction systems did not affect the fatty acid profile of pistachio oil (Rabadán et al., 2017a). In other oils obtained from rice germ (Kim et al., 2002), red pepper seeds (Jung et al., 1999) or Pistacia terebinthus seeds (Durmaz and Gökmen, 2011), even under severe roasting processes, the fatty acid composition also remained unalterable.

Some of the health benefits of nut oils are attributed to their sterol content (Catalán et al., 2017; Cuesta et al., 2017; Rabadán et al., 2017b) due to their potential to reduce the risk of coronary heart disease (Awad and Fink, 2000). Within considered nut oils, pistachio oil shows the highest sterol content. The highest sterol content reported in our study was 3431 ± 283 mg/kg for oils from pistachios extracted at the lowest temperature (50˚C). Lower concentrations were reported in almond and walnut oils, with maximum values of 2264 ± 48 mg/kg and 1490 ± 51 mg/kg, respectively. Attending to specific sterols, aparent β-sitosterol was clearly dominant with percentages larger than 94.23% in all oils. Small concentrations of campesterol were found in walnut (4.27 – 4.40%) and pistachio (4.17 - 4.20%) been even lower in almond oil (2.60 -2.67%).

Even if sterols in oils are sensible to high temperatures, oil matrix can have a main influence in protecting them from degradation (Barriuso et al., 2015). Unsaturated oil matrix is able to limit the degradation of sterols with heating (Ansorena et al., 2013), and unsaturated fatty acids compound more than 85% of the fatty acid profile in almonds, walnuts and pistachios. Our results support that conclusion as no effect of the extraction temperature or extraction speed on the total content of sterols in oils was found (table 3 and 4).

Although the total content of sterols was not affected by the extraction conditions, the content of some specific sterols show significant differences depending on the temperature selected for oil extraction. In almond and walnut oils extraction at higher temperatures caused a reduction in the content of Δ7-stigmasterol. This could be the result of possible thermal degradation of specific sterols at high temperatures. The reduction of Δ7-stigmasterol with high temperatures has been previously

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Influence of temperature in oil extracted with the crew press reported in olive (Koutsaftakis et al., 1999) and grape seed oils (Pardo et al., 2009) when the products have been exposed to drying processes. Before oil extraction.

Significant concentrations of polyphenols were reported in nuts oils. However, high variability in the total content of polyhpenols was found, rangin from 18.93 to 26.60mg/kg in almond oil and 20.27 to 29.27mg/kg in pistachio oil. These values agree with previous studies (Rabadán et al., 2017a; Rabadán et al., 2017b). For almond and pistachio oil, significant differences in the content of polyphenols were not found for the different extraction conditions considered. However, the increase of temperature in the heating ring and in the rotational speed caused a significant increase in the total polyphenol content of walnut oils (figure 5). This can be the result of the increase of availability of phenolic precursors (Que et al., 2008) or caused by the release of phenolic compounds from bound structures or chemical alteration of phenolics (Wijesundera et al., 2008), contributing the transfer of the plyphenols in the oil (Vujasinovic et al., 2012). The formation of phenolic compounds at high temperatures had been previously reported in apricots (Madrau et al., 2009), prunes (Del Caro et al., 2004), red pepper (Vega-Gálvez et al., 2009) and also pistachios when drying at temperatures over 50˚C (Sena-Moreno et al., 2015).

Figure 5. Polyphenols in walnut oil attending to the different combination of temperatures and rotational speeds considered for oil extraction

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4. Conclusions

Temperature plays a crucial role when the screw press is used for nut oil extraction. Even when using low extraction temperatures, the temperature of extracted oil can reach 50˚C. This effect is even more intense when low rotational speeds are used. The reported increase in oil temperature can cause a reduction of oil quality, generally reflected in increases of free acidity, peroxide index and values of K232 and

K270. Attending to the chemical analysis of oils, oils subjected to high temperatures show slight differences in the content of specific sterols while no effects on the fatty acid profiles were found. On the other hand, oil extraction performed at higher temperatures can promote the development of Maillard reaction products, causing slight increments in the oxidative stability of oils. In walnut oil, an increase in the total concentration of polyphenols is observed when oils are extracted at higher temperatures.

References

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and hydraulic presses

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Optimization of pistachio oil extraction regarding processing parameters of screw and hydraulic presses

ABSTRACT Keywords: Optimization of pistachio oil extraction with hydraulic and screw presses Functional foods regarding yield, physicochemical and sensory analysis of obtained oils was Fatty acids developed. In addition, pistachio defatted flour was also analysed. In the Antioxidants Thermal hydraulic press extraction, no differences were found regarding yield and processing physicochemical characteristics in oils. However, sensory analysis suggested

extraction using roasted pistachios (100 ºC / 30 minutes). In the screw press differences were found in yield and sensory attributes. Oil extraction with lower rotational speed (17 rpm) resulted in higher yields and the oil was more valued by consumers. Pistachio flour obtained with the two presses presented differences in moisture, fibre, nitrogen, protein and ash content. The lack of influence of extraction parameters in physicochemical characteristics of oil suggested to change the focus to yields and sensory evaluation of oils. Pistachio flour as a by-product had elevated protein content.

1. Introduction

The pistachio (Pistacia vera) is a tree native of the Middle East that has become a popular crop in arid areas of the Mediterranean basin and the United States due to its tolerance to hot and dry conditions. The annual growth rate of the harvested area has increased 2.15 % per year and the production up to 5.70 % yearly in a period of twenty years (1994-2014) (FAOSTAT, 2016). As a result, its global production has increased from less than 40,000 Mt in 1993 to exceed for the first time 100,000 Mt in 2012 (FAOSTAT, 2016).

Pistachio contains about 50% of oil (Tsantili et al., 2010) with some variations depending on the cultivar. It is a healthy oil according to the fatty acids profile with predominance of unsaturated fatty acids, mainly oleic acid, linoleic acid, and to a

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Optimization of screw press and hydraulic press extractions lesser extent linolenic acid (Arena et al., 2007; Tsantili et al., 2010; Sena-Moreno et al., 2014). In addition, pistachios present high contents of phytosterols and phenolic compounds (Tomaino et al., 2010) with many health benefits according to their antioxidant properties (Gentile et al., 2007). Phytosterols have been proposed as blood cholesterol reducing agents and have been associated to a decrease in the risk of several types of cancer (Awad and Fink, 2000; Ostlund, 2004). Due to the higher content of unsaturated fatty acids and the presence of important quality micronutrients, it is important to control the extraction process to obtain high-quality pistachio oil.

Pistachio oil extraction can be done using different methods that result in different yields and pistachio oil qualities. Solvent extraction provides the highest yields but the obtained oils present lower quality due to the appearance of undesirable flavours and odours and the inactivation or disappearance of vitamins and other bioactive substances (Satil et al., 2003; Miraliakbari et al, 2008; Abdolshahi et al., 2015). In recent years, the use of supercritical fluids has emerged as an alternative to solvent extraction (Palazoglu and Balaban, 1998; Jokić et al., 2014). The use of pressurized

CO2 allows obtaining high quality oils due to the extraction at lower temperatures (Abbasi et al., 2008; Chan and Ismail, 2009). However, oil quality and yield need to be balanced as the reduction of temperature, pressure, and CO2 mass flow rate influence the extraction yield of oil (Jokić et al., 2012). High production costs are the main constraint of this method, limiting its use to high valued products.

Pressure systems allow obtaining high quality oils at an affordable price. Presses produce a pleasant product that may be directly consumed and that conserve all the health benefits associated to pistachios consumption (Álvarez-Ortí et al., 2012). Presses enable the obtainment of high quality oils with yields that allow a viable production of pistachio oil.

Pistachio roasting is a primary step in pistachio oil extraction that could increase consumer preference (Kashani and Valadon, 1984) due to the appearance of Maillard reaction products responsible of pleasant odours and colours. Pistachio roasting also contributes to reduce the main health risk of pistachio consumption as it reduces the

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After pistachio oil extraction, a solid by-product remains. By grinding, it becomes pistachio defatted flour, a highly valuable product that can be used in human nutrition, or with other purposes such as animal nutrition or as a nutritional supplement in mushroom cultivation (Pardo-Giménez et al., 2016).

The main objective of this study was to compare and optimize pressure extraction conditions using a screw press and a hydraulic press, in order to obtain high quality oils from the physical-chemical and sensory points of view. In addition, the changes in the defatted flours originated in the process were evaluated.

2. Materials and Methods

2.1. Sampling

Pistachios from Kerman cultivar (Pistacia vera L. var. Kerman) were provided by the Centro de Mejora Agraria El Chaparrillo (Ciudad Real, Spain). Pistachios were shelled in controlled conditions before immediate drying at room temperature for three days. Dried pistachios were vacuum packed and refrigerated until processing.

2.2. Pistachio oil extraction

Oil was extracted with two different presses: hydraulic press (MECAMAQ Model DEVF 80, Vila-Sana, Lleida, Spain) and a screw press (Komet Oil Press CA59G – IBG Monforts Oekotec GmbH & Co. KG, Mönchengladbach, Germany). In the hydraulic press nine different extraction conditions were performed regarding different pressures applied and extraction times. Three different pressures (7.84 MPa, 11.77 MPa, 15.69 MPa) combined with three different extractions times (10min, 12min and 15min) were considered. In the screw press fifteen different extraction conditions were evaluated regarding different temperature and speed extractions. The influence of four

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Optimization of screw press and hydraulic press extractions different temperatures (50ºC, 75ºC, 100ºC, 150ºC) and three rotational speed conditions (17 rpm, 49 rpm and 96 rpm) were tested.

Oil extraction was performed using 200g of pistachios for each processing condition. For the hydraulic press, the pistachios were ground and placed in the press using a filter. For the screw press, the pistachios were introduced directly into the press once the barrel was heated to ensure the correct extraction procedure. For each of the 24 extraction conditions tested, oil extraction was performed in triplicate.

After oil extraction, a centrifugation step was carried out in order to eliminate the remaining solid residues from the samples. Oil and defatted flour were stored under refrigeration conditions.

2.3. Pistachio roasting

An additional pistachio roasting step was considered for the extraction with the hydraulic press. To identify the better conditions of pistachio roasting, three temperatures (50ºC, 100ºC and 150ºC) and three roasting times (30 min, 60 min and 120 min) were considered. Roasting was performed in an oven, where pistachios were placed in a monolayer.

2.4 Physicochemical analysis of pistachio oil

Free acidity, given as % of oleic acid, was determined by titration of a solution of oil dissolved in ethanol/ether (1:1) with 0.1 mol/L potassium hydroxide ethanolic solution (EEC, 1991).

K270 and K232 extinction coefficients were calculated from absorbance of a 10 l/ml solution of oil in cyclohexane at 270 and 232 nm, respectively, with a UV/VIS spectrophotometer Jasco V-530 (Jasco Analitica Spain, Madrid, Spain), and a path length of 1 cm (EEC, 1991).

Oxidative stability was evaluated by the rancimat method (Gutiérrez, 1989). Stability was expressed as the oxidation induction time (hours), measured with the Rancimat

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743 apparatus (Metrohm Co., Basel, Switzerland). An oil sample of 3.5 g was used, warmed to 100°C under an air flow of 10 l h-1.

In order to determine fatty acids composition (%), the methyl-esters were prepared by vigorous shaking of a solution of oil in hexane (0.2 g in 3 ml) with 0.4 ml of 2 mol equi/L methanolic potassium hydroxide solution, and analysed by GC with a Hewlett- Packard (HP 6890) chromatograph equipped with a FID Detector. A fused silica column (50 m length x 0.25 mm i.d.), coated with SGL-1000 phase (0.25 µm thickness; Sugerlabor), was used. Helium was employed as a carrier gas with a flow through the column of 1 ml min-1. The temperatures of the injector and detector were set at 250°C with an oven temperature of 210°C. An injection volume of 1µL was used (Regulation EEC 2568/91, corresponding to AOCS method Ch 2–91).

The concentration of total polyphenols (ppm) was estimated using the Folin– Ciocalteau method, the absorption of the solution was measured on a spectrophotometer Hewlett-Packard 8450 A UV/Vis.

Sterols (%) were determined with a Hewlett-Packard (HP 6890) gas chromatograph with a capillary column (25 m length x 0.25 mm i.d.) coated with SGL-5 (0.25 µm thickness; Sugerlabor). Working conditions were as follows: carrier gas, helium; flow through the column, 1.2 ml min-1; injector temperature, 280°C; detector temperature, 290°C; oven temperature, 260°C; injection volume 1 µl (Regulation EEC 2568/91, corresponding to AOCS method Ch 6–91). Apparent β-sitosterol was calculated as the sum of β-sitosterol, Δ5,23-stigmastadienol, chlerosterol, sitostanol, and Δ5,24-stigmastadienol.

Analytical tests were performed in triplicate.

2.5. Analysis of pistachio defatted flour

Main nutritional components of pistachio flour extracted with the hydraulic and the screw presses were determined. The method used to determine the water content consisted on measuring the loss of weight after oven drying at 105ºC for 72h at least (Lau, 1982). Flour protein content was calculated by multiplying the total nitrogen

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Optimization of screw press and hydraulic press extractions content, obtained by the Kjeldahl method (FOSS, 2003), by a conversion factor of 4.38 (Miles and Chang, 1997). To determine ash content, flours were ashed at 540°C for at least 6 h, to constant weight (Lau, 1982). Crude fat (ether extract) was estimated gravimetrically by filter bag technique after petroleum ether extraction of the dried sample in an extraction system Ankom XT10 (ANKOM, 2009). To determine the content of crude fiber, Weende technique adapted to the filter bag technique was applied. This method determines the organic residue remaining after digestion with solutions of sulfuric acid and sodium hydroxide, using an Ankom 220 Fiber Analyzer (ANKOM, 2008). Total carbohydrate content was calculated by subtracting the sum of the crude protein, total fat, water and ash from the total weight of the flours (Sullivan, 1993). Available carbohydrates content (nitrogen-free) is calculated by subtracting the crude fibre from the total carbohydrate content (Lau, 1982). The energy value of flours was estimated from the relative content of protein, fat and carbohydrates using the modified Atwater factors (Lau, 1982).

2.6. Sensory analysis

Two kinds of sensory analysis were performed on pistachio oils. First, an affective test, where oils were randomly labelled and 108 consumer type panellists were asked to evaluate smell, taste, and oil colour to rank oil samples regarding their degree of acceptability. A nine-point hedonic scale was used (-4 = dislike extremely, 0 = neither like nor dislike, +4 = like extremely). The mean scores for the three sensory properties were calculated separately for each evaluated oil.

The second sensory analysis consisted in simple preference tests. In this case, the consumer type panellists were asked to choose for each characteristic (colour, odour and flavour) between a pair of oil samples in a forced choice test (ISO, 2005). Thus, consumers were asked to choose only the preferred sample. This method was used to compare the preference for two oil pairs: oil extracted with the screw press and oil extracted with the hydraulic press; and the natural and roasted pistachio oils extracted with the hydraulic press.

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2.7. Statistical analysis

Physicochemical parameters, chemical composition, and sensory attributes were processed using variance analysis (ANOVA). Differences between means were compared using a Duncan test with a 95% significant level (p<0.05). Principal component analysis (PCA) was carried out using SPSS 23.0 (SPSS, Chicago, IL).

3. Results and discussion

3.1. Oil yield

Pistachio kernels from the Kerman variety contain about 50 % of fat (Tsantili et al., 2010; Ling et al., 2016). However, when pressure systems are used to obtain the oil, the yields are reported to be around 30 % (Alvarez-Ortí et al., 2012; Ling et al., 2016) with small differences depending on the press type.

Results showed differences in the proportion of oil extracted with both presses. Minor differences appeared in the oil yield obtained using the hydraulic press at different extraction conditions while these differences were higher when using the screw press.

The oil yield ranged between 27.0 – 31.1 g kg -1 when the hydraulic press was used (Figure 1a). Regarding the two considered variables (pressure and time) both caused small increases in pistachio oil yield. However, individual consideration of variables resulted in significant differences in oil extraction depending on the pressures applied with no affection of the extraction time. Thus, the use of high pressures at shortened extraction times could lead to a more efficient extraction.

Yields obtained when the screw press is used are shown in figure 1b. Extraction temperature had little effects on oil yield with larger effect of extraction speed. Lower rotational speed (17 rpm) resulted in higher oil yield, while extractions done at the fastest speed (96 rpm) led to the lower yields. Significant differences were found for the three proposed rotational speeds.

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a

b

Figure 1. a) Oil yield (g kg -1) that results of extraction using different pressures 7.84 MPa, 11.77 MPa and 15.69 MPa) and extraction times (10 min, 12 min and 15 min) with the hydraulic press. b) Oil yield (g kg -1) that result of extraction using different temperatures (50ºC, 75ºC, 100ºC, 150ºC and 200ºC) and rotational speeds (17 rpm, 49 rpm and 96 rpm) with the screw press.

A parallel study to identify the effects of the temperature applied on the screw press barrel in oil was developed, concluding that the temperature applied on the barrel was not directly translated to oil, which never reached temperatures higher than 70˚C. The continuous provision of raw pistachio in the screw press, which increases as rotational speed does, led to more intense cooling effect. Oil temperature and the rotational speed appeared inversely correlated. Rotational speed of the screw press had a more direct effect on pistachio oil temperature than the temperature applied

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These results show that in order to maximize oil yield with the screw press, lower speeds should be considered while extraction temperature has low influence.

3.2. Physicochemical characteristics of extracted oil

No significant differences were found in oils regarding different extraction conditions with the same press. The six extraction conditions analysed with the hydraulic press resulted in similar pistachio oils and the same happened with regard the fifteen extraction conditions analysed for the screw press. However, some parameters showed differences between presses (table 1).

Pistachio oils obtained with both presses showed an elevated proportion of oleic acid which constitute 54.80 - 55.17 g/100g of the fatty acids. Linoleic acid was the second in importance reaching 30.93 - 31.52 g/100g of the total fatty acid profile. Kerman cultivar has been described as one on the varieties that present higher content of linoleic acid (Tsantili et al., 2010). Values for linolenic acid were around 0.47 - 0.50 g/100g. On the other hand, stearic and palmitic acid were the main saturated fatty acids that appear in a proportion of 11.59 – 12.31 g/100g. Both extraction systems led to the same fatty acid profile with no significant differences.

Oxidative stability was higher when the screw press was used due to the possible influence of higher temperatures in the increase of antioxidant compounds (Durmaz & Gökmen, 2011). Seed exposure to heat have proved to be useful in improving the oxidative stability of canola and mustard seed oils (Wijesundera et al., 2008) and pumpkin oil (Vujasinovic et al., 2012). This is attributed to the possible increase in the availability of phenolic precursors (Que et al., 2008) and their easiest pass into the oil phase (Durmaz & Gökmen, 2011) or due to the formation of Maillard reaction products that improve antioxidant properties of food (Borrelli et al., 2002; Papetti et al., 2006). Higher drying temperatures of pistachio kernels have shown increases in the polyphenol concentration of obtained pistachio oil (Sena-Moreno et al., 2015)

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Optimization of screw press and hydraulic press extractions which could also explain the increase of oxidative stability in oil extracted with the screw press.

Table 1. Average composition of pistachio oil obtained with each press at the different extractions conditions. Parameter Screw press Hydraulic press

K232 1.65a ± 0.02 1.53b ± 0.06 K270 0.15a ± 0.01 0.09b ± 0.01 Acidity 0.29a ± 0.04 0.20b ± 0.04 Palmitic acid (C16:0) 10.89a ± 0.13 11.05a ± 0.09 Palmitoleic acid (C16:1) 1.03a ± 0.03 1.06a ± 0.02 Stearic acid (C18:0) 1.00a ± 0.05 1.07a ± 0.03 Oleic acid (18:01) 54.80a ± 0.55 55.17a ± 0.12 Linoleic acid (C18:2) 31.52a ± 0.95 30.93a ± 0.11 Linolenic acid (C18:3) 0.50a ± 0.02 0.47a ± 0.01 Oxidative stability (h) 30.91a ± 1.75 26.40b ± 0.37 Total sterols (mg/kg) 3371b ± 144 3818a ± 147 Total polyphenols (ppm) 24.31a ± 5.20 25.63a ± 3.17 Cholesterol (%) 0.10a ± 0.00 0.10a ± 0.00 Campesterol (%) 4.18a ± 0.04 4.13a ± 0.05 Stigmasterol (%) 0.59a ± 0.05 0.55a ± 0.04  sitosterol (%) 94.23a ± 0.13 91.09a ± 10.01  7 stigmasterol (%) 0.37b ± 0.05 0.50a ± 0.04 Mean values (± standard deviation) were the averages of the different extraction conditions proposed. Means followed by different superscript letter represent significant differences in the measured components (ρ<0.05).

On the other hand, free acidity and extinction coefficients (K270 and K232) were higher in the oils obtained with the screw press. Although the screw press is considered a cold extraction method, heat needs to be applied on the barrel to ensure correct oil extraction. Primary oxidation products tended to increase with the use of high temperatures due to the action of hydrolytic enzymes involved in the hydrolysis of fatty acids (Malheiro et al., 2009). Differences in the temperature applied in the

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barrel did not cause significant differences in free acidity, K270 and K232 as differences of temperature on the barrel did no translate directly to oil.

Attention should be provided to the elevated content of sterols of pistachio oil (Arena et al., 2007). The presence of sterols and Δ-7-Stigmasterol was higher in the oils extracted with the hydraulic press. Although sterols in oils were sensible to heating, oil matrix could have main influence in protecting them of degradation (Barriuso et al., 2015). Degradation of sterols with heat decrease in more unsaturated oil matrix (Ansorena et al., 2013) and unsaturated fatty acids in pistachio oil represented around 85% of the fatty acid profile. Pistachio fatty acid profile may prevent further sterol degradation when using the screw press, although significant reductions in sterol concentration in screw press oil appeared.

Principal Component Analysis (PCA) (figure 2) showed differences in oils obtained with the two presses. Oils obtained with the hydraulic press had higher sterol content and Δ-7-stigmasterol while oils extracted with the screw press were more stable. The effects of heat during extraction, also resulted in higher free acidity, K270 and K232. Results suggest that two different oils can obtained depending on the type of press used.

Figure 2. Principal component analysis of the physicochemical parameters and chemical composition in oils obtained from pistachios subjected to different extraction conditions ( hydraulic press,  screw press).

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3.3. Sensory analysis

The sensory evaluation of oils extracted with the hydraulic press showed no significant differences regarding the variations of the considered extraction parameters. This led to the selection of intermediate values in the extraction parameters (pressure of 11.77 MPa applied for 15 minutes) as representative extraction conditions for posterior comparison with the screw press.

The sensory analysis of pistachio oils obtained using the screw press showed significant differences regarding consumers’ evaluation (figure 3). Pistachio oils extracted with higher temperatures (150 ºC and 200 ºC) and lower rotational speed (17 rpm) were more valued. In the screw press the higher temperatures resulted in oils with characteristics that recalled light roasting, which was positively valuated by consumers.

Figure 3. Consumer’s acceptance (nine-point hedonic scale) of colour (black bars), odour (grey bars) and flavour (white bars) of oils obtained using the screw press at different conditions of temperature (50ºC, 75ºC, 100ºC, 150ºC and 200ºC) and rotational speed (17 rpm, 49 rpm and 96 rpm). Error bars represent standard deviation. x: median value from 108 consumer-judges.

When the oils extracted with both presses were compared, only colour showed significant differences (figure 4a). The screw press produced greener oils due to the increase in the temperature, while the oils obtained in the hydraulic press were yellow. As regards odour and flavour, consumers appreciated similarly both oils. This means that both products could be interesting from the commercial point of view.

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Figure 4. Consumer’s acceptance (nine-point hedonic scale) of colour (black bars), odour (grey bars) and flavour (white bars) of oils obtained using the hydraulic press with pistachios roasted at different conditions of temperature (50˚C, 100˚C, 150˚C) and time (30 min, 60 min and 120 min). Error bars represent standard deviation. x: median value from 108 consumer-judges.

In general, oil obtained with the hydraulic press showed low intensities of odour and flavour. The previous roasting of pistachios was included to provide an improvement in these parameters when the oil is extracted with the hydraulic press, causing a better consumers’ evaluation.

Thus, roasting conditions were also evaluated by analysing the consumer preference for the oil extracted using the previously defined extraction conditions (11.77 MPa/15 minutes). In this case, significant differences were observed. Best evaluations were obtained by the oil extracted from pistachios that had been previously roasted at 100 ºC for 30 minutes (figure 5).

The oils obtained from roasted pistachios were significantly better valued than the oils from unroasted pistachios when the hydraulic press was used (figure 4b). Consumers valued more positively the three sensory parameters of the oil obtained from roasted pistachios. Therefore, the previous roasting step improved the sensory parameters of pistachio oil and led to a better-valued oil, although oil from unroasted pistachios could be an interesting product for consumers who enjoy products with a minimal processing.

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a

b

Figure 5. a) Number of favourable opinions of consumers (108 consumers) for the selected sample of hydraulic press (white bars) and screw press (black bars) for the parameters colour, odour flavour. b) Number of favourable opinions of consumers (108 consumers) for the oils obtained with the hydraulic press (11.77 MPa and 15 min) depending on the characteristics of pistachio: natural (white bars) and roasted at 100˚C 30 min (grey bars), for the parameters colour, odour and flavour.

3.4. Characteristics of flour

The pressing cake resulting from the oil extraction process can be grinded to form a defatted flour, which can be considered as a by-product. As in the case of the oils, the processing conditions may influence the characteristics of the defatted flours. Thus, significant differences were found for some main nutritional components when the two presses are compared (table 2) although differences did not appear when the flours from the same press were compared. Due to the previous oil removal, the flours presented reduced lipid content compared to full fat pistachio flours. However, an important part of crude fat remained (between 20 and 25g/100g), and although no significant differences were found, it was slightly higher in the flour that results from hydraulic press extraction due to the lower oil yields obtained in the extraction process. The fat content in pistachio defatted flour contributes to increase its

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Table 2. Average defatted flour components depending on the press used (g/100g). Average parameters Screw press flour Hydraulic press flour

Moisture content 11.35a ± 0.74 9.52b ± 0.61

Nitrogen 6.42a ± 0.44 5.11b ± 0.12

Protein 40.15a ± 2.77 35.96b ± 0.77

Ashes 4.02a ± 0.23 4.80b ± 0.09

Crude fibre 3.24a ± 0.20 3.54b ± 0.13

Crude fat 22.88a ± 3.87 24.48a ± 2.01

Carbohydrate content 32.96a ± 1.16 33.76a ± 1.69

Available carbohydrates content 29.72a ± 1.11 30.22a ± 1.66

Energy value (kcal/100g dm) 464.93a ± 19.15 470.57a ± 9.16

Mean values (± standard deviation) were the averages of the different extraction conditions proposed. Means followed by different superscript letter represent significant differences in the measured components (ρ<0.05).

Moisture content was higher in the screw press flour, but with values lower to wheat flour moisture limits defined by Codex Alimentarious (CODEX STAN 152-1985). The moisture content of pistachios used for oil extraction is usually lower than 5g/100g, but this concentration increases in the defatted flour when the oil is subtracted. In a similar way, the concentration of the rest of the components of the flour increases when the oil extraction yield is high.

Both protein and nitrogen content showed significant differences with lower values for the hydraulic press flour. In any case, the values of protein content of pistachio defatted flour obtained with both presses were higher than those obtained for other nut defatted flours (Pineli et al., 2015). High protein content in pistachio flour could be interesting due to the positive influence of the protein content to the structure of dough when baking, enabling a higher proportional replacement of wheat flour (Pineli et al., 2015). In addition, the high protein content of pistachio defatted flours

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Optimization of screw press and hydraulic press extractions allows to use it as nutritional substrate supplement to cultivate mushroom species (Pardo-Giménez et al., 2016).

Ashes and fibre contents were significantly higher in the flour obtained after hydraulic press. Pistachios are a rich source of many minerals, and ashes content in non-defatted pistachio flours is reported to be generally higher than in other non- defatted flours like almond (Joshi et al., 2015). On the other hand, the fibre content in pistachio flour was lower than the values obtained in traditional flours such as wheat or corn flours (USDA, 2016). This lower fibre content may be related to lower capacity for hold oil of doughs (Sharma and Gujral, 2014).

No significant differences were found regarding carbohydrates concentration and the energy value. The energy value obtained in pistachio defatted flours is higher than the medium values of wheat flour, mainly for the higher content of lipids that remain even after oil extraction.

Pistachio defatted flours, independently of the extraction system, are an interesting food from the nutritional point of view regarding protein content, fat content and energy value.

4. Conclusions

Optimization of pistachio oil extraction using two different presses has been performed regarding different functioning parameters: pressure and extraction time in the hydraulic press and temperature and rotational speed in the screw press. Higher extraction yields are obtained with the screw press, especially when using low rotational speed.

When comparing the two presses, clear differences are observed in the obtained oils.

Oils from the screw press showed higher values of acidity, K232, K270 and stability due to the effect of the temperature originated during the extraction process in this press. On the other hand, oils extracted with the hydraulic press seemed to have a higher content in sterols.

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When the oils from the same press extracted under varied conditions were evaluated, the differences were not so clear. The different extraction conditions did not affect physicochemical or sensory characteristics of oils extracted with the hydraulic press, although oil yield increased with pressure and extraction time. Higher variability appeared on the screw press due to the influence of temperature. Lower rotational speed originates higher temperatures due to friction, leading to higher oil extraction yield. When the rotational speed is high, the continuous input of fresh raw material contributes to the cooling of the system, decreasing the effects of temperature. Although screw press is considered as a cold extraction procedure, different processing conditions may cause changes in the extraction temperature, resulting in differentiated products.

Regarding consumers’ preference, the temperature also plays an important role, as it increases the intensity of sensory properties of the oils. Thus, the best valued oils were those extracted with higher temperatures and low rotational speed in the screw press and the oils from roasted pistachios extracted with the hydraulic press.

Pistachio defatted flour obtained as a by-product of oil extraction with the two presses showed significant differences in moisture, fibre, nitrogen, protein and ash content, related with the higher oil extraction yield of the screw press. Anyway, the high protein content and the oil fraction that remains after oil extraction make pistachio defatted flours an interesting ingredient to be included in the diet.

References

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Storage stability and composition changes of nuts oils under refrigeration and room temperature

conditions

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Evaluation of storage conditions for nuts oils

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Storage stability and composition changes of nuts oils under refrigeration and room temperature conditions

ABSTRACT Keywords: Chemical composition and stability parameters of three nut oils were Almond Walnut monitored during storage of the oils at refrigerated and room temperature Pistachio conditions. Almond, walnut and pistachio kernels were cold-pressed using Cold-pressed oil Storage stability a hydraulic press to extract the nut oils. Oil storage was carried out for up

to 16 months at three refrigerated temperatures (5ºC, 10ºC and 20ºC) in the dark, at room temperature in the dark and at room temperature with exposure to daylight. At the beginning of the storage period, pistachio oil had lower peroxide value than almond oil and higher induction period than almond and walnut oils, indicating a higher stability. The increase of peroxide values during the storage period was faster for room temperature than for refrigerated conditions, especially in the case of pistachio oil stored exposed to daylight. The induction period decreased during the storage period for all three nut oils, regardless of the storage conditions. Pistachio oil remained the most stable oil at the end of the storage period, followed by almond oil. The percentage of polyunsaturated fatty acids decreased slightly throughout the storage.

1. Introduction

Almonds (Prunus dulcis), walnuts (Juglans regia) and pistachios (Pistacia vera) are some of the most important nuts in terms of commercial production and human consumption and are considered as healthy products within the Mediterranean diet. Fat content in these nuts amounts to 30-63% for almonds (Roncero et al., 2016b), 52- 70% for walnuts (Gharibzahedi et al., 2014); and 48-54% for pistachios (Tsantili et al., 2010). These high lipid concentrations, together with their fatty acid profiles, presence of bioactive compounds and favourable sensory properties make these nuts a likely source of valuable oils.

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It is of prime importance to preserve the positive properties of these oils during storage to achieve wide commercialization. Cold-pressed almond oil (AO), walnut oil (WO) and pistachio oil (PO) have been studied widely, since high quality oils can be obtained from these nuts at affordable prices. The oil extraction method influences oil quality and its later susceptibility to oxidation. Niewiadomski (1990) claimed that high quality raw materials, an optimized pressing process, immediate cleaning of the crude oil and appropriate storage of the oil are all necessary conditions to obtain high-quality cold-pressed oils. Oxidation appears as a major constraint for the production and marketing of nut oils, so optimal storage conditions are a key factor to ensure the sensory and nutritional quality of the final product.

The oxidative stability of edible oil is affected by processing and storage conditions, namely temperature, light and oxygen availability, by its fatty acid composition and by the presence of minor components in the oil, such as free fatty acids, mono- and diacylglycerols, phospholipids, chlorophylls, tocopherols, phytosterols, phenolic compounds, carotenoids, and transition metals (Choe and Min, 2006). Some of these compounds can have both antioxidant and prooxidant activity, as two kinds of oil oxidation can take place: autoxidation and photosensitized oxidation (photooxidation). Autoxidation requires the presence of lipid radicals, while photooxidation requires the presence of a photosensitizer, like chlorophyll, to initiate.

Oil oxidation needs energy to occur and that energy is mainly obtained from light or heat. The energy needed to cause oxidation depends on the hydrogen position in the molecules. Photooxidation is not highly influenced by temperature as it requires low activation energy, but temperature is a major concern to control autoxidation in oils (Choe and Min, 2006). An increase in the oil temperature leads to an increase in the autoxidation and the decomposition of hydroperoxides. This is the main cause of the appearance of undesirable flavours in the oxidized oil (Ling et al., 2016). Mexis et al.

(2009) found that the effect of temperature was more important than O2 barrier and lighting conditions in the lipid oxidation of shelled walnuts stored at 4ºC and 20ºC.

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However, light is a major factor to control photooxidation, although its importance reduces as temperature increases. Early studies showed that the rate of oxidation increases with decreasing wavelength (Sattar et al., 1976) and that higher light intensity also lead to faster deterioration of oil (Chahine and deMan, 1971). A reduction in minor components, such as carotenes, chlorophylls and total phenols, was observed in oils contained in clear glass bottles (Gargouri et al., 2015). Light exposure was identified as the main factor that affects shelf life of pumpkin cold- pressed seed oils (Naziri et al., 2016).

Oxygen concentration is another factor that affects the oxidation of oils. A higher concentration of oxygen dissolved in oil is related to faster oil deterioration. Small oil samples are more prone to react with oxygen, since they have a high ratio of surface to volume, which promotes oxidation (Kanavouras et al., 2005). The effect of oxygen concentration on the oxidation of oil was more significant with increasing temperatures and with exposure to light (Choe and Min, 2006).

The extraction method also influences oil oxidative stability. The oxidative stability of

WO obtained by supercritical CO2 extraction is significantly lower than for WO obtained by press systems (Crowe and White, 2003). The higher stability of cold pressed oils is attributed to their richness in bioactive compounds such as phenolics and tocopherols (Ramadan, 2013). The exposition of seeds to high temperatures during drying, roasting or extraction can increase oil oxidative stability, as some Maillard reaction products are reported to be antioxidants (Choe and Min, 2006).

Triglycerides are the main compounds of oils and their oxidation is mainly determined by their fatty acid profile. The iodine value, as a measure of oil unsaturation, can be a useful indicator of oil’s tendency to oxidation. Studies show that the more unsaturated an oil is, the higher the oxidation rate (Parker et al., 2003). Autoxidation is highly influenced by the fatty acid profile, while photooxidation is less affected (Choe and Min, 2006).

Free fatty acids, mono-and diacyl-glycerols, phospholipids and thermally oxidized compounds act as prooxidants, as they decrease the surface tension of the oil, which increases the diffusion rate of oxygen. However, phospholipids can also act as

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Evaluation of storage conditions for nuts oils antioxidants by sequestering metals. Chlorophylls and phenolic compounds have proved to reduce oil autoxidation in dark conditions. Conversely, in the presence of light, chlorophylls and other pigments can perform as photosensitizers for the formation of singlet oxygen that lead to oil oxidation (Ayu et al., 2016). Under light exposure, tocopherols, carotenoids and squalene prevent photosensitized oxidation (Ayu et al., 2016; Naziri et al., 2016), but tocopherols can also act as prooxidants when the oil is stored at high temperatures (Liu et al., 2016). Arranz et al. (2008) showed that tocopherols are responsible for most of the radical scavenging capacity in nut oils. However, according to Alamed et al. (2009), free radical scavenging assays have a limited value in predicting the antioxidant activity in food. The effect of carotenoids and phytosterols is also variable and depends on the oil storage conditions. Phytosterol concentration was found to be negatively correlated with the induction period in grape seed oil (Yang et al., 2013).

Polyphenols have been shown to increase the oxidative stability of cold pressed oils (Ramadan, 2013; Yang et al., 2013), and the addition of tea polyphenols retarded lipid oxidation in walnut beverage emulsions exposed to heat and UV light during the storage (Liu et al., 2016). The protection of polyphenols against oxidation is related to their capacity to donate one hydrogen atom to a free radical and reduce propagation of the radical chain reaction (Gallego et al., 2013).

Most studies on oil stability have been performed in conditions of accelerated oxidation, at temperatures far above the usual storage temperatures or at high light intensities. Nevertheless, the oxidation kinetics of oil components has been shown to be very dependent on temperature and light conditions. In this study, three cold- pressed nut oils (almond, walnut and pistachio) were stored at refrigerated or room temperature for up to 16 months to investigate the influence of storage conditions on oil stability and composition.

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2. Materials and Methods

2.1. Sampling

Almonds (Prunus dulcis L.) from cultivar Marcona, walnuts (Juglans regia L.) from cultivar Pedro and pistachios (Pistacia vera L.) from cultivar Kerman were provided by Instituto Técnico Agronómico de Albacete, Spain. All the nuts were dried at room temperature for three days and subsequently cracked. The kernels were separated from the shells, vacuum packed and stored under refrigerated conditions.

2.2. Oil extraction

Pistachio kernels were roasted at 100ºC for 30 min before extraction and almonds were roasted at 150ºC for 30 min. Pistachio roasting has been shown to improve the sensory properties and antioxidant capacity of PO (Rabadán et al., 2017b). Almond roasting was also shown to cause a decrease in the acidity index and improve the oxidative stability of AO (Roncero et al., 2016a). Nut oils were extracted with a hydraulic press (MECAMAQ Model DEVF 80, Vila-Sana, Lleida, Spain). Applied pressure and extraction time were fixed at 20 MPa and 10 min, respectively, based on previous work (Rabadán et al., 2017a). The nuts were ground with a blender (GM200-RETSCH) and placed in the press using a filter cloth. Oil extraction was performed using 200 g of nuts for each processing extraction. After oil extraction, a centrifugation step was carried out to eliminate any remaining solid residues from the samples using a centrifuge (Centronic-BL, Geberlab).

2.3. Storage conditions

Oil samples were kept for up to 16 months under different temperature conditions (5ºC, 10ºC, 20ºC and Room Temperature (RT) in dark glass bottles stored in the darkness. RT oscillated between 19ºC and 25ºC, with an average of 22ºC. Another set of samples were stored at RT in transparent bottles exposed to daylight.

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2.4. Physicochemical analysis

Free acidity, given as % of oleic acid, was determined by the titration of a solution of oil dissolved in ethanol:ether (1:1) with 0.1 mol·L-1 potassium hydroxide ethanolic solution (Anonymous, 1991). Peroxide value (POV), expressed as meq of active oxygen per kg of oil, was determined as follows: a mixture of oil and chloroform- acetic acid was left to react with a solution of potassium iodine in darkness; the free iodine was then titrated with a sodium thiosulfate solution (Anonymous, 1991). K270

-1 and K232 extinction coefficients were calculated from the absorbance of a 10 µL·mL solution of oil in cyclohexane at 270 and 232 nm respectively, with a UV/VIS spectrophotometer Jasco V-530 (Jasco Analitica Spain, Madrid, Spain) and a path length of 1 cm (Anonymous, 1991).

Oxidative stability was evaluated by the Rancimat method (Gutiérrez, 1989). Stability was expressed as the oxidation induction period (IP) measured with the Rancimat 743 apparatus (Metrohm Co., Basel, Switzerland). An oil sample of 3.5 g was used, warmed to 100ºC under an air flow of 10 L·h-1.

The fatty acid composition (%) was determined as explained elsewhere (Rabadán et al., 2017a). Briefly, the fatty acid methyl esters were prepared by vigorously shaking an oil solution in hexane (0.2 g in 3 mL) with 0.4 mL of 2 mol equi/L methanolic potassium hydroxide solution, and analyzed by GC with a Hewlett-Packard (HP 6890) chromatograph equipped with an FID detector.

The concentration of total polyphenols (ppm) was estimated using the Folin- Ciocalteau method (Gutfinger, 1981) and expressed as ppm of cafeic acid.

Sterols (%) were determined with a Hewlett-Packard (HP 6890) gas chromatograph with a capillary column (25 m length x 0.25 mm i.d.) coated with SGL-5 (0.25 µm thickness; Sugerlabor). Working conditions are detailed in Rabadán et al. (2017a). Apparent -sitosterol was calculated as the sum of -sitosterol, 5,23-stigmasterol, chlerosterol, sitostanol and 5,24 -stigmastadienol.

All analyses were performed in triplicate.

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2.5. Statistical analysis

Analysis of variance (ANOVA) was performed to search for significant differences among means with a 95% significant level (p<0.05) using SPSS 23.0 (SPSS, Chicago, IL).

3. Results and discussion

3.1. Properties of extracted oils

Mean oil yields were 31.2% for almonds, 56.0% for walnuts and 28.6% for pistachios. They were within the usual range for these nuts when they are extracted with a hydraulic press (Sena-Moreno et al., 2016).

Table 1 shows the physicochemical parameters, stability and chemical composition of freshly pressed nut oils. Crude oil contains free fatty acids that are more susceptible to autoxidation than esterified fatty acids (Choe and Min, 2006). PO showed higher acidity values than AO and WO, but all the acidity values were remarkably low at the start of the storage period, below 0.5% of oleic acid. AO had a significantly higher POV than WO and PO at the beginning of the storage time, but all the values were well below the maximum value of 15 meq/kg established by the

Codex Alimentarius for non-refined oils. The specific extinction coefficient values K232 and K270 are used as an indication of the formation of conjugated dienes and trienes, respectively, which are found in primary oxidation products. The initial values of K232 and K270 were also low for all the samples. Total polyphenol values were highest for AO and lowest for WO. They were much lower than those reported by Arranz et al. (2008). This discrepancy could be attributed to the fact that they worked with solvent-extracted nut oils.

The IP values determined by the Rancimat test at 100ºC at the beginning of the storage period were in the following order: PO>AO>WO (Table 1). This is in accordance with existing literature, where PO is reported to be one of the most stable nut oils, whereas WO is one of the least (Arranz et al., 2008; Miraliakbari and Shahidi, 2008). The IP values concur with those reported by Arranz et al. (2008) for solvent

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Evaluation of storage conditions for nuts oils extracted AO and WO, and were below the values reported for PO by these authors. Rabadán et al. (2017a) and Sena-Moreno et al. (2015) reported lower IP values than ours for cold-pressed AO obtained from roasted almonds and for cold-pressed PO from roasted pistachios, respectively. Savage et al. (1999) extracted WO with solvents and determined the IP using the Rancimat method at 110ºC. They reported IP values in agreement with our results, ranging from 3.9 to 7.8 h.

Table 1. Physicochemical and stability parameters and fatty acid and sterol composition of cold-pressed almond, walnut and pistachio oils. Parameter Almond Walnut Pistachio Acidity (% oleic acid) 0.13  0.05a 0.12  0.01a 0.39  0.03b

b a a Peroxide value (meq O2/kg) 2.6  0.7 1.3  0.5 0.9  0.4

b a b K232 1.82  0.09 1.59  0.07 1.76  0.04

a a b K270 0.07  0.01 0.09  0.02 0.15  0.03 Total Polyphenols (ppm) 44.5  1.1c 16.9  0.7a 34.0  0.9b Induction period (h) 21.0  0.3b 4.7  0.1a 31.3  0.5c Miristic C14:0 (%) ND ND 0.08  0.00 Palmitic C16:0 (%) 6.61  0.01b 5.97  0.01a 10.76  0.02c Palmitoleic C16:1 (%) 0.49  0.00b 0.11  0.00a 1.01  0.01c Margaric C17:0 (%) 0.06  0.00b 0.05  0.00a ND Margaroleic C17:1 (%) 0.09  0.01a ND 0.09  0.00a Stearic C18:0 (%) 2.15  0.00b 2.39  0.01c 1.05  0.00a Oleic C18:1 (%) 68.38  0.06c 14.47  0.02a 54.59  0.02b Linoleic C18:2 (%) 22.00  0.08a 61.51  0.01c 31.33  0.03b Linolenic C18:3 (%) ND 15.14  0.02b 0.50  0.00a Arachidic C20:0 (%) 0.09  0.00b 0.08  0.00a 0.12  0.00c Gadoleic C20:1 (%) 0.05  0.00a 0.21  0.02b 0.33  0.01c Behenic C22:0 (%) ND ND 0.08  0.00 SFA (%) (%) 8.91  0.01b 8.49  0.01a 12.09  0.02c MUFA (%) 69.01  0.07c 14.79  0.01a 56.02  0.02b PUFA (%) 22.00  0.08a 76.65  0.03c 31.83  0.03b Total sterols (mg/kg) 2294  19b 1009  08a 3555  16c Campesterol (%) 2.5  0.1a 5.4  0.1c 4.2  0.1b Stigmasterol (%) 0.4  0.0b 0.3  0.0a 0.6  0.0c Apparent -Sitosterol (%) 95.7  0.2b 93.6  0.2a 94.1  0.2a -7-Stigmastenol (%) 0.4  0.0b 0.1  0.1a 0.4  0.0b Different letters on the row indicate significant differences among oils, Duncan test (p<0.05)

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Saturated fatty acids (SFA) were highest in PO and lowest in WO. Palmitic acid was the most abundant SFA in the three nut oils, followed by stearic. Stearic acid proportion for AO was above that of the range reported by Roncero et al. (2016b), but was in accordance with the findings of Arranz et al. (2008). Monounsaturated fatty acids (MUFA) were the main type of fatty acids for the three nut oils. Oleic acid was predominant in AO and PO, while linoleic was the main fatty acid in WO. Polyunsaturated fatty acids (PUFA) accounted for more than 76% of fatty acids in WO and only 22% in AO. Oleic and linoleic acid values for PO concurred with those reported by Catalán et al. (2017). Most of our fatty acid composition data is in accordance with the values reported by Arranz et al. (2008), except that they give a negligible linolenic acid content for WO. The variety of nut tested was not specified in that study, which makes comparison difficult.

PO showed the highest values of total sterols and WO the lowest. The three nut oils showed high content of -sitosterol and low stigmasterol and -7-stigmastenol content. The highest campesterol content was found for WO and the lowest for AO.

3.2. Evolution of oil properties during storage

Acidity did not increase significantly during storage, regardless of storage conditions (Table 2), except for a slight increase in AO stored at RT (p=0.022). Other authors (Maskan and Karataş, 1998) have found a significant increase in the percentage of free fatty acids of PO after three months of storage of pistachio nuts at refrigerated and ambient temperatures.

The POV of AO and WO increased during the initial stages of storage and showed a maximum value at month 12. The presence of this maximum was less clear for PO (Figure 1). The decrease in POV at the end of storage could be due to the destruction of peroxides as secondary oxidation takes place. AO stored at RT showed the highest

POV during storage, and exceeded 15 meq O2/kg after three months. Only some samples of WO and PO stored at 20ºC or at room temperature reached this POV threshold during storage. PO stored at RT in daylight showed a high increase in POV throughout storage and reached 44 meq O2/kg of oil after 16 months (Table 2).

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Maskan and Karataş (1999) found that POV of PO from pistachio kernels stored at ambient conditions increased suddenly after 6-9 months of storage, but it took up to 15 months to reach 15 meq/kg oil. Ling et al. (2016) found that this threshold was reached in just 30 and 75 days for PO stored at RT in daylight and dark conditions, respectively. Bellomo et al. (2009) found no significant differences for pistachio kernels stored at 10, 25 and 37ºC for up to 14 months in their POV, K232 and K268 indexes. WO from walnut kernels stored at 20ºC in daylight and dark conditions reached 15 meq/kg oil in 6 and 10 months, respectively (Mexis et al., 2009). ANOVA of POV data at the end of the storage period showed that the influence of storage conditions was significant for AO (p=0.03) and PO (p=0.002), but not for WO.

Table 2. Acidity, specific UV extinctions (K232 and K270) and total polyphenols (TP) of cold-pressed almond, walnut and pistachio oils after 16 months of storage.

Nut oil Tª Exposure to light Acidity (%) K232 K270 TP (ppm)

Almond 5ºC dark 0.13  0.05a 2.68  0.15ab 0.09  0.01a 18.6  1.1b

10ºC dark 0.12  0.04a 3.18  0.05bc 0.10  0.02 a 14.5  0.8ab

20ºC dark 0.11  0.06a 4.34  0.07bc 0.12  0.02 a 15.5  1.0ab

RT dark 0.20  0.12b 3.25  0.09bc 0.12  0.01 a 15.5  0.5ab

RT light 0.23  0.07b 3.25  0.11bc 0.12  0.02 a 13.5  1.0ab

Walnut 5ºC dark 0.10  0.03a 2.78  0.08ab 0.13  0.02ab 13.5  0.5ab

10ºC dark 0.13  0.06a 2.35  0.08ab 0.13  0.02ab 6.3  0.9 a

20ºC dark 0.12  0.05a 2.85  0.07ab 0.16  0.03ab 15.5  1.3ab

RT dark 0.11  0.08a 2.94  0.10ab 0.19  0.03ab 13.5  1.2ab

RT light 0.13  0.07a 2.66  0.09ab 0.18  0.02ab 10.4  1.0ab

Pistachio 5ºC dark 0.39  0.04c 1.86  0.08a 0.16  0.03ab 19.6  1.5b

10ºC dark 0.40  0.04c 1.97  0.07a 0.16  0.03ab 17.6  1.3b

20ºC dark 0.39  0.05c 1.98  0.10a 0.16  0.02ab 18.6  1.8b

RT dark 0.42  0.07c 2.24  0.11ab 0.17  0.03ab 23.1  1.5bc

RT light 0.42  0.08c 3.40  0.09bc 0.36  0.05c 16.5  0.9b Different letters on the column indicate significant differences among oils, Duncan test (p<0.05)

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Figure 1. Evolution of peroxide value (POV) at each storage condition for AO (a), WO (b) and PO (c).

The trend of the specific extinction coefficient K232 followed a similar pattern than that of POV, with increasing values during storage, but the values at month 12 were not significantly different from the values at month 16. K270 also increased during storage for all three oils. Storage conditions did not have any influence on the K232 or

K270 extinction coefficients at the end of storage (p>0.05), except for PO stored at RT exposed to daylight (Table 2).

The total polyphenols content decreased steadily during the storage period for all three nut oils, but storage conditions did not have an influence on the final amount (Table 2). AO showed a higher decrease in polyphenol levels than WO or PO, and the amounts after 16 months of storage were not significantly different for any of the three nut oils.

The IP decreased slightly for AO and PO during storage, and remained unchanged for WO (Figure 2). Storage conditions did not significantly influence the value of IP at the end of storage (p<0.05). After 3 months of storage at room temperature, the values

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Evaluation of storage conditions for nuts oils for AO were in close agreement with the data published by Rabadán et al. (2017a). Throughout storage, the IP of PO was the highest, while the IP of WO was the lowest.

Figure 2. Evolution of the Induction Period (IP) at each storage condition: 5ºC (*), 10ºC (), 20ºC (), RT in the dark () and RT in the light () for PO, AO and WO. Lines show the evolution of average IP values for each nut oil and error bars indicate SD limits for those averages.

The fatty acid composition changed slightly, but significantly, during the storage of all three nut oils. These changes were unaffected by the storage conditions. The fatty acid composition of the main fatty acids at the end of the storage period is shown in Table 3. The proportion of PUFA decreased during storage, probably due to a higher rate of oxidation of these fatty acids. A reduction in the proportion of polyunsaturated fatty acids during storage of pistachio nuts was observed by Maskan and Karataş (1998). Linoleic acid proportion decreased by 1% for AO and by 0.5% for PO, while the reduction was not significant for WO. Linolenic acid proportion decreased by 0.4% for WO. The total PUFA decreased by 1% for AO, 0.2% for WO and 0.5% for PO. Note that although WO has a high proportion of PUFA, it is highly stable during the storage compared with AO and PO. The relative content of SFA and MUFA

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Product and process innovation increased during the storage due to the reduction of PUFA. The SFA increased by about 0.8-0.9% for all three nut oils. The proportion of MUFA (mainly oleic acid) experienced a smaller increase (0.2-0.3%), which was not significant for PO.

Table 3. Main fatty acid and sterol composition of cold-pressed almond, walnut and pistachio oils after 16 months of storage (mean of samples at different storage conditions  SD) Parameter Almond Walnut Pistachio Palmitic C16:0 (%) 6.66  0.01 6.03  0.00 10.86  0.03 Palmitoleic C16:1 (%) 0.50  0.00 0.12  0.00 1.02  0.00 Stearic C18:0 (%) 2.17  0.00 2.41  0.00 1.06  0.00 Oleic C18:1 (%) 68.52  0.05 14.52  0.01 54.61  0.02 Linoleic C18:2 (%) 21.78  0.06 61.42  0.00 31.19  0.07 Linolenic C18:3 (%) ND 15.08  0.01 0.48  0.00 SFA (%) 8.98  0.01 8.57  0.00 12.20  0.04 MUFA (%) 69.16  0.05 14.85  0.01 56.05  0.02 PUFA (%) 21.78  0.06 76.50  0.01 31.67  0.07 Total sterols (mg/kg) 2363  19 1030  12 3505  45 Apparent -Sitosterol (%) 95.8  0.1 93.5  0.0 94.2  0.1

Total sterols increased slightly during storage for AO and WO, while the change was not significant for PO. The change in apparent -sitosterol was not significant for any of the three nut oils.

4. Conclusion

Pistachio oil was more stable than almond and walnut oils according to its lower induction period, and it remained the most stable after the storage time, followed by almond oil. The changes on composition and stability parameters of all three nut oils were not affected by the storage temperature in the range 5-20ºC for up to 16 months. The induction period decreased during the storage time for all three nut oils, regardless of the storage conditions. The peroxide values increased faster at room temperature than at lower temperatures during the storage time, and the highest increase was for PO stored at room temperature exposed to daylight. The percentage of polyunsaturated fatty acids decreased slightly throughout the storage. These

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Evaluation of storage conditions for nuts oils results are valuable in determining optimal storage conditions for nut oils and lower storage costs while maintaining maximum quality.

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3.1. Product innovation

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Changes in physical parameters, chlorophyll and carotenoids in pistachio oil with

previous roasting

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Effect of previous pistachio roasting on pistachio oil

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Changes in physical parameters, chlorophyll and carotenoids in pistachio oil with previous roasting

ABSTRACT Keywords: Pistachio oil is considered a health-promoting product due to their fatty acid Pistacia vera Temperature profile and the presence of a battery of antioxidant compounds. Pistachio Lutein roasting before extraction increases consumer preference, but at the same Chlorophyll degradation time causes changes in pistachio oil physical parameters, oil oxidative stability products and oil pigment concentration. Regarding physical parameters, density values

of pistachio oil reduce with severe roasting conditions, while viscosity increases. Severe roasting conditions increases the oxidative stability of oils due to the antioxidant activity of Maillard reaction products. In accordance with the apparent colour of obtained oils, they can be classified into two groups. The first group of oils shows yellow colours and are obtained from natural pistachio or low temperature roasted pistachios (50 or 75 ºC), while a second group of oils with green colour, are obtained after more severe roasting (100 or 125 ºC). Regarding pigments, carotenoids are transferred from pistachio to oil in greater proportion than chlorophylls in all samples. Therefore, the ratio between the chlorophyll and the carotenoid fraction is reduced considerably in oils. While in the natural pistachio the ratio between the chlorophyll and the carotenoid was 2.76, in oil this ratio fell to 0.18. Higher roasting temperatures favoured the solubilisation of pigments in the oil, mainly in the fraction of chlorophyll derivatives. Thus, the chlorophyll/carotenoid ratio in these oils remained balanced around the unit. Higher roasting temperatures promote the degradation of chlorophylls to form pheophytins and pyropheophytins. Within carotenoids, higher roasting temperatures increase significantly the concentration of β-carotene in oils.

1. Introduction

Pistachio nut (Pistacia vera L.) is one of the most popular nuts in the world. It is widely cultivated across arid zones in the world due to the ability of pistachio tree to grow

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Effect of previous pistachio roasting on pistachio oil in hot and dry areas. The production of pistachio worldwide has increased from 440.000 tons in 2004 to exceed 850.000 in 2014. Iran, the United States and Turkey are the major pistachio producers (FAO, 2016). The escalation of pistachio production has been the cause of an important increase of pistachio demand due to the health- promoting compounds of nuts in general and pistachio particularly.

Pistachios have a high nutritional value, becoming a good source of vegetable proteins (Harmankaya et al., 2014), fiber with positive effects on gut microbiota associated to its consumption (Ukhanova et al., 2014) and phytosterols linked to the reduction of total plasma cholesterol and low-density lipoprotein cholesterol (Rabadán et al., 2017b). In addition, pistachios contain between 50 and 62 % of oil composed mainly by unsaturated fatty acids, mainly oleic (52 – 81%) and linoleic (8 - 31%) (Catalán et al., 2017). Due to its high lipid content, the extraction of pistachio oil appears a viable procedure (Rabadán et al., 2017b). Within oil extraction methods, cold pressed extraction has been widely studied as it allows the production of high quality oils (Álvarez-Ortí et al., 2012).

Pistachios are usually sold and consumed roasted, so roasting process is responsible for the characteristic aroma and taste of pistachios (Nicoli et al., 1997). Pistachio aroma after roasting has been identified as determinant for consumer acceptance (Aceña et al., 2010). However, the appearance and evolution of main components of sensory odour and flavour aromas with roasting also differ depending on the pistachio cultivar considered (Vázquez-Araújo et al., 2009). The preference of consumers for roasting has been also described in pistachio oil when roasted pistachios are used for oil extraction (Rabadán et al., 2017a). Moreover, roasting process decrease the risk of aflatoxin contamination when consuming pistachio (Yazdanpanah et al., 2005). This is an important factor to consider as pistachio has been identified as the raw food with the highest risk of aflatoxin contamination in humans (Pittet, 1998).

Roasting increases the concentration of volatiles and originates changes in the color of pistachios as a result of Maillard reaction (Hojjati et al., 2013; Ling et al., 2015). The Maillard reaction causes the decrease of sugars and can lead to the formation of

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Product and process innovation aroma compounds that cause consumer preference. The antioxidant activity of Maillard reaction compounds is considered responsible of the increase of oxidative stability values in oils obtained from thermally treated foods (Veldsink et al., 1999; Rabadán et al., 2017). Pistachio roasting could even be able to inactivate enzymes responsible for pistachio rancidity (Durmaz and Gökmen, 2011; Pumilia et al., 2014a), increasing pistachio quality and shelf-life.

Pistachios are particularly appreciated for its emerald-green color, originated by the pigments present in the kernel. A wide range of studies have reported the important roles that plant pigments play in health (Mayne et al., 1994), with special attention to the potential health benefits of a carotenoid-rich diet, due to their role as antioxidants (Landrum and Bone, 2001). The most important pigments in the raw pistachio kernel are chlorophylls and lutein. Chlorophylls a and b, may reach 150 mg/kg in raw pistachio (Bellomo and Fallico, 2007), however lower concentrations have been reported in pistachio kernels exposed to severe roasting conditions (Pumilia et al., 2014a). Still, extractable chlorophylls a and b were higher in pistachio kernels slightly roasted than in the raw kernel. Chlorophylls are heat labile and can degrade to form pheophytins and pyropheophytins (Pumilia et al., 2014a). Beyond temperature, pistachio long term storage also causes the formation of pheophytins (Bellomo et al., 2009b). The degradation of chlorophylls has been reported in thermally processed green fruits and vegetables causing the appearance of undesirable grey-brown compounds that dull the bright green color of fresh vegetable products (Heaton and Marangoni, 1996).

On the other hand, the predominant carotenoid in pistachios is lutein, a xanthophyll with a characteristic yellow colour (Giuffrida et al., 2006). Lutein content in pistachios ranges from 18 to 52 mg/kg depending on the pistachio origin and the kernel degree of ripeness (Bellomo and Fallico, 2007; Bellomo et al., 2009a). In contrast with chlorophylls, lutein seems to be more resistant to high temperatures (Khachik et al., 1992). In addition, lutein dissolved in oil is more resistant to thermal processing than other carotenoids (Henry et al., 1998), with studies even reporting an increase in the content of lutein after thermal processing of food products due to inactivation of the enzymes able to oxidize the carotenoids (Kirk and Tilnet-Basset, 1978).

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The complexity of food systems due to the co-oxidant or protective effect that the numerous compounds can show, encourages the study of the evolution of pigments in every oil matrix individually (Henry et al., 1998). In this study, the changes in the concentration of a wide range of chlorophylls and carotenoids in cold pressed oil from pistachios subjected to different roasting conditions are analyzed. In addition, physical parameter of oil (color, density and viscosity) are also analyzed.

2. Materials and Methods

2.1. Pistachio samples and oil extraction

Pistachios of the Kerman variety were collected manually and selected according to its sanitary state from orchards of the Instituto Técnico Agronómico Provincial de Albacete (ITAP), in Albacete (Spain). Then, the pistachios were dried at room temperature under controlled conditions until a humidity percentage lower than 5 % was reached.

The roasting of pistachios was done in a hot air oven. Eight homogeneous pistachio batches were subjected to roasting under different conditions of temperature (50, 75, 100 and 125 ºC) and time (15 and 30min). Additionally, the analysis on the natural pistachio and natural pistachio oil were also performed.

To extract the oil, a hydraulic press was used (Mecamaq DEVF 80, Vila Sana, Lleida, Spain). This press allows the oil extraction at room temperature so any additional effect of heat on oil characteristics is expected. Samples of 250 g of each batch were ground and subjected to a pressure of 200 bar for 15 minutes. The resultant oil was centrifuged to eliminate residual solid particles.

2.2. Physical characteristics

Oil samples were filtered and the colour of the oil samples was measured using a spectrophotometer UV/Vis Jasco V-530 (Jasco Analytical, Madrid, Spain). Oil was placed in quartz cuvettes (1cm path length) for their analysis. N-hexane was used as blank reference. The values obtained were used to calculate the CIELAB chromatic

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Product and process innovation coordinates: L∗ (brightness), a∗ (red–green component), b∗ (yellow–blue component) as recommended by the Commission Internationale de l’Eclairage (CIE, 1986).

Oxidative stability was evaluated by the rancimat method (Gutiérrez, 1989). Stability was expressed as the oxidation induction time (hours), measured with the Rancimat 743 apparatus (Metrohm Co., Basel, Switzerland). An oil sample of 3.5 g was used, warmed to 100 °C under an air flow of 10 l h−1.

Oil density was measured by dividing the mass in grams by the volume in milliliters. Viscosity was measured by a rotary viscometer test method (Visco Basic Plus, Fungilab S.A.) using the method described by Xu et al. (2007).

2.3. Pigments determinations

Pigments were determined using the method described by (Aparicio-Ruiz and Gandul-Rojas, 2014).

2.4. Statistical analyses

Determinations in this study are means of triplicate measurements from three independent samples. Statistical differences were estimated from ANOVA test at the 5% level of significance. All statistical analysis were carried out using the SPSS program, release 23.0 for Windows.

3. Results and Discussion

3.1. Oil yield

Roasting originates a loss of humidity in the pistachio samples. Humidity of the samples is an important parameter that affects oil yield (figure 1). The pistachios that were subjected to higher roasting treatments suffered a decreased in the humidity level, from 4.28 % to 2.10 %, which was related to a decrease in the oil obtained, from 39.08 % in the raw pistachios to 35.46 % in the oil from pistachios subjected to a roasting treatment of 125ºC for 30 min. Similar results have been found for other oils

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Effect of previous pistachio roasting on pistachio oil extracted by pressure methods (Ezeh et al., 2016; Savoire et al., 2013), which demonstrates the importance of performing the drying operation in a controlled manner, to avoid the over drying of the samples.

Figure 1. Correlation between pistachio kernel humidity and pistachio oil yield obtained.

3.2. Physical parameters

Pistachio roasting influence the physical parameters of oils (Table 1). While density values of pistachio oil reduce with severe roasting conditions, viscosity seems to increase. Density values range from 0.909 g/ml in oil from unroasted pistachios to 0.835 g/ml in oil from pistachios roasted at 125˚C for 30min. Within oil parameters, viscosity is related to oil processing and oil quality. Research has proved that oil viscosity is related to the temperature of the oil (Kumar et al., 2013), the degree of unsaturation (Kim et al., 2010; Oliveira et al., 2016) and the triglyceride composition of the vegetable oil (Geller and Goodrum, 2000). Variations in these parameters can be the responsible of the differences in the viscosity values with roasting conditions.

3.3. Oxidative stability

The increase of roasting temperature and time increases the oxidative stability of pistachio oils (Figure 2). The lowest value appears in the oil obtained from unroasted pistachios (24.66 h) while the highest value is reported in oil from pistachios previously roasted at 125˚C for 30 min (32.16 h). Previous studies support our results as oils obtained from roasted seeds and nuts are more resistant to oxidation processes (Elizalde et al., 1991; Rabadán et al., 2017c). This may be the result of the

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Product and process innovation appearance of Maillard reaction compounds with antioxidant capacity (Durmaz and Gökmen, 2011). In addition, higher stability in oils extracted from roasted seeds compared to unroasted ones can be attributed to the facilitation of antioxidant extraction along with oil (Chiou and Tsai, 1989), including phenolic compounds (Wijesundera et al., 2008), tocopherols (Kim et al., 2002) and pigments (Pumilia et al., 2014a).

Table 1. Density and viscosity of pistachio oils obtained after different roasting conditions.

Density Viscosity Roasting conditions (g/ml) (mPa·s) Unroasted 0.909 ± 0.021 55.66 ± 0.71 50˚C 15min 0.910 ± 0.031 55.79 ± 0.23 50˚C 30min 0.900 ± 0.022 55.71 ± 0.75 75˚C 15min 0.904 ± 0.021 56.22 ± 0.85 75˚C 30min 0.876 ± 0.014 56.19 ± 0.93 100˚C 15min 0.873 ± 0.018 56.18 ± 0.82 100˚C 30min 0.842 ± 0.018 55.88 ± 0.91 125˚C 15min 0.854 ± 0.024 56.45 ± 1.00 125˚C 30min 0.835 ± 0.010 57.31 ± 0.90

Figure 2. Oxidative stability values of pistachio oils obtained after different roasting conditions.

3.3. Chlorophylls and carotenoids

Pistachio oils contained, to a greater or lesser extent, pigments typical of a green fruit and those formed during thermal processing or in the oil extraction process by physical methods (Table 2). The chlorophyll fraction consisted mainly of chlorophylls

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Effect of previous pistachio roasting on pistachio oil and pheophytins (a and b), their respective epimers (a and b), pyropheophytin a and 132-OH-pheophytin a. Other compounds, such as pyropheophytin b, 132-OH- pheophytin b, 132-OH-chlorophylls (a and b) and pheophorbide a were only present in certain samples of oil. Regarding carotenoid fraction, lutein was the major compound, along with β-carotene, neoxanthin, violaxanthin, antheraxanthin, luteoxanthin, auroxanthin, neochrome, mutatoxanthin and β-cryptoxanthin. Other unidentified minor carotenoids with an absorption spectrum similar to that of lutein were also detected and quantified together as lutein equivalents. These peaks disappeared from the chromatogram when the oil was saponified, suggesting that they could be carotenoids (probably luteins) esterified with fatty acids.

In accordance with the apparent color of the oils, they can be classified into two groups. A first group with yellow color (Group Y), obtained from natural pistachio or low temperature roasted pistachios (50 or 75 ºC), and a second group with green color (Group G), from high temperature roasted pistachios (100 o 125 ºC). The oils from each group showed a quantitative composition of pigments of the same order, with the green oils having a total pigment content between 2.3 and 4 times higher than the oils of the yellow group.

The content of carotenoid pigments in the green group, which varies between 28.36 to 38.76 mg/kg, is approximately double (between 1.6 and 2.7 times) than in the oils of the yellow group (14.59 to 18.05 mg/kg). In the fraction of chlorophyll pigments greater differences were observed. Chlorophylls content in green oils ranged from 26.08 to 39.09 mg/kg, which was between 5.5 and 13 times greater than the chlorophylls measured in the yellow oils (2.93 to 4.75 mg/kg).

Therefore, the percentage represented by chlorophyll pigments with respect to total pigments in oils of group Y (15.39 to 20.83 %), increases considerably (46.99 to 54.60 %) when oils are obtained from pistachios roasted at 100 or 125ºC. The increase in temperature of the pistachios favors the extractability of the pigments together with the oil. The increase in the roasting temperature of the pistachios may originate a greater breakage of the pigment-protein complexes as indicated by Pumilla et al. (2014). Thus, the migration and solubilization of these compounds in the oil is favored

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Product and process innovation given its lipophilic nature. This effect is much more pronounced in the case of chlorophyll pigments than in carotenoids because the latter are easier to extract with the oil under the other conditions of the study, i.e. from pistachios roasted at 50 or 75 ºC, or natural pistachios and, although the roasting of pistachios at 100 or 125 ºC also favors their transfer to the oil, the differences found are lower than in the case of chlorophylls.

Carotenoids are transferred from pistachio to oil in greater proportion than chlorophyll pigments in all samples. Therefore, the ratio between the chlorophyll and the carotenoid fraction is reduced considerably in oils. While in the natural pistachio this ratio was 2.76, in the corresponding oil this ratio fell to 0.18. This result is in agreement with those obtained in studies carried out with virgin olive oils, where the transfer of pigments from olives to the oil is always greater for carotenoid pigments than for chlorophyll pigments. The roasting of the pistachio at low temperature (50- 75 ºC) did not improve the extractability of the pigments, maintaining the ratio between fractions. In the same way, the pigment content of the oils remained in the same order as in the oil extracted from the natural pistachio. On the other hand, higher roasting temperatures (100-125 ºC) favored the solubilization of pigments in the oil, mainly in the fraction of chlorophyll derivatives. Thus, the chlorophyll/carotenoid ratio in these oils, although decreasing with respect to that of the natural pistachio, remains balanced around the unit, reaching a value of 1.20 in the oil obtained from roasted pistachios at 125 ºC during 30 min.

Within the chlorophyll fraction, although the roasting of pistachios over 100 ºC significantly increases the chlorophyll transformation in their free Mg derivatives (brown and gray tones), the extractability of native chlorophylls (chlorophyll a and b) is also favored (bright green). Thus, the percentage of chlorophylls a and b that can be found in the oils obtained from pistachios roasted at lower temperatures (24% of the total chlorophyll pigments) increased when the oils are obtained from pistachios roasted at 100 ºC for 15 or 30 min or at 125 ºC for 15 min (up to 55% of the total chlorophyll pigments). However, this percentage fell to 31% when the pistachios were roasted at 125 ºC for 30 minutes, since in these conditions, in addition to the pheophytinization reaction, the C-13 decarbomethoxylation of the chlorophyll

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Effect of previous pistachio roasting on pistachio oil structure is produced, with the consequent formation of the pyroderivatives, mainly pyropheophytin a.

From the correlation between the chromatic coordinates measured in the oils and the pigment content, differentiated by fractions of chlorophylls and carotenoids, additional conclusions could be drawn. For example, with respect to the influence of the roasting time at 125 ºC, it would be expected that an increase in the roasting time would produce a decrease in the degree of greenery, measured from the chromatic coordinate -a *, as a consequence of the decrease in the percentage of native chlorophylls of green color.

3.4. Color

The temperature has a clear impact on the color of the oils extracted, changing from yellow in oils from raw or minimally roasted pistachios (50-75ºC) to brilliant green in oils from pistachios subjected to greater roasting treatments (100-125ºC). Previous studies suggest that roasting originates the breakdown of chlorophyll-protein complexes, improving the extractability of the pigments from pistachio (Pumilia et al., 2014b). This fact could explain the peak observed at 670 nm in the transmittance spectrum of oil from roasted pistachios (figure 2). Measuring the absorbance of oils at 670 nm through the use of appropriate molar absorption coefficients has been proposed as an appropriate method to measure the chlorophyll fraction in olive oils (Moyano et al., 2008). By adapting this method, the measurement of the chlorophyll fraction in pistachio oil may be performed.

As regards CIELab color coordinates, there is a clear difference in the oils (figure 3). Oils from pistachios roasted at 100 and 125ºC show an increase in the b* parameter (green-red component) and a decrease in the a* (yellow-blue component). Commercial pistachios are usually subjected to a drying treatment to avoid fungal growth and increase shelf-life, which may originate changes in pistachio oil characteristics and pigment concentration when the temperature is high (Sena‐ Moreno et al., 2015).

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Table 2. Concentration of chlorophylls and carotenoids in natural pistachio and pistachio oils obtained from roasted kernels Pigment 50ºC 50ºC 75ºC 75ºC 100ºC 100ºC 125ºC 125ºC3 NP NPO (mg/kg ± SD) 15´ 30´ 15´ 30´ 15´ 30´ 15´ 0´ Chlorophylls

24.65 ± 0.25 ± 0.27 ± 0.39 ± 0.57 ± 0.83 ± 8.97 ± 11.71 ± 12.67 ± 8.17 ± Chlorophyll aa 0.06 0.02 0.01 0.04 0.01 0.08 0.28 2.3 1.59 0.21 0.04 ± 0.04 ± 0.03 ± 132-OH-chlorophyll a 0.24 ± 0 ------0.01 0.01 0 151-OH-lactone 0.02 ± 0.04 ± 0.11 ± 0.13 ± 0.56 ± 0.81 ± 0.08 ± 0 - - - chlorophyll a 0 0.01 0.02 0.03 0.03 0.03 2.39 ± 2.08 ± 2.26 ± 2.3 ± 2.14 ± 3.04 ± 11.39 ± 12.25 ± 13.74 ± 10.95 ± Pheophytin aa 0.08 0.04 0.14 0.02 0.08 0.26 1.02 0.45 0.45 0.66 0.11 ± 0.14 ± 0.08 ± 0.11 ± 0.14 ± 0.22 ± 0.16 ± 0.19 ± 0.27 ± 132-OH-Pheophytin a 0.12 ± 0 0 0.01 0.01 0 0.03 0.02 0.02 0.04 0.02 0.06 ± 0.06 ± 0.07 ± 0.06 ± 0.08 ± 0.17 ± 0.44 ± 1.64 ± 13.91 ± Pyropheophytin a 0.03 ± 0 0 0 0 0 0.01 0.01 0.04 0.1 0.91 0.01 ± 0.01 ± 0.02 ± 0.01 ± Pheophorbide a 0.04 ± 0 - - - - - 0 0 0 0 0.17 ± 0.15 ± 0.24 ± 0.23 ± 4.56 ± 4.44 ± 4.09 ± 2.08 ± Chlorophyll ba 10.8 ± 0.1 0.2 ± 0 0.01 0.02 0 0.02 0.07 0.44 0.41 0.08 0.04 ± 0.09 ± 0.4 ± 0.86 ± 132-OH-chlorophyll b 0.04 ± 0 - - - - - 0.01 0.02 0.03 0.03 0.22 ± 0.25 ± 0.22 ± 0.22 ± 0.35 ± 0.57 ± 0.55 ± 0.73 ± 0.51 ± Pheophytin ba 0.2 ± 0.01 0.01 0.01 0.01 0 0.06 0.05 0.04 0.06 0.02 0.03 ± 0.04 ± 0.03 ± 0.03 ± 0.04 ± 0.04 ± 0.04 ± 0.05 ± 0.08 ± 132-OH-Pheophytin b 0.02 ± 0 0 0 0 0 0 0.01 0 0 0 0.24 ± 1.4 ± Pyropheophytin b ------0.04 0.04 Carotenoidsb

8.96 ± 12.16 ± 12.35 ± 11.25 ± 11.61 ± 13.83 ± 22.21 ± 26.46 ± 29.74 ± 24.61 ± Lutein 0.08 1.23 1.48 0.28 0.13 2.88 1.32 0.58 1.82 0.82 2.96 ± 2.67 ± 2.00 ± 2.18 ± 2.15 ± 2.55 ± 2.68 ± 2.83 ± 4.11 ± 3.93 ± β-carotene 0.17 0.37 0.01 0.15 0.08 0.31 0.22 0.39 0.1 0.37 1.02 ± 0.24 ± 0.23 ± 0.19 ± 0.24 ± 0.29 ± 1.35 ± 1.52 ± 2.31 ± 1.58 ± Neoxantin 0.04 0.06 0.07 0.01 0.03 0.08 0.11 0.08 0.31 0.16 0.03 ± 0.03 ± 0.02 ± 0.06 ± 0.07 ± 0.04 ± 0.04 ± 0.24 ± 0.06 ± Neochrome 0.03 ± 0 0 0 0 0.01 0.02 0 0 0.31 0 0.38 ± 0.24 ± 0.21 ± 0.18 ± 0.24 ± 0.31 ± 0.65 ± 0.78 ± 0.9 ± 0.64 ± Violaxanthin 0.02 0.04 0.06 0.01 0 0.08 0.08 0 0.07 0.02 0.21 ± 0.2 ± 0.18 ± 0.19 ± 0.23 ± 0.36 ± 0.42 ± 0.42 ± 0.39 ± Luteoxanthin 0.1 ± 0 0.03 0.04 0 0 0.06 0.04 0.02 0.13 0.01 0.05 ± 0.04 ± 0.04 ± 0.04 ± 0.06 ± 0.08 ± 0.09 ± 0.11 ± 0.1 ± Auroxanthin - 0.01 0.01 0 0 0.01 0.01 0.01 0.02 0.01 0.1 ± 0.09 ± 0.08 ± 0.11 ± 0.13 ± 0.19 ± 0.23 ± 0.25 ± 0.19 ± Antheraxanthin 0.13 ± 0 0.01 0.02 0 0 0.03 0.02 0 0.11 0 0.04 ± 0.03 ± 0.03 ± 0.03 ± 0.04 ± 0.05 ± 0.06 ± 0.07 ± 0.06 ± Mutatoxanthin - 0.01 0 0 0 0 0 0 0 0 0.01 ± 0.02 ± 0.02 ± 0.01 ± 0.01 ± 0.02 ± 0.02 ± 0.02 ± 0.03 ± β- ryptoxanthin 0.01 ± 0 c 0 0 0 0 0 0 0 0 0 No identified 0.37 ± 0.49 ± 0.44 ± 0.44 ± 0.53 ± 0.72 ± 0.69 ± 0.6 ± 0.92 ± 0.26 ± 0 carotenoids c 0.07 0.03 0 0.03 0.05 0.04 0.07 0.04 0.03 38.61 ± 2.93 ± 3.17 ± 3.31 ± 3.36 ± 4.75 ± 26.08 ± 29.82 ± 34.36 ± 39.09 ± Total chlorophylls 0.06 0.1 0.14 0.08 0.1 0.47 0.95 3.14 2.49 1.99 13.85 ± 16.11 ± 15.69 ± 14.59 ± 15.12 ± 18.05 ± 28.36 ± 33.14 ± 38.76 ± 32.51 ± Total carotenoids 0.18 1.48 1.69 0.24 0.26 3.49 1.69 0.67 1.81 0.93 52.46 ± 19.04 ± 18.86 ± 17.9 ± 18.48 ± 22.79 ± 54.44 ± 62.96 ± 73.13 ± 71.61 ± Total pigments 0.12 1.57 1.6 0.2 0.32 3.96 0.96 3.76 4.29 2.91 a Data include the value of respective epimer; b Data include the value of isomers cis if present; c Data are the sum of up to six compounds. Abreviations: NP, natural pistachio; NPO, natural pistachio oil.

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Figure 2. Transmittance spectrum of oil from roasted pistachios

Figure 3. Graphical representation of pistachio oil values for CIELab coordinates a* and b*.

4. Conclusion

Roasting can be considered a beneficial step previous to pistachio oil production due to the positive effect that has on pistachio oil characteristics. Severe roasting conditions increase the oxidative stability of pistachio oils. This is attributed to the larger extraction of antioxidant along with oil and the production of Maillard reaction compounds with antioxidant activity. Regarding pigments, total chlorophylls and carotenoids increase with roasting. This can be the result of the inactivation of the enzymes able to oxidize some pigments but also for the effect of higher roasting temperatures improving the solubilization of pigments in the oil.

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Mayne, S.T., Janerich, D.T., Greenwald, P., Chorost, S., Tucci, C., Zaman, M.B., Melamed, M.R., Kiely, M., McKneally, M.F., 1994. Dietary beta carotene and lung cancer risk in U.S. Nonsmokers. Journal of the National Cancer Institute 86, 33-38.

Moyano, M.J., Meléndez-Martínez, A.J., Alba, J., Heredia, F.J., 2008. A comprehensive study on the colour of virgin olive oils and its relationship with their chlorophylls and carotenoids indexes (II): CIELUV and CIELAB uniform colour spaces. Food Research International 41, 513-521.

Nicoli, M.C., Anese, M., Manzocco, L., Lerici, C.R., 1997. Antioxidant properties of coffee brews in relation to the roasting degree. LWT - Food Science and Technology 30, 292-297.

Oliveira, P.D., Araujo, L.A., Silva, L.H.M., Rodrigues, A.M.C., 2016. Predicting Temperature-Dependent Viscosity of Amazonian Vegetable Oils and Their Mixtures from Fatty Acid Composition. International Journal of Food Properties 19, 1972-1982.

Pittet, A., 1998. Natural occurrence of mycotoxins in foods and feeds - An updated review. Revue de Medecine Veterinaire 149, 479-492.

Pumilia, G., Cichon, M.J., Cooperstone, J.L., Giuffrida, D., Dugo, G., Schwartz, S.J., 2014a. Changes in chlorophylls, chlorophyll degradation products and lutein in pistachio kernels (Pistacia vera L.) during roasting. Food Research International 65, 193-198.

Pumilia, G., Cichon, M.J., Cooperstone, J.L., Giuffrida, D., Dugo, G., Schwartz, S.J., 2014b. Changes in chlorophylls, chlorophyll degradation products and lutein in pistachio kernels (Pistacia vera L.) during roasting. Food research international 65, 193-198.

Rabadán, A., Álvarez-Ortí, M., Gómez, R., Alvarruiz, A., Pardo, J.E., 2017a. Optimization of pistachio oil extraction regarding processing parameters of screw and hydraulic presses. LWT - Food Science and Technology 83, 79-85.

Rabadán, A., Álvarez-Ortí, M., Pardo, J.E., Gómez, R., Pardo-Giménez, A., Olmeda, M., 2017b. A comprehensive approach to pistachio oil production. British Food Journal 119, 921-933.

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Sena‐Moreno, E., Pardo, J.E., Catalán, L., Gómez, R., Pardo‐Giménez, A., Alvarez‐Ortí, M., 2015. Drying temperature and extraction method influence physicochemical and sensory characteristics of pistachio oils. European Journal of Lipid Science and Technology 117, 684-691.

Ukhanova, M., Wang, X., Baer, D.J., Novotny, J.A., Fredborg, M., Mai, V., 2014. Effects of almond and pistachio consumption on gut microbiota composition in a randomised cross-over human feeding study. The British journal of nutrition 111, 2146.

Vázquez-Araújo, L., Verdú, A., Navarro, P., Martínez-Sánchez, F., Carbonell- Barrachina, A.A., 2009. Changes in volatile compounds and sensory quality during toasting of Spanish almonds. International Journal of Food Science and Technology 44, 2225-2233.

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Effect of roasting conditions on the composition and antioxidant properties of defatted walnut

flour

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Effect of roasting conditions on the composition and antioxidant properties of defatted walnut flour

ABSTRACT Keywords: The walnut oil extraction by pressure systems, produces a press cake as a Pistacia vera Antioxidant by-product, with many of the beneficial walnut properties. The objective of properties this work was to evaluate the composition and the antioxidant properties By-product Nutritional of walnut flours submitted to different roasting protocols (50, 100 and 150 composition ºC during 30, 60 and 120 minutes). All walnut flours had about 42% of

protein and a significant amount of dietary fibre (17%), not being affected by the roasting process. Nonetheless, the fat content increased around 50% in walnuts flours subjected to longer and higher roasting temperatures (150 ºC). The lipid fraction showed a good nutritional quality with a high vitamin E content (mainly γ-tocopherol) and fatty acid profile rich in linoleic and linolenic acids. The high phenolic content also provides great antioxidant capacity to the flours. Mild roasting of the walnuts did not affect the quality of the flours that could be used as functional ingredient in the food industry.

1. Introduction

Walnut (Juglans regia L.) is the most widespread tree nut in the world. It is native from Central Asia, but nowadays it is cultivated commercially throughout southern Europe, northern Africa, eastern Asia, United States of America, and western South America (Martínez et al., 2010). The production of walnut (with shell) has risen continuously in the last decade to reach a current World production of about 3.5 x 106 tons (FAO, 2015). The consumption of walnuts is recommended due to their high nutritional value, and are associated to improved blood lipoprotein profile, antiatherogenic effect, antioxidant activity, reduction of tumour initiation or promotion, regulation of cell differentiation and proliferation, repair of DNA damage, regulation of immunological activity and inflammatory response, induction of phase

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II metabolic enzymes (Yang et al., 2009; Sze-Tao et al., 2001). With a content of around 20% of highly digestible protein and with a balanced content of essential amino acids, walnuts are an interesting source of vegetable proteins (Sze-Tao and Sathe, 2000). Walnuts contain about 50-70% of oil rich in polyunsaturated fatty acids (about 70% of total fatty acids), especially linolenic acid (Amaral et al., 2003). These nuts are also rich in magnesium, and contain a battery of phytochemicals, like phenolic compounds with antioxidant properties (Slantar et al., 2015), and are also an excellent source of tocopherols, containing four forms of tocopherols (α, β, , and δ), sterols, carotenoids, and aliphatic alcohols (Abdallah et al., 2015).

The production of cold pressed walnut oil is in great demand due to its strong aromatic flavour and high nutritional value (Zhou et al., 2016). Although the pressure systems lead to lower oil recovery, it is the preferred method in order to obtain virgin oils with the highest quality (Prescha et al., 2014). To overcome this, the use of mechanical and thermal pre-treatment of the nuts are common to enhance the extraction performances (Savoire et al., 2013). The main by-product resulting from oil extraction is a press cake that can be ground to form flour, maintaining almost the beneficial composition of the whole walnut. Presently, the resulting defatted walnut flour has been already used as an ingredient of several food products, like meat, dairy and bakery products, as a way to increase the nutritional and sensory quality of those products (Martínez et al., 2010).

Roasting treatments can be applied to nuts prior to oil extraction to provide differentiated sensory characteristics to the oils (Sena-Moreno et al., 2015). However, these treatments may originate changes in the physicochemical composition of the oils and the resulting press cakes, as it can cause breaking of cell walls, protein clotting by denaturation, sterilization and deactivation of thermos sensitive enzymes (Savoire et al., 2013; Vaida et al., 2013).

Several walnut samples have been subjected to different roasting protocols, and the oil has been extracted by means of hydraulic pressure. The resulting press cake has been ground and changes in nutritional composition, vitamin E and fatty acid profile

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Product and process innovation were evaluated. Antioxidant capacity, phenolic and flavonoid contents of the walnut flours were also assessed.

2. Material and Methods

2.1. Plant Material and roasting treatment

Shelled walnuts from Pedro variety were kindly provided by Albaga S.A.T. from their own cultivation fields located in Hellín (Albacete, Spain). They were carefully selected to eliminate fruits with dark spots or with a deficient sanitary condition in order to obtain a high quality raw material. Prior to oil extraction, walnuts were subjected to a roasting treatment with different temperatures and times. The used temperatures were 50 ºC, 100 ºC and 150 ºC during 30, 60 and 120 minutes. A natural walnut sample without roasting was used as control, giving a total of ten different flour samples after oil extraction.

2.2. Oil extraction

500 gr of walnuts from each of the ten conditions previous described were grinded and subjected to a pressure of 40 tons in a hydraulic press (MECAMAQ DEVF 80, Vila- Sana, Lleida, Spain) for 15 min. The extraction yield was calculated for each roasting condition tested. After oil extraction, the press cake was grinded to obtain a defatted walnut flour with a particle size <0.5 mm. The walnut flours were stored at 4ºC in plastic hermetic recipients until analysis.

2.3. Nutritional Composition

Water (AOAC method nº 945.15), protein (AOAC method nº 920.54), fat (AOAC method nº 920.39), ash (AOAC method nº 942.05) and total dietary fibre (AOAC method nº 985.29) content were determined according to the AOAC methods in the different samples of walnut flour (AOAC, 2012). Protein content (N x 5.30) was assessed by a Kjeldahl method, fat by a Soxhlet extraction of a known weight of powdered sample with petroleum ether, the ash content was determined by incineration at 600 ± 15 °C and dietary fibre by an enzymatic gravimetric method.

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All values were presented as g/100 g product (dry weight basis (d.w).), being the available carbohydrates calculated by difference accordingly to the following equation: 100 – (protein + fat + water + ash + dietary fibre in 100 g of flour). All proximate composition analyses were done, at least, in triplicate. Energy was calculated according Atwater Factors (namely a factor of 4 for protein and available carbohydrates, a factor of 2 for dietary fibre, and a factor of 9 for fat content) (Otten et al., 2006).

2.4. Vitamin E determination

Vitamin E profile was determined by HPLC analysis in the lipid fraction of the walnut flours, obtained by Soxhlet extraction with petroleum ether (3 h). Briefly, about 20 mg of the obtained oil were diluted in 1 mL of n-hexane (HPLC-grade, Merck, Darmstadt, Germany) with 20 µg/mL of tocol (internal standard). 20 µL were injected in a HPLC system (Jasco, Tokyo, Japan) equipped with AS-2057 automated injector, a PU-2089 pump and a MD-2018 multi-wavelength diode array detector (DAD) coupled to a fluorescence detector FP-2020 (Jasco, Japan), programmed for excitation at 290 and emission at 330 nm. The chromatographic separation was achieved on a normal phase SupelcosilTM LC-SI column (3 µm; 75 x 3.0 mm; Supelco, Bellefonte, PA, USA) according to Alves, Casal and Oliveira (Alves et al., 2009). The compounds identification was accomplished by comparison with commercial standards, which were also used in the construction of external calibration curves used for quantification purposes. The analyses were done in duplicate and the results expressed as mg/kg of fat.

2.5. Fatty acid profile

First, fatty acid (FA) methyl esters were obtained by cold transmethylation with methanolic potassium hydroxide, by adding two mL of n-hexane to 0.02 g of oil previously extracted from the flour samples by Soxhlet extraction with petroleum ether (3 hours) (EC, 2002). Then, 200 µL of methanolic potassium hydroxide solution (2 N) was added and vigorously mixed. After the supernatant was transferred to a glass vial and analysed by gas chromatography in a Shimadzu GC-2010 Plus Gas Chromatograph equipped with a split-splitless injector, a FID detector and an

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Product and process innovation autosampler Shimadzu AOC-20i (Shimadzu, Tokyo, Japan). The column used was a CPSil 88 fused silica capillary column (Varian, Middelburg, Netherlands; 50 m x 0.25 mm i.d., 0.20 µm film thickness) and helium was used as gas carrier (120 kPa). The following temperature program was used: 5 min at 140 °C, followed by an increase of 5 °C/min from 140 °C to 220 °C and maintained at 220 °C for 15 min. The temperature of the injector and detector were 250 °C and 270 °C, respectively, and the split ratio was of 1:50 with an injection volume of 1 µL. Each FAME (fatty acid methyl ester) was identified by direct comparison with a standard mixture (FAME 37, Supelco, Bellefonte, PA, USA). The analyses were done in duplicate and the results expressed in relative percentage of each FA, based on the relative peak areas.

2.6. Determination of soluble phenolic and flavonoid contents

Total contents of soluble phenolics and flavonoids were determined in hydroalcoholic extracts of the walnut flours. Briefly, 1 g of ground sample and 25 mL of 50% ethanol solution were stirred at 40 °C, 600 rpm, for 1 hour. Then it was filtered through Whatman No. 4 paper and the extract stored in amber glass vials at -18ºC until analysis (Costa et al., 2014). Three extracts from each flour sample were analysed.

The total soluble phenolic content of the walnut flours was determined according to the Folin-Ciocalteu procedure (Singleton and Rossi, 1965) with minor modifications (Alves et al., 2010). Briefly, 500 µL of the diluted hydroalcoholic flour extract was mixed with 2.5 mL of the Folin-Ciocalteu reagent (1:10) and 2 mL of a sodium carbonate solution (7.5% m/v). The mixture was incubated for 15 min at 45º C, followed by 30 min period at room temperature, protected from light exposure. The absorbance was measured at 765 nm (Synergy HT Microplate Reader, BioTek Instruments, Inc., USA). The total soluble phenolics were quantified through a gallic acid (GA) calibration curve and reported as mg gallic acid /g of walnut flour (d.w.).

Flavonoids content was determined according to Soares, Alves, Pires, Oliveira and Vinha (Soares et al., 2013). Four mL of ultrapure water and 300 μL of sodium nitrite solution (5% m/v) were added to a 1 mL of diluted sample extract. After 5 min, 300 μL of aluminum chloride solution (10%) and after 1 min, 2 mL of NaOH (1M) and 2.4 mL of ultrapure water were also added. The absorbance was read at 510 nm (Synergy

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HT Microplate Reader, BioTek Instruments, Inc., USA). Catechin was used as standard for quantification purposes and the flavonoids content was expressed as mg of catechin /g of walnut flour (d.w.).

2.7. Antioxidant capacity assays

The antioxidant activity of the hydroalcoholic walnut flour extracts was determined by two different assays. The first was a modification of the 2,2-diphenyl-1- picrylhydrazyl (DPPH) radical-scavenging assay described by Santos, Herrero, Mendiola, Oliva-Teles, Ibanez, Delerue-Matos and Oliveira (Santos et al., 2014). 50 µL of five different concentrations of each flour extract (from 5 to 0.32 mg/mL) were allowed to react with 250 µL of a DPPH methanolic solution (9.5 x 10-5 mol/L) on the well of the 96-wells microplate. The reduction of the DPPH radical was monitored at 517 nm until it reached a stable absorbance value (30 minutes). The % DPPH inhibition obtained in each extract concentration was used to calculate the samples EC50 (mg/mL), used to express the obtained results.

The ferric reducing antioxidant power assay (FRAP) was also applied to all extracts (Benzie and Strain, 1996). Briefly, 90 µL of diluted extract were mixed with 270 µL of distilled water and 2.7 mL of the FRAP solution (containing 0.3 M acetate buffer, 10 mM TPTZ(2,4,6-Tris(2-pyridyl)-s-triazine) solution, and 20 mM of ferric chloride in a10:1:1 proportion), and incubated at 37 °C for 30 minutes, protected from light exposure. After, 300 μL of the reaction mixture were transferred to a 96-wells microplate and the absorbance read at 595 nm in Synergy HT Microplate Reader (BioTek Instruments, Inc., USA). FRAP values were obtained by comparison with a ferrous sulphate solution calibration curve. The results were expressed as µmol of ferrous sulphate/ g of walnut flour (d.w).

2.8. Statistical Analysis

Data were expressed as mean ± standard deviation and the differences between the flours submitted to 3 different temperatures and 3 drying times were tested by one- way ANOVA analysis followed by post hoc Tukey HSD test. Normal distribution of data in the different samples was assessed by Kolmogorov–Smnirnov test. Also, a

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Product and process innovation correlation study between the analysed parameters was performed. Statistical significance was defined for a p<0.05 (95% confidence level). The statistical analyses were carried out using the Statistica 8.0 software (Statsoft Inc., Tulsa, USA).

3. Results and discussion

3.1. Nutritional composition

The walnut flour nutritional composition is presented in Figure 1 and the moisture content of the flours is presented on Table 1. As expected, the moisture of the flours showed a clear decrease with the intensity of the roasting procedure (at 150 ºC during 120 min the walnut flour had less 70% of moisture than the control sample) (see Table 1).

Figure 1. Proximate composition of walnut flour samples according to the roasting conditions (time and temperature) (mean ± standard deviation; different letters represent significant differences p<0.05 between samples)

Usually, to preserve quality, walnuts must be dried to under 8% of moisture as soon as possible after the harvest. Although it is normal an over drying to ensure the absence of microbiological growth (Khir et al., 2013). Prior to the roasting, the raw walnuts used showed moisture content of 3.96%, decreasing to values under 1.5% when submitted to a roasting treatment of 150 ºC for 120 min (figure 2). Moisture content of the walnuts also affected the oil extraction yield, with a clear relation

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Antioxidant properties of walnut flour from roasted walnuts between walnuts moisture and oil recovery from the sample. In the raw walnuts, the oil yield was about 62%, while for the walnuts roasted at 150 ºC for 2 hours, the oil yield decreased to about 59%. The influence of the nut’s water content on the oil yield is dependent on the raw material, and contrarily to what happen in the walnuts, in some seeds the roasting process enhances the oil yield (Savoire et al., 2013).

Table 1. Moisture content of the walnuts flours (mean ± standard deviation; different letters in the same column represent significant differences p<0.05 between samples). Roasting Conditions Moisture content (%) Control 11.43 ± 0.36a 50˚C 30’ 9.91 ± 0.22b 60’ 9.45 ± 0.18bc 120’ 8.80 ± 0.12c 100˚C 30’ 6.56 ± 0.3d 60’ 5.29 ± 0.01e 120’ 5.05 ± 0.16e 150˚C 30’ 4.08 ± 0.16f 60’ 3.50 ± 0.09fg 120’ 3.22 ± 0.18g

Figure 2. Effect of the initial sample moisture on the oil yield obtained with hydraulic press. The roasting conditions are shown for each sample

The roasting process also had a clear effect on the fat content of the resulting walnut flours, that increased significantly (p<0.05) when the walnuts were subjected to more intense roasting procedures (figure 1). After oil extraction from the raw walnuts, 18% of fat remained in the press cake. This content increased to values over 28% when

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Product and process innovation the walnuts were roasted at 150 ºC. In order to obtain the desired fat content in the flour, the moisture of the walnuts should be adjusted by choosing the most favourable roasting conditions. Our results show that moisture contents of 6-7% guarantee a higher oil yield, with a consequent lower fat content in the resulting flour. Another aspect that comes from the higher fat content of the walnut flours roasted at and above 100 °C is their higher energy, when compared to the others defatted flours (see figure 1).

Contrarily to the significant changes between samples roasted at different conditions on fat and moisture content, the protein, ash, total dietary fibre and sugar contents were more constant throughout the different samples. The ash content did not show a significant variation (p>0.05) within the defatted flours (figure 1), showing a mean value of 5%. Walnuts are a known source of minerals, especially manganese, iron, copper, potassium and magnesium, so the inclusion of walnut flour as an ingredient in the food formulations may lead to the increase of the nutritional value (Serrano et al., 2005). Regarding the protein content, the walnut flours showed values around 39-44%, and significant changes (p<0.05) were only registered when more drastic roasting was performed. These values were higher than those reported in previous studies with similar extraction methods (Labuckas et al., 2014), however the differences could be due to the different factors as the variety of the walnut used, geographic origin and environmental conditions (Uribe et al., 2013). Almost of the flours roasted above 100 °C showed a slight lower protein value that could resulted from denaturation process (Uribe et al., 2013).

Walnut proteins are mainly composed by glutelins, with a balanced content of essential amino acids, with exception for methionine (Martínez et al., 2010; Mao et al., 2014). Another characteristic of the walnut proteins is their lower lysine/arginine ratio, related to the reduction of atherosclerosis progression (Martínez et al., 2010). Thus, they have been proposed as a good alternative for vegetal protein intake, representing one of the main nutritional advantages for the use of the defatted walnut flours. The mean 17% dietary fibre content (comprising the soluble and insoluble fractions of the walnut flours fibre) is also one main nutritional advantage of the walnut flours that gains even more relevance in these defatted products. The

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Antioxidant properties of walnut flour from roasted walnuts positive role of dietary fibre in the maintenance of human health is recognized due to its potential to control weight, constipation, serum lipids and glycemic index and reduce cholesterol and the risk of coronary heart disease (O’sea et al., 2012). Due to their insoluble and soluble properties, dietary fibre confers a range of technological attributes such as water binding, gelling, and structure building and it can be used as a fat replacer (O’sea et al., 2012). In this case, the different roasting conditions did not a cause any significant (p<0.05) change of this fraction of the flours composition.

The roasting process has been associated with the formation of melanoidins that are dark brown-coloured polymers, of high molecular weight, produced by Maillard reactions between sugars and amino acids (Wang et al., 2011). These compounds are associated with the increased of browning coloration that was registered in the flours roasted at higher temperatures. In the defatted walnut flours, we could see that the sugars content (determined by difference) diminish when high roasting temperatures were used (above 100 ºC) (figure 1). Although that value was mostly affected by the higher fat content, it is also probable that the amino acids release during protein denaturation had reacted with the sugars present to form this kind of compounds.

The compositional changes seen in the walnuts flours were primarily related to roasting temperature used, with these features showing higher and significant (p<0.05) correlation scores with this parameter than with the duration of the process (see table 2).

Due to all changes that were seen in the composition of flours, mainly in their fat and moisture content, and in a smaller scale in their protein and sugar content, it is possible to expect an overall alteration of the structure of the defatted flours submitted to more drastic roasting treatment. This will lead different textural properties that should be taken in consideration when choosing the most suitable roasting conditions (Uribe et al., 2013).

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3.2. Vitamin E

Vitamin E is a group of fat-soluble compounds including , ,  and  tocopherols and , ,  and  tocotrienols. Each isomer has unique physiological functions, so it is advisable to know the vitamin E profile of each product (Amaral et al., 2005). In the oil fraction of walnut flour samples, the four tocopherol isoforms and one tocotrienol (-tocotrienol) were detected (see Table 3).

Table 2. Pearson’s correlation coefficient obtained for the relation between the walnut flour composition and the two parameters tested during roasting (*marked correlations are significant at p < 0.05) Time Temperature Nutritional Composition Protein -0.23 -0.64* Fat 0.34 0.93* Moisture -0.45 -0.97* Ash -0.53 -0.82* Total dietary fibre 0.43 -0.03 Sugars -0.48 -0.90* Vitamin E α-tocoferol 0.26 0.42* β-tocoferol 0.45* 0.49* γ-tocoferol 0.32 0.54* γ-tocotrienol 0.43 0.60* δ-tocoferol 0.36 0.78* Total Vit E 0.42* 0.69* Phenolic Fraction Total phenolics 0.03 -0.81* Flavonoids -0.29 -0.60* Antioxidant Capacity FRAP -0.3 -0.89* DPPH 0.03 0.22

The predominant form is -tocopherol, representing 76.5% of total tocols in the control sample (6.39 ± 0.05351.5 mg/kg of fat), followed by -tocopherol (51.9 mg/kg of fat) and α-tocopherol (29.5 mg/kg of fat), which agree with previous studies (Miraliakbari and Shahidi, 2008; Abdallah et al., 2015). The total vitamin E content of the walnut flour was higher than other previously found (Alasalvar and Pelvan, 2011; Abdallah et al., 2015). Tocopherol content depends on walnut variety and

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Antioxidant properties of walnut flour from roasted walnuts environmental conditions, and many studies described tocopherol differences in oils from walnuts grown in several countries (Amaral et al., 2005; Kornsteiner et al., 2006; Miraliakbari and Shahidi, 2008).

The walnut flours studied were obtained after oil extraction by means of a hydraulic press, which is a method that is not influenced by temperature and leads to high levels of vitamin E isomers in its lipid fraction. However, previous thermal treatments can influence the tocol levels, justifying the significant differences recorded between samples (p<0.05) (see Table 3), especially those submitted to higher roasting temperatures and times that showed, in most cases, a decreased content of tocopherols. However, one exception occurred in the flour obtained after roasting the walnuts at 150 ºC for 120 min. This sample showed an increased content of β- tocopherol and γ-tocotrienol, being those values higher than the ones found in the control sample (see Table 3). Usually, the roasting procedures have been associated to a decrease in the tocopherols content (Schlormann et al., 2015). However, in these flours the content of tocols remained high in all samples, especially -tocopherol, which is one of the main antioxidant compounds of plants and exhibits more potent and effective antioxidant effect compared to α-tocopherol and a higher intake of this compound was successfully correlated to a lower risk of cancer and cardiovascular diseases (Aggarwal et al., 2010; Camara and Schlegel, 2016). Thus, tocols content can contribute to increase the flour antioxidant properties and for that matter, their oxidative stability, even when walnuts have been subjected to a roasting treatment. According to Vanhanen and Savage (2006) the high levels of vitamin E protect walnut flour from oxidation reactions, especially of their polyunsaturated fatty acids present in high levels.

The walnut flours studied were obtained after oil extraction by means of a hydraulic press, which is a method that is not influenced by temperature and leads to high levels of vitamin E isomers in its lipid fraction. However, previous thermal treatments can influence the tocol levels, justifying the significant differences recorded between samples (p<0.05) (see Table 3), especially those submitted to higher roasting temperatures and times that showed, in most cases, a decreased content of

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Product and process innovation tocopherols. However, one exception occurred in the flour obtained after roasting the walnuts at 150 ºC for 120 min. This sample showed an increased content of β- tocopherol and γ-tocotrienol, being those values higher than the ones found in the control sample (see Table 3). Usually, the roasting procedures have been associated to a decrease in the tocopherols content (Schlormann et al., 2015). However, in these flours the content of tocols remained high in all samples, especially -tocopherol, which is one of the main antioxidant compounds of plants and exhibits more potent and effective antioxidant effect compared to α-tocopherol and a higher intake of this compound was successfully correlated to a lower risk of cancer and cardiovascular diseases (Aggarwal et al., 2010; Camara and Schlegel, 2016). Thus, tocols content can contribute to increase the flour antioxidant properties and for that matter, their oxidative stability, even when walnuts have been subjected to a roasting treatment. According to Vanhanen and Savage (2006) the high levels of vitamin E protect walnut flour from oxidation reactions, especially of their polyunsaturated fatty acids present in high levels.

Table 3. Tocopherols and tocotrienols content (mg/kg of fat) of the walnut flour samples submitted to different roasting procedures (mean ± standard deviation; different letters in the same column represent significant differences p<0.05 between samples).

α-tocopherol β-tocopherol γ-tocopherol δ-tocopherol γ-tocotrienol Total Vitamin E Control 29.5 ± 2.8a 7.3 ± 0.2c 351.5 ± 2.7a 51.9 ± 0.3a 19.2 ± 0.7c 459.4 ± 6.7a 50° 30’ 25.1 ± 1.0b 8.6 ± 0.2b 324.5 ± 6.1ab 47.2 ± 1.6ab 22.2 ± 0.6b 427.6 ± 9.5ab ab b ab ab bc ab 60’ 26.2 ± 0.6 7.8 ± 0.1 320.7 ± 0.7 46.5 ± 0.6 21.1 ± 0.3 422.3 ± 1.0 ab b ab ab bc ab 120’ 28.7 ± 0.8 7.9 ± 0.0 316.8 ± 3.9 45.9 ± 0.1 20.7 ± 0.4 420.0 ± 5.3 100° 30’ 26.3 ± 1.4ab 8.0 ± 0.4b 225.3 ± 30.5c 44.1 ± 5.3c 20.8 ± 1.1bc 324.5 ± 22.1c c c b b d b 60’ 21.7 ± 0.0 6.1 ± 0.0 298.6 ± 3.9 41.2 ± 0.9 15.3 ± 0.1 383.0 ± 2.9 c c b b cd b 120’ 20.4 ± 0.2 6.9 ± 0.4 303.6 ± 0.3 43.9 ± 0.9 17.6 ± 1.3 392.3 ± 3.1 150° 30’ 20.9 ± 0.1c 6.9 ± 0.4c 321.9 ± 4.6ab 43.3 ± 1.4ab 17.0 ± 1.3cd 409.9 ± 7.6b c c c c cd c 60’ 19.6 ± 1.2 7.3 ± 0.3 230.1 ± 8.8 38.0 ± 2.1 16.7 ± 1.1 311.6 ± 13.5 a a b b a a 120’ 27.8 ± 0.9 27.7 ± 0.8 295.0 ± 29.1 48.9 ± 2.7 26.0 ± 0.9 425.3 ± 30.8

Regarding the effect of both roasting parameters tested, once more, there was a noticed influence of the temperature used, although in the case of the total content of vitamin E we could also register a significant relation with the duration of the process (see Table 2).

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3.3. Fatty acids

The FA composition of walnut oil is known for its high percentage of polyunsaturated FA, especially linoleic (C18:2n6, about 62-63%) and linolenic (C18:3n3, about 14-15%) acids and a n6/n3 ratio close to 4:1, which was associated to a decrease the incidence of cardiovascular diseases via the reduction of total plasma cholesterol and low density-lipoprotein cholesterol (Crews et al., 2005; Labuckas et al., 2011). A similar FA profile was found in all defatted walnut flours studied (see Table 4), in agreement with previous studies (Amaral et al., 2003).

As expected and desired the extraction process did not affect the overall FA composition of the oil (Martínez et al., 2010). As no significant changes (p>0.05) were noticed between the flours samples roasted at the same temperature, only the mean values of each temperature are presented in Table 4.

Table 4. Fatty acid composition of the walnut flour samples submitted to different roasting process (mean ± standard deviation; different letters in the same line represent significant differences p<0.05 between samples). Control 50° 100° 150° C14:0 0.03 ± 0.01a 0.02 ± 0b 0.02 ± 0b 0.02 ± 0b C15:0 0.02 ± 0a 0.01 ± 0b 0.01 ± 0b 0.01 ± 0b C16:0 6 ± 0.01 5.89 ± 0.01 5.85 ± 0.02 5.96 ± 0.01 C16:1 0.06 ± 0a 0.05 ± 0b 0.05 ± 0b 0.05 ± 0ab C17:0 0.05 ± 0a 0.04 ± 0b 0.04 ± 0b 0.04 ± 0b C17:1 0.03 ± 0a 0.02 ± 0b 0.02 ± 0bc 0.02 ± 0b C18:0 2.26 ± 0.01a 2.09 ±0.01b 2.08 ± 0.01b 2.12 ± 0.02b C18:1n9 13.54 ± 0.05b 13.51 ± 0.37 13.88 ± 0.03a 14.11 ± 0.05 C18:2n6 62.52 ± 0.09 62.87 ± 0.31 63.12 ± 0.12 62.74 ± 0.01 C20:0 0.13 ± 0 0.1 ± 0a 0.09 ± 0.01 0.15 ± 0.01b C18:3n3 15.01 ± 0.01a 14.88 ± 0.07b 14.26 ± 0.02c 14.15 ± 0.02c C20:1 0.32 ± 0.01b 0.5 ± 0.05b 0.55 ± 0.05a 0.6 ± 0.07a C20:2 0.02 ± 0a 0.02 ± 0b 0.02 ± 0b 0.02 ± 0b C22:0 0.02 ± 0a 0.01 ± 0b 0.01 ± 0b 0.01 ± 0b ΣSFA 8.5 ± 0.04 8.14 ± 0.04 8.22 ± 0.14 8.36 ± 0.27 ΣMUFA 13.95 ± 0.07 14.22 ± 0.13 14.67 ± 0.24 14.35 ± 0.38 ΣPUFA 77.53 ± 0.13 77.63 ± 0.15 77.1 ± 0.37 77.27 ± 0.49

Some small changes were noticed between the control and the roasted samples, especially in the saturated and monounsaturated FA, however no significant correlation was found between the FA and the roasting temperature used. The

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Product and process innovation percentage of saturated FA, like the C14:0, C15:0, C17:0, C18:0 and C22:0 was lower in the roasted samples, but, as these are minority compounds (represent less than 2.2% of the total FA profile) it could be assumed that the walnut FA were preserved in the roasted samples. The preservation of the FA profile during the roasting may be due to the inactivation of lipoxygenase enzymes of walnut, responsible for catalysing the oxidation of polyunsaturated FA containing cis,cis-1,4-pentadiene units (present in the major FA of walnut oil) to their conjugated unsaturated FA hydroperoxides (Robinson et al., 1995; Buranasompob et al., 2007). Buranasompob, Tang, Powers, Reyes, Clark and Swanson (1995) had reported that a heat treatment at 55 °C for 2 min or greater was able to reduce the activity of lipoxygenase, delaying the development of oxidative rancidity, thus maintaining the FA profile. On the other hand, due to a high percentage of polyunsaturated FA in the walnut lipid fraction, the presence of higher amounts of fat in the flours roasted above 100 °C may represent a risk for its oxidative stability, considering the large surface area exposed to air (Vanhanen and Savage, 2006), so it must be conveniently stored (Labuckas et al., 2011). Then again, the health benefits of walnut oil composition have been extensively demonstrated (Rajaram, 2014), and the fat content may contribute to increase the nutritional value of the flours in order to their inclusion in the formulation of novel products where unhealthy fats could be substituted (Ayo et al., 2007).

3.4. Phenolic fraction and Antioxidant analysis

The walnuts are recognized for their high antioxidant capacity when compared with other nuts, being normally associated to this property to a high phenolic content (Arcan and Yemenicioglu, 2009; Yang et al., 2009; Bakkalbasi et al., 2015). Regarding the phenolic fraction and the antioxidant capacity of the ethanolic extracts of the defatted walnut flours, figure 3 show the obtained results. The determined values were similar to those already published (Arranz et al., 2008; Yang et al., 2009).

Concerning the roasting effect on the phenolic fraction and antioxidant capacity of the flours, all assays showed smaller values for the samples submitted to a more drastic roasting (at 150 ºC for more than 1 hour), with exception for the results

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Antioxidant properties of walnut flour from roasted walnuts obtained in the DPPH method (figure 3c). In more moderate roasting conditions, the flours kept a phenolic and flavonoid content similar to the control sample, showing all a high phenolic content and a higher capacity to reduce the ferric ion. About to the ability to reduce DPPH, the flour that show the best results was the one submitted to a roasting process at 100 ºC for 30 min. Contrarily to the results obtained for the phenolic, flavonoid and in the FRAP antioxidant assay, where a significant correlation was found between those values and the temperature of roasting, the results obtained by DPPH method did not showed a significant correlation with the roasting process (see table 2).

Figure 3. Total phenolic (a) and Flavonoid (b) contents, and antioxidant capacity (DPPH (c) and FRAP (d) assays) of the ethanolic extracts of the walnut flour samples (mean ± standard deviation) (mean ± standard deviation; different letters represent significant differences p<0.05 between samples).

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The antioxidant capacity of the walnut kernel is mostly associated to their phenolic content, being the contribution of oil to the total antioxidant capacity of walnut (mostly due to their vitamin E content) less than 5% (Arranz et al., 2008). In this sense, the defatted walnut flours could keep most of the antioxidant properties of the walnut, which will improve the nutritional profile of this product. It can also help in the preservation of flour, during storage, due to strong free radical-scavenging ability of the phenolic compounds present (Buranasompob et al., 2007; Bakkalbasi et al., 2015). The hydrolysable tannins, ellagitanins (especially glansreginin A), are reported as the major phenolic compounds of walnuts followed by flavanols like catechin and procyanidins and hydroxybenzoic acids like ellagic and gallic acids (Slatnar et al., 2015). Those compounds are more abundant in the kernel coat of the walnut (Arranz et al., 2008; Arcan and Yemenicioglu, 2009), that was kept in the walnuts used in this study, and that certainly contributed to enhance the antioxidant properties of the product.

On the other hand, the use of high temperatures of roasting will not be favourable for the walnut flours antioxidant capacity, as the tannin content could be reduced (Sxe-Tao et al., 2001). On the other hand, the melanoidins formed during roasting have showed some in vitro antioxidant protective effects on lipids (Wang et al., 2011), which will be important in preventing oxidative damage of the walnut flour oil (Zhou et al., 2016). Therefore, melanoidins are probably contributors to the antioxidant activity in the roasted flour samples.

4. Conclusions

The results obtained in this study confirmed the nutritional richness of the defatted flour obtained as a by-product of the walnut oil production. These flours had about 40% of protein and a significant amount of dietary fibre. Although submitted to an oil extraction process, the remaining lipid fraction in the flours had a high nutritional quality confirmed by their vitamin E content and FA profile. The phenolic compounds present in this by-product also represent an advantage for their use, as they can confer high antioxidant capacity. The used roasting procedures did not affect the quality of the flour when more mild conditions were used. At 150 ºC for more than

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60 min, some of the quality traits of the flour could be compromised. In overall, this by-product showed valuable potentials to be further used as a functional ingredient in the food industry for enrichment purposes of food products.

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Effect of almond roasting and addition of different garlic cultivars on oil stability

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Addition of garlic cultivars to almond oil

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Effect of almond roasting and addition of different garlic cultivars on oil stability

ABSTRACT Keywords: The oxidation of commercial oils is a major concern nowadays for producers Prunus dulcis Storage and consumers. In this study, we evaluate the influence on almond oil Oxidative stability of previous almond roasting, light exposure and the addition of four stability Purple garlic different garlic cultivars by two different addition methods. Results show

that previous almond roasting and oil storage in darkness conditions reduce

significantly the oxidation of almond oil over time. The direct addition of garlic shows slight reductions on oils stability. However, differences appear depending on the garlic cultivar and the way of garlic addition. The addition of the whole clove, cause lower reductions on oil stability than the addition of crushed garlic, but only on roasted oils. Within the considered garlic cultivars, the “Purple garlic from Las Pedroñeras” (European Protected Geographical Indication) shows higher protection to oxidation, especially if the whole garlic clove was added into oil from roasted almonds.

1. Introduction

Lipid oxidation causes changes in sensory quality (changes in taste, odour, viscosity and colour) and nutritional value of foods (Tan and Che Man, 1999). During storage, two kinds of oil oxidations occur in oils: photosensitized oxidation and autoxidation (Choe and Min, 2006). In photosensitized oxidation, chlorophylls perform as

1 photosensitizers for the formation of singlet oxygen ( O2) that leads to oil oxidation.

3 On the other hand, in autoxidation, triplet oxygen ( O2) reacts with oil. Autoxidation require lipid radicals (Min and Wen, 1983) and it accelerates if the fatty acids content is high. Studies show that oils that are more unsaturated are oxidized more quickly than less unsaturated oils (Parker et al., 2003). Oxidative stability in oils is mainly determined by its fatty acid profile (Roncero et al., 2016a) and the concentration of

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Addition of garlic cultivars to almond oil minor components with antioxidant properties as phenols and flavonoids (Barreira et al., 2008).

Almond oil is a high valued product that contains bioactive compounds with health promoting properties. Almond fatty acid profile is mainly composed by unsaturated fatty acids as oleic acid (57-79%) and linoleic acid (12-34%). Within saturated fatty acids, palmitic acid is dominant (5-7%) (Roncero et al., 2016b). Thus, according to its profile of unsaturated fatty acids, almond oil is susceptible to suffer lipid oxidation that may lead to changes in the quality and sensory characteristics of the oil in a relatively short period.

The oxidative stability of nut oils has been effectively increased with seed exposition to high temperatures. Some operations made during processing of almond oil like drying, roasting or extraction may increase oil oxidative stability due to the formation of some Maillard reaction products that are reported to be antioxidants (Choe and Min, 2006; Sena-Moreno et al., 2015). Other way to protect oils from oxidation is the addition of antioxidants (Navas et al., 2006; Bodoira et al., 2017). Synthetic antioxidants, such as butylated hydroxytoluene (BHT), have been widely used with that purposes, but recently major interest is focused on the development of natural antioxidants. Although no health risks are associated with regular consumption of artificial antioxidants (Botterweck et al., 2000; Pokorný, 2007), the development of natural antioxidants is expected to be positively valued by consumers. However, compared to synthetic antioxidants, natural antioxidants are less active, so they need to be added in larger amounts to achieve the same effect (Pokorný, 2007). Within the studied natural antioxidants, traditional herbs and spices, mainly from the Mediterranean region, have shown the most promising results (Hraš et al., 2000; Zheng and Wang, 2001; Gramza-Michalowska et al., 2011; Bag and Chattopadhyay, 2015).

The garlic (Allium sativum L.) is an annual herbaceous plant native from Asia that has been widely cultivated around the Mediterranean Basin. Its health promoting characteristics, together with a strong and unique flavour, have made garlic a popular condiment in the Mediterranean diet. Garlic possesses characteristics that make it an

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Product and process innovation interesting product to be added to food products. It shows antifungal, antibacterial, antiviral, antiaggregatory, immunostimulatory, antimicrobial, anticarcinogenic, antimutagenic, antiasthmatic, hypotensive, and antioxidant properties (Corzo- Martínez et al., 2007; Iciek et al., 2009). Garlic antioxidant properties have been attributed to the presence of organosulfur compounds, in particular allicin. Allicin appears when fresh garlic is chopped or crushed and the enzyme alliinase breaks alliin (Rybak et al., 2004). The biological properties from garlic are associated to the apparition of organosulfur compounds derived from allicin (Iciek et al., 2009). Regarding food storage, garlic antioxidant and antimicrobial properties are considered useful to improve the storage conditions of food products (Gheisari and Ranjbar, 2012). As a natural product, it possesses the advantage of being readily accepted by consumers concerned about the use of synthetic food additives (Pokorný, 1991). Beyond its benefits for health and food storage, garlic confers to the food new organoleptic characteristics that may offer new possibilities to consumers.

Garlic can be added to food products directly (Gheisari and Ranjbar, 2012), using macerations (Navas et al., 2006), in the form of extracts obtained by previous infusion (Gambacorta et al., 2007), as ethanolic extract (Epaminondas et al., 2015) or even as supercritical fluid extract (Bravi et al., 2017). Among considered forms of garlic addition, garlic herb extracts showed the less promising results as they have the lowest antioxidant activity between the Mediterranean herbs and spices considered (Gramza-Michalowska et al., 2011). On the other hand, supercritical fluid garlic extracts have proved their effectivity in protecting sunflower oil from oxidation. Garlic extracts effectivity was even higher than the reported with synthetic antioxidants (Bravi et al., 2017). Similar results were also obtained for linseed oil (Epaminondas et al., 2015). The addition of fresh garlic and powder garlic was useful in preserving meat products (Gheisari and Ranjbar, 2012). Maceration of refined corn oil with garlic also improved oil stability at 55 ˚C, but not at 140 ˚C (Navas et al., 2006).

The effect of adding an antioxidant on a specific product must be individually checked, as antioxidants activity changes when they are added to different products. Antioxidant activity depends on factors such as solubility, stability during processing and possible interactions with other components (Gramza-Michalowska et al., 2011).

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Addition of garlic cultivars to almond oil

In this study, we evaluate the effects of maceration of different garlic cultivars in the stability of almond oil obtained from natural and roasted almonds. In addition, the effect of a previous roasting treatment to almonds and the light is also considered.

2. Materials and methodology

2.1. Roasting and oil extraction

Almonds from Guara cultivar were collected at the experimental orchard in the Instituto Técnico Agronómico Provincial of Albacete in the southeast Spain in 2015. Almonds were collected manually, shelled under controlled conditions and selected to eliminate those with dark spots. Then, almonds were dried at room temperature until they reached a moisture content lower than 6%. To evaluate the effect of roasting in the stability of oil, a batch of almonds was placed in a monolayer in an oven and subjected to a thermal treatment at 150˚C for 30 minutes.

Oil was extracted with a hydraulic press (MECAMAQ Model DEVF 80, Vila-Sana, Lleida, Spain). Almonds were previously ground and placed on the press at a pressure of 150 bar for 20 minutes. After oil extraction, a centrifugation step was carried out to eliminate the remaining solid residues from the samples. The oil was stored in 50 mL bottles at room temperature to simulate standard storage conditions. To evaluate the effect of light, three aliquots were stored in transparent glass bottles, while the rest was stored in dark glass bottles.

2.2. Garlic cultivars and maceration

Garlic samples were kindly provided by the Garlic Cooperative San Isidro El Santo from Las Pedroñeras, Cuenca (Spain). To evaluate the effect of garlic addition to the almond oil, four different garlic types were evaluated: common white garlic, violet or Chinese garlic, purple garlic from the Protected Geographic Indication (PGI) “Ajo Morado de Las Pedroñeras” (EC, 2007) and black garlic (fermented purple garlic) (Kimura et al., 2017).

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The use of maceration of unprocessed garlic in oil was selected as the best way for garlic addition as it was considered the most natural way to add this product. Maceration consisted in the addition of 2 g of unshelled garlic into 50 mL of oil from unroasted and roasted almonds. In addition, garlics were added in two forms: whole or chopped. Three replicas were made for each sample. All the samples were stored in dark glass bottles with a reduced head space at room temperature.

2.3. Oxidative stability

Oxidative stability of the oils was measured every 15 days during a 75 days period by the rancimat method (Gutiérrez, 1989). Stability was expressed as the oxidation induction time (hours), measured with the Rancimat 743 apparatus (Metrohm Co., Basel, Switzerland). An oil sample of 3.5 g was used, warmed to 100°C under an air flow of 10 l h-1. By analysing the oxidative stability, we are able to provide information about a parameter of main interest, especially for food industry, as it measures the quality of the oil and the shelf life of the product. The oxidative stability collets the effects and interactions of numerous antioxidants and provide a specific value about the current state of the oil.

2.4. Statistical analysis

The data were tested for statistical significance using the ANOVA (analysis of variance) test from the statistical package SPSS 23 version (SPSS Inc, Chicago, IL, USA). Differences between means of multiple measurements were separated with the Duncan's test (p < 0.05) and T-test (p < 0.05). To evaluate the effects of roasting, light, garlic cultivar added and influence of garlic structure (whole/crushed), the statistical and graphical analysis of groups within the different oil samples were performed.

3. Results and discussion

3.1. Effect of roasting and light exposure on oil stability

Almond roasting, previous to oil extraction, increases the stability of almond oils due to the development of Maillard reaction products with potential antioxidant activity

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(Sena-Moreno et al., 2015). Figure 1 shows the results of oxidative stability of oils from unroasted and roasted almonds. In the first measurement, made immediately after oil extraction, stability of oil obtained from roasted almonds is significantly higher (31.2 h) than the reported in oil from unroasted almonds (26.4h).

A decrease in the oxidative stability is observed during the storage period, significantly greater when light is considered. As expected, light is an important parameter to consider when analysing oil stability over time, as it has been previously reported for pumpkin, pistachio or chia oils (Bodoira et al., 2017; Naziri et al., 2016; Sena-Moreno et al., 2015). If oils are stored in darkness conditions, the reduction in the stability values is smaller. In oil from roasted almonds stored in darkness, after 75 days of storage, the accumulated reduction of oil stability was 1.1 hours, while in oil from unroasted almonds stored in darkness was 4.3 hours. However, when oils were stored exposed to light a sharp fall in the stability values was observed. When oils were exposed to light the reduction was similar, regardless roasting, reaching up to 13.4h in oil from unroasted almond and 14.1h in oil from roasted almonds.

35.0

30.0

25.0

20.0

15.0 Oxidative Stability Stability (hours) Oxidative

10.0 Day 0 Day 15 Day 30 Day 45 Day 60 Day 75

RO NO(L) NO RO(L)

Figure 1. Evolution of oxidative stability values in almond oils obtained from roasted and unroasted almonds and stored in light and darkness conditions. RO oil from roasted almonds stored in darkness; RO(L) oil from roasted almonds stored in light conditions; NO oil from natural almonds stored in darkness; RO(L) oil from roasted almond stored in light conditions.

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3.2. Effect of garlic addition on oil stability

The addition of garlic to oil has been reported to increase oil stability (Bravi et al., 2017; Navas et al., 2006). However, when garlic was added to almond oil, opposite results were obtained (figure 2). Stability of almond oils without added garlic barely changed in the studied period; however, the addition of garlic reduced the stability of almond oil during the storage. This reduction may be attributed to hydrolysis processes together with the activity of garlic enzymes in almond oil components. It should be considered that the direct addition of unprocessed garlic involves the addition of significant quantities of water (about 63% of fresh garlic weight) (Kimura et al., 2017). Moreover, natural garlic has a wide range of enzymes that could interact with oil components, accelerating oxidation processes. Although direct addition of garlic increases shelf-life of products as meats (Gheisari and Ranjbar, 2012), direct addition of garlic into oil (maceration) has received minor attention compared to garlic extracts (Bravi et al., 2017; Epaminondas et al., 2015). Maceration by adding natural garlic into almond oil failed to improve oil stability. Previous studies have reported the effectiveness of garlic for improving the stability of refined corn oil at high temperatures by limiting the formation of peroxides (Navas et al., 2006). Those high temperatures could affect water content or enzymatic activity, which are proposed as responsible for the reduction in stability (Calín-Sánchez et al., 2014). Moreover, different behaviour is expected depending on the oil analysed as solubility and interactions of garlic with oil components may vary (Gramza-Michalowska et al., 2011) so the study of every single oil need to be developed.

Some differences appear on oil stability depending on the garlic cultivar added. Previous in vitro experiments have shown different antioxidant activities for the garlic cultivars considered (Bhandari et al., 2014; Chen et al., 2013; Denre et al., 2013), but those differences need to be evaluated when garlic is added to a product. When the different garlic cultivars were added to almond oil, significant differences in the oxidative stability of the oils were found after 75 days stored (p<0.05, Duncan test) but not before. Higher stability values were found for the purple garlic from PGI “Ajo Morado de las Pedroñeras” although significant differences with white garlic were not reported. The higher stability values of oils with purple garlic could be attributed

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Addition of garlic cultivars to almond oil to its higher content of allicin (EC, 2007). However, the medians of stability values of garlics white and violet were higher than the reported for purple garlic (Figure 2). High antioxidant activity was expected for black garlic (Toledano Medina et al., 2016), however it showed the lower medium stability values.

Figure 2. Evolution of almond oils stability during storage without garlic addition and with different garlic cultivars added. Different letters on the same storage times indicate significant differences between samples (Duncan Test, p<0.05).

3.3. Effect of roasting and garlic addition method on oil stability

Figure 3 shows the effect of garlic maceration (whole or crushed garlic) in the stability of oils from roasted and uroasted almonds. No significant differences were found in the oil stability of oils from unroasted almonds depending on the addition of whole or crushed garlic. However, when the garlic was added to oil from roasted almonds, the oxidative stability was significantly higher after 75 days of storage if the whole garlic was added (T-test, p<0.05). The garlic crushing is reported to convert alliin into allicin, with the development of organosulfur compounds with antioxidant properties (Rybak et al., 2004). However, the degradation effect in almond oil expected due to enzymes that appear because of garlic crushing may be higher that the protective effect of the resulting organosulfur compounds.

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Further analysis of the influence of whole garlic addition to oil stability showed differences among garlic cultivars (figure 4). Within the considered garlic types, the purple garlic from PGI “Ajo morado de Las Pedroñeras” showed the highest stability values. Further statistical analysis showed significant differences (Duncan test, p<0.05) between purple garlic addition with the rest of garlic cultivars in which differences were not reported.

Figure 3. Effects of adding the whole garlic or crushed garlic on the oxidative stability of oils obtained from natural and roasted almonds after 75 days stored.

Figure 4. Evolution in the oxidative stability values (hours) of roasted almond oils. RO roasted almond oil (control sample); RWW Roasted oil Whole White garlic; RWV Roasted oil Whole Violet garlic; RWP Roasted oil Whole Purple garlic; RWP Roasted oil Whole Black garlic.

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4. Conclusion

A previous roasting of almonds increased the oxidative stability of oils. During storage period, the light plays a crucial role to maintain stability on both kind of oils, from roasted or unroasted almonds. Thus, almond oil stored under light conditions showed a quick decrease in oxidative stability while in the oil stored in the dark the reduction in stability was moderate. Maceration with garlic reduces the oxidative stability of almond oil. However, when the oil is elaborated from roasted almonds, this reduction is lower. Among garlic cultivars, purple garlic offered the best results, as the reduction of oil stability was lower, especially when the whole garlic is used for maceration on oil from roasted almonds.

References

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ABSTRACT Keywords: The interest of pistachio trade relies in the strict food safety standards that are US Iran applied on pistachio exports due to aflatoxin contamination. Furthermore, the Exports two major pistachio producers (the US and Iran) are countries with a different Aflatoxins Legislation level of development of sanitary control on this production. The aim of this

study is to identify if the aflatoxin standards have acted as a catalyst or as a barrier in the pistachio exports from the US and Iran during the period 1996 to 2013. The influence of technological and non-technological innovation on exports has been also considered to analyse if innovation is driven by aflatoxin control measures. Results confirm that both countries obtain benefits from stricter food safety standards all over the world. However, some differences appear in the drivers of pistachio exports between the main producers, as US exports are not negatively affected by the distance with the importer (higher differentiation) and they also obtain higher prices (quality premium). Attending to the results it can be concluded that stricter requirements regarding food safety are positive for both producer countries, regardless of their level of economic development, and for consumers, resulting in a “win-win” scenario.

1. Introduction

The influence of food safety standards on agricultural products trade has attracted wide interest of policy makers in the last years. Since the creation of World Trade Organization (WTO), policy barriers as tariffs have lost importance in favour of other commercial obstacles: the non-tariff barriers (NTBs). These NTBs, mainly Sanitary and Phyto-Sanitary Measures (SPS) and Technical Barriers to Trade (TBT), have been used by importing countries to restrict global trade in the last decades (Chen et al., 2008; Ardakani et al., 2009).

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In the context of trade liberalization and globalization, agricultural and food markets are more affected for these regulatory measures than other markets due to the increasing demand of “safe” foodstuffs by consumers (Li and Beghin, 2014). The rise of economic development has drastically improved living conditions and, consequently, consumer demand pressure and consciousness about health food issues has increased. In this regard, food scares explain strict SPS regulations all over the world. The Spanish Toxic Oil Syndrome in the 1980s; the mad cow disease at a global scale in the 1980s-1990s; the Belgium animal feed contaminated with dioxins in the 1990s; or most recent food scandals as the Baby milk scandal in China or the E. coli outbreak in US are well-known contamination food incidents with important economic and social consequences to be evaluate by academic researchers.

This new situation justifies the use of health and food safety motives to restrict the trade of agri-food merchandises. Since food safety must be guaranteed, public organisms and regulatory agencies are in charge of supervising the accomplishment of minimum quality standards in domestic and international trade flows of fresh and processed food and beverages. Although several studies have showed that strict food standards cause important economic losses to poorest exporting countries (Chen et al., 2008; Jongwanich, 2009; Bao and Qiu, 2012; Drogué and DeMaria, 2012; Li and Beghin, 2014; Melo et al., 2014), policy makers impose standards to guarantee a minimum level of food safety to their inhabitants in developed and less-developed economies. The main reason that justifies this decision is that participation in international trade requires compliance with food safety standards. Thus, farmers and food industry producers -from developed and developing countries- are required to comply with food safety regulation to avoid the loss of demand and consumer´s trust.

Technically, food may be accidentally or deliberately contaminated by microbiological, chemical or physical hazards, but there are some sensitive agricultural products with a higher sanitary risk. This is the case of pistachio that has been identified as the raw food that presents the highest risk of aflatoxin contamination (Pittet, 1998). To prevent the mentioned risk and protect their

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Economics citizens, most of the countries have established regulatory limits of allowable aflatoxin levels over imported products.

In general, developed countries have more restrictive standards while less-developed countries have no limits or they establish high limits for aflatoxins. From 10 µg/kg in the EU to 40 µg/kg or any limitation in some developing countries, the main pistachio exporter countries face a dissimilar level of stringency depending on the market destination. The stricter standards established mainly by developed countries, and especially by the European Union (EU), have been accused of acting as a trade barrier to developing countries exports (Otsuki et al., 2001).

Given the discussion above the main objective of this study is to examine the influence of food safety regulation on pistachio exports from US and Iran taken into account the stringency in maximum concentration of aflatoxins in each of these importing nations. For this purpose, the different patterns of the two main pistachio exporters in the world (US and Iran) have been taken account. Beyond that, different measures based upon aflatoxins standards have been used to calculate the level of food stringency in the market for this crop. Finally, the study estimates the gravity equation using different measures of food safety regulation and econometrical procedures to compare the results for US and Iran.

2. Literature review and theoretical framework

2.1. Pistachio Market

Native from the Middle East, the pistachio (Pistacia vera) has long been a popular crop in arid areas of the Mediterranean basin, middle East and US due to is tolerance to hot and dry conditions. World pistachio production has increased sharply in the last twenty years (with a 2% and a 5.7% of annual growth in harvested area and production) (FAO, 2016; IPA, 2016).

The major producers of pistachio in the world are five countries: United States (US), Iran, Turkey, China and Syria. In the year 2013, US at 196,930 tonnes and 32.4% of the world’s production, Iran at 170,000 tonnes (28.0%), Turkey at 88,600 tonnes

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(14.6%), China at 74,000 tonnes (12.2%) and Syria at 54,516 tonnes (9.0%) (FAO, 2016; IPA, 2016). Regarding this data, the US and Iran show clearly a dominant position in the world pistachio production as both of them comprise the 70.7% of world’s production in the period 1996–2013. However, the production quotas and the export destinations have changed drastically during this time.

Important fluctuations in the volume of pistachio exports from the two main producers have been observed. Despite that Iran led pistachio exports with 100,000- 150,000 tonnes each year in the past, the continuous increase of the US exports in the 2000’s, together with the reduction of Iran exports from 2007 (due to a food safety incident at the EU borders) explains that US exports surpassed Iranian exports for the first time in 20091. Nowadays, the US exports keep growing and the gap with Iran increases every year.

2.2. Food Safety and Aflatoxins

Aflatoxins comprise a group of secondary metabolites of toxins produced by some species of Aspergillus (mainly Aspergillus flavus and Aspergillus parasiticus). Aflatoxin contamination primarily occurs in maize, spices, groundnuts, tree nuts (pistachios, almonds, hazelnuts, pecans, etc.) and milk, and can appear almost at any production or commercialization stage, in the field, at harvest, during post-harvest operations and in storage.

Although numerous residues have been described, four aflatoxins are recognised in legislation: B1, B2, G1 and G2. These aflatoxins were long ago identified as carcinogenic to humans for the International Agency for Research on Cancer (Anttila et al., 2002). Among them, aflatoxin B1 is considered the most toxic and the strongest chemical liver carcinogen known (Wu and Guclu, 2012).

Aflatoxins represent a risk for human health both in the short and in the long run. Acute aflatoxicosis, caused by consumption of a high dose of aflatoxins in a short time, cause severe gastrointestinal symptoms and even death (Wu and Guclu, 2012).

1 In the years 2010 and 2011, the stagnation of US exports enabled Iran to leader again the world pistachio exports, but that situation did not last long. 326

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One of the largest documented aflatoxicosis outbreaks arose in rural Kenya in the year 2004 with 317 cases and 125 deaths (Wild et al., 2015). Although acute aflatoxicosis is not a risk in developed countries, continuous ingestion of low levels of aflatoxins over a long period causes liver cancer and other disorders as cirrhosis, chronic hepatitis or jaundice (Kuniholm et al., 2008).

The reduction of aflatoxins sanitary risk has been a major challenge in global food security in the world. However, as aflatoxins can hardly be completely eliminated of some foods, it has been assumed that absolute safety is never achieved. Developed countries reduce harmful exposure by imposing regulatory limits of aflatoxins concentration on the commodities that present the higher risks (as pistachio) food monitoring and the implementation of optimal drying and storage practices (Brown et al., 1999; Strosnider et al., 2006). On the other hand, fewer advances have been made in developing countries where more than 5 billion people are at risk of chronic exposure to aflatoxins through contaminated foods (Strosnider et al., 2006).

To prevent risks caused for aflatoxin contamination in pistachio, most of the countries establish maximum concentration levels of aflatoxins in imported nuts. Different limits appear in the legislation of each country depending on the risk they “want to assume”. In general, less-developed countries have no limits or put high limits for aflatoxins while developed countries, and especially the EU, have more restrictive standards.

Restrictive standards cause economic problems to the countries that export vulnerable and high-risky products due to greater control needed (Otsuki et al., 2001). The rejection of shipments in the borders not only increases economic losses, they also cause social alarm and damage the exporter reputation. However, restrictive food safety standards benefit high-quality producers motivated by the achievement of high prices due to premium-quality related to food safety. Furthermore, restrictive limits could even benefit lower-quality export markets by forcing them to adopt technologies and methods to control food quality resulting in a “win-win” situation (Wu, 2008).

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2.3. Influence of Food Safety Standards on Food and Pistachio Trade

In the last decades, food markets have changed from price-based to quality-based competition. Increases in income and living standards in the developed countries led to a demand intensification of high quality and safe food products (Kinsey, 1993). These characteristics exert a prominent role in global food markets (Hooker, 1999). The study of how food safety affects trade has become an actual topic of debate.

Maskus et al. (2001) claim the lack of empirical evidence about how food safety legislation affects trade. Since this research, numerous empirical studies have analysed the effects of food safety regulation on trade. Different perspectives have been developed, with studies focusing on the effects of specific regulations in the trade of individual products (Wilson and Otsuki, 2004; Dal Bianco et al., 2016;), the consequences of different regulatory frameworks on trade (Schlueter et al., 2009) or the influence of regulation on the exports of countries distinguishing different levels of economic development (Disdier et al., 2008; Karandagoda et al., 2014; Liu and Yue, 2012; Melo et al., 2014).

Regarding food safety measures, most of studies have used the Maximum Regulatory Limits (MRL) of regulation in each country as a measure of food safety for affected products (Wilson and Otsuki, 2004; Wei et al., 2012; Xiong and Beghin, 2014). Additional variables as the number of border rejections (Jaud et al., 2013) or the number of restrictive applicable regulations affecting a product (Disdier et al., 2008; Kareem, 2016) have also been considered. Beyond raw data, diverse stringency indexes also have been proposed to capture the effect of food safety regulation on food trade (Melo et al., 2014).

The results of the empirical studies have proved that food safety regulation has benefits or no effects on the exports from developed countries (Disdier et al., 2008; Liu and Yue, 2012). On the other hand, the effect on the exports from developing countries have been identified as negative in most of the studies (Chen et al., 2008; Jongwanich, 2009; Bao and Qiu, 2012; Drogué and DeMaria, 2012; Li and Beghin, 2014; Melo et al., 2014), but opposite results have also been described (; Xiong and Beghin, 2014, Dou et al., 2015; Ishaq et al., 2016). Differences in the product

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Economics analysed, the exporter country and the food safety variable used seem to lead to different results in the empirical analysis.

Regarding food safety in pistachio exports, there are important differences between the two main producing countries. Historically, Iranian pistachios have contained a high average aflatoxin concentration while US pistachio production had levels below 10 ppb on average (JECFA, 2007). As far as we know, only five studies have tried to evaluate the importance of food safety in pistachio trade (Wu, 2008; Ardakani et al., 2009; Alaeibakhsh and Ardakani, 2012; Zheng et al., 2012; Bui-Klimke et al., 2014).

The first study about the influence of the EU aflatoxin standard on the pistachio exports is made by Wu (2008). This author analyses the impact of the strict EU aflatoxin standard on the US almond and pistachio exports during the period 1998- 2004. Based upon a straightforward analysis, the author argues that producing countries could benefit from strict aflatoxin standards if producers are able to achieve a high-quality good. Otherwise, the consequences on pistachio exports will be very negative. In this regard, negative effects of NTBs in pistachio exports from Iran were proved by Alaeibakhsh and Ardakani (2012) for the period 1996-2008. In an early study, the same authors showed that an increase of 1 percentage point in the total agricultural NTBs decreases Iran pistachio exports 2.24 percentage points, being this reduction more important than in other Iranian products - raisins and shrimp (Ardakani et al., 2009). Thus, NTBs matter in Iran exports.

The influence of rejections and food alarms in US pistachio trade has been also analysed. Zheng et al. (2012) showed that food safety shocks in Iranian pistachio had a negative and significant impact in the US pistachio consumer confidence. This fact was related to the US consumers’ association of Iran pistachio safety problems with the product itself. On the contrary, US pistachio safety shocks showed a positive effect on the export demand function. This unexpected result can be explained by the importance of reputation: US consumers value positively the identification and notification of low impact incidents of US pistachios as it shows that control safety mechanisms are efficient.

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Finally, Bui-Klimke et al. (2014) is the last study about pistachio trade regarding food safety. Using network analysis, they represent the volume of pistachio trade from US and Iran in the years 1996, 1997, 2004, 2008 and 2010 to prove the different influence of aflatoxin legislation depending on the origin of the pistachio (Iran or US). But even in the light of this pistachio world market segregation, their findings about the influence of aflatoxin limits in the importer country on pistachio trade are not conclusive. This conclusion is achieved because countries without aflatoxin limits import pistachios from the US (China) while other countries with a restrictive legislation (i.e., Russia with 10 ppb) import pistachios from Iran. Thus, further research is needed to assess the incidence of aflatoxin legislation on trade. In this sense, other influencing factors as consumer preference, cultural proximity and information cost related to the two major producing countries (US and Iran) must be considered.

2.4. The role of technological and non-technological innovations in the pistachio market.

To gather the level of introduction of technological innovation by pistachio farmers is a complex issue. Although technological innovations improve efficiency in the agricultural production along the time, each crop requires a special focus. The continuous mechanization of agriculture practices together with the introduction of new chemicals (from fertilizers to phytosanitary products) boosts agricultural yields of all products in the world (including pistachios). Although researchers agree about the positive relationship between inputs use intensification and yields (Reidsma et al., 2009), some authors argue that there is also a decoupling between inputs and output due to environmental concerns mainly in developed countries (Levers et al., 2016). This observed reduction on the use of inputs in agriculture would be determined by the introduction of new technologies and more efficient production systems (for example, precision agriculture systems). Regarding the pistachio production, the level of technological innovation has substantially increased yield in the last decades. The introduction of technological innovations in the sector affects the production per hectare and, consequently, the competitiveness and position of the two main producing countries in the world pistachio market. On one hand, Iran

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Economics has an average annual yield of about 1000 kg/ha, while the annual yield in the US is usually about 3000 kg/ha, occasionally reaching up to 4000 kg/ha (FAO, 2016; IPA, 2016). Changes in US pistachio yields are mainly attributed to alternative bearing of pistachio tree. This situation shows the high agro-technological level of intensification of this production in which agricultural practices enhancing a high production one year usually result in lower production in the following year, intensifying the alternative bearing effect. Thus, US pistachio production is more capital-intensive (machinery, efficient irrigation, fertilization…) while Iran pistachio production is more labour-intensive.

However, non-technological innovations must also be considered. Marketing and organizational innovations that happen at macro and micro-level must be taking into account (Garcia and Calantone, 2002). Using the Oslo Manual by OECD (2005), non- technological innovations are “all the innovation activities of firms which do not relate to the introduction of a technologically new or substantially changed good or service or to the use of a technologically new or substantially changed process… (but includes) the organisational and managerial innovations” (OECD, 2005). Thus, the implementation of advanced management techniques, i.e., the introduction of Total Quality Management systems for control aflatoxin contamination, new organisational structures with economic impact could be considered as non- technological innovations.

In this regard, the Iran Pistachio Association (IPA) was established in 2007 to promote and protect the interests of the pistachio industry of Iran. This association is formed by private owners of pistachio business, from growers and processors to service providers and exporters. The main objective of the association is to promote good agricultural and manufacturing practices, storage and transportation facilities, and the extension of credit for trade and export promotion. For this purpose, three different commissions were created to cover the main concerns of Iranian pistachio industry: horticulture, processing and trade (IPA, 2016). IPA main projects are related with the modernization of pistachio production in Iran and, especially, with the improvement of pistachio food safety to ensure profitable exports.

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In the case of US, the California Pistachio Commission was the first organization created to boost US pistachio production and quality in the year 1981. Financed by a California producers payment of $0.035 per pound of pistachios, this Commission provides support to California pistachio producers through public relations, research, government relations and promotion of pistachio exports in international markets (with additional funding from the US Market Access Program). Twenty-five years later, Federal Marketing Order for Pistachio is created following USDA’s Agricultural Marketing Service recommendations. After this decision, the production of safe and high quality pistachios, with special attention to aflatoxin standards in the pistachio exports become one of the main tasks of this organization. The consequences of this approval, beyond the increase in the US pistachio production and exports, are analysed by Gray et al. (2005). Based upon stochastic dynamic simulations of the industry under different scenarios, they show that this decision has been beneficial for producers from the US and the world in cost/benefit terms.

2.5. Influence of Food Safety Standards and Innovation on Pistachio trade.

The influence of food safety stringency and different types of innovation are going to be considered in this work. According to the review of literature in the section, the influence of food safety standards on trade does not depend on the level of economic development of exporting country. In this regard, authors as Wu (2008) argues that “even lower-quality export markets may benefit […], because it forces them to adopt technologies and methods to control food quality”. This means that the adoption of food safety standards leads to positive results for all the concerned parties (importing and exporting countries). To achieve this positive outcome, technological and non- technological innovation are needed to increase the perceived-quality of internationally traded pistachio production. If innovation is driven by control aflatoxin (market-push or regulatory pull-push), pistachio producers could increase their exports and profitability. Moreover, consumers have safer foods both domestically and abroad.

Both theoretical explanations are going to be considered in this work. On the one hand, technological innovations enable to achieve the quality standards

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Economics requirements to satisfy the requirements in food international markets. On the other hand, non-technological innovation facilitates the control, monitoring and accomplishment of these food safety standards taken into account the regulatory stringency on import markets.

3. Data and Methodology

3.1. Data

The data used to study the influence of aflatoxin standards on pistachio trade are now presented (table 1). Trade data come from the United Nations Commodity Trade Statistics Database (COMTRADE). We focus on bilateral imports of pistachio (HS1992: 080250 Pistachios). Information from the two main exporting and producer countries of pistachio, namely US and Iran, are selected and taken separately to analyze their exports to all importing countries over the period 1996-2014. Due to missing values for some years in this database for Iran, information provided by the Iran Pistachio Association (IPA) has been also used.

Regarding aflatoxin regulation for pistachios, information comes from FAO (FAO, 1997, 2004), Bui-Klimke et al. (2014) and national legislations for importing countries. Similar to Bui-Klimke et al. (2014), unchanged standards during the period without news about changes on aflatoxin standard are assumed. Most of countries usually establish the aflatoxin standards by the maximum allowable concentration of total aflatoxin that can appear on foodstuff taking into account the sum of all aflatoxins (B1, B2, G1, and G2). However, some countries only legislate about the most dangerous aflatoxin, the B1. To solve this potential bias in the aflatoxin indicator, if this is the case, the maximum concentration of aflatoxin B1 standard is multiplied by two (Wu and Guclu, 2012). Moreover, if the country does not give information about pistachio maximum aflatoxin concentration, the legislation reference regarding to nuts is considered.

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Table 1. Definition of variables and source

Variable Description Source Exports value Exports (constant US$) COMTRADE data GDP Country income (constant US$) World Bank data Distance between capitals Distance CEPII data (kilometers) Own elaboration using COMTRADE Price Constant US$ by kg data Common =1 if countries share language CEPII data language =0 otherwise =1 if countries has a colonial past Colonial links CEPII data =0 otherwise =1 if the exporting and the destination Country belong to the RTA Own elaboration using WTO data same trade agreement =0 otherwise UNCTAD - Trade Analysis Tariffs Average tax on pistachio imports Information System (TRAINS) Own elaboration using FAO (FAO, Maximum level of total aflatoxins AflatStand 1997, 2004), Bui-Klimke et al. permitted (ppb) (2014) and national legislations. Own elaboration based in the Restrictiveness purpose made by Ferro et al. index 1 (2015) Restrictiveness Own elaboration index 2 Technological Own elaboration using FAO and innovation in Average Kg/hectare of pistachio IPA data the production =1 after the creation of the Own elaboration based in the Management Federal Marketing Law purpose made by Zheng et al. innovation US =0 otherwise (2012). Management =1 after the creation of the Iranian innovation Pistachio Association Iranian Pistachio Association (IPA) Iran =0 otherwise

To consider the mediating role of technological process innovations on the influence of food safety stringency on pistachio exports, the annual average crop yield of pistachio in US and Iran are considered. For its calculation, data of pistachio production for US and Iran from the FAOSTAT are consulted. Furthermore, some corrections of FAO data are made using IPA data when pistachio yields for Iran are implausible. US and Iran pistachio prices are also estimated by dividing the pistachio export value by the quantity of export to each importing country. Data quantities (in value and in volume) for these prices estimations come from COMTRADE. Finally, the influence of the introduction of non- technological innovation related to the control

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Economics of quality and safety on pistachio exports from US and Iran are proxied with two dummies that capture the creation of the Federal Marketing Order for US pistachios in the year 2005 and the creation of the Iranian Pistachio Association in the year 2007.

Tariffs have been obtained from the UNCTAD - Trade Analysis Information System (TRAINS). The tariff included is the simple average tariff rates of included HS 6-digit subheading products. Similar to Ferro et al. (2015), tariffs will be introduced in the gravity model as (1 + tariff) to the sample attrition due to zero values for tariffs in some cases. For the identification of the influence of Free Trade Agreements, the information from the World Trade Organization (WTO) website (www.wto.org) has been used.

The data on GDP of importing countries were compiled from the World Bank’s World Development Indicators. The data required for other gravity variables, like distance, common border, common language, and colonial tie are from CEPII (http://www.cepii.fr/CEPII/en/bdd_modele/presentation.asp?id=6). The total sample for this study has 2,071 observations; of these 1,035 and 1,272 are zeros for US and Iran, respectively. Some of these zeros may be due to the incompleteness of the trade data, while the others represent true zero trade among countries.

3.2. Methodology: Gravity Model

In a traditional gravity model of trade, the volume of exports between pairs of countries depends on their GDP or disposable income, their geographical distances and other variables as a common language, border or religion. These last variables are often dichotomous, analysing the influence of factors as adjacency, have common language or religion or be part of a regional trade agreement on trade flows. Trade is expected to be positively related to GDPs and negatively related to distance. Most of studies have used the gravity model to assess the effect of the stringency of the SPS standards on trade flows. Nevertheless, it is interesting to discuss how each variable could affect international trade.

GDP is one of the fundamental variables in gravity models. When economies grow, trade expands in the extensive margin (more products are traded) and at the

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Influence of food safety on pistachio trade intensive one (larger quantities of products are traded) (WTO and UNTACD, 2012). Trade is also positively influenced by other variables associated to the size of economies such the exporting infrastructure and/or quality control facilities (García- Álvarez-Coque and Martí Selva, 2007). GDP in gravity models is especially significant when trade flows of aggregated data are considered, but when working with specific products the increase of countries GDPs may not be significant.

The role of geographical distance is also essential in gravity models. Haveman and Thursby (1999) observe a decline in the importance of distance variables on trade pattern in the last decades. This trend has been attributed to technology-induced reductions in transportation costs. However, the effect of distance on international trade of food depends on each agricultural product (Wang et al., 2000). The reduction of the distance effect has been smaller in bulk commodities, as their transportation had experienced less development than in the processed food (Jongwanich, 2009; Wang et al., 2000). Considering 243 different food products, Ferro et al. (2015) conclude that greater distances restrict both the probability of trade as well as trade intensity. Even for the trade of non-perishable products such as wine (Dal Bianco et al., 2016) and cereals, dried fruits and nuts (Otsuki et al., 2001) the geographical distance shows a significant and negative effect on trade.

Given the difficulties to take into account the quality differences of trade exports, price estimations for each market destinations are considered in the analysis of agricultural products trade. In this regard, Curzi and Pacca (2015) proved that quality and price in European manufactured food products were imperfectly correlated using the model purposed by Khandelwal (2010). However, this result could be not considered conclusive because these models are implemented for various products and not for a specific food product. In the case of foodstuffs, quality is related to different characteristics of the product: its organoleptic value, its nutritional value and its fulfilment of sanitary or phytosanitary measures. Products accomplishing with the strict standards of food safety achieve a quality premium. Furthermore, the production of safer products is considered to be more expensive than the production of less safer products as the marginal cost of an additional unit of food safety is very high (Swinbank, 1993). Since high food safety increase the income elasticity of food

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Economics products, price and quality must be correlated. In this direction, Asche et al. (2015) used the price as a quality proxy to study the seafood trade among developed and developing countries. Moreover, the origin of products itself constitutes a quality indicator for consumers in some products. In this case, the consumers understand that product quality is closely related with the income of the producer country (Hudson and Jones, 2003). Thus, a high price could be associated to high quality.

The difficulties to find data about bilateral tariffs over time justify that researchers use dummies of Regional Trade Agreements (RTA) to capture tariff barriers effects (WTO and UNTACD, 2012). RTAs will reduce the tariffs that cut-off trade flows, but this reduction does not affect equally to all products. Therefore, the influence of the RTA on the trade of specific products is very limited. Furthermore, the RTAs rarely are going to affect the tariffs imposed to primary food products due to the reduction of commercial protectionism of agricultural trade products in the last period (Anderson, 1979). Empirical studies show that effect of RTAs on agricultural trade is uneven and depends on geographical zones. For example, the NAFTA did not reduce significantly the border effect for agricultural trade among Canada, US and Mexico (Furtan and Van Melle, 2004) while an important mitigation of the border effect in the EU-intra trade has been achieved for the European countries, at least on the trade of fruits and vegetables (García-Álvarez-Coque and Martí Selva, 2007).

Regarding the inclusion of tariffs, trade liberalization has diminished the interest of this variable in an international context in which NTBs dominate policy barriers. Haveman and Thursby (1999) proved that in agricultural and processed food sectors the reduction effects on trade of NTBs is greater than tariff reduction effects. The same result was found by Olper and Raimondi (2008) for the trade of food-processed products between the US, Japan and Canada. Similar conclusions were also found in the research focused on specific products: NTBs have a larger effect on trade reduction than import tariffs in the Chinese exports of vegetables and fish and aquatic products (Chen et al., 2008) and in Iranian exports of pistachio and shrimp (Ardakani et al., 2009).

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Influence of food safety on pistachio trade

To capture the influence of information cost on trade dummies for common language, adjacency or other relevant cultural characteristics such as religion or had shared a common colonial past are used in gravity models. These proxies have proved its utility for capturing the differences in information costs on trade (Rauch, 1999). For countries with similar business practices, competitiveness and delivery reliability, lower search costs are also expected (WTO and UNTACD, 2012). The information- related costs or the consumer preferences compared to the role of NTBs on trade has also been included in the trade analysis (Anderson and Van Wincoop, 2004). Results show that in processed food trade, information-related costs and consumer’s preferences matter more in explaining the border effect than policy barriers (Olper and Raimondi, 2008). If all agricultural products are considered, non-trade distortionary factors result especially important (Olper and Raimondi, 2008).

The possibility of combining the use of binary variables together with continuous variables makes gravity method especially useful for the purpose theoretical framework for understanding the trade flow between US-Iran and the rest of the world.

Regarding the specification, the gravity model in its generalized form is:

푉푖푗푡 = 푓 (푋푖푡, 퐼푗푡, 푅푖푗푡) (1)

where Vijt represents the export value of pistachio from country i (i= US or Iran) to country j in the year t, while Xit represent the attributes depending on exporting country i, Ijt represent the characteristics of importing countries and Rijt the other variables to be included in the model during the period.

The log-linear version of gravity model is:

푙푛푉푖푗푡 = 훽0 + 훽1 ∗ 푙푛퐺퐷푃푗푡 + 훽2 ∗ 푙푛퐷퐼푆푇푖푗 + 훽3 ∗ 푙 푛(1 + 푇퐴푅퐼퐹퐹푖푗푡) + 훽4 ∗

퐿퐴푁퐺푖푗 + + 훽5 ∗ 푅푇퐴푖푗푡 + 훽6 ∗ 퐶푂퐿푂푁푖푗 + 훽7 ∗ 퐴퐹퐿푆푖푗푡 + 훽8 ∗ 푙푛푃푅퐼퐶퐸푖푗푡 + 훽9 ∗

푙푛푌퐼퐸퐿퐷푖푡 + 훽10 ∗ 푀퐼푁푁푂푉푖푡 + 퐹퐸푖푡 + ԑ푖푗푡 (2)

where Vijt is the export value from i (US or Iran) to country j in the year t; GDPjt is the gross domestic product of the destination countries in t; DISTjt is the geographical

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Economics distance in kilometres between the exporting country i and the importing countries j; TARIFFijt is the simple average tariff rates of country j to country i in the year t; RTAijt is a zero-one dummy for the existence of a regional trade agreement between country i and country j in the year t; LANGij is a zero-one dummy (= 1 if country i and country j have common language); COLONij is a zero-one dummy (=1 if country i and country j have a common colonial past). AFLS is the measure of food safety related to aflatoxin in pistachio production for each importing country j along the time t.

PRICEijt is the unitary pistachio export price from i to country j in the year t. YIELDit is the average pistachio yield in the corresponding producer country i in the year t;

MINNOVit is the dummy reflecting the influence of the Federal Marketing Law for i= US and the influence of Iranian Pistachio Association for i=Iran. Finally, we include a set of year dummies to capture time-specific effects FEt.

Regarding food safety related to aflatoxin contamination in pistachio export, three distinct measures are used in the analysis. First, maximum aflatoxin limit (StandAflat) by each importing country is used. Some countries have not specific legislation about maximum allowable concentration of aflatoxins. To avoid the reduction of sample, the maximum restriction for pistachio in the world in the corresponding year t is assigned for these countries. This solution is similar to the purpose made by Ferro et al. (2015) for 243 agricultural products. It is based upon the assumption that these countries are more permissive (less restrictive) than the countries that have easily available their legislation to public access (the maximum aflatoxin standard means that they are the least stringent in food safety standards for pistachio imports). A zero cannot be assigned because it would mean a maximum aflatoxin restrictive index for these countries, and the case is the opposite (Ferro et al., 2015). These standards of aflatoxins have been used by Otsuki et al. (2001) for cereals, dried fruits and nuts and Wu and Guclu (2012) for maize exports.

The second variable for food safety related to aflatoxin in pistachio (Restrict1jt) is a measure of production for each importing country i in year t similar to the stringency index proposed by Ferro et al. (2015). It is defined as follows:

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푀퐴푋푗푡 − 퐴푓푙푎푡푗푡 푅푒푠푡푟𝑖푐푡 1푗푡 = ( ) 푀퐴푋푗푡 − 푀퐼푁푗푡

where MAXt is the maximum standard of aflatoxins for pistachio and year t across all importing countries and MINt is the minimum standard of aflatoxins for pistachio in the world in the year t. Aflatit is the maximum allowable concentration of aflatoxins in each importing country j in the year t. By using this index we can identify how restrictive is the legislation of the country j with regard the rest of the world through the time. Changes in the country legislation, but also changes in the world legislation, affects the restrictiveness of every importer country j. This index will be between zero and one, zero being the least restrictive and one the most restrictive.

A third measure is a reformulation based on the previous index taken into account the share of importing country in the total pistachio international trade flows

(Restrict 2jt). By including the import share corresponding to each importing country j from i=USA or i=Iran, a weighted measure of stringency is purposed under the assumption that the effects of the food safety standards corresponding to the most important partners for each producer country i must be identified.

푀퐴푋푗푡 − 퐴푓푙푎푡푗푡 푅푒푠푡푟𝑖푐푡 2푗푡 = ( ) ∗ 𝑖푚푝표푟푡 푠ℎ푎푟푒푖푗푡 푀퐴푋푗푡 − 푀퐼푁푗푡

The use of the Ordinary Least Squares (OLS) is the simplest technique to the estimation of gravity models. The limitations of OLS estimates for economic interpretation have been showed in some recent studies (Xiong and Beghin, 2013). To avoid the bias related panel data, estimation techniques have been used to include the effects of unobserved individual heterogeneity (Ferro et al., 2015).

The statistical analysis program Stata 12.0 version has been used to estimate random and fixed effects models to analyze the impact of food safety stringency on pistachio imports from US and Iran. The unobserved heterogeneity among the different importing countries is considered to avoid biased estimations. In this sense, some authors note the advantages of fixed effects model related to random effects in these gravity models. However, this choice does not allow getting information about the

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Economics influence of non-time varying effects traditionally considered in gravity models (distance, language, religion, border, and distance among other).

Both the random and fixed effects models estimator are used to contrast the impact of the food safety stringency on the US and Iran pistachio exports. Year dummies are also included in all estimations and the correlation of errors across years for the same country-pair is taken into account by clustering. Heteroscedasticity is corrected with White’s (1980) method.

4. Results and discussion

Model (1) provides a reduced-gravity model including GDP of importing country, distance and the first measure of food stringency on pistachio trade (standard of aflatoxins). Model (2) adds the typical variables in gravity models related to tariffs, common language, RTA and colonial ties. Model (3) includes also the variables that proxy the quality (price) and (yield) related to the introduction of technological innovations in the producer country i (US or Iran). Finally, Models (4) include the dummy variables refereed to Pistachio Federal Marketing Order in US and the beginning of the Iran Pistachio Association in Iran. The same variables are included from Model (5) to Model (8) (but considering fixed-effects). Since three measures have been proposed to consider the level of stringency in food safety for pistachio, identical specifications are presented considering two additional restrictiveness indexes. The interpretation of each of these food safety measures on pistachio trade exports must be done carefully. As it was explained above (see 3.2. Methodology), the first measure considered is the maximum concentration of aflatoxins defined by the importers regulations (StandAflat). The higher the aflatoxin standard, the less restrictive is the country regulation (it allows pistachio imports with a higher concentration of aflatoxins). The sign expected for Restrict 1jt and Restrict 2jt in the following specifications must be the contrary: the higher the restrictiveness in aflatoxins, the more restrictive is the regulation in the importing country (it allows only pistachio imports with a low concentration of aflatoxins). Differences in the results obtained considering random and fixed effects are presented for each

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Influence of food safety on pistachio trade country. Thus, we estimate 4x2x3x2 models for US and Iran (random and fixed effects). All estimations are carried to try to check the robustness of our results.

The influence of Aflatoxin Standards on Pistachio Exports

US

Table 2 shows the regression results of aflatoxins standards to US pistachio exports considering random and fixed effects. The results confirm that GDP of importing country influence positively on US pistachio export value. Moreover, pistachio export value increases faster than the importer’s GDP in all the models. This result confirms the arguments regarding that large economies import more than small economies at the intensive and in the extensive margin (WTO and UNTACD, 2012). Although the effect of GDP variations of importing partner on exports of a specific agricultural production could not be significant, this variable exerts a high influence on US pistachio exports. The effect could be more pronounced due to the perception of pistachio as a luxury product by consumers.

The geographical distance is not significant for explaining US pistachio exports. This result disagrees with most of empirical gravity models estimated for analysing the trade flows of different agricultural and food products (Otsuki et al., 2001; De Frahan and Vancauteren, 2006; Jongwanich, 2009; Winchester et al., 2012; Ferro et al., 2015;). The coefficients corresponding to tariffs and the existence of RTA are not significant. Similar result is found for the rest of variables related to information costs (common language and colonial links).

The coefficient for US pistachio price is statistically significant in the models (3), (4), (7) and (8). Since product differentiation in US pistachio could be very important, distance is not significant and price seems to act as a signal of premium quality for pistachio importers. The proxy used to gather the introduction of technological innovations in the US pistachio crops (yield) is not significant. However, the implementation of the Federal Marketing Order is significant and positive (only with random-effects). This result confirms the positive effects of the Pistachio Marketing Law described by Gray et al. (2005). Non-technological innovations seem to be for US

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Economics

pistachio exports more determinant than the introduction of technological innovation in the pistachio sector.

Table 2. Influence of aflatoxins standards on US Pistachio bilateral trade.

Random effects Fixed effects (1) (2) (3) (4) (5) (6) (7) (8) LnGDP 0.839*** 0.843*** 0.858*** 0.858*** 1.860*** 1.874*** 1.880*** 1.880*** (0.0721) (0.0738) (0.0739) (0.0739) (0.205) (0.207) (0.198) (0.198) Lndistance 0.194 0.207 0.279 0.279 - - - - (0.249) (0.259) (0.262) (0.262) Lntariff 0.0173 0.0446 0.0446 0.0408 0.0691 0.0691 (0.048) (0.0462) (0.0462) (0.0483) (0.0463) (0.0463) Common -0.391 -0.465 -0.465 - - - languaged (0.417) (0.421) (0.421) RTAd 0.0801 0.12 0.12 -0.0737 -0.0228 -0.0228 (0.187) (0.18) (0.18) (0.196) (0.188) (0.188) Colonial linksd 0.0952 0.104 0.104 - - - (0.359) (0.362) (0.362) StandAflat -0.0317*** -0.0324*** -0.0320*** -0.0320*** -0.0344*** -0.0346*** -0.0341*** -0.0341*** (0.00605) (0.00614) (0.00597) (0.00597) (0.00721) (0.00725) (0.00693) (0.00693) Lnprice 0.605*** 0.605*** 0.621*** 0.621*** (0.0671) (0.0671) (0.0661) (0.0661) Lnyield -0.0149 -0.0149 -0.0238 -0.0208 (0.0868) (0.0868) (0.0728) (0.0717) FedMarkOrdd 1.018*** 0.0109 (0.252) (0.274) Constant -9.030*** -9.216*** -11.24*** -12.26*** -33.86*** -34.21*** -35.37*** -35.41*** (2.761) (2.870) (2.996) (3.007) (5.426) (5.465) (5.293) (5.224) Observations 1036 1036 1036 1036 1036 1036 1036 1036 Number of 95 95 95 95 95 95 95 95 countries R-squared 0.474 0.469 0.476 0.476 0.45 0.45 0.498 0.498 ***p < 0.01, **p < 0.05, *p < 0.1. Superscript d is for dummy variables.

Finally, we get the expected sign for the coefficient corresponding to aflatoxin standards: negative. Thus, a declining standard level of aflatoxin has a significant export promoting effect on US pistachio exports. The increase in the maximum concentration allowed (less restrictive regulation) affects negatively the US pistachio exports.

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Influence of food safety on pistachio trade

Iran

Table 3 shows the regression results of aflatoxins standards to Iran pistachio for random and fixed effects. The results confirm that GDP of importing country influence positively on Iran pistachio export value. This result is similar to the estimations for US pistachio exports. However, the main difference among US and Iran estimated gravity models is related to the influence of distance. The geographical distance is very significant for explaining Iran pistachio exports while it was not significant for US. This means that transport costs are very important for Iran pistachio exports and the remoteness of the importing countries influence negatively Iranian exports (Otsuki et al., 2001; De Frahan and Vancauteren, 2006; Jongwanich, 2009; Winchester et al., 2012 Ferro et al., 2015).

Table 3. Influence of aflatoxins standards on Iran Pistachio bilateral trade.

Random effects Fixed effects (1) (2) (3) (4) (5) (6) (7) (8) LnGDP 0.342*** 0.354*** 0.358*** 0.358*** 0.653*** 0.638*** 0.665*** 0.665*** (0.103) (0.106) (0.106) (0.106) (0.245) (0.245) (0.246) (0.246) - Lndistance 1.031*** -1.074*** -1.079*** -1.079*** - - - - (0.309) (0.309) (0.321) (0.321) Lntariff 0.110* 0.110* 0.110* 0.107* 0.107* 0.107* (0.0627) (0.0627) (0.0627) (0.0648) (0.0648) (0.0648) Common languaged 0.891 0.906 0.906 - - - (1.658) (1.658) (1.658) RTAd 0.514 0.51 0.51 0.979* 0.971* 0.971* (0.399) (0.4) (0.4) (0.558) (0.558) (0.558) StandAflat -0.0199** -0.0228*** -0.0230*** -0.0230*** -0.0185** -0.0208** -0.0212** -0.0212** (0.00774) (0.00785) (0.00786) (0.00786) (0.00864) (0.0087) (0.00871) (0.00871) Lnprice 0.261 0.261 0.366 0.366 (0.326) (0.326) (0.33) (0.33) Lnyield -0.663 -0.663 0.0286 0.0286 (0.912) (0.912) (0.269) (0.269) IPAd 0.133 1.080** (0.272) (0.423) Constant 13.89*** 13.77*** 18.89** 18.76** -2.084 -3.073 -4.7 -4.7 (3.45) (3.587) (8.792) (8.902) (6.29) (6.095) (6.474) (6.474) Observations 799 799 799 799 799 799 799 799 Number of countries 100 100 100 100 100 100 100 100 R-squared 0.121 0.127 0.125 0.125 0.2 0.207 0.208 0.208 ***p < 0.01, **p < 0.05, *p < 0.1. Superscript d is for dummy variables.

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Common language does not affect the Iran pistachio exports while the existence of RTA is significant only at 0.1 level if fixed effects are considered (from Model 6 to Model 8). Although these coefficients are not very significant, the positive effect of the RTA in Iran pistachio exports could be attributed to the Iran membership to the Global System of Trade Preferences among Developing Countries (GSTP). Regarding the influence of tariffs on Iran pistachio exports, we also find a positive significance at 0.1 level (Model 2 to Model 4 and Model 6 to Model 8 in Table 7). The positive effect observed on tariffs could be attributed to the establishment of higher tariffs of some importing countries located in the Middle-East that are trying to protect their national pistachio productions of Iranian exports.

Not prices neither yields (technological innovations) are significant to explain the evolution of Iran pistachio exports. Opposite results are found for the influence of the innovation management proxied by the IPA creation. The creation of this organism in the year 2007 made possible the implementation of a specific programme to improve the technological and non-technological innovation in pistachio crops, quality controls, and, consequently, to try to enhance Iran pistachio exports. The implementation of the IPA is positively significant at 95% level if fixed effects are considered (Model 8).

Similarly to the US models, a maximum standard of aflatoxins (less restrictive) decrease the Iran pistachio exports. The aflatoxin regulation has a negative effect (at 95% level of significance) on Iran exports. Thus, a less strict standard hinders pistachio exports from the US and Iran. Both countries are obtaining benefits when they export to market destinations with more restrictive standards (high standard of aflatoxin). This result was expected for US pistachio exports, but also Iran exports are incentivized for strict standards of aflatoxins. Better performance is expected from developed countries in which more restrictive measures are implemented (Disdier et al., 2008), but poor countries also can benefit from food safety standards. As it has been confirmed in recent studies, developing countries can obtain gains from a stricter food safety regulation imposed by importing countries (Dou et al., 2015; Ishaq et al., 2016; Xiong and Beghin, 2014). According to Wu (2008), if consistently high-

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quality good exists and the global scene allows market shifts, export markets can benefit from stricter standards.

Strictness index 1 (Restrict 1jt)

US

Table 4 shows the regression results to evaluate the influence of aflatoxin regulation

strictness (Restrict 1jt) to US pistachio exports considering random and fixed effects. The results confirm previous findings. All variables show similar evolution that in the former models with the aflatoxin standards variable (StandAflat).

Table 4. Influence of Strictness Index 1 (Restrict 1jt) on US Pistachio bilateral trade.

Random effects Fixed effects (1) (2) (3) (4) (5) (6) (7) (8) lnGDP 0.839*** 0.843*** 0.858*** 0.858*** 1.860*** 1.874*** 1.880*** 1.880*** (0.0721) (0.0738) (0.0739) (0.0739) (0.205) (0.207) (0.198) (0.198) Lndistance 0.194 0.207 0.279 0.279 - - - - (0.249) (0.259) (0.262) (0.262) Ltariffs 0.0173 0.0446 0.0446 0.0408 0.0691 0.0691 (0.048) (0.0462) (0.0462) (0.0483) (0.0463) (0.0463) Common -0.391 -0.465 -0.465 - - - languaged (0.417) (0.421) (0.421) RTAd 0.0801 0.12 0.12 -0.0737 -0.0228 -0.0228 (0.187) (0.18) (0.18) (0.196) (0.188) (0.188) Colonial linkd 0.0952 0.104 0.104 - - - (0.359) (0.362) (0.362) Lnprice 0.605*** 0.605*** 0.621*** 0.621*** (0.0671) (0.0671) (0.0661) (0.0661) Lnyield -0.0149 -0.0149 -0.0238 -0.0208 (0.0868) (0.0868) (0.0728) (0.0717) FedMarkOrdd 1.018*** 0.0109 (0.252) (0.274) Restrict 1 1.269*** 1.295*** 1.278*** 1.278*** 1.374*** 1.386*** 1.362*** 1.362*** (0.242) (0.245) (0.239) (0.239) (0.289) (0.29) (0.277) (0.277) Constant -10.30*** -10.51*** -12.52*** -13.54*** -35.23*** -35.60*** -36.73*** -36.77*** (2.736) (2.847) (2.974) (2.987) (5.413) (5.451) (5.28) (5.209) Observations 1036 1036 1036 1036 1036 1036 1036 1036 Number of 95 95 95 95 95 95 95 95 countries R-squared 0.474 0.469 0.476 0.476 0.45 0.45 0.498 0.498 ***p < 0.01, **p < 0.05, *p < 0.1. Superscript d is for dummy variables.

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The expected sign is found for the coefficient corresponding to strictness in the aflatoxin standards: positive. The increase in the restrictiveness of the regulation increases the US pistachio exports controlling by importing country characteristics. Yield (technological innovation) does not affect US, but the Federal Marketing Order is positive and significant if random effects are considered (Model 4).

Iran

Table 5 shows the regression results of the influence of aflatoxin regulation strictness

(Strict 1jt) on Iran pistachio exports considering random and fixed effects. The results confirm previous findings although small differences appear comparing with US results.

Table 5. Influence of Strictness Index 1 (Restrict 1jt) on Iran Pistachio bilateral trade.

Random effects Fixed effects (1) (2) (3) (4) (5) (6) (7) (8) lnGDP 0.342*** 0.354*** 0.358*** 0.358*** 0.653*** 0.638*** 0.665*** 0.665*** (0.103) (0.106) (0.106) (0.106) (0.245) (0.245) (0.246) (0.246) Lndistance -1.031*** -1.074*** -1.079*** -1.079*** - - - - (0.309) (0.319) (0.321) (0.321) Lntariffs 0.110* 0.110* 0.110* 0.107* 0.107* 0.107* (0.0627) (0.0627) (0.0627) (0.0648) (0.0648) (0.0648) Common languaged 0.891 0.906 0.906 - - - (-1.658) (-1.667) (-1.667) RTAd 0.514 0.51 0.51 0.979* 0.971* 0.971* (-0.399) (-0.4) (-0.4) (0.558) (0.558) (0.558) Lnprice 0.261 0.261 0.366 0.366 (-0.326) (-0.326) (0.33) (0.33) Lnyield -0.663 -0.663 0.0286 0.0286 (-0.912) (-0.912) (0.269) (0.269) IPAd 0.133 1.080** (-0.272) (0.423) Restric 1 0.795** 0.912*** 0.920*** 0.920*** 0.739** 0.830** 0.846** 0.846** (0.31) (0.314) (0.314) (0.314) (0.346) (0.348) (0.348) (0.348) Constant 13.09*** 12.86*** 17.97** 17.84** -2.822 -2.785 -5.547 -5.547 (3.404) (3.545) (8.772) (8.883) (6.319) (6.307) (6.497) (6.497) Observations 799 799 799 799 799 799 799 799 Number of countries 100 100 100 100 100 100 100 100 R-squared 0.121 0.127 0.125 0.125 0.2 0.207 0.208 0.208 ***p < 0.01, **p < 0.05, *p < 0.1. Superscript d is for dummy variables.

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We find a positive sign for the coefficient corresponding to strictness in the aflatoxin standards. However, the regulatory stringency of aflatoxins only is significant at 95% level if fixed effects are considered (from Model 5 to Model 8 in Table 6). The rest of the variables show a similar evolution that in the models using the aflatoxin standards variable (StandAflat). These results confirm the robustness of results.

Strictness index 2 (Restrict 2)

US

Table 6 shows the results of the influence of aflatoxin regulation strictness (Strict 2jt) to US pistachio exports considering random and fixed effects.

Table 6. Influence of Strictness Index 2 (Restrict 2jt) on US Pistachio bilateral trade.

Random effects Fixed effects (1) (2) (3) (4) (5) (6) (7) (8)

lnGDP 0.782*** 0.786*** 0.797*** 0.797*** 1.736*** 1.748*** 1.755*** 1.755*** (0.0565) (0.0577) (0.0581) (0.0581) (0.198) (0.2) (0.191) (0.191) Lndistance 0.199 0.206 0.274 0.274 - - - - (0.191) (0.198) (0.201) (0.201) Lntariffs -0.0249 0.00265 0.00265 0.013 0.0414 0.0414 (0.0457) (0.044) (0.044) (0.0464) (0.0443) (0.0443) Common languaged -0.34 -0.407 -0.407 - - - (0.322) (0.325) (0.325) RTAd 0.103 0.138 0.138 -0.0759 -0.0258 -0.0258 (0.176) (0.17) (0.17) (0.188) (0.18) (0.18) Colonial linksd 0.0436 0.048 0.048 - - - (0.277) (0.28) (0.28) Lnprice 0.588*** 0.588*** 0.611*** 0.611*** (0.0652) (0.0652) (0.0635) (0.0635) Lnyield 0.00267 0.00267 -0.0199 -0.0213 (0.0848) (0.0848) (0.0699) (0.0688) FedMarkOrdd 1.061*** 0.174 (0.243) (0.263) Restrict 2 0.216*** 0.216*** 0.210*** 0.210*** 0.180*** 0.180*** 0.176*** 0.176*** (0.018) (0.0181) (0.0174) (0.0174) (0.0181) (0.0181) (0.0173) (0.0173) Constant -8.467*** -8.586*** -10.60*** -11.66*** -31.49*** -31.78*** -33.00*** -33.16*** (2.127) (2.209) (2.359) (2.386) (5.223) (5.263) (5.086) (5.017) Observations 1036 1036 1036 1036 1036 1036 1036 1036 Number of countries 95 95 95 95 95 95 95 95 R-squared 0.578 0.577 0.585 0.585 0.491 0.491 0.538 0.538 ***p < 0.01, **p < 0.05, *p < 0.1. Superscript d is for dummy variables.

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The results confirm previous findings; all variables show similar evolution that in the models developed using the aflatoxin standards variable (StandAflt) and the

Strictness Index 1 (Restrict 1jt).

Iran

Table 7 shows the results of the influence of strictness of aflatoxin regulation (Strict

2jt) on the evolution of Iran pistachio exports considering random and fixed effects. Variables show a similar evolution that in the previous models using the aflatoxin standards (StandAflat) and the Restrictiveness Index 1 (Restrict 1jt) as food safety measures.

Table 7. Influence of Strictness Index 2 (Restrict 2jt) on Iran Pistachio bilateral trade.

Random effects Fixed effects (1) (2) (3) (4) (5) (6) (7) (8)

lnGDP 0.363*** 0.377*** 0.380*** 0.380*** 0.660*** 0.642*** 0.671*** 0.671*** (0.0912) (0.0935) (0.094) (0.094) (0.24) (0.24) (0.241) (0.241) Lndistance -0.987*** -1.010*** -1.014*** -1.014*** - - - - (0.268) (0.275) (0.277) (0.277) Lntariffs 0.098 0.0977 0.0977 0.0912 0.0908 0.0908 (0.0609) (0.0609) (0.0609) (0.0631) (0.0631) (0.0631) Common languaged 0.788 0.802 0.802 - - - (1.425) (1.433) (1.433) RTAd 0.396 0.389 0.389 0.797 0.787 0.787 (0.368) (0.369) (0.369) (0.549) (0.549) (0.549) Lnprice 0.297 0.297 0.404 0.404 (0.321) (0.321) (0.325) (0.325) Lnyield -0.628 -0.628 0.0276 0.0276 (0.903) (0.903) (0.264) (0.264) IPAd 0.0903 1.104*** (0.269) (0.41) Restrict 2 0.348*** 0.351*** 0.352*** 0.352*** 0.283*** 0.280*** 0.283*** 0.283*** (0.0472) (0.0472) (0.0472) (0.0472) (0.0516) (0.0516) (0.0517) (0.0517) Constant 12.30*** 11.96*** 16.71** 16.61* -2.902 -2.689 -5.594 -5.594 (2.963) (3.07) (8.499) (8.614) (6.174) (6.172) (6.365) (6.365) Observations 799 799 799 799 799 799 799 799 Number of countries 100 100 100 100 100 100 100 100 R-squared 0.282 0.293 0.291 0.291 0.229 0.233 0.235 0.235 ***p < 0.01, **p < 0.05, *p < 0.1. Superscript d is for dummy variables.

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Again, the expected sign is found for the coefficient corresponding to strictness in the aflatoxin standards: positive (significance at 99% level of significance for all proposed models, Table 8). In Model (8) the implementation of the IPA is more significant than in previous models (p < 0.01). R2 in Model (8) reaches 0.235; lower than in the similar models proposed for the US. This fact shows that common variables included in the gravity models are more useful to explain the exports of developed countries (i.e. US) that the export performance of developing countries.

To control for heterogeneity among countries, cross-section gravity models for consecutive years have been estimated. In this regard, the Breusch-Pagan LM tests are performed to test if the random-effects panel model is better than the pool variance of the unobserved fixed effects is zero, i.e. var(u) = 0. In all the cases this hypothesis is rejected. Thus, random-effects estimation is selected instead of pooled OLS. Moreover, Hausman tests are calculated to test fixed versus random effects. All estimations (from Table 3 to Table 8) provides the random-effects and fixed-effects models for the influence of the three measures of strictness on US and Iran pistachio exports. Although the coefficients of the random effects are not significantly different from the fixed-effects estimations, Hausman tests confirms that fixed effects are the better choice for the US Models while random-effects would be a better option for the Iran Models.

5. Conclusions

The main aim of this paper is to analyse the influence of product standards on pistachio bilateral trade flows on the two main producing and exporting countries: US and Iran. Taken into account the strict food safety legislation applied to pistachio exports due to aflatoxin contamination, the effects of these food safety standards on pistachio international market during the period 1996-2014 are considered. For this purpose, different measures of food stringency regulation in this merchandise are considered.

Based upon panel gravity models, results confirm that aflatoxin regulation influences significantly on Iran and US pistachio trade. In particular, food safety regulation acts

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Economics as a catalyst for pistachio global trade in both countries. Despite it could be expected even the less-developed producer country, Iran, had accomplished the food safety requirements at international level during the period 1996-2014. However, there are important differences in the drivers of the Iran and US pistachio exports trade. In particular, distance only affects to Iran pistachio exports while the geographical distance among US and the potential importers does not affect US pistachio exports. This conclusion is especially relevant because it could be interpreted as the consequence of the higher differentiation achieved by US pistachio and macroeconomic factors related to the US political hegemony in the international economy. Moreover, it shows that consumer´s trust on US pistachio is higher than confidence on Iran pistachio or other less-developed countries with high sanitary and health risk.

Traditional trade barriers (tariffs), cultural differences and information costs also play a secondary role to explain pistachio trade flows (variables as common language or colonial ties are not significant). This result shows the effects of trade liberalization and tariffs reduction on agricultural products as pistachio in the last decades.

In regard to the role of technological and non-technological innovations, the findings show that exports are not related with the level of technological change in the production processes in US neither in Iran. On the contrary, the introduction of non- technological innovation (management innovations related to the creation of Federal Marketing Order and the IPA) exerts a positive influence on the pistachio exports.

Despite the evidence about that stricter food safety regulation benefits pistachio importers and exporters in the world, further research is needed. In this regard, one of the limitation of this research is that is only an exploratory study of the influence of food safety stringency due to aflatoxin contamination on one agricultural product: pistachio. Further studies should be conducted through using proper estimation strategies with more detailed data to mitigate the endogeneity problems and fill the questions that remains unsolved. In this regard, the use of dummies to capture the influence of information cost has been very limited by the correlation between some variables traditionally used in the gravity models (i.e., dummies as religion and

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Influence of food safety on pistachio trade common borders have been removed by their high correlation with other variables such as the distance). Another important limitation is that the excessive number of zeros in the database has not been considered. In this regard, the Poisson Pseudo Maximum Likelihood (PPML) methods or Negative Binomial (NB) models similar to Ishaq et al. (2016) and Heckman models to control sample selection as in Ferro et al. (2015) must be used in future research. The advance on new results based upon proper estimation strategies could provide suggestions for farmers, food producers and policy-makers all over the world.

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Zheng, Z., Saghaian, S., Reed, M.R., 2012. Factors Affecting the Export Demand for U.S. Pistachios. International Food and Agribusiness Management Review 15, 139- 154.

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Analysis of production costs for the industrial production of pistachio oil by using two different production

lines

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Analysis of production costs for the industrial production of pistachio oil by using two different production lines

ABSTRACT Keywords: One of the primary constraints for pistachio oil production is the lack of Pistacia vera Quality information regarding oil extraction from an industrial perspective. To ensure Consumer the success of pistachio oil production at a commercial scale, attention should preference Extraction be paid to the effect of main extraction procedures on oil characteristics, the

consumer acceptance of these oils and their production cost. Comparison and

evaluation of the physicochemical and sensory characteristics and production cost of oil extracted using two different production lines (hydraulic press and screw press) are here considered. Slight differences were found in the physicochemical analysis, but significant differences were identified in the sensory analysis. Consumer-judges preferred the oil extracted with the hydraulic press. According to production costs, the breakeven value that makes screw press extraction sustainable is 70.4 €/litre, while for the hydraulic press is 91.0 €/litre, due mainly to a lower extraction yield and the larger extraction time required. As production costs of both methods are high, pistachio oil quality should prevail, making the use of the hydraulic press more advisable. This study provides a comprehensive approach to high quality pistachio oil production from an industrial perspective.

1. Introduction

The pistachio (Pistacia vera L.) is a native tree of Middle East and Central Asia (Whitehouse, 1957) that has been successfully introduced in the Mediterranean basin and the US. Its tolerance to dry and hot climates makes possible its cultivation in arid areas of Spain where its implantation has intensified in the last years. Pistachio global production increases each year, reaching 916.921t in 2013 (FAO, 2015), as consumption steady rises due to its consideration of health promoting nut within the Mediterranean diet.

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Oil content in pistachio kernels reaches about 45-57%. This makes pistachio an interesting fat source to be included in a healthy diet (Tsantili et al., 2010). Pistachio fatty acids profile is characterized by the dominance of unsaturated fatty acids like oleic, linoleic and linolenic acids (Arena et al., 2007; Tsantini et al., 2010; Tavakolipour et al. 2010). In addition, pistachio oil presents high contents of phytosterols (Phillips et al. 2005), which have been proved to reduce blood cholesterol and decrease the appearance of certain types of cancer (Award and Fink, 2000; Ostlund, 2004); and phenolic compounds that provide pistachio with antioxidant properties (Gentile et al., 2007; Tomaino et al., 2010; Dreher, 2012; Ling et al., 2016).

Attending to pistachio high content of oil and the health benefits associated to its chemical characteristics, oil extraction from pistachio can be identified as a commercial opportunity. Pistachio oil can be used in the food industry, as a condiment to provide pistachio taste and smell in food, creating new culinary perspectives based on its unique sensorial properties (Uriarte et al., 2011). In addition, pistachio oil can also be used in the cosmetic and pharmaceutical industry due to its emollient properties and vitamin content (Fàres et al., 2011).

Pistachio oil extraction can be done by using different methods. Higher oil yields can be extracted using solvents as hexane or diethyl ether (Bellomo et al. 2009; Conte et al. 2011), but solvent extraction originates oil with unpleasant smells and tastes which make mandatory the refining if it is destined to human consumption (Roncero et al., 2016). Supercritical CO2 extraction technology is the alternative to solvent extraction, and it is able to provide high oil yields without the problem of solvent traces appearance. The downside of this method is that it presents high cost of investment, constraining its use to high valuable oils (Rosa and Meireles, 2005).

The pressing systems provide high quality oils at an affordable price, and are considered as environmental friendly processes (Khoddami et al., 2014). The presses that have been widely used for nut oil extraction are screw press and hydraulic press. Oil extraction with hydraulic press demands high labour, however, oils obtained by using this method present better maintenance of their physicochemical and sensory properties. On the other side, oil extraction with screw presses usually demands a

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Economics previous thermal conditioning of material, which may lead to a reduction of the final product quality if extraction parameters are not carefully selected and controlled.

Pistachio is one of the most expensive nuts in the market, and pistachio oil extraction is presented as an expensive process. Little attention has been provided in the bibliography to the study of pistachio oil production cost and the comparison of extraction procedures to evaluate commercial viability of the product.

The extraction of pistachio oil using two pressing systems, screw press and hydraulic press, is here compared. The yield and the physicochemical and sensory quality of oil extracted using both presses have been evaluated in order to determine possible differences. In addition, the cost of production of pistachio oil with the two presses at a small-scale processing unit has been estimated.

2. Materials and methods

2.1. Raw material

Pistachios collected during the 2013/2014 season were provided by the Centro de Mejora Agraria El Chaparrillo (Ciudad Real, Spain). The shell of pistachios was removed in controlled conditions for immediate drying. Pistachios were dried at room temperature until they reached a moisture content lower than 6%. After drying, pistachios were vacuum packed and refrigerated until extraction time to reduce the influence of environmental conditions on the results. Six samples of pistachios (Pistacia vera L. var. Kerman) were used for the study. Two production lines were tested and three replicates were performed for each line.

2.2. Oil extraction

The whole extraction process was done in the small-scale processing unit at an university center. The process equipment needed in a pistachio oil extraction plant may vary according to the extraction press used, defining two possible ways that are shown in the flowchart in Figure 1.

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Figure 1. Flow chart of hydraulic and screw extraction systems

Previous roasting is considered in the process of hydraulic press extraction to improve the sensory properties of the oil. Roasting also increase the total phenolics content and the antioxidant capacity of oil (Ling et al., 2016). In the case of the screw press, this operation is not considered because of the functioning of the press itself, which may use high temperatures to avoid blockage and low yields, providing the oil with characteristics that recall roasting (Sena-Moreno et al., 2015). Similar characteristics to those obtained in oil from roasted pistachios using the hydraulic press (Ling et al., 2016) have been reported for pistachio oil obtained with the screw press from natural pistachios (Sena-Moreno et al., 2016).

Although in both cases pressing system was considered, differences in the optimal input material needed for the proper function of the two presses was taken into account. Whole raw pistachios were directly used in the case of the screw press, while pistachios were previously ground in the case of the hydraulic press. The grinding leads to highest yields when the hydraulic press is used. Thus, an additional step of pistachio grinding will be needed in the case of hydraulic press extraction. For pistachio grinding a blender (GM200-RETSCH) was used.

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The oil was, therefore, extracted with two different pressure systems: hydraulic press (MECAMAQ Modelo DEVF 80, Vila-Sana, Lleida, Spain) and a screw press (Komet Oil Press CA59G – IBG Monforts Oekotec GmbH & Co. KG, Mönchengladbach, Germany).

After oil extraction, in both cases, a centrifugation step was carried out in order to eliminate the remaining solid residues from the samples. A centrifuge (Centronic-BL de Geberlab) was used with this purpose. Residues and centrifuged oil were weighted and oil was collected and stored in dark glass bottles under refrigeration until analysis.

2.3. Comparative oil extraction

Operating conditions were ensured to be optimal based on preliminary experiments of oil extraction conditions with both presses. Selected conditions were based on the functional parameters of presses. For the hydraulic press extraction was carried out at a pressure of 160 kg/cm2 during 12 minutes and in the case of screw press, extraction at 100ᵒC and 49 rpm engine speed was considered. Temperature in the screw press was calculated for the exterior part of the tip. For the two pressing systems used, time needed to obtain one litre of pistachio oil was calculated. In both cases, three replicates were considered to obtain the oil that was used in posterior quality analysis.

2.4. Oil recovery

The throughput of each pressing system was calculated as the ratio of the weight of pistachios processed in kilograms to the time taken in minutes. The oil yield was taken as the ratio of the weight of oil after eliminating solid impurities with centrifugation over the weight of pistachio processed. The extraction efficiency was calculated as the ratio of pure oil yield to the oil content in the raw pistachio.

2.5. Analytical methods

Free acidity, given as % of oleic acid, was determined by titration of a solution of oil dissolved in ethanol/ether (1:1) with 0.1 M potassium hydroxide ethanolic solution (EEC, 1991). Peroxide value, expressed in milliequivalents of active oxygen per

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Production costs of pistachio oil kilogram of oil (meq kg-1), was determined as follows: a mixture of oil and chloroform- acetic acid was left to react with a solution of potassium iodide in darkness; the free iodine was then titrated with a sodium thiosulfate solution (EEC, 1991).

K270 and K232 extinction coefficients were calculated from absorbance of a 1% solution of oil in cyclohexane at 270 and 232 nm, respectively, with a UV/VIS spectrophotometer Jasco V-530 (Jasco Analitica Spain, Madrid, Spain), and a path length of 1 cm (EEC, 1991). Total phenol compounds were isolated by extraction of a solution of oil in hexane three times with a water/methanol mixture (60:40). Folin– Ciocalteau reagent and sodium molybdate, 5% in 50% ethanol (Merck), were added to a suitable aliquot of the combined extracts, and the absorption of the solution at 725 nm was measured. Values were given as mg of caffeic acid per kg of oil (Vázquez et al., 1973; Gutfinger, 1981).

In order to determine fatty acids composition (%), the methyl-esters were prepared by vigorous shaking of a solution of oil in hexane (0.2 g in 3 ml) with 0.4 ml of 2 N methanolic potassium hydroxide solution, and analysed by GC with a Hewlett- Packard (HP 6890) chromatograph equipped with a FID Detector. A fused silica column (50 m length x 0.25 mm i.d.), coated with SGL-1000 phase (0.25 µm thickness; Sugerlabor), was used. Helium was employed as a carrier gas with a flow through the column of 1 ml min-1. The temperatures of the injector and detector were set at 250°C with an oven temperature of 210°C. An injection volume of 1µL was used (Regulation EEC 2568/91, corresponding to AOCS method Ch 2–91).

Oxidative stability was evaluated by the rancimat method (Gutiérrez, 1989). Stability was expressed as the oxidation induction time (hours), measured with the Rancimat 743 apparatus (Metrohm Co., Basel, Switzerland). An oil sample of 3.5 g was used, warmed to 100°C under an air flow of 10 l h-1.

In order to determine fatty acids composition (%), the methyl-esters were prepared by vigorous shaking of a solution of oil in hexane (0.2 g in 3 ml) with 0.4 ml of 2 N methanolic potassium hydroxide solution, and analyzed by GC with a Hewlett- Packard (HP 6890) chromatograph equipped with a FID Detector. A fused silica column (50 m length x 0.25 mm i.d.), coated with SGL-1000 phase (0.25 µm thickness;

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Sugerlabor), was used. Helium was employed as a carrier gas with a flow through the column of 1 ml min-1. The temperatures of the injector and detector were set at 250°C with an oven temperature of 210°C. An injection volume of 1µL was used (Regulation EEC 2568/91, corresponding to AOCS method Ch 2–91).

Sterols (%) were determined with a Hewlett-Packard (HP 6890) gas chromatograph with a capillary column (25 m length x 0.25 mm i.d.) coated with SGL-5 (0.25 µm thickness; Sugerlabor). Working conditions were as follows: carrier gas, helium; flow through the column, 1.2 ml min-1; injector temperature, 280°C; detector temperature, 290°C; oven temperature, 260°C; injection volume 1 µl (Regulation EEC 2568/91, corresponding to AOCS method Ch 6–91). Apparent β -sitosterol was calculated as the sum of β-sitosterol, Δ5,23-stigmastadienol, chlerosterol, sitostanol, and Δ5,24-stigmastadienol.

The oil content of pistachios was determined by a Soxhlet extractor (BUCHI Labortechnik AG, Flawil Switzerland) using petroleum ether as solvent, for an extraction period of 6 hours (AOAC, 1990).

Analytical tests were performed in triplicate.

Two kinds of sensory analysis were performed on pistachio oils. In affective test, oils were randomly labelled and 108 consumer type panellists were asked to smell, taste, and evaluate oil colour in order to rank oil samples according to their degree of acceptability. Each hedonic description was assigned a nine-point scale (-4 = dislike extremely, 0 = neither like nor dislike, 4 = like extremely). The mean scores for the three sensory properties were calculated separately. This analysis was complemented with a descriptive analysis by nine trained judges to evaluate the possible appearance of negative characteristics in oils. Presence of recurrent oil negative attributes plastic, burnt and rancid was also evaluated using continuous non-structured scales. In scales the left side corresponded to the lowest intensity of the defect (value 0) and the right side corresponded to the highest intensity (value 10).

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2.6. Economic analysis

The economic analysis of the two extraction procedures was based on the study of the material, equipment and labour input against returns for the small-scale processing unit at ETSIAM.

For its condition of small scale production unit, only the work of one man was considered for each production line proposed. Pressing time was considered the limiting time for both extraction processes, by assuming that the rest of operations: roasting, grinding and centrifugation in the case of hydraulic press extraction, and only centrifugation in the case of screw press extraction, do not require extra time. Only two exceptions were considered, the roasting of pistachios destined to the hydraulic press with the first extraction every day, and the preheating of screw press needed before extraction starts daily.

Taking into account previous considerations, the yearly processing cost and revenue were based in a considered work of 20 days per month, giving a total of 240 days for the yearly processing period as the raw pistachio can be easily conserved for one year time. Furthermore, working day at ETSIAM is established in 8 h/day, as it is a public establishment. Considering pre-processing operations, mentioned above, and other non-productive times, the calculation of daily throughput was done considering an effective extraction time of 6 h in the screw press processing time and 5.5 h in the case of the hydraulic press.

The total cost of equipment including running cost, maintenance cost and labour cost were calculated for one year period. The estimated production cost of one litre of pistachio oil was calculated attending to the annual oil production of the considered processing unit at the mentioned functional parameters for both processing lines.

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3. Results and discussion

3.1. Oil yield, processing time and extraction efficiency

Table 1 shows the differences in the processing system on pressing time, oil yield, extraction efficiency and solid impurities in oil. The results show moderate differences in pressing time and oil yield obtained by using the different presses. The quantity processed, as a reference unit in the research, has been based in the quantity of ground pistachio that can be processed at a time in the hydraulic press (1.2Kg) when extraction is replicated twice, as the screw press presents continuous oil extraction. Pressing is considered in both productions the time limiting factor in the production. In the case of hydraulic press, although the effective pressing time has been estimated in 12 minutes, charging and discharging of the press was also considered as part of the time need for each extraction, increasing the time to 28 min. Therefore, the processing of 2.4Kg of pistachio with the hydraulic press is settled at 56 min. In the case of screw press, the time required to process 2.4 kilograms of pistachios was established in 48 minutes. As it was expected, pressing time is higher when using the hydraulic press to process the same quantity of pistachios.

Table 1. Effect of processing system on processing time, oil yield, extraction efficiency and solid impurity in oil in processing 2.4 kg of pistachios. System processing Hydraulic press Screw press parameter Pressing time 56 min 48 min Oil yield (%) 31.02 34.80 Extraction efficiency (%) 57.40 64.40 Solid impurity in oil (%) 8.38 10.70

Oil yield obtained also presents differences. By using the screw press, oil yield reaches 34.8% while the hydraulic press yield is 31.0%. The yields obtained with the hydraulic press are in accordance with the results obtained by Ling et al. (2016) as roasting does not affect extraction efficiency. This represents a yield 3.8% higher when the screw press is used. Considering an initial oil content of pistachios of 54% (Soxhlet extractor), extraction efficiencies replicates the previous results, reaching efficiencies of 64.4% in the case of the screw press and 57.4% with the hydraulic. Larger

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Production costs of pistachio oil extraction efficiencies have been reported by using the screw press when high quality pistachios are used (Álvarez-Ortí et al., 2015). To increase extraction efficiency a second pressing could be projected, but the increase in oil yield could hardly compensate invested time, expecting also a reduction in oil quality.

Solid impurities in oil appear to be lower in the hydraulic press, 8.38%, possibly because of filtration effect that takes place during oil extraction procedure. The continuous extraction in the screw press, together with the absence of incorporated effective filtration methods, results in the occurrence of more solid impurities in oil with a percentage of 10.7%.

3.2. Physicochemical and sensory quality of pistachio oils

Physicochemical and sensory analysis were performed in triplicate to evaluate the quality of pistachio oil obtained (table 2). Physicochemical analysis shows little differences comparing oils obtained with the two proposed methods in accordance with the results obtained by Sena-Moreno et al., (2015). Pistachio oil presents a healthy fatty acid profile with predominance of unsaturated fatty acids that is not susceptible to the press system used (Álvarez-Ortí et al., 2012) or the roasting process (Martínez et al., 2016). Oleic and linoleic acids account for more than 85% of total fatty acids profile in both oils (Arena et al., 2007; Tsantili et al., 2010; Ling et al., 2016). The peroxide value is reduced and similar in both oils. A storage time of 75 days in dark conditions would be expected for the obtained oils attending to Ling et al. (2016) findings.

Pistachio nut has a high antioxidant capacity among nuts (Arranz et al., 2008). Significant differences appear attending to total sterols and polyphenols with higher concentration in oil extracted with the hydraulic press. Previous roasting of pistachios could be the responsible of the increase observed in the polyphenols content in oil extracted with the hydraulic press (Sena-Moreno et al., 2015) although not all studies agree in this effect of temperature (Martínez et al., 2016). The stigmasterol content is lower than the values reported in other studies that used Soxhlet extraction (Arena et al., 2007) but similar to the concentrations obtained in studies that use presses for pistachio oil extraction (Sena-Moreno et al., 2015). This reduction was expected as

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Economics stigmasterol content is sensitive to high temperatures (Koutsaftakis et al., 1999; Pardo et al., 2009; Sena-Moreno et al., 2015).

Table 2. Values of physicochemical analysis of oils obtained using the lines that include the hydraulic and the screw presses Presses Quality parameter Hydraulic Screw A. Palmitic C16:0 (%) 11.2 a ± 0.7 10.6 a ± 0.8 A. Palmitoleic C16:1 (%) 1.1 a ± 0.2 1.0 a ± 0.1 A. Stearic C18:0 (%) 1.1 a ± 0.1 1.0 a ± 0.1 A. Oleic 18:1 (%) 55.34 a ± 1.0 54.6 a ± 0.7 A. Linoleic C18:2 (%) 30.1 a ± 0.8 31.5 a ± 0.9 A. Linolenic C18:3 (%) 0.5 a ± 0.0 0.5 a ± 0.1 Total sterols (mg/kg) 3465 a ± 86 3245 b ± 102 Cholesterol (%) <0.1 0.1 a ± 0.1 Brasicasterol (%) 0.1 a ± 0.1 <0.1 Campesterol (%) 4.2 a ± 0.1 4.2 a ± 0.2 Estigmasterol (%) 0.6 a ± 0.1 0.6 a ± 0.0  sitosterol (%) 94.2 a ± 0.2 94.3 a ± 0.1 7 estigmasterol (%) 0.4 a ± 0.1 0.3 a ± 0.0 a a K232 1.51 ± 0.08 1.58 ± 0.12 a a K270 0.11 ± 0.2 0.13 ± 0.4 Acidity 0.17 a ± 0.3 0.25 a ± 0.8 Peroxide index 0.5 a ± 0.1 0.4 a ± 0.0 Total polyphenols (mg/kg) 32.2 a ± 4.5 18.6 b ± 7.1 Oxidative stability (h) 26.8 a ± 0.9 28.9 a ± 1.2 Different letters on the row indicate significant differences (p<0.05)

Pistachio oil presents a higher oxidative stability that most of nuts (Miraliakbari and Shahidi, 2008). Althought polyphenols have proved to increase oxidative stability in sunflower oil (Ramadan, 2013) and grape seed oil (Yang et al., 2013), the higher polyphenol content of the pistachio oil extracted with the hydraulic press does not translate in a significant increase in the oxidative stabiblity. The similar fatty acid profile can be key to understand the reason of the similar values in the oxidative stability paramethers. Acidity and oxidative stability are higher in oil extracted with screw press, but as reported in Sena-Moreno et al., (2015) differences are not significant. Attending to their physicochemical quality, oils extracted using both presses present high and similar nutritional value.

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Differences were greater when analysing the results of sensory analysis (Figure 2). Both oils obtained a positive valuation from consumer-judges, with values higher than 1.15 for all parameters at a nine point scale with extreme values -4 and 4.

Differences are larger attending to flavour parameter, with mean values of 1.89 for the hydraulic extracted oil and 1.15 for the screw press oil. Consumer-type panellist valued positively the intense pistachio flavour of both samples. Pistachio roasting flavour was appreciated in both oils, derived from previous roasting in the hydraulic extraction process and from the operation of the screw press itself in the second. Previous studies had reported a consumer preference for oils extracted by the screw press (Sena-Moreno et al., 2015), but previous roasting of pistachios used in the hydraulic press appear to increase the preference for these oils.

Figure 2. Results of sensory evaluation of pistachio oil samples obtained by hydraulic and screw press

Odour parameter is also more appreciated in the oil extracted with the hydraulic press (1.73) compared to the oil extracted with the screw press (1.15). The higher valuation of the oil extracted with the hydraulic press is directly attributed to roasting. The roasting is responsible for the development of volatile compounds as pyrazines that contribute to the roasting aroma (Ling et al., 2016) and improve the consumer acceptance. The effect of the temperature applied on the exterior part of the tip in the screw press does not improve the development of this components in the same proportion.

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Smaller differences appear in the colour parameter, with values of 1.87 in the oil obtained with the hydraulic press and 1.62 in oil extracted with the screw press. Higher temperatures increase the content of chlorophylls in pistachio oil (Martínez et al., 2016) intensifying the green colour of oil. Greener oils are reported to be more valuated by panellist than yellowish oils (Sena-Moreno et al., 2015).

Sensory analysis made by trained judges did not identify any negative characteristics on oils extracted with the hydraulic press. Minor plastic attributes of 0.6 (at a 0 to 10 valued scale) were found on oils extracted with the screw press.

The results obtained from the physicochemical and sensory analysis show that both extraction systems produce high quality oils, with few differences in their physicochemical quality and some differences attending to the evaluation that consumer-type panellist do about them. Thus, although both oils obtain positive evaluation from potential customers, the oil obtained with the proposed hydraulic press extraction system was the preferred option.

3.3. Economic analysis

3.3.1. Cost related to fresh pistachio

Pistachio is a high valuable nut and its market prize tends to be one of the highest among nuts. For the pistachio oil extraction, high quality pistachios have been used in order to obtain high quality oils. The market price of dried and vacuum packed pistachios used in this study was stabilised in 18€/kg based on the previous experiences of pistachio purchase.

3.3.2. Investment cost

The investment cost is defined by the purchase of needed equipment. As two extraction processes were compared, two different investment needs were calculated. In the case of hydraulic press extraction, the cost of the hydraulic press, the blender, laboratory ovens and centrifuge were considered. For the screw press extraction only screw press and centrifuge were taken into account. In both cases costs related to basic extraction instruments were also considered. Estimated

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Production costs of pistachio oil investment cost was 8,201.38€ for the screw extraction process and 19,620.78€ for the hydraulic extraction process. By comparison it can be observed that the more equipment needed, and the higher cost of the press, makes the investment needed to implement the hydraulic extraction process 2.4 times more expensive.

Assuming that the equipment has to be completely paid back in 10 years, with a cost of money of 7% year, the annual cost for the repayment of the initial investment was calculated for each extraction system proposed. French amortization, straight-line capital consumption allowance, was used for both systems.

3.3.3. Operating cost

Yearly operating cost of proposed extraction methods are indicated in table 3. A single operator was considered to control the whole extraction process. Industrial optimization of oil extraction with the equipment was not considered, describing an 8 hours workday as it is expected from a government establishment. Industrialization of the process could be implemented by extracting 24h a day using shift-workers, reducing the impact of fix capital in final oil price.

Table 3. Comparison of yearly cost for the proposed pistachio production lines Screw press extraction Typology Yearly cost (€/year) Labour cost 35,000.00 Electrical power (variable portion) 281.10 Maintenance (10% fixed capital) 820.14 Investment cost for equipment 1,050.92 Pistachio purchase 77,760.00 Total cost 114,912.16 Hydraulic press extraction Typology Yearly cost (€/year) Labour cost 35,000.00 Electrical power (variable portion) 517.40 Filters 2,817.13 Maintenance (10% fixed capital) 1,972.08 Investment cost for equipment 2,527.02 Pistachio purchase 61,098.02 Total cost 103,931.65

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Regarding power consumption, cost of electrical power used for the equipment was considered. Market cost for electric power was estimated as 0.14 €/kWh. Fixed and variable portion of electricity use were both considered. Two consumptions were calculated, one for each extraction process.

In the case of the hydraulic press extraction, the cost of filters needed for extraction were also calculated. Six filters were used in each extraction and each one of them was reused five times. The market price for these filters, cut and ready to used, is 0.7 €/filter. This cost was considered only in the case of the hydraulic press, as the screw press does not require any filters.

To identify maintenance cost, the differences in the equipment used makes necessary to consider the maintenance as a proportion of fixed capital. The cost of maintenance and spare parts was considered as the 10% of fixed capital for each extraction process. The percentage considered is based in Owolarafe (2002) and validated by the experience with the utilization of the considered equipment. As a result, the differences observed in the cost for equipment were directly translated to maintenance cost.

3.3.4. Product and return

The annual quantity of oil extracted with the two proposed extraction processes results to 1,638 litres for the screw press extraction and 1,147 litres with the hydraulic press extraction. Higher oil yield and reduced pressing time needed to extract oil with the screw press result in an increase of obtained oil of 42.8% compared to hydraulic press. This higher extraction efficiency, both in oil quantity and extraction time, causes a reduction in the production cost of pistachio oil extracted using the process described for the screw press.

Yearly production costs show small differences with 114,912 € needed to implement the hydraulic extraction process and 103,932€ to implement the process considered for the screw press. Differences of equipment cost are balanced by the higher quantity of raw pistachio processed with the screw press process, that results in higher quantities of oil obtained.

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Production cost is calculated as the minimum value of the product (litre of pistachio oil) to make the extraction process economically sustainable in both processes. Taking into account the initial investment, the yearly costs and the quantity of oil produced, the breakeven value that makes the described extraction processes sustainable is 70.4 €/litre for the oil obtained with the screw press and 91.0 €/litre for the oil obtained with the hydraulic press. Differences in oil yield extraction and extraction times seem determinant to make hydraulic press extracted oil more expensive to produce. Production costs are heavily influenced by raw pistachio price that represents 67.4% of total cost in the screw press extraction and a bit less in the hydraulic extraction process (58.5%). Labour is the second cost in importance, having the repayment of the initial investment little influence in final oil prices.

As a result of pistachio oil extraction, valuable pistachio partially deffated flour appears as a secondary product in a percentage higher than 60% of raw pistachio. To be conservative, in the calculated production costs, the selling of this by-product has not been considered. To our knowledge, pistachio flour is an available product at local and e-commerce at a price around 15-40€/kg. The use of pistachio flour has focused major attention lately due to its potential to improve human health (Martínez et al., 2016). The selling of this by-product would reduce the production cost of pistachio oil significantly.

Physicochemical analysis shows little differences in obtained oils, considering both high quality products with a healthy fatty acids profile and high concentrations of sterols. Sensory analysis has evidenced the positive evaluation that potential customers make of pistachio oil. However pistachio oil extracted by using the process that involves the hydraulic press has obtained a better evaluation in the sensory analysis in colour, odour and specially flavour.

Roasting of fresh pistachio has been proved to be one of the factors that makes oil extracted with the hydraulic press more valuated by potential customers due to the appearance of improved sensory characteristics originated by Maillard reaction. The functioning of the screw press itself, although provides oil with significant roasting characteristics, is not equally valuated. The controlled roasting that laboratory ovens

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Economics provides, previous to hydraulic press extraction, cannot be compared to that of screw press as it presents difficulties to be controlled. On the other hand, the screw press continuous extraction enables the obtainment of higher quantities of oil, but also reduces the control that can be done on oil quality, making even possible the appearance of undesirable flavours originated by pistachio residues that remain in the press barrel during continuous operation, with the consequent overheating.

Calculated prices define a product which is more expensive than other oils that are widely consumed. Pistachio oil extracted with presses can be a consumer oriented product if it presents a high level of quality and differentiation. Identified prices makes advisable the sale of 100 ml glass bottles as it is a high valuable product that, foreseeably, presents a distinctive consumption to conventional oils.

Both oils present good qualities to be on the market, but the sensory analysis determines that consumers, in general, prefer pistachio oil extracted with the hydraulic press with a previous roasting. As the production prices of both oils are high, achieving the higher quality should became the goal of producers.

4. Conclusion

A comparative evaluation of costs and quality of pistachio oil obtained by means of two production lines, including two different presses, has been described. By considering a previous roasting before the hydraulic press extraction the differences between the oils obtained with the hydraulic and the screw press are minimized as temperature effect is similarly included. Both production lines have proved to be useful to obtain high quality pistachio oil with minor differences attending to its physicochemical quality. Larger differences have been identified by comparing their sensory properties with more favourable valuations for oil obtained using the hydraulic press.

Attending to the industrial opportunities of pistachio oil production, the breakeven value that makes screw press extraction sustainable is 70.4 €/litre of oil, while the proposed hydraulic extraction is convenient if pistachio oil price reaches 91.0 €/litre.

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It should be noted that yields, extractions times and costs have been identified for a small scale processing unit at an university centre, enabling adjustments and improvements that could reduce production costs although economies of scale are not expected as the extraction requires accurate control and high labour to obtain a differentiated oil with optimal quality able to persuade the consumer to pay for it.

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Martínez, M.L., Fabani, M.P., Baroni, M.V., Huaman, R.N.M., Ighani, M., Maestri, D.M., Wunderlin, D., Tapia, A., Feresin, G.E., 2016. Argentinian pistachio oil and flour: a potential novel approach of pistachio nut utilization. Journal of Food Science and Technology 53, 2260-2269.

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Conclusions/Conslusiones

Conclusions

Regarding the influence of raw material in oil characteristics:

 Almond, pistachio and walnut kernels from different cultivars show significant differences in physical parameters as weight, width, length and kernel colour. Hardness values show significant differences in almond and pistachio cultivars but not in walnuts cultivars. Within physical parameters of nuts oils, viscosity is a valuable parameter as it is related to fatty acids and triglycerides profiles.

 Nuts cultivars compose a huge genetic resource of plant material that can be used to obtain oils and defatted flours with different characteristics. Results indicate the potential of nuts oils and flours as functional ingredients that could be consumed directly or be used for enrichment purposes of other food products. Almond, pistachio and walnut oils show extensive health promoting properties due to their fatty acid profile, and the presence of minor components of interest as tocopherols, phytosterols and polyphenols. Partially defatted flours resulting from extraction have been identified as a source of high-quality proteins and essential minerals. However, high variations in the composition and properties of oils and flours obtained from different cultivars is reported, therefore the selection of the cultivar must always be considered as a crucial factor. An extensive amount of data regarding the physicochemical characteristics of the main almond, pistachio and walnut cultivars is provided.

 The analysis of the source of variability in nuts oils concludes that nut oil chemometrics are significantly affected by the genotype, the crop year and the interaction of both. On the one hand, oil content and fatty acid profile are predominantly determined by the genotype. However, the crop year must also be considered as an important parameter causing variability. In nuts oils, the crop year affects the concentration of some minor components of great interest, such as polyphenols and phytosterols. Genotype has been identified as the main source of variability for the concentration of tocopherols in almonds and pistachio, but a

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Conclusions/Conclusiones

greater influence was reported of crop year in the content of tocopherols in walnut oil.

Regarding the product and process innovation:

 For oil extraction using the screw press, even when low temperatures are selected on the heating ring, the temperature of extracted oil can reach 50˚C, due to the friction of the raw material itself within the crew. This effect is even more intense when low rotational speeds are used. By increasing the screw seed, the continuous supply of fresh raw material in a faster way contributes to the cooling of the system, avoiding an excessive increase of the temperature. The reported increase in oil temperature can cause a reduction of oil quality, but also the appearance of Maillard reaction compounds with antioxidant capacity that can contribute to increase the oxidative stability of the oils. In walnut oil, an increase in the total concentration of polyphenols is observed when oils are extracted at higher temperatures.

 In the analysis of extraction parameters using the hydraulic press, no differences were found regarding yields and physicochemical characteristics in pistachio oils. In the screw press differences were found in oil yields and sensory attributes. Oil extraction with lower rotational speed (17 rpm) resulted in higher yields and the oil was more valued by consumers. Pistachio flour obtained with the two proposed presses showed differences in moisture, fibre, nitrogen, proteins and ash content. The lack of influence of screw press and hydraulic press extraction parameters in physicochemical characteristics of pistachio oil suggest to change the focus to yields and sensory evaluation of oils.

 By using previous roasting of pistachio kernels, carotenoids are transferred from the kernel to oil in greater proportion than chlorophylls. While in the natural pistachio the ratio between the chlorophyll and the carotenoid was 2.76, in oil this ratio fell to 0.18. Higher roasting temperatures favoured the solubilisation of pigments in the oil, mainly in the fraction of chlorophyll derivatives, and promote the degradation of chlorophylls to form pheophytins and pyropheophytins. As a

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result, the temperature and time of roasting affects the concentration of pigments and determines the colour of obtained oil that changes from yellow to bright green colours.

 In walnut, previous roasting of the kernels did not affect the quality of the flour when mild roasting conditions were used. The lipid fraction showed a good nutritional quality with a high vitamin E content (mainly γ-tocopherol) and high phenolic content that provides great antioxidant capacity to the flours.

 Regarding storage, pistachio oil was more stable than almond and walnut oils according to its lower induction period, and it remained the most stable after the storage time, followed by almond oil. The changes on composition and stability parameters of all three nut oils were not affected by the storage temperature in the range 5-20ºC for up to 16 months. The induction period decreased during the storage time for all three nut oils, regardless of the storage conditions. The percentage of polyunsaturated fatty acids decreased slightly throughout the storage.

 Almonds oils obtained from roasted kernels showed higher oxidative stabilities in all cases while the direct addition of garlic caused slight reductions on almond oils stability over time. However, differences appear depending on the garlic cultivar added and the way of garlic addition. Within the considered garlic cultivars, the “Purple garlic from Las Pedroñeras” (European Protected Geographical Indication) shows the most promising results for further study.

Regarding economics:

 Stricter requirements regarding food safety in pistachio trade are positive for producer countries (US and Iran), regardless of their level of economic development. However, some differences appear in the drivers of pistachio exports between the main producers, as US exports are not negatively affected by the distance with the importer (higher differentiation) and they also obtain higher prices (quality premium). Higher food safety standards might not be a promising

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Conclusions/Conclusiones

driver of Spanish pistachio exports as undeveloped countries are also able to comply with the restrictive legislation.

 Regarding the industrial production of pistachio oils, the study of production costs concludes that the breakeven value that makes pistachio oil industrial production sustainable is 70.4 €/litre and 91.0 €/litre for the screw press and the hydraulic press extraction lines, respectively. The higher consumer’s valuation of pistachio oils obtained with the hydraulic press, encourage the use of this production line, even if selling prices would need to be higher.

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Conclusiones

Con respecto a la influencia de la materia prima en las características del aceite:

. Los frutos secos procedentes de diferentes variedades de almendra, pistacho y nuez muestran diferencias significativas con respecto a sus parámetros físicos, incluyendo su peso, su anchura, su longitud y su color. La dureza de los frutos presenta diferencias significativas en las variedades de almendra y pistacho estudiadas, pero no en las de nuez. Entre los parámetros físicos del aceite considerados, la viscosidad aparece como un parámetro de gran interés, al estar relacionada con el perfil de ácidos grasos y el perfil de triglicéridos de los mismos.

. Las diferentes variedades de frutos secos constituyen un enorme y valioso recurso genético de material vegetal que puede ser utilizado para producir aceites y harinas parcialmente desengrasadas con diferentes características. Los resultados indican que estos productos tienen un gran potencial como ingredientes funcionales, tanto para ser consumidos directamente, como al ser utilizados para mejorar otros alimentos. Los aceites de almendra, pistacho y nuez presentan una gran cantidad de propiedades beneficiosas para la salud debidas a su perfil en ácidos grasos y a la elevada presencia de componentes minoritarios de gran interés como tocoferoles, fitosteroles y polifenoles. Por su parte, las harinas parcialmente desengrasadas, que aparecen como subproducto de la extracción del aceite, han sido identificadas como una interesante fuente de minerales esenciales y de proteínas de elevada calidad. Sin embargo, la composición de los aceites y de las harinas muestra grandes diferencias en función de la variedad considerada, por lo que la selección de la misma debe ser considerada como un factor fundamental. Por todo ello, esta tesis doctoral presenta una gran cantidad de datos sobre las diferentes variedades de almendra, pistacho y nuez que pueden ser utilizadas con este objetivo.

. El estudio sobre el origen de variabilidad en los parámetros fisicoquímicos de los aceites de frutos secos concluye que el genotipo, la campaña y la interacción entre ambos afectan significativamente a los mismos. Parámetros de gran interés, como el contenido en aceite y el perfil de ácidos grasos, están determinados

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Conclusions/Conclusiones

mayoritariamente por la variedad; sin embargo, la campaña también debe ser considerada como un parámetro fundamental a tener en cuenta, ya que afecta a la concentración de componentes minoritarios, como los polifenoles o los fitosteroles. Con respecto a la concentración de tocoferoles, el genotipo ha sido identificado como el principal factor de variabilidad en los aceites de almendra y pistacho, mientras que en el aceite de nuez, la campaña ha sido el factor que mayor influencia ha mostrado.

Con respecto a la innovación del proceso y el producto:

. En la extracción de aceite con la prensa de tornillo, incluso cuando se utilizan temperaturas bajas en el anillo calefactor, la temperatura del aceite extraído puede llegar a alcanzar fácilmente los 50˚C, debido a la fricción de la propia materia prima en el tornillo. Este efecto es aún más intenso cuando se utilizan velocidades de rotación bajas. Sin embargo, cuando se aumenta la velocidad de rotación del tornillo, el aporte continuo de materia prima contribuye a la refrigeración del sistema, evitando un incremento excesivo de la temperatura. El aumento de temperatura identificado en el aceite puede reducir su calidad, pero también da lugar a la aparición de compuestos resultantes de la reacción de Maillard con capacidad oxidante, capaces de aumentar la estabilidad oxidativa de los mismos. Además, concretamente en el aceite de nuez, se ha identificado un aumento en la concentración de polifenoles totales en los aceites extraídos a temperaturas más altas.

. En el estudio de los parámetros de extracción utilizando la prensa hidráulica no se han encontrado diferencias en el rendimiento de aceite de pistacho obtenido o en las características fisicoquímicas del mismo. Sin embargo, en la prensa de tornillo, las diferentes condiciones de extracción dieron lugar a diferencias en el rendimiento y en las valoraciones sensoriales. Así, la extracción de aceite a velocidades de rotación más bajas (17 rpm), dio lugar a mayores rendimientos y a aceites más valorados por los consumidores. La harina de pistacho parcialmente desengrasada, obtenida con las dos prensas, presentó diferencias significativas en la humedad y en los contenidos de fibra, de nitrógeno, de proteínas y de cenizas.

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Conclusions/Conslusiones

La falta de influencia de los parámetros de extracción de las prensas hidráulica y de tornillo en los parámetros fisicoquímicos de los aceites, indica la necesidad de cambiar el enfoque de estudio hacia las diferencias que aparecen en el rendimiento y en las valoraciones sensoriales.

. Mediante el tostado previo de los pistachos, se ha observado como los carotenoides se transfieren del pistacho al aceite en una mayor proporción que las clorofilas. Mientras en el pistacho natural el ratio entre clorofilas y carotenoides es de 2,76, en el aceite este ratio se reduce a 0,18. El uso de temperaturas de tostado más elevadas favorece la solubilización de los pigmentos en el aceite, fundamentalmente de la fracción de los derivados de las clorofilas, favoreciendo a su vez la degradación de las clorofilas a feofitinas y pirofeofitinas. Por todo ello, se concluye que la temperatura y el tiempo de tostado afectan de manera determinante a la concentración de pigmentos y al color del aceite de pistacho.

. El tostado previo de la nuez no afecta a la calidad de la harina parcialmente desengrasada, obtenida tras la extracción de aceite, cuando se utilizan temperaturas de tostado bajas o medias. La fracción lipídica de las harinas muestra una elevada calidad nutricional, con un contenido elevado de vitamina E (fundamentalmente γ-tocoferol) y un elevado contenido fenólico, que proporcionan a estas harinas gran capacidad antioxidante.

. En relación al almacenamiento, el aceite de pistacho es más estable que los aceites de almendra y de nuez, si tenemos en cuenta el menor periodo de inducción de estos últimos. Durante el almacenamiento, la composición y los parámetros de estabilidad de los tres aceites de frutos secos considerados no fueron afectados significativamente por temperaturas de almacenamiento en el rango de los 5 a los 20ºC. Así, el periodo de inducción decreció durante el almacenamiento en los tres aceites, independientemente de las condiciones de almacenamiento. Se observa sin embargo, una reducción ligera, pero significativa, del porcentaje de ácidos grasos poliinsaturados de todos los aceites tras su almacenamiento durante 16 meses.

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Conclusions/Conclusiones

. El tostado previo de las almendras da lugar a aceites con una mayor estabilidad oxidativa. Por el contrario, la adición directa de ajo causa ligeras reducciones en la estabilidad de los mismos durante el almacenamiento. Sin embargo, aparecen diferencias en función de la variedad de ajo añadida y el método de adición del mismo. Así, de entre las variedades de ajo consideradas, la variedad “Ajo Morado de Las Pedroñeras” (Indicación Geográfica Protegida) ha mostrado los resultados más prometedores para ser incluida en futuros estudios.

Con respecto a los factores económicos:

. La normativa más restrictiva con respecto a la seguridad alimentaria en el comercio internacional de pistacho tiene un efecto positivo para los dos principales países productores (EEUU e Irán), independientemente de su nivel de desarrollo económico. Sin embargo, sí se observan diferencias en algunos factores que afectan a las exportaciones de estos dos países. Así, las exportaciones de EEUU no aparecen afectadas negativamente por la distancia con el importador (mayor diferenciación) y también obtienen precios más altos (calidad premium). Unos estándares de seguridad alimentaria muy restrictivos a nivel internacional podrían no ser así un factor determinante para favorecer las exportaciones de pistacho españolas, ya que incluso los principales países subdesarrollados productores serían capaces de cumplir con esa legislación restrictiva.

. Con respecto a la producción industrial de aceite de pistacho, el estudio de las dos líneas de producción planteadas concluye que el coste de producción de aceite de pistacho sería de 70,4 €/litro y 91,0 €/litro para las líneas de producción que incluyen la prensa de tornillo y la prensa hidráulica, respectivamente. La valoración más positiva que los consumidores hacen de los aceites obtenidos con la prensa hidráulica animan a utilizar esta última, a pesar de los mayores costes de producción que redundarían en un precio de venta más elevado.

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Artículos:

Rabadán, A., Álvarez-Ortí, M., Gómez, R., Pardo-Giménez, A., Pardo, J.E. (2017). Suitability of Spanish almond cultivars for the industrial production of almond oil and defatted flour. Scientia Hortinulturae, 225, 539-546. Scientia Horticulturae, 225: 539- 546. Factor de impacto: 1,624 (2016). Categoría: Horticulture. Posición: 8 de 36 (Q1).

Rabadán, A., Álvarez-Ortí, M., Gómez, R., Alvarruiz, A., Pardo, J.E. (2017). Optimization of pistachio oil extraction regarding processing parameters of screw and hydraulic presses. LWT-Food Science and Technology, 83, 79-85. Factor de impacto: 2,329 (2016). Categoría: Food Science & Technology. Posición: 32 de 129 (Q1).

Rabadán, A., Pardo, J.E., Gómez, R., Alvarruiz, A., Álvarez-Ortí, M. (2017). Usefulness of physical parameters for pistachio cultivar differentiation. Scientia Horticulturae, 222, 7-11. Scientia Horticulturae, 225: 539-546. Factor de impacto: 1,624 (2016). Categoría: Horticulture. Posición: 8 de 36 (Q1).

Rabadán, A., Pardo, J.E., Gómez, R., Álvarez-Ortí, M. (2017). Effect of almond roasting, light exposure and addition of different garlic cultivars on almond oil stability. European Food Research and Technology, 1-7. Aceptado. Factor de impacto: 1,664 (2016). Categoría: Food Science & Technology. Posición: 57 de 130 (Q2).

Rabadán, A., Álvarez-Ortí, M., Pardo, J.E., Gómez, R., Pardo-Giménez, A., Olmeda, M. (2017). A comprehensive approach to pistachio oil production. British Food Journal, 119 (4), 921-933. Factor de impacto: 1,206 (2016). Categoría: Food Science & Technology. Posición: 75 de 130 (Q3).

Santos, J., Álvarez-Ortí, M., Sena-Moreno, E., Rabadán, A., Pardo, J.E., Oliveira, B. (2017). Effect of roasting coditions on the compostiion and antioxidant properties of defatted walnut flour. Journal of the Science of Food and Agriculture. Aceptado. Factor de impacto: 2,463 (2016). Categoría: Agriculture, Multidisciplinary. Posición: 4 de 56 (Q1).

Rabadán, A., Álvarez-Ortí, M., Gómez, R., de Miguel, C., Pardo, J.E. Influence of genotype and crop year in the chemometrics of almond and pistacho oils. Journal of the Science of Food and Agriculture. Aceptado. Factor de impacto: 2,463 (2016). Categoría: Agriculture, Multidisciplinary. Posición: 4 de 56 (Q1).

Catalán, L., Álvarez-Ortí, M., Pardo-Giménez, A., Gómez, R., Rabadán, A., Pardo, J.E. (2017). Pistachio oil: a review on its chemical composition, extraction systems and uses. European Journal of Lipid Science and Technology, 119 (5). Factor de impacto: 2,145. Categoría: Food Science & Technology. Posición: 38 de 130 (Q2).

Cuesta, A., Álvarez-Ortí, M., Pardo-Giménez, A., Gómez, R., Rabadán, A., Pardo, J.E. (2017). Walnut virgin oil: a different but high quality vegetable oil. Rivista Italiana delle Sostanze Grasse, 94, 9-17. Factor de impacto: 0.125 (2016). Categoría: Food Science & Technology. Posición: 123 de 130 (Q4).

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Publicaciones

Pardo, J.E., Rabadán, A., Gómez-Cantó, C.M., Pardo-Giménez, A., Gómez, R., López, E., Cuesta, M.A., Álvarez-Ortí, M. (2016). Como aprovechar al máximo los aceites de frutos secos. Revista Alimentaria, 474, 98-115.

Roncero, J.M., Álvarez-Ortí, M., Pardo-Giménez, A., Gómez, R., Rabadán, A., Pardo, J.E. (2016). Virgin almond oil: extraction methods and composition. Grasas y Aceites, 67 (3). Factor de impacto: 0.910. Categoría: Food Science & Technology. Posición: 86 de 130 (Q3).

Roncero, J.M., Álvarez-Ortí, M., Pardo-Giménez, A., Gómez, R., Rabadán, A., Pardo, J.E. (2016). Virgin almond oil: parameters of regulated physicochemical quality and stability. Rivista Italiana delle Sostanze Grasse, 93, 237-243. Factor de impacto: 0.125. Categoría: Food Science & Technology. Posición: 123 de 130 (Q4).

Rabadán, A., Álvarez-Ortí, M., Gómez, R., Pardo-Giménez, A., Pardo, J.E. Chemometric characterization of pistachio oils and defatted flours regarding cultivar and geographic origin. Journal of Food Composition and Analysis. Enviado para su publicación.

Rabadán, A., Pardo, J.E., Gómez, R., Pardo-Giménez, A., Álvarez-Ortí, M. Effect of genotype and crop year in the nutritional value of walnut virgin oil and defatted flour. Journal of Agriculture and Food Chemistry. Enviado para su publicación.

Rabadán, A., Pardo, J.E., Gómez, R., Álvarez-Ortí, M. Evaluation of physical parameters of walnut and walnut products obtained by cold pressing. LWT-Food Science and Technology. Enviado para su publicación.

Rabadán, A., Triguero, A. Influence of food safety on trade: evidence for pistacho. Foodborne Pathogens and Disease. Enviado para su publicación.

Rabadán, A., Pardo, J.E., Gómez, R., Álvarez-Ortí, M. Influence of the temperature in the extraction of nuts oils by means of screw press. LWT-Food Science and Technology. Enviado para su publicación.

Rabadán, A., Álvarez-Ortí, M., Pardo, J.E., Alvarruiz, A. Storage stability and composition changes of three cold-pressed nut oils under refrigeration and room temperature conditions. LWT-Food Science and Technology. Enviado para su publicación.

Rabadán, A., Álvarez-Ortí, M., Gómez-Cantó, C.M., Pardo, J.E. Matching physical parameters of almond and pistachio kernels with consumer preference. International Journal of Food Science & Technology. Enviado para su publicación.

Rabadán, A., Álvarez-Ortí, M., Gallardo, L., Gandul, B., Pardo, J.E. Changes in physical parameters, chlorophyll and carotenoids products in pistachio oil during roasting. Journal of Food Composition and Analysis. Enviado para su publicación.

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Publicaciones

Capítulo de libro:

Rabadán, A., Pardo, J.E., Gómez, R., Álvarez-Ortí, M. (2017). Aprendizaje basado en proyectos en la planta piloto de extracción de aceites vegetales de la ETSIAM. University-Vocational Training Network. Herramientas docentes en energía y medioambiente. pp. 90-99. Chartridge Books Oxford. Withney, Reino Unido. ISBN: 978-1-911033-31-8.

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