Centro de Investigación en Alimentación y

Desarrollo, A.C.

EFECTO DE LA ADICIÓN DE EXTRACTOS DE

SUBPRODUCTOS DE DÁTIL SOBRE LOS PRODUCTOS DE
OXIDACIÓN DE LÍPIDOS Y PROTEÍNAS LIBERADOS
DURANTE LAS ETAPAS DE DIGESTIÓN
GASTROINTESTINAL SIMULADA DE UN PRODUCTO
CÁRNICO
_______________________________________

Por:

María de los Ángeles de la Rosa Alcaraz

TESIS APROBADA POR LA

COORDINACIÓN DE TECNOLOGÍA DE ALIMENTOS DE ORIGEN ANIMAL

Como requisito parcial para obtener el grado de

DOCTORA EN CIENCIAS

Hermosillo, Sonora Septiembre 2019

 APROBACIÓN

Los miembros del comité designado para la revisión de la tesis de María de los Ángeles de
la Rosa Alcaraz la han encontrado satisfactoria y recomiendan que sea aceptada como requisito
parcial para obtener el grado de Doctorado en Ciencias.

______________________________
Dra. Armida Sánchez Escalante
Directora de Tesis

______________________________
Dr. Gastón R. Torrescano Urrutia
Integrante del comité de tesis

______________________________
Dr. Humberto Astiazarán García
Integrante del comité de tesis

______________________________
Dr. José Ángel Pérez Álvarez
Integrante del comité de tesis

______________________________
Dra. Juana Fernández López
Integrante del comité de tesis

2
 DECLARACIÓN INSTITUCIONAL

La información generada en la tesis “Efecto de la adición de extractos de subproductos de

dátil sobre los productos de oxidación de lípidos y proteínas liberados durante las etapas de
digestión gastrointestinal simulada de un producto cárnico” es propiedad intelectual del Centro de
Investigación en Alimentación y Desarrollo, A.C. (CIAD). Se permiten y agradecen las citas breves
del material contenido en esta tesis sin permiso especial de la autora, María de los Ángeles de la
Rosa Alcaraz, siempre y cuando se dé crédito correspondiente. Para la reproducción parcial o total
de la tesis con fines académicos, se deberá contar con la autorización escrita de quien ocupe la
titularidad de la Dirección General del CIAD.

La publicación en comunicaciones científicas o de divulgación popular de los datos contenidos en

esta tesis, deberá dar los créditos al CIAD, previa autorización escrita del manuscrito en cuestión
de la directora de tesis.

3
El presente trabajo de investigación fue realizado en el Laboratorio de Investigación en Carne y
Productos Cárnicos del Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD) en
Hermosillo, Sonora, México.
Durante la tesis de doctorado se realizó una estancia de investigación, auspiciada por el Programa
de Becas Mixtas del Consejo Nacional de Ciencia y Tecnología (CONACYT) en la Universidad
Miguel Hernández, en Orihuela, España; bajo la conducción de los Doctores Juana Fernández
López y José Ángel Pérez Álvarez.

4
 AGRADECIMIENTOS

Al Consejo Nacional de Ciencia y Tecnología (CONACyT), por el financiamiento económico para

la realización del Doctorado.
Al Centro de Investigación en Alimentación y Desarrollo, A. C. (CIAD), por brindarme las
facilidades para realizar mis estudios de Doctorado y por ser como un segundo hogar durante mi
formación.
Mención especial merece la Dra. Armida Sánchez Escalante, por la dirección de este trabajo, por
su apoyo incondicional, su tiempo y la confianza depositada en mí para llevar a cabo este proyecto.
Mi más sincero agradecimiento.
A los miembros del comité Dr. Gastón R. Torrescano Urrutia, Dr. Humberto Astiazaran García,
Dra. Juana Fernández López y Dr. José Ángel Pérez Álvarez, por el apoyo brindado para la
culminación de esta investigación, su entusiasmo y disposición durante cada etapa de este trabajo,
no sin mencionar las palabras de aliento, así mismo por ser mi ejemplo como investigadores.
A los C.P. Ignacio García Fierros y Blanca Almada, les expreso mi gratitud por la confianza
brindada y por abrirme las puertas de su empresa I.B. MISIONEROS (Empresa Socialmente
Responsable), para obtención de la materia prima utilizada en este proyecto, sin ustedes no hubiera
sido posible este trabajo.
A mis Maestros, quienes me formaron durante los ocho semestres del doctorado y que sin duda
sembraron semilla para mi futuro en el área de investigación.
A la Dra. Rosario Maribel Robles Sánchez, por las facilidades de su laboratorio para llevar acabo
parte experimental del trabajo de investigación.
A la empresa NORSON (Empresa Socialmente Responsable), por el apoyo brindado para la
obtención de la materia prima que fue parte fundamental de este trabajo de investigación.
A mis compañeros de laboratorio, Dr. Rey David Vargas Sánchez, MC. Alejandro Felician Vega
Lic. Carlos Sampieri Jiménez, MC. Brisa del Mar Torres Martínez y a las estudiantes del Verano
de la Investigación Científica y Tecnológica del Pacífico: Cassandra Guadalupe Machain Meza,
Araceli Manzano Gómez, Dayanara Soto López, Mariana Torrecillas Ramírez y Kenya Mitzel
Tirado Barajas, quienes contribuyeron en el trabajo experimental, mil gracias porque de cada uno
me llevo un gran aprendizaje, y sobre todo gratos momentos.

5
Al Laboratorio de Química y Biotecnología de Productos Lácteos por abrirme las puertas y
brindarme las facilidades de sus equipos en determinada etapa de la investigación. A la Dra.
Belinda Vallejo Galland, Dr. Aarón González Córdova y Dra. María de Jesús Torres Llanez.
Especialmente a la MC. María del Carmen Estrada Montoya, por su valioso apoyo técnico durante
el análisis de muestras.
Al grupo de investigación IPOA de la Universidad Miguel Hernández en Orihuela España, quienes
me acogieron de la manera más amable y profesional durante mi estancia de investigación, en
especial al Dr. Manuel Viuda Martos, Dra. Estrella Sayas Barberá y a la doctoranda Raquel Lucas
González, mi más sincera gratitud por su amistad y su ejemplo del trabajo en equipo. Además, a
los doctores Carmen Ballester Costa y José María Sánchez Larrosa por su apoyo técnico y amistad.

6
 DEDICATORIA

A Dios, porque no hay mayor expresión de amor. En los momentos de tensión o angustia me diste
fuerza, paciencia y sabiduría para sobrellevar cada reto, gracias por tantas bendiciones y
provisiones durante estos años en Hermosillo, Sonora.

A mi madre María de los Ángeles Alcaraz Reyes, por el gran amor que me profesas todos los días,
por tus enseñanzas y por ser mi mejor ejemplo de fortaleza que día a día reconozco como tu mayor
virtud. Te amo.

A mi hija Isabella Ortiz de la Rosa, quien se ha convertido en mi fuerza e inspiración para ser una
mejor persona. Eres sin duda sinónimo de alegría y dulzura, y me siento afortunada porque siempre
expresas el cariño hacia el trabajo que hacen tus padres en CIAD.

A Ángel Ortiz Estrada, por tu apoyo incondicional durante el doctorado, porque formaste parte
de este proyecto familiar que sin duda sorteaste con amor y paciencia.

A mis Hermanos, por siempre confiar en mí, por su apoyo moral y su gran cariño, el cual, a pesar
de la distancia me lo transmiten y me motivaban a seguir creciendo personal y profesionalmente.

A mis Sobrinos, gracias por ser tan lindos conmigo y por impulsarme a seguir hacia adelante y
permitirme ser su ejemplo, los amo.

A mis Amigos entrañables, Brenda Rivera Soto, María Isabel Jiménez, Andrés Pacheco, Rigoberto
Hernández, Martín Moreno, Elizabeth Reyes, Monserrat Portillo, José Alfredo Quintana, Aline
Reyes Díaz, Erick Gabriel Valenzuela, Mayra Yazmín Sánchez, Georgina Alba, Dafne Velázquez
Jiménez y familia, Alejandro Felician Vega, Elena Carrasquer, Carlos Sampieri, Guadalupe Cota
Verdugo, Elizabeth Cota Verdugo, Gerardo Reyna, Mariela Menchaca Armenta y Marcela Becerra
Castro, quienes cerca o a la distancia siempre estuvieron conmigo brindándome sus consejos y su
amistad sincera.

A los chicos ERASMUS, quienes durante mi estancia en España me brindaron muchos momentos
felices, y por enseñarme que, el idioma no es una barrera para cosechar grandes amistades, Giulia

7
Bazzocchi, Paola Uda, Francesca Gargiulo, Nalan Demirbaş, Fernanda SLP, Estefany Otero, Frank
Toledo y Luce Michel.

8
 CONTENIDO

APROBACIÓN............................................................................................................................... 2
DECLARACIÓN INSTITUCIONAL .......................................................................................... 3
AGRADECIMIENTOS ................................................................................................................. 5
DEDICATORIA ............................................................................................................................. 7
CONTENIDO ................................................................................................................................. 9
RESUMEN .................................................................................................................................... 11
ABSTRACT .................................................................................................................................. 12
1. SINOPSIS.................................................................................................................................. 13
1.1 Justificación ......................................................................................................................... 13
1.2 Antecedentes ........................................................................................................................ 13
1.3 Hipótesis .............................................................................................................................. 16
1.4 Objetivo General.................................................................................................................. 16
1.5 Objetivos Específicos .......................................................................................................... 17
1.6 Sección Integradora del Trabajo .......................................................................................... 17
1.7. Literatura Citada .................................................................................................................20
2. USE OF SEED BYPRODUCT AS A SOURCE OF FOOD INGREDIENTSFOR
MEAT AND MEAT PRODUCTS: A REVIEW.................................................................... 22
3. EVALUACIÓN DE FITOQUÍMICOS Y ACTIVIDAD ANTIOXIDANTE DE
SUBPRODUCTOS DE DÁTIL (Phoenix dactylifera L.) PRODUCIDOS EN EL
ESTADO DE SONORA ........................................................................................................... 83
4. RECOVERY AND CHARACTERIZATION OF BIOACTIVE COMPOUNDS OF
DATE (Phoenix dactilyfera L.) SEED USING ULTRASOUND ASSISTED
EXTRACTION ......................................................................................................................... 91
5. EFFECT OF INCLUSION OF DATE SEED OIL AND EXTRACT ON THE
FATTY ACID PROFILE, PHYTOSTEROLS AND LIPID OXIDATION OF RAW
AND COOKED PORK MEAT ............................................................................................. 116
6. BIOACCESIBILIDAD DE UN EXTRACTO DE COPRODUCTOS DE DÁTIL
(Phoenix dactylifera L.) UTILIZADO COMO ADITIVO NATURAL EN
HAMBURGUESA DE CERDO ............................................................................................ 139
7. BIOACCESSIBILITY OF POLYPHENOLS OF DATE SEED EXTRACT USED
AS NATURAL ANTIOXIDANT AGAINST LIPID OXIDATION DURING
IN VITRO DIGESTION OF PORK PATTIES.................................................................... 151
8. ASSESSMENT OF LIPID OXIDATION INHIBITION, TOTAL ANTIOXIDANT
ACTIVITY, COLOR AND SENSORY ATTRIBUTES OF PORK PATTIES
INCORPORATE WITH DATE BYPRODUCT DURING REFRIGERATED
STORAGE............................................................................................................................... 170

9
9. CONCLUSIONES GENERALES ........................................................................................ 195
10. RECOMENDACIONES ...................................................................................................... 196
11. ANEXOS ............................................................................................................................... 197
11.1. Evaluación del Contenido de Fitoquímicos y Actividad Antioxidante de Extractos
de Dátil en sus Diferentes Etapas de Madurez Obtenidos con el uso de Diferentes
Solventes y Métodos de Extracción ..………………………………………………….199
11.2. Evaluación de la Actividad Antimicrobiana de Extractos de Subproductos de Dátil
Obtenidos Utilizando Diferentes Solventes y Métodos de Extracción…...……………205

10
 RESUMEN

El aprovechamiento de subproductos agroindustriales para obtener moléculas funcionales,

como antioxidantes, es una tendencia actual en la industria cárnica. Su inclusión puede contribuir
a la inhibición en la formación de los productos de oxidación de lípidos (LOX) y proteínas (POX),
reduciendo las pérdidas de calidad de los productos cárnicos. El objetivo fue evaluar el efecto de
la adición de extractos de subproductos de dátil en un producto cárnico, sobre los metabolitos
potencialmente dañinos liberados durante su digestión gastrointestinal in vitro (DGI). Se
obtuvieron extractos de subproductos de dátil [fruto (EFD) y semilla (ESD)] con diferentes
solventes y métodos de extracción. También se extrajo aceite de la semilla para su adición en
hamburguesa de cerdo (HC), sustituyéndolo por grasa y comparando con aceite de oliva,
evaluándose composición química, color, LOX y POX, ácidos grasos y esteroles. Adicionalmente
se evaluó vida útil en refrigeración, comparando ESD (0.2% y 2.0%) y control, analizando
composición química, pH, color, LOX, actividad antioxidante, composición de fitoquímicos y
calidad microbiológica. Para la evaluación de DGI, se extrajeron los fitoquímicos de la HC
conteniendo ESD (0.2% y 2.0%), e identificándose por HPLC; además, se evaluó actividad
antioxidante y peroxidación lipídica (gástrica e intestinal). El análisis sensorial del producto
incluyó nivel de agrado y aceptación. Los resultados muestran que los extractos retardan la LOX a
partir de 0.2%, durante la vida útil de HC. La inclusión de aceite de semilla de dátil en HC modificó
la composición de los ácidos grasos, incluyendo fitoesteroles (β-esterol, estigmaesterol y
campesterol); con la adición del ESD se retardó la oxidación (LOX y POX). Durante la DGI el
ESD (2.0%) evidenció los mejores resultados de bioaccesibilidad, y los análisis por HPLC
revelaron la presencia de ácidos fenólicos (ferúlico y protocatécuico) y un flavonoide (catequina)
los cuales se mantuvieron estables durante la DGI del producto. La inhibición de LOX (gástrica e
intestinal), fue detectada en el tratamiento con ESD 2.0%; sin embargo, en el control los valores
de malondialdehído fueron 4.09 y 4.60 mg/kg, en las fases gástrica e intestinal, respectivamente.
Sensorialmente, al incluir 2.0% de ESD, el color y la apariencia de la HC fresca fueron afectados;
sin embargo, en el producto cocinado la aceptación fue de 74%, y el nivel de agrado fue alto. De
acuerdo a las tendencias actuales por alimentos sin aditivos sintéticos, los ESD pueden considerarse
candidatos idóneos para su inclusión en el diseño de productos cárnicos apetitosos y más seguros.

Palabras clave: dátil, oxidación, digestión in vitro, hamburguesa de cerdo

11
 ABSTRACT

The use of agro-industrial byproducts to obtain functional molecules, like antioxidants, is

one of the most promising trends in the meat industry. Their inclusion can contribute to lipid and
protein oxidation products (LOX and POX) inhibition, which reduce the loss of quality of meat
products. The aim was to evaluate the effect to date seed byproducts addition to meat products on
potentially harmful metabolites released during in vitro gastrointestinal digestion (GID). Extracts
of the date byproducts were obtained [fruit (DFE) and seed (DSE)] using different solvents and
extraction methods. Also, was extracted date seed oil for inclusion in pork pattyes (PP), substituting
by pork fat and comparing with olive oil, chemical composition, color, LOX and POX, fatty acids
and sterols were evaluated. Additionally, was evaluated shelf life during refrigerated storage,
comparing ESD (0.2% and 2%) and control, chemical composition, pH, color, LOX, antioxidant
activity, phytochemicals, and microbial quality was analyzed. For GID, phytochemicals were
extracted from PP containing DSE (0.2% and 2%), and identified by HPLC; also, antioxidant
activity and lipid peroxidation (gastric and intestinal) were evaluated. The sensorial analyses of
product included two tests (hedonic and acceptability). Results showed that extracts delay LOX
from 0.2% during shelf life of PP. The date seed oil inclusion in PP modified fatty acid
composition, and confer phytosterols (β-sterols, stigmasterol, and campesterol); and when DSE
was included oxidation delayed (LOX and POX). During GID the DSE (2.0%) evidenced the better
results of bioaccessibility, and HPLC analysis revealed presence of phenolic acids (ferulic and
protocatechuic) and one flavonoid (catechin), which were stables through GID of product. The
LOX inhibition (gastric and intestinal), was not detected in DSE 2.0%: however, in control
malondialdehide values were 4.09 and 4.60 mg /Kg, gastric and intestinal, respectively. Sensory,
when 2.0% DSE was included, color and appearance of PP were impacted, although, in cooked
products acceptability was 74% and hedonic scores were high. According to actual tendencies
towards less synthetic additives foods, DSE can be considered suitable candidates for inclusion for
the design of appetizing and safer meat products.

Keywords: date, oxidation, in vitro digestion, pork pattie

12
 1. SINOPSIS

1.1 Justificación

La carne es una fuente importante de nutrientes y proteínas de alta calidad. Sin embargo, durante
su procesamiento y preparación culinaria se da lugar a una serie de procesos oxidativos que afectan
principalmente a lípidos y proteínas, generando compuestos tóxicos como malondialdehído (MDA)
y compuestos nitrosos, mismos que comprometen la salud del consumidor. En la última década, el
uso de antioxidantes obtenidos a partir de plantas, especias, frutos y sus subproductos se ha
planteado como una estrategia viable para disminuir dicho problema. Sin embargo, estos últimos,
han centrado la atención de los investigadores en la última década derivado de la enorme cantidad
que se generan a nivel mundial a través de los eslabones de la cadena agroalimentaria. Su
aprovechamiento, por un lado, provee de moléculas funcionales como antioxidantes que ayudan a
inhibir, retardar o disminuir los procesos oxidativos en matrices alimentarias como la carne y
productos cárnicos y por el otro, se disminuye la contaminación que genera su disposición al medio
ambiente.

1.2 Antecedentes

El diseño de alimentos funcionales en las últimas décadas se ha convertido en uno de los mercados
más dinámicos. El incremento en la prevalencia de enfermedades crónico degenerativas como son
diabetes, obesidad, hipertensión y algunos tipos de cáncer, ha promovido que la industria de
alimentos sea más innovadora y brinde opciones viables de alimentos, que, además de nutrir,
ofrezcan otras propiedades. En este sentido, la adición de fitoquímicos ha sido una de las estrategias
para intentar mitigar algunos problemas derivados del consumo de alimentos que comprometan la
salud del consumidor. Entre estos alimentos, la carne y los productos cárnicos han sido
ampliamente sometidos al escrutinio, debido a los riesgos para la salud, principalmente los

13
asociados con citotoxinas que potencialmente pueden ser generadas durante su procesamiento. Lo
anterior se ha convertido en un reto, pero también en una oportunidad para la comunidad científica
(Jiang y Xiong, 2016).
En particular, los fitoquímicos son moléculas multifuncionales y hay evidencia de que pueden
ayudar a proteger al organismo frente a enfermedades crónicas, principalmente a través de la
reducción del daño oxidativo de algunas biomoléculas, principalmente lípidos y proteínas. El
mecanismo antioxidante, se cree que ocurre a través de dos vías: 1) mediante la donación de
electrones para romper y terminar el ciclo de oxidación en la etapa de propagación y, por lo tanto,
evitar la formación de radicales libres; 2) eliminando el iniciador de radicales libres (ROS) para la
inhibición del catalizador que inicia la cadena (radical) o limitando el iniciador de radicales uniendo
metales como hierro y cobre como quelantes de metales para estabilizarlos en una forma inactiva
o soluble (Allen y Cornforth, 2010; Rice-Evans, Miller y Paganga, 1996).
Los frutos son una fuente excelente de estas biomoléculas; sin embargo, la literatura científica ha
mostrado que, no sólo los frutos son fuente de materia prima para la obtención de dichas moléculas.
También los subproductos generados durante las etapas de su producción (recolección, selección,
almacenamiento y comercialización) como son, cáscaras, semillas y pulpa, representan una fuente
potencial y excelente de moléculas que aún conservan sus funciones biológicas (Galanakis, 2012).
A nivel mundial, se generan enormes cantidades de desechos los cuales representan 1.3 billones de
toneladas por año. Dichos desechos se producen a través de la cadena de suministro de alimento y
se han distinguido cinco eslabones: producción agrícola, manejo post-cosecha y almacenamiento,
proceso, distribución y consumo (Gustavsson, Cederberg, Sonesson, VanOtterdijk, y Meybeck,
2011) lo anterior conduce a serios problemas ambientales junto con una gran pérdida de materia
prima.
A partir de subproductos agroalimentarios se puede obtener fibra dietaria, antioxidantes,
antimicrobianos, minerales, ácidos grasos, ácidos orgánicos y proteínas, los cuales, debido a su
bajo costo, es posible utilizarlos como materia prima para su adición a otras matrices alimentarias.
En el caso de los antioxidantes, se asume que su uso puede retardar la oxidación de biomoléculas
funcionales como lípidos y proteínas, proceso que limita la calidad y aceptabilidad, así como
también afecta la producción de radicales libres, sabor, textura, color y componentes nutricionales
del alimento. Por lo anterior, la industria de alimentos ha puesto un gran interés en este tema, y
demanda aditivos provenientes de fuentes naturales, que no comprometan la seguridad del

14
alimento, así como las características sensoriales que son atractivas para el consumidor, y que
además determinan el poder de compra (Shah et al., 2014; Ahmad et al., 2015).
Uno de los frutos que ha sido utilizado en la elaboración de productos cárnicos con valor agregado
es el dátil. Este, provee un amplio rango de nutrientes esenciales (minerales, vitaminas,
aminoácidos, fibra dietaria, que incluye pectinas y β-D-glucanos, entre otros); también tiene
importantes implicaciones como auxiliares en terapéuticas para ciertas enfermedades como son
diabetes, enfermedades coronarias y obesidad (Baliga et al., 2011). En la literatura está bien
documentado que el fruto del dátil y su semilla en forma de polvo, pulpa e infusión, son
ampliamente utilizados en el tratamiento de dichas afecciones. Asimismo, extractos obtenidos a
partir de este fruto y su semilla han mostrado resultados interesantes en la prevención de daño
oxidativo a nivel in vitro e in vivo (Subash et al., 2015, Hasan y Mohieldein 2016).
En el año 2015, en México la producción nacional de dátil fue de 7,427.10 T, cifra producida en
los estados de Sonora, Baja California y Coahuila, siendo Sonora el estado con mayor
participación, con 4,845.40 T y un valor de la producción de 180,128.33 miles de pesos, con ello
Sonora se posiciona como el principal productor a nivel nacional, como resultado de las
condiciones climatológicas idóneas para su cultivo exitoso y único en el país
(http://infosiap.siap.gob.mx/aagricola_siap_gb/icultivo/index.jsp). Sin embargo, se ha
documentado que, de la cantidad producida de este fruto se descarta como subproducto alrededor
del 10%, considerando la semilla como el principal subproducto de la industria datilera, la cual
representa 10% del fruto, y misma que ha sido poco explorada como una fuente valiosa de
ingredientes bioactivos (Sirisena et al., 2015). A pesar de su importancia económica, es necesario
destacar que el conocimiento respecto a la composición y características funcionales de los
subproductos de dátil cultivado en México es limitado; por ello la importancia de llevar a cabo
investigación en este campo.
En otros grupos de trabajo, se han incluido productos alimentarios intermedios (PAI) como fibra
dietética o pulpa, provenientes de subproductos de dátil, para la formulación de productos cárnicos
como paté de hígado de cerdo, salchicha seca curada, salchicha bologna, dando como resultado
que, al incluirlos mejoran las propiedades tecnológicas como incorporación de humedad y fibra
dietaria, incrementando en la capacidad de emulsificación, disminución del contenido de grasa y
oxidación de lípidos, no encontrando diferencias de estos productos con el producto control

15
(Sánchez Zapata et al., 2011; Martín Sánchez et al., 2013; Martín Sánchez et al., 2014; Martín-
Sánchez et al., 2017).
Sin embargo, la inclusión de extractos a partir de subproductos de dátil en matrices cárnicas no se
ha explorado, a pesar de que pueden tener un efecto potencial en la mejora de las propiedades
tecnológicas de los productos cárnicos formulados, poco se conoce de la bioaccesibilidad de los
compuestos presentes en los extractos y productos alimentarios intermedios que se incluyen en la
formulación de matrices cárnicas. El término bioaccesibilidad se define como, la cantidad de un
compuesto que es liberado de la matriz y es considerado disponible para su absorción a través de
la pared intestinal. Para conocer este parámetro, existen herramientas como los modelos de
digestión gastrointestinal simulada que nos acercan a conocer los eventos fisiológicos que suceden
una vez que el alimento ha sido consumido, así como la cinética de los compuestos adicionados a
la matriz alimentaria, como pueden ser los fitoquímicos (Gil-Izquierdo et al., 2002, Bouayed et al.,
2011; Minekus et al., 2014). Sin embargo, poco se ha explorado en matrices complejas como la
carne y los productos cárnicos bajo este enfoque.

1.3 Hipótesis

La adición de extractos subproductos de dátil variedad Medjool a un producto cárnico provee de

compuestos con propiedades biológicas, que al ser liberados durante la digestión gastrointestinal
simulada tienen un efecto protector contra los metabolitos potencialmente dañinos presentes en la
matriz alimentaria.

1.4 Objetivo General

Evaluar el efecto de la adición de extractos de subproductos de dátil variedad Medjool a un

producto cárnico, sobre los metabolitos potencialmente dañinos liberados durante la digestión
gastrointestinal simulada de la matriz alimentaria.

16
 1.5 Objetivos Específicos

• Evaluar la composición química proximal de los subproductos de dátil.

• Obtener extractos de subproductos de dátil utilizando diferentes solventes y métodos de
extracción, para determinar su capacidad antioxidante y antimicrobiana.
• Caracterizar y cuantificar los compuestos presentes en los extractos obtenidos a partir de
subproductos de dátil.
• Evaluar el efecto de la adición de extractos de subproductos de dátil en hamburguesas de
cerdo sobre la composición química, propiedades fisicoquímicas, calidad microbiológica y
sensorial, así como oxidación de lípidos y proteínas, durante su almacenamiento en
refrigeración.
• Evaluar la liberación de compuestos biológicamente activos durante la digestión simulada
in vitro del producto cárnico adicionado con extractos de subproductos de dátil.

1.6 Sección Integradora del Trabajo

La presente tesis está integrada por siete capítulos, los cuales, a su vez corresponden a documentos
que se sometieron o someterán para su publicación en revistas nacionales e internacionales
indizadas y de divulgación. Asimismo, cubren parte de los objetivos particulares de este trabajo.
Dichos documentos, se presentan en el formato, estatus (publicado, aceptado o revisión interna) e
idioma en los que fueron escritos.
En el artículo 1, se presenta un artículo de revisión que tuvo como objetivo conocer el estado del
arte del uso de semillas, principal subproducto de la transformación industrial de frutos, para la
obtención de aditivos con uso potencial en la industria cárnica. En dicha revisión, se plasma la
cantidad de desechos agroalimentarios que se generan a nivel mundial y las implicaciones que tiene
su disposición al medio ambiente, las principales fuentes y las estadísticas de su producción, así
como su caracterización química. Por otro lado, se presentan los métodos para la recuperación de
moléculas bioactivas y los principales hallazgos de su uso e impacto de su utilización como

17
antioxidantes, antimicrobianos, sustitutos de grasa, emulsificantes, y colorantes en carne y
productos cárnicos, además del efecto sobre la aceptación sensorial cuando estos subproductos son
incluidos en la elaboración de un nuevo producto. Finalmente, se enfatiza en las nuevas tendencias
de los consumidores hacia la demanda de productos más “naturales” y la aceptación sensorial de
los productos cárnicos elaborados con aditivos provenientes de semillas, debido a que este es un
parámetro determinante en la decisión de compra del consumidor y que tiene implicaciones
importantes para el desarrollo de alimentos cárnicos más saludables, seguros y apetitosos.
En el artículo 2, se presenta un artículo de investigación original, en el cual se evaluó el contenido
fitoquímico y actividad antioxidante de subproductos de dátil (fruto no comercial) producidos en
el estado de Sonora. Como materia prima se utilizaron dátiles no comerciales de la región de
Caborca, Sonora, y considerando que el método de extracción y el tipo de solvente son factores
que afectan el rendimiento, actividad biológica y riqueza de moléculas. La extracción de
compuestos fitoquímicos se llevó a cabo mediante extracción asistida por ultrasonido (EAU) (1 h)
y maceración (48 h); en ambos casos, se utilizaron como solventes agua, acetona: agua, y etanol:
agua. Respecto al contenido fitoquímico actividad y antioxidante se encontró que el solvente afecta
el contenido de fenoles y flavonoides totales, captación del radical DPPH y el poder reductor. En
cuanto a los métodos de extracción, la EAU mostró ser la más eficiente debido al corto tiempo de
extracción y la mezcla de acetona fue el solvente más adecuado para la extracción de compuestos
de esta materia prima.
En el artículo 3, se presentan los resultados de un artículo de investigación original, en el que se
identificaron y cuantificaron fitoquímicos de extractos de semilla de dátil y se evalúo su actividad
antioxidante. A partir de polvo de semilla de dátil, se elaboraron extractos utilizando extracción
asistida por ultrasonido y maceración. Se utilizaron diferentes mezclas de solventes (acetona: agua,
etanol, agua, metanol: agua y agua). Tanto el método como el solvente fueron determinantes en la
concentración de fitoquímicos, la mezcla de acetona-agua reveló la mayor concentración de fenoles
totales, flavonoides y actividad antioxidante (ABTS, DPPH, y poder reductor). A pesar de que la
región geográfica y las condiciones climáticas son factores importantes en el contenido de
fitoquímicos, nuestros resultados de identificación mediante HPLC revelaron ocho compuestos del
grupo de los ácidos fenólicos hidroxibenzóicos e hidroxicinámicos y un flavonoide, mismos que
concuerdan con lo reportado en otros estudios.

18
En el artículo 4, se presentan los resultados de un artículo de investigación original, en el cual se
evaluó la sustitución parcial de grasa de cerdo por aceite de semilla de dátil y como antioxidante
natural extracto de semilla de dátil. Al reemplazar parcialmente los aceites se encontró una mayor
oxidación de lípidos (TBARS), sin embargo, al adicionar el extracto de semilla de dátil (ESD)
interesantemente los valores disminuyeron, especialmente en el tratamiento con aceite de oliva,
mismo que había reportado los valores más altos, observándose que la oxidación de lípidos y
proteínas disminuyó, por lo que se infiere que el ESD retarda la oxidación de lípidos y está en
correspondencia con el menor contenido de hidrazonas proteicas en los tratamientos con respecto
al control. Al incluir aceite de oliva en las hamburguesas, se encontró un incremento en los ácidos
grasos monoinsaturados (MSFA) especialmente del ácido graso oleico, mientras que el tratamiento
con aceite de semilla de dátil afectó positivamente el contenido de esteroles (β-esterol,
estigmasterol y campesterol). De manera contundente, se observó el efecto antioxidante del
extracto de semilla de dátil y un cambio positivo en el perfil de ácidos grasos al incluir aceite de
oliva y fitoesteroles por parte del aceite de dátil. Por lo anterior, se sugiere que el extracto de semilla
de dátil puede ser utilizado para inhibir oxidación de otras matrices cárnicas y fuentes de aceite
susceptibles a oxidación.
En los artículos 5 y 6, se muestran los resultados de dos artículos (divulgación y científico)
derivados de investigación original. En los cuales, se evalúo la bioaccesibilidad de un extracto de
semilla de dátil adicionado a una hamburguesa de cerdo durante la digestión gastrointestinal (DGI)
in vitro y la inhibición de peroxidación lipídica. Se evaluaron tres etapas de la DGI; oral, gástrica
e intestinal y se pudieron identificar y cuantificar fitoquímicos presentes en el extracto hasta la fase
intestinal de la digestión. Por lo que, se puede inferir que el extracto es bioaccesible y por tanto
disponible para su absorción. Los resultados de peroxidación lipídica tienen relación con lo anterior
ya que la inclusión de extracto de semilla de dátil mostró un efecto inhibitorio de la peroxidación
lipídica (gástrico e intestinal), comparado con el control donde se logró cuantificar
malondialdehído (fases gástrica e intestinal). Uno de los principales fitoquímicos presentes en el
extracto de dátil es la catequina, la cual interactúa con proteínas y lípidos y es capaz de alterar la
cinética de absorción, favorablemente. Lo anterior, plasma el potencial del uso de extracto de
semilla de dátil como un aditivo antioxidante para su uso en matrices susceptibles a la oxidación y
el desarrollo de productos cárnicos más saludables.

19
En el artículo 7, se plasman los resultados de un artículo de investigación original, en el cual se
evaluó el efecto de la adición de extracto de semilla de dátil sobre la oxidación de lípidos, actividad
antioxidante, contenido de fitoquímicos, atributos sensoriales y calidad microbiológica de
hamburguesas de cerdo durante su almacenamiento en refrigeración. En el estudio se evaluaron
tres tratamientos diferentes incluyendo dos niveles de ESD (0.2% y 2.0%) y un control, los cuales
se almacenaron durante 9 días (2 ºC), periodo durante el que se evaluó, pH, color, oxidación de
lípidos, actividad antioxidante, contenido de fitoquímicos y calidad microbiológica. Además, se
realizó la evaluación sensorial de los atributos del producto fresco (color, olor y apariencia) y
cocinado (olor, sabor y jugosidad). El ESD 0.2% y 2.0% mostró efectividad para retardar la
oxidación de lípidos; particularmente, la inclusión de 2.0% incrementó la actividad antioxidante y
el contenido de fenoles y flavonoides en la hamburguesa de cerdo. No obstante, afectó el color del
producto en fresco; mientras L* disminuye, a* aumenta debido a los pigmentos del extracto, a
pesar de ello, el producto cocinado con 2.0% de ESD fue aceptado por 74% de los panelistas,
respecto a 88% y 87% del tratamiento 0.2% ESD y control, respectivamente. Por lo anterior se
sugiere, la inclusión de ESD en productos cárnicos como un potente aditivo natural con
propiedades antioxidantes y que confiere biomoléculas como fitoquímicos que podrían contribuir
al desarrollo de productos cárnicos más saludables, no afectando la calidad sensorial del producto.

1.7. Literatura Citada

Allen, K., y Cornforth, D. 2010. Comparison of spice-derived antioxidants and metal chelators on
fresh beef color stability. Meat Sci, 85(4), 613-619.
Ahmad S, Gokulakrishnan P, Giriprasad R y Yatoo M. 2015. Fruit-based Natural Antioxidants in
Meat and Meat Products: A Review. Crit Rev Food Sci Nutr. 55: 1503-1513.
Bouayed, J., Hoffmann, L., y Bohn, T. 2011. Total phenolics, flavonoids, anthocyanins and
antioxidant activity following simulated gastro-intestinal digestion and dialysis of apple
varieties: Bioaccessibility and potential uptake. Food Chem. 128(1), 14-21.
Galanakis CM. 2012. Recovery of high added-value components from food wastes: Conventional,
emerging technologies and commercialized applications. Trends Food Sci Tech. 26: 68-87.
Gil-Izquierdo, A., Zafrilla, P., y Tomás-Barberán, F. A. 2002. An in vitro method to simulate
phenolic compound release from the food matrix in the gastrointestinal tract. Eur Food Res
Technol. 214(2), 155-159.

20
Gustavsson, J., Cederberg, C., Sonesson, U., Van Otterdijk, R., y Meybeck, A. (2011). Global food
losses and food waste (pp. 1-38). Rome: FAO.
Hasan M y Mohieldein A. 2016. In Vivo Evaluation of Anti Diabetic, Hypolipidemic,
Antioxidative Activities of Saudi Date Seed Extract on Streptozotocin Induced Diabetic
Rats. J Clin Diagn Res: JCDR 10: FF06.
Jiang, J., y Xiong, Y. L. (2016). Natural antioxidants as food and feed additives to promote health
benefits and quality of meat products: A review. Meat Sci. 120, 107-117.
Martín‐Sánchez, A. M., Ciro‐Gómez, G. L., Zapata‐Montoya, J. E., Vilella‐Esplá, J., Pérez‐
Álvarez, J. A., y Sayas‐Barberá, E. 2014. Effect of Date Palm Coproducts and Annatto
Extract on Lipid Oxidation and Microbial Quality in a Pork Liver Pâté. J Food Sci. 79(11),
M2301-M2307.
Martín-Sánchez, A. M., Ciro-Gómez, G., Vilella-Esplá, J., Pérez-Álvarez, J. Á., y Sayas-Barberá,
E. (2017). Physicochemical and Sensory Characteristics of Spreadable Liver Pâtés with
Annatto Extract (Bixa orellana L.) and Date Palm Co-Products (Phoenix dactylifera
L.). Foods. 6(11), 94.
Minekus, M., Alminger, M., Alvito, P., Ballance, S., Bohn, T., Bourlieu, C., . . . Dupont, D. (2014).
A standardised static in vitro digestion method suitable for food–an international consensus.
Food Funct. 5(6), 1113-1124.
Sánchez-Zapata, E., J. Fernández-López, M. Peñaranda, E. Fuentes-Zaragoza, E. Sendra, E. Sayas
y J. A. Pérez-Alvarez. 2011. Technological properties of date paste obtained from date by-
products and its effect on the quality of a cooked meat product. Food Res Int. 44(7):2401-
2407.
Shah MA, Bosco SJD y Mir SA. 2014. Plant extracts as natural antioxidants in meat and meat
products. Meat Sci. 98: 21-33.
SIAP. (2015). http://infosiap.siap.gob.mx/aagricola_siap_gb/icultivo/index.jsp.
Sirisena S, Ng K y Ajlouni S. 2015. The Emerging Australian Date Palm Industry: Date Fruit
Nutritional and Bioactive Compounds and Valuable Processing By‐Products. Compr Rev
Food Sci F. 14: 813-823.
Subash S, Essa MM, Al-Asmi A, Al-Adawi S, Vaishnav R y Guillemin GJ. 2015. Effect of dietary
supplementation of dates in Alzheimer's disease APPsw/2576 transgenic mice on oxidative
stress and antioxidant status. Nutr Neurosci. 18: 281-288.

21
2. Use of Seed Byproduct as a Source of Food Ingredients for Meat and Meat
Products: A Review

Ma. Ángeles De la Rosa-Alcaraz, Gastón R. Torrescano-Urrutia, Rey David Vargas-Sánchez,

Nelson O. Huerta-Leidenz, Armida Sánchez-Escalante

Comprehensive Reviews in Food Science and Food Safety

(Enviado a corrección de idioma)

22
 Use of Seed Byproduct as a Source of Food Ingredients for Meat and Meat products: A
Review
Ma. Ángeles De la Rosa-Alcaraz1, Gastón R. Torrescano-Urrutia1, Rey D. Vargas-Sánchez1,
Nelson O. Huerta-Leidenz2, Armida Sánchez-Escalante1*

1
Centro de Investigación en Alimentación y Desarrollo, A.C. Coordinación de Tecnología de
Alimentos de Origen Animal. Ctra. Gustavo Enrique Astiazarán Rosas No. 46, Col. La Victoria.
Hermosillo, Sonora, 83304, México.
2
Texas Tech University, 2500 Broadway, Lubbock, Texas, 79409, United States of America.

Abstract
Currently the meat industry face major challenges principally derived of association its components
as lipids with chronic-degenerative diseases development. The strategies to mitigate this problem
are in constant evolution/change. Thus, the trends to use natural products as additives have led to
researchers to find novel ingredients to development safe, nutritious, and appetizing meat and meat
products. In this context the right strategies for recovery of functional molecules from agro-
industrial byproducts that can use as raw material to design of functional products is an attractive
option, not only by great diversity of molecules that contain but also their use contributed to delay
environmental problems together with a great loss of raw material. The rational disposal of seeds
represents no only a resource problem but also an environmental and economic one, efforts are
necessary for authorities, academic, and agro-industrial producers to carry out more research
focused on seed as a bioactive rich agro-industrial byproducts, from which compound can be
recovered, characterized and used for the development of raw materials for inclusion in complex
food matrices as meat and meat products giving added value and improving their safety and
nutritive value.

Keywords: seeds, agro-industrial byproducts, functional ingredients, meat

23
 1. Introduction
According to FAO, roughly one third of the edible parts of food produced for human consumption,
gets lost or wasted globally, which is about 1.3 billion tons per year. Food is wasted throughout the
food supplied chain (FSC), and five system boundaries are distinguished: agricultural production,
postharvest handling and storage, processing, distribution and consumption from initial agricultural
production down to final household consumption (Gustavsson, Cederberg, Sonesson, Van
Otterdijk, & Meybeck, 2011). The above, results in the generation of a large amount of byproducts,
however, according to Mirabella, Castellani, and Sala (2014) notwithstanding specific statistics on
food waste quantify are quite difficult to be collect, the best available estimates account for a total
food loss in the EU (European commission, 2010) 42% of food waste is produced by households,
39% losses occur in the food manufacturing industry, 14% pertains to food sector (ready to eat
food, catering and restaurants), while 5% lost along distribution chain (agricultural food loss is not
included in this estimation). In addition, food waste is expected to rise of 89 Mt to about 126 Mt
by 2020 if additional prevention policy or activities are not undertaken.
Byproduct is the term used by scientific in order to notify that food waste are the ultimate substrates
for the recapture of functional compounds, this are considered source of component of low cost
because exists technologies that allow of key compounds recovery and their integration in the food
chain. Thus new aspects concerning the use of these byproducts for further exploitation on the
production of food additives or supplements with high nutritional value and economically attractive
have gained increasingly interest (Faustino et al., 2019; Galanakis, 2012).
Considering the above, this review is focused in the use of recovery by-products generated during
some popular fruit and vegetable processing, specifically seeds, which remaining and represent a
serious problem for disposal. Recent study showed that seeds are byproducts rich in bioactive
compounds such as phytochemicals, fatty acids, dietary fiber and minerals their use could be
improve foods lacking of them (M Freire et al., 2017).
In meat industry, the search of natural ingredients that contribute to production of safe, nutritious,
appetizing, and healthy is one of the key challenges for the global meat industry of the 21 st
(Estévez, 2017). Thus, the use of this bioactive molecules that contributes significantly to improve
meat and meat products quality, is one of the central focus (Oostindjer et al., 2014). Per example
phytochemicals could be applied as natural antioxidants and antimicrobial since they extend the
shelf life of the product by delaying the formation of off-flavors and rancidity, antimicrobials

24
capable of diminishing or eradicating microorganisms of sanitary interest, fatty acids, whose use
in changes in fat contents and lipids profile could contribute to development of foods more healthy
and well-balanced diet (M Freire et al., 2017), and dietary fiber that could be improving
technological and healthy properties used as emulsified, and gelling agent (López-Marcos, Bailina,
Viuda-Martos, Pérez-Alvarez, & Fernández-López, 2015). For the above the objective of this
review is to project to scientific community the potential use of seeds byproducts as raw material
in meat and meat products, tendency in extraction methods by recovery bioactive compounds from
these material, the inclusion in meat matrix, as well as sensory acceptability and finally are
described future perspectives based on results of scientific research.

2. Principals source of seed byproducts: global statistic and chemical composition

According to the data on global fruit and vegetable production, the amount of residues with
potential utilization after processing has been estimated in millions tons every year. Of greatest
importance is the byproduct potential because of the content of biocomponents, which may be used
for innovative food production (Kowalska, Czajkowska, Cichowska, & Lenart, 2017). Processing
of fruits and vegetables produces various types of byproducts such as solid residues of peel/skin,
seeds, stones, stem and pulp (Amaya-Cruz et al., 2015). From the environmental protection point
of view safe disposal of residue from processing is highly important in order to limit this quantity,
e.g. by integrated approaches for complete utilization of the byproduct in recovery valuable
byproducts or ingredients (Goula & Lazarides, 2015).
The seed byproducts are known as a good natural source of bioactive molecules composed
principally of oil, protein, dietary fiber, common characteristic between different sources of seeds
fruit byproducts. In the past, these had been described as devalued products only used in animal
feed, however, in the last decade the recovery of chemical molecules from these source that can
applied in pharmaceutical, food industry, cosmetically areas have raised great interest, in fact, in
addition to contributing to the overall process sustainability, natural ingredients used in daily life
products have greater acceptability for most people over their synthetic counterparts (Kowalska et
al., 2017). Following are described the nutritional value of some traditional seed byproducts.

Grape

25
The grape (Vitis vinifera L.,) is one of the major fruit crops in the world, according to the FAO
over 77 million metric tons. About 80 % of the grape production is used in juice making, during
this process seeds and skins remaining as byproducts. An extensive review about nutritional and
chemical composition of grape seed has been done by (Rodríguez & Ruiz, 2016). Basically grape
seed contains (w/w) between 5.02% and 7.16% of moisture, 40% fiber, 5.8 to 16% oil. The latter,
is characterized by a very interesting profile of fatty acids: 90% poly-and monounsaturated fatty
acids, particularly of linoleic acid (about 70%) followed by oleic acid, also has unique attributes
respect to others oils don’t have effect on foods flavor (Bialek et al., 2017).
Regarding proteins content values range from 7.5 to 16.5%, and 7% complex phenolic compounds,
including tannins, flavonoids particularly flavan 3-ol (catechin, epicatechin, and epectechin-3-o-
gallate monomers) and their polymers. Non-phenolic antioxidants such as tocopherols and β-
carotene are mainly concentrated in grape oil, where also sterols are presents (Rodríguez & Ruiz,
2016). Other researchers have reported that extracts from grape seed possess an antioxidant
potential 20 and 50 times higher than vitamin E and C, respectively (R. Carpenter, M. O’grady, Y.
O’callaghan, N. O’brien, & J. Kerry, 2007; Shi, Yu, Pohorly, & Kakuda, 2003). However, the
chemical composition in all seed depends on variety of fruit and where it comes.

Tomato
As one of the most popular vegetables in the world, tomatoes are rich in lycopene, phenolics,
organic acids, vitamins and many others beneficial compounds. In addition to being served as fresh
vegetable, tomato is also consumed in the form of various processed products, such as paste, juice,
sauce, puree and ketchup. During processing industrial of afore mentioned products are generating
large amounts of waste mainly in the form of seeds and peels (Z. Lu, Wang, Gao, Ye, & Zhao,
2019). The tomato seed commonly accounted for approximately 45% of the resulting tomato
pomace (TP). Their composition is characterized by the high levels of biologically active
compounds such as oil and protein, as mentioned by Fuentes et al. (2013), in this study the seed
present 9% of moisture, 32% of protein, 22 % of fat, ash 5 %, carbohydrates 25%, crude fiber 16%.
The protein isolate obtained from tomato seed contain all the essential amino acids (including
lysine) and meets the minimum requirements of the reference protein by specific groups of
population. Therefore the flour from tomato seeds may be used as an ingredient in various
preparation foods (Sarkar & Kaul, 2014). Up to now, tomato seed into meat products not has been

26
used, due richness of lycopene in peel and pomace tomato that have been used as colorant that
favored the appearance of meat products, especially those from red meats. However as fed
ingredient have been exploited with cows, cattle, sheep, goats, ewes, pigs, rabbits, hamsters,
chickens, roosters, hens, and carp. Overall, the feeding of seed component could lead to significant
changes not only in animal growth (weight gain) but also in the yields and nutritional quality of the
resulting foods (mainly meat, milk and eggs) (Yadav, Malik, Pathera, Islam, & Sharma, 2016).

Date seed
Date palm is a popular and high-revenue crop, and the consumption of date fruits is no longer
limited to the Middle Eastern countries. Due to nutritional value and taste has gained interest. Date
seed is an abundant byproduct of date processing and excellent source of valuable functional
ingredients as dietary fiber, oil, minerals, resistant starches and antioxidants even above of pulp
and peel (Al-Farsi & Lee, 2008; Mrabet et al., 2015; Sirisena, Ng, & Ajlouni, 2015). Habib and
Ibrahim (2009) search eighteen varieties and found significate difference (p<0.05) between
varieties, the moisture ranged 8.64-12.45%, protein of 4.81-5.83%, fat 5.71-7.92% and ash content
of 0.82-1.14%. According to Besbes, Blecker, Deroanne, Drira, and Attia (2004) in lipid fraction
of date seed the major unsaturated fatty acid was oleic acid (41.3-47.7%), the main saturated acid
was lauric (17.8%) palmitic (15%), capric, myristic, myristoleic, palmitoleic, stearic, linoleic and
linolenic acids were also found. In the same way, hydroxytyrosol, alpha tocopherol and β-sitosterol
has been reported, even the oxidative stability was better than that of most vegetable oil and
comparable to that of olive oil, that distinguish them from other vegetable oils, and they could
easily be conserved and used in cosmetic, pharmaceuticals and food products.

Pomegranate
Pomegranate is an important fruit of tropical and subtropical region, which originated in the Middle
East and India (Basiri, 2015). The fruit can be divided into several anatomical compartments: (1)
outside peel, (2) inside peel (pellicles), and (3) arils (pulp and seed). Arils are usually used fresh
consumption, juice, jams and jellies production, represents 50 to 70% of total fruit and comprises
of 78% juice and 22% seeds. Pomegranate seed of pomegranate processing show average content
of about 37-143 g/kg of fruit (Kalamara, Goula, & Adamopoulos, 2015). Have found that extracts
from pomegranate seed may have the potential to be a good source of antioxidants 353.2 mg of

27
gallic acid equivalents (GAE) in 1 g of extract (Jing et al., 2012), and are mainly composed of fiber
and lipids, with an oil content varying from 12% to 20% on a dry weight basis (gained relevance
because great potential for utilization in pharmaceutical industry). Several studies shown that
pomegranate seed oils are good source of polyunsaturated fatty acids (90.3%), especially linoleic
(3523 and 10586 mg/100g of seed, punicic acid omega-5 (ω-5) (86.2%) and tocopherol 135 to 525
mg/100 g of oil, concerning sterols, β-sitosterol was the most predominant (364 to 553 mg/100 g)
(Fernandes et al., 2015; Jing et al., 2012).

Citrus
The genus Citrus, belonging to the Rutaceae family comprise 140 genera. Citrus sinensis (Orange),
Citrus paradisi (Grapefruit), Citrus lemon (lemons), Citrus reticulate (tangerine), and Citrus
aurantifolia (lime) are among the most popular fruits in the world especially in tropical and
subtropical countries (Kamal, Anwar, Hussain, Sarri, & Ashraf, 2011). According to FAO, 2016
annual citrus production was estimated at over 124 thousand tones; orange whit over 54% of the
total production worldwide, constituted the most important citrus species followed by 26.5%
tangerine, 12.8% lemon and lime, and 6.7% grapefruit. Brazil, United State, Japan, China, México,
and countries of the Mediterranean region were the main producer.
Although, citrus fruit are consumed as fresh fruit, juice, flavored citrus beverages, marmalade, jams
and other food products. The main purpose of citrus fruits is to produce citrus juice, during the
citrus juice extraction process, large quantities of byproducts are produced (55% of the weight
products). They are constituted by peels, seeds, remaining after juice extraction and are a serious
environmental problem for disposal. Residues of citrus juice production are composed principally
by water, soluble sugar, fiber, organic acids, amino acids and proteins, minerals, oils and lipid, and
also contain flavonoids and vitamins. All of these components are found in different amount
depending on the fraction of the fruit (juice, alberdo, flavedo, rag and pulp and seeds) and therefore
their proportion in citrus juice residues depends on the juice extraction system used (Anwar et al.,
2008; Fernández-López et al., 2004).
Citrus seeds contain about 36% oil and 14% protein, the oil content varies greatly with the
particular citrus variety. C. sinensis, C. paradise, and C. reticulate show palmitic, oleic and linoleic
acids, also lesser amounts of stearic and linoleic acids are the most common fatty acids in these
seed oils. The range of oil content of citrus seeds in the present analysis was found to exceed those

28
of three conventional oilseeds crops: cotton (15.0-24%), soybean (17.0-21.0%), and olive (20.0-
25.0%) (Anwar et al., 2008). After seed oil extraction, the remaining as citrus seed meal had an
excellent amino acid profile, higher glycine, cysteine, methionine and tryptophan and lower lysine
than soybean meal, and also serve as a good source of K, Ca, Na, Fe and Mg, for above the citrus
seed can be an ingredient with great potential for inclusion in foods (El‐Adawy, El‐Bedawy,
Rahma, & Gafar, 1999).
In other way, seeds can be used to recovery limonoids, which are typical citrus fruit terpenoids.
Within the different parts of the fruit, limonin was found to be 6 to 12 times higher in the seeds
than in the peel and juice, respectively (Yahia, 2017). In lemon for instance, the seed contains
principally eriocitrin and hesperidin. Neoericcotrin and naringin have similar concentration in the
peel, whereas in the seed ariocitrin is 40 times more abundant than naringin. Four phenolic acids
are found in the citrus seed; caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid (Bocco,
Cuvelier, Richard, & Berset, 1998).

Mango
The Mangifera genus comprises about 40 species, the most common species in this genus is
Mangifera indica, or the common mango, which is cultivated in more than 103 countries worldwide
(Solís-Fuentes & María del Carmen, 2011). According to FAO in 2017 was main tropical fruit
produced; in terms of regional distribution, approximately 75% of mangoes are produced in Asia,
while 15% and 10% are produced in Africa and Latin America and the Caribbean countries,
respectively (Altendorf, 2019). The mango is consumed fresh principally, although, exist a variety
of processed products that include syrups, juices, jelly, among others. During industrial processing,
is used mainly the pulp and bagasse, whilst seed and peel are discarded as byproducts, these
represent between 40% to 60% of total weight of fruit; 12-15% pulp, peel and bagasse 15-10%,
and mango seed 15-20%. Above, represent loss by producer and in other way due to high sugar
content and water, the byproducts quickly decompose becoming hotspot of contamination
(Kodagoda & Marapana, 2017).
The mango seed has aroused interest in scientific community due to it has been reported as a
byproduct whit high content of bioactive compounds (phenolic compounds, carotenoids, vitamin
C, and dietary fiber) that improve human health (M. H. A. Jahurul et al., 2015). It is a good source
of carbohydrates (58-80%) and protein (6-13%) and has an important profile of essential amino

29
acids (Diarra, 2014). In M. pajang has been reported highest amount of phenolic acids (gallic,
coumaric, sinapic, caffeic, ferulic, chlorogenic) and flavonoids (naringin, hesperidin, rutin,
diosmin) (Fadzelly Abu Bakar, Mohamed, Rahmat, Burr, & Fry, 2010) and potent antioxidant
activity (1738.2 mg trolox/g and IC50 (0.078 mg/mL) (Torres-León et al., 2016). Fat content like
other vegetable fats, are mainly constituted of palmitic (3 to 18%), stearic and oleic acids (34 to
56%), and represent 7.1-15% on a dry basis, and phenols 98.7 mg/g. It is a promising safe and
natural source of edible fats, as it does not contain any trans fatty acids, which has great potential
for use in the food industry (M. Jahurul et al., 2018; M. H. A. Jahurul et al., 2015; Tapia, Pérez,
Cavazos, & Mayett, 2013). Respect to fiber content, flours from India and Indochinese cultivars
had 11 and 15% respectively, and presented functional properties as water absorption capacities,
oil absorption capacity and foaming capacity (Okpala & Gibson-Umeh, 2013).

Avocado
The avocado (Persea americana Mill.) is a tropical and subtropical fruit, native to southern Mexico
and Central America, produced in more than 60 countries, mainly México, Israel, Colombia and
Dominican Republic (Altendorf, 2019). Hass variety is the most produced and dominates the
international market due its quality, productivity, resistance to commercial management and
constant availability. The avocado processing industry produces essential oils, and once pulp is
processed, seed, peel and exhausted pulp are discarded, resulting in thousands of tons of waste
byproduct. Specify avocado seed represent up to 16% the total weight of the fruit because between
peel and seed corresponding to 30-33 % of the fruit (López-Cobo et al., 2016).
The chemical composition of seed varies between cultivars and sometimes varies between the same
cultivar, i.e., Haas seed: moisture content, 7.66-54.45%, ash content, 1.29-3.85%, lipids 3.32-
14.7%, protein 0.14-3.44, fibers 3.98, carbohydrates 79.54 (Araújo, Rodriguez-Jasso, Ruiz,
Pintado, & Aguilar, 2018). Contain great amount of phenolic compounds and display a higher
antioxidant activity than the pulp (J. G. Rodríguez-Carpena, Morcuende, & Estévez, 2011), the
levels of these compounds in the seeds vary whit variety of avocado, conditions of growth and
stage of maturity (Dabas, M Shegog, R Ziegler, & D Lambert, 2013). In Hass variety has been
found B-type procyanidins with A type procyanidins as minor components. Monomers, oligomers
and polymers with a degree of polymerization (Wang, Bostic, & Gu, 2010). Other study of avocado
seed and peel reported the presence of phenolic compounds belonging to five groups, i.e., catechin,

30
hydroxybenzoic acids, hydroxiccinamic acids, flavonols, and procyanidins (J. G. Rodríguez-
Carpena et al., 2011). The confirmation by mass spectra analysis revealed phenolic and other polar
compounds: quinic acid, citric acid, hydroxytyrosol glucosidase, 1-caffeoylquinic acid, tyrosol
glucosidase, 3-O-caffeoylquinic acid, 3-O-p-coumaroylquinic acid, 4-caffeoylquinic acid, vanillic
acid glucosidase procyanidin trimer A (I) procyanidin trimer A (II) catequina/epicatechin gallate
(Kosińska et al., 2012; López-Cobo et al., 2016). Figueroa, Borrás-Linares, Lozano-Sánchez, and
Segura-Carretero (2018) using a powerful instrumental technique identified forty five phenolic
compounds in avocado seed that confirm the potential of avocado seed as a source of bioactive
ingredients for its use in the food sector.
Avocado seeds present high proportions of soluble dietary fiber and neutral detergent fiber, their
fibrous residues retain four times their weight in water and six time their weight in oil due it
avocado seed fibrous residues properties make them promising technological ingredients (Barbosa-
Martín, Chel-Guerrero, González-Mondragón, & Betancur-Ancona, 2016). For the above, is
crucial developing strategies that lead to integral use of this byproducts. In Table 2 are summarized
any investigation about application of avocado seed extracts in muscle foods.

Apple
The mass fraction of the seed in apple pomace account for 4-7% , according to (Soong & Barlow,
2004) apple seed present more antioxidant activity that edible part of fruit, also an important protein
quantity (~50%) and oil (~24%, exhibited high stability include oleic and linoleic acid) minerals,
and phytochemicals (chlorogenic acid and phloridzin, epicatechin, catechin, and cyaniding-3-
galactosides). Unfortunately, contain a cyanogenic glycoside, amygdalin, the degradation of which
by glucosidase naturally present in human intestine can lead to the formation of cyanide causing
human severe toxicity therefore these compounds or whole seeds must be separated (Bolarinwa,
Orfila, & Morgan, 2014; Rabetafika, Bchir, Blecker, & Richel, 2014). An study conducted by
Walia, Rawat, Bhushan, Padwad, and Singh (2014) evaluated in vitro cytotoxic activity of apple
seed oil against a specific cell line (CHOK1 (Chinese hamster), A549 (human lung carcinoma) and
SiHa (human cervical cancer cell)), and exhibited, its potential as an anticancer agent.

Tamarind
Tamarind (Tamrindus indica L.) is a tree that can reach up a high of 30 m. This tree is better known

31
for the pod pulp (approx. 40%), which is rich in vitamin C, tartaric, malic, citric acids and sugars.
The worldwide distribution includes, Bangladesh, India, Myanmar, Malaysia, Sri Lanka, Thailand,
Australia and several African, Central American and South American countries (Alpizar-Reyes et
al., 2017).
Tamarind seed is an available and underutilized byproduct tamarind pulp industry, consisting of
30% of hard brown seed coat containing up to 70% of polysaccharides, mainly composed by a
galactoxyloglucan polysaccharide (Khounvilay & Sittikijyothin, 2012), which is composed of β-
(1,4)-D-glucan back-bone substituted with side chains of α-(1,4)-d-xylopyranose and (1,6) linked
[β-D-galactopyranosyl-(1,2)-α-D-xylapiranosyl] to glucose residues, where glucose, xylose and
galactose units are present in the ratio of 2.8:2.25:1.0 as the monomer units and with a molecular
weight of 720-880 kDa (Sharma, Mondal, Mukesh, & Prasad, 2014).
The tamarind seed contained 6.54 g/kg total phenolics predominantly: 2-hydroxy-3´4´-
dihydroxyacetophenone, methyl 3,4-dihydroxybenzo-ate, 3,4-dihydroxyphenylacetate, follow by
procyanidins; tetramer (30.2%), hexamer (23.8%), trimer (18.1%), and pentamer (17.6%) with
lower amounts of procyanidin B2 (5.5) and (-)- epicatechin (4.8%) (Sudjaroen et al., 2005; Tsuda
et al., 1994). Tamarind seed extract has been shown strong antioxidant scavenging activity against
hydroxyl radicals, peroxyl radical generated by ABTS/H2O2/peroxidase and
ABTS/H2O2/myoglobin system, and superoxide anions produced by the ABTS/H2O2/FeCl3 (fenton
reaction) (Hemshekhar, Kemparaju, & Girish, 2011), which is very important in muscle foods.
On the other hand, from tamarind seed any materials have been characterized as; tamarind seed
gum that contains approximately 12.7-22.7% of protein, 3-7.5% of fat, 0.7-8.4% of crude fiber, 61-
79.8% of carbohydrates and 0.7-3.3 of ash (depending to botanical source and method used) and
mucilage extracted from seeds that represent a source of natural hydrocolloids with low cost
offering a low-calorie intake turning it in an ideal product for the development/improvement of
health products with beneficial properties to human consumption, making them a potential option
for application in the food (Alpizar-Reyes et al., 2017; Nayak, Pal, & Santra, 2015).

Guava
Guava (Psidium guajava L.), is a popular exotic fruit in tropical and subtropical countries,
according to FAO in 2017, 75 percent of guava production originated in Asia, 15 percent in Africa
and 10 percent in Latin America and the Caribbean in 2017 (Altendorf, 2019). The guava cultivars

32
planted around the world, including Red Land, Supreme, and Ruby. Guava fruits are commonly
consumed in natura, but also in the form of jellies, juices, pie filling, ice creams and other products.
Since guava is extremely rich in vitamins A, B, and C (180-300 mg of vitamins C per 100 g of
fruit), showing higher concentration than orange and lemons, this fruit is highly appreciated by
populations in developing countries both as a functional food and as a natural medicine (Patrícia
Barbosa Pelegrini & Franco, 2011).
Guava processing waste (seed, peel, and pulp leftovers) can represent up to 30% of the fruit volume
(Lima, Ferreira, Vitali, & Block, 2019) and guava seed are 6 to 12 % of the fruit weight. Their
proximate composition, on a wet weight basis is: 7.6 % protein, 16.0 % fat, 0.93% ash and 4.1 %
water. The fat contains 11.8 % saturated fatty acids and 87.3 % unsatured fatty acids (76.5 %
polyunsaturates). The fatty acids are 0.1 % myristic, 6.6 % palmitic, 4.6 % stearic, 10.8 % oleic,
76.4 % linoleic, 0.3 % arachidonic and 0.1 % linolenic (Prasad & Azeemoddin, 1994). Respect to
dietary fiber contain, seed contain: total dietary fibre 78.4 g/100 g, soluble dietary fibre 4.3 g/100
g and insoluble dietary fibre 74.4 g/100 g, the high content of insoluble dietary fiber was associated
to the greatest antiobesogenic effect respect to mango and peach byproducts, in murin model
(Amaya-Cruz et al., 2015).
El Anany (2015), extracted the phenolic compounds from raw guava seeds and found a total phenol
content of 973.80 mg GAE/ 100 g, total flavonoids content 290.30 mg/100 g DPPH radical
scavenging 63.74 (%),reducing power 0.56 (absorbance at 700 nm). Also antinutrients: tannin
325. 67 mg/100 g and phytic acid 273.63 mg/100 g (El Anany, 2015).
Phytic acid, has been reported to exert hepatotoxic activity, however, in a study directed by
(Amaya-Cruz et al., 2015), using guava byproducts that included peel, remnants of pulp and seeds,
founded that supplementation whit seed containing guava byproducts did not have hepatotoxic
effect.
From guava seed was isolates a new peptide whit antimicrobial activity against gram-negative
bacteria causing gastrointestinal infection in humans, unlike many members of the glycine rich
family, it is characterized by its low molecular weight and three-dimensional structure similar to
antimicrobial peptides from several other families (Patricia B. Pelegrini et al., 2008).

Melon
According to the FAO, the world melon production is 784, 205 tons and the cultivated area is 893,

33
855 ha (FAO, 2013). The chemical composition of three varieties seed (Honeydew, Dessert 5 and
Hybrid 1) were as follows: fat content ranged from 41.6-44.5%, protein 34.4-39.8%, crude fiber
4.5-8.5, carbohydrates 8.2-12.7, soluble sugar 3.7-4.2% and minerals 4.6-5.1%.
Melon seed is rich in biologically active substances such as larger quantity of tocopherols (345 and
304 mg/kg), sterols (0.3 %), and phospholipids (0.3-07%) from varieties Honeydew and Hybrid 1,
respectively. The seeds are a waste product are rich in glyceride oil, globally, they are used to
produce glyceride oil by cold pressing. It possesses high nutritional value due to its high content
of polyunsatured fatty acids (PUFA): linoleic acid (31.0-69.0%), followed by oleic (12.1-31.0%),
palmitic (7.8-39.36%) and stearic acid (4.9-10.45%), ΣSFA 28.8-38% and ΣUFA 62.0-71.2. The
principal sterols in melon seed oil were β-Sitosterol and ∆5 –Avenasterol free and esterified,
phospholipids, phosphatidylinositol (24.4-33.9), phosphatidylcholine (23.0-33.1), and
phosphatidic acid (13.5-16.3). Because of their composition, melon seed can be successfully as an
alternative source in the food industry, respect to melon seed oil in spite of not widespread in food
industry are not inferior in quality from the common oils in the market (Mariod & Matthaeus, 2008;
Petkova & Antova, 2015).

3. Extraction of bioactive compounds from seed fruit byproducts

The extraction process can be defined as a separation based on differences in solubility in which
a solvent is used to solubilize and separate a solute from a material with lower solubility in the
solvent employed (solid-liquid extraction). While, the extraction process based on differences in
solubility in which a solvent is used to extracted a solute from a solution in a specific solvent knows
like liquid-liquid extraction (Berk, 2018). Therefore, based on the solubility properties, in various
research works have been used solvents of different polarity to obtain compounds mainly
phytochemicals from natural sources, including roots, woods, barks and leaves of plants, as well
as from the peel, pulp and seed of their fruits (Falowo, Fayemi, & Muchenje, 2014; J.-G.
Rodríguez-Carpena, Morcuende, Andrade, Kylli, & Estévez, 2011; Shah, Bosco, & Mir, 2014).
Moreover, several methods have been developed from the past in order to increase the extraction
of phytochemicals, for example homemade methods such as cooking or infusion, in order to treat
certain diseases such as conjunctivitis, diarrhea, diabetes, flu, among others (Azmir et al., 2013;
Hasan & Mohieldein, 2016). In the last decade, a large number of investigations have been
developed to obtained phytochemicals using different extractions methods (Table 1), among which

34
conventional (extraction by maceration, Soxhlet and hydro distillation) and unconventional
methods (extraction assisted by ultrasound, enzymes, microwave, pressurized liquid and
supercritical fluid) (Azmir et al., 2013).
The extraction by maceration is considered a solid-liquid extraction, due in the system the three
components necessary for the extraction are included: A, the extractable solute (phytochemicals);
B, the insoluble matrix (commonly fiber or another insoluble component; and C, the liquid solvent
(water, ethanol, hexane, among others) (Berk, 2018). Soxhlet extraction is based on phytochemical
extraction at the boiling point of the solvent used (Luque de Castro & García-Ayuso, 1998), while
hydro distillation relies the extraction of volatile compounds through the steam trawl (Berk, 2018).
Additionally to the solvent used for the extraction, the phytochemical yields can be influence by
several factors including solvent-solute ratio, temperature, time and number of extraction used (Al-
Farsi & Lee, 2008).
In relation to unconventional methods, the ultrasound-assisted extraction involve the inter-
particle collisions of the solute and shockwaves created for collapsing cavitation bubbles in the
solvent, which reduce the particle size and increase surface area of the solute increasing the mass
transfer (Chemat et al., 2017; Vilkhu, Mawson, Simons, & Bates, 2008). The enzyme-assisted
extraction is based on the hydrolysis of the organic material with enzymes (lipases, proteases,
carbohydrases, among others) to increase the release of phenolic compounds from the solute (Puri,
Sharma, & Barrow, 2012). During the microwave-assisted extraction, the non-ionizing
electromagnetic waves exert a considerable amount of pressure in the organic material improving
the porosity, which allows better solvent extraction (Routray & Orsat, 2012). Furthermore,
pressurized liquid extraction works in accordance with the principle of static extraction using
superheated liquids, in order to improve the diffusivity of the solvent and increase the speed and
efficiency of extraction (Benthin, Danz, & Hamburger, 1999). In addition, the supercritical fluid
extraction relies the extraction with a substance gas and/or liquid in conditions of pressure and
temperature above its critical point, which increase their diffusion coefficient and reduce the
viscosity and surface tension than a liquid solvent, leading high penetration and mass transfer to
the solute (Azmir et al., 2013).

4. Use of seed byproducts in meat and meat products

In the past, the seed from agro-foods byproducts were considered low value material, however,

35
currently their use has gained great interest, because it represents widely source of natural
compounds that may be regarded as a possible source of new antioxidant and preservatives based
on antimicrobial activity when are applying in meat and meat products as food ingredients (Table
2), likewise have shown great potential as animal feed supplements, improving the final quality
characteristics of meat, a recompilation of this studies are show in Table 3. Their use in daily life
have greater acceptability for most people over their synthetic counterparts which could potentially
be translated into an industrial application if the appropriate regulatory body gives a positive
opinion (Faustino et al., 2019; Kowalska et al., 2017). Following are present any examples about
specific activities.

4.1 Antimicrobial
Microbial contamination is a major safety concern in meat products, because it is a nutrient dense
food that provide ideal conditions for microbes to grow and defines its perishable nature (Aiyegoro,
2014). In order to satisfy consumers demands and recover its confidence in the safety of food
products, the food industry was motivated to look for natural alternatives that exhibit strong
antimicrobial and or antioxidants properties (Fernández-López, Zhi, Aleson-Carbonell, Pérez-
Alvarez, & Kuri, 2005).
Plant phenolic compounds are believed to be promising candidates to exert antimicrobial activity
since they can modify bacterial behavior by affecting microbial cell by a number of antimicrobial
mechanism, including attacking the phospholipid bilayer of the cell membrane, disrupting enzyme
system, compromising the genetic material of bacteria, quorum sensing and production of virulence
determinants (Maisuria, Hosseinidoust, & Tufenkji, 2015; Tajkarimi, Ibrahim, & Cliver, 2010).
Some phytochemicals from seeds with antimicrobial activity have been described, per example,
the monomeric procyanidins, major compounds in grape seed extract, was associated to fully
inhibition of Gram-positive bacteria at one dose of 850-1000 ppm, while Gram-negative bacteria
at one doses of 1250-1500 ppm, (Jayaprakasha, Selvi, & Sakariah, 2003) similar to founded by
Juhee Ahn, Grün, and Mustapha (2007) using commercial grape seed extracts decreased E. coli
O157:H7, S. typhimurium, L. monocytogenes, and Aeromonas hydrophila in 1 Log compared to
the control, this decrement could be associated to synergistic activity of phytochemicals compound
such as phenolic acids, caffeic acid, quercetin, myricetin, proanthocyanidins, catechin, epicatechin,
and resveratrol presents in the extract (J Ahn, Grün, & Fernando, 2002). Thus Anastasiadi,

36
Chorianopoulos, Nychas, and Haroutounian (2008) compared the antilisterial properties of grape
seed extract vs individual polyphenols and found that antibacterial activity of the isolated
polyphenolic molecules was significantly lower compared to the extract. On the other hand,
different results were obtained when comparing two fraction of white grape seeds extract,
separated fraction of grape seed extract, one containing aliphatic aldehydes, fatty acids and their
derivate, and sterols was more effective than the fraction composed of catechin, epicatechin, and
gallic acid (Badet, 2011). The above, may be related to the chemical structure-function of
phytochemical molecules, has been reported that, in general the antimicrobial effect of natural
extracts decreases when these extracts are applied to food system, specify due to solubility of plant
extracts that play an important part in their antimicrobial activity in meat products. The
antimicrobial effect of plant extracts increases with increasing solubility in certain foods (Burt,
2004). Therefore, the polar compounds present in grape seed extract shown potent antimicrobial
effect in the study afore mentioned.

4.2 Antioxidant
Oxidation of lipids, proteins, and other food constituents, such as heme pigments and fat-soluble
vitamins remains a major concern regarding sensory quality, nutritional value, chemical safety, and
economic value of meat products. Oxidative stress occurs due to uneven generation of free radical
reactive oxygen species (ROS) and reactive nitrogen species (RNS) which triggers oxidative and/or
nitrosative stress and damage of macromolecules including the lipid and protein fractions (Estévez,
2017).
The rate and extent of oxidative deterioration can be reduced through various strategies such as;
curing, vacuum packaging, modified atmosphere packaging, and most importantly adding synthetic
or natural antioxidants (Ahmad, Gokulakrishnan, Giriprasad, & Yatoo, 2015). Some synthetic
additives commonly used by the food industry are e.g., ascorbic acid, generally recognized as safe
(GRAS) when used in accordance with good manufacturing practice, butylated hydroxyanisole
(BHA) and butylated hydroxytoluene (BHT) that, in spite of have been used extensively, their use
is questionable due to potential toxic effects as damage and mutation DNA, as well as nitrites and
nitrates, involved in etiology of stomach, bowel and food allergy (Barlow, 1990; Ribeiro et al.,
2019). For the above, natural polyphenols compounds represent a novelty and viable option to the
use of synthetic antioxidant, them could play a vital role in delay and inhibit of the formation of

37
toxic compounds (e.g., malondialdehyde, heterocyclic amines and protein carbonyls) in meat and
meat products as well as function as boost in endogenous antioxidant against oxidative stress in
farm animals, Table 3 and 4 show diverse studies using seed byproducts as potent antioxidants in
animals and muscle foods.
Due to plant polyphenols are multifunctional molecules, the antioxidant mechanism is believed to
occur through two major pathways. First is by donating electrons to break and terminate the
oxidation cycle at the propagation step and thereby preventing additional lipid and protein radical
forming. Second is by removing free radicals (ROS) initiator in order to quench chain-initiating
catalyst (radical) or limiting the radicals initiator by binding metals such as iron and copper as
metal chelators to stabilize them in an inactive or soluble form (Allen & Cornforth, 2010; Rice-
Evans, Miller, & Paganga, 1996). On the other hand, the chemical properties of polyphenols in
terms of the availability of hydrogen-donating predicts their antioxidant activity. Specific
polyphenols from seeds byproducts have revealed potent antioxidant activity as is the case of,
procyanidins from tamarind seed, catechin, epicatechin, gallic acid, resveratrol, from grape seed,
and more of 40 phenolic compounds identified in avocado seed whiting eight class group (tannins,
phenolic acids and flavonoids) to mention just a few examples.

4.3 Colorants
According definition by FDA (Food & Drug Administration) a color additive is any dye, pigment,
or other substance that can impart color to a food, drug, or cosmetic for to the human body. Color
plays a key role in determining the expectation and perception of consumer with respect to food. It
has interrelation with other parameter as flavor and may also indicate the safety of a food. Although
artificial colorants have a long history of use, and are easy to produce, stable, less expensive, and
have increasingly begun to consider synthetic colorants undesirable. In this context, there is an
industrial interest in natural colorants and has increased effort to discovery new natural alternatives
in scientific community (Náthia-Neves & Meireles, 2018; Socaciu, 2007).
A natural pigment can be defined as one pigment synthesized by and accumulated and/or excreted
by living cells. It may also synthesize by cells under stress or by dying cells. Historically, natural
pigments were used to color food products. Currently, 26 natural colorants including anthocyanin,
curcumin, carminic acid, lycopene, betanin, paprika, and saffron are permitted for use as exempt
colorants in the United State (Amiot-Carlin, Babot-Laurent, & Tourniaire, 2007).

38
The seed byproducts as natural colorants have been few explored, however for give an example
about the potential use we will emphasize avocado seed and annatto seed colorants (Figure 1).
Dabas, Elias, Lambert, and Ziegler (2011) have observed that avocado seed when ground with
water and incubated in presence of air produces a bright orange color, this colorant from avocado
seed may be usable at higher pH. Comparatively, anthocyanins, common natural colorant, give a
stable hue only at pH values lower than 5. The color change with increased pH under oxygen was
irreversible. Therefore, for use in foods pH and storage condition must be considered. The color
extract was stable at pH 7.5 for 2 months when stored at -18 °C. At higher temperatures significant
changes in the color were observed over shorter time periods. About safety, preliminary
investigation has been carried out using murino model (Ozolua, Anaka, Okpo, & Idogun, 2009), in
spite of this further safety studies for use in food are needed to assess. Likewise, annatto is a natural
colorant derived from the seeds of a tropical bush (Bixa orellana L.), with a yellow-orange-red
color due to its high content of carotenoids, mainly bixin and norvixin, which has antimicrobial
and antioxidant activity, as it is a vital source of phenolic and flavonoid compounds (Chisté,
Yamashita, Gozzo, & Mercadante, 2011; Shahid ul, Rather, & Mohammad, 2016). Annatto extract
was added to spreadable type pork liver pâtés, this being very rich in carotenoids is known to give
a yellowish color, therefore led a redder product (improve coordinate a*), increased b*, Chroma
and ∆E*, pointed to evident visual differences between the control and treatment, these differences
were not perceived by the panel (Martín-Sánchez, Ciro-Gómez, Vilella-Esplá, Pérez-Álvarez, &
Sayas-Barberá, 2017).
Assay with specific natural pigments including norbixin, lycopene, zeaxanthin, β-carotene have
been investigated to substituted sodium erythorbate (ERY) in sausage stored under refrigeration.
The best color stability after 45 days at 4 °C was observed in control and ERY samples, however
samples containing natural pigments showed significant reduction in redness (norbixin and
lycopene) and increased in yellowness (carotene and zeaxanthin). Regarding the sensory data,
ANOVA showed that the panelist not differences were observed between the scores reported by
the same samples (Mercadante, Capitani, Decker, & Castro, 2010).

4.4 Fat replacer

Actually, the consumers desire healthier foods, therefore limit their consumption of high-fat
convenience meat products. However, fats are an essential source of energy and carrier for fat

39
soluble vitamins in these products, and contribute significantly to sensorial attributes as flavor,
tenderness, appearance and texture, of meat products. Therefore, development low fat meat
products are a challenge. One of the strategies proposed to improve fatty acid profile, is to
incorporate ingredients that are potentially health-enhancing (functional) MUFAs and PUFAs
(Jiménez-Colmenero, Carballo, & Cofrades, 2001).
In this sense, the seed agro-industrial byproducts constitute an adequate source to improve the fatty
acid profile (Table 4) in meat and meat products, due to is rich oil and bioactive compounds as
tocochromanols, essential fatty acids, phytosterols, carotenoids and squalene (Górnaś &
Rudzińska, 2016). In the last decade, this subject has emerged in a relevant way, and various studies
have focused in the use of vegetable oil from seed byproducts as fat replacers in muscle foods and
animal feeding.
Bialek et al. (2017) asses the influence of pomegranate seed oil and grape seed oil on cholesterol
content and fatty acid profile in livers of chickens. They found that, the modification with grape
see oil and grape seed oil influences the FA profile in livers, detecting the presence of punic acid
(cis-9 trans-11, cis-13 C18:3, PA) in livers of chicken fed with pomegranate seed oil and
substantial amounts of rumenic acid (cis-9, trans-11 C18:2, RA), the major isomer of conjugated
linoleic acids (CLA), did not change total cholesterol content, SFA not differ significantly among
groups. In other study, Choi et al. (2010) replaced pork back fat with grape seed oil and rice bran
fiber and developed reduced fat met emulsion systems. They concluded that addition of grape seed
oil and rice bran fiber improved cooking characteristics and successfully reduced the animal fat
content in the final product.

4.5 Emulsifier
The incorporation of ingredients as proteins, carbohydrates and synthetic in meat products, has
been planted as strategy. Among of these, the use of hydrocolloids offers technological and
functional advantages including water holding capacity, stability to emulsion, and yield (Abbasi et
al., 2019; M Freire et al., 2017; Jiménez-Colmenero et al., 2001) (Figure 1). Plant seeds coming
out from agro-food processing industry are an interesting cheap alternative source of hydrocolloids
with a wide variety of functions useful for production value added products (Chandra Mohan et al.,
2018; Ravindran & Jaiswal, 2016). One example is the mucilage, which offer low-cost, low calorie
intake turning in an ideal product for the development/improvement of health products with

40
beneficial properties to human consumption, making them a potential option for application in the
food and pharmaceutical industry (Alpizar-Reyes et al., 2017; Huanbutta, Sangnim, &
Sittikijyothin, 2016; Nayak et al., 2015). In this regards, mucilage extracted from tamarind
(Tamarindus indica L.) seed has shown ability of forming viscous solutions, with high thermal and
chemical stability edible, biodegradable, non-carcinogenic, biocompatible and nontoxic properties
(Joint, Organization, & Additives, 2017; Sharma et al., 2014) similarly, tamarind seed powder is
widely used in the food industry as an emulsifier and stabilizer food grade
https://www.altrafine.com/blog/category/tamarind-kernel-powder/, due to retain high viscosity for
the longer period time and water absorption capacity, moreover it is rich in protein, carbohydrates,
fiber, and is odorless. Other example is mango seed, rich source of starch, oil and phytochemical,
which could be incorporated to improve oxidative stability, yields and better fatty acid profile of
meat and meat products (Melo et al., 2019).

5. Sensorial acceptance
The consumer perception, which refers to the process of selecting, organizing, and interpreting
information related to the new meat products, plays an important role in shaping consumer
acceptance, purchase and future consumption, and industry competitiveness by extension (Grunert,
Verbeke, Kügler, Saeed, & Scholderer, 2011; Kotler & Armstrong, 2013). Driven by the increasing
market for functional foods, foods enriched to provide health benefits, one the innovations are to
incorporate healthy ingredients into processed meat (Grasso, Brunton, Lyng, Harrison, &
Monahan, 2016). The sensorial evaluation later than inclusion of new ingredient from seed
byproducts in meat and meat products little has been research. There is documented that, addition
of natural ingredients may have direct impact on color, texture, flavor and purchasing decisions,
thus the ultimate success of innovative food products depends on consumer acceptance, which
translate to economic losses for the producer (Mancini & Hunt, 2005; Shan et al., 2017; Van Kleef,
Van Trijp, Luning, & Jongen, 2002). Therefore, this type of studies is especially relevant,
considering the potential technological and functional of seed inclusion in meat and meat products
(Table 3 and 4). Hung, de Kok, and Verbeke (2016) showed attitude towards processed meat
products with natural compounds including Belgium, Netherlands, Italy and Germany and
purchase intention was associated positively with: attitude; preference for natural over chemical
additives; perceived harmfulness of chemical additives; risk importance; domain specific

41
innovation, awareness of nitrite added; education; general health interest; and processed meat
consumption frequency. However, more studies are needed by specific ingredients, and specific
geographic area.

6. Conclusion and recommendations

This study contributes to better understanding of seeds as potential agro-industrial byproducts for
recovery of functional molecules for meat and meat products. Since they can act as natural
antimicrobial, antioxidant, colorants and fat replacer additives. Today, few residues from the
processing of fruit and vegetables are used appropriately in the food industry as new attractive with
natural ingredients enriching food. This has particularly been reflected in the enormous quantities
of waste around the world, therefor, effort is need in food processing sector industrial to applied
the symbiosis concept to integral management (including the concept make more of foods) of
resource that allowed recovery, identification, quantification, characterization of residues, through
sustainable technology. The impact of these actions is important for the current food economy, as
well as the future state of the environmental (bio-economy for the cities).
Important progress has been achieving in investigation of specific seeds including: grape, avocado,
tamarinds and mango, however is necessary more assay in complex matrices as muscle foods, to
stablish adequate dosage, and delay negative effects that have been described in some studies such
as off-flavors or color changes reflects in appearance. As well as, those related to safety and health.
Considering that, natural ingredients of products used in daily life have greater acceptability for
most people over their synthetic counterparts their potentially can be translated into an industrial
application if the appropriate regulatory body gives a positive opinion.

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Table 1. Extraction method for seed byproducts.

Method/byprodu Extracted Treatment Extraction Reference

ct compounds conditions improvement
Maceration
Avocado seed Alkaloids, Solvent (hot The phytochemicals
glycosides, water, cold alkaloids, glycosides,
saponins and water, polyphenols (Nwaoguikpe,
polyphenols methanol (flavonoids and Braide, &
and tanins) and saponins Ujowundu, 2011)
ethanol), were identified in
stirring (at extracts
room
temperature
during 24 h)
Avocado seed Polyphenols Solvent In both varieties, the (Rodríguez-
(ethyl- highest polyphenol Carpena et al.,
acetate, contents were 2011)
acetone- obtained for acetone
water 7:3, > methanol > ethyl-
and acetate solvent. The
methanol- compounds
water 7:3), catechins,
stirring hydroxybenzoic acid,
(conditions hydroxycinnamic
not acid and
specified), procyanidins were
and number identified in avocado
of extraction seed
(2 times)
Avocado seed Polyphenols Solvent Highest polyphenol (Gómez et al.,
(ethanol at content was obtained 2009)
26, 40, 60, at the following
80 and conditions: 80-93.6
93.6%), °C, ethanol 60-80%
temperature and 5-25 min.
(at 26, 40,
60, 80 and
93.6 °C) and
time (2.7, 5,
25, 45 and
55.2 min)
Avocado seed Glucosides Variety Two glucosylated (del Refugio
(Hass), abscisic acid Ramos et al.,
solvent derivates were 2004)
(methanol), isolated and
stirring identified (1′S,6′R)-

61
 (conditions 8′-hydroxyabscisic
not acid β-d-glucoside
specified). and
(1′R,3′R,5′R,8′S)-
epi-dihydrophaseic
acid β-d-glucoside
Date seed Polyphenols Solvent Solvent (acetone > (Suresh et al.,
(methanol- ethanol = methanol > 2013)
water 1:1, water) and
ethanol- temperature (60 °C >
water 1:1, 45 °C > 22 °C)
acetone- increase the
water 1:1 or polyphenol content
water), time
(2 h) and
temperature
(22, 45 and
60 °C)
Date seed Polyphenols Experiment Experiment 1 (Al-Farsi & Lee,
1 Acetone 1:1 > water 2008)
Solvent extraction increased
(water and the phenolic acid
acetone- content, in
water 1:1), combination with a
solvent- high solvent-solid
sample ratio ratio, temperature,
(10:1, 20:1, time and number of
40:1, 60:1 extraction
and 80:1),
temperature Experiment 2
(25, 35, 45, Acetone 1:1 >
55 and 65 methanol 1:1 =
°C), time methanol = ethanol >
(0.5, 1, 2, 3 ethanol 1:1 > water >
and 4 h) and acetone increased the
number of flavonoid content
extraction
(1, 2, 3 and
4 times)

Experiment
2
Solvent
(Acetone
1:1,
methanol
1:1,
62
 methanol,
ethanol,
ethanol 1:1,
water,
acetone),
solvent ratio
(60:1),
temperature
(45 °C),
time (1 h)
and number
of extraction
(2-stage).
Grape seed Polyphenols Solid-liquid The optimal (Nawaz, Shi,
ratio (0.1 to conditions for Mittal, & Kakuda,
0.25 g/mL), polyphenol 2006)
number of extraction were 0.2
extractions g/mL solid-liquid
(1 to 3), ratio, and double
time (1 h), stage extraction.
solvent
(ethanol)
Grapefruit seed Polyphenols Solvent These results showed
(ethanol and that aqueous extract (Giamperi,
water), showed high Fraternale,
number of biological activity, Bucchini, & Ricci,
extractions which was associated 2004)
(not with the presence of
specified), phenolic constituents
time (not such as naringin,
specified) neonaringin,
hesperidin, poncirin,
and quercetin
Guava seed Polyphenols Solvent The results indicate (Li et al., 2013)
(ethanol- the presence of gallic
water 1:1), and homogenistic
solvent- acid
sample ratio
(3:1),
number of
extractions
(1 time),
time (24 h)
Mango seed Polyphenols Solvent The highest phenolic (Lim, Cabajar,
(water, content was obtained Lobarbio,
ethanol and in the ethanolic- Taboada, &
combination water extract (50%). Lacks, 2019)
63
 s at 10, 25, In addition, gallic
50 and acid, caffeic acid,
75%), rutin and penta-O-
solvent- gallolyl-β-D-glucose
sample ratio were the identified
(10:1), compounds
temperature
(25 °C),
time (1 h),
stirring (200
rpm) and
number of
extraction (1
times)
Tamarind seed Polyphenols First Methanol extraction (Siddhuraju, 2007)
extraction showed the highest
process: total phenolic and
solvent tannins content
(methanol),
solvent-
sample ratio
(5:1),
temperature
(25 °C),
time (48 h),
stirring (not
specified)
and number
of extraction
(2 times).
Second
extraction
process: the
residue
obtained
from the
first
extraction
was re-
extracted
with solvent
(acetone
70%),
temperature
(25 °C),
time (48 h)
and pressure
64
 (52-56
KPa).
Tamarind seed Polysaccharide Solvent It was reported the (Choi et al., 2009)
s (water). presence of
Solvent- polysaccharides in
sample ratio tamarind seed
(10:1), extracts
temperature
(not
specified
°C), time
(20 min),
stirring (not
specified
rpm) and
number of
extraction (1
times)
Tomato seed Polyphenols For The highest (Chandra &
and polyphenols polyphenol contents Ramalingam,
carotenoids extraction: was obtained for 2011)
solvent PKM1 and CO3
(methanol), cultivars, while for
solvent ratio lycopene content the
(5:1), highest value was
temperature showed in Sindhu
(not and Shalimar.
specified),
time (not
specified)
and number
of extraction
(1-stage).

For
carotenoids
extraction:
solvent
(hexane-
methanol-
acetone,
2:1:1),
solvent ratio
(not
specified),
temperature
(not
65
 specified),
time (not
specified)
and number
of extraction
(1-stage).
Soxhlet
Avocado seed Alkaloids, Solvent (n- The highest alkaloid (Temitope et al.,
polyphenols, hexane, content was obtained 2017)
saponins, and methanol, when methanol
steroids and ethyl- solvent was used,
acetate), while the highest
temperature saponin, flavonoid
(not and tannin content
specified), was obtained when
and time (3 ethyl-acetate and n-
h) hexane were used as
solvent extraction
Avocado seed Alkaloids, Solvent The phytochemical (Leite et al., 2009)
polyphenols, (hexane and analysis revealed the
sterols, and methanol), presence of phenolic
triterpenes temperature compounds such as
(not anthocyanins,
specified), condensed tannins
and time (6 and flavonoids, as
h) well as alkaloids and
triterpenes for
methanol extracts. In
addition, sterols (β-
sitosterol) and
triterpenes were
identified in hexane
extracts
Date seed Polyphenols, Solvent Hydroxytirosol, (Besbes, Blecker,
sterols, and (petroleum gallic acid, Deroanne, Bahloul
tocopherol ether), protocatechuic acid, et al., 2004)
temperature p-coumaric acid and
(not oleuropein were the
specified), main phenolic of
and time (1 Deglet Nour seed oil.
h) While the
compounds 3,4-
dihydroxyphenylacet
ic acid, tyrosol, and
caffeic acid were the
main constituents of
Allig seed oil
66
 Cholesterol,
stigmasterol, β-
sitosterol were the
main sterols of
Deglet Nour seed oil.
While the sterols
campesterol, Δ5-
avenasterol and
Δ5.2,4-
stigmastadienol were
the main sterols of
Allig seed oil

α-tocopherol, γ-
tocopherol, and δ-
tocopherol were
indentified in date
seed oil (Allig >
Deglet Nour)
Grape seed Polyphenols Solvent The highest (Jayaprakasha,
(acetone, monomeric Singh, &
ethyl flavonoids, Sakariah, 2001)
acetate, and procyanidins and
67ethanol), total flavanols were
solid- obtained in the ethyl
solvent ratio acetate extract
(1:3), time
(10 h) and
temperature
(60-70 °C)
Grapefruit seed Polyphenols Solvent The results showed (Cvetnic &
(ethanol at the presence of Vladimir-
70%), time phenolic constituents Knezevic, 2004)
(6 h) such as flavonoids
Guava seed N/A N/A N/A N/A
Mango seed Polyphenols Solvent The highest total (Castro-Vargas et
(methanol), phenolic and al., 2019)
pressure flavonoid content
(0.3 atm), were obtained in
temperature sugar mango seed
(35 °C), extract
time (24 h)
Mango seed Polyphenols Solvent Gallic acids and its (Melo et al., 2019)
(methanol), derivatives were
solvent- obtained in the
sample ratio mango seed extract.
67
 (7:1),
temperature
(not
specified),
and time (24
h)
Tamarind seed Polyphenols Solvent The results indicate (Sundaram et al.,
(ethanol), the presence of 2014)
solvent- procyaninds
sample ratio
(3:1),
temperature
(not
specified),
and time
(not
specified)
Tomato seed Carotenoids Solvent The results showed (Knoblich,
(petroleum the presence of the Anderson, &
ether), carotenoids Latshaw, 2005)
temperature lycopene, lutein,
(60 °C), and zeaxanthin, α-
time (6 h) carotene, β-carotene
and cis-β-carotene
Ultrasound
Avocado seed Polyphenols Solvent Increasing (Segovia, Corral-
(water), temperature and Pérez, &
temperature ultrasound power Almajano, 2016)
(20, 40 and results in higher
60 °C) and polyphenol content
ultrasound
(0-140 W)
Date seed Polyphenols Solvent Acetone 1:1 (Al-Farsi and Lee,
(water and increased the 2008)
acetone extraction of gallic,
1:1), p-hydroxybenzoic,
solvent- caffeic, p-coumaric,
sample ratio and ferulic acids
(40:1),
ultrasound Water increased the
(30 min) extraction of vanillic
acid and m-coumaric
acid,
Grape seed Polyphenols Frequency The optimal (Ghafoor, Choi,
and power conditions for total Jeon, & Jo, 2009)
(40 kHz and phenolic content
250 W), were 53.15% of
68
 solvent ethano, 56.03 °C and
(ethanol at 29.03 min. While for
25 and anthocyanins the
50%), time optimal conditions
(5, 15 and were 52.35% of
25 min), ethanol, 55.13 °C
temperature and 29.49 min
(30, 40 and
50 °C)
Grapefruit seed N/A N/A N/A N/A
Mango seed Polyphenols Solvent Optimal polyphenols (Gao, Wang, Luo,
(propyl- extraction was Dai, & Li, 2012)
alcohol and showed at 59.7% of
water from propyl-alcohol,
40 to 80%; 23.4% of ammonium
ammonium sulfate, 27 min of
sulfate from extraction and liquid-
0.05 to 30 solid ratio of 23
mL/g), time mL/g.
(10 to 50
min),
temperature
(45 °C),
ultrasound
(frequency
20 kHz at
400 W)
Guava seed N/A N/A N/A N/A
Tamarind seed Polyphenols Solvent The results showed (Kothari et al.,
(methanol highest total phenolic 2012)
and ethanol and total flavonoid
50%), time content, when
(2 min), compared with
temperature maceration method
(not
specified),
ultrasound
(frequency
25 kHz at
720 W)
Tomato seed Polyphenols Solvent The phytochemical (Fuentes et al.,
(Solanum (water and analysis revealed that 2013)
lycopersicum) petroleum the highest phenolic
ether), content was found in
solvent- red tomato extract
sample ratio (aqueous >
(1:1),
69
 temperature petroleum ether
(not extracts)
specified),
and
ultrasound
(5 min)

Enzymes
Avocado seed N/A N/A N/A N/A
Date seed N/A N/A N/A N/A
Grape seed Polyphenols Enzymes The enzymatic (Chamorro,
and (pectolytic hydrolysis increase Viveros, Alvarez,
polysaccharide enzyme the polyphenolic Vega, & Brenes,
s complex, constituents, and the 2012)
hydrolysis presence of gallic
β-glucans acid, gallocatechin,
enzyme epigallocatechin,
complex, catechin, epicatechin,
and procyanidin B1 and
pentosans), B2, epigallocatechin
pH (5.5.), O-gallate,
temperature gallocacetechin O-
(35 °C), gallate, and
time (24 h at epicatechin O-gallate
100 rpm)
Grapefruit seed N/A N/A N/A N/A
Guava seed N/A N/A N/A N/A
Mango seed N/A N/A N/A N/A
Tamarind seed Polysaccharide Enzymes The highest (Poommarinvarak
s (protease at polysaccharide ul et al., 2010)
0.16, 0.48 extraction was
and 0.80 obtained at 0.8 U/mL
U/mL), pH and 5 h
(not
specified),
temperature
(37 °C),
time (1, 3
and 5 h)
Tomato seed Carotenoids Time (1-6 h) The highest lycopene (Ranveer, Patil, &
and 0-2.5% extraction was when Sahoo, 2013)
of enzyme both enzymes were
(celullase used at 1.5% during
1470 U/mL 4h
and
pectinase
160 /mL).
70
Microwave
Avocado seed N/A N/A N/A N/A
Date seed N/A N/A N/A N/A
Grape seed Polyphenols Solvent The optimal (Li,
(ethanol at extraction conditions Skouroumounis,
10, 30, 50, were ethanol Elsey, & Taylor,
70 and concentration 47.2%, 2011)
90%), time liquid-solid ratio
(2, 4, 8, 16 45.3:1, and time 4.6
and 32 min), min
liquid-solid
ratio (10:1,
20:1, 30:1,
40:1 and
50:1),
temperature
(40, 45, 50,
55 and 60
°C), power
(100, 125,
150, 175
and 200 W).
Grapefruit seed N/A N/A N/A N/A
Mango seed Polyphenols Particle size The optimized (Torres-León,
(150 to 450 conditions were Rojas, Serna-
µm), pH solid-liquid ratio Cock, Belmares-
water (2 to (1/60 g/mL), 75 °C, Cerda, & Aguilar,
8), solvent and 2 cycles. In 2017)
(ethanol 30 addition, analysis of
to 90%), polyphenols revealed
solid-liquid the presence of ethyl
ratio (1/50 gallate, penta-O-
to 1/10), galloyl-glucoside and
extraction rhamnetin-3-[6ʺ-2-
time (0 to butenoil-hexoside]
60 min),
temperature
(25 to 75
°C),
extraction
cycle (1 to
3), potency
(600 W)
Guava seed N/A N/A N/A N/A
Tamarind seed Polyphenols Solvent The results showed (Kothari et al.,
(methanol. highest total phenolic 2012)
ethanol and and total flavonoid
71
 water), time content, when
(1.5 min), compared with
temperature maceration method
(not
specified),
potency
(720 W)
Tomato seed N/A N/A N/A N/A
Supercritical
fluid
Avocado seed N/A N/A N/A N/A
Date seed Polyphenols Time (1, Optimal SFE (Liu et al., 2013)
1.5, 2, 2.5, conditions for
and 3 h), maximum yield of
pressure polyphenol content
(250, 300, were 50 °C, 350 bar,
350, 400 and two-repeated
and 450 extraction, each for 2
bar), h. The phenolic
temperature compounds detected
(40, 50 and were chlorogenic
60 °C), flow acid, rutin, ellagic
rate ( ), and acid and quercetin
number of
extraction
(1, 2 and 3
times)
Grape seed Polyphenols Supercritical When CO2- (Palma, Taylor,
fluid (CO2 Methanol was used Varela, Cutler, &
and CO2- as fluid, the presence Cutler, 1999)
Methanol of phenolic
5:1), constituents
temperature (catechin,
(35 °C), epicatechin, and
pressure gallic acid) was
(450 atm), increased
time (15
min)
Grapefruit seed Polyphenols Fluid (CO2 The highest (Yu, Dandekar,
and ethanol flavonoid extraction Toledo, Singh, &
as co- (naringin) was Patil, 2007)
solvent), obtained at 41.4
flow rate (5 MPa, 60 °C, 20% of
L/min), ethanol, and 20 min
pressure
(34.5, 41.4
and 48.3
72
 MPa),
extraction
(20, 40 and
60 min),
temperature
(40, 50 and
60 °C),
number of
extraction (2
times)
Guava seed Fluid (CO2, The highest total (Castro-Vargas et
as co- phenol content was al., 2010)
solvent obtained at 10 and 20
ethanol and MPa
ethyl
acetate),
flow rate
(not
specified),
pressure
(10, 20 and
30 MPa),
temperature
(40, 50 and
60 °C)
Mango seed Polyphenols Fluid (CO2 The optimal (Ghafoor, Al-
and ethanol extraction conditions Juhaimi, & Choi,
at 5, 6, 7 were 44.46 °C, 153- 2012)
and 8%), 161 bar CO2
flow rate (2 pressure with ethanol
mL/min), at 7%
time (30
min),
temperature
(37, 40, 43
and 46 °C),
pressure
(137, 147,
157 and 167
bar)
Tamarind seed Polyphenols Fluid (CO2, The use of ethanol (Luengthanaphol
as co- 10% as co-solvent et al., 2004)
solvent results in higher
ethanol and extraction of (–)-
ethyl acetate epicatechin, under 40
at 10%), °C and 10 MPA
flow rate (5
73
 mL/min),
time (5 h),
temperature
(35-80 °C),
pressure
(10-30
MPa)
Tomato seed Carotenoids Fluid (CO2), The optimal (Rozzi, Singh,
temperature conditions for Vierling, &
(23-86 °C), lycopene extraction Watkins, 2002)
flow rate ( ), (61% of yield) were
and pressure 86 °C, 34.47 MPa,
(13.78- 500 mL of CO2 at
48.26 MPa) flow rate of 2.5
mL/min.
Tomato seed Carotenes Fluid (CO2), The optimal (Sabio et al.,
temperature conditions for 2003)
(60 and 80 lycopene and β-
°C), flow carotene extraction
rate (0.792 (80 and 88% of
and 1.35 yield, respectively)
kg/h), and were 300 bar, 80 °C
pressure at lower flow rate.
(250-300
bar)

74
Table 2. Integration of seed byproduct to improving quality and shelf life of meat and meat
products
Meat Amount
Source Form Effect Author
product (%)
Beef and Inhibitory effects on
Powder (Keşkekoğlu &
chicken 0.5 % formation of heterocyclic
extract Üren, 2014)
meatballs aromatic amines
Reduction of TBAR respect to
the control, and patties not
Pomegranate show any significant
Goat (Devatkal,
10 difference for L and b values
meat liquid Narsaiah, &
g/100ml different to a value. However
patties Borah, 2010)
sensory evaluation did not
reveled any significant
difference.
Decreased lipid oxidation
(TBARS) at 9 days, in raw
(R. Carpenter,
pork patties stored 12 days at
M. N. O’Grady,
0-1000 4 °C, antioxidant activity of
Pork Powder Y. C.
µg/g of GSE cooked pork patties was
patties extract O’Callaghan, N.
mucle observed, indicating stability
M. O’Brien, & J.
of GSE. Sensory properties of
P. Kerry, 2007)
cooked pork patties were
unaffected.
(Pateiro,
The GSE reduced lipid
Pig liver 1000 Powder Lorenzo,
oxidation, and permits obtain
pâté mg/kg extract Amado, &
more attractive product.
Franco, 2014)
The aw values were affected
by the addition of GSE than
Grape
the control, L*, a* and b* (Lorenzo,
was affected positive at the González-
Dry cured
1000 end of process, and more Rodríguez,
sausage
ppm effective against TBARS Sánchez,
“chorizo”
formation than BHT. The Amado, &
microbial quality (TVC, Franco, 2013)
LAB) was adequate in GSE
treatment
Lag time (LT) was 95 h in
control samples at 15 °C,
Sous vide
whereas 1-3% GSE
cooked Liquid (Cosansu &
1 to 3% significantly extended LT to
ground form Juneja, 2018)
244 h or longer. Extended the
beef
period need to reach 6 log
UFC Clostridium perfringens

75
 at 15 or 20 °C, while 3% of
GSE was required at 25°C.
The GSE rapidly decreased
the numbers of E. coli
O157:H7 in the first days of
stored (1 log reduction), S.
typhimorium and delayed the
growth of L. monocytogenes
and Aeromonas hydrophila,
Raw and
Powder compared with BHT/BHA. (Juhee Ahn et al.,
cooked 1.0%
extract The TBARS were 2007)
beef
significantly lower than those
for the control.The color of
cooked beef treated with
GSE was less light (L), more
red a*) and lees yelow (b*)
than those treated with
BHA/BHT.
High correlation between
Hexanal, pentanal, octanal, 2-
octenal, 1-octen3-ol, 2-octen-
(Mielnik, Olsen,
Turkey 0.4 to 1-ol, and 1-penten-3-ol and
Liquid Vogt, Adeline, &
breast 1.6 g/kg TBARS values (r>0.95). The
Skrede, 2006)
ability of GSE to prevent
lipid oxidation was
concentration dependent.
Rancid odor and flavor
scores of GSE containing
samples were lower than (Kulkarni,
100, 300 those of controls after 4 DeSantos,
Beef Spry
and 500 months of storage. The L* Kattamuri,
Sausages dried
ppm increased during storage and Rossi, & Brewer,
TBARS of the control and 2011)
ascorbic acid containing
samples increased.
GSE was use as sodium
nitrite substitute and the
Dry- addition not affect the overall
(Aquilani et al.,
fermented 10 g/kg Extract aroma profile and
2018)
sausage acceptability, except for color
related traits, underscore in
GSE.
Avocado extracts reduced the
(Rodríguez-
Pork 10 g/50 loss of redness, inhibiting
Avocado Liquid Carpena et al.,
patties ml discoloration the chilled
2011)
patties, significantly inhibited

76
 the formation of protein
carbonyls and patties treated
had lower amount of TBARS
than control.
The lychee seed water extract
(LSWE) retarded lipid
0.1%, oxidation, the antioxidative (Qi, Huang,
Raw meat Freeze-
Lychee 0.5% or effect were dose-dependent Huang, Wang, &
paste dried
1.0% and did not adversely affect Wei, 2015)
the sensory properties of the
meat paste.
Decreased saturated fatty
acid and cholesterol content
Increased PUFA from
10 %, 2.51%(control) to (Gök, Akkaya,
Ground Beef Seed
15 % or 46.43%(20%GPS) Obuz, & Bulut,
Poppy seed patties powder
20 % Improved linoleic acid from 2011)
2.12(control) to 45.2(20%
GPS)

Shelf life extension through

the release of clove and
cinnamon active compounds
fused into the film, which
was about 40%, shelf life
increased by 1 week at
Mutton
storage temperature of 10°C
meat
and 3 weeks at store
(biceps 5 g/100 Edible (Chandra Mohan
Tamarind temperature of 4°C.
femoris- ml film et al., 2017)
Significant differences in
leg
total viable count,
muscle)
pseudomonas, lactic acid
bacteria, yeast and moulds,
Brochothrix thermosphacta
and enterobacteria growth
were noted between control
and test mutton samples
The date seed extract delay
protein oxidation, and
TBARS lower than control
(De la Rosa-
Pork Ethanol and lipid oxidation inhibition
Date 0.2 % Alcaraz et al.,
patties extract during in vitro digestion. The
2019)
total viable count, lactic acid
bacteria and psicrophylic
bacteria have given an

77
acceptable microbial limit for
all meat and meat products.

78
Table 3. Use of seed byproducts as a strategy to improve health status and animal product
quality.
Effect
Source Model Doses Reference
Health status Meat quality
Lamb 50 mg GSE/Kg-1 No differences Although not (Guerra-
after 10 days of attributable to significant, an Rivas et al.,
adaptation to the experimental improvement in 2016)
experimental treatment were TBARS values of
diet. observed. about 20% was
observed in
treatment
compared with
control, from day
7 of storage.
Porcine 100, 300, 700 GSE not Supplementation (O’Grady,
Grape seed mg/kg feed for significantly of porcine diet Carpenter,
extract 56 days reduce lipid with GSE did not Lynch,
oxidation in significantly O’Brien, &
tissue (liver, improve fresh Kerry, 2008)
heart and pork quality.
kidney)
homogenates.
Excretion of
compounds
present in GSE
in the animal
urine or faeces
was also
possible.
Grape Chicken 30 and 60 g/kg Dietary GPC Inclusion of 60 (Sáyago-
pomace during 21 days did not affect g/kg was more Ayerdi,
(GPC: chicken effective to Brenes,
seeds, performance reduce TBARS Viveros, &
stems and when values of raw (up Goñi, 2009)
peels) administered to 35%) and
up to 60 g/kg. coked (up to 28%)
But GPC are breast chicken
distributed, patties, and
retained and increased ABTS
remained values compared
functional in with control
muscle. samples at 20
days 4 °C,
extended the store
life.

79
Grape seed Chicken 0.1% of GSE Chickens In thigh meat 14 (Muñoz-
extract during 35 day showed phenolic González et
(GSE) significantly metabolites were al., 2019)
higher identified but no
plasmatic differences were
concentration found between
of 21 phenolic diets.
metabolites
Improvement
plasma
antioxidant
status
Avocado Porcine 30% during Pigs fed with Significant (Hernández-
(seed and fattening period paste had impact on López,
pulp) higher levels intramuscular fat, Rodríguez-
of linoleic acid lower lipids and Carpena,
(C18:2 n-6), protein oxidation Lemus-
and DHA than in fresh loin Flores,
control Longissimus Grageola-
counterparts. thoracis et Nuñez, &
lomborum (LTL) Estévez,
muscle treated, 2016)
than the muscle
control.
Subjective color
L* were higher
than control, a*
no revealed
differences and
b* was higher in
control than
treated.
Whole Cattle 30% during 104 In WCS diet Increased the (Ferrinho et
cotton seed days finishing the linoleic level of PUFA al., 2018)
(WCS) period. acid (18:2 n-6) and sensory
and level n- attributes were
6/n-3 ratio better than control
increased. (more tender and
juicier).
DHA: docosahexaenoic acid

80
Table 4. Seed byproducts as potential fat replacers for meat and meat products.

Parameter (%) Pomegranate Grape Mango Date

Yield 12-20 48.9-73 10-15 5.89-9.1
Lauric (C12:0) — — — 15.93-18.58
Myristic (C14:0) — — — 9.74-12.31
Palmitic (C16:0) 2.0 7 4-12 9.49-10.12
Stearic (C18:0) 1.6 2-4 31-48 2.44-3.23
Arachidic (C20:0) 3.0 — — —
Oleic acid (C18:1 ω-9) 3.7 10-20 38-50 45.40-50.76
Linoleic acid (C18:2 ω-6 3.3 66-75 3-6 7.41-8.91
Araquidonic acid (C20:4 ω-6) — — 2-6 —
Punic acid
57.3 — — —
(C18:3-9cis,11trans, 13 cis)
Catalpic acid
7.6 — — —
(C18:3.9trans, 11trans, 13cis)
α-eleostearic acid
21.1 — — —
(C18:3-9cis, 11trans, 13trans)
*CLNA: conjugated linoleic acid: (Özgül-Yücel, 2005) (Lutterodt, Slavin, Whent, Turner, & Yu,
2011) (Gunstone, 2011)

81
 Colorants
Annato seed Avocado seed

Bixin

Catechin Epicatechi

Norbix

Luteolin Trimeric procyanidin (Type

Emulsifiers

Basil seed Tamarind seed Mango seed

Figure 1. Potential use of seed byproducts as natural ingredients in meat and meat products.

82
3. Evaluación de Fitoquímicos y Actividad Antioxidante de Subproductos de Dátil (Phoenix
dactylifera L.) Producidos en el Estado de Sonora

María de los Ángeles de la Rosa-Alcaraz, Gastón Ramón Torrescano Urrutia, José Ángel Pérez
Álvarez, Juana Fernández López, Armida Sánchez Escalante

Biotecnia (2017) Volumen XIX, Número 3

(Publicado)

83
84
85
86
87
88
89
90
4. Recovery and Characterization of Bioactive Compounds of Date (Phoenix dactilyfera L.)
Seed Using Ultrasound Assisted Extraction

Ma. Ángeles De la Rosa-Alcaraz, Gastón R Torrescano-Urrutia, José A Pérez-Álvarez, Juana

Fernández-López, Raquel Lucas-González, Manuel Viuda-Martos, Humberto Astiazarán- García,
Armida Sánchez-Escalante

Journal of the Science of Food and Agriculture

(Enviado para publicación)

91
Recovery and characterization of bioactive compounds of date (Phoenix dactilyfera L.)
seed using ultrasound assisted extraction

Ma. Ángeles De la Rosa-Alcaraz a, Gastón R Torrescano-Urrutia a, José A Pérez-Álvarez b,

Juana Fernández-López b, Raquel Lucas-González b, Manuel Viuda-Martos b, Humberto
Astiazarán- García a, Armida Sánchez-Escalante a*

a
Centro de Investigación en Alimentación y Desarrollo, A.C. Coordinación de Tecnología
de Alimentos de Origen Animal. Ctra. Gustavo Enrique Astiazarán Rosas No. 46, Col. La
Victoria. Hermosillo, Sonora, 83304, México.
b
Universidad Miguel Hernández, Escuela Politécnica Superior de Orihuela, Departamento
de Tecnología Agroalimentaria, Ctra. A Beniel Km 3.2 (03312) Orihuela, Alicante, España.

*Corresponding author: Dra. Armida Sánchez Escalante, E-mail: armida-sanchez@ciad.mx

Abstract

BACKGROUND: Date seed is an abundant byproduct of date processing; this contains bio-
active molecules having an impact in quality of food and health of the consumer. Actually,
the recovery of high valuable compounds using greener and cleaner methods has been
promoted, ultrasound assisted extraction (UAE) has gained relevance because it adapts to
this trend. In the current study date seed extracts from Mexican date byproducts were
obtained, characterized, and assessed their phytochemical composition and antioxidant
activity.

RESULTS: The UAE was the most efficient extraction method, due to low time to operation.
The aqueous extract evidenced the best value for total phenols (TP), using maceration method
(38.96 mg GAE/ g), and while for total flavonoids (TFlvC) acetone: water [50:50 v/v]
extracts showed the highest values, but not differences were found between methods. The
antiradical efficiency was presented by acetone and methanol extracts independently of
method, in agreement with reducing power (RP) (Abs700=0.8 to 0.98). All extracts contained
hydroxycinnamic acids principally, including ferulic and chlorogenic acid (2395.32-117.05
and 962.95-172.70 µg/g of extract, respectively), hydroxybenzoic acid, as protocatechuic

92
39.07-248.93 µg/g of extract, and catechin (982.69-454.61 µg/g of extract), being the only
one flavonoid found in the extracts.

CONCLUSION: Date seed extracts possess a great potential to be used in the future as
potential antioxidant natural additives that could be counteracting the excessive use of those
of the synthetic origin.

Keywords: industrial byproduct; antioxidant activity; date seed; phytochemicals;

Ultrasound-Assisted Extraction.

93
INTRODUCTION

In the last decade the design and development of functional food has become dynamic
market. Prevalence of chronic degenerative disease as diabetes, obesity, hypertension, and
cancer, have been fomented to the food industry to be more innovative and give viable
options of foods that in addition to nourish offer other properties. In this sense, phytochemical
addition has been a strategy to mitigate problems derivate of intake foods that compromises
the health. Since it has been shown that, phytochemical compounds play role as mediators in
the prevention or reduction of oxidative damage, which has been implicated in the pathogenic
processes and probably the most investigate molecules of nutritional interest. (1)

Fruits, vegetables and their byproducts are the most studied substrates for extraction of
phytochemicals. The disposal of these materials usually represents a problem and has
negative effects on the environment. According to the recent research the amount of food
that is wasted each year will rise by a third by 2030, when 2.1 billion tons will either be lost
or thrown away (https://www.theguardian.com/global-development/2018/aug/20/food-
waste-alarming-rise-will-see-66-tonnes-thrown-away-every-second), about 1.3 billion tons
of food has been wasted worldwide per year, which represents one third of the total food
industry production. (2) Thus, new aspects in relation to the use of these byproducts for further
application on the production of food ingredients with high nutritional value have showed
increasing interest. Therefore, their recovery as high-value products may provide an
economic benefit. (3)

Many byproducts originated in plant food processing have been studied and recognized as
source of bioactive compounds, however has been evidenced in several studies that seeds
show more compounds than, even exceeding the edible portion depending on the fruit in
(4-6)
question, for example; grapes and avocado seeds. In the case of date seed, previous
studies have evaluated phytochemicals, antioxidant activity, biological activity of date seed
extract, powder or infusion, and has been found positive effect to treat lung diseases, diabetes,
atherosclerosis and cancer using in vitro and in vivo models, which has been related mainly
to its antioxidant activity. However, in these studies to obtain bioactive extracts maceration
has been most used method. (7, 8)

For obtaining functional molecules as antioxidants derived of agricultural byproducts, exist

94
several methods and extraction solvents, such as water, acetone, methanol, ethanol or the mix
of these, which have been considerate suitable and are the more used for get them. However,
it is not possible considerate a single solvent that can be standard in relation to its chemical
nature and polarities of these molecules, so that, an important factor to consider is that it will
(9)
safe for its incorporation as food ingredient. Regarding to extraction methods, the
ultrasound assisted extraction (UAE) has gained relevance because is friendly to the
environment, easy to operate, efficient and low cost, provides a high recovery of compounds
of interest, with a low solvent consumption and short times, which overcoming to the
investment of time and solvent used in the maceration method. (10) For the above, the aims of
this research were i) recovery of bioactive compounds of date seed using different methods
and solvents and ii) characterize and assess of phytochemical and antioxidant activity.

MATERIALS AND METHOD

Sample collection

Date seeds were obtained from no-commercial date of variety Medjool (54 kg), in maturity
stage Tamar and harvested in October 2015 in the region of Caborca, Sonora, northwestern
México. Caborca is a city located at latitude 30° 42 ´N; length 112° 09 ´east, at an elevation
of 289 m above sea level. The seeds used were fist cleaned to remove any pulp residue.
Subsequently, they were dried (50° C) for 48 h, ground and sieved with a mesh of 1-2 mm in
a hammer mill (Thomas Wiley, model 4), and next the powder was stored at -20° C until
analysis.

Chemical composition and physical properties of date seed

The chemical composition was evaluated according to AOAC methodology. (11) The moisture
content was determined by weight difference method (934.06), protein according to the
Kjeldahl nitrogen content (method 920.152), ash (method 940.26) and fat by continuous
solvent extraction (goldfish). The percentage of crude protein was estimated by multiplying
the total nitrogen content by the factor 6.25. Total carbohydrates were estimated subtracting
(12)
from the total percentage of the rest of the determinations. The results of chemical
composition analysis were expressed as g/100 g of dry weight. The physical measurements
of date seed were carried out using one hundred seed of Medjool variety selected randomized,
each seed was subjected to physical measurement of length and weight was obtained using

95
digital Vernier and analytical balance, respectively. (13)

Extraction of phenolic compounds

The date seed powder (1 g) was extracted with 10 mL of solvent system; water, 50% aqueous
methanol, 50% aqueous ethanol and 70% aqueous acetone. All homogenized were subjected
to two different process, 1) ultrasound assisted extraction (UAE) during 60 min at 40 kHz of
frequency and temperature of 25 ° C ± 3°C (Ultrasound Bath Branson, Model 1800, Series
CPXH) and 2) maceration during 48 h at constant stirring rate using a magnetic stirrer plate.
Solids were separated by centrifugation (15,000 rpm, 15 min at 4 °C) and filtration trough
Whatman 1. Extracts were evaporated to dryness at 60 °C under vacuum using rotatory
evaporator (RE301, Yamato, Japan). Finally, the dry extract was lyophilized and stored in
dark conditions at -20 °C.(14, 15)

Total phenol (TP) and flavonoids (TFlv) content

The total phenols content (TPC) in date seed extracts was evaluated according to Singleton
(16)
and Rossi , using Folin-Ciocalteau as oxidizing agent. The extracts (10 µL) were mixed
with 40 µL of reagent, 160 µL of distilled water; immediately 60 µL of sodium carbonate
(5%) were added to the mix. After 60 min at room temperature, the absorbance was measure
TM
at 750 nm using spectrophotometer UV/VIS Multiskan GO (Thermo Scientific). Total
phenolic compounds were calculated using a standard curve of gallic acid (considering six
different concentrations, 0-1 mg/mL), the results were expressed as milligrams of gallic acid
equivalents (GAE) per gram of extract.

Total flavonoids content (TFlvC) were determined following the established by Zhishen,
(17)
Mengcheng, Jianming, with some modification. The extract (100 µL) was mixed with
430 µL of NaNO2 (5%), and kept at rest during 5 min. Next, 30 µL of AlCl3 (10%) was added
and remain at rest for one minute to carry out the reaction, finally, 440 µL of NaOH (1 M)
was added. Previous reaction mixture was taken 150 µL in triplicate and the absorbance was
measured at 510 nm using spectrophotometer UV/VIS Multiskan TM GO (Thermo Scientific).
The results of TFlv were expressed as milligrams of rutin equivalents (RE) per gram of
extract.

Antioxidant activity

96
ABTS assay

The radical scavenger activity of ABTS was determined after the radical cation (ABTS•+) is
produced by the reaction of a stock solution of ABTS (7 mM) with persulfate of potassium
(2.45 mM), both dissolved in water, this mixture is kept in rest 12-16 h in dark conditions at
room temperature before of use. The ABTS solution is prepared with ethanol until reach 0.70
of absorbance at 734 nm. The absorbance reading is taken exactly 5 min after the reaction
mixture of the radical with the sample. The absorbance of the reaction samples was compared
to the Trolox standard curve (ranging from 60-300 µM of Trolox in 80% methanol), and the
results were calculated and expressed as micromole Trolox equivalents per milligram of
extract. All determinations were carried out at least three times.(18)

DPPH

The 1,1´-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity test was realized

using 100 µL of extract and mixing 100 µL of radical DPPH 300 µM in ethanol. After 30
min in the darkness absorbance at 517 nm was recorded. (19) A standard curve was obtained
by using acid ascorbic solution at various concentration (ranging from 3 to 180 µM) in
ethanol. The absorbance of the reaction samples was compared with the Trolox standard
curve, and the results were calculated as µM of AAE/mg of extract. All determinations were
carried out at least three times.

Reducing power

The reducing power was evaluated mixing 200 µL of extract (5 mg/mL), 500 µL of phosphate
buffer 50 mM (pH 6.6) and 500 µL of potassium ferricyanide (1%). The mixture was
incubated at 50 °C for 20 min; next was added 500 µL of trichloroacetic acid (10%). The
mixture was centrifuged at 2800 XG for 10 min; the supernatant was taken 500 µL and mixed
with 500 µL of milliQ water and 100 µL de FeCl3 (0.1 %). Finally, was taken 100 µL of the
mixture and absorbance at 700 nm was measured. The presence of reducing agents triggers
the reduction of complex Fe3+/ferricyanide to ferrous form, which is measured for the average
of the formation of color Prussia blue pearls (Perl´s Prussian). The increased of the
absorbance in the mixture reaction indicates an increment of the reducing power of the
sample. (20)

HPLC quantification and identification

97
HPLC conditions

The analysis of the compounds presents in date seed extracts was realized using High
Performance Liquid Chromatography, using Hewlett-Packard HPLC series 1200 instrument
(Woldbronn, Germany), equipped with UV-visible diode array detector. Separations were
achieved on C18 Teknokroma column (Mediterranea sea18, 25×0.4 cm, 5 μm particle size;
Teknokroma, Barcelona, Spain) and the chromatograms were recorded at 270, 280, 320, 325
and 360 nm. Phenolic compounds were analyzed, in standard and sample solutions with the
following gradient program: T0 5% B, min 20 25% of B, min 40 50% B, min 45 5% B. The
mobile phases (A) were composed of formic acid 1% in water and (B) methanol. The
temperature of injection was 35 °C, the flux of movil phase was 1 mL/min, the injection
volume of standards and extract was of 20 µL. The identification of compounds was carry
out by comparing UV absorption spectra and retention times of each compound with those
of pure standards injected under the same conditions. The polyphenolic compounds were
quantified through calibration curves of standard compounds (phenolic acid standard:
clorogenic, cafeic, coumaric, sinapic, ferulic, protocatechuic, flavonoid standard: catequin
and quercetin). High linearity (r2 > 0.995) was achieved for each standard curve for each
compound. (21)

Statistical analysis

The analysis of the data consisted of a linear model GLM-ANOVA of two ways, the effects
of the treatment were considered, and all statistics assay were performed using NCSS (2011
version) software. The significant differences were estimated at a probability level of p<0.05,
if differences were found between treatments, a mean comparison test was performed by
Tukey-Kramer.

RESULTS

Chemical composition and physical properties

The chemical composition values and physicochemical properties of the date seed are
presented in Table 1, carbohydrates (79.36 g/100 g) were present in the highest concentration
on date seed powder, followed by fat (6.56 g/100 g) and protein (5.28 g/100g). The weight
and length of date were 1.10 and 2.41, respectively.

98
Yield of extraction

The yield extraction is shown in Table 2. In the present study were used four solvent systems
for obtaining of date seed extract and the results reflect that the extraction yield was
depending of solvent and extraction method; for UAE was as following:
methanol:water>acetone:water >water>ethanol: water; while with maceration method, the
yield was different: ethanol: water> methanol: water> acetone: water> water. However, is
necessary considered the efficiency and operating time between methods; 48 h maceration
against 1 h UAE. Likewise, it is important to consider the solvents that offering a minor
residue level and them use is unrestricted for use in the preparation of food ingredients.

Total phenol content (TPC)

The TPC in the lyophilized extracts of date seed was dependent (p<0.05) of extraction
method and solvent as shown in Table 3. The highest TPC was presented in aqueous extract
using maceration method (38.96 mg EAG/g of extract), followed by the acetone extract using
UAE (33.02 mg EAG/g of extract).

Total flavonoids content (TFlvC)

The TFlvC in date seed extract was affected (p<0.05) for the extraction method and solvent
as shown in Table 3. The highest flavonoid content was presented in acetone extract (50.00
mg RE/mg of extract) independently of extraction method used, followed for the methanol
extract obtained for maceration (31.95 mg RE/g of extract) and ethanol extract for both
methods (UAE and maceration) (32.54 y 31.68 mg RE/g of extract, respectively). The
aqueous extract, for both methods result with the lowest value (UAE: 12.95, maceration:
15.19 mg RE/g of extract, respectively).

Antioxidant capacity

ABTS

The free radical scavenging capacity of date seed extracts againts ABTS radical is shown in
Figure 1A. Significant differences were found (p<0.05) between solvent and extraction
method. Using acetone, methanol and ethanol, were found higher (p<0.05) values of Trolox
equivalents (TE). Interestingly, between maceration and UAE method not significant
differences were found. Whilst, water extracts present the lower values independently of the

99
method.

DPPH

Capacity of scavenging DPPH radical varied with the polarity of solvent. All the solvents
exhibited moderates to good activity by DPPH method. The highest DPPH scavenging
activities were given by acetone, methanol, and ethanol extracts and UAE method presented
the higher values; 29.65, 29.94. 29.61 µM of AAE/g of extract respectively. Aqueous extracts
have the lowest values, 23.96 and 17.53 µM of AAE/g, using UAE and maceration,
correspondingly as presented in Figure 1B.

Reducing power

The RP of date seed extract at a concentration of 5 mg/ml was determinate and the results is
shown in Figure 1C, significant differences were found (p<0.05) between method extraction
and solvent. The higher RP were presented in acetone and ethanol extracts (Abs 700nm=
0.85 and 0.93, respectively), although not affected by the extraction method. Opposite case
in aqueous extract for which the RP was different between the extraction method (UAE: Abs
700nm= 0.48 y maceration: Abs 700nm= 0.8). The lower value was presented by methanol
extract, independently of extraction method, with Abs 700nm= 0.5.

Identification and quantification of phytochemicals using HPLC

The HPLC results recorded at 270, 280, 325, nm showed a large diversity of compound as
show in Table 4. Examination of the UV-visible spectra allowed detection of different
families of polyphenolic compounds, namely phenolics acids and flavonoids. The profile at
325 nm shows the presence of five hydroxycinnamic acids eluted at 21.4-22, 23, 29.1, 31.1
and 32 min, while at 270 nm one hydroxybenzoic acid eluted at 12 min, and at 280 nm one
type flavonoid eluted at 18.8 min. The use of different solvent and extraction methods
allowed found the solvent and method most suitable for this material, in general acetone
extracts independently of the extraction methods revealed and ethanol and methanol extracts
with maceration method presented the major amount phytochemical compounds.

DISCUSSION

Chemical composition and physical properties

100
 (13)
The proximate composition was similar to previously report by Habib and Ibrahim for
date seed Medjool variety. Particularly, the protein and fat contents of date seed were greater
than avocado and mango seed, while ash content was similar (22). The chemical composition
can be influenced by factor such as variety and geographical origin, a clear example is fat
content that show a range 0.218 to 10.19 between varieties, however have been found
variation even between same variety of seed (13, 23). The dimensions of date seed were found
according to stablished by others authors,(23) who evaluated three varieties, including
Medjool variety and indicated that seeds from Medjool variety show the larger dimensions
and relation mass seed/fruit. This characteristic is widely used for producer to evaluate the
quality of fruit varieties, therefore this information can be used as useful parameter to
calculate yield extraction of phytochemical compounds and other components of the seed,
considering extrinsic factors as weather, fertilization, temperature, irrigation, duration of the
growth period, postharvest treatment, variety, that can effect in physical properties of the
fruit and seed.(24)

Yield of extraction

The extraction with solvents is frequently used to recovery of antioxidants compounds; and
both, the yield and the antioxidant activity of the extracts are dependents of solvents, because
to different polarity of antioxidant compounds, the time and temperature, sample to solvent
(5, 25)
ratio as well as the chemical composition and physical characteristics of the sample .
Solvents, such as methanol, ethanol, acetone, ethyl acetate, and their combinations have been
used for the extraction of phenols from plant materials, often with different proportion of
water, selecting the right solvent affects the amount and rate of polyphenols extracted (26). In
the present study was clear the effect of method and solvent used, specifically in ethanolic
extract. For recuperation of phytochemical from green peas, grape waste and avocado seed
has been reported that aqueous acetone 70% was the most efficient solvent. Although, for
olive seed has been described that ethanol is an excellent solvent followed by water (9.2 and
(27)
4.0 g/100 g, respectively) which, is in according with our results for ethanolic extract
using maceration method. For phytochemical extraction from black tea were evaluated
different solvents (water, acetone, ethanol, methanol and N-N-dimethylformamide) and
found that aqueous acetone 50% showed the best efficiency of extraction.(28) For the above,
can be confirmed the importance of method extraction and solvent with relation to yield of

101
extract obtained. However, it is difficult compared the results with other investigation group,
for the great difference in the methods used. Although, our results can be used as a reference
for the exploitation of matrix with similar characteristics to the date seed.

Total phenols content (TPC)

The TPC in the present investigation, surpass those found in cetonic extract of date seed of
khalas variety (21.62 mg GAE/g) (14) and Mabseeli, Um-sellah and Shahal variety (31 to 44.3
(29)
mg GAE/g . The phytochemical content in date seed evidently is affected by agro
ecological factors such as cultivar, year of production (i.e., climatic conditions year to year),
production place (geographical origin), soil (chemistry soil and fertilization) and maturation
degree(30). When comparing the phenols content of date seed with other fruits seeds described
as good source of antioxidants compounds, was found that date seed was above the values of
(5)
avocado seed, jackfruit seed and mango seed . In same way, other study using different
grape parts, seeds showed the higher antioxidant activity followed by peel and pulp (31). The
above emphasizes the relevance of using by-products of agro-food industry to obtain natural
phytochemical compound.

Total flavonoids content (TFlvC)

The TFlvC in date seed extract was affected (p<0.05) for the extraction method and solvent
as shown in Table 3. The highest flavonoid content was presented in acetone extract (50.00
mg RE/g of extract) independently of extraction method used, followed for the methanol
extract obtained for maceration (31.95 mg RE/g of extract) and ethanol extract for both
methods (UAE and maceration) (32.54 y 31.68 g RE/g of extract, respectively). The aqueous
extract, for both methods result with the lowest value (UAE: 12.95, maceration: 15.19 mg
RE/g of extract, respectively). In study directed by Teng, Chen (32), they found in three date
seed varieties (Boufgous, Bousthammi y Madhoul) values of 122.4, 165.9 y 184.4 mg
RE/100, respectively. The above, indicates that the values for methanol extract in the present
study were found above of values afore mentioned. These flavonids compounds has been
relationed with positive effects in health because have a modulatory actions signalling
(33)
phatways do not act as convetional hydrogen-donating antioxidant . The foregoing must
be considered for future studies in the seed since it is well known that the flavonoid content
exceeds the fruit. For this reason is interesting investigate independently this molecules in

102
date seed.

Antioxidant capacity

ABTS

The free radical scavenging capacity of date seed extract could be attributed to the dense
quantity of phenols and flavonoids present in the extracts. In some studies, a correlation has
(34)
been established between antioxidant capacity and phenols concentration . In our study,
the lower values were presented by the aqueous extract using UAE method. The above
mentioned could be influenced by the experimental conditions used, principally temperature
(25 °C) and solvent, because when water is used as solvent, low extraction efficiency is found
for phytochemicals, elevated temperatures can be much more efficient, to achieve better
extraction considering also non polar compounds that were not extracted.(35) The values
found for ethanol, methanol and acetone extracts in our study are similar to fund in other
studies with methanol apple seed extracts (6) ABTS values ranged from 220.52 to 708.02 µM
of TE/g, values affected by the variety of the fruit. (35). In other way, acetone and methanol
extract from avocado seed ´Hass´ variety presented low values, 158.29 and 78.93 mmol
(36)
Trolox/g fresh matter respectively, that the presented in this study for acetone and
methanol extracts 267.26 and 259.14 µM Trolox/mg of extract, individually.

DPPH

The antioxidant using four solvents and two methods were evaluated. Have been reported
that the polarity of solvent as well as the variety of date seed have an impact in the % of
inhibition of DPPH radical. Using acetone as solvent extraction date seed fardh variety
presented 78.25%, khalas 56.83%, khinazi 70.92%, khasab 73.50% and neghal 17.21% (37).
In the present study, acetone, methanol and ethanol display better activity, this could be due
to 1) phenolic acids that included ferulic acid, p-coumaric acid, chlorogenic and flavonoid,
catechin found in this study and 2) others water soluble compounds such as oligomers. (12, 38)

Reducing power

The reducing power in date seed extracts using acetone and ethanol as solvent was better
than aqueous and methanol extracts, both methods reflected remarkable results. When the
RP of date seed was compared with others seeds from fruits byproducts, for example

103
mandarin seed (Abs 700nm=0.31), litchi (Abs 700nm=0.64), grape (Abs 700nm= 0.23), and
(39)
synthetic antioxidant BHT (Abs 700nm=0.56) , was concluded that the values in the
present study exceeded the RP of all previously mentioned source, this are in corresponding
whit the antioxidant activity. For the above, date seed byproducts can be considered as a raw
material to obtained antioxidant compounds, at the same time the negative environmental
effects attributed to dispose them can be diminished.

Identification and quantification of phytochemicals using HPLC

Phenolic acids constitute one of main classes of secondary metabolites and in recent years
have been a subject of intense study. It contains a hydroxylated benzene ring with one or
more carboxyl group attached directly to it. In this study, catequin, a flavan 3-ol antioxidant
part of flavonoid family was the most predominant. These results are in accordance with
(40, 41)
other studies , these authors indicated that when used acetone 80%, catechin, ferulic
acid, caffeic acid, vanillic acid and p-coumaric acid were found as well as using acetone
50% gallic acid, p-hydroxybenzoic acid, caffeic acid, p-coumaric acid and ferulic acid, the
above is similar to found in the present study. The difference in concentration and presence
of compounds identified can be differing considerably due to variations in the extraction
methods and conditions used. Aqueous acetone showed capability of dissolving many
hydrophilic compounds and high molecular weight bioactive compounds as compared to
only aqueous solvent, such as the case of catechin, which is reflected in this paper. The
mixing of different solvents with varied degree of polarity can increase the extraction of
(14, 42)
polyphenols by dissolving compounds with varied degrees of polarity . However,
selection of better solvent affects the amount and rate of polyphenols extracted. In particular,
methanol has been generally found to be more efficient in extraction of lower molecular
weight polyphenols, while the higher molecular weight flavonols are better extracted with
aqueous acetone. The HPLC screening of phytochemicals evidenced that date seed
constitutes one of the highest source of total polyphenols, surpassing tea, grapes, flaxseed,
nut seeds and even date flesh, and as in grape seed, flavan 3-ol were found to be the most
abundant polyphenol in date seed extracts. The determination of polyphenol profile is of great
importance in terms of future potential values of date seed as functional ingredient, their use
as a low cost raw material open the door to explore their antioxidant activities and
mechanisms of action against chronic degenerative diseases that involving damage oxidative

104
of the principal biomolecules of the cell.

CONCLUSION

In the present investigation, the UAE was identified as an appropriate method for obtaining
phytochemical compounds from date seed, mainly at short times compared with maceration.
Regarding the solvents, the study of several solvent systems allowed to consider those
extracts that presented the highest phytochemical content, antioxidant activity, and that its
use is allowed in the food industry (acetone and water). In this sense, it is important to
continue exploring the antioxidant potential not only in vitro but in food matrices, to establish
its effectiveness.

ACKNOWLEDGEMENT

The first author thanks CONACyT (México) for support with Ph.D. fellowship during the
realization of this work. Authors also thank the technical assistance of María del Carmen
during research stay in Universidad Miguel Hernández, Orihuela, España.

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Table 1. Chemical composition and physical properties of date seed.

Chemical Composition Mean ± DS Reference†

Moisture 7.66 ± 0.05 8.26 ± 0.27

Protein 5.28 ± 0.15 5.66 ± 0.28

Fat 6.56 ± 0.08 6.14 ± 0.07

Ash 1.14 ± 0.01 1.09 ± 0.05

Carbohydrates 79.36 ± 0.07† 77.032‡

Physical properties

Seed/date ratio 5.92 ± 0.21 7.29 ± 0.17

Weigth 1.10 ± 0.13 1.35‡

Length 2.41 ± 1.87 2.75 ± 0.00

†(42, 43)

‡The carbohydrates were calculated subtracting from the total percentage of the rest of the determinations.

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Table 2. Yield of date seed extracts.

Solvent UAE Maceration

Acetone: water 5.72 6.66

Methanol: water 6.00 8.00

Ethanol: water 4.71 9.68

Water 5.67 3.22

The yield extract was expressed as g per 100 g of date seed powder

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Table 3. Phytochemicals in extract lyophilized of date seed byproducts.

Phytochemical Method Acetone: water Methanol: water Ethanol: water Water

Total phenols content UAE 33.02 ± 0.67aX 29.49 ± 0.42bX 32.23 ± 0.52aX 13.28 ± 1.16cX

(TPC) Maceration 33.11 ± 0.80bX 27.31 ± 0.51cY 32.34 ± 0.76bX 38.96 ± 2.9aY

Total flavonoids content UAE 50.00 ± 0.42aX 24.62 ± 1.17cX 32.54 ± 0.42bX 12.93 ± 0.2dX

(TFlvC) Maceration 49.58 ± 0.68aX 31.95 ± 1.96bY 31.68 ± 3.08bX 15.19 ± 0.46cX

TPC are expressed as mg GAE/g of extract, TflvC are expressed as mg RE/g of extract. Mean values with different lowercase letter within a row are significantly
different (p < 0.05) between solvents. Different uppercase letter in same column indicate differences (p < 0.05) between methods.

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 300 A)
A B T S (m M o f T E /g o f e x tra c t) aA aA
aA aA bA UAE

M a c e r a tio n
bB
200
cA

cB

100

0
A ceto n e M eth an o l Eth a n o l W ater

40
B) UAE
D P P H (µ M o f A A E /g o f e x tra c t)

aA aA aA
aA M a c e r a tio n
30 aB
aB
bA

20 bB

10

0
A ceto n e M eth an o l Eth a n o l W ater

1 .5
UAE
C)
M a c e r a tio n
A b so rb a n c e to 7 0 0 n m

aA
aA
1 .0 bB
bA
bA

dA dA cB
0 .5

0 .0
A ceto n e M eth an o l Eth a n o l W ater

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Figure 1. Results corresponding to A) ABTS, B) DPPH and C) reducing power. Lowercase
letter indicates differences (p<0.05) between solvents. Uppercase letter indicate differences
(p<0.05) between methods.

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Table 4. Identification and quantification by HPLC of phytochemical compounds from date seed extracts using different solvent and
methods of extraction.

Acetone:water Ethanol:water Methanol:water Water

Phytochemicals λmax (nm) Rt (min)
UAE Maceration UAE Maceration UAE Maceration UAE Maceration

Chlorogenic acid 325 21.4-22 962.95 855.4 172.70 874.46 220.81 845.84 nd 299.24

Caffeic acid 325 23 nd nd 134.57 nd nd nd nd nd

Coumaric acid 325 29.1 507.87 454.74 190.94 303.16 46.03 441.02 321.6 23.91

Ferulic acid 325 31.1 2395.32 2232.6 434.7 1962.33 256.14 1664.87 141.06 482.49

Sinapic acid 325 32 117.05 136.97 52.01 23.62 nd 79.37 52.45 64.89

Protocatechuic acid 270 12 nd 75.59 248.93 nd 71.25 79.33 nd 39.07

Catechin 280 18.8 907.53 737.45 982.69 823.82 661.18 1090.99 454.61 548.93

The results are expressed as µg/g of extract.

UAE: ultrasound assisted extraction.

nd: not detected.

Rt, retention time.

λmax, Absorbance.

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5. Effect of Inclusion of Date Seed Oil and Extract on the Fatty Acid Profile, Phytosterols
and Lipid Oxidation of Raw and Cooked Pork Meat

De la Rosa-Alcaraz MA, Estrada-Montoya MC, Torrescano-Urrutia GR, Astiazarán-García H,

González-Córdova AF, Vallejo-Córdoba B, Pérez-Álvarez JA, Fernández-López J, Sánchez-
Escalante A

Meat Science

(Revisión Interna)

116
Effect of inclusion of date seed oil and extract on the fatty acid profile, phytosterols and lipid
oxidation of raw and cooked pork meat

De la Rosa-Alcaraz MA1, Estrada-Montoya MC1, Torrescano-Urrutia GR1, Astiazarán-García H1,

González-Córdova AF1, Vallejo-Córdoba B1, Pérez-Álvarez JA2, Fernández-López J2, Sánchez-
Escalante A1*

1
Centro de Investigación en Alimentación y Desarrollo, A.C. Coordinación de Tecnología de
Alimentos de Origen Animal. Ctra. Gustavo Enrique Astiazarán Rosas No. 46, Col. La Victoria.
Hermosillo, Sonora, 83304, México.
2
Universidad Miguel Hernández, Escuela Politécnica Superior de Orihuela, Departamento de
Tecnología Agroalimentaria, Ctra. A Beniel Km 3.2. Orihuela, Alicante 03312, España.

*Corresponding author. Tel.: +52 (662) 2892400, Ext 361; fax +52 (662) 2800421. E mail address:
armida-sanchez@ciad.mx

Highlights

Treated cooked patties have more MUFA and phytosterols than control

Olive patties plus date seed extract have lower oxidation products

Date seed oil is a potential source of phytosterols and fatty acids for pork patties

Date seed extract can use in other food matrix with high oxidative instability

Abstract

The purpose of present study is known the effect of partial substitution of fat by date seed oil plus
natural extract of date seed as antioxidant on fatty acids profile, phytosterols and oxidative stability
of pork patties. The results showed that compared to control, the addition of oils increase TBARS
values, although treatments with extract diminishes this parameter (p<0.05). The fatty acid profile
of pork patties was modified with olive oil, increased oleic acid and ΣMUFA and while date seed
oil inclusion increased (p<0.05) 39.25%, 27.28%, 55.81% in β-sterol, stigmasterol and

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campesterol, respectively compared with olive treatment. Hence, it is concluded that extract and
oil from date seed byproduct can be used as rich-antioxidant and phytosterols ingredient to meat
products

Keywords: date seed, byproducts, oil, phytosterols, antioxidant, meat

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1. Introduction

The evidence of role fat has in the diet and development of chronic degenerative diseases and
diabetes type 2 is growing. The promotion of health trough of nutrition has been established as an
objective important in many countries. In agreement with World Health Organization (WHO), the
recommendations of daily optimal intake of unsaturated fatty acids not be exceed of 10-15 % of
intake of energy (Jarosz and Bułhak-Jachymczyk, 2008). These recommendations are in the diet in
general, however, meat and meat products stand out as the source of fat more important (Jiménez-
Colmenero et al., 2015).

Change in quantity of fat and lipid profile of meat products would be help to improve their
nutritional quality, beside can be developed not only altering the amount and type of fat but also
including other type of ingredients that enhance health for example antioxidants (Jiménez-
Colmenero et al., 2000) and contribute to delay lipid oxidation that play very important role, since
it is during this process a wide range secondary compounds derived of aldehydes and ketones are
produced with toxic potential for organism, example malondialdehyde, they are inversely related
to oxidative stability of rest of lipids of meat matrix (Armenteros et al., 2012).

In previous studies, where vegetable oils have been added to dry-fermented sausages,
frankfurter and pork patties, have revealed conflict because their inclusion modifies the fatty acid
profile of monounsaturated and polyunsaturated fatty acid (MUFA and PUFA), which increases
the oxidative process (Ansorena and Astiasarán, 2004; Bloukas, Paneras, and Fournitzis, 1997;
Choi, Choi, Han, Kim, Lee, Jeong, et al., 2010; Freire et al., 2017). For the above, use of
antioxidants as butylated hydroxyanisole (BHA) butylated hydroxytoluene (BHT), propyl gallate
(PG) and tert-butylhydroquinone (TBHQ) have been used for years to control oxidation reactions
in products of meat origin, since can be an effective and economic alternative. However, its use is
questioned from the point of view of food safety as various clinic studies have proven the toxic
effect of these synthetic additives. Whereby, the meat industry has shown increasing interest for
search of other compounds that have antioxidant activity and not compromise the health of the
consumer. In this sense, after searching natural and safety alternatives the investigation in natural
antioxidants has acquired relevance (Armenteros et al., 2012; Shah et al 2014; Sousa 2019).

In this sense, date fruit byproduct specifically seeds have proven to be excellent source of dietary
fiber and antioxidant compounds present in high quantities such as hydroxytyrosol, protocatechuic

119
acid, gallic acid, caffeic acid, p-coumaric acid and oleuropein, which have biological activity.
However, its use has been limited to animal feed (camels, cattle, sheep and birds) and production
of a drink type coffee free caffeine “Herbal coffee”, to which has been attributed many benefits
(Sirisena et al., 2015). With respect to, date seed oil fatty acids profile has been characterized,
resistance to thermal processes and oxidative stability (Besbes et al., 2004: Rahman et al., 2007),
finding very important results, although application in foods is scarce. For the above, the objective
of this work was replaced partially pork fat for date seed oils and including date seed extract to
reduce lipid and protein oxidation in pork patties.

2. Materials and methods

2.1. Materials

The commercial Olive oil (La Española trademark) was purchased from a local supermarket
in Mexico, and stored at 4 °C until analysis and pork patties manufacture. Pork meat
(Semimembranosus, Semitendinosus, Bicepsfemoris) and fat were purchased with a local provider,
fat and connective tissue were removed in meat, and visible tissue to fat. The raw material was
stored at -20 °C, and 24 h before to making pork patties, remained at -1 °C (process temperature).

2.1.1. Pork patties manufacture

As shown Table 1, the experiment consisted of four treatments and control: T1 consisted of
partial substitution (30%) of pork back fat for olive oil: T2 the partial substitution of pork back fat
(30%) for date seed oil: T3 conserved the characterisics of T1 plus the addition of date seed extract
(DSE):T4 conserved the characteristics of T2 plus DSE, and was included a control additionated
only with pork back fat (75:25, meat:fat). Six patties (30 g) per treatment were elaborated in two
independents batches, twelve in total, meat and fat were grinder using plate 3/16”, when all
ingredients were mixed, the fat content was verified in each formulation using Univex FA-73
equipment. Cooking of pork patties was realiced usisng plate grill (George Foreman, GR144B) to
achieve a meat core tempeture of 70 °C.

2.2. Obtention of date seed sample

Date seeds Medjool variety were selected directly from 50 Kg of date fruit not commercial
and collected in stage Tamar (fully ripe). After, seeds were washed with water removing with the
hands rest of fruit adhered, air dried 48 h and transfer to oven (Yamato, DX302/402/602, model,

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JP) 24 h at 50 °C. Also, date seeds were milled and tamized using 1-2mm mesh in a hammer mill
(Thomas Wiley, model 4, USA), the powder resulting was conserved at -20 °C under vacuum
conditions (Nehdia et al., 2010).

2.2.1. Date seed oil extraction

The extraction was in according to Besbes et al. (2004) using a solver extractor 148 SER (Velp
Scientifica, IT). Sample 15 g of date seed (1-2 mm) was placed on post Soxhlet, and petroleum
ether was used as solvent of extraction (40 °C). The operating conditions were the following:
immersion time, 30 min the thimble with solvent; washing time 60 min, 8 reflux to sampling
washing. After, solvent was removed using a rotatory evaporator at 40 °C, 5 min, and oil was dried
under a nitrogen stream and storage at -20 °C until analysis. The relative percent of lipids was
compared with the weight of dried seed powder (resulting in 10 % of yield).

2.2.2. Obtention of date seed extract

The extract was obtained in according to Al Farsi et al. (2005), with slight modifications. The
seed powder (1 g) was mixed with 10 mL of ethanol:water [1:1, v/v] and was subjected to
ultrasound-assisted extraction (UAE) during 60 min at 40 kHz and temperature of 25 °C ± 3 °C
(Ultrasound water bath, Branson, 1800 Model, Series CPXH, USA), centrifugates to 11, 000 x g/15
min/ 4 °C (Beckman coulter, GX-IM-5AB Model, US). The supernatant was filtered through filter
paper Whatman 1, concentrated at 60 °C (rotatory evaporator, RE301 Model, YAMATO, JP), and
dried (Freeze dryer, DC401/800 Model, JP). The extract was stored at -20 °C under dark, until
analysis.

2.3. Chemical proximal composition

The chemical proximal analysis of the raw and cooked samples was determined in according
to established by AOAC (1995), using the following methods: moisture (950.46 methods) ash
(900.02 methods), fat using continuum extraction with solvent (Goldfish) 991.36 methods, and
protein nitrogen was realized following the Kjeldahl (976.05, methods) 6.25 value was utilized as
factor of conversion to raw protein. The results were expressed as g of each component per 100 g
of sample.

2.4. Physicochemical analysis

2.4.1. Instrumental Color

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 The superficial color of raw patties was evaluated using a Minolta colorimeter CR-300
(Minolta Camera Corp., Metar division, USA). The following coordinates were determinate: L*
lightness, a* for green-red, b* for blue-yellow, Chroma and Hue*. Before each measurement, the
colorimeter was calibrated in the color space system, CIE Commission Intl. of I´Eclairage (1978)
using a white tile. The measure of color was realized in the of each raw pattie per tenfold at room
temperature (~22 °C) whit illuminate D65 and angle of 10°C in the observer.

2.4.2. Cooking loss weight (CLW)

For evaluation of CLW, the patties were grilled in a George Foreman Grill (GR144B). The
CLW was expressed in percentage and calculated as following: CLW = [(Wb-Wa)/Wb]*100,
where Wb and Wa are the weight of patties before and after of cooked, respectively. After cook,
the patties were subsequently cooled at room temperature, and stored at -20 °C, until analysis.

2.5. Determination of thiobarbituric acid reactive substance (TBARS)

The TBARS were determinate using the method of Pfalzgraf et al. (1995). Briefly, each sample
(10 g) was mixed (30 sec at 6000rpm) with trichloacetic acid (10%, w/v) using a homogenizer
(Ultra-Turrax modelo IKA-T25, US) the tube is submerged in ice, next centrifuged at 2,300 x g for
20 min to 5 °C (Beckman coulter, GX-IM-5AB Model, USA), and the supernatant was filtered
using Whatman paper No. 4. After, 2 ml of the filtrated was mixed with 2 mL of thiobarbituric acid
(20 mM), placed in a boiling water bath (97 °C, during 20 min) and subsequently cooled at room
temperature. The absorbance was read at 531 nm (Spectronic Genesys 5, Thermo Electron Corp.
336001 Model). The concentration of malondialdehyde (MDA) in the sample was calculated using
standard curve of 1, 1, 3, 3-tetraethoxypropano (TEP). The results were expressed as mg of
MDA/Kg of sample.

2.6. Total protein carbonyls quantification

The protein oxidation was measure for the total content of carbonyls and evaluated for 2, 4-
Dinitrophenylhydrazine (DNPH) derivatization, following the method of Oliver, Ahn, Moerman,
Goldstein, & Stadtman (1987) with a slight modifications. The patties (1 g ) were homogenized
with sodium phospahe buffer 20 mM (1:10, w/v) contained 0.6 M of NaCl (pH 6.5) using a
homogenizer Ultra-Turrax during 30 s. Two equal aliquotes of 0.2 mL were taken of homogenized
and put in microtubes of 2 mL. The proteins were precipitated with TCA at 10% (1 mL) and
subsequently centrifugated (at 17 000 x g/5 min/ 4 °C). After, one pellet was treated with 1 mL of
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HCL 2 M (to measure protein concentration) and the other with an equal volume of 0.2% (p/v)
DNPH in 2 M of HCl (to measure carbonyl concentration). Both samples were incubated during 1
h at room temperature. In addition, the sample was precipitated with TCA 10% (1 mL), and washed
two fold with ethanol:ethyl acetate (1:1, v/v) (1mL) to remove DNPH excess. The pellets were
dissolved in 1.5 mL of sodium phosphate buffer with 6 M of guanidine (pH 6.5), shaking and
centrifuged (3500 x g/ 2 min/4 °C) to remove fragments insoluble. The protein concentration was
calculated from absorption at 280 nm (Mutiskan Go, Thermo Scientific, Fl) using BSA as standard.
The number of carbonyls was expressed as nmol of carbonyls per mg of protein using the
absorption coefficient of 21.0 nM-1 cm-1 at 370 nm for protein hydrazones.

2.7. Profile of fatty acids of vegetable oils, pork back fat and patties

The analysis of the fatty acids was realized per triplicate. About 200 mg of fat from burger or
vegetable oils was weighed, and then 2 mL of sodium methoxide 0.5 N in methanol were used for
saponification during 15 min at 70 °C. After, were added 2 mL of boron trifluoride in methanol at
14% (v/v) during 30 min at 70 °C for residue methylation. The samples were cooled to added 4
mL of distilled water and 8 mL of hexane, the mix was vigorously shaked in a vortex. Of the layers
formed the upper one containing the methyl esters was taken, to which were added 1 g of sodium
sulfate anhydrous and mixed, to subsequently transfer te hexane to other assay tube containing 0.25
mg of C9:0 (perlagonic acid methyl ester), used as an internal standard (Kramer et al., 2001;
Shantha et al., 1993). The samples were stored under dark conditions and kept an inert atmosphere
of nitrogen at -20 °C, until the chromatographic analysis was carried out.

For the chromatographic analysis of the extract containing the methyl esters was evaporated
and resuspended in 0.2 mL hexane. After, the sample 1.0 µL was injected into the gas
chromatograph coupled with a flame ionization detector (FID) (VARIAN Saturn 2100D). The
chromatographic separation carried out in a column Thermo science TR-5MS (30 m x 0.25 mm
I.D. x 0.25 µm film). The operating condition were as follow: initial temperature (T1) 60 °C/min,
with a initial ramp (R1) of 10 °C /min; medium temperature (T2) of 190 °C sustained for 30 min,
with a medium ramp (R2) of 5 °C/min, to reach final temperature (T3) of 220 °C, keeping this
temperature 25 min. Total run time was 1 h approximately, helium was used as carrier gas at 1.3
psi. Finally, the identification of fatty acids of the samples was perform throught the comparison
of retention times (FAME peaks) respect to a internal standard. The quantitative composition was

123
made relating to the areas under the curve of the interest peaks of the sample with the area of the
standard peaks (Sampugna et al., 1982).

2.8. Statistical analysis

The data obtained of proximal composition, physicochemical analysis, fatty acids and
cholesterol were analyzed using one-way analysis of variance (ANOVA). The effects of the
treatment were considered and all statistics assay were performed using NCSS version 2012
(Number Cruncher Statistical System, 2012) software. The significant differences were estimated
at a probability level of 0.05, if differences were found between treatments, a mean comparison
test was performed by Tukey Kramer.

3. Results and discussion

3.1 Chemical proximate composition

As shown in Table 2, the results of this work showed that the chemical proximal composition of
raw and cooked patties was similar to previous reported works (Choi et al., 2010; Rodriguez-
Carpena et al., 2012; Valenzuela-Melendres et al., 2018), where pork backfat was partially replaced
for vegetable oils, not significative differences (p<0.05) was found between treatments.

3.2 Physicochemical analysis

3.2.1 Instrumental color measurement

The meat color is a quality parameter important to purchase decision of the consumer (Moon et al.,
2012). The partial substitution of vegetable oils affect the color of patties, as show in Table 3, the
value L* increased in all treatments compared to the control, in terms of a* (red) values, DSO and
DSO + DSE patties presented the lowest values (7.6 and 7.8 respectively) with respect to control
(10.4), it is known that antioxidant in the vegetable oils, as E vitamin could inhibit oxymyoglobin
oxidation resulting in a higher red color in the final product (Rodriguez-Carpena et al., 2012),
however, the own color of DSO can be affected this parameter. The lowest value of b* (yellow)
were presented in the control and DSO + AOX, 18.65 and 17.86, respectively, O (Olive) and O +
DSE revelated the highest values, this was observed in another studies where was added olive oil
the authors that increased in the yellow color can be part of the original color of olive oil (Moon et
al., 2012; Muguerza, Fista, Ansorena, Astiasaran, & Bloukas, 2002). On the other hand, C* values
were presented in the range of 19.55-26.41, treatments added with date seed oil (DSO, DSO +

124
DSE), exhibited the lowest value, different to h* value where the same treatments (DSO, DSO +
DSE) shown the highest values. These values are in according to reported for Bañón, Díaz,
Rodríguez, Garrido, and Price (2007), this author indicate that while C* decreases h* increases,
this change usually asociated with a low value of red and low stability of meat color. However the
changes in te color by the effect of the addition of oil can depend of meat product (Moon et al.,
2012).

3.2.2 Cooking loss weight (CLW)

In the present study not found differences (p>0.05) in CLW for the effect of treatment, the values
oscillated between 27.55-30.89. These values are within the reported value 11-38% for others
authors (Brugiapaglia & Destefanis, 2012; Moon et al., 2012; Serrano, Librelotto, Cofrades,
Sánchez-Muniz, & Jiménez-Colmenero, 2007). The cooking loose depends of the mass
transference during the process and thermic treatment and, cooking method can mark differences
in the loss of water (Cheng & Sun, 2008). Likewise, in meat products the CLW is multifactorial
since many factor have impact in this parameter such as meat quality, precooking treatment,
cooking technique, rank of heat, cooking temperature and final temperature in the center, this can
explain most of the variation in the cooking loss (Lorenzo et al., 2015).

3.3 Evaluation of lipids and proteins oxidation

3.3.1 Determination of thiobarbituric acid reactive substances (TBARS)

The content of thiobarbituric acid reactive substances expressed as malondialdehyde (MDA) is

shown Table 4. In raw product, all treatments were found for below of suggested value 0.5 mg od
MDA/Kg of meat, TBARS numbers above about this value are critical since they indicate a level
of lipid oxidation products wich produce a rancid odor and taste which can be detected by consumer
(Wood et al., 2008). However, when samples were cooked lipid oxidation increase quickly, except
in treatment whit DSE inclusion, particularly in O + DSE treatment compared with control (0.59 ±
0.02 and 2.68 ± 0.38 mg MDA/Kg respectively). In a study realized by Gray and Pearson (1987)
explain that lipid deterioration as reflect in sensorial characteristics of the product, since the flavor
to rancid taste is detected in meat product with TBARS values between 0.5 and 2.0 mg MDA/Kg.
In according to (Boles, 1990), values higher than 1.0 can be perceived as over warmed flavor odor
in meat products. Some study shows that the replacement of pork fat for oil promotes the formation
of lipid oxidation secondary products, samples with addition of vegetal oil reflect higher value of

125
TBARS, due to their fatty acids composition are more susceptible given its chemical structure
(Choi, Choi, Han, Kim, Lee, Jeong, et al., 2010; Freire et al., 2017; Rodríguez-Carpena et al.,
2012). However, it seems that the factor that most favors the formation of secondary products of
oxidation is quantitative (quantity of fat) more than qualitative (insaturation level) (Freire et al.,
2017). In the present study, O + DSE and DSO + DSE showed the lowest TBARS values, which
indicate that DSE can protect from oxidation to olive and date oil and possibly had potential for
application in other meat products. Because, the temperature is the principal factor that determines
phytochemicals degradation and consequently loss functionality (Turturică, Stănciuc, Bahrim, &
Râpeanu, 2016), in cooked patties effectiveness of DSE was proven, because in treatments where
it was added had acceptable values (<1.0) (Bloukas et al., 1997).

3.3.2 Quantification of total protein carbonyl

In the present study, protein carbonyls content was reduced in an important way (p<0.05) in cooked
patties by effect of inclusion of vegetable oils and extract compared with control (Fig. 1), the lower
values were presented in O + DSE and DSO + DSE 2.07 nM of hydrazones/mg of protein and 1.8
nM of hidrazones/mg of protein respectively, conferring 65.27 and 69.80% of protection versus
control products. Similar values were reported (Rodríguez-Carpena et al., 2012) to pork patties
with remplaced of 50% of fat by vegetable oils: avocado, sunflower, and olive. In addition, the
protector effect of oils can be attributed to the minors components as tocopherols, that contribute
to the oxidative stability of oil and have an important paper in remove free radicals in vivo (Estévez,
Kylli, Poulanne, Kivikari, & Heinonen, 2008b). The above, in synergy with phytochemicals
components of date seed extract, by acting against lipid oxidation is expected to contribute to the
reduction of protein oxidation to some extent minimizing the formation of secondary products lipid
oxidation and blocked your effect prooxidative in proteins. Although, it is necessary indicate that
some compounds that can prevent lipid oxidation can not be effective for inhibit the formation of
protein carbonyls (Haak, Raes, & De Smet, 2009). The effect of phenols against protein oxidation
is governed by interaction of specific mechanisms between phenolic compounds and proteins.
These interactions are dependent of the quantity and chemical state of phenolic compounds, size,
conformation, general charge of proteins, which must be taken into account (Kroll & Rawel, 2001).

3.4 Fatty acids compositions

126
In Table 5, is presented fatty acid profile of pork patties with partial substitution of back pork fat,
the inclusion of vegetables oils improved the fatty acids profile in pork patties (p<0.05). . The fatty
acids present in all treatments were oleic acid, palmitic acid, and stearic acid. The greater presence
of oleic acid (C18:1N-9) was observed in O and O + DSE. The above mentioned, has also been
reported by other authors when is replaced olive oil to improve fatty acids profile of meat products
(Choi, Choi, Han, Kim, Lee, Jeong, et al., 2010; Freire et al., 2017; Rodríguez-Carpena et al.,
2012). The olive oil is a vegetable oil with a hight level of fatty acids monounsaturated (MUFA)
and has attracted the attention as a substitute for animal fat in processed meat products, principally
because to favorable mix of MUFA, E and K vitamins, carotenoids and polyphenols as
hydroxytyrosol, tyrosol and oleuperin (Boskou, 2009). The positive influence of olive oil in health
include an improvement in lipoproteins profile, blood pressure, sugar metabolism, oxidative stress
reduction, antithrombotic profile and colon and breast cancer prevention (Moon et al., 2012).
Respect to the addition of date seed oil in meat products, up to our knowledge, the application is
limited, different to olive oil that has been very studied in meat matrices (Bloukas et al., 1997; Choi
et al., 2009; López-López, Cofrades, Yakan, Solas, & Jiménez-Colmenero, 2010; Rodríguez-
Carpena et al., 2012).

3.5 Sterols quantification

The sterols content was affected (p<0.05) treatment as indicated in Table 6, compared with
control DSO + DSE revealed a decrease. In accordance with others investigations (Choi et al.,
2010; Kayaardi 2003; Muguerza et al 2001), the partial replaced of animal fat for olive oil and
sunflower impact in decreased of cholesterol in meat products. Although, others authors, not have
found an impact on the content of cholesterol, after replacing 50% and 25% of vegetable oil for
animal fat in patties and fermented sausages respectively (Rodriguez-Carpena et al. 2012;
Muguerza, Ansorena y Astizaran., 2003). The implications of cholesterol in the diet associated
with fat content has developed a negative perception in the consumer. However, for the stricter
scientific standards, the evidence of cholesterol dietetic itself is associated with heart illnesses risk
it not definitive (Armenteros et al., 2012). Even with it, the effort in the manufacturing of meat
products with modifications in fat content is important.

Respect to phytosterols, the treatment in which the date oil was added show high β-sitosterol,
stigmasterol and campesterol than olive oil treatments. The phytosterols/stanols are used as new

127
food ingredients with biological activity to reduce plasma cholesterol, of this have substantial
evidence of that the sterols and stanols of plants diminished the cholesterol LDL-total, due to
partial inhibition of cholesterol, and your effect is additional compared with others strategies (for
example, low-fat diet, use of drugs to diminished cholesterol) (Pennisi Forell et al., 2010). There
is an extensive variety of phytosterol structures, being β-sitosterol and stigmasterol are the most
frequently found in the nature. In olive oil, the total sterols are presents in esters form of fatty acids,
the sitosterol represents from 75-90% of the total fraction of sterols, for its part the sitosterol is
found only in traces or small quantities, which agrees with our results.

3.6 Atherogenic index

The atherogenic index (AI) assess the risk of atherosclerosis and was proposed by Ulbricht
and Southgate (1991), so it is recommended low values of 0.5 (AI) (De Lorenzo et al., 2001). In
the Tabla 5 are presented the results of this parameter for cooked patties, treatments where was
added date seed oil (DSO, DSO +DSE) showed high AI values, 0.6 againts the control. López-
López et al. (2011) found AI of 0.68 in control pork patties and when was added wakame (Undaria
pinnatifida) 0.67, however, patties with olive oil and wakame presented values of 0.44, likewise,
the found our study when was added the same source of oil. Inversely, in the treatment where only
wakame was added, the AI increased to 0.67, results similar to those found our treatment with
DSO. The above could be in relation to the per se composition of fatty acids of the DSO, it seems
that the myristic fatty acids (C14:0) and palmitic (C16:0) and to a lesser extent C18:0 the more
atherogenic which are present in a significant amount in date oil.

4. Conclusion

This study allowed to advance in the knowledge of the contribution that the inclusion of date seed
oil has in fatty acids profile and phytosterols of a meat product in comparison with another common
source that is olive oil, also the date extract showed to be effective against lipid oxidation that is
one of the main problems for the meat industry. Which opens the doors to its application not only
in this product but also in meat matrices with high susceptibility to oxidation.

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Tables and Figures

Table 1

Formulation of control and fat reduced pork patties.

Ingredients represented as g/Kg

Treatment
Lean pork leg Oil Pork back fat DSE (%) Salt Water

Control 750 0 250 0 20 180

T1 750 75 175 0 20 180

T2 750 75 175 0 20 180

T3 750 75 175 0.1 20 180

T4 750 75 175 0.1 20 180

Control pork back fat (30%); fat replaced with olive oil1; fat replaced with date seed oil2; fat replaced with olive oil +
DSE (date seed extract)3; fat replaced with date seed oil + DSE4.

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Table 2

Chemical composition of pork patties reduced pork fat (g/100 g).

Item Treatment Moisture Fat Ash Protein

Control 62.54 ± 0.26ªX 16.76 ± 0.14bX 2.65 ± 0.02aX 14.50±0.04aX

T1 62.17 ± 1.13ªX 16.28 ± 0.62bX 3.12 ± 0.07aX 15.02±0.02aX

Raw T2 62.72 ± 0.65ªX 16.45 ± 0.37bX 2.55 ± 0.02aX 14.53±0.04aX

T3 62.56 ± 0.35ªX 18.68 ± 0.04cC 2.16 ± 0.01aX 13.13±0.06X

T4 61.99 ± 0.43ªX 17.04 ± 0.87bX 2.56 ± 0.01aX 14.68±0.01aX

Control 58.75 ± 2.06bY 14.97 ± 0.62aY 3.22 ± 0.11bX 20.67±0.07bY

T1 55.05 ± 1.02bY 16.99 ± 0.45bX 3.40 ± 0.01bX 21.99±0.05bY

Cooked T2 56.20 ± 2.01bY 16.96 ± 0.63bX 3.15 ± 0.01bY 21.12±0.05bY

T3 57.83 ± 0.71bY 16.19 ± 0.22abD 2.89 ± 0.05bY 20.33±0.13bY

T4 56.45 ± 1.91bY 15.09 ± 0.14aY 3.35 ± 0.03bY 22.34±0.01bY

Control pork back fat (30%); fat replaced with olive oil1; fat replaced with date seed oil2; fat replaced with olive oil +
DSE (date seed extract)3; fat replaced with date seed oil + DSE4. Lower case in same column indicate significant
difference (p<0.05), different letter a and b indicate difference between treatments and X, Y indicate difference
between raw or cooked.

133
Table 3

Instrumental color of fresh pork patties whit partial fat substitution for vegetable oils.

Treatment L* a* b* C* H*

Control 60.83 ± 4.02ª 10.40 ± 1.39a 18.65 ± 1.74ª 21.37 ± 2.02a 60.91 ± 2.52ª

T1 66.62 ± 2.23b 12.17 ± 1.98b 23.41 ± 1.22b 26.41 ± 1.93b 62.70 ± 2.83ª

T2 66.90 ± 2.21b 7.65 ± 0.80c 19.34 ± 1.02a 20.79 ± 1.30c 68.68 ± 2.25b

T3 64.43 ± 2.11b 9.60 ± 0.89a 21.27 ± 1.46c 23.34 ± 1.56a 65.70 ± 1.71b

T4 62.40 ± 2.36c 7.89 ± 1.33c 17.86 ± 1.12a 19.55 ± 1.37c 66.27 ± 3.13b

Control pork back fat (30%); fat replaced with olive oil1; fat replaced with date seed oil2; fat replaced with olive
oil + DSE (date seed extract)3; fat replaced with date seed oil + DSE4. Lower case in same column indicate
significant difference (p<0.05), different letter a and b indicate difference between treatments.

Table 4

TBARS in pork patties with partial substitution of back pork fat for vegetable oils.

Item Control T1 T2 T3 T4

Raw 0.33 ± 0.05a 0.17 ± 0.01b 0.24 ± 0.04c 0.14 ± 0.02b 0.22 ± 0.05c

Cooked 2.68 ± 0.38a 0.92 ± 0.20b 0.92 ± 0.09b 0.59 ± 02c 0.85 ± 0.19b

Control pork back fat (30%); fat replaced with olive oil1; fat replaced with date seed oil2; fat replaced with olive oil +
DSE (date seed extract)3; fat replaced with date seed oil + DSE4. Values expressed as mg MDA/ Kg of sample. Row
whit different latter indicate differences (p<0.05).

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Table 5

Fatty acids content in pork patties with partial substitution of back pork fat (mg/100 g).

Fatty acid Control T1 T2 T3 T4

C8 0.06 ± 0.00a 0.00 ± 0.00 0.09 ± 0.01a 0.06 ± 0.00a 0.12 ± 0.02b

C10 0.16 ±0.01a 0.09 ± 0.01b 0.18 ± 0.00a 0.10 ± 0.01c 0.17 ± 0.02a

C12 0.31 ±0.02b 0.24 ± 0.01c 5.20 ± 0.14a 0.12 ± 0.02d 4.94 ± 1.13a

C14 1.29 ±0.07b 1.18 ± 0.05b 3.85 ± 0.09a 1.26 ± 0.16b 3.76 ± 0.51a

C16 21.88 ±0.40a 18.42 ± 2.22b 0.00 ± 0.00 19.25 ± 0.40b 18.76 ± 0.37b

C16:1 1.99 ±0.25a 1.91 ± 0.08a 1.53 ± 0.05b 1.77 ± 0.04c 1.52 ± 0.08b

C17 0.31 ±0.02a 0.25 ± 0.01b 0.26 ± 0.02b 0.26 ± 0.02b 0.24 ± 0.01b

C17:1 0.26 ±0.02a 0.23 ± 0.04a 0.20 ± 0.02a 0.23 ± 0.03a 0.20 ± 0.01a

C18 11.18 ±0.29a 8.54 ± 0.25c 9.49 ± 0.54b 9.20 ± 0.48b 8.93 ± 0.47c

C18:1N9c 37.09 ±4.60a 47.96 ± 1.40c 40.10 ± 1.76b 47.33 ± 1.14c 41.03 ± 2.48a

C18:1N7 3.13 ±0.38a 2.92 ± 0.13a 2.59 ± 0.20b 2.71 ± 0.32b 2.33 ± 0.25b

C18:2N6c 22.94 ±0.15a 15.54 ± 0.32c 16.27 ± 0.51b 15.08 ± 0.86c 15.84 ± 0.51c

C20 0.23 ±0.02a 0.18 ± 0.00b 0.24 ± 0.02a 0.18 ± 0.00b 0.17 ± 0.01b

C20:1 0.86 ±0.09b 0.55 ± 0.42d 0.76 ± 0.06c 0.97 ± 0.05a 0.79 ± 0.08c

C18:3N3 0.75 ±0.04a 0.46 ± 0.03b 0.44 ± 0.03b 0.46 ± 0.04b 0.70 ± 0.07a

C20:2 0.67 ±0.01a 0.47 ± 0.01b 0.54 ± 0.03b 0.51 ± 0.02b 0.50 ± 0.03b

C22 0.36 ±0.01a 0.15 ± 0.00b 0.19 ± 0.01a 0.14 ± 0.02a 0.18 ± 0.00a

C20:4N6 0.27 ±0.01b 0.50 ± 0.01a 0.53 ± 0.02a 0.48 ± 0.05a 0.44 ± 0.01a

ΣSFA 35.36 ±0.84b 29.05 ± 2.56c 39.02 ± 1.84a 30.57 ± 1.14c 37.25 ± 2.54a

135
ΣMUFA 43.34 ±5.35c 53.57 ± 2.07a 45.19 ± 2.09b 53.01 ± 1.58a 45.87 ± 2.89b

ΣPUFA 23.95 ± 0.22a 16.98 ± 0.38b 17.25 ± 0.57b 16.02 ± 0.97c 16.98 ± 0.59b

n6/n3 31.13 ± 0.22c 34.95 ± 0.38b 38.06 ± 0.38a 37.70 ± 0.97a 23.25 ± 0.59d

IA* 0.41 ± 0.01b 0.33 ± 0.03c 0.58 ± 0.07a 0.35 ± 0.004a 0.62 ± 0.07a

Control pork back fat (30%); fat replaced with olive oil1; fat replaced with date seed oil2; fat replaced with olive oil
+ DSE (date seed extract)3; fat replaced with date seed oil + DSE4. Results are expressed as mean ± standard
deviation. Values with a different letter within a row are significantly different (p<0.05). *Atherogénic Index (C1:0
+ 4 x C14:0 + C16:0)/(ΣMUFA+ΣPUFA).

136
Table 6

Sterols content in pork patties with partial substitution of back fat (mg/100 g).

Treatment Cholesterol β-Sterol Stigmasterol Campesterol

T1 62.37 ± 5.81a ------- ------ ------

T2 51.23 ± 2.02 b 4.38 ± 0.28c 0.47 ± 0.03c 0.48 ± 0.05b

T3 45.61 ± 0.35 c 11.16 ± 0.31b 1.71 ± 0.10b 0.86 ± 0.02a

T4 47.74 ± 1.56 c 4.99 ± 0.14c 0.58 ± 0.12c 0.48 ± 0.05b

T5 45.36 ± 0.89 c 13.07 ± 0.50a 2.06 ± 0.22a 0.97 ± 0.11a

Control pork back fat (30%); fat replaced with olive oil1; fat replaced with date seed oil2; fat replaced with olive
oil + DSE (date seed extract)3; fat replaced with date seed oil + DSE4. Different letter in same column indicate
significant difference (p<0.05).

137
 7.00
a)
6.00 a
nM hidrazones/mg protein

a
5.00
4.00
b
b
3.00 b
2.00
1.00
0.00
Control Olive DSO Olive + DSO+DSE
DSE

7.00 a b)
6.00
nM hidrazones/mg protein

5.00
4.00
3.00 b
c c c
2.00
1.00
0.00
Control Olive DSO Olive + DSO +
DSE DSE

Figure 1. Protein hydrazones in raw (a) and cooked (b) pork patties with partial substitution of pork
back-fat. DSO: date seed oil; DSE: date seed extrac

138
 6. Bioaccesibilidad de un Extracto de Coproductos de Dátil (Phoenix dactylifera L.)
Utilizado como Aditivo Natural en Hamburguesa de Cerdo

Ma. de los Ángeles de la Rosa-Alcaraza, Gastón Ramón Torrescano-Urrutiaa, Humberto

Astiazarán-Garcíaa, Juana Fernández-Lópezb, José Ángel Pérez-Álvarezb, Manuel Viuda-Martos,
Armida Sánchez-Escalante*a

EUROCARNE

(en prensa)

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7. Bioaccessibility of Polyphenols of Date Seed Extract Used as Natural Antioxidant Against
Lipid Oxidation During in vitro Digestion of Pork Patties

Ma. de los Ángeles de la Rosa-Alcaraza, Gastón Ramón Torrescano-Urrutiaa, Juana Fernández-

Lópezb, José Ángel Pérez-Álvarezb, Manuel Viuda-Martos, Raquel Lucas-Gonzálezb, Humberto
Astiazarán-Garcíaa and Armida Sánchez-Escalante*a

Food and Function

(Revisión Interna)

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 Bioaccessibility of Polyphenols of Date Seed Extract Used as Natural Antioxidant Against
Lipid Oxidation During in vitro Digestion of Pork Patties

Ma. de los Ángeles de la Rosa-Alcaraza, Gastón Ramón Torrescano-Urrutiaa, Juana Fernández-

Lópezb, José Ángel Pérez-Álvarezb, Raquel Lucas-Gonzálezb, Humberto Astiazarán-Garcíaa and
Armida Sánchez-Escalante*a
a
Centro de Investigación en Alimentación y Desarrollo, A.C. Coordinación de Tecnología de
Alimentos de Origen Animal. Ctra. Gustavo Enrique Astiazarán Rosas No. 46, Col. La Victoria.
Hermosillo, Sonora, 83304, México.
b
Universidad Miguel Hernández, Escuela Politécnica Superior de Orihuela, Departamento de
Tecnología Agroalimentaria, Ctra. A Beniel Km 3.2 (03312) Orihuela, Alicante, España.

ABSTRACT

Lipid oxidation in meat and meat products is one of the major processes responsible for generation
of cytotoxic and genotoxic compounds such as malondialdehyde (MDA). During digestive process
has been established that lipid oxidation increasing. The aim of the investigation was to evaluate
the bioaccessibility of date seed (Phoenix dactylifera L.) extracts and inhibition of lipid
peroxidation throughout in vitro gastrointestinal digestion of pork patties. During experimental
phase were manufactured two batches of pork patties: 1) control and 2) with date seed extract (DSE,
2%), cooked at 70 °C for static in vitro digestion, according to cost action INFOGEST.
Consecutively, polyphenols were identified at 270, 280, 320 y 325 nm using HPLC, while lipid
peroxidation was determined by MDA accumulation in the gastric and intestinal phase (37 °C, 120
min). The chromatographic analysis showed the presence of phenolic acids (ferulic,
protocatechuic) and a flavonoid (catechin) in the DSE. The phenolic compounds were kept stable
through gastrointestinal digestion of pork patties added with DSE. The lipid peroxidation in the
gastric and intestinal phase after intake of pork patties was not detected in treatment with DSE,
however, in the control MDA values were 4.09 mg/Kg of sample and 4.60 MDA mg /Kg of sample,
gastric and intestinal, respectively. The use of date seeds extract can be proposed as a
viable alternative to overcome use of synthetic antioxidant in foods and may be included
in the formulation of healthier foods, such as in the case of meat products

Introduction

152
 The meat and meat products are an important source of high quality protein, vitamins, and
many other micronutrients. Consumption of meat, particularly red meat (beef, pork, and lamb), is
dated back to antiquity and remains to be a dominant lifestyle and usually a nutritionally
indispensable form of life in the modern society (Jiang & Xiong, 2016). According to FAO is
expected that meat consumption to double by the year 2050, which represent a challenge for meat
industry (FAO, 2016). In spite of growing projected of this industry, in the last decade, meat
products have been submitted to scrutiny because diverse epidemiological studies, as well as
experimental data suggest that high intake of high-fat red meat and processed meat, contribute to
development of cardiovascular diseases (CVD), diabetes, colon cancer and other degenerative
diseases (J Kanner, Selhub, Shpaizer, Rabkin, Shacham, & Tirosh, 2017).

Particularly, have been described that secondary products from lipid oxidation such as
malondialdehyde are the causative agents of affections above mentioned. The oxidation is an
irreversible and complex process where all nutrients and other bioactive molecules are impacted.
They occurrence and proportion of the LOX in food system are determinate by many factors, for
the example, composition of food, time and type of storage, process and culinary preparation
(Estévez, 2017). In addition, the subsequent digestion of the food, during which the food matrix is
exposed to different compartments of the gastrointestinal system each one with its own specific
environment and impact on the oxidative process (Joseph Kanner & Lapidot, 2001).

Has been observed that consumption of a meal rich in oxidizable fat together with a rich
source of antioxidants, such as procyanidins reduces the absorption of lipid hydroperoxides and
their secondary lipid peroxidation products as consequence of antioxidant activity of this molecules
(Tagliazucchi, Verzelloni, Bertolini, & Conte, 2010).

The digestion model has been proposed to approaching to know the events that happen after
the ingestion of specific food and interaction between food constituents (Minekus, Alminger,
Alvito, Ballance, Bohn, Bourlieu, et al., 2014; Parada & Aguilera, 2007). For the above, it is
essential know the balance between pro and antioxidant reaction specify when are used of
antioxidant rich ingredients. For the above, the present research was conducted to assess the

153
bioaccessibility of date seed (Phoenix dactylifera L.) extract added to pork patties and lipid
peroxidation during in vitro gastrointestinal digestion (GID).

Materials and methods

Plant material

The date seed Medjool variety were selected of 50 Kg of no commercial fruit and collected in
Tamar stage. Next, were washed with tap water to eliminate any adhered fruit, dry at room
temperature during 48 h and after put on oven at 50 °C during 24 h. The date seeds were milled
and sieving using a 1-2mm mesh (Thomas Wiley, model 4), and the resulting powder was kept in
storage at -20°C.

Manufacture of patties

The meat and porcine back-fat was purchased in a local marked. The meat was freed from
visible fat, whereas the back fat was cleaned and freed from the skin. Two batches were prepared
1) control and 2) DSE 2%. Patties (90 g) where then prepared using a manual burger former six
per treatment. Patties were cooked 150 ± 5 °C in a contact grill up to reach 70 °C (centre of
product).

Phytochemicals extraction from patties

The extraction was realized using two methods, 1) Ultrasound assisted extraction and 2) alkaline-
acid extraction. For 1): undigested sample (3 g) were mixed with 30 mL of methanol-water (80:20,
v/v) during 60 min, then, the sample was centrifuged 10 min, 8000 g at 4°C. The supernatant was
collected, and the pellet was mixed with 30 ml of acetone-water (70:30, v/v) and the same step was
repeated again. The supernatants were combined and vapored to dryness in a rotatory evaporator
with reduced pressure (40 °C). Five millilitres of methanol were added to residue, and the
methanolic extract was filtered with a 0.45 µm filter and stored at -20 °C until further analysis. For
2): was followed the described method by Hirawan, Ser, Arntfield, and Beta (2010).

154
Simulated gastrointestinal digestion in vitro

The simulated gastrointestinal digestion was realised according to stablished by (Minekus, et al.,
2014). The method consists of three sequential steps: an initial α-amylase 2 min at 37 °C to
simulated mouth conditions followed by pepsin/HCl for 2 h at 37 °C and finally to simulate small
intestine conditions bile salt/pancreatin for 2 h at 37 °C. The simulated digestion fluids: Simulated
Salivary Fluid (SSF), Simulated Gastric Fluid (SGF), and Simulated Intestinal Fluid (SIF) were
prepared following to Minekus, et al. (2014). After simulated gastrointestinal digestion in vitro
process, each fraction was centrifuged 12 min to 8000 g, 4 °C, the supernatant was frozen at -80
°C and subsequently lyophilized for further analysis.

Measurement of individual phenolic compounds

The profile of polyphenols in each sample obtained in each phase of digestion was evaluated using
high performance liquid chromatography according to methodology described by (Lucas-
Gonzalez, Navarro-Coves, Pérez-Álvarez, Fernández-López, Muñoz, & Viuda-Martos, 2016). The
polyphenols compounds were identified by comparing UV absorption spectra and retention time
of each compound with those of pure standard injected in the same conditions.

Phytochemicals and antioxidant activity

The total phenols concentration of the sample before and after each phase gastrointestinal digestion,
were evaluated according to Singleton and Rossi (1965) using as oxidizing agent the Folin-
Ciocalteu reagent and gallic acid (GA) was the reference standard and the results were expressed
as mg GA equivalents/g sample. Whereas total flavonoids content was based in the methodology
of Blasa, Candiracci, Accorsi, Piacentini, Albertini, and Piatti (2006). The assays were realised in
methanolic solutions of lyophilized samples. For quantification, different concentrations of rutin
(8.5-170 µg/mL) were used for calibration curve. The results were expressed in mg rutin
equivalents (RE)/g of sample.

DPPH and ABTS assay

155
The scavenging radical activity DPPH of the samples before and after each phase gastrointestinal
digestion, were evaluated according to Brand-Williams, Cuvelier, and Berset (1995), this method
is based on discoloration of radical when is put in contact with a reducing agent. The absorbance
of the reaction was measured by spectrophotometry at 517 nm. The results were expressed as µg
of Trolox equivalents (TE) per g of sample.

The ABTS assay was determinate following to Re, Pellegrini, Proteggente, Pannala, Yang, and
Rice-Evans (1999) with slight modifications. ABTS radical cation (ABTS•+) was produced by
reacting ABTS stock solution 7 mM with 2.45 mM potassium persulfate and allowing the mixture
to stand in dark at room temperature for 12-16 h before of use. The solution of ABTS•+ was
dissolving to reach 0.70 (± 0.02) of absorbance at 734 nm. The reaction was done added 990 µL of
ABTS solution to 10 µL of each extract. The lecture of absorbance was take exactly 5 min after of
the mix between radical and sample. The results were expressed as mM of Trolox equivalents (TE)
per g of sample.

Malondialdehyde in gastric and intestinal fluid

The gastric MDA was carry out following to J Kanner, Selhub, Shpaizer, Rabkin, Shacham, and
Tirosh (2017), and was adapted to (Minekus, et al., 2014) protocol, this allowed evaluate not only
MDA in gastric phase but also in intestinal phase, which was done in order to know the
concentration of MDA that could be absorbed. In each phase evaluated the sample was divided
into several tubes for evaluation at different times (0, 30, 60, 120 min), in a shake and temperature
constant (37 °C, 80 rpm). The quantification of MDA was determined according to established by
Pfalzgraf, Frigg, and Steinhart (1995).

Statistical analysis

The analysis of data consisted of a lineal model GLM-ANOVA of two ways, using the Number
Cruncher Statistical System 2011 (NCSS, Kaysville, UT, USA) software (Hintze, 2007).
Significant differences were estimated at a probably of p<0.05, when significant differences were
found between treatments Tukey Kramer test was performed.

156
3 Results and discussion

3.1 Polyphenolic compounds in date seed extract

The HPLC analysis evidenced a total of seven phenolic compounds on date seed extract, six
phenolic acids (ferulic, cafeic, clorogenic, sinapic and protocateic) and one flavan 3-ol flavonoid
(catechin). The principal components were catequin, ferulic, and protocatehuic (982.69, 434.7
248.93 µg/g of extract respectively). These results are in agreement to others authors (Al-Farsi &
Lee, 2008; Habib, Platat, Meudec, Cheynier, & Ibrahim, 2014; Sirisena, Zabaras, Ng, & Ajlouni,
2017) however, the presence of these compounds are obtained using acetone:water as solvent. In
spite of, acetone is an unrestricted solvent permitted for use in the preparation of food ingredients
in the European Union, in applying the principles of green chemistry clearly ethanol (used as
solvent in the present study) represent one of the safer solvents and the use of a renewable feedstock
as well as having the widest acceptability (Marriott, 2010).

Bioaccessibility of phenols during simulated in vitro digestion

According to other studies, bioactive polyphenols can be release of food microstructure to exert
biological effect in the human health. In this sense, bioaccessibility is defined as the amount of an
ingested nutrient that is available for absorption in the gut after digestion (Hedrén, Mulokozi, &
Svanberg, 2002; Parada & Aguilera, 2007). In the present study after in vitro GID, were found
phenolic acids as chlorogenic, coumaric and ferulic in oral, gastric and intestinal phases and
catequina only in intestinal phase (113.27 µg/g of sample), whereas that, control treatment was not
found mentioned compounds, as show in Table 2.

Cocoa is rich in polyphenols, particularly catechins (flavan-3-ols) and procyanidins. In a study

directed by Serafini, Bugianesi, Maiani, Valtuena, De Santis, and Crozier (2003) reported that
absorption of chocolate flavonoids into the bloodstream was 69% less in milk chocolate compared
to absorption from dark chocolate alone, insinuating that these compounds interacted with milk
proteins during manufacturing or in the gut. However Keen and others (2005) suggested that
differences in flavonol activity and its bioavailability between dark and milk chocolate were the

157
results of the food matrix altering the kinetics of absorption rather than due to flavonol-milk protein
interaction. In we research the presence of catechin only on intestinal phase can be due to
interaction whit other complex molecules as lipids and proteins.

Evidently, in the present study, the concentration of polyphenols in the DSE was higher to found
after in vitro GID. Is known that catechin is susceptible to deterioration under digestive conditions
and present low stability in the fluid intestinal simulated (pH> 7) (Aditya, Aditya, Yang, Kim,
Park, & Ko, 2015; Ortega, Reguant, Romero, Macià, & Motilva, 2009). According to Jakobek
(2015) lipids interact with polyphenols, they capture polyphenols and protect them in their passage
through the gastrointestinal tract after digestive enzymes (lipase and proteases) acts, it facilitates
exposure in this phase of the GID. Food lipid rich could be protector effect probably related with
a better micellization to favour stability of the polyphenols during digestion. Meat and meat
products can be vehicles of extracts with catechins like the DSE that protect them, on the one hand,
during the shelf life and later they can exert their biological action once they are consumed.

Phytochemicals and antioxidant activity during in vitro digestion

The total phenols content after in vitro GID of patties is presented in the Table 3, in oral, gastric
and intestinal phases its quantification was possible, and however the greatest amount was
presented in the gastric phase, both for control and DSE treatment. Other studies suggested that
Folin-Ciocalteu used as reducing agent for quantification of phenols, also react with aromatic
amino acids presents in the pork meat, although the assess is quick, it is a nonspecific trail, so
results like these can be observed (Singleton & Rossi, 1965). In regard to, total flavonoids content
(Table 3), the mayor proportion was presented in DSE treatment. During in vitro GID an increased
was found as follow: oral (1.80 ± 0.11), gastric (3.70 ± 0.25) and intestinal (6.14 ± 0.76). These
may be related to catequina content in extract. In spite of, great susceptibility of catechin mentioned
in the scientific literature, in this research using patties as model, can be established that
phytochemical compounds present in DSE are bioaccessible.

In the present study, the antioxidant assay is based on different mechanism that measure the
antioxidative effect of phenolic compounds. DPPH assay is based on the ability of antioxidant to

158
act as radical scavengers and ABTS is based on the interaction with hydrogen or electron donor
species. The results of antioxidant activity are shown in Table 3, in the intestinal phase differences
(p>0.05) was found between treatments, DSE have the highest values different to the control.
Which can be attributed to the OH groups present in phytochemicals compounds such phenolic
acids and catechin that exert a potent antioxidant activity. Besides, have been demonstrate that
alkaline pH increases free radical scavenging activity of phenol compounds. The change in pH
from an acidic to an alkaline medium favours the deprotonation of the hydroxyls presents in the
aromatic rings of said compounds, resulting in the alteration of their chemical structure, which
impact in different bioavailability and biological activity (Bermúdez-Soto, Tomás-Barberán, &
García-Conesa, 2007).

The results obtained in this work for ABTS assay are presented in Table 2, intestinal phase showed
a higher (p < 0.05) antioxidant activity, which agrees with (Pellegrini, Lucas-Gonzalez, Sayas-
Barberá, Fernández-López, Pérez-Álvarez, & Viuda-Martos, 2018) for chia and defatted chia seed.
This behaviour after the intestinal phase was hypothetically related to the release from the matrix
of unextractable bioactive compounds and/or their chemical transformation into compounds with
a greater antioxidant activity. In this step could be due to the hydrolysis under digestion conditions
of the glycoside forms yielding the aglycone forms (Ortega, Reguant, Romero, Macià, & Motilva,
2009).

Gastric and intestinal lipid peroxidation

The lipid peroxidation determinate by MDA accumulation in gastric and intestinal phase only was
quantified in control treatment 4.09 y 4.60 g/Kg of pattie in gastric and intestinal respectively at
120 min, 37 °C, whilst with the addition of DSE (2%) not was quantified MDA. According to
others studies the acidity of the gastric flow increase the lipid peroxidation catalysed by
metamyoglobin or iron ions, which are presents in meat matrices (Guéraud, Taché, Steghens,
Milkovic, Borovic-Sunjic, Zarkovic, et al., 2015; Joseph Kanner & Lapidot, 2001; Marnett, 1999).
In the case of, intestinal phase others components as bile also will play an important role due to
phospholipids and cholesterol composition, which can also be oxidized and contribute to MDA
quantity quantified in this phase of the digestion (Steppeler, Haugen, Rødbotten, & Kirkhus, 2016).

159
In same way Hur, Lee, and Lee (2015) found that after in vitro human digestion the lipid oxidation
trend to exponentially increases because chemical composition of fatty acids of meat that prone to
oxidation in addition to digestive enzymes and pH conditions in each compartment during
digestion. In the other hand, the inhibition of lipid oxidation in patties added whit DSE, can be
attributed to diversity of polyphenols compounds presents in DSE, particularly to catechin that
represent one the most important. Joseph Kanner and Lapidot (2001) evaluated the polyphenols
dietary capacity to inverter catalysis of prooxidation and antioxidation and found the catechin and
polyphenols presents in red wine can inhibit in totality lipid peroxidation and co-oxidation on β-
carotene. Additionally, Tagliazucchi, Verzelloni, Bertolini, and Conte (2010) investigates coffee
melanoidins on lipid oxidation during simulated gastric digestion of turkey meat and found that
3mg/ml reversed the reaction and broke down hydroperoxides to concentration lower than initial
value provided antioxidant activity and protect during gastrointestinal digestion against lipid
oxidation.

Date seed have been used to make traditional herbal coffees in turkey and some Arabic countries
with many protective effects (Sirisena, Ng, & Ajlouni, 2015). Moreover, Habib and Ibrahim (2011)
observed significantly lower levels of malondialdehyde formation in the liver and serum of rats fed
date seed power compared with the control group. The same authors found reduced lactate
dehydrogenase (LDH) and creatine Kinase (CK) enzyme levels in serum, which indicated a
possible effect of date seed against cellular damage. For the above, we can infer that the addition
of DSE in pork patties could inhibit toxic compounds as MDA and increased the protection against
oxidation and provided the phytochemicals compounds to muscle foods and consumer. However,
it would be worthwhile investigating, with base on previous studies.

Conclusions

The DSE (2%) provides phytochemicals compounds to pork patties such as hydroxycinnamic acids
and flavonoles that confer antioxidant protection against lipid oxidation during gastrointestinal
digestion simulated in vitro. Which was evidenced when MDA was inhibited simulated the gastric
and intestinal fluids in patties with DSE. For the above, is necessary explore the mechanisms
involved in the protection that the extract exert in other meat matrices whit high susceptibility to

160
oxidation and ability to inhibit others cytotoxic molecules involved in oxidative damage associated
to ingested of meat and meat products.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We thank CONACyT (México) for support with a Ph. D. fellowship to the first author.

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 Tables and figures

Table 1. Identification and quantification by HPLC-DAD of phytochemical compounds from date

seed extracts.

Table 2. Phytochemical content of pork patties added with date seed extract before in vitro
gastrointestinal digestion.

165
Table 3. Phytochemical content in each phase after in vitro digestion process of pork patties with
date seed extract.

166
Table 4. Phytochemical content and antioxidant activity of each phase during in vitro digestion
process of pork patties with date seed extract.

167
 6 Section a, 325

4 5

2
1

Section b. 325

4 5
3 6
1 2

Figure 1. Chromatograms at 325 nm of date seed extract (section a) and intestinal phase of In vitro
digestion process of pork patties added with date seed extract (section b). Peak identifications are
given in table 1 and 3.

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 b

b b b
a

a a
a

Figure 2. Lipid peroxidation during In vitro digestion process of pork patties added with date seed
extract.

169
8. Assessment of Lipid Oxidation Inhibition, Total Antioxidant Activity, Color and Sensory
Attributes of Pork Patties Incorporate with Date Byproduct During Refrigerated Storage

Ma. Ángeles de la Rosa-Alcaraza, Gastón Ramón Torrescano-Urrutiaa, Juana Fernández Lópezb,

José Ángel Pérez Álvarezb, and Armida Sánchez Escalantea*

Food Chemistry

(Revisión Interna)

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 Assessment of lipid oxidation inhibition, total antioxidant activity, color and sensory
attributes of pork patties incorporate with date byproduct during refrigerated storage

Ma. Ángeles de la Rosa-Alcaraza, Gastón Ramón Torrescano-Urrutiaa, Juana Fernández Lópezb,

José Ángel Pérez Álvarezb, Armida Sánchez Escalantea*
a
Centro de Investigación en Alimentación y Desarrollo, A.C. Coordinación de Tecnología de
Alimentos de Origen Animal. Ctra. Gustavo Enrique Astiazarán Rosas No. 46, Col. La Victoria.
Hermosillo, Sonora, 83304, México.
b
Universidad Miguel Hernández, Escuela Politécnica Superior de Orihuela, Departamento de
Tecnología Agroalimentaria, Ctra. A Beniel Km 3.2. Orihuela, Alicante 03312, España.

*Corresponding author, E mail: armida-sanchez@ciad.mx; Telephone number +52 (662)

2892400; Fax number +52 (662) 2800421

Highlights

• The addition of date seed extract delay lipid oxidation in pork patties.
• Use of date seed extract contributes to phytochemicals content of pork patties.
• Sensorial attributes of pork patties are not affected with date seed inclusion.
• Date seed extract may be a sustainable and cheap antioxidant additive.

ABSTRACT

Lipid oxidation produces nutritional, safety and sensorial deterioration in meat products.
The incorporation of date seed extract (DSE) on lipid oxidation, antioxidant activity, and sensorial
properties was evaluated in order to know their potential use as natural antioxidant in pork patties
during refrigerated storage. The effectiveness of DSE on delay of lipid oxidation was demonstrated,
decreased more than 50% compared to control, over nine days. Total phenols and flavonoids
increased more than two folds; total antioxidant activity increased in modified patties respect to
control. Instrumental color measured by the CIE L*a*b* system showed significant difference by
DSE inclusion, principally high a* value, in relation to the control. Sensorial acceptability was
confirmed by hedonic test, resulted in positive scores. However, extract has no antimicrobial
activity. The DSE can be used as antioxidant additive or phytochemical-rich ingredient to improve
safety and nutritional of pork patties.

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Keywords: date seed extract, byproducts, pork patties, lipid oxidation, antioxidant, sensorial
properties.

1. Introduction

Food and oxygen the two fundamentals of human life. Their interaction during the production,
processing, distribution and storage preceding actual consumption, food undergoes various modes
of deterioration that involve biological changes by microbes as well chemical changes When
absorbed, these compounds may participate in vivo oxidative stress, inflammation, and various
other pathological conditions (Namiki, 1990).

Deterioration of foods by oxidation is an irreversible and complex process, during which nutrients
and another bioactive molecule are impacted. Particularly, in meat products, sensorial attributes as
color (cooxidation of pigments), odor (volatile compounds) and texture (protein oxidation) are
affected by oxidation of principal components like lipids and proteins. In the same way, nutritional
quality and safety are affected negatively by oxidation due to losses in antioxidant vitamins,
polyunsaturated fatty acids (PUFAs), or essential amino acids. Which impact safety, and economic
value of meat products (Estevez, 2017).

Generally, in industrial processing, four commonly used synthetic antioxidant butylated

hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), propyl gallate (PG) and tributyl
hydroquinone (TBHQ), are the most widely used, due to stability, low cost, and other practical
advantages. However, their use may constitute a potential health hazard for consumer. For the
above interest in natural antioxidants and search on naturally occurring compounds with
antioxidant activity has increased dramatically (Moure, 2000).

The use of natural antioxidants from plants and byproducts as peel, seed, and pulp, represent a
viable option to recovery functional molecules that contributed to delay or inhibit oxidation, due
to ability as radical scavengers work either as hydrogen donors or as electron donors. Considering
the enormous quantity or agro-industrial byproducts produced around the world through supply
chain of agro-foods (Gustavsson, Cederberg, Sonesson, Van Otterdijk, & Meybeck, 2011) and that
disposition represent contamination focus due to richness in water and carbohydrates, as well as
loss of opportunity to obtain economic benefits since they do not use integrally.
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The recovery of antioxidants from byproducts and their inclusion on complex matrices such as
meat represents an ideal alternative to improve quality of traditional meat products. However, is
important to consider the implications that have in sensorial attributes due to their pigment contain,
such as the case of date seed, which can influence color or flavor, and impact on the purchase
decision of consumer, therefore concentration selected is imperative. In spite of, date seed extracts
in previous studies have demonstrated high antioxidant power used as powder or infusion, positive
effect has been found to treat lung diseases, diabetes, atherosclerosis, delay lipid peroxidation,
cancer using in vitro and in vivo models, up to knowledge in meat has not been evaluated yet. For
the above, hypotheses, the inclusion of elevated quantities of date seed extract in pork patties can
have prooxidant and affect sensorial attributes of products, as well as, physicochemical properties
through storage time.

2. Materials and Methods

2.1 Materials

Chemicals: 2-Thiobarbituric acid as obtained from J.T. Baker, 1,1,3,3-Tetramethoxypropane, Folin

ciocalteu, gallic acid, rutin hydrate, 1,1-Diphenyl-2-picrylhydrazyl radical, (±)-6-Hydroxy-2,5,7,8-
tetramethylchromane-2-carboxylic acid,were purchased from Sigma-Aldrich (Mexico). For the
pork patties, fresh lean meat and pork back fat was obtained from local meat market.

2.2 Date seed extract manufacture

Date seed powder was extracted in relation 1:1 with 50% aqueous ethanol using ultrasound-assisted
extraction (EAU) during 60 min at 40 kHz and 25 °C ± 5 (Ultrasound Bath Branson, Model 1800,
Series CPXH. The homogenized was centrifuged (10, 000 rpm, 15 min, 4 °C), filtrated through
Whatman 1, and concentrated using rotatory evaporator to dryness (60 °C) (RE301, Yamato,
Japan). Finally, the dry extract was lyophilized and stored in dark conditions at -20 °C.

2.3 Incorporation of date seed extract into pork patties

2.3.1 Pork patties manufacture

Three different formulations of pork patties were manufacture, each formulation was carried out
twice, each time on a different day (3 kg batch per formulation were prepared each day). For the
control formulation, fresh lean minced meat (80%) and minced pork back fat (20%) were used. In
the modified formulation DSE 0.2% and DSE 2% were added. In addition, both formulations also
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included the following common ingredients per kilogram of minced pork meat: 15 g salt, black
pepper, white pepper, garlic powder, water.

2.4 Proximal composition of pork patties

Proximate composition analyses were done on each replicate and formulation, with three
measurements per sample. Quantification of moisture, protein, ash, and fat was performed
following the official methods (AOAC, 2002).

2.5 Physicochemical properties

The pH of samples was measured in a suspension resulting from blending sample with water, 1:9
ratio during 30 s, using a pH meter (Hanna Instruments). All samples were assessed for triplicate.
The color was performed in surface of raw pork patties using a Minolta colorimeter CR-300
(Minolta Camera Corp., Metar division, USA). Before each measuring session the colorimeter was
calibrated on the color space system, CIE Commission Intl. of I´Eclairage (1978) using a white
tile. The following coordinates were determinate: L* value indicates lightness; a* value indicates
redness and b * value indicates yellowness. Color measurements were made on the surface of each
patty in triplicate at three randomly selected locations. Color measurements were made at room
temperature with illuminant D65 and 0° angle observer.

2.6 Lipid oxidation (TBARS)

In order to assess the oxidation status of the pork patties, malondialdehyde content was determinate.
The evaluation was assessed in duplicate by the 2-thiobarbituric acid (TBA) method of Pfalzgraf,
Frigg, & Steinhart, (1995), using 10 g of sample. TBARS value was calculated from a standard
curve of malondialdehyde (MDA) and expressed as mg of MDA per Kg-1 meat.

2.7 Microbial analysis

For each patty (raw) 10 g was obtained and located in a sterile plastic bag with 90 ml of peptone
water (0.1%) and 0.85% NaCl. After serial 10-fold dilution was prepared by diluting 1 ml in 9 ml
of peptone and appropriate decimal dilution were pour plate on plate count agar (PCA) for total
viable count (TVC) (37 °C for 48 h) and for psychrotrophic bacteria (7 °C, 10 days). The results
were expressed as logarithms of colony-forming units per gram (Log CFU/g) (Pierson, Zink,
&Steinhart, 1995).

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2.8 Extraction of phytochemicals of pork patties

The extraction was realized using Ultrasound-Assisted Extraction (UAE), sample (3 g) was mixed
with 30 mL of methanol-water (80:20, v/v) during 60 min, then, the sample was centrifuged 10
min, 8000 g at 4°C. The supernatant was collected and the pellet was mixed with 30 ml of acetone-
water (70:30, v/v) and the same step was repeated again. The supernatants were combined and
vapored to dryness in a rotatory evaporator with reduced pressure (40 °C). Five milliliters of
methanol were added to residue, and the methanol extract was filtered with a 0.45 µm filter and
stored at -20 °C until further analysis. For 2): was followed the described method by Hirawan, Ser,
Arntfield, & Beta, 2010.

2.9 Determination of phytochemicals and antioxidant activity in pork patties extracts

2.9.1 Total phenols and flavonoids

The total phenols content (TPC) in pork patties extracts was evaluated according to Singleton &
Rossi (1965), using Folin-Ciocalteau as an oxidizing agent. The extracts (10 µL) were mixed with
40 µL of reagent, 160 µL of distilled water; immediately 60 µL of sodium carbonate (5%) were
added to the mix. After 60 min of repose at room temperature, the absorbance was measure at 750
TM
nm using spectrophotometer UV/VIS Multiskan GO (Thermo Scientific). Total phenolic
compounds were calculated using a standard curve of gallic acid (considering six different
concentrations, 0-1 mg/mL), the results were expressed as mg of gallic acid equivalents (GAE)/ g
of sample.

Total flavonoids content (TFlvC) were determined following the established by Zhishen,
Mengcheng, & Jianming (1999), with some modifications. The extract (100 µL) was mixed with
430 µL of NaNO2 (5%), and kept at rest for 5 min. Next, 30 µL of AlCl3 (10%) was added and
remain at rest for one minute to carry out the reaction, finally, 440 µL of NaOH (1 M) was added.
The previous reaction mixture was taken 150 µL in triplicate and the absorbance was measured at
TM
510 nm using spectrophotometer UV/VIS Multiskan GO (Thermo Scientific). The results of
TFlv were expressed as milligrams of rutin equivalents (RE)/g of sample.

2.9.2 DPPH

The antiradical activity of the samples was based on the scavenging activity of stable radical 1,1´-
diphenyl-2-picrylhydrazyl (DPPH). The analysis was realized using 100 µL of extract and mixing
175
100 µL of radical DPPH 300 µM in ethanol. The reaction mixture was stirred and allowed to stand
at room temperature in the dark for 30 min and the absorbance was immediately recorded at 517
nm. The percent inhibition was calculated as follows: [(A0-A1)/A0] x 100, where A0 corresponding
to control absorbance, and A1 to extract absorbance (Brand-Williams, Cuvelier, & Berset, 1995).

2.9.3 Reducing power

The reducing power was evaluated mixing 200 µL of extract, 500 µL of phosphate buffer 50 mM
(pH 6.6) and 500 µL of potassium ferricyanide (1%). The mixture was incubated at 50 °C for 20
min; next was added 500 µL of trichloroacetic acid (10%). The mixture was centrifuged at 2800
XG for 10 min; the supernatant was taken 500 µL and mixed with 500 µL of Milli-Q water and
100 µL de FeCl3 (0.1 %). Finally, was taken 100 µL of the mixture and absorbance at 700 nm was
measured. The presence of reducing agents triggers the reduction of complex Fe3+/ferricyanide to
ferrous form, which is measured for the average of the formation of color Prussia blue pearls (Perl´s
Prussian). The results are expressed as TE/ g of sample (Kumaran & Karunakaran, 2007).

2.10 Sensorial analysis

A hedonic test was performed in order to evaluate the overall acceptability of the raw and cooked
patties. 50 no-trained panelist participated in this study, which was carried out into session. For the
evaluation, each panelist scored three samples, control, DSE 0.2%, DSE 2%. The score ranged
from 0 to 10 (0 dislikes extremely and 10 like extremely). Each point marked was converted to
numerical value (1 to 10) for future statistical analyses of data.

2.11 Statistical analysis

Data obtained for color, TBARS, phytochemical and antioxidants were analyzed by mean of two-
ways analysis of variance test. Whilst, proximate composition, and sensorial analysis were
evaluated using one-way analysis of variance (ANOVA) in order to evaluate differences among
for formulation (p< 0.05). All statistical analyses were performed using NCSS software (NCSS,
2011). All assessment of the experiment was carried out in duplicate. All test were considered
statistically significant at a probability level of 0.05, and significant differences were identified
with Tukey Kramer range test.

3. Results and discussion

3.1 Proximate composition
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The proximal composition of pork patties added with date seed extract is given in Table 1. As
expected, the addition of different concentrations of extract does not affect the chemical
composition. The chemical composition of the patties of the present study is different other studies
(Alejandre, Ansorena, Calvo, Cavero, & Astiasaran, 2019; Rodriguez-Carpena, Morcuende, &
Estevez, 2011), which could be related with the difference in formulation.

3.2 Physicochemical

3.2.1 pH

The pH values of pork patties added with date seed extract are presented in Table 2. Initially, the
values of 0.2 and 2% of date seed extract treatments surpass the range suggested for pork muscle
postmortem (5.8-5.4). However, from day 3 to 9, all values stabilized. This property plays an
important role in food quality, for example, meat color and development microorganisms, or
oxidation reaction depend on this physical-chemical property in foods (Musavu Ndob & Lebert,
2018). In our study, values were adequate by this product type.

3.2.2 Color

The instrumental color values of pork patties added with date seed extract through storage are given
in Table 2. The lowest L* value presented the treatment 2% however 0.2% and control, presented
similar values. In the same way, the a* value was affected by date seed extract addition, specify
when 2% was added, both results L* and a* can be related to natural pigment (brown in color) of
extract that impact in patties color. For the above, different study indicate that when selecting a
natural antioxidant as an ingredient of food formulation, the concentration should be considered to
achieve desired traits due to theme can impact in flavor and color of food. These attributes are
determinants of whether a consumer will purchase a specific type of meat or not (Barriuso,
Ansorena, Calvo, Cavero, & Astiasarán, 2015; Karre, Lopez & Getty, 2013). The b* values
oscillated between 13.89 to 17.16 through storage time, which are similar to reported by Pateiro et
al., (2018) in pork patties added with guarana seed extract.

3.2 Lipid oxidation (TBARS)

The lipid peroxidation expressed as malondialdehyde (MDA) in pork patties through storage is
presented in Figure 1. The results when 0.2 and 2% of the extract were added is overwhelming.
The addition of date seed extract delay lipid oxidation compared with the control samples, in the
177
which increased with storage time. The values for control at 9 days were 1.24 followed 0.39, and
0.25 in 0.2% and 2% respectively. Respect to percent inhibition at 9 days 0.2 % and 2% inhibited
70.89% and 81.34 respectively. The high content of antioxidant molecules as phenols in date seed
could be related to oxidative stability of pork patties during storage. Other authors have described
that natural extract had more antioxidant power in real meat products than synthetic antioxidants,
thus suggesting the possibility of using these extracts as replacers of commercial additives
(Fernandes et al., 2017; Zhang, Wu, & Guo, 2016; Radha Krishnan et al., 2014). The above
represents an opportunity to include natural extracts in meat matrix contribute to delay lipid
oxidation which represents serious problems in meta quality parameters as color, flavor, odor,
texture and even nutritional value (Estevez, 2017). Finally, in order to verify that carbohydrates,
which are the preponderant components in the extract, did not mask the MDA value, a spectral
sweep was performed, results revealed that carbohydrates not affect this value as shown in Figure
2.

3.3 Microbial quality

Total viable count (TVC) and psychotropic microorganism were affected by formulation through
storage time. Initially, all microbial counts were presented below 7 Log UFC/g of sample as shown
in Figure 7., however at finally of storage (9 days) all treatment exceed established value by
International Commission on Microbiological Specifications for Foods (ICMSF). In the same way,
in other studies when natural compounds are added in meat products, antioxidant activity has been
found but, not antimicrobial delay (de Ameida et al., 2015; Freire et al., 2017). These results
confirmed that natural antioxidant differs in vitro assessment and when they are tested in meat or
other real models (Pateiro et al., 2018). In spite of, date seed extract is rich in phytochemicals such
as flavonoids which have shown antibacterial activity, through inhibition virulence factors,
quorum-sensing signal receptors, enzymes, and toxins (Nikmaram et al., 2015), in this study extract
was not effective to delay TVC and psychrotrophic microorganism but not potentiate growth.

3.4 Phytochemicals content and antioxidant activity of extract of pork patties

3.4.1 Total phenols and flavonoids

The total phenols in pork patties during storage, are presented in Figure 3. Clearly, samples of pork
patties with 2% of date seed extract presented the high content different to control and 0.2%

178
treatments at each period of storage. The presence of phenolic compound in control samples may
be attributed to the phenolic structures of aromatic amino acids. In case of treatments with 0.2 and
2.0% of extract, is known that date seed extract is a good source of phenolic acids of
hydroxybenzoic and hydroxycinnamic group such as caffeic acid, gallic acid, coumaric acid,
sinapic acid, vanillic acid, chlorogenic acid, protocatechuic acid. The results in our study, are in
according with reported by Ergezer & Serderoglu (2018), where artichoke (Cynarascalymus L.)
byproducts extract was added to patties.

In respect to total flavonoids, like total phenols, the highest values were found in 2% samples and
were stable during the storage. Thus, we can infer that these compounds could be related to stability
oxidative of pork patties during storage, however, is necessary know the mechanism involved.
Catechin is a majority flavonoid in date seed extract, include several isomers (epicatechin,
epicatechingallate, epigallocatechingallate, and epigallocatechin), and has strong antioxidant
properties that have been attributed to several mechanisms, such as interaction with free radicals,
prevention of radical chain initiation, and metal chelation (Kaewprache et al., 2018). Dietary intake
of flavonoids rich foods has been connected with reduced risks of some cancers (lung, colorectal,
and gastric) and also with low incidences of coronary heart diseases (Kim et al., 2013). The above,
open the door to study if the inclusion of natural extracts with flavonoids compound in meat as
date seed extract also can contribute to better health status when the product is consumed.

3.4.2 DPPH

The results of scavenging activity in pork patties with extracts through storage are given in Figure
5. The 2.0% samples exhibited the strongest scavenging activity against DPPH radical for 9 days.
The results presented in this study are similar with reported on other studies where natural extracts
showed strong radical scavenging activity in diverse muscle foods, beef patties with guarana extract
(Ergezer & Serderoglu, 2018), goat meat patties with pomegranate seed powder extract (Devatkal,
Narsaiah, & Borah, 2010), and grape seed extract in raw and cooked muscle foods (Hygreeva,
Pandey, & Radhakrishna, 2014) broccoli powder extract goat meat nuggets (Banerjee et al., 2012).

3.4.3 Reducing power

The reducing power of pork patties extracts during storage time are shown in Figure 6. The values
are in corresponding with total phenols and flavonoids and DPPH inhibition. The reducing power

179
in samples with 2% of date seed extract evidenced the higher concentration (expressed as µg of
Trolox) during the storage. Reducing ability of polyphenols seems to be an important factor
dictating the antioxidant and free radical-scavenging capacity of these compounds When
polyphenols (phenolic acids and flavonoids) are presented in foods naturally or as a food
ingredient, an increased reducing ability with time might signify a longer protecting effect
scavenging of free radicals of polyphenols against oxidative damage of the food material such lipid
oxidation (Pulido, Bravo, & Saura-Calixto, 2000). Because natural extracts are not composed of a
single molecule, in particular, the synergistic effect of all compound potentiates the reducing
power, this property could be taken as an advantage of these new natural compounds.

3.5 Sensory attributes

The sensorial attributes and purchase intention of raw and cooked pork patties added with date seed
extract are shown in Table 3 and Figure 9. The incorporation of plant extracts at unsuitable levels
may result in unpleasant sensory characteristics of meat products as patties. Therefore, control pork
patties and modified product were subjects to sensory evaluation. In raw product, color, odor, and
appearance attributes were evaluated, whilst in cooked product odor, taste, and juiciness, all by
means of a hedonic test. These attributes are determinants of whether a consumer will purchase a
specific type of meat or not. In our study, the score for all attributes of raw patties oscillated 6-7,
and 7-8 for cooked patties overpass to obtained by Alejandre, Ansorena, Calvo, Cavero, &
Astiasaran, (2019) in beef patties with microalgal oil and blackthorn (Prunus spinosa L.) branch
extract, the authors indicate that “general acceptability” was by control patties 6.88, whereas
modified patties had 6.58 points and consider as a positive evaluation of the new product. In another
study, Freire at al., (2017) evaluate sensorial attributes of pork patties added with hydroxytyrosol
and fatty acids, attributes aroma, taste, color has 65.87% of correlation with overall acceptability.
In our study, the attributes oscillated between 7-8 in cooked patties and acceptability percent was
as following 87% control, 88 % 0.2% and 74% 2%. Values adequate for this type product. In spite
of date seed extract confer color to pork patties in raw product, the panelist evaluated positively in
cooked patties. And indicated that raw pork patties are similar to beef patties, therefore date seed
extract can be used also as natural color for design of new meat products.

4. CONCLUSION

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The DSE could be used as a powerful additive antioxidant and bioactive ingredient since contribute
to increased phytochemical content and delay lipid oxidation in pork patties. Due to natural
pigments of DSE content, this can be used as a colorant in meat products without altering sensorial
attributes and function as a new additive to design healthier muscle products aimed at specific niche
market. The integral use of agro-industrial byproducts is an emergent topic, by which is necessary
that academy, industry and productive sector work together, with the purpose of contribute to the
circular economy in agro-food sector.

Acknowledgment

The authors gratefully thank CONACyT (México) for support with a Ph. D. fellowship to the first
author.

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Declaration of interests

The authors declare that they have no conflict of interests.

184
FIGURE LEGENDS

Figure 1. Figure. Lipid peroxidation expressed as MDA in pork patties added with date seed extract
during refrigerated storage.

Figure 2. Spectral sweep of MDA and carbohydrates at nine days of storage of pork patties.

Figure 3. Total phenols of extract from pork patties added with lyophilized date seed extract during
refrigerated storage.

Figure 4. Total flavonoids of extract from pork patties added with lyophilized date seed extract
during refrigerated storage.

Figure 5. Scavenging activity of extract from pork patties added with lyophilized date seed extract
during refrigerated storage.

Figure 6. Reducing power of extract from pork patties added with lyophilized date seed extract
during refrigerated storage.

Figure 7. Total viable count of pork patties added with lyophilized date seed extract during
refrigerated storage.

Figure 8. Count of Psychotropic microorganism of pork patties added with lyophilized date seed
extract during refrigerated storage.

Figure 9. Purchase intention of pork patties added with date seed extract, expressed as percentage.

185
Table 1. Chemical composition of pork patties added with lyophilized date seed extract.

Treatment Moisture Fat Protein Ash

Control 59.13 ± 0.27a 19.31 ± 1.49a 23.16 ± 4.27a 0.58 ± 0.20a
0.20% 57.79 ± 0.47b 19.55 ± 1.14a 22.99 ± 3.45a 0.89 ± 0.10b
2% 59.01 ± 0.80a 18.78 ± 0.57a 23.48 ± 2.80a 0.60 ± 0.21a
Results are expressed main ± standard deviation (g/100g). Different letter in column indicate
significant differences between treatment.

186
Table 2. Comparison of pH and instrumental color of pork patties added with lyophilized date seed
extract during refrigerated storage.

Treatment
Parameter Time
control 0.2 % 2%

0 5.71 ± 0.22bX 6.00 ± 0.22abX 6.23 ± 0.33aX

3 5.69 ± 0.06aX 5.62 ± 0.02aY 5.71 ± 0.07aY

pH
6 5.56 ± 0.21aX 5.61 ± 0.26aY 5.58 ± 0.07aY

9 5.71 ± 0.20aX 5.71 ± 0.23aX 5.64 ± 0.08aY

L* 0 60.15 ± 3.40aX 60.59 ± 3.54aX 51.64 ±2.76bY

3 61.66 ± 4.78aY 59.62 ± 3.02aX 50.46 ± 2.25bY

6 59.70 ± 5.57aX 58.75 ± 3.26abX 53.33 ± 2.39cX

9 58.91 ± 3.76aX 59.43 ± 2.65aX 50.04 ± 3.07bY

a* 0 5.10 ± 0.50cX 8.01 ± 0.51bX 12.93 ± 1.32aX

3 4.98 ± 0.37bX 4.86 ± 0.37bY 11.80 ± 0.76aY

6 4.99 ± 0.56bX 5.55 ± 0.41bY 11.43 ± 0.91aY

9 4.58 ± 0.34bX 4.91 ± 0.49bY 11.24 ± 1.02aY

b* 0 16.27 ± 1.54abX 17.16 ± 1.77aX 15.47 ± 1.08bX

3 14.68 ± 1.48aX 14.66 ± 1.32aY 14.22 ± 0.62aY

6 14.65 ± 1.28aX 13.89 ± 1.16bY 14.87 ± 0.88aY

9 14.22 ± 1.41aX 14.58 ± 1.89aY 13.87 ± 1.05bZ

187
Results are expressed main ± standard deviation. Different letters (a-c) in same row indicate
differences (p<0.05) between treatments, while different letters (X-Z) in same column indicate
differences (p<0.05) between storage time.

188
Table 3. Sensorial attributes of raw and cooked pork patties added with lyophilized date seed extract.

Fresh product Cooked Product

Treatment
Color Odor Appearance Odor Taste Juiciness

Control 7.21 ± 2.01a 7.50 ± 2.17ª 7.16 ± 2.26ab 7.85 ± 2.07a 8.46 ± 1.78a 8.22 ± 1.92a

0.2 % 7.44 ± 1.84a 6.93 ± 2.03b 7.40 ± 2.03a 7.41 ± 2.05a 8.34 ± 1.68a 7.87 ± 2.04a

2% 6.48 ± 2.37b 6.79 ± 2.01b 6.71 ± 2.21b 7.54 ± 2.03a 7.84 ± 1.97b 7.21 ± 2.14b

Results are expressed main ± standard deviation. Different letters (a,b) in same column indicate differences (p<0.05) between treatments.

189
 2
1.8

TBARS (mg of MDA/Kg sample)

1.6
1.4
1.2
1 Control
0.8 0.2%
0.6 2%
0.4
0.2
0
0 3 6 9
Storage time

F1

0.45
0.40
0.35
0.30
Absorbance

0.25
Control
0.20
0.2%
0.15
2%
0.10
0.05
0.00
400 450 500 550 600
Wavelenght (nm)

F2

190
 120
c
100 a
c
mg GAE/g sample a
80

60 Control
b 0.2%
b b
40 b b
b b 2%
b
20

0
0 3 6 9
Storage time

F3

12 a
a
a c
10

8
mg RE/g sample

6 Control
b b 0.2%
4 b b b b b
b
2%

0
0 3 6 9
Storage time

F4

191
 100
90 a a a
80 a
DPPH inhibition (%)

70
60
b b
50 b Control
c b
40 c 0.2%
d
30 2%
20 e
10
0
0 3 6 9
Storage time

F5

2000
1800
a a
1600
a
µg of Trolox/g sample

1400 a
1200
1000 Control
b
800 b b 0.2%
b c
600 c c 2%
c
400
200
0
0 3 6 9
Storage time

F6

192
 10
9
8
TVC (Log ufc/g sample) 7
6
5 Control
4 0.2%
3 2%
2
1
0
0 3 6 9
Storage time

F7

10
Psychrotrophic (Log UFC/g sample)

9
8
7
6
5 Control
4 0.2%
3 2%
2
1
0
0 3 6 9
Storage time

F8

193
F9

194
 9. CONCLUSIONES GENERALES

El aprovechamiento de subproductos de la industria datilera (fruto y semilla) es una

alternativa para la recuperación de fitoquímicos que puedan ser incluidos como aditivos
antioxidantes en el desarrollo de productos cárnicos más seguros.

Particularmente, los extractos obtenidos a partir de semilla de dátil revelan el mayor

potencial, considerando su contribución con la disminución de la oxidación de lípidos y proteínas,
procesos que representan un desafío para la industria cárnica del siglo XXI debido a que afectan la
calidad nutricional, sensorial, y la seguridad de los productos cárnicos se ve comprometida.

Por otra parte, es relevante que el extracto de semilla de dátil a una concentración de 2%,
además de tener potencial antioxidante durante la vida útil de hamburguesas de cerdo, sus
compuestos fenólicos sean bioaccesibles, y contribuyan a la inhibición de la peroxidación lipídica
en la fase gástrica e intestinal determinada in vitro. Por otra parte, las cualidades sensoriales al
adicionar 2% de extracto no afectan negativamente los atributos del producto, lo cual es interesante
para la industria cárnica, desde que el uso de concentraciones elevadas de aditivos naturales
frecuentemente impacta en el color y sabor debido a sus propiedades per se.

Con el presente estudio se da a conocer el potencial antioxidante de extractos de

subproducto de dátil adicionados a una matriz alimentaria compleja como es la carne a una
concentración de 0.2% y 2 %.

Tomando en consideración las tendencias del consumidor hacia la búsqueda de alimentos

con menos aditivos sintéticos, los extractos obtenidos a partir de fuentes naturales como
subproductos de dátil se posicionan como candidatos idóneos para su inclusión en el diseño de
alimentos para nichos de marcado específicos y se abren las puertas para futuras investigaciones
respecto a su biodisponibilidad, cinética de absorción y estatus oxidativo in vivo.

Asimismo, reviste importancia conocer el impacto en la microbiota colónica tras el

consumo de estos nuevos alimentos y los metabolitos producidos, haciendo uso de herramientas

195
disponibles como los modelos ex vivo en conjunto con un abordaje multidisciplinario como el uso
de la proteómica y metabolómica.

10. RECOMENDACIONES

El aprovechamiento de subproductos agroindustriales como parte de la economía circular,

es un tema de importancia actual alrededor del mundo que involucra a la academia, industria y
dependencias gubernamentales. Sin embargo, en México se ha puesto poca atención al potencial
que tiene esta materia prima (subproductos).

La inversión en infraestructura e implementación de tecnología para el aprovechamiento y

recuperación de moléculas bioactivas, asimismo desde la academia proponer modelos de negocio
e innovación en el desarrollo de alimentos con el uso de aditivos elaborados a partir de los
subproductos agroindustriales de mayor producción en nuestro país, lo anterior se plasma como la
principal recomendación.

196
 ANEXO I

Evaluación del contenido de fitoquímicos y actividad antioxidante de extractos de dátil en sus

diferentes etapas de madurez obtenidos con el uso de diferentes solventes y métodos de
extracción

197
 CONTENIDO

Figura 1. Contenido de fenoles (a) y flavonoides (b) totales en extractos de fruto de dátil en sus
diferentes estados de madurez.

Figura 2. Porcentaje de inhibición de los radicales (a) DPPH y ABTS (b) de extractos de fruto de
dátil en sus diferentes estados de madurez.

Figura 3. Poder reductor de extractos de fruto de dátil en sus diferentes estados de madurez.

198
Figura 1. Contenido de fenoles a) y flavonoides b) totales en extractos de fruto de dátil en
sus diferentes estados de madurez.

199
Figura 2. Porcentaje de inhibición de los radicales (a) DPPH y ABTS (b) de extractos de
fruto de dátil en sus diferentes estados de madurez.

200
Figura 3. Poder reductor de extractos de fruto de dátil en sus diferentes estados de
madurez.

201
 CONCLUSIÓN

Los extractos de subproductos de dátil (5 mg/ml) presentaron contenido fenólico y actividad

antioxidante igual al extracto de dátil comercial, a excepción de que en la primera etapa de

maduración (Khalal) se encontró el contenido más alto de fenoles y actividad antioxidante, con el

uso de una mezcla de acetona-agua al 70% como solvente.

Debido a lo anterior, el aprovechamiento de los subproductos de dátil se presenta como una opción

viable para su inclusión en matrices alimentarias carentes de estas moléculas bioactivas, las cuales

hacen frente a los procesos oxidativos que se presentan sin excepción en todas las matrices

alimentarias durante su manufactura y almacenamiento.

202
 ANEXO II

Evaluación de la actividad antimicrobiana de extractos de subproductos de dátil obtenidos

utilizando diferentes solventes y métodos de extracción

203
 CONTENIDO

Tabla 1. MIC de extractos liofilizados del fruto de dátil frente a cepas bacterianas por el método de
microdilución en caldo.

Tabla 2. MIC de extractos liofilizados de semilla de dátil frente a cepas bacterianas por el método
de microdilución en caldo.

204
Tabla 1. MIC de extractos liofilizados del fruto del dátil frente a cepas bacterianas por el método
de microdilución en caldo.

Sonicación Maceración
Extracto Organismo de prueba Inhibición MIC50 Inhibición MIC50
(%) (mg/ml) (%) (mg/ml)
Salmonella choleraesuis 58.73 ≤ 4 mg/ml 38.16 > 4 mg
Listeria innocua 64.52 ≤ 4 mg/ml 52.18 ≤ 4 mg/ml
Acuoso Escherichia coli 27.27 > 4 mg 10.97 > 4 mg
Staphylococcus aureus 54.55 ≤ 4 mg/ml 62.13 ≤ 4 mg/ml
Pseudomonas aeruginosa 55.22 ≤ 4 mg/ml 52.21 ≤ 4 mg/ml

Salmonella choleraesuis 49.60 4 mg/ml 4.52 > 4 mg

Listeria innocua 57.38 ≤ 4 mg/ml 75.73 ≤ 4 mg/ml
Etanólico Escherichia coli 14.03 > 4 mg 32.72 > 4 mg
Staphylococcus aureus 46.84 > 4 mg 59.56 ≤ 4 mg/ml
Pseudomonas aeruginosa 42.31 > 4 mg 0.46 > 4 mg

Salmonella choleraesuis 54.08 ≤ 4 mg/ml 54.08 ≤ 4 mg/ml

Listeria innocua 60.58 ≤ 4 mg/ml 57.35 ≤ 4 mg/ml
Acetónico Escherichia coli 28.06 > 4 mg 27.88 > 4 mg
Staphylococcus aureus 61.53 ≤ 4 mg/ml 64.06 ≤ 4 mg/ml
Pseudomonas aeruginosa 47.64 > 4 mg 41.40 > 4 mg

205
Tabla 2. MIC de extractos liofilizados de semilla de dátil frente a cepas bacterianas por el método
de microdilución en caldo.

Sonicación Maceración
Extracto Organismo de prueba MIC50
Inhibición Inhibición MIC50
(%) (µg/mL) (%) (µg/mL)
Salmonella choleraesuis 19.55 > 1000 0.79 > 1000
Listeria innocua 25.88 > 1000 6.87 > 1000
Acuoso Escherichia coli 2.93 > 1000 20.38 > 1000
Staphylococcus aureus 11.08 > 1000 4.57 > 1000
Pseudomonas aeruginosa 24.51 > 1000 37.75 > 1000

Salmonella choleraesuis 27.03 > 1000 3.66 > 1000

Listeria innocua 14.91 > 1000 15.64 > 1000
Etanólico Escherichia coli 34.55 > 1000 23.03 > 1000
Staphylococcus aureus 10.33 > 1000 2.99 > 1000
Pseudomonas aeruginosa 24.51 > 1000 1.67 > 1000

Salmonella choleraesuis 38.95 > 1000 32.59 > 1000

Listeria innocua 14.91 > 1000 14.91 > 1000
Acetónico Escherichia coli 0.37 > 1000 7.86 > 1000
Staphylococcus aureus 11.08 > 1000 11.08 > 1000
Pseudomonas aeruginosa 24.51 > 1000 24.51 > 1000

206
 CONCLUSIÓN

Los extractos de subproductos de dátil obtenidos con el uso de diferentes métodos de

extracción y solventes presentaron efectividad baja, expresada como MIC50, contra diferentes
bacterias Gram + y Gram -, específicamente los obtenidos a partir de semilla de dátil (1mg/mL).
Diferente a lo que se encontró para los extractos del fruto de dátil, ya que en los ensayos realizados
la concentración de 4 mg/mL logró inhibir más del 50% del crecimiento bacteriano (Salmonella
Choleraesuis, Listeria innocua, Staphylococcus aureus, Pseudomonas aeruginosa),
particularmente cuando se obtuvieron los extractos por sonicación y con el uso de agua y acetona
como solvente.

Cuando se evalúa la efectividad de extractos naturales frente a bacterias de interés sanitario o

clínico, debe considerarse su solubilidad como un factor clave ya que a medida que se incrementa
la concentración puede presentarse sedimentación y alterar los resultados cuando se evalúa la
turbidez espectrofotométricamente después de incubar.

207