universidad de murcia...la presente tesis doctoral ha sido realizada para optar al grado de doctor...
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UNIVERSIDAD DE MURCIA
ESCUELA INTERNACIONAL DE DOCTORADO
Application of Mediterranean Ingredients for
the Bromatological Improvement of Clean Label Animal
Origin Products
Aplicación de Ingredientes Mediterráneos para
la Mejora Bromatológica de Alimentos Clean Label
de Origen Animal
Dª Lorena Martínez Zamora
2019
UNIVERSITY OF MURCIA
INTERNATIONAL DOCTORAL SCHOOL
Lorena Martínez Zamora
DOCTORAL DISSERTATION
“Application of Mediterranean ingredients for the bromatological
improvement of Clean Label animal origin products”
“Aplicación de ingredientes mediterráneos para la mejora
bromatológica de alimentos Clean Label de origen animal”
Supervisors:
Gema Nieto Martínez
Gaspar Francisco Ros Berruezo
“International mention”
“Thesis in another language”
“Thesis as a compendium of publications”
2019
This Doctoral Thesis has been carried out to obtain the degree of Doctor in "Food Technology,
Nutrition and Bromatology" by the University of Murcia. This Doctoral Thesis has been written
in another language and has been carried out by compendium of publications. Likewise, this thesis
is proposed for International Doctorate Mention by virtue of the predoctoral stay carried out under
the supervision of Professor Leif Horsfelt Skibsted in the "Department of Food Science (UCPH
FOOD)" located in "University of Copenhagen" of Copenhagen (Denmark).
La presente Tesis Doctoral ha sido realizada para optar al grado de Doctor en “Tecnología de
los Alimentos, Nutrición y Bromatología” por la Universidad de Murcia. Esta Tesis Doctoral ha
sido redactada en otro idioma y se ha realizado por compendio de publicaciones. Asimismo, dicha
Tesis se propone para Mención de Doctorado Internacional en virtud de la estancia predoctoral
realizada bajo la supervisión del Profesor Leif Horsfelt Skibsted en el “Department of Food
Science (UCPH FOOD)” situado en “University of Copenhagen” de Copenhague (Dinamarca).
ACKNOWLEDGEMENTS
I would like to thank to my directors, Gaspar Ros Berruezo, PhD, and Gema Nieto Martínez,
PhD for all the knowledge provided to me and for guiding me during this long stage. Team GG.
My scientific parents. Specially thanks to Gema, for taking care of me and giving to me so many
opportunities. After almost five years together, I have learnt everything I know from you. And of
course, thank you Gaspar for having the power to fix everything with a simple scheme. You are
the best.
Also, thanks to Julián Castillo, from Nutrafur-Frutarom, S.A., who has supported great part of
this work and who has provided all the studied natural extracts. Without your help I could not
have carried out this docthoral thesis.
I also want to express my gratitude to the Centro Tecnológico de la Conserva, and to the
Servicio de Cultivo de Células Animales (SACE) and all their members, for teaching and helping
me in the development of this docthoral thesis.
Otherwise, I would like to thank to Campus Mare Nostrum for the financial support through
the Convocatoria de ayudas para estancias en el extranjero de jóvenes investigadores y alumnos
de doctorado en las líneas de actuación de Campus Mare Nostrum Curso 2017/2018 (-47/2018).
Finally, deepest and faithful thanks to Leif H. Skibsted and Sisse Jongberg for the financial
support and all the knowledge provided to me during the most productive, intense and satisfying
period of my life.
A mi abuelo, mi abuela y mis padres.
“Tú tienes gran poder, solo quiérete, puedes lograr
cualquier cosa esforzándote. […] Tú puedes cambiar la
percepción de lo que vives. La belleza está en los ojos del
que mira. Todo es del color de la luz que recibe.”
Javier Ibarra Ramos
“The future belongs to those who believe in the beauty of their dreams”
Eleanor Roosevelt
“The food you eat can either be the safest and most
powerful form of medicine, or the slowest form of poison”
Ann Wigmore
ABSTRACT
Trends in food are changing rapidly in recent years and food businesses need to put in place
strategies that compassionate or anticipate these new ways of thinking about food choice and
consumption. Knowing how food has been produced or what impact it has on our body, our well-
being or the environment will have an increasing weight in consumption decisions.
Meat and animal products and their derivatives are perishable foods that suffer a gradual loss of
bromatological quality during their conservation, both in refrigeration, in a controlled atmosphere,
and in freezing. It is for this reason that since the last century and up to the present day the
widespread use of synthetic additives (sulphites, BHA, BHT, and nitrifying agents) has been
extended in order to extend the useful life of this type of product. However, excess consumption
of this type of ingredients has reported the possibility of having health effects from excessive
exposure.
Antioxidant compounds, both natural and synthetic, are substances that retard the oxidation of
food products by inhibiting the formation of free radicals or interrupting this pathway through
some specific mechanisms. One of these pathways is the transfer of hydrogen atoms, when the
antioxidant compounds (AH) gives an H to a free radical (R-), generating a more stable radical
(A-) (R- + AH → RH + A-), while the other way is the transfer of electrons, when AH gives an
electron to reduce the free radical (R- + AH → R- + AH-). In parallel, in terms of their chemical
nature and origin, these compounds could also prevent bacterial development by inhibiting
several functions, such as maintenance of the cell wall of bacteria, protein synthesis, transport or
DNA replication, as the main mechanisms of antimicrobial action.
In the present Doctoral Thesis, the development of strategies for obtaining "Clean label" animal
origin products (by reducing the concentration of certain synthetic additives associated with "E"
numbers) has been addressed. The strategies followed for their bromatological improvement aim
to contribute especially to the knowledge within the field of antioxidant and antimicrobial agents
of natural origin. To this end, two ways of incorporating antioxidant compounds have been
studied, one endogenous and the other exogenous, and the following objectives have been
pursued, which will form the five trials that have been developed during this Thesis Dissertation.
The aim of this work is to disseminate basic knowledge about the production of “Clean label”
animal origin products following different treatments and the organoleptic, oxidative and
microbiological changes that occur during the conservation of this kind of products. To this end,
a bibliographic review was carried out on products of animal origin, including all oxidative and
degradation processes resulting from their conservation. This bibliographic review also focused
on the use of synthetic additives and the possible substitution by Mediterranean ingredients with
potential health benefits for consumers. Subsequently, the experimental part of the PhD project is
described from the materials, methods, and analytical techniques used and developed throughout
the experimental part. Finally, the results obtained in the project have been properly presented
and discussed. The main conclusions of the project and the future perspectives within “Clean
label” animal origin products allow to give the final conclusion to this project.
As a consequence, on the one hand, a mineral bioavailability test was carried out on Caco-2 human
intestine cells in the presence of HXT. For this purpose, endogenously enriched meat emulsions
in Zn and Se minerals, of both organic and inorganic forms, and exogenously enriched in
hydroxytyrosol (HXT) and extra virgin olive oil (EVOO) were subjected to in vitro digestion and
subsequently incorporated into a human intestine cell line (Caco-2). In this cell line the
bioavailability of both mineral and natural extract (HXT) absorbed by the enterocytes was
measured.
On the other hand, all the extracts used with significant antioxidant and antimicrobial capacity
(hydroxytyrosol, grape seed, harpagophyte, rosemary, pomegranate, citrus, acerola, paprika,
oregano, garlic, beet, lettuce, rocket, watercress, spinach, chard, and celery) were characterized.
For these measures, known methods of measuring antioxidant capacity were applied such as:
FRAP (Ferric Reducing Antioxidant Power), ORAC (Oxygen Radical Absorbance Capacity),
DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-
sulphonic acid)); or antimicrobial capacity, such as the disk diffusion technique using different
bacterial strains such as Staphilococcus aureus, E. Coli, Listeria monocytogenes or Clostridium
perfringens. In addition, the quantity of phenolic compounds in each extract was also evaluated,
following the method described by Folin-Ciocalteu, as well as the quantity in nitrate, in the case
of natural nitrate sources.
Similarly, the products made using these extracts were characterized, so a complete nutritional
analysis was made of each of them, taking into account the content of protein, fat, and minerals.
When these foods were enriched in fatty acids, the fatty acid profile was also determined,
evaluating the content of saturated, mono- and polyunsaturated fatty acids (SFA, MUFA, and
PUFA).
Each of the processed products (Frankfurt sausages, chicken nuggets, dry-cured Spanish
“chorizo”, and fish preparations type hamburger) was subjected to a shelf life study in which
different deterioration parameters were evaluated, such as colour (CIELab), pH, lipid oxidation
(Thiobarbituric Acid Reactive Substances (TBARs) or determination of volatile compounds by
GS-MS), protein oxidation (free thiol groups), microbiological development (total viable count
of mesophilic bacteria, total count of coliform micro-organisms, E. Coli, Salmonella, Listeria
monocytogenes or Clostridium perfringens), and autolytic changes, in the case of fish
(trimethylamine (TMA), ammonia (Nessler procedure), or Total Volatile Basic Nitrogen (TVB-
N) content).
Finally, the sensory quality of the processed products was also assessed by a panel of tasters
trained according to established ISO standards, in order to produce food that was organoleptically
pleasing and thus assess the possibility of its marketing in the future.
The tests described above led to at least five new ways of obtaining food of animal origin "Clean
label":
- Study of bioavailability of Zn and Se minerals in an in vitro Caco-2 cell model through
endogenous enrichment through diet and exogenous enrichment through the
incorporation of HXT and EVOO in chicken meat emulsions.
- Study of exogenous enrichment of frankfurters by incorporating HXT, nuts, and EVOO.
- Shelf-life study (365 days) of frozen chicken nuggets enriched endogenously in Zn and
Se minerals and exogenously in natural extracts (pomegranate, grape seed,
hydroxytyrosol, rosemary, and harpagophyte).
- Shelf-life study (150 days) of dry-cured Spanish “chorizo” exogenously enriched in
natural extracts:
o Determination of the antioxidant and antimicrobial capacity of all the extracts
used: citrus, rosemary, acerola, paprika, garlic, oregano, lettuce, arugula, spinach,
chard, celery, beetroot, and watercress.
o Study of protection against protein oxidation of an oxidised pork meat model
system, after incorporation of the Mediterranean ingredients under study.
o Shelf-life study and evaluation of the bromatological loss during 150 days (25
days of ripening and 125 days under refrigerate storage).
- Shelf-life study of hake preparations "hamburger" type (14 days) exogenously enriched
in natural extracts:
o Determination of the antioxidant and antimicrobial capacity of the extracts used:
citrus, rosemary, acerola, Hydroxytyrosol, and pomegranate.
o Shelf-life study and evaluation of the bromatological loss during 14 days under
refrigerated and aerobiosis storage.
However, after the development of each of the experiments described, the following results have
been obtained:
Firstly, the organic forms of the minerals Zn and Se have been shown to be more bioavailable in
a chicken meat emulsion enriched endogenously with these minerals and also exogenously by the
incorporation of EVOO and HXT using an in vitro Caco-2 cell model system. An important result
is related to the degradation of HXT, which was minimal during in vitro digestion, leading to the
idea that at least 90% of HXT consumed may be available at the intestinal level.
As expected, the use of EVOO and nuts as ingredients improves the fatty acid profile in chicken
meat emulsions, providing a good nutritional profile with a higher concentration of MUFA and
PUFA. At the same time, the exogenous use of HXT extract prevents the oxidation of proteins
and lipids for 21 days in sausages, while maintaining organoleptic quality in combination with
EVOO and nuts.
The addition of phenolic compounds such as natural extracts of seeds, herbs and fruits, together
with organic forms of Zn and Se, slows down microbial growth (longer LAG phase, bacteria adapt
to growing conditions), reduces the oxidation time of proteins and lipids, and does not modify
sensory quality, which, as a general conclusion, prolongs the shelf life of chicken nuggets for one
year in frozen (-18 ºC).
As for the antioxidant and antimicrobial capacity of natural extracts used as an ingredient for the
production of Spanish “chorizo”, rosemary showed the compound with the highest antimicrobial
activity followed by natural sources of nitrates (beet, lettuce, rocket, spinach, chard, celery and
watercress) and spices such as paprika, garlic and oregano. Of all the natural extracts, citrus fruits
(herperidin) were the only ones that showed the highest antioxidant capacity, as well as the lowest
antimicrobial activity. However, the combination of citrus extract with nitrate-rich green leafy
plant extracts showed increased antimicrobial power. Consequently, the sources of hesperidin and
natural nitrate showed synergistic behaviour, but did not show the same effectiveness in
combination with the monoterpenes of rosemary extracts (carnosic acid and carnosol). This
combination of extracts allows the samples of dry-cured Spanish chorizo to be kept for 150 days
in cold rooms without modifying their sensory quality.
Citrus fruits, as well as lettuce and spinach, protect almost completely against the loss of thiol
protein in the meat model system, initiated by the hydrophilic initiator, OXHydro and by the
lipophilic initiator, OXLip. The same components also showed efficient radical scavenging activity
as determined by ESR spectroscopy. In addition, it was found that natural sources of nitrate
protect against oxidation of the thiol protein and were able to eliminate radicals in the meat
oxidation system. The possible substitution of synthetic or phenolic antioxidants with natural
sources of nitrates from green leafy vegetables in the production of meat products for protection
against oxidation and prolongation of shelf life is indicated by the results obtained.
The natural extracts analysed (pomegranate, olive, rosemary, and citrus) are also suitable for
prolonging the shelf life of fish hamburgers up to 11 days, with mechanisms that slow down the
autolytic phases (degradation of non-protein nitrogen components), as well as the microbiological
growth of decomposition, and any oxidation of lipids or proteins, maintaining the same high
sensory acceptability for panelists, and without detection of abnormal tastes (smell or taste).
As a final comment on the current doctoral thesis, the strategies followed provide a useful tool to
"Clean Label" animal origin products (based on meat or fish), in which synthetic additives with
analogical effect have been replaced by natural extracts produced from traditional products of
animal origin.
Mediterranean ingredients are rich in bioactive compounds. For this reason, their consumption
can lead to significant improvements in the health of the human body. In addition, this change did
not affect the sensory properties of the product, which showed a high acceptance avoiding
oxidative damage and microbiological growth.
Finally, in this Doctoral Thesis, synthetic additives have been substituted by various means,
especially through the use of natural extracts obtained as by-products of the Food Industry, from
traditional ingredients of the Mediterranean Diet rich in bioactive compounds, whose
consumption has shown a significant improvement.
RESUMEN
Las tendencias en alimentación están cambiando de un modo vertiginoso en los últimos años y
las empresas alimentarias tiene que establecer estrategias que se compasen o adelanten a estas
nuevas formas de pensar en la elección y consumo de alimentos. Saber cómo se han producido
los alimentos o qué impacto tienen en nuestro cuerpo, nuestro bienestar o en el entorno, tendrán
un peso cada vez mayor en las decisiones de consumo. Estas tendencias ya han empezado en otros
países y nos van llenando de términos en inglés que es bueno ir conociendo y asimilando para la
producción de alimentos.
La carne y los productos de origen animal y sus derivados son alimentos perecederos que sufren
una pérdida gradual de calidad bromatológica durante su conservación, tanto en refrigeración, en
atmósfera controlada, como en congelación. Es por ello, que desde el siglo pasado y hasta la
actualidad se ha extendido el uso generalizado de aditivos sintéticos (sulfitos, BHA, BHT y
agentes nitrificantes) con el fin de alargar la vida útil de este tipo de productos. Sin embargo, el
consumo excedido de este tipo de ingredientes ha reportado la posibilidad de tener efectos sobre
la salud una exposición excesiva.
Los compuestos antioxidantes, tanto naturales como sintéticos, son sustancias que retardan la
oxidación de los productos alimenticios inhibiendo la formación de radicales libres o
interrumpiendo esta vía a través de algunos mecanismos específicos. Una de estas vías es la
transferencia de átomos de hidrógeno, cuando el compuesto antioxidante (AH) da un H a un
radical libre (R-), generando un radical más estable (A-) (R- + AH → RH + A-). Mientras que la
otra vía es la transferencia de electrones, cuando AH da un electrón para reducir el radical libre
(R- + AH → R- + AH-). Paralelamente, en cuanto a su naturaleza química y origen, estos
compuestos también podrían prevenir el desarrollo bacteriano mediante la inhibición de varias
funciones, como el mantenimiento de la pared celular de las bacterias, la síntesis de proteínas, el
transporte o la replicación del ADN, como principales mecanismos de acción antimicrobiana.
En la presente Tesis Doctoral se ha abordado el desarrollo de estrategias de obtención de
productos de origen animal “clean label” o “etiqueta limpia” (reduciendo la concentración de
ciertos aditivos sintéticos asociados a los números “E”). Las estrategias seguidas para la mejora
bromatológica de los mismos pretenden contribuir especialmente al conocimiento dentro del
campo de los agentes antioxidantes y antimicrobianos de origen natural. Para ello, se han
estudiado dos vías de incorporación de compuestos antioxidantes, una endógena y otra exógena,
y perseguido los siguientes objetivos que van a conformar los cinco ensayos que se han
desarrollado durante la presente Tesis.
Este trabajo tiene por objetivo difundir los conocimientos básicos sobre la elaboración de
productos de origen animal “Clean label”, siguiendo diversos tratamientos y los cambios
organolépticos, oxidativos y microbiológicos que se producen durante la conservación de este
tipo de productos. Para ello, se realizó una revisión bibliográfica sobre los productos de origen
animal, incluyendo todos los procesos oxidativos y de degradación que resultan de su
conservación. Esta revisión bibliográfica también se centró en el uso de aditivos sintéticos y la
posible sustitución por ingredientes mediterráneos con beneficios potenciales para la salud de los
consumidores. Posteriormente, se describe la parte experimental del proyecto de doctorado a
partir de los materiales, métodos y técnicas analíticas utilizadas y desarrolladas a lo largo de la
parte experimental. Finalmente, los resultados obtenidos en el proyecto han sido presentados y
discutidos adecuadamente. Las principales conclusiones del proyecto y las perspectivas de futuro
dentro de los productos de origen animal “Clean label” permiten dar el broche final a este
proyecto.
Como consecuencia, se llevó a cabo un ensayo de biodisponibilidad mineral en células Caco-2 de
intestino humano en presencia de HXT. Para ello, emulsiones cárnicas enriquecidas de forma
endógena en minerales Zn y Se, de origen tanto orgánico como inorgánico en hidroxitirosol
(HXT) y aceite de oliva virgen extra (AOVE) fueron sometidas a una digestión in vitro para
posteriormente ser incorporadas a una línea celular de intestino humano (Caco-2). En esta línea
celular se midió la biodisponibilidad tanto de mineral como de extracto natural (HXT) absorbida
por los enterocitos.
Por otra parte, se caracterizaron todos los extractos utilizados de significativa capacidad
antioxidante y antimicrobiana (hidroxitirosol, semilla de uva, harpagofito, romero, granada,
cítrico, acerola, pimentón, orégano, ajo, remolacha, lechuga, rúcula, berros, espinaca, acelga, y
apio). Para dichas medidas se aplicaron conocidos métodos de medida de la capacidad
antioxidante como: FRAP (Ferric Reducing Antioxidant Power), ORAC (Oxygen Radical
Absorbance Capacity), DPPH (2,2-difenil-1-picrylhydrazyl), ABTS (2,2'-azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid)); o de la capacidad antimicrobiana, como la técnica de
difusión de disco utilizando distintas cepas bacterianas como Staphilococcus aureus, E. Coli,
Listeria monocytogenes o Clostridium perfringens. Además, también se valoró la cantidad de
compuestos fenólicos de cada uno de los extractos, siguiendo el método descrito por Folin-
Ciocalteu, así como la cantidad en nitrato, en el caso de las fuentes naturales de nitrato.
De igual modo, los productos elaborados usando dichos extractos fueron caracterizados, por lo
que se realizó un análisis nutricional completo de cada uno de ellos, contemplando el contenido
en proteínas, grasas y minerales. Cuando dichos alimentos fueron enriquecidos en ácidos grasos
también se determinó el perfil de ácidos grasos, valorando el contenido en ácidos grasos
saturados, mono y poliinsaturados.
Cada uno de los productos elaborados (salchichas Frankfurt, nuggets de pollo, chorizo curado y
preparados de pescado tipo hamburguesa) fue sometido a un estudio de vida útil en los que se
valoraron distintos parámetros de deterioro como el color (CIELab), pH, oxidación lipídica
(Sustancias reactivas del ácido thiobarbitúrico (TBARs) o determinación de compuestos volátiles
mediante GS-MS), oxidación proteica (grupos tioles libres), desarrollo microbiológico (recuento
total de bacterias mesófilas, recuento total de microorganismos coliformes, E. Coli, Salmonella,
Listeria monocytogenes o Clostridium perfringens) y cambios autolíticos, en el caso del pescado
(contenido en trimetilamina (TMA), amoníaco (procedimiento de Nessler) o contenido en
Nitrógeno Básico Volátil Total (NBVT)).
Por último, la calidad sensorial de los productos elaborados también fue valorada por un panel de
catadores entrenado según las normas ISO establecidas, con el fin de producir alimentos que
fuesen agradables organolépticamente y así valorar la posibilidad de su comercialización en un
futuro.
Los ensayos previamente descritos dieron lugar a, al menos, cinco nuevas vías de obtención de
alimentos de origen animal “Clean label”.
- Estudio de biodisponibilidad de minerales Zn y Se en un modelo celular in vitro Caco-2
a través del enriquecimiento endógeno, mediante la dieta de las aves, y exógeno, mediante
la incorporación de HXT y AOVE en emulsiones cárnicas de pollo.
- Estudio del enriquecimiento exógeno de salchichas tipo Frankfurt mediante la
incorporación de HXT, nueces y AOVE.
- Estudio de vida útil (365 días) de “Nugget” de pollo congeladas enriquecidas en minerales
Zn y Se de forma endógena y en extractos naturales (granada, semilla de uva, HXT,
romero y harpagofito) de forma exógena.
- Estudio de vida útil (150 días) de chorizo sarta curado enriquecido en extractos naturales
de forma exógena.
o Determinación de la capacidad antioxidante y antimicrobiana de todos los
extractos utilizados: cítrico, romero, acerola, pimentón, ajo, orégano, lechuga,
rúcula, espinaca, acelga, apio, remolacha y berros.
o Estudio de protección contra la oxidación proteica de un modelo cárnico oxidado
elaborado a base de carne de cerdo, tras la incorporación de los ingredientes
mediterráneos objeto de estudio.
o Estudio de vida útil y valoración de la pérdida de calidad bromatológica durante
150 días (25 días de curado y 125 de conservación en refrigeración).
- Estudio de vida útil de preparados de merluza, tipo “hamburguesa” (14 días) enriquecidas
en extractos naturales de forma exógena.
o Determinación de la capacidad antioxidante y antimicrobiana de los extractos
utilizados: cítrico, romero, acerola, HXT y granada.
o Estudio de vida útil y valoración de la pérdida de calidad bromatológica durante
14 días en refrigeración y aerobiosis.
Con todo, tras el desarrollo de cada uno de los experimentos descritos se han podido obtener los
siguientes resultados:
En primer lugar, las formas orgánicas de los minerales Zn y Se han demostrado ser más
biodisponibles en una emulsión de carne de pollo enriquecida endógenamente con esos minerales
y también exógenamente mediante la incorporación de AOVE y HXT utilizando un sistema de
modelo celular in vitro Caco-2. Un resultado importante está relacionado con la degradación de
HXT, que fue mínima durante la digestión "in vitro", que llevó a la idea de que al menos el 90 %
de HXT consumido puede estar disponible a nivel intestinal.
Como era de esperar, el uso de AOVE y nueces como ingredientes, mejora el perfil de ácidos
grasos en las emulsiones de carne de pollo, proporcionando un buen perfil nutricional con una
mayor concentración de AGM y AGP. Al mismo tiempo, el uso exógeno del extracto HXT evita
la oxidación de proteínas y lípidos durante 21 días en salchichas, manteniendo al mismo tiempo
la calidad organoléptica en combinación con AOVE y frutos secos.
La adición de compuestos fenólicos como extractos naturales de semillas, hierbas y frutos, junto
con formas orgánicas de Zn y Se, retrasa el crecimiento microbiano (fase LAG más larga, las
bacterias se adaptan a las condiciones de crecimiento), reduce el tiempo de oxidación de proteínas
y lípidos, y no modifica la calidad sensorial, lo que, como conclusión general, prolonga la vida
útil de los nuggets de pollo durante un año en congelado (-18 ºC).
En cuanto a la capacidad antioxidante y antimicrobiana de los extractos naturales utilizados como
ingrediente para la producción de chorizo español, el romero mostró el compuesto de mayor
actividad antimicrobiana seguido de fuentes naturales de nitratos (remolacha, lechuga, rúcula,
espinaca, acelga, apio y berros) y especias, como el pimentón, el ajo y el orégano. Entre todos los
extractos naturales, los cítricos (herperidina) fueron los únicos que mostraron la mayor capacidad
antioxidante, al mismo tiempo que la menor actividad antimicrobiana. Sin embargo, la
combinación de extracto cítrico con extractos vegetales de hoja verde ricos en nitratos mostró un
mayor poder antimicrobiano. En consecuencia, las fuentes de hesperidina y nitrato natural
mostraron un comportamiento sinérgico, pero no presentaron la misma efectividad en
combinación con los monoterpenos de extractos de romero (ácido carnósico y carnosol). Esta
combinación de extractos permite mantener las muestras de chorizo español curado en seco
durante 150 días en cámaras frigoríficas sin modificar su calidad sensorial.
Los cítricos, así como la lechuga y la espinaca, protegen casi completamente contra la pérdida de
proteína tiol en el sistema de modelos de carne, iniciada por el iniciador hidrofílico, OXHydro y por
el iniciador lipófilo, OXLip. Los mismos componentes mostraron también una eficiente actividad
de barrido de radicales según lo determinado por la espectroscopia ESR. Además, se encontró
que las fuentes naturales de nitrato protegen contra la oxidación de la proteína tiol y fueron
capaces de eliminar los radicales en el sistema de oxidación de la carne. La posible sustitución de
antioxidantes sintéticos o fenólicos con fuentes naturales de nitratos de hortalizas de hoja verde
en la producción de productos cárnicos para la protección contra la oxidación y la prolongación
de la vida útil se señala con los resultados obtenidos.
Los extractos naturales analizados (granada, olivo, romero y cítricos) también son adecuados para
prolongar la vida útil de las hamburguesas de pescado hasta 11 días, con mecanismos que
ralentizan las fases autolíticas (degradación de los componentes de nitrógeno no proteínico), así
como el crecimiento microbiológico de la descomposición, y cualquier oxidación de lípidos o
proteínas, manteniendo la misma alta aceptabilidad sensorial para los panelistas, y sin detección
de sabores anormales (olor o sabor).
Como comentario final de la actual tesis doctoral, las estrategias seguidas proporcionan una
herramienta útil para "Etiquetar de forma limpia" los productos de origen animal (a base de carne
o pescado), en los que los aditivos sintéticos con efecto analógico han sido sustituidos por
extractos naturales producidos a partir de productos tradicionales de origen animal.
Ingredientes mediterráneos ricos en compuestos bioactivos, cuyo consumo conlleva importantes
mejoras en la salud del cuerpo humano. Además, este cambio no afectó a las propiedades
sensoriales del producto, que mostraron una alta aceptación evitando el daño oxidativo y el
crecimiento microbiológico.
Con todo, en la presente Tesis Doctoral, los aditivos sintéticos han sido sustituidos mediante
distintas vías, sobre todo mediante el uso de extractos naturales obtenidos como sub-productos de
la Industria Alimentaria, a partir de ingredientes tradicionales de la Dieta Mediterránea ricos en
compuestos bioactivos, cuyo consumo ha demostrado una mejora significativa de la salud en
humanos, tales como compuestos fenólicos. Además, esta sustitución no afectó a la calidad
sensorial de los productos desarrollados, evitando el daño oxidativo y el deterioro microbiológico.
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TABLE OF CONTENTS
1. INTRODUCTION ......................................................................................................................... 1
2. ANIMAL ORIGIN PRODUCTS ................................................................................................. 5
2.1. Meat .......................................................................................................................................... 7
2.1.1. Definition and chemical composition of meat ..................................................................... 7
2.1.2. Meat emulsions: frankfurter-type sausages .......................................................................... 8
2.1.3. Pre-fried products: chicken nuggets ..................................................................................... 9
2.1.4. Dry-cured products: Spanish “chorizo” ............................................................................... 9
2.1.4.1. Dry-curing chemistry ............................................................................................ 10
Nitrate and nitrite ............................................................................................................... 10
Colour development ........................................................................................................... 10
Microbiology of dry-cured process .................................................................................... 11
2.2. Fish ......................................................................................................................................... 12
2.2.1. Definition and chemical composition of fish ..................................................................... 12
2.2.2. Fish degradation mechanisms ............................................................................................ 13
2.2.3. Fish patties ......................................................................................................................... 15
3. OXIDATIVE DETERIORATION IN ANIMAL ORIGIN PRODUCTS .............................. 17
3.1. Lipid oxidation ....................................................................................................................... 19
3.2. Protein oxidation .................................................................................................................... 20
4. USE OF ANTIOXIDANT AND ANTIMICROBIAL COMPOUNDS TO PRESERVE
ANIMAL ORIGIN PRODUCTS ................................................................................................... 23
4.1. Synthetic adidtives, their antioxidative mechanisms, and health risks ................................... 25
4.2. Mediterranean ingredients, their antioxidative mechanisms, and health benefits .................. 27
4.2.1. Hydroxytyrosol .................................................................................................................. 29
4.2.2. Extra Virgin Olive Oil ........................................................................................................ 30
4.2.3. Nuts .................................................................................................................................... 30
4.2.4. Spices and herbs ................................................................................................................. 31
4.2.5. Fruits .................................................................................................................................. 32
4.2.6. Green leafy vegetables ....................................................................................................... 32
4.2.7. Harpagophyte ..................................................................................................................... 32
4.2.8. Antioxidant mechanisms of phenolic compounds ............................................................. 32
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5. DEVELOPMENT OF CLEAN LABEL ANIMAL ORIGIN PRODUCTS ........................... 35
5.1. Ante-mortem antioxidant strategies ....................................................................................... 38
5.2. Post-mortem antioxidant strategies ........................................................................................ 39
6. JUSTIFICATION AND OBJECTIVES .................................................................................... 41
7. EXPERIMENTAL DESIGN ...................................................................................................... 45
7.1. Assay I: Study of endogenous enrichment of meat products through animal diet ................. 49
7.2. Assay II: Study of the exogenous enrichment of cooked meat product through the addition of
natural antioxidant extracts ........................................................................................................... 50
7.3. Assay III: Study of endogenous and exogenous enrichment of frozen pre-cooked meat
products, through the incorporation of Zn and Se to animal feed and natural antioxidant extracts
during the elaboration of chicken nuggets .................................................................................... 53
7.4. Assay IV: Study of exogenous enrichment of dry-cured meat products through the addition
of natural antioxidant and nitrate source extracts .......................................................................... 55
7.4.1. Characterization of natural extracts and application in Spanish “chorizo” ........................ 55
7.4.2. Study of protein oxidation in pork meat after application of natural extracts .................... 56
7.4.3. Shelf-life study of Spanish “chorizo” enriched in natural extracts .................................... 57
7.5. Assay V: Study of exogenously enrichment of processed fish products through the addition
of natural antioxidant extracts ....................................................................................................... 59
7.5.1. Characterization of natural extracts and application in fish patties .................................... 59
7.5.2. Shelf-life study of fish patties enriched in natural extracts ................................................ 60
8. RESULTS AND DISCUSION .................................................................................................... 65
8.1. Assay I: Obtained results of endogenous enrichment of meat products through animal diet 66
8.1.1. Study of mineral bioavailability ......................................................................................... 69
8.2. Assay II: Obtained results of the exogenous enrichment of cooked meat product through the
addition of natural antioxidant extracts ......................................................................................... 72
8.2.1. Proximate composition and improve of fatty acid profile .................................................. 72
8.2.2. Shelf life study of chicken frankfurters .............................................................................. 78
8.3. Assay III: Obtained results of endogenous and exogenous enrichment of frozen pre-cooked
meat products, through the incorporation of Zn and Se to animal feed and natural antioxidant
extracts during the elaboration of chicken nuggets ....................................................................... 87
8.3.1. Shelf life study of frozen chicken nuggets ......................................................................... 88
8.4. Assay IV: Obtained results of exogenous enrichment of dry-cured meat products through the
addition of natural antioxidant and nitrate source extracts ............................................................ 94
8.4.1. Characterization of natural extracts and application in Spanish “chorizo” ........................ 94
8.4.2. Obtained results of protein oxidation in pork meat after application of natural extracts . 102
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8.4.3. Shelf-life study of Spanish “chorizo” enriched in natural extracts .................................. 110
8.5. Assay V: Obtained results of exogenously enrichment of processed fish products through the
addition of natural antioxidant extracts ....................................................................................... 122
8.5.1. Characterization of natural extracts and application in fish patties .................................. 122
8.5.2. Shelf-life study of fish patties enriched in natural extracts .............................................. 129
9. CONCLUSIONS ....................................................................................................................... 135
10. PERSPECTIVES FOR FURTHER RESEARCH ACTIVITIES ....................................... 139
11. REFERENCES ........................................................................................................................ 143
12. SCIENTIFIC PRODUCTION ............................................................................................... 161
12.1. Publications ........................................................................................................................ 163
12.2. Book chapters ..................................................................................................................... 163
12.3. Scientific congress .............................................................................................................. 163
12.4. Prizes .................................................................................................................................. 164
13. ANNEXES ................................................................................................................................ 165
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TABLE INDEX
Table 2.1. Summary of post-mortem autolytic changes in refrigerated fish (Source: FAO, 1992). . 14
Table 7.1. Ingredients (g) of chicken emulsion samples elaborated in Assay I................................ 49
Table 7.2. Ingredients (g) of chicken frankfurters samples elaborated in Assay II; Paper II and III.51
Table 7.3. Ingredients (g) of frozen chicken nuggets samples elaborated in Assay III. Paper IV. ... 54
Table 7.4. Ingredients (g) of dry-cured Spanish “chorizo” samples elaborated in Assay IV, Papers
V and VII........................................................................................................................................... 58
Table 7.5. Ingredients (g) of fish patties samples elaborated in Assay V, Paper VIII.. .................... 60
Table 7.6. Ingredients (g) of fish patties samples elaborated in Assay V, Paper IX. ....................... 61
Table 7.7. Summary of material and methods followed in the present thesis dissertation ............... 62
Table 8.1. HXT concentration in emulsions (soluble fraction added to Caco-2 cells) (M ± SD)
measured by HPLC. .......................................................................................................................... 68
Table 8.2. Fe retention, transport, and cellular uptake (M ± SD) in enriched chicken emulsions. ... 69
Table 8.3. Zn retention, transport, and cellular uptake (M ± SD) in enriched chicken emulsions. .. 70
Table 8.4. Se retention, transport, and cellular uptake (M ± SD) in enriched chicken emulsions. ... 71
Table 8.5. Retention time and abundance of the main phenolic present in hydroxytyrosol extracts
(HXT1, HXT2, and HXT3) ................................................................................................................. 72
Table 8.6. Nutritional composition (%) and fatty acid profile (%) of chiken meat, walnut paste, and
olive oil.............................................................................................................................................. 73
Table 8.7. Chemical composition of cooked chicken frankfurters elaborated with hydroxytyrosol,
walnut, and olive oil. ......................................................................................................................... 73
Table 8.8. Mineral content (mg/100 g) of chicken frankfurters elaborated with hydroxytyrosol,
walnut, and olive oil. ......................................................................................................................... 74
Table 8.9. Fatty acid profile (% of the most abundant) of chicken frankfurters elaborated with
hydroxytyrosol, walnut, and olive oil. ............................................................................................... 75
Table 8.10. Evolution of storage time on the fatty acid composition and nutrioncal index of chicken
frankfurters elaborated with hydroxytyrosol, walnut, and olive oil stored under modified
atmosphere during 21 days. ............................................................................................................... 76
Table 8.11. Effects of olive oil, hydroxytyrosol, extracts and walnut on colour (L∗ = lightness, a∗ =
redness, b∗ = yellowness) in frankfurters stored in modified atmosphere packaging (MAP: 70%
O2/20% CO2/10%N2) at day 0 of storage. ....................................................................................... 78
Table 8.12. Effects of olive oil, hydroxytyrosol extracts and walnut on thiobarbituric acid-reactive
substances (TBARs, mg MDA/kg product) in frankfurters stored in modified atmosphere packaging
(MAP: 70% O2/20% CO2/10%N2) during 21 days. ........................................................................ 81
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Table 8.13. Effects of olive oil, hydroxytyrosol extracts and walnut on concentration of protein
thiols in frankfurters stored in modified atmosphere packaging (MAP: 70% O2/20% CO2/10%N2)
during 21 days. .................................................................................................................................. 83
Table 8.14. Effect of storage time on the odour, flavour, acceptability of frankfurters stored in
modified atmosphere CO2/10 % N2) during 21 days ....................................................................... 85
Table 8.15. Proximal composition of chicken frozen nuggets enriched in Zn, Se, and phenolic
compounds from natural extracts.. .................................................................................................... 87
Table 8.16. Results of pH values and colour CIELab (M ± SD) in chicken frozen nuggets for
twelve months under frozen storage... ............................................................................................... 89
Table 8.17. Results of microbiological analysis (M ± SD cfu/g) in chicken frozen nuggets for
twelve months under frozen storage.... .............................................................................................. 91
Table 8.18. Total phenolic content (TPC) (mg GAE/100 g) and total nitrate content (TNC) (ppm
NO3-) in natural extracts (M ± SD)... ............................................................................................... 94
Table 8.19. Antioxidant activity of natural extracts by measuring their ABTS, and DPPH radical
scavenging activity, together with their ORAC and FRAP (µM TE/100 g) (M ± SD)... .................. 95
Table 8.20. Average values and standard deviations of volatile compounds /mg/g meat) in chorizo
for 0, 25, 50, and 125 days, under retail conditions... ....................................................................... 99
Table 8.21. Microbiological results (cfu/g) of Spanish chorizo analysis after 50 days under
refrigerated storage.. ........................................................................................................................ 101
Table 8.22. Proximate composition (g/100 g), airing losses (%), nitrate (ppm), and nitrite (ppm)
content (M ± SD) in Spanish “chorizo” enriched with natural extracts... ....................................... 110
Table 8.23. Results of pH, water activity (aw), and colour CIELab (M ± SD) in Spanish “chorizo”
enriched with natural extracts for 150 days of refrigerated storage... ............................................. 113
Table 8.24. Results of microbiological analysis (cfu/g) in Spanish “chorizo” after 50 days of
refrigerated storage... ....................................................................................................................... 114
Table 8.25. Results of protein oxidation related with thiol group loss (nmol thiol/mg protein),
respectively, for 150 days of refrigerated storage (M ± SD)... ........................................................ 115
Table 8.26. Evolution of volatile compounds of Spanish “chorizo” samples for 150 days of
refrigerated storage (M ± SD)... ...................................................................................................... 116
Table 8.27. Pearson correlations between different measured parameters... .................................. 121
Table 8.28. Total phenolic content (TPC) of natural extracts (mg GAE/g) (M ± SD) and their
antioxidant activity by measuring their ABTS, and DPPH radical scavenging activity, together to
their ORACHP, and FRAP (µM TE/g) (M ± SD)... ........................................................................ 124
Table 8.29. Antimicrobial activity of natural extracts measured by the disc difussion method (mm ±
SD)... ............................................................................................................................................... 125
Table 8.30. Average values and standard deviations of organic compounds (mg/g) in fish patties
stored for 11 days, under retail conditions... ................................................................................... 127
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Table 8.31. Microbiological results (cfu/g) of fish patties analysis at days 0, 4, 7, and 11 under
refrigerated storage... ....................................................................................................................... 128
Table 8.32. Proximal composition (M ± SD) of fish patties samples.... ......................................... 130
Table 8.33. Mineral content (M ± SD) (mg/100 g) of fish patties and RDA percent that supposes
consumption of 100 g per day. ........................................................................................................ 130
Table 8.34. Obtained results of pH and colour (CIELab) (M ± SD) evolution of fish patties for 14
days under refrigerated storage..... .................................................................................................. 131
Table 8.35. Obtained results of lipid oxidation (TBARs), protein oxidation (thiol loss), and fish
degradation (TMA and TVB-N) (M ± SD) of fish patties for 14 days under refrigerated storage..132
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FIGURE INDEX
Figure 2.1. General explanation of carcinogenic substances produced through Maillard reaction. ... 8
Figure 2.2. Nitrate and nitrite role in the dry-cured meat products. ................................................. 10
Figure 2.3. Autolytic changes of carbohydrates in muscle tissue of fish (Source: FAO, 1998). ..... 13
Figure 2.4. Trimethylamine formation during degradation of fish ................................................... 15
Figure 3.1. Scheme of different phases in lipid oxidation (modified from Guyon, Meynier &
Lamballerie, 2016). ........................................................................................................................... 20
Figure 3.2. Pathways for the oxidation of thiol groups in presence of different prooxidant agents
and effect in myofibrillar proteins (modified from: Ellgaard, Sevier & Bulleid, 2017; and Estévez,
2011). ................................................................................................................................................ 21
Figure 4.1. Chemical structures of commercial synthetic antioxidants. BHA (A), BHT (B), sodium
sulfite (C), nitrate (D), and nitrite (E). .............................................................................................. 27
Figure 4.2. Mediterrean and non-Mediterranean ingredients as source of natural extracts used in the
present thesis dissertation: EVOO (A), HXT (B), nuts (C), oregano (D), rosemary (E), garlic (F),
paprika (G), citrus (H), grape seed (I), pomegranate (J), lettuce (K), arugula (L), spinach (M), chard
(N), celery (O), watercress (P), beet (Q), acerola (R), harpagophyte (S). ......................................... 29
Figure 4.3. Chemical structures of TYR and HXT: phenolic compound from olive leave and olive
oil. TYR: tyrosol (left); HXT: hydroxytyrosol (right). ..................................................................... 30
Figure 4.4. Functional groups of phenolic compounds structure. (A) Phenol, (B) Catechol, (C)
Gallol. ................................................................................................................................................ 33
Figure 5.1. Strategies to improve the bromatological quality of animal origin products. Clean label
food production. ................................................................................................................................ 37
Figure 7.1. Graphical abstract of the development of the present thesis dissertation ...................... 48
Figure 7.2. Graphical abstract Assay I. Paper I ................................................................................ 50
Figure 7.3. Graphical abstract Assay II. Paper II and III ................................................................. 52
Figure 7.4. Graphical abstract Assay III. Paper IV .......................................................................... 53
Figure 7.5. Graphical abstract Assay IV. Paper V ........................................................................... 56
Figure 7.6. Graphical abstract Assay IV. Paper VI .......................................................................... 57
Figure 7.7. Graphical abstract Assay IV. Paper VII ......................................................................... 59
Figure 7.8. Graphical abstract Assay V. Paper VIII ......................................................................... 60
Figure 7.9. Graphical abstract Assay V. Paper IX ........................................................................... 61
Figure 8.1. Negative mycoplasma test in Caco-2 cell line. .............................................................. 67
Figure 8.2. Caco-2 cell development for 20th days of seeding (A: day 0; B: day 3; C: day 5; D: day
7; E: day 12; F: day 20). .................................................................................................................... 68
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Figure 8.3. Effect of addition of olive oil, walnut or hydroxytyrosol on cooking losses of cooked
frankfurters. a, b, c: Different letters between rows indicate significant differences (p<0.05). ........ 79
Figure 8.4. Scanning electron micrographs (magnification, 500×) of backfat (Control),
hydroxytyrosol extract1+ 2.5% walnut (HXT1) or hydroxytyrosl 1 + 2.5% walnut+ 20 g/100 g olive
oil (HXT1OLW). ................................................................................................................................... 86
Figure 8.5. Results of lipid oxidation, TBARs (mg MDA/kg) (A); protein oxidation, thiol groups
(nmol thiol/mg protein) (B) of chicken frozen nuggets for twelve months of storage.. .................... 90
Figure 8.6. Results of sensory evaluation (A: at time 0, and B: at month 12) of chicken frozen
nuggets for twelve months of storage.. ............................................................................................. 95
Figure 8.7. Antimicrobial activity of natural extracts expressed by bacterial growth (cfu) at
different concentrations in Clostridium perfringens NCTC 8237 CECT 376 after 48 h incubation at
37 °C under anaerobic conditions. (A) obtained results for Ct: Citric; R: Rosemary; Ac: Acerola;
(B) obtained results for Paprika, Garlic, and Oregano; (C) obtained results for L: Lettuce; A:
Arugula; S: Spinach; Ch: Chard; Ce: Celery; W: Watercress. Superscript letters indicate significant
differences (p < 0.05) between samples. Control sample represents the normal bacterial growth
without any extract. ........................................................................................................................... 98
Figure 8.8. Percentage thiol groups in meat model systems oxidized by AAPH (OXHydro) or
AMVN (OXLip) after addition of phenolic extracts (Citrus (500 ppm), Acerola (250 ppm) and
Rosemary (500 ppm)) (A), traditional ingredients (Paprika (30000 ppm), Garlic (4000 ppm) and
Oregano (4000 ppm)) (B), or natural nitrate sources (1500 ppm Beet, Lettuce, Arugula, Spinach,
Celery, Chard or Watercress) (C) relative to a control meat model system without oxidant (C-
NoOX). All data points represent the mean ± sd of triplicated determinations. Different letters (a-i)
indicate significant differences between samples (p<0.05). ............................................................ 103
Figure 8.9. Radical signal intensity in meat model systems oxidized by AAPH (OXHydro) or
AMVN (OXLip) after addition of phenolic extracts (Citrus (500 ppm), Acerola (250 ppm) and
Rosemary (500 ppm)) (A), traditional ingredients (Paprika (30000 ppm), Garlic (4000 ppm) and
Oregano (4000 ppm)) (B), or natural nitrate sources (1500 ppm Beet, Lettuce, Arugula, Spinach,
Celery, Chard or Watercress) (C) relative to a control meat model system without oxidant (C-
NoOX). All data points represent the mean ± sd of triplicated determinations. Different letters (a-g)
indicate significant differences between samples (p<0.05). ............................................................ 105
Figure 8.10. Percentage thiol groups in meat model systems oxidized by AAPH (OXHydro) or
AMVN (OXLip) after addition of 0, 0.001, 0.5, 37.5, 375, 1500, and 6000 ppm of NaNO2. All data
points represent the mean ± sd of triplicated determinations. Different letters (a-e) indicate
significant differences (p<0.05) between OXHydro samples and C-NoOX. Different letters (A-H)
indicate significant differences (p<0.05) between OXLip samples and C-NoOX. ......................... 107
Figure 8.11. Radical signal intensity in meat model systems oxidized by AAPH (OXHydro) or
AMVN (OXLip) after addition of 0, 0.001, 0.5, 37.5, 375, 1500, and 6000 ppm of NaNO2. All data
points represent the mean ± sd of triplicated determinations. Different letters (a-e) indicate
significant differences (p<0.05) between OXHydro samples and C-NoOX. Different letters (A-H)
indicate significant differences (p<0.05) between OXLip samples and C-NoOX. ......................... 108
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Figure 8.12. Relevant bioactive compounds from phenolic extracts, traditional ingredients, and
natural nitrate sources...................................................................................................................... 109
Figure 8.13. Results of organoleptic analysis, colour (A), odour (B), flavour (C), texture (D), and
Aceptability of Spanish “chorizo” at 50 days of chilled storage. (F) represents the hardness in
Newton (N) measured by a texturometer TA-XT2i (ANAME, Madid, Spain). RLAW: 500 ppm
Rosemary extract + 250 ppm Acerola + 3000 ppm Lettuce, Arugula, and Watercress; RSCe: 500
ppm Rosemary extract + 250 ppm Acerola + 3000 ppm Spinach and Celery; RChB: 500 ppm
Rosemary extract + 250 ppm Acerola + 3000 ppm Chard and Beet; CLAW: 500 ppm Citric extract
+ 250 ppm Acerola + 3000 ppm Lettuce, Arugula, and Watercress; CSCe: 500 ppm Citric extract +
250 ppm Acerola + 3000 ppm Spinach and Celery; CChB: 500 ppm Citric extract + 250 ppm
Acerola + 3000 ppm Chard and Beet. ............................................................................................. 120
Figure 8.14. HPLC chromatograms for natural extract. (a) RA: Rosemary extract rich in
Rosmarinic Acid, (b) NOS: Rosemary extract rich in diterpenes and NOVS: Rosemary extract rich
in diterpenes and with lecitin as emulsifier, (c) P: Pomegranate extract, (d) HYT-F: Hydroxytyrosol
extract obtained from olive fruit, (e) HYT-L: Hydroxytyrosol extract obtained from olive leaf. ... 122
Figure 8.15. Obtained results of organoleptic analysis of fish patties enriched in phenolic
compounds and essential fatty acids. .............................................................................................. 134
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List of Publications
1. Nieto, G., Martínez, L., Castillo, J., Ros, G. (2017). Effect of hydroxytyrosol, walnut and
olive oil on nutritional profile of low-fat chicken frankfurters. European Journal of Lipid
Science and Technology, 119: 1600518. DOI: 10.1002/ejlt.201600518,
2. Nieto, G. Martínez, L., Castillo, J., Ros, G. (2017). Hydroxytyrosol extracts, olive oil and
walnuts as functional components in chicken sausages. Wiley Online Library. DOI:
10.1002/jsfa.8240.
3. Martínez, L., Ros, G., Nieto, G. (2018). Fe, Zn and Se bioavailability in chicken meat
emulsions enriched with minerals, hydroxytyrosol and Extra Virgin Olive Oil as measured
by Caco-2 cell model. Nutrients, 10(8): E969. DOI: 10.3390/nu10080969.
4. Martínez, L., Castillo, J., Ros, G., Nieto, G. (2019). Antioxidant and antimicrobial activity
of rosemary, hydroxytyrosol, and pomegranate natural extracts in fish patties. Antioxidants,
8(4): 84. DOI: 10.3390/antiox8040086.
5. Martínez, L., Bastida, P., Castillo, J., Ros, G., Nieto, G. (2019). Green alternatives to
synthetic antioxidants, antimicrobials, nitrates, and nitrites in Clean Label Spanish chorizo.
Antioxidants, 8(6): E184. DOI: 10.3390/antiox8060184.
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Abbreviations
AAPH: 2,2-Azobis(2-methylpropionamidine)
dihydrochloride
ABTS: 2,2’-Azino-bis(3-ethylbenzthiazoline-
6-sulfonic acid)
ANOVA: Analysis of variance
AOAC: Association of Official Agricultural
Chemists
aw: Water activity
BHA: Butylated hydroxyanisole
BHT: Butylated hydroxytoluene
Ca: Calcium
CAE: Código Alimentario Español (Spanish
Food Code)
CDVs: Cardiovascular diseases
CFU: Colony forming units
Cl: Chlorine
DMA: Dimethylamine
DPPH: 2,2-Diphenyl-1-picrylhydrazyl
DHA: Docosahexanoic acid
EPA: Eicosapentanoic acid
EVOO: Extra Virgin Olive Oil
FA: Formaldehyde
FAO: Food and Agriculture Organization
Fe: Iron
FRAP: Ferric reducing antioxidant power
GC: Gas chromatograph
GC-MS: Gas chromatograph-mass
spectrometer
HCAs: Heterocyclic amines
HDL: High-density lipoprotein
HPLC: High performance liquid
chromatography
HXT: Hydroxytyrosol
IARC: International Agency for Research on
Cancer
ICP-OES: Inductively coupled plasma-
optical emisión spectroscopy
ISO: International Organization for
Standardization
K: Potassium
LAB: Lactic acid bacteria
LDL: Low-density lipoprotein
Mg: Magnesium
MALDI-TOF/TOF: Matrix-Assisted Laser
Desorption/Ionization - Time-Of-Flight
MAP: Modified atmosphere packaging
Mb: Myoglobin
MD: Mediterranean Diet
MHC: Myosin Heavy Chain
MMb: methamyoglobin
MMbNO: Nitrosylmethamyoglobin
MbNO: Nitrosomyoglobin
MDA: Malondialdehyde
Meq: Milliequivalents
MUFA: Monounsaturated fatty acid
Na: Sodium
Nd: No data
Ns: No significant
ORAC: Oxygen radical absorbance capacity
P: Phosphorus
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PAHs: Polyaromatic hydrocarbons
PBS: Phosphate-buffered saline
PCA: Plate count agar
PUFA: Polyunsaturated fatty acid
RDA: Recommended dietary allowances
ROS: Reactive oxygen specie
RT: Room temperature
S: Shulfur
SD: Standard deviation
Se: Selenium
SEM: Standard error mean
SFA: Saturated fatty acid
TBA: Thiobarbituric acid
TBARS: Thiobarbituric acid-reactive
substances
TBVN: Total Basic Volatile Nitrogen
TCA: Trichloroacetic acid
TCC: Total coliform count
TE: Trolox equivalents
TMA: trimethylamine
TMAO: trimethylamine oxide
TPC: Total phenolic content
TVC: Total viable count
USDA: United States Department of
Agriculture
WHO: Whorl Health Organization
Zn: Zinc
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1. Introduction
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Nowadays, consumer concerns have increased in order to demand new healthy and safer foods.
One reason is the potential risk of the consumption of synthetic additives such as BHA, BHT,
sulphites or nitrites, that are used as ingredients or additives in most of the animal products that
are frequently consumed in the diet globally and also in the Spanish diet (AESAN/MARM, 2011).
Based on this concern on heath perception, there is a new research trend to achieve the reduction
and/or replacement of these synthetic preservatives by natural extracts or essential oils from fruits,
plants or spices (Karre et al., 2013; Ahmad Shad et al., 2014; Jiang & Xiong, 2016). Additionally,
most of these natural extracts shown to be antioxidants in meat and fish, but they have a negative
impact on organoleptic characteristics of foods due to their high concentration in terpenoids and
phenolic. For this reason, its commercial application would not be viable, despite being focused
on a population increasingly aware of its health and that demands products free of synthetic
additives (Hung et al., 2016).
In this sense, one of the research fields is to study the different strategies to produce, select
and combine natural extracts that do not modify sensory parameters of animal origin food
products and to maintain their antioxidant, antimicrobial and preservative potential. Therefore,
the main objective of this project was to achieve a variety of food products of animal origin free
of artificial preservatives using natural plant and fruits extracts obtained from food industry by-
products, specially from traditional Mediterranean ingredients, among others. After a prelaminar
screening of the efficiency and effectiveness of a wide range of natural ingredients made by the
research group in combination with food and ingredients enterprises, the extracts selected for the
current PhD Thesis were citrus, grape seed, pomegranate, green leafy vegetables, Hydroxytyrosol
(from olive leave), acerola and rosemary, which were selected because of their richness in
bioactive compounds with promising antioxidant and antimicrobial activities.
Additionally, the strategy of adding these extracts to feed the animals was used in this PhD
Thesis. Organic forms of antioxidant minerals, such as Zn and Se, were used as a new way of
endogenous enrichment of the meat of ungulates and poultry (Calvo et al., 2016) directly related
to the resistance of the skin to external agents, of the carcasses and the bioavailability of these
trace minerals in the meat.
The incorporation of natural extracts and organic minerals at both endogenous level (in the
chicken diet) and exogenous level (in the elaboration of animal origin products) in order to
improve their bromatological quality, is a very interesting alternative when replacing synthetic
preservatives.
The experimental design of the present PhD Thesis is presented in Papers and future papers,
numbered I to IX, which are included as Annexes of the Thesis. Paper I (Assay I), which was
initiated during the master Thesis of the doctorate and completed during the current PhD Thesis,
describes the bromatological improvement through the endogenous incorporation of organic and
inorganic forms of Zn and Se, and the exogenous incorporation of natural ingredients from olive
leaves. This paper proposes the potential increasing of Fe, Zn and Se bioavailability measured in
vitro in a cell model Caco-2. Paper II and III are an extension of the previous study (Assay II),
where poultry meat emulsions were exogenously enriched with EVOO, HXT and walnuts to
improve the lipid profile, and shelf life during 21 days based on the oxidative. Paper IV (Assay
III) describes the maintenance of the shelf life of pre-cooked (fried) meat products (chicken
nuggets) endogenously enriched with organic and inorganic forms of Zn and Se, and exogenously
enriched with natural extracts obtained from grape seed, olive tree, rosemary, pomegranate, and
harpagophytum. Papers V, VI and VII were included in the Assay IV and were focused in the
production of traditional dry-cured meat products (Spanish “chorizo”) exogenously enriched with
Lorena Martínez Zamora PhD Thesis, 2019
4
phenolic rich extracts, from citrus, rosemary and acerola, and the traditional Spanish ingredients,
such as paprika, garlic and oregano, and natural nitrate sources from green leafy vegetables
(lettuce, arugula, spinach, chard, celery, watercress, and beet). In Paper V, antioxidant and
antimicrobial activities of natural extracts were tested and applied in the described food matrix.
Paper VI evaluates how affecting each extract by separated to protein oxidation in pork meat,
while Paper VII presents the shelf-life study carried out with the dry-cured meat samples for 150
days, focusing in the study of the organoleptic, oxidative and microbiological quality. Finally,
Papers VIII and IX (Assay V) are focused in the development of fish patties exogenously also
enriched with Mediterranean antioxidant extracts obtained from pomegranate, rosemary and olive
tree. Results of characterization of these extracts have been showed in Paper VIII, while the
Paper IX is a manuscript where the organoleptic, oxidative and microbiological changes of fish
patties for 14 days under refrigerated storage are showed.
The composition of the PhD Thesis was structured to disseminate basic knowledge on the
elaboration of Clean Label animal origin products, by following several treatments and the
organoleptic, oxidative and microbiological changes that are produced during the preservation of
this type of products. For that purpose, a literature review or state of the art about animal origin
products was carried out, including all the oxidative and degradation processes that result from
their preservation. This literature review was also focused in the use of synthetic additives and
the possible substitution by Mediterranean ingredients with potential benefits for health
consumers. Subsequently, the experimental part of the PhD Thesis is explained at the materials
and methods section, with a detailed description of the analytical techniques used and performed
throughout the experimental part. Finally, the results obtained in the project have been presented
in the Papers I to V, and properly discussed based on the previous chapters. Main conclusions of
the project and future perspectives within Clean Label animal origin products allow to give the
final remark to this PhD Dissertation.
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2. Animal origin products
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Animals have been the principal food source of proteins for humans since 5 million years ago.
However, in last century, the excessive intake of animal protein has influenced on human health,
due to the fact that population is more sedentary than before and combined with the high fat food
intake, an increment in heart diseases is produced (Larsen, 2003). The two most important food
groups into animal origin products are meat and fish.
2.1. Meat 2.1.1. Definition and chemical composition of meat.
Meat is defined by the Codex Alimentarius as “All parts of an animal that are intended for or
have been judged as safe and suitable for human consumption”. Notwithstanding, the CAE
defines meat as “the edible part of the muscles of healthy cattle, sheep, goat, pig, horse, and camel,
slaughtered in hygienic conditions, which is also applicable to poultry and marine mammals”.
Meat tissues are mainly composed by water (moisture), which constitutes approximately 75
%. Apart from that, proteins approximately constitute 19 % of the total weight, followed by lipids
(2.5 %), carbohydrates and inorganic matter (ash). Non-protein nitrogen compounds, such as
nucleotides, peptides, creatine, creatine phosphate, inosine monophosphate, dinucleotides,
nicotinamide-adenine and urea (1.5 %), together with non-nitrogenous compounds, such as
vitamins and organic acids (1 %) and inorganic matter (1 %) represent the remaining part
(Dikeman & Devine, 2014).
The water, protein and lipid content of meat depend on several factors like species, age,
anatomical location of meat piece and skin or bone presence, as well as the processing or the
incorporation of additional ingredients to manufactured meat products, such as, salt, alkaline
phosphates, nitrate or nitrites, sulphites, BHA (butylated hydroxyanisole), BHT (butylated
hydroxytoluene), sugars, spices and seasonings (Dikeman & Devine, 2014).
In addition, meat is an important source of 25 essential and non-essential elements. These
compounds are oxygen, carbon, hydrogen, nitrogen, minerals: Fe–heme, Ca, P, K, S, Na, Cl, Mg,
Zn and Se; and vitamins: A, thiamine, riboflavin, niacin, retinol, B6, folic acid, B12, D and K
(Dikeman & Devine, 2014). In this way, meat and meat product consumption provides high-
quality proteins and important substances necessary for a balanced diet.
However, these products are usually rich in saturated fatty acids and recently, the IARC
(International Agency for Research on Cancer), under the WHO (World Health Organization),
has classified processed meat as a carcinogen (Group I) and red meat as possible carcinogen
(Group 2A) (IARC, 2015). In fact, carcinogenic compounds in meat could be added during their
processing (synthetic additives), but they also can be formed during their storage through lipid
and protein oxidation, or during cooking through the Maillard reaction (Figure 2.1.) (Lund & Ray,
2017; Capuano & Fogliano, 2011).
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Figure 2.1. General explanation of carcinogenic substances produced through Maillard reaction.
Source: modified from Lund & Ray (2017) and Capuano & Fogliano (2011).
If the consumption of a reasonable amount of meat is evaluated as part of balanced human
diet, it is important to note that is an excellent source of minerals, vitamins, proteins and essential
amino acids. The consumption of fresh and processed meat is increasing worldwide, for example
the consumption of pork is 115.5 million tonnes and 108.7 million tonnes for poultry (USDA,
2017). Parallelly, in Spain, after milk and cereals, meat and meat products are the food group
most consumed. Actually, 23 % of consumed meat products are manufactured and 28 % chicken
meat (AESAN/MARM, 2011). Therefore, if the influence of meat consumption on human health
is taking into account and that among the most frequently consumed meat products are
frankfurter-type sausages, chicken nuggets and dry-cured meat products, the development of
healthier manufactured meat products containing lower amounts of fat, salt and with natural
ingredients is a good strategy to improve human health. Actually, in this fact lies the state of art
of the present doctoral thesis. The need of develop manufactured animal products free of synthetic
additives with potential carcinogenic activity through the incorporation of natural extracts
obtained from traditional ingredients of the Mediterranean Diet.
2.1.2. Meat emulsions: frankfurter-type sausages
Among the most frequently consumed meat products are frankfurter-type sausages. The meat
emulsions that form sausages are finely comminute and cooked products composed of fat, muscle
proteins (which serve as natural emulsifiers), salt, water, ice and non-meat ingredients (Nieto et
al., 2014). During the emulsification process, the chemical interactions between fat and protein
and their respective concentrations affect emulsion stability and therefore the quality of the final
products. In such products, the most critical aspect is protein and fat stabilisation, an aspect that
affects subsequent cooking losses, texture, lipid and protein oxidation (Nieto et al., 2009). Bearing
in mind that the proportions of fat and protein must be suitable to stabilise the fat inside the protein
Lorena Martínez Zamora PhD Thesis, 2019
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matrix (through the formation of a protein film around the fat particles), an excessive reduction
of fat particle size or formulations that contain new ingredients such as fat replacers may result in
an inadequate fat: protein ratio or soluble protein ratio. According to Barbut (1998), all these
aspects that affect frankfurter stability reduce the quality of the final product.
In addition, preservative synthetic additives (BHT, BHA, phosphates and sulphites) are added
to meat emulsions in order to prolong their shelf-life at vacuum packed. Therefore, the future
perspectives of this kind of products are focused in the development of new products made of
natural ingredients rich in bioactive compounds as functional foods.
2.1.3. Pre-fried products: chicken nuggets
As it has been previously cited, poultry meat consumption has been increasing at a rapid rate
over the past 50 years (USDA, 2017) and it is expected to further increase in the next decade, as
the world population is growing. The rapid increase in poultry meat consumption has been due to
several factors, such as healthy image, low price and the availability and development of new
products made of poultry meat. Actually, the poultry industry has also focused on the
development of new products (e.g. chicken frankfurters or turkey ham). Ready-to-eat products of
easy preparation, which also helps the industry to maintain the sales during all the year.
Chicken nuggets are an example of this development, which had a significant impact on raising
consumption. This product was initially introduced in the Western world (Europe and USA) and
prepared from whole muscle white meat. Currently, it is sold by fast food restaurants and
purchased at stores all over the world. This kind of convenience products represent an overall
growing market, due to the reduction of time spent in food preparation, which has supposed a
huge opportunity for the food industry to develop and market food products ready-to-eat or
convenience products which require minimal preparation time (Barbut, 2011).
During chicken nugget manufacturing different batters, flavourings and breading materials are
included to achieve different appearance, texture and commercial value, among other type of
synthetic additives (BHT and BHA), phosphates, water, salt and sugars, with the purpose of
improve juiciness, yield and add flavour to the product.
In this way, this kind of products have been included into the group of processed meat
products, whose consumption results carcinogenic (IARC, 2015), as it has been previously cited.
Then, there is a need to develop free synthetic additives chicken nuggets in order to improve these
products.
2.1.4. Dry-cured products: Spanish “chorizo”
Dry-cured meat products are produced by selection, cutting and mincing of meat, fat and
condiments, spices and authorised additives, as the most of manufactured meat products.
However, during this type of elaborations dry-meat products are dried and ripened for a period of
time when dehydration produces biochemical and microbiological changes that develop their
characteristic odour and flavour.
Spanish “chorizo” is a traditional fermented sausage which is elaborated with pork meat,
curing salts, paprika (as the main spice among other ones, like garlic, and oregano) and starter
cultures that control the presence of microorganisms that can alter their quality. Once these
products are mixed and stuffed into natural or artificial casings, they are subjected to a curing-
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ripening process, that usually includes the fermentation. When their manufacture depends on the
action of microorganisms (added in the form of starter cultures, or present in the meat), these are
called fermented sausages (Marín-Juárez, 2005).
2.1.4.1. Dry-curing chemistry
Nitrate and nitrite
Nitrates and nitrites are authorised additives that act as the curing agents and carry out several
essential functions for the correct development of the product. In this way, nitrates are reduced to
nitrites by fermenting microorganisms (Micrococcus and Staphylococcus) that possess the
enzyme nitrate reductase. Then, nitrites can be reduced to nitrous oxide (NO) (Figure 2.2.)
(Alahakoon et al., 2015).
Figure 2.2. Nitrate and nitrite role in the dry-cured meat products. Source: modified from
Alahakoon et al. (2015)
Colour development
Colour is one of the factors that most affects the general appearance of meat and its alteration
is used by consumers to define the acceptability of the product (Erkmen & Bozoglu, 2016).
Manufactured meat products that contain nitrate and nitrite in their formula have a characteristic
colour due to the interaction of NO (derived from nitrite) with myoglobin (Mb). Nitrosylation of
myoglobin can occur in two pathways (Figure 2.2.). The direct way, in which Mb reacts with NO
by producing the pigment nitrosomyoglobin (MbNO); or the indirect way, in which myoglobin
oxidized (MMb) reacts with NO by producing nitrosylmethamyoglobin (MMbNO), that is also
reduced to form nitroso myoglobin (MbNO) (Erkmen & Bozoglu, 2016).
This function of maintenance of the colour in meat products is part from the antioxidative
activity of those compounds. The mechanisms of action of this antioxidant effect includes the
chelating effect of nitrites on free iron ions from heme-group degradation, protecting them to the
catalysis of lipid oxidation reactions. In addition, nitrite may react with amino acids containing
thiol groups (-SH) to form nitroso thiols (e.g. nitroso cysteine from reaction between cysteine and
free thiol groups), which also constitutes a reservoir of NO in cured meat products (Gaston, 1999).
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Nonetheless, generated NO from nitrites can be oxidized in presence of oxygen and form
nitrogen dioxide (NO2). This reaction can be understood as a protection mechanism in which NO
acts as oxygen scavenger (Erkmen & Bozoglu, 2016).
Microbiology of dry-cured process
There are microorganisms that are technologically important due to the fact that they help to
provide the aroma, texture, colour and final flavour characteristic of meat processing by
modifying its basic components (carbohydrates, proteins and lipids). In addition, they are used to
avoid possible defects in the ripening process produced by other microorganisms that normally
grow in the meat or comes from the manipulation. This group of microorganisms is formed by
“starter cultures”, which are usually freeze-dried in a powder preparation and carry out various
functions when they are added to the mass of meat and ingredients (Martín-Juárez, 2005).
During the ripening, water activity (aw) is decreasing from 0.99 to 0.96 due to the presence of
salt, curing agents, sugars, nitrate and nitrite. Once the mix of meat is stuffed into the casings
(artificial or naturals), sausages are maintained in a room with the temperature (12–25ºC) and
humidity (90–95 %) controlled in a short period of time (24–72 h). During this phase,
microorganisms, both from the meat and from “starter cultures”, metabolize sugars to produce
lactic acid and pH decreases to 5.0, approximately (around the isoelectric point of the meat
proteins (Demeyer, 1992)). This reduces the water retention capacity of the mass, making easier
the subsequent drying process, as well as promoting the coagulation of meat proteins, which gives
the characteristic texture parameters to the final product.
Together with the fermentation of sugars, meat proteins (actin and myosin) begin to be
degraded to peptides, which results in an increase of free nitrogen, due to the action of the
muscular proteases (cathepsin D). Parallelly, lipid hydrolysis or lipolysis is initiated, which also
affect to the organoleptic quality of the dry-cured sausages (Ordoñez et al., 1999).
Also, during this phase the LAB carry out the reduction of nitrates to nitrites resulting in the
formation of nitroso myoglobin, as it has been previously explained.
Nevertheless, it must be taken into account that with the incorporation of “starter cultures” and
the control of the dry-curing process it also avoids the growth of pathogenic bacteria, such as
Clostridium perfringens. This strain is an anaerobic gram-positive pathogenic bacterium, that has
the capacity to form spores being very ubiquitous in nature and in the intestinal tract of many
animals (Jackson et al., 2011).
In raw-cured products the role of nitrites in inhibiting the growth of this type of bacteria,
together with the decrease in pH and water activity, has been demonstrated by Jackson et al.
(2011). The inhibitory effect of nitrites can be explained by the interaction between the NO
produced from the degradation of these compounds by LAB and the Clostridium sulfoproteins.
This fact is due to the action of the enzyme’s ferredoxin and/or ferredoxin-pyruvate
oxidoreductase, which causes a decrease in intracellular levels of ATP of these bacteria (Hospital
et al., 2016).
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2.2. Fish 2.2.1. Definition and chemical composition of fish
Fish is defined by FAO as “any of the cold-blooded (ectothermic) aquatic vertebrates, without
including amphibians and aquatic reptiles”. Notwithstanding, the CAE defines fish as “any
vertebrate animal, marine or freshwater (fish, mammals, cetaceans, and amphibians), fresh or
preserved by approved procedures”. Hence, fresh fish is defined by FAO as “fish or fishery
products that have received no preserving treatment other than chilling”, while frozen fish is
defined as “fish that have been subjected to a freezing process sufficient to reduce the temperature
of the whole product to a level low enough to preserve the inherent quality of the fish and that
have been maintained at this low temperature (-18 / -20 ºC) during transportation, storage and
distribution up to and including the time of final sale”. Into the definition of fish, it can be
appreciated the difference with ready-to-eat fish products obtained from fresh fish and through
technological and adequate procedures, as in the case of fish patties.
This food group is known to be highly nutritious and one of the fundamental supports of the
Mediterranean diet. Fish generally have low calorie content, the moisture represents a variable
percentage (53-96 %) and are an important source of proteins of high biological value (18–20 %),
vitamins, minerals (Se, P, Fe, Mg and K). Nonetheless, this kind of products are rich in
monounsaturated and polyunsaturated fatty acids (Ω-3, 6, and 9), as DHA (docosahexaenoic acid)
and EPA (eicosapentaenoic acid), which give them an important role in human nutrition (Hosomi,
Yoshida & Fukunaga, 2012). Actually, their continued consumption contributes to normal heart
function and maintain normal blood cholesterol levels. In addition, fatty acids DHA and EPA are
essentials for the develop of the central nervous system during the first stages of life, but also to
avoid neurodegenerative chronic diseases (Hosomi, Yoshida & Fukunaga, 2012).
According to FAO, global per capita fish consumption has increased from 9.9 kg in the 60s to
20 kg in 2015 (FAO, 2016). However, unless worldwide fish consumption has increased in last
50 years, in Spain, the contrary occurs. In our country, the consumption of fish and fish products
has decreased from 26.4 kg per person to 25.5 kg in last 10 years. If these data are analysed, it
can be proved that the consumption by people under 35 years of age is more reduced that the
reference one above mentioned, especially by children under 15 years old (Martín-Cerdeño,
2017). In addition, the Region of Murcia is the 3rd Spanish autonomous community with the
lowest consumption of fish, preceded by the Canary Islands and the Balearic Islands. Therefore,
new fish products are needed to encourage the consumption of this food group. In this sense, the
development of healthy ready-to-eat products, such as fish patties could be a good strategy to
stimulate fish consumption, especially among young people (Martín-Cerdeño, 2017). Taking into
account that functional food is one that has shown to provide benefit (beyond its nutritional
effects) to specific functions of the human body, maintaining a correct state of health and well-
being and/or reducing the risk of disease (Palou, Serra & Pico, 2003).
Actually, fish itself can be considered as a functional food, due to the fact that it is rich in
components that improve the health of those who consume it. Fish is a source of long chain,
polyunsaturated fatty acids (PUFA) (Ω-3, 6 and 9), in particular, eicosapentaenoic acid (EPA),
docosahexaenoic acid (DHA) and, to a lesser extent, docosapentaenoic acid (DPA). These act in
the body by increasing HDL cholesterol, decreasing LDL cholesterol, triglycerides and blood
pressure and are a protective factor against autoimmune, inflammatory and cardiovascular
diseases, as it has been also previously explained (Hosomi, Yoshida & Fukunaga, 2012).
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2.2.2. Fish degradation mechanisms Although fish muscle can be considered sterile when it is still alive, its deterioration can occur
rapidly after capture (enzymatic autolysis) and during subsequent stages of production, processing
and storage (lipid oxidation and bacterial growth) (Uchyama & Ehira, 1974).
Autolytic changes of carbohydrates and nucleotides are the first that occur in the muscle tissue
of the fish and they begin with the degradation of glucose to lactic acid through the aerobic and
anaerobic intracellular breathing processes, which produces a decrease of the pH (Figure 2.3.).
Nevertheless, proteins are also degraded by muscle enzymes, which produces great changes in
texture properties of the meat of fish. Collagenases degrade muscle collagen, while digestive
enzymes, such as trypsin, chymotrypsin and carboxypeptidase, can produce the bursting of the
stomach during times of abundant feeding (FAO, 1998).
Figure 2.3. Autolytic changes of carbohydrates in muscle tissue of fish (Source: FAO, 1998).
Nevertheless, post-mortem changes of fish also include the hydrolysis of lipids that can form
diglycerides and free fatty acids due to the action of microbiological enzymes and lipases, which
is increased by high temperatures. Free fatty acids are oxidized by an autolytic mechanism,
through which hydroperoxides are formed and secondary products from this oxidation, such as
aldehydes, ketones, alcohols and short chain fatty acids, that produce the rancid flavour to the
product (FAO, 1998). This is a reaction of special relevance in fish, due to the high content of
polyunsaturated fatty acids that they contain, being responsible for changes in texture, aroma and
taste as well as alterations in their nutritional properties.
Lipid oxidation occurs after a chain reaction of free fatty acids, in which molecular oxygen
participates and three phases can be distinguished: initiation (formation of the lipid radical),
propagation (formation of the peroxyl radical) and termination (creation of oxidation secondary
products responsible for the alterations associated with rancidity) (Secci & Parisi, 2016).
On the other hand, trimethylamine oxide (TMAO) is an osmoregulatory compound present in
marine fish and its reduction is usually due to bacterial action, but some fish species present in
muscle tissue an enzyme (TMAO-ase) able to break down TMAO into dimethylamine (DMA)
and formaldehyde (FA) (Figure 2.4.).
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Visceral tissues have a high activity of the TMAO-ase enzyme, for this reason is really
important to eviscerate and clean the fish before freezing. If not, it has been demonstrated that the
accumulation of FA produces hardening in hake muscle, which is also increased at high
temperaturas under frozen storage (Gill et al., 1979). In next table (Table 2.1.), a summary
including the main post-mortem autolytic changes in refrigerated fish is presented.
Table 2.1. Summary of post-mortem autolytic changes in refrigerated fish (Source: FAO, 1992).
Enzyme Sustrate Observed changes
Glucolytic enzymes Glucogen Lactic acid production: decrease of pH.
Texture changes
High temperatures increase this
reaction.
Trypsin,
chymotrypsin and
carboxypeptidase
Proteins and peptides Bursting of the stomach
Lipase Free fatty acids Lipid oxidation and production of
rancid flavour.
Collagenase Connective tissue Softening of muscle tissue
TMAO-ase TMAO Hardening induced by FA production,
even under frozen storage.
Unless autolytic changes precede growth of microorganisms, this last is the main cause of
deterioration (25–30 % of the origin of the loss of quality). This fact is due to fish have high water
content, free amino acids, a high post-mortem pH level and the most marine species contain high
levels of TMAO, which promotes the bacterial growth (both Gram-positive and Gram-negative)
(Ghaly et al., 2010).
Actually, when fish has been just captured, the muscle tissue is sterile. However, once post-
mortem autolytic changes are carried out, skin and visceral bacteria start to grow and invade
muscle tissue. In addition, fish is deteriorated at different rates depending on storage conditions
and the type of skin of the fish (Ghaly et al., 2010). Otherwise, this bacterial growth is the
responsible of the increase of volatile compounds, such as trimethylamine (TMA), volatile
sulphurous compounds, aldehydes, ketones, hypoxanthine, as well as basic volatile nitrogen
compounds (FAO, 1998).
The reduction of TMAO is also associated with the bacterial growth of Photobacterium, Vibrio
and Shewanella putrefaciens, but it is also carried out by Aeromonas and Enterobacteriaceae.
During both the anaerobic and aerobic growth, S. putefraciens uses the cycle of Krebs, where
electrons are generated by a metabolic route (serine route) from carbon sources (acetate or
succinate) (Figure 2.4.).
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Figure 2.4. Trimethylamine formation during the degradation of fish. Source: modified from
Surette et al. (1988).
The production of TMA is carried out at the same time that the production of hypoxanthine,
from autolysis of nucleotides. However, hypoxanthine can also be formed under the bacterial
action of Pseudomonas spp., S. putrefaciens and P.phosphoreum (Surette et al., 1988).
TMA conform the major part of total basic volatile nitrogen (TBVN). Hence, TMAO is
decreasing in the fish, while TMA and TBVN reach the maximum level, due to the formation of
ammonia (NH3) and other volatile amines. Once fatty acids and proteins are degraded, they are
used as substrate of anaerobic bacteria that produce high quantities of ammonia. Even after that,
biogenic amines, such as histamine, putrescine and/or cadaverine, are formed from
decarboxylation of free amino acids as histidine, ornithine and lysine, leading to rotten smell
(FAO, 1998).
Reached this point, when TMA exceeds values of 15 mg TMA-N/100 g of fish, while levels
of TBVN and NH3 are increasing, it is completely deteriorated, it has lost all its organoleptic
quality and it results unpalatable (Dalgaard et al., 1993).
2.2.3. Fish patties
As it has been previously exposed, fish consumption per capita has decreased in our country
in last 20 years and more among young population. For this reason, new processed fish products
have been developed to increase the demand of this food group, base of our Mediterranean Diet.
However, this kind of products are rich in synthetic additives to prolong their shelf-life and many
alternatives for their replacement have not already been studied.
Yerlikaya, Gokoglu & Uran (2005) studied quality changes of fish patties produced from
anchovy during refrigerated storage, when these new formulas began to be found in supermarkets.
Sehgal et al. (2011) also studied the changes of microbiological growth and organoleptic quality
of fish patties prepared from carp, but they did not use natural antioxidants to preserve them. More
recently, Salgado et al. (2013) published their results of their study using sunflower protein films
enriched with clove essential oil and its potential application for the preservation of sardine
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patties, but they did not apply these functional ingredients directly to the formula. López-
Caballero (2005) and Nowzari et al. (2013) studied the incorporation of chitosan-gelatine blend
as a coating for fish patties and rainbow trout, respectively.
Beside the use of natural extracts, different techniques are used today to extend the shelf life
of fish and to postpone its deterioration. These techniques are based on the control of temperature,
water activity, oxygen and microbial load, or a combination of all of them. Refrigeration (storage
in temperatures between 0-4°C) is an efficient and simple method of preserving fish. Although it
cannot prevent microbiological spoilage or enzymatic activities, it slows down these processes,
as it has been previously explained (Sampels, 2015). Nevertheless, the most common control
procedure used to preserve the characteristics and quality of fish intact is freezing (Hall, 2011;
Jessen, Nielsen & Larsen, 2014). Additionally, the use of modified atmosphere packaging is
receiving special attention, as the reduction in oxygen content and the increase in carbon dioxide
and nitrogen leads to a longer shelf life of the product (Noseda et al., 2014). Nonetheless, it has
been considered that in order to appreciate how natural extracts act in the preservation of this kind
of products, it would be better to have a view in aerobic conditions, only using natural extracts as
antioxidants, without the incorporation of modified atmosphere packaging. Finally, the use of
high pressures has also become particularly important in recent years due to its high capacity to
inhibit the growth of microorganisms and autolytic enzymes, thus extending the shelf life of fish
(Santiago, 2017). However, this kind of procedures have not been studied in the present thesis.
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3. Oxidative deterioration in
animal origin products
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The oxidative degradation of animal origin products consists of reactions proceeding
successively. Protein and lipid oxidation mechanisms are able to reduce the shelf life of this food
group, decrease the nutritional value and alter the bromatological and organoleptic quality.
Actually, cutting, mincing, irradiation, handling, packaging, storage and cooking procedures
promote chemical and enzymatic processes in animal products that stimulates oxidative reactions.
These degradation reactions of lipids and proteins in meat promote the appearing of abnormal
odours, tastes, colours and even toxic compounds that can decrease the consumer acceptance
(Papuc et al., 2017). This present chapter gives an overview of the oxidation processes occurring in animal origin
products during storage and interactions between compounds derived from these oxidative
reactions.
3.1. Lipid oxidation
Lipid oxidation is considered one of the most important ways of deterioration in food products.
Lipid oxidation depends on fat content and composition (polyunsaturated fatty acids, triacyl
glycerides, phospholipids and sterols), but also it depends on the processing and the storage
conditions. The metal-catalysed lipid oxidation is a radical-derived chain reaction which takes
place in three simultaneous phases: 1) initiation, 2) propagation and 3) termination (Figure 3.1.).
1) Initiation: R• + LH → RH + L•
2) Propagation: L• + O2 → LOO•
LOO• + LH → LOOH + L•
3) Termination: LOO• + LOO• → LOOL + O2
LOO• + L• → LOOL
Lipid peroxidation in meat and meat products happens through the radical chain reaction
mechanism, although oxygen presence accelerates this process. This oxidation is due to several
factors such as polyunsaturated fatty acids concentration (PUFA), the deficit of antioxidants in
animal feed (tocopherol, rosmarinic acid) and a high concentration of prooxidants, free radicals
or added salt (NaCl). At the same time, these reactions produce reactive oxygen species (ROS)
like hydroxyl radical, superoxide anion, ferryl and perferryl species, lipid peroxyl radical and
secondary products like reactive carbonyl species (MDA (malondialdehyde) and 4-HNE (4-
hydroxynonenal)) responsible for the rancid flavour in animal products.
Initiation (1) of lipid oxidation starts in a double bond in an unsaturated fatty acid, through H
abstraction leaving a carbon centred radical on the fatty acid carbon chain. During propagation
(2), the carbon radical forms a lipid peroxyl radical (ROO•) due to the presence of molecular
oxygen. Then lipid oxidation propagates as the lipid peroxyl radical abstracts hydrogen atoms
forming lipid hydroperoxides and new lipid radicals. For this reason, the lipid hydroperoxides are
determined as primary lipid oxidation products. However, they are also decomposed in free
radicals and secondary lipid oxidation products, such as aldehydes, hydroxyl (HO•), per hydroxyl
(HOO•), alkoxyl radicals (RO•) and/or volatile compounds that alters the quality of the product.
Finally, lipid oxidation chain ends when oxygen is depleted or lipid radical species are increased
(3). The chain of oxidative reactions concludes when the radicals L•, LO• and LOO• react with
each other or with free radicals to generate non-radical stable compounds (3) (Papuc et al., 2017).
Nevertheless, lipid oxidation can also carry out by enzymatic action. Lipoxygenases are the
responsible of this reaction and it is carried out in four steps:
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The first one consists in the reduction of Fe3+ ion bound enzyme to Fe2+ and the subtraction of
a hydrogen. During second phase a delocalization of a double bound is carried out. Deoxygenation
of the lipid radical and formation of peroxyl radicals (ROO•) is occurred in the third step. In the
last phase, ROO• is reduced by action of Fe2+ and the protonation of the peroxyl anion is produced
(Papuc et al., 2017).
Figure 3.1. Scheme of different phases in lipid oxidation (modified from Guyon, Meynier &
Lamballerie, 2016).
Produced substances from lipid oxidation can be divided into two groups: primary and
secondary products. Lipid hydroperoxides conform the first group and they promote DNA
synthesis and begin the ornithine decarboxylase activity in the colonic mucosa, indicating an
improvement in tumorigenesis. However, secondary products from lipid oxidation, such as
carbonyls, alcohol, hydrocarbons and furans, are related with cytotoxic and mutagenic effects
(Papuc et al., 2017). Once these substances are in the circulatory system, they may affect vaious
organs, such as liver, kidneys, lungs and intestine (Kanner, 2007). Studies based on animal
experimentation have suggested that the ingestion of secondary products from lipid oxidation may
promote oxidative stress, LDL oxidation and generates dysfunction of red blood cells due to the
beginning of the oxidative cascade (Tesoriere et al., 2002; Papuc et al., 2017).
3.2. Protein oxidation mechanisms
Although protein oxidation has received less attention, it has a huge influence on quality of
meat (Nieto et al., 2013). Protein oxidation has been defined as a covalent modification of protein
induced either directly by reactive species or secondary products of oxidative stress (Xiong,
2010). The same oxidants that induce the lipid peroxidation produce this alteration and carbonyl
formation or thiol loss are common reactions in protein oxidation. Furthermore, proteins can react
with secondary products of lipid peroxidation like aldehydes and ketones to produce complexes
between proteins, proteins and carbonyls or proteins and lipids. In muscle fibres, hydroxyl radical
(OH•) in presence of Fe or Cu or ROS causes modifications of amino acids, like methionine,
lysine, arginine, histidine, tryptophan, valine, serine and proline. This reaction increases
proteolytic enzymes and protein polymerization, which produces soluble aggregates, that
promotes gelation and emulsification that modifies the texture and toughening of the meat (Xiong,
2010; Xiong et al., 2010; Estévez, 2011). But this not only is critical for organoleptic quality, but
it might have an impact on human health and safety. For example, during cooking it increases
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free radical generation while it decreases the antioxidant compounds in meat, which contribute to
protein oxidation.
In general, protein oxidation begins by a hydrogen atom abstraction from a susceptible protein
and as a result it generates a protein radical, which in presence of oxygen it will form a protein
peroxyl radical (POO•) that may decompose in the presence of transition metal ions and propagate
the oxidation processes. In the present thesis dissertation, protein disulphides as protein oxidation
products have been considered. Figure 3.2. represents the different reactions that can be occurred
during the oxidation of thiol groups in presence of different prooxidant agents, together with the
effect of the thiol oxidation in myofibrillar proteins.
The formation of disulphide bonds involves a series of thiol and disulphide reactions, which
may be oxidized in the presence of metal ions, which can also decompose into cross-linked
structures. As it has been showed in Figure 3.2. this reaction can be increased by presence of small
molecules, that includes hydrogen peroxide, hydrogen sulphide, nitric oxide and glutathione and
which mediate sulfenylation, sulfhydration, nitrosylation, and glutathionylation, respectively.
The greater oxidation processes take place in different locations into proteins. In the side
chains of amino acid residues oxidation causes solubility loss, essential amino acid loss and an
increment of protein aggregation. Otherwise, the oxidation of the backbone of a protein promotes
modification in the atoms of the polypeptide chain, fragmentation, aggregation and
polymerization of the proteins (Papuc et al., 2017). Myosin is the most affected protein, among
Figure 3.2. Pathways for
the oxidation of thiol groups
in presence of different
prooxidant agents and effect
in myofibrillar proteins
(modified from: Ellgaard,
Sevier & Bulleid, 2017; and
Estévez, 2011).
Lorena Martínez Zamora PhD Thesis, 2019
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other amino acids that are also especially sensitive to ROS, such as arginine, cysteine, histidine,
lysine, methionine, phenylalanine, proline, tryptophan and tyrosine (Lund et al., 2011).
The main reaction products of this oxidation are the protein disulphides that have demonstrated
to have a strong impact on the quality of meat, which can be appreciated as an increase of the
toughness of meat and a decrease of tenderness (Xiong et al., 2009). Otherwise, oxidation of
myofibrillar proteins by hydroxyl radicals (OH•) shows that cross-link formation and consequent
disulphide formation, which is related with an increased myosin heavy chain (MHC). The cross-
linked MHC has been correlated with a significant decrease in tenderness by several authors in
pork steaks (Lund et al., 2007) and beef steaks (Zakrys-Waliwander et al., 2012; and Delles,
Xiong & True, 2011).
Thiol groups can be oxidized producing the protein disulphides (RSSR), the main reaction
products, that have a large impact on the quality of meat (Lund et al., 2007). The polymerization
or aggregation of the myofibrillar proteins may lead to poor protein solubility and alteration of
other functional properties of the meat proteins (Tang et al., 2018).
Oxidatively modified meat proteins show altered protein functionalities as presented in Figure
3.2. These altered functionalities can be used to manufacture meat products with desired
properties, such as dry-cured meat products. However, for fresh meat products the impaired
protein functionalities are mainly considered damaging to the overall quality.
Actually, the problem with secondary products resulted from protein oxidation. According
with the results obtained by Rutherfurd, Montoya & Moughan (2014), protein oxidation produced
an increase of the protein denaturation and a decrease of the formation of non-digestible peptides
along with digestion process. This decrease of protein digestibility increased the amount of
protein substrate available for microbial enzymes in the colon, which indicates the potential harm
for human health. In addition, toxic ammonia, phenols, acyclic amines, cyclic amines, N-nitroso
compounds and sulphides are formed in the colon as a consequence of protein oxidation. For
instance, ammonia generated due to amino acid deamination is suspected to promote tumour
formation following several pathways, such as modification of the morphology and metabolism
of intestinal cells, alteration on the pattern of DNA replication and early death of intestinal cells
(Papuc et al., 2017). Nonetheless, acyclic amines, such as tyramine, pyrrolidine, piperidine,
cadaverine, putrescine, are precursors of N-nitroso compounds, that have been classified as
potentially carcinogenic, as well as cyclic amines also are potential carcinogenic compounds that
are also produced by hydrolysis and decarboxylation of amino acids. Some harmful effects related
with these substances were observed in the organism, such as cancer, ulcerative colitis, alteration
of cellular homeostasis, modulation of gene expression and increase of inflammatory and DNA
repair responses (Papuc et al., 2017). For these reasons, the inadequate integration of oxidized
amino acids in the protein leads to structural problems in the molecule and, as a consequence, in
human health.
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4. Use of antioxidant and
antimicrobial compounds to
preserve animal origin products
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Oxidation of animal origin products can be reduced by addition of synthetic or natural
additives with antioxidant properties. In this present chapter, a description about antioxidants
compounds, both synthetic and natural origin and their role in the preservation of animal products
is shown. In addition, a study of the different reactions between bioactive compounds from
additives and nutrients from food (lipids and proteins) has been carried out.
According to the Codex Alimentarius, a food additive is any substance with no nutritional
value that is incorporated into a food solely for technological or organoleptic purposes during the
production of that food.
Antioxidant compounds are substances that delay the oxidation on food products by inhibiting
the free radical formation or interrupting this pathway through some specific mechanisms. One
of these pathways is the hydrogen atoms transference, when the antioxidant compound (AH) gives
a H to a free radical (R•), generating a more stable radical (A•) (R• + AH → RH + A•). While the
other pathway is the electron transference, when AH gives an electron in order to reduce the free
radical (R• + AH → R- + AH•) (Brewer, 2011). Parallelly, regarding to their chemical nature and
origin, these compounds could also prevent against the bacterial development through the
inhibition of several functions, such as the bacteria cell wall maintenance, the protein synthesis,
transport or the DNA-replication, as principal antimicrobial mechanisms of action (Li et al.,
2017). On the other hand, nitrate and nitrite salts are used in food products for the control and
prevention of C. Botullinum growth. However, the consumption of this additive is regulated
because this substance is naturally present in soil, vegetables, water and animals and the normal
levels have increased in recent years due to the use of nitrogen fertilizers. For this reason, the use
of natural sources of nitrate from green leafy vegetables could prevent the abuse of synthetic
nitrates and develop Clean Label food products (Jiang & Xiong, 2016).
4.1. Synthetic additives, their antioxidative
mechanisms and health risks
For the processing of meat and fish products, food industry normally uses synthetic additives
as an efficient and economic system to reduce oxidative damage. According with their function,
additives authorised by the European Union are divided into: colourings, preservatives,
antioxidants, metal sequestrants, gelling agents, stabilisers, emulsifiers, thickeners, flavour
enhancers, waxes, sweeteners, products for the treatment of flours and starch derivatives. This
chapter is focused in preservatives and antioxidants additives, from which the most used in animal
origin products are BHT, BHA, sulphites, nitrates and nitrites. Their use in food is restricted to a
maximum amount marked by legislation, which is due to their potential toxic effects. Hence,
synthetic additives such as sulphites, BHT (butylated hydroxytoluene) and BHA (butylated
hydroxy anisole) are added in meat product formulation to preserve them. The use of these
synthetic additives has given rise to social concern by consumers, due to studies that correlates
their consumption with disease development (asthma, hyperactivity, cancer, etc.) (Soubra et al.,
2007; Chang & Pan, 2008; Clough, 2014).
BHA (E-320) is a monophenolic antioxidant produced by the mixture of two isomeric
compounds: 2-tert-butyl-4-hydroxyanisole (90 %) and 3-tert-butyl-4-hydroxyanisole (10 %). In
fact, this compound is efficient controlling oxidation reactions in products with short chain fatty
acids, for this reason it has been widely used as antioxidant in food, food packaging, animal feed,
cosmetics, rubber, cosmetics and medicines. Its role consists on inhibition of reaction brought
about by dioxygen or peroxides (Shahidi & Ambigaipalan, 2015). However, its extensive
Lorena Martínez Zamora PhD Thesis, 2019
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application and consumption have produced the development of allergic reactions cause of
dermatitis (Clough, 2014; Weber, 2014).
BHT (E-321) is also a monophenolic antioxidant with lipophilic properties. Nevertheless,
effectiveness of BHT is lower than BHA due to the presence of two tert-butyl groups, which
provide a higher steric hindrance, for this reason their combination has been widely used for the
protection against oxidation in soybean oil, but also in fuel and other materials where free radicals
must be controlled (Shahidi & Ambigaipalan, 2015). Otherwise, its application has increasing
adverse reactions in humans, such as dermatitis of chronic urticaria (Weber, 2014).
Sulphites (from E-220 to E-228) are widely distributed throughout the food sector. In fact,
they are the most commonly group of additives used in the meat industry, capable of maintaining
the red colour of meat for longer and extending its shelf-life (even when it is not in perfect
freshness). Sulphites are sulphur derivatives that are used as preservative additives in foods in
order to prevent lipid oxidation, maintain their original colour, prolong their shelf-life and prevent
the growth of bacteria, moulds and yeasts, especially in an acidic environment. In wine, sulphites
are naturally found at low levels, although they are also added artificially to ensure inhibition of
the growth of bacteria, preventing oxidation of the wine and preserving its aroma. In addition,
they are also applied in commercial sauces, fruit derivatives and vegetable or seafood preserves
(Clough, 2014). However, in the human body this additive is metabolized by the enzyme sulphite
oxidase. In subjects with a deficient enzymatic activity, such as asthmatics, their consumption can
produce harmful reactions, such as shortness of breath, wheezing, coughing, dermatitis, headache,
irritation of the gastrointestinal tract and even anaphylactic shock or serious brain damage
(Clough, 2014).
One of the latest publications that has demonstrated the prooxidant and altering capacity of
sulphites is the study carried out by Parmeggiani et al. (2015). In this research, they conducted an
study in vitro on various areas of the cerebral cortex of rats has been shown how sulphites and
thiosulphites, applied at low concentrations (from 10 to 500 mM), accumulated in deficiency of
sulphite oxidase, which reduces the uptake of glutamate, inhibits the activity of glutamine
synthetase and other enzymes related to glutathione metabolism, contributing to brain damage
and impairing glutamatergic neurotransmission and redox homeostasis in the cerebral cortex. This
aspect makes it possible to clarify why in patients with rare diseases such as Sulphite oxidase
deficiency (SOX) there is severe neurological dysfunction accompanied by convulsions. Taking
into account the relationship between respiratory conditions and consumption of sulphites,
Ranguelova et al. (2013) examined oxidative damage caused by sulphite-derived free radicals in
human neutrophils in vitro by the formation of protein radicals, which demonstrated damage to
myeloperoxidase radicals, a heme protein secreted by activated neutrophils that plays a central
role in allergic reactions.
Otherwise, nitrates are also very important in the preservation of meat products by obtaining
the reddish and pink colours typical of cured and cooked products, respectively. Its active
component is nitrite, in which it is converted by enzyme catalysed reduction of bacteria from the
ripening, as it has been previously described (2.1.4.1.). However, its prolonged use presents
certain risks for the health of the consumer. The first risk is acute toxicity, where two grams of
nitrite can cause the death of one person (Özen et al., 2014). For this reason, the admissible
maximum dose added for cured and untreated meat products is set at 100 and 150 mg/kg
respectively. Whereas the maximum number of nitrates added in fresh and cured meat products
has been set at 250 and 150 mg/kg, respectively. Moreover, these compounds can lead to food
poisoning in more vulnerable population groups. In fact, carcinogenic compounds are formed
Lorena Martínez Zamora PhD Thesis, 2019
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during the cooking of meat products, such as N-nitrosamines, which are formed when nitrites are
combined with biogenic amines present in fermented dry sausages, which reach our organism
through their consumption to bioaccumulate and cause alterations in healthy cells (De Mey et al.,
2014; Herrmann et al., 2015; Crews 2014). On the other hand, when nitrite reaches the
bloodstream it reacts with haemoglobin oxidizing it and forming methaemoglobin, reducing the
capacity of this compound to transport oxygen and may cause serious health problems (Jang &
Chen, 2015). In Figure 4.1., general molecular structure of described synthetic additives is
presented.
Figure 4.1. Chemical structures of commercial synthetic antioxidants. BHA (A), BHT (B),
sodium sulphite (C), nitrate (D) and nitrite (E).
The extensive consumption of synthetic antioxidants may produce allergic reactions and
chronic diseases, among other health risks. For this reason, there is a search for green alternatives
with antioxidant and antimicrobial properties obtained from new natural extracts and essential
oils from fruits, vegetables, herbs and spices, which have been studied in last twenty years for
their application in food industry.
4.2. Mediterranean ingredients, their
antioxidative mechanisms and health benefits
The term Mediterranean Diet (MD) has been recognised as Intangible Cultural Heritage of
Humanity by UNESCO in 2010. It refers to the dietary pattern followed by people who live in the
olive growing areas of the Mediterranean Sea and includes not only the diet, also the lifestyle,
with a cultural, social, territorial and environmental character (Trichopoulou et al., 2014). In
addition, the adherence to this dietary pattern has demonstrated to have potential health benefits,
such as cardioprotective, neuroprotective, antioxidant, anti-inflammatory and anticarcinogenic
(Trichopoulou et al., 2014). These beneficial effects are due to the presence of foods rich in
polyunsaturated fatty acids in the diet, from fish, Extra Virgen Olive Oil (EVOO) and nuts, as
well as vitamins, minerals and phenolic compounds from spices and herbs, such as oregano, olive
tree, rosemary, garlic, paprika, among other fruits and vegetables.
In fact, natural antioxidants obtained from MD can prevent lipid peroxidation on different
ways: preventing chain inhibition by scavenging initiating radicals, breaking chain reaction,
decomposing peroxides, decreasing localized oxygen concentrations and binding chain initiating
Lorena Martínez Zamora PhD Thesis, 2019
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catalyst such as metal ions. Therefore, the use of natural preservatives to keep the shelf life of
animal origin products has exhibited similar antioxidant properties compared to some synthetic
additives. For this reason, it is a promising tool due to that many fruits (grapes, grape seed,
pomegranate, date, kinnow mandarin), vegetables (broccoli, potato, drumstick, pumpkin), herbs
(olive leaf, acerola, grape seed, cocoa, green coffee, Ginkgo biloba, etc.) and spices (rosemary,
green tea, black pepper, garlic, oregano, cinnamon, sage, thyme, mint, ginger, clove) have
reported antioxidant properties in animal origin products (Jian & Xiong, 2016; Ahmad-Shah et
al., 2014; Nieto et al., 2010; Nieto et al., 2011).
Therefore, the main objective of this doctoral thesis has been to study natural extracts obtained
from the MD, such as, hydroxytyrosol (HXT), nuts, EVOO, rosemary (Rosmarinus officinalis L.),
pomegranate (Punica granatum), grape (Vitis vinífera) seed, garlic (Allium sativum), oregano
(Oreganum vulgare), paprika (from red peppers Capsicum annuum), citrics (Citrus sinensis) and
leafy green vegetables, such as lettuce (Lactuca sativa), arugula (Eruca vesicaria), spinach
(Spinacia oleracea), chard (Beta vulgaris subsp. vulgaris), celery (Apium graveolens), watercrees
(Portulaca oleracea) and beet (Beta vulgaris).
In addition, in the present thesis dissertation, acerola (Malpiguia emarginata) and
harpagophyte (Harpagophytum procumbens) from South America and Africa, respectively, have
been also studied due to their richness in bioactive compounds.
Figure 4.2. shows as scheme of the natural extracts from the Mediterranean Dieto f not that
have been studied during this doctoral thesis.
Lorena Martínez Zamora PhD Thesis, 2019
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Figure 4.2. Mediterranean and non-Mediterranean ingredients as source of natural extracts used
in the present Thesis dissertation: EVOO (A), HXT (B), nuts (C), oregano (D),
rosemary (E), garlic (F), paprika (G), citrus (H), grape seed (I), pomegranate (J),
lettuce (K), arugula (L), spinach (M), chard (N), celery (O), watercress (P), beet (Q),
acerola (R), harpagophyte (S).
4.2.1. Hydroxytyrosol One of most potent natural antioxidant extracts in MD is hydroxytyrosol (or 4-(2-
dihydroxyphenyl) ethanol) (HXT), just below gallic acid (Lee-Richard, 2014). This compound is
ten times more antioxidant than green tea and two times more than coenzyme Q10 (Lee-Richard,
2014). Additionally, HXT scavenging ability is comparable to oleuropein and catechol. HXT is a
phenylethanoid with demonstrated antioxidant properties in vitro, it is obtained from olive leaf
and oil from this fruit, responsible for intense flavour and aroma, being oleuropein its precursor
(Yadav & Singh, 2004; Wang et al., 2013). In addition, it has demonstrated this antioxidant
Lorena Martínez Zamora PhD Thesis, 2019
30
capacity in vivo in several studies in rats, such as Merra et al. (2014) or Lemonakis et al. (2017),
that showed the power of HXT to reduce the risk to suffer metabolic syndrome. In its chemical
structure, this compound has an additional hydroxyl group in its benzene ring, compared to the
tyrosol (TYR) (Figure 4.3.). Therefore, it obtains a greater function as a free radical scavenging,
increasing its antioxidant power, as well as its efficacy under stress conditions (Lemonakis et al.
2017).
Figure 4.3. Chemical structures of TYR and HXT: phenolic compound from olive leave and olive
oil. TYR: tyrosol (left); HXT: hydroxytyrosol (right).
This extract has demonstrated during the monitoring of this thesis project its antioxidant
capacity in meat products rich in unsaturated fatty acids like sausages and frankfurters with added
HXT, nuts and extra virgin olive oil (Assay II) (Nieto et al., 2017a; Nieto et al., 2017b). Moreover,
HXT is an antioxidant compound linked to certain minerals, such as gluconate Fe (II) in black
olives, which catalyzes the oxidation of this compound, so it is possible that HXT influence on
biological bioavailability of some minerals and trace elements (Wang et al., 2013).
4.2.2. Extra Virgin Olive Oil (EVOO) The main source of HXT is EVOO, once of the principal ingredients of MD, that is used as
cooking fat and salad dressing. EVOO is rich in unsaturated fatty acids (especially oleic) and
phenolic groups, as antioxidant substances, followed by tocopherols and carotenes, that are also
present. The phenols detected in EVOO can be divided into alcohols, acids, flavonoids, lignans
and psecoiridoids. In fact, HXT is the most important psecoiridoid in EVOO.
Great variations in the concentration of these antioxidant compounds exist according to one
olive oil or another (0.02-600 mg/kg), which can occur due to factors such as the olive variety,
ripening, processing or the region and cultivation technique used (Cicerale et al., 2009). These
compounds are characterised by their antimicrobial, antioxidant, anti-inflammatory and anti-
cancer biological properties. Numerous studies have demonstrated the capacity of EVOO
phenolic groups to reduce the excess of free radicals that can cause oxidative damage (De la Torre
Carbot, et al., 2010; Machowetz et al., 2007; Deiana et al., 2010; Loru et al., 2009; Visioli et al.,
2005; Trichopoulou & Dilis, 2007).
4.2.3. Nuts Otherwise, nuts are essential ingredients in MD and they are composed of water, proteins,
lipids (Ω-3 and Ω-6), carbohydrates, fibre, an excellent lipid profile and their antioxidant
compounds content. Thus, oleic and α-linolenic fatty acids account for 75 % of total lipids, while
SFAs do not exceed 7 %. This is because of they can be considered, after oily fish, the most
important source of ALA (Albert et al., 2002). Nuts are also very rich in antioxidants: vitamin E
(low levels of α-tocopherol, 2 mg, although very high levels of γ-tocopherol, 45 mg), polyphenols,
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31
Se, Zn, Mg and folic acid. For instance, Blomhoff et al. (2006) and López Uriarte et al. (2009)
demonstrated in vitro how lipid peroxidation or oxidative damage to animal DNA was reduced
by introducing tocopherols and polyphenols from nut extracts, as well as increased antioxidant
enzyme activity and decreased cholesterol oxidation products. Moreover, Torabian et al. (2009)
observed that incorporating nuts into meals created a decreasing effect on plasma oxidative
biomarkers on study subjects.
4.2.4. Spices and herbs MD is also characterized by the use of herbs and spices in its traditional recipes. Rosmarinus
officinalis is a natural woody perennial green herb from the Mediterranean region, which is rich
in phenolic compounds with anti-inflammatory, antioxidant, anti-aging, antibacterial and
anticancer properties (Alu’datt et al., 2018). The polyphenolic profile of this herb is characterized
by the presence of carnosic acid, carnosol, rosmarinic acid and hesperidin, as major components
(Tai et al., 2012). Among the most effective antioxidant constituents of rosemary, the cyclic
diterpene diphenols, carnosolic acid and carnosol have been identified. In addition, its extract
contains carnosic acid, epirosmanol, rosmanol, methylcarnosate, and isorosmanol (Tai et al.,
2012; Hölihan et al., 1984; Bozin et al., 2007). Rosmarinus officinalis, L. is a rich source of
phenolic compounds and their properties are derived from its extracts (Gao et al. 2014) and
essential oils (Olmedo et al., 2013). Both are used for the treatment of illnesses and in the food
preservation.
The chemical composition of oregano is divided into two groups: essential oils, such as thymol
or trans-Sabinene hydrate, with hydrophobic properties, and phenolic compounds, such as
phenolic acids (rosmarinic acid) and flavonoids (kaempferol, catechin or epicatechin, among
others), with hydrophilic properties. Actually, phenolic compounds are responsible of
characteristic flavour of this herb and the USDA database established the total phenolic content
of this herb at 3789 mg GAE per 100 g product (Haytowitz & Bhagwat, 2010). This fact can be
compared with current values obtained by us in one of our last study of 1439.7 mg GAE per 100
g oregano (water as solvent) or 5500 mg GAE per 100 g oregano (70 % methanol as solvent)
showed by Skendi, Irakli & Chatzopoulou (2017). Previous reports from different oregano species
have shown as the most common flavonoids found in oregano are flavones, flavonols, flavanones,
and flavanols.
Otherwise, paprika is a red powder condiment made from red peppers Capsicum annum. It is
one of the most commonly used species and natural colorant in the preparation of cured sausages
due to its characteristic aroma, colour, flavour, and antioxidant power. In addition, paprika is an
important source of bioactive compounds, such as carotenoids (β-carotene and β-cryptoxanthin
as major), vitamin E, C and phenolic compounds (feruloyl glycosides, luteolin and quercetin
glycosides) with excellent antimicrobial and antioxidant properties, among other health benefits
(Škrovánková et al., 2017; Molnár et al., 2018; Serrano et al., 2018).
Garlic (Allium sativum) has been studied, emphasizing its antioxidant and antimicrobial
properties, which have been associated with the high concentration of allicin and other
organosulfuric compounds, such as thiosulfinates and phenolic compounds, as flavonoids and
pherulic acids (Petropoulos et al., 2018). Likewise, oregano (Origanum vulgare) is also
commonly used as a flavouring in cured or fresh meat products with excellent antibiotic and
antioxidant properties related to its principal components as rosmarinic acid and monoterpenes,
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32
carvacrol and thymol, among other phenolic compounds, such as γ-terpinene and p-cymene
(Baranauskaite et al., 2017).
4.2.5. Fruits MD is also known by including fruits into its dietary pattern. Into this group, grape (Vitis
vinifera), pomegranate (Punica granatum) and citrics (Citrus sinensis), as well as, acerola
(Malpiguia emarginata) have been included.
Grape (Vitis vinifera) seed extract is obtained from wine production and it also has a high
content of phenolic compounds (eg. flavanols, ellagitannins, anthocyanins, stilbenes) that can act
as therapeutic antioxidant, anti-inflammatory and anticancer agents (Nowshehri et al., 2015).
Another such extract is Punica granatum, due to its high content of punicalagin, among other
phenolic compounds. This extract is obtained from peels of this fruit and its consumption also has
beneficial effects for the human body as an antioxidant, anti-inflammatory, antibacterial and
anticancer agent (Khwairakpam et al., 2018).
Citric extracts, obtained from a mix of sweet orange (Citrus sinensis) and bitter orange (Citrus
aurantium) rich in bioactive compounds, such as naringin and hesperidin, both glycosides of
flavanones that act as antioxidants for their great ability to chelate iron and activity of sweeping
their hydroxyl groups (Franco-Vega et al., 2016).
In addition, acerola, also known as Malpiguia emarginata, is a plant native to Central and
South America and one of the most important sources of vitamin C (ascorbic acid), along with
carotenoids and bioflavonoids as anthocyanins and flavonols that increase its antioxidant power
(Moura et al., 2018).
4.2.6. Green leafy vegetables
Green leafy vegetables are also grown in Mediterranean region and widely consumed by it
population. Natural nitrate sources have been studied in order to find potential substitutes of
synthetic nitrates and nitrites used in dry-cured food products, such as green leafy vegetables rich
in nitrates (beet, lettuce, arugula, watercress, celery, spinach, and chard) (Bahadoran et al., 2016;
Alahakoon et al., 2015). However, regular consumption of nitrites can affect to the human body
by different ways, such as, causing allergic problems, reacting with haemoglobin to produce
methaemoglobin in blood (reduce the transport capacity of oxygen) (Sindelar & Milkowski,
2012). Synthetic nitrate incorporation in cured meat product can produce a similar effect, because
nitrate is gradually reduced to nitrite in meat, unless in a minor concentration that human body
can metabolize and eliminate before being affected. In addition, green leafy vegetables are also
rich in phenolic compounds (both phenolic acids and flavonoids) able to act as antioxidant
compounds together with nitrates (Bahadoran et al., 2016; Alahakoon et al., 2015).
4.2.7. Harpagophyte Finally, Harpagophytum procumbens is an herb grown in southern Africa with a great anti-
inflammatory power (Mancwangui et al., 2012) due to its high content of iridoid and
phenylpropanoid glycosides that can contribute added value to the meat products made with it.
Lorena Martínez Zamora PhD Thesis, 2019
33
However, this plant has not reported an important antioxidant activity (Mancwangui et al., 2012),
neither it has not been included into food formula.
4.2.8. Antioxidant mechanisms of phenolic compounds
As it has been explained throughout this chapter, the antioxidant capacity of studied extracts
in the present doctoral project lies in bioactive compounds content, such as nitrates, vitamins and
phenolic compounds.
Phenols and polyphenols are secondary metabolites of plants that are essential for their growth
and development. In addition, they have an important defensive function, therefore they are
protective agents against the attack of pathogens. However, in their molecular structure, they are
formed by one aromatic ring with one or various hydroxyl groups. For this reason, they are
responsible for organoleptic properties and the antioxidant activity, which follows several modes
of actions: radical scavenging activity and metal chelating activity (Brewer, 2011).
For their classification, plant derived phenolics can be separated into phenolic acids
(Hydroxybenzoic and hydroxycynnamic acids), phenolic diterpenes, flavonoids, and volatile oils
(Brewer, 2011).
In fact, phenolic compounds have been classified by their structure and the presence of
functional groups, due to their antioxidant capacity depends on their presence. The antioxidative
efficiency depends on the number of hydroxyl groups (OH), but also on the order that OH groups
follow around the aromatic ring (ortho-, para- and meta-). In this way, phenol (A), catechol (B)
and gallol (C) groups are represented in Figure 4.4., as functional regions on the molecular
structures of phenolic compounds.
Figure 4.4. Functional groups of phenolic compounds structure. (A) Phenol, (B) Catechol, (C)
Gallol.
As it was explained by Jongberg (2012), polymerization, nucleophilic interactions and
regeneration of phenolics is the key of the antioxidative activity by which the pool of oxidizable
hydroxyl groups is reinforced. In fact, this regeneration is accelerated through reduction by
ascorbic acid, sulphur dioxide, citric acid and erythorbate (Waterhouse & Laurie, 2006). This
synergistic effect involves a reaction between phenol with a radical and reduces stable phenoxyl
radicals to regenerate the hydroxyl group (OH) (Uri, 1961; Singleton, 1987; Kroll et al. 2003).
For this reason, it can be justified the combination of phenolic compound sources with vitamin C
(e.g. acerola extract) in order to preserve animal origin products, as it has been carried out in the
present PhD Thesis.
Lorena Martínez Zamora PhD Thesis, 2019
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Lorena Martínez Zamora PhD Thesis, 2019
35
5. Development of Clean label
animal origin products
Lorena Martínez Zamora PhD Thesis, 2019
36
Lorena Martínez Zamora PhD Thesis, 2019
37
Nowadays and in a society destined for obesity and chronic and degenerative diseases, the
concept of "functional food" has become powerful. According to the consensus document
elaborated by FUFOSE (Functional Food Science in Europe): "A food may be considered
functional if it has been satisfactorily demonstrated that it has a beneficial effect on one or more
specific functions in the human body, beyond the usual nutritional effects and this being relevant
for the improvement of health and/or in the reduction of the risk of disease" (ILSI Europe, 1998).
Increasingly, these foods are a quick and easy option for those population groups that tend to
show dietary deficits or for people who wish to fortify their diet. For this reason, the food industry
has chosen to research into this field and the relationship between nutrients and diseases in order
to bring to the market new products that respond to the needs of a current population concerned
about their health.
Some possibilities exist in the design of potential functional animal origin products in order to
facility the presence of beneficial compounds and / or limit those that can produce harmful effects
on the consumer health (e.g. saturated fatty acids).
These strategies firstly focus on animal production (endogenously enrichment) and, secondly,
on technological systems (exogenously enrichment) (Figure 5.1.). Endogenously enrichment of
animal origin products can be commonly carried out throughout genetic and nutritional
modifications, such as animal feeding. Otherwise, the exogenously enrichment can be carried out
with the direct transformation of the raw material or the formulation of processed animal origin
products by incorporating potential functional ingredients.
Then, the oxidative stress in animal products could be avoided through two ways: ante-mortem
antioxidative strategies (endogenously enrichment) and post-mortem antioxidative stress
(exogenously enrichment), by application of extracts or essential oils, obtained as food industry
by-product, directly to the animal before slaughtering or after it during the elaboration or
packaging of manufactured animal origin products (Figure 5.1.).
Figure 5.1. Strategies to the improve bromatological quality of animal origin products. Clean
label food production.
Lorena Martínez Zamora PhD Thesis, 2019
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5.1. Ante-mortem antioxidant strategies
In recent years, the meat products improvement has focused on modifying its composition in
fatty acids and increasing the presence of antioxidants compounds (Higgs, 2000). Strategies
employed for this purpose, which are applied in animal production, are focused on breed selection,
genetic lines and changes in livestock feed. The results of these procedures have shown great
changes in the lipid fraction of carcasses, allowing a significant reduction of fat, reach up 30% in
pork, 15% in beef and 10% in sheep (Jiménez-Colmenero et al., 2001; Channon & Trout, 2002).
Then, genetic selection has getting reducing the amount of fat in the carcass, producing leaner
animals, meat with less fat marbling and a higher proportion of polyunsaturated fatty acids (Fortin
et al., 2005; Jiménez-Colmenero et al., 2010). Similar results were obtained by providing in the
diet of porks with vegetable and fish fats rich on MUFA and PUFA (Ω-3, 6 and 9) (Higgs, 2000;
Channon & Trout, 2002).
Nonetheless, the improvement of the fatty acids profile leads to increase the concentration of
double bonds, which enhances the susceptibility to meat oxidation. One of the most important
and pioneering studies in this area could be the supplementation of the diet of birds, porcine and
bovine with vitamin E (Decker et al., 2000). In the case of meat, Delles et al. (2014) verified that
the endogenous enrichment of chicken meat with Zn, Se and vitamin E decreased lipid and protein
oxidation, making it a good strategy for reducing the concentration of synthetic additives in meat
and meat products. Moreover, mineral supplementation in broilers increases their performance,
antioxidant enzyme activities and the bioavailability of minerals, which also improves the
nutritional quality of the meat (De Marco et al., 2017; Kakhi et al., 2016).
Early studies in porcine with a diet rich on vitamin E showed an improvement on immune
response (Ellis & Vorhies, 1976; Babinsky et al., 1994; Nemec et al., 1994). Along the same way,
Daza et al. (2000) demonstrated as supplementation with vitamin E and Se in weaned piglets
improved productivity of meat, the weight gain and increased the antibody formation in them.
Moreover, Cabrera et al. (2010) showed a significant improvement of the technological properties
of beef and minerals retention in different meat cuts of this animal after application of feed rich
in minerals like Se, Cu, Zn, Fe and Mn.
More recent studies, such as Lalpanmawia et al. (2014), demonstrated the effectiveness of
adding phytase on growth, nutrient utilization and bone mineralization in broilers. This is because
phytases act denaturing phytic acid from cereal, allowing intestinal absorption of minerals retain
by phytate (Ca, Zn, Se and Fe). In this dynamic, in order to enhance the absorption of nutrients
and improve the lipid profile in broilers, Mondal et al. (2007) conducted a study which showed
that the addition of Cu and soybean oil potentiated this effect. It was also showed as a smaller
amount of organic copper proteinate was more effective than inorganic copper sulphate, using
doses of 200 and 400 mg/kg.
Otherwise, Jaskiewicz et al. (2014) also presented an improvement of fatty acid profile and
the content of fat-soluble vitamins in broiler chickens after application of Camelina sativa oil,
rich on alpha-linolenic acid (ALA). In addition, Dvorska et al. (2007) demonstrated protective
and antioxidant effect of the addition of organic Se and glucomannans on feeds opposite the T-2
mycotoxin that affects the liver of these animals.
Lorena Martínez Zamora PhD Thesis, 2019
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5.2. Post-mortem antioxidant strategies
Technological strategies to optimize the composition of meat products often focus on
reformulating them through reduction or elimination of harmful compounds by the addition of
substances with positive implications for health, promoting the functional nature of these
derivatives. Thus, when reducing or eliminating a harmful compound, food industry focuses in
fat, calories and sodium nitrite, among other synthetic additives, such as sulphites, BHA or BHT,
that can cause damage to the health if the consumer eats large amounts or has a disease that makes
him vulnerable.
Meanwhile, in order to development of meat products, some ingredients are used for different
purposes, focusing on improving the technological properties during processing (colorants,
flavourings, sweeteners, acidulates, seasonings and spices, emulsifiers, stabilizers, salts,
phosphates, preservatives, antioxidants, humectants and fat or salt substitutes). However, the use
of natural ingredients is a strategy that is being developed during the last decade, due to the fact
that addition of natural ingredients endogenously and exogenously in food products with positive
implications for health.
The incorporation of these substances has been carried out directly or as a constituent of some
of its ingredients (extracts, flours, concentrates, homogenized, etc.). Some of them have been or
are being subjected to different studies through which it is a question of evaluating the
consequences on the processes of transformation, conservation, commercialization. and the
conditions of consumption. The most studied components for addition to functional foods have
been lipids, proteins, peptides and amino acids, probiotics, prebiotics or symbiotics, various
antioxidant compounds, minerals, phytosterols, phytoestrogens and other compounds, such as
polyphenols, soy isoflavones or compounds sulfurized from garlic or onion, among others.
The production of animal origin products with a healthier lipid profile has been carried out
through the substitution of animal fat by fish or vegetable oils, giving rise to products with a lower
SFA and cholesterol content, a higher amount of MUFA and PUFA and improvements in the Ω-
6/Ω-3 ratio.
The bibliography of those who have carried out studies on this effect is extensive. For example,
ingredients such as olive oil (López-López et al., 2009), nuts (Jiménez-Colmenero et al., 2010),
canola oil (Álvarez et al., 2011), grape seed essential oil (Choi et al., 2009), rosemary essential
oil (Estévez & Cava, 2005), rice fibre (Álvarez et al., 2011; Choi et al., 2009), linseed oil (Lunn
& Theobald, 2006), or fish oil (He, 2009); León et al., 2008), among others, have been
incorporated to animal origin products improving their bromatological quality and maintaining
their shelf-life.
In addition, there is also evidence that antioxidants ingested in the diet contribute to preventing
oxidative damage to the body, limiting the oxidation of lipids in food and reducing the risk of
certain diseases, such as CVDs, some types of cancer, Alzheimer's and cataracts, among others
(Lee et al., 1998; Chowdhury et al., 2014).
On the other hand, food industry generates an enormous amount of waste in form of skins,
seeds and leaves, whose disposal is a problem for the environment and expensive for companies
concerned. However, many residues from fruits are rich in phenolic compounds that can be
extracted and used by food industries as antioxidant and antimicrobial preservatives, as is the case
of the present work.
Lorena Martínez Zamora PhD Thesis, 2019
40
In this way, during the development of this doctoral project, the improvement of animal origin
products through both strategies has been conscientiously studied, also through the redaction of
exposed publications, among which two reviews about the use of natural extracts obtained from
oregano and HXT can be highlighted (Annexes) and which are going to be discussed in detail
below.
Lorena Martínez Zamora PhD Thesis, 2019
41
6. Justification and Objectives
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Lorena Martínez Zamora PhD Thesis, 2019
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The present Doctoral Thesis contributes to the knowledge in the field of elaboration of
new Clean Label animal origin products by different nutritional strategies. Once of them is
the incorporation of natural antioxidants obtained as food industry by-products applied to
animal origin products destined for human consumption. While the other is the enrichment of
animal diet by mineral fortification.
Nowadays, a general demand exists for the study of new antioxidant sources to avoid the
use of synthetic additives in foods, being able to maintain their shelf-life in a more sustainable
way.
Therefore, the general objective of this Doctoral Thesis was to study the incorporation of
new natural extracts and organic minerals both endogenously (through animal diet) and
exogenously (in the elaboration of manufactured products) in order to develop animal origin
foods with beneficial properties for health, decreasing and/or replacing the percentage of
animal fat, salt and synthetic additives, maintaining their shelf-life without modifying their
sensory characteristics. This main objective was achieved following the next:
1. Study of mineral bioavailability of inorganic and organic Zn and Se in
endogenously enriched chicken meat emulsions measured in vitro in a Caco-2 cell model.
2. Study the antioxidant and antimicrobial potential of natural extracts obtained as
food industry by-products from Mediterranean ingredients, acquiring knowledge about
their composition of bioactive compounds and their molecular structure.
3. Evaluation of the inhibitory effect of extracts in the bromatological quality
(physical-chemical, organoleptic and microbiological) of different animal origin
products:
a. To study of the synergistic combination of natural extracts
(hydroxytyrosol, rosemary, grape seed, pomegranate and harpagophyte) with
inorganic and organic Zn and Se to preserve chicken frozen pre-fried products.
b. To study of the combination of fat replacers (EVOO and nuts) with
natural extracts from olive tree (hydroxytyrosol) to produce Clean Label chicken
meat emulsions.
c. To study of the combination of natural nitrate (beet, lettuce, arugula,
spinach, celery, chard and watercress) and phenolic sources (citrus, rosemary,
acerola, paprika, garlic and oregano) to produce Clean Label traditional pork dry-
cured products.
d. To study of the combination of fat replacers (essentials oils from algae
and lindseed) with natural extracts (pomegranate, rosemary, hydroxytyrosol,
citrus and acerola) to produce Clean Label manufactured fish products.
The achievement of these objectives led to the results presented in this research work,
which has derived in the publication of several papers whose references are attached in the
annexe included at the end of the present manuscript and are also showed below:
I. Nieto, G., Martínez, L., Castillo, J., Ros, G. (2017). Effect of hydroxytyrosol,
walnut and olive oil on nutritional profile of low-fat chicken frankfurters. European
Journal of Lipid Science and Technology, 119: 1600518
II. Nieto, G. Martínez, L., Castillo, J., Ros, G. (2017). Hydroxytyrosol extracts, olive
oil and walnuts as functional components in chicken sausages. Wiley Online Library.
DOI: 10.1002/jsfa.8240
Lorena Martínez Zamora PhD Thesis, 2019
44
III. Martínez, L., Ros, G., Nieto, G. (2018). Fe, Zn and Se bioavailability in chicken
meat emulsions enriched with minerals, hydroxytyrosol and Extra Virgin Olive Oil as
measured by Caco-2 cell model. Nutrients, 10.
IV. Martínez, L., Castillo, J., Ros, G., Nieto, G. (2019). Antioxidant and
antimicrobial activity of rosemary, hydroxytyrosol and pomegranate natural extracts in
fish patties. Antioxidants, 8.
V. Martínez, L., Bastida, P., Castillo, J., Ros, G., Nieto, G. (2019). Green alternatives
to synthetic antioxidants, antimicrobials, nitrates and nitrites in Clean Label Spanish
“chorizo”. Antioxidants, 8(6).
In addition, future papers currently under review have been also drafted from the work carried
out during this doctoral project. They are exposed below and also attached at the end of this thesis
dissertation as future publications:
i. Martínez, L., Ros, G., Nieto, G. (2019). Effect of natural extracts obtained from
food industry by-products on nutritional quality and shelf-life of chicken nuggets
enriched with organic Zn and Se provided in broiler diet.
ii. Martínez, L., Jongberg, S., Skibsted, L., Ros, G., Nieto, G. (2019). Plant derived
ingredients rich in nitrates or phenolics for protection of pork against protein
oxidation.
iii. Martínez, L., Bastida, P., Ros, G., Nieto, G. (2019). Development of Clean Label
dry-cured meat products (Spanish “chorizo”) enriched with antioxidant
compounds and nitrates from fruits and vegetables.
iv. Martínez, L., Lloret, P., Ros, G., Nieto, G. (2019). Development of Clean Label
fish patties enriches in Omega-3 and natural extracts from fruits and herbs.
Lorena Martínez Zamora PhD Thesis, 2019
45
7. Experimental design
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Lorena Martínez Zamora PhD Thesis, 2019
47
This chapter provides an overview of the experiments, which have been conducted to address
the objective of the present thesis, which has been previously exposed in Chapter 6. Materials and
methods of each experiment are described in Papers and future Papers I-IX. The main focus of
the present thesis is the incorporation of natural antioxidant extracts rich in phenolic compounds,
among other bioactive compounds. Nonetheless, the Paper I describe other way to improve the
quality of animal origin products: the increase of bioavailability of antioxidant minerals (Zn and
Se) in combination with phenolic compounds (exogenously incorporated). In the present chapter,
all the assays included in this thesis dissertation are going to be exposed in order to relate them to
the common purpose for which they have been developed: the production of Clean Label animal
origin products through different approaches and possibilities (Figure 7.1.).
In addition, this experimental design has been structured in such a way that publications and
works pending publication follow the next logical order, which will be developed in the present
thesis dissertation.
I. Martínez, L., Ros, G., Nieto, G. (2018). Fe, Zn and Se bioavailability in chicken
meat emulsions enriched with minerals, hydroxytyrosol and Extra Virgin Olive Oil as
measured by Caco-2 cell model. Nutrients, 10.
II. Nieto, G., Martínez, L., Castillo, J., Ros, G. (2017). Effect of hydroxytyrosol,
walnut and olive oil on nutritional profile of low-fat chicken frankfurters. European Journal
of Lipid Science and Technology, 119: 1600518
III. Nieto, G. Martínez, L., Castillo, J., Ros, G. (2017). Hydroxytyrosol extracts, olive
oil and walnuts as functional components in chicken sausages. Wiley Online Library. DOI:
10.1002/jsfa.8240
IV. Martínez, L., Ros, G., Nieto, G. (2019). Effect of natural extracts obtained from
food industry by-products on nutritional quality and shelf-life of chicken nuggets enriched
with organic Zn and Se provided in broiler diet. Poultry Science.
V. Martínez, L., Bastida, P., Castillo, J., Ros, G., Nieto, G. (2019). Green alternatives
to synthetic antioxidants, antimicrobials, nitrates and nitrites in Clean Label Spanish chorizo.
Antioxidants, 8(6).
VI. Martínez, L., Jongberg, S., Skibsted, L., Ros, G., Nieto, G. (2019). Plant derived
ingredients rich in nitrates or phenolics for protection of pork against protein oxidation.
VII. Martínez, L., Bastida, P., Ros, G., Nieto, G. (2019). Development of Clean Label
dry-cured meat products (Spanish “chorizo”) enriched with antioxidant compounds and
nitrates from fruits and vegetables.
VIII. Martínez, L., Castillo, J., Ros, G., Nieto, G. (2019). Antioxidant and
antimicrobial activity of rosemary, hydroxytyrosol and pomegranate natural extracts in fish
patties. Antioxidants, 8.
IX. Martínez, L., Lloret, P., Ros, G., Nieto, G. (2019). Development of Clean Label
fish patties enriches in Omega-3 and natural extracts from fruits and herbs.
Lorena Martínez Zamora PhD Thesis, 2019
49
Figure 7.1. Graphical abstract of the development of the present thesis dissertation.
Lo
rena M
artín
ez Za
mo
ra
P
hD
Th
esis, 201
9
48
Lorena Martínez Zamora PhD Thesis, 2019
49
7.1. Assay I:
Study of endogenous enrichment of meat products through animal
diet
In this first Assay, two batches of chicken meat were used (from animals fed with an organic
or inorganic mineral enriched diet) to elaborate meat emulsions, whose formulation incorporated
HXT and EVOO, according to Table 7.1. Six different chicken emulsions were elaborated. Three
were made with chicken meat from broilers fed a diet supplemented with inorganic Zn and Se:
control (C), 50 ppm HXT (CHXT) and 50 ppm HXT and EVOO (9.5%) (CHXTOl); and three were
made with chicken meat supplemented with organic Zn and Se: control (SZ), 50 ppm HXT
(SZHXT) and 50 ppm HXT and EVOO (9.5%) (SZHXTOl).
Table 7.1. Ingredients (g) of chicken emulsion samples elaborated in Assay I.
Chicken meat emulsion
Ingredients (g) Enriched forms of Zn and Se
Inorganic Organic
C CHXT CHXTOl SZ SZHXT SZHXTOl
Chicken meat (g) 713 713 616 713 713 616
HXT (ppm) 0 50 50 0 50 50
EVOO (ml)1 0 0 100 0 0 100
Water (ml) 172 172 172 172 172 172
Ice (g) 100 100 100 100 100 100
Salt (g NaCl) 15 15 15 15 15 15
Total 1000 1050 1053 1000 1050 1053
HXT: Hydroxytyrosol (23% extract from vegetation waste water). EVOO: Extra Virgin Olive Oil. C: Control; CHXT: 50 ppm HTX; CHXTOl: 50 ppm HXT + 10% EVOO; SZ: Control fortified with Zn and Se meat; SZHXT: SZ + 50 ppm
HXT; SZHXTOl: SZ + 50 ppm HXT + 10% EVOO. 1Emulsion made with olive oil and 3% soy lecithin (see materials and methods section for further details).
After mixing all the ingredients, trimmed chicken meat was placed in a cutter and homogenised
for 1 min or until a final temperature of 15°C in a room at 4ºC (knife and bowl speeds 3000 and
10 rpm, respectively). Then samples were cooked in a bath to reach an internal temperature of
75°C. After cooking, they were left to cool at 4ºC.
Once samples were elaborated, scheme represented in Figure 7.2. was followed in order to
reach the main objective of this assay: measuring the in vitro mineral bioavailability of chicken
meat emulsion endogenously enriched in organic and inorganic forms of Zn and Se and
exogenously enriched in hydroxytyrosol and EVOO, in Caco-2 cells. Followed methods are also
explained in chapter 8 and in Paper I.
Lorena Martínez Zamora PhD Thesis, 2019
50
Figure 7.2. Graphical abstract Assay I. Paper I. HXT: Hydroxytyrosol (23% extract from vegetation waste water). EVOO: Extra Virgin Olive Oil. C: Control;
CHXT: 50 ppm HTX; CHXTOl: 50 ppm HXT + 10% EVOO; SZ: Control fortified with Zn and Se meat; SZHXT: SZ + 50 ppm HXT; SZHXTOl: SZ + 50 ppm HXT + 10% EVOO. ICP-OES: Inductively coupled plasma optical
emisión spectrometry.
7.2. Assay II:
Study of the exogenous enrichment of cooked meat product through
the addition of natural antioxidant extracts
In Assay II, the incorporation of three different extracts of HXT, from different origins,
combined with EVOO and walnuts to preserve and improve the quality of the fatty acids profile
in chicken frankfurters was studied for 21 days of chilling storage.
Formula used for each sample is described in Table 7.2. Pork fat and chicken meat were
purchased from a local butcher. The HXT extracts (HTX1, HXT2, HXT3) were obtained from
Nutrafur-Frutaron group (Alcantarilla, Murcia, Spain). Walnuts and virgin olive oil (Hacendado,
Spain) were purchased in a local supermarket.
The three HXT extracts used in this study are from olive plant materials obtained using
different extraction process: HXT 23% (HXT1) was obtained from olive waters during fruit
processing (separating the oil from wet centrifugation), using a solvent extraction and purification
process, including, crystallization and clarification steps. For that, the original plant material
(vegetation water) is dried under vacuum at 50-60°C until a solid is obtained. This solid is
suspended in 96% ethanol in a 1: 2 w/v ratio. The suspension is stirred for about 30 minutes at
room temperature. It is filtered through laboratory filter paper. A 1:1 ratio of water is added to the
obtained alcoholic solution. The obtained precipitate is filtered and removed. The filtered
hydroalcoholic solution is concentrated under vacuum until obtain a syrup with an HTX
Lorena Martínez Zamora PhD Thesis, 2019
51
percentage around 20-25%. Among the characteristic polyphenolic compounds from olive oil that
this extract contains are large quantities of fulvic acids.
HXT 7% (HXT2) was obtained from olive leaves (dehydrated) by hydroalcoholic extraction
and subsequent hydrolysis. In a first stage, ethanol extraction of 70% of the milled leaves is
carried out for 1 hour at 40°C of temperature. The extraction is filtered through a polypropylene
cloth. The hydroalcoholic solution obtained contains as main active compound the oleuropein that
is present in the leaves. This solution is concentrated to remove all ethanol. To the obtained
aqueous medium, sulfuric acid is then added until reaching a concentration of 0.5 N.
Ta
ble
7.2
. In
gre
die
nts
(g)
of
chic
ken
fra
nkfu
rter
s sa
mple
s el
abora
ted i
n A
ssa
y I
I. P
ap
ers
II a
nd
III
Ch
ick
en m
eat
fra
nk
furt
ers
Sa
mp
les:
C
H
XT
1
HX
T2
H
XT
3
Cw
C
OL
O
Lw
H
XT
1O
LW
Ch
ick
en m
eat
(g)
71
3
68
8
68
8
68
8
68
8
61
3
58
8
58
8
Ice
(g)
10
0
10
0
10
0
10
0
10
0
10
0
10
0
10
0
Wa
ter
(ml)
1
72
17
2
17
2
17
2
17
2
17
2
17
2
17
2
Wa
lnu
t p
ast
e (g
) 0
2
5
25
25
25
0
25
25
EV
OO
(m
l)a
0
0
0
0
0
10
0
10
0
10
0
HX
T (
pp
m)
0
50
50
50
0
0
0
50
Sa
lt (
g N
aC
l)
15
15
15
15
15
15
15
15
To
tal
10
00
10
00
10
00
10
00
10
00
10
00
10
00
10
00
C:
Co
ntr
ol;
HX
T1:
50
pp
m H
yd
rox
yty
roso
l (2
3 %
extr
act
fro
m v
eget
atio
n w
aste
wat
er)
+ 2
.5 %
wal
nu
t; H
XT
2:
50 p
pm
Hyd
roxy
tyro
sol
(7 %
ex
trac
t fr
om
oli
ve
leaf
) +
2.5
% w
aln
ut;
HX
T3:
50
ppm
Hyd
rox
yty
roso
l (7
% e
xtr
act
from
veg
etat
ion
was
te w
ater
) +
2.5
% w
alnu
t; C
w:
Co
ntr
ol
wal
nu
t 2
.5 %
; C
OL:
Contr
ol
Oli
ve
Oil
; O
LW
: O
liv
e oil
+
2.5
% w
alnu
t; H
XT
1O
LW
: 50
pp
m H
ydro
xy
tyro
sol
(23
% e
xtr
act
from
veg
etat
ion w
aste
wat
er)
+ o
live
oil
+ 2
.5 %
wal
nut.
aE
mu
lsio
n m
ade
wit
h o
live
oil
and
3%
so
y l
ecit
hin
(se
e m
ater
ials
and
met
hod
s se
ctio
n f
or
furt
her
det
ails
).
Lorena Martínez Zamora PhD Thesis, 2019
52
Subsequently, the process of hydrolysis of oleuropein takes place for 2 h at 50°C of temperature.
The medium is neutralized with calcium carbonate until a pH of 5.0-5.5. The calcium sulphate
formed is filtered and separated. The filtered aqueous solution is concentrated and dried under
vacuum at a maxim of 70°C of temperature. The solid obtained is a hygroscopic product with
HXT percentage around 7%.
Olive oil was used as pre-emulsified fat. For the emulsification process, 8 parts of hot water
were mixed for 2 min with one part of isolated egg yolk lecithin and 10 parts of olive oil, in a
TissueRuptor (Qiagen, Hombrechtikon, Switzerland) at 18000 rpm.
HXT 7% from olive waters (fruit processing) (HXT3) was obtained by liquid- liquid extraction
with ethanol. For that, the original plant material (vegetation water) is concentrated in vacuum at
temperature of 50-60ºC until a syrup of 65-70% solids (ºBrix) is obtained. This syrup is suspended
in 96% ethanol in a 1: 4 w/v ratio. The suspension is stirred for about 30 minutes at room
temperature. Two phases are generated and the mixture is allowed to decant for about 3 hours at
room temperature. The supernatant is removed, which is concentrated in vacuum and finally a
hygroscopic solid with an HTX percentage of around 7% was obtained.
Walnuts were processed according to the method described by Ayo et al. (2008). For that,
eight different chicken frankfurter formulations (each containing 1.5% salt) were prepared in a
cooler room (6 - 8ºC) to obtain 1 kg of batter for each formulation (Table 7.2.). Trays containing
the raw emulsion were put into an industrial pot and were cooked for 3 h at 72ºC. After cooking,
the sausages were immediately cooled with cold water for 2 min, packed in polystyrene trays
using modified atmosphere packaging (MAP) with an 10/20/10 of O2/CO2/N2 gas composition in
BB4L bags of low gas permeability (8-12 cm3/m2 per 24 h) (Cryovac, Fuenlabrada, Spain). The
sausages were stored in a cabinet illuminated with white fluorescent light (620 lux) simulating
retail display conditions at 4ºC for up to 21 days. After elaboration, material and methods
followed for the development of Paper II and III in Figure 7.3. They are also explained in this
section of Papers II and III.
Figure 7.3. Graphical abstract Assay II, Papers II and III.
EVOO: Extra Virgin Olive Oil; HXT: Hydroxytyrosol; TBARs: Thiobarbituric acid reactive substances.
Lorena Martínez Zamora PhD Thesis, 2019
53
7.3. Assay III: Study of endogenous and exogenous enrichment of frozen pre-
cooked meat products, through the incorporation of Zn and Se to
animal feed and natural antioxidant extracts during the elaboration of
chicken nuggets
For the development of Assay III, eight different chicken nugget samples were elaborated,
before they were separated into two batches: four samples were enriched endogenously with
inorganic Zn and Se (C) and four samples enriched endogenously with organic Zn and Se (SZ).
Chicken nugget samples were enriched exogenously with natural extracts obtained from plants
(Rosemary (RH and RL), Pomegranate (P), Grape Seed (GS), Hydroxytyrosol (HXT) and
Harpagophytum (H)), according to Table 7.3.
After mixing all the ingredients, trimmed chicken meat was placed in a cutter and homogenised
for 1 min or until reaching a temperature of 15°C in a room at 4ºC (knife and bowl speeds 3000
and 10 rpm, respectively). Chicken nuggets were prepared in characteristic shapes of 5 x 3 x 1
cm, each weighting 25 g and frozen at -18ºC.
Subsequently, all the nugget batches were pre-fried using a household fryer (Taurus S.L.,
Lérida, Spain) for 30 s at 165ºC in sunflower oil (Koipesol Semillas S.A., Sevilla, Spain). The
pre-fried nuggets were packaged in polyethylene bags and stored at -18ºC until analysis at month
0, 3, 6, 9 and 12 by triplicated.
In addition, experimental design of this Assay is represented in Figure 7.4. and followed
methods are explained in Paper IV.
Figure 7.4. Graphical abstract Assay III. Paper IV. TBARs: Thiobarbituric acid reactive substances; TVC: Total Viable Count; TCC: Total Coliform Count.
Lorena Martínez Zamora PhD Thesis, 2019
54
Ta
ble
7.3
. In
gre
die
nts
(g
) of
froze
n c
hic
ken
nugget
sam
ple
s el
abora
ted i
n A
ssay I
II.
Ing
red
ien
ts (
g)
Ch
ick
en m
eat
emu
lsio
n
E
nri
ched
wit
h i
no
rga
nic
fo
rms
of
Zn
an
d S
e E
nri
ched
wit
h o
rga
nic
fo
rms
of
Zn
an
d S
e
C
C
RH
+P
C
RL
+G
S
CH
YT
+P
+H
S
Z
SZ
RH
+P
SZ
RL
+G
S
SZ
HY
T+
P+
H
Ch
ick
en m
eat
(g)
67
5
67
2.5
6
72.5
6
72.2
5
67
5
67
2.5
6
72.5
6
72.2
5
Pla
nt
extr
act
(p
pm
)
•R
H
1
000
1
000
•P
15
00
1
500
1
500
1
500
•R
H
10
00
1
000
•G
S
15
00
1
500
•H
YT
75
0
7
50
•H
50
0
5
00
Wate
r (m
l)
15
0
15
0
15
0
15
0
15
0
15
0
15
0
15
0
Ice
(g)
10
0
10
0
10
0
10
0
10
0
10
0
10
0
10
0
Co
mm
erci
al
mix
® (
g/k
g)
75
75
75
75
75
75
75
75
To
tal
10
00
10
00
10
00
10
00
10
00
10
00
10
00
10
00
RH:
Ro
sem
ary
extr
act;
P:
Po
meg
ran
ate;
RL:
Ro
sem
ary
extr
act
(Nutr
ox
OS
); G
S:
Gra
pe
See
d;
HY
T:
Hyd
rox
yty
roso
l; H
: H
arpag
op
hytu
m.
C:
Co
ntr
ol;
CR
H+
P:
10
00
pp
m R
ose
mar
y e
xtr
act
+ 1
500
pp
m P
om
egra
nat
e ex
trac
t; C
RL
+G
S :
100
0 p
pm
Nu
trox
OS
+ 1
50
0 p
pm
Gra
pe
seed
ex
trac
t; C
HY
T+
P+
H:
150
0 p
pm
Po
meg
ran
ate
extr
act
+ 7
50
pp
m H
yd
roxy
tyro
sol
+ 5
00
pp
m H
arp
agoph
ytu
m;
SZ
: C
on
tro
l fo
rtif
ied
wit
h Z
n a
nd S
e m
eat;
SZ
RH
+P:
10
00
pp
m R
ose
mar
y e
xtr
act
+ 1
500
pp
m
Po
meg
ran
ate
extr
act;
SZ
RL
+G
S :
1000
pp
m N
utr
ox
OS
+ 1
500
ppm
Gra
pe
seed
extr
act;
SZ
HY
T+
P+
H :
15
00
pp
m P
om
egra
nat
e ex
trac
t +
75
0 p
pm
Hyd
rox
yty
roso
l +
50
0 p
pm
H
arp
agoph
ytu
m
Co
mm
erci
al m
ix ®
: m
ix o
f sp
ices
fo
r th
e p
repar
atio
n o
f ch
ick
en n
ugget
s, w
itho
ut
pre
serv
ativ
es n
eith
er s
ynth
etic
co
lou
rs,
supp
lied
by
Pim
urs
a S
.L.
(Murc
ia, S
pai
n).
Lorena Martínez Zamora PhD Thesis, 2019
55
7.4. Assay IV:
Study of exogenous enrichment of dry-cured meat products
through the addition of natural antioxidant and nitrate source
extracts For the development of this Assay, experiments were divided into three phases. In the first
one, antioxidant and antimicrobial capacities of several natural extracts were evaluated for they
application in a dry-cured Spanish “chorizo”. The second one was focused in the study of these
extracts in an oxidized pork meat model system in order to study how affect them to protein
oxidation. In the third phase, a shelf-life study of a dry-cured Spanish “chorizo” was carried out
for 150 days in order to know how affect the incorporation of natural extracts to the quality of
this kind of product.
7.4.1. Characterization of natural extracts and application in
Spanish “chorizo”
Firstly, natural antioxidant extracts (from citric, rosemary and acerola) together with
traditional ingredients of cured meat products (paprika, garlic and oregano) and natural sources
of nitrates obtained from green leafy vegetables (beet, lettuce, arugula, spinach, chard, celery and
watercress) were tested for their potential use in the food industry as antioxidant and antimicrobial
extracts. In addition, in this first phase, these extracts in combination were also used for the
formulation of eight different dry-cured products.
For that, eight different batches (10 samples per batch) of Spanish chorizo were manufactured
using the recipe shown in Table 7.4. Minced meat was purchased in a local supermarket, Hipercor,
S.A. (Murcia, Spain). Dextrose, meat protein and the commercial mix of additives and spices
composed of spices, salt, dextrose, lactose, milk protein, emulsifiers (triphosphates E-451,
diphosphate E-450), flavour enhancer (monosodium glutamate E-621), preservative (sodium
nitrate E-251), antioxidant (sodium ascorbate E-301) and colouring (carminic acid E-120), was
used as the control sample (C). A commercial starter culture composed of Pediococcus (50 g per
100 g culture), Staphylococcus xylosus (25 g per 100 g culture) and Staphylococcus carnosus (25
g per 100 g culture). The lyophilised cultures were rehydrated (50 g in 750 ml of chlorinated-free
water) for 8 h and then sown in the mass at a rate of 6 × 107 CFU/g. Traditional ingredients of
Spanish chorizo (paprika, garlic powder and oregano) were purchased in a local supermarket,
Hipercor, S. A. (Murcia, Spain).
The meat was then mixed with the starter cultures, additives, spices and natural extracts. The
paste was stuffed into swine casing, slightly curved, 40 to 43 mm calibre and 300 to 400 mm of
length, using an automatic stuffer (Silvercrest ® kitchen tools, Barcelona, Spain). The casing was
supplied by Tripas De Murcia, S.L. (Alhama de Murcia, Murcia, Spain) and was previously
desalted and washed with chlorinated-free water. The Spanish chorizo samples were labelled and
placed in an air-drying chamber, Binder 115 redLine RI (Tuttlingen, Germany), set at 22 ± 1°C
and 90 ± 5% R.H. for two days. After that, the temperature and humidity were adjusted to 14 ±
1°C and 70 ± 5% R.H. for 20 days. Analysis were carried out at 0 and 50 days. After the curation
process, to study the shelf life, the Spanish chorizo samples were vacuum packaged and then
stored at 5 ± 1°C and 65 ± 5% R.H. for 125 days. Analyses were carried out at days 0, 25, 50, 75
and 125 from elaboration. Microbiological growth was determined at day 50, while volatile
compounds were measured at days 0, 25, 75 and 125.
Lorena Martínez Zamora PhD Thesis, 2019
56
The experimental design of this phase, whose results were published in Paper V, is showed
in Figure 7.5. Material and methods are explained in next chapter and also in this publication.
Figure 7.5. Graphical abstract Assay IV. Paper V. DPPH: 2,2-diphenyl-1-picrylhydrazyl; ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); FRAP: ferric
reducing antioxidant power; ORAC: oxygen radical absorbance capacity.
7.4.2. Study of protein oxidation in pork meat after application of
natural extracts
This study was carried out in the Faculty of Food Science in the University of Copenhagen as
a part of this international PhD programme. In this way, the second phase of this Assay was
carried out in order to study protein oxidation in a oxidized pork meat model system. Material
and methods followed are represented in Figure 7.6. and explained in Paper VIII.
For the elaboration of the oxidized pork meat model system, three kg of pork loin were
purchased from a local Danish supermarket (Coop A/S, Frederiksberg, Copenhagen, Denmark).
Initially, fat was removed and the meat was cut into cubes of 1 × 1 × 1 cm, vacuum-packed in
bags of 50 g and kept at -18ºC until analysis. Frozen vacuum-packed meat was thawed and minced
using a grinder (12ºC, 2 min, 500 rpm). 1.5 g were homogenized in 12.5 ml of 0.05 M MES
buffer, pH = 5.8, together with phenolic extracts (Citrus, Rosemary, Acerola), traditional Spanish
ingredients (Paprika, Garlic, Oregano), or natural nitrate sources (Beet, Lettuce, Arugula,
Spinach, Celery, Chard and Watercress). The concentrations were selected based on the
concentrations of ingredients applied in traditional Spanish “Chorizo” and given in ppm based on
the weight of the meat model system (meat and buffer). During homogenization using an Ultra
Turrax T25 at 11,600 rpm for 30 sec samples were kept on ice to reduce oxidation. Subsequently,
the azo-initiators, 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) diluted in MilliQ
Lorena Martínez Zamora PhD Thesis, 2019
57
water (0.54 mM final concentration) or 2,2’-azobis(2,4-dimethylvaleronitrile (AMVN) diluted in
99.9 % EtOH (3 mM as final concentration) were added as hydrophilic oxidation initiator
(OXHydro), or lipophilic oxidation initiator (OXLip), respectively. Immediately after addition of
the azo-initiators, samples were placed in a water bath under agitation at 37ºC for 200 min to
oxidize the meat model system. After oxidation, thiol groups were quantified and the remaining
meat model system was frozen to -80ºC and lyophilized for the analysis of protein radical intensity
by ESR spectroscopy. All samples were prepared in minimum triplicates and on all days of
analysis a non-oxidized control (C-NoOX) was included. The non-oxidized controlled contained
only meat and no oxidation initiator, but was otherwise treated similarly to the samples.
Therefore, next experimental design was carried out to study the antioxidant protection of
ingredients used against protein oxidation (Figure 7.6.).
Figure 7.6. Graphical abstract Assay IV. Paper VI. AAPH: 2,2'-Azobis(2-amidinopropane) dihydrochloride; AMVN: 2,2'-azobis (2,4-dimethylvaleronitrile); ESR: Electron Paramagnetic Resonance.
7.4.3. Shelf-life study of Spanish “chorizo” enriched in natural
extracts
This last experiment of Assay IV is the continuation of study started with Paper V. In this
way, the same procedure and formula was followed for the elaboration of dry-cured Spanish
“chorizo” samples that in 7.4.1. (Table 7.4.).
Lorena Martínez Zamora PhD Thesis, 2019
58
Nonetheless, in this last step, a shelf-life study for 150 days was carried out for this experiment.
All the material and methods are exposed in Figure 7.7. and also explained in next chapter, as well
as, in Paper VII.
Ta
ble
7.4
. In
gre
die
nts
(g)
of
dry
-cu
red
Sp
an
ish
“ch
ori
zo” s
am
ple
s el
ab
ora
ted
in
Ass
ay I
V,
Pa
per
s V
an
d V
II
S
am
ple
s en
rich
ed w
ith
Ro
sem
ary
ex
tra
ct
Sa
mp
les
enri
ched
wit
h C
itru
s ex
tra
ct
Ing
red
ien
ts:
Co
ntr
ol
RL
AW
R
SC
e R
Ch
B
CL
AW
C
SC
e C
Ch
B
Po
rk m
eat
(g)
87
5
87
5
87
5
87
5
87
5
87
5
87
5
Po
rk f
at
(g)
13
50
13
50
13
50
13
50
13
50
13
50
13
50
Wa
ter
(ml)
7
5
75
75
75
75
75
75
Co
mm
erci
al
mix
(g/k
g)
65
Pa
pri
ka
(g/k
g)
3
0
30
30
30
30
30
Ore
ga
no
(g
/kg
)
3
3
3
3
3
3
Ga
rlic
(g
/kg
)
3
3
3
3
3
3
Dex
tro
se (
g/k
g)
3
3
3
3
3
3
Sa
lt (
g/k
g)
5
5
5
5
5
5
Mea
t p
rote
in
(g/k
g)
2
3
23
23
23
23
23
Fer
men
t (m
l)
20
20
20
20
20
20
20
Ex
tra
cts
(pp
m):
•C
5
00
50
0
50
0
•R
50
0
50
0
50
0
•A
cero
la
2
50
25
0
25
0
25
0
25
0
25
0
•L
AW
30
00
+15
00
+1
50
0
30
00
+15
00
+1
50
0
•S
Ce
30
00
+30
00
30
00
+30
00
•C
hB
30
00
+30
00
30
00
+30
00
C:
Cit
ric;
R:
Ro
sem
ary;
LA
W:
Let
tuce
+ A
rugula
+ W
ater
cres
s; S
Ce:
Sp
inac
h +
Cel
ery;
ChB
: C
har
d +
Bee
t
Lorena Martínez Zamora PhD Thesis, 2019
59
Figure 7.7. Graphical abstract Assay IV. Paper VII.
Aw: water activity; TVC: Total Viable Count.
7.5. Assay V:
Study of exogenous enrichment of processed fish products through
the addition of natural antioxidant extracts
In addition, two studies were carried out, which focused in the development of manufactured
fish products enriched in antioxidant extracts.
7.5.1. Characterization of natural extracts and application in fish
patties
For that, firstly, several natural extracts from Mediterranean ingredients, such as, pomegranate,
rosemary and hydroxytyrosol were tested for their potential antioxidant and antimicrobial
capacities, following the described methods in next chapter and in Paper VIII.
Once all the extracts were tested, their antioxidant and antimicrobial capacities were also
comprobed after application in fish patties for 14 days. For the elaboration of fish product
samples, formula represented in Table 7.5 were followed.
Then, ultra-frozen skinless hake fillets (Merluccius capensis, Merluccius paradoxus)
(Pescanova España S.L.U.) were bought in a local supermarket and thawed in refrigeration for 24
h before elaboration fish patties. Fish was minced in an electric mincer (Bosch, Germany) for 2
minutes, mixing all the ingredients of each one of seven samples, represented in Table 1.
Afterwards, fish patties were formed (50 g) and packed in aerobic conditions. Samples were
stored at 4ºC until analysis, at day 0, 4, 7 and 11 from elaboration. 20 fish patties were formed for
each batch and analysis were carried out by triplicated.
Lorena Martínez Zamora PhD Thesis, 2019
60
Table 7.5. Ingredients (g) of fish patties samples elaborated in Assay V, Paper VIII Ingredients Control P RA NOS NOSV HYT-L HYT-F
Hake (g) 852 875 875 875 875 875 875
Water (ml) 100 100 100 100 100 100 100
Commercial mix (g/kg) 48
Fibers (g/kg) 25 25 25 25 25 25
Natural extracts (ppm) 200 200 200 200 200 200
Commercial mix®: supplied by Catalina Food Solutions, S.L. (El Palmar, Murcia, Spain) and composed by: vegetables fibers, salt,
potato starch, stabiliser (Pocessed euchema seaweed (PES) E-407-a), acidity correctors (sodium citrate E-331 and sodium acetate E-262), spices, spice extracts and antioxidant (sodium ascorbate E-301). P: Pomegranate extract, RA: Rosemary extract rich in
Rosmarinic Acid; NOS: Rosemary extract rich in diterpenes; NOVS: Rosemary extract rich in diterpenes and with lecitin as emulsifier;
HYT-L: Hydroxytyrosol extract obtained from olive leaf; HYT-F: Hydroxytyrosol extract obtained from olive fruit.
In addition, experimental design for this study is represented in Figure 7.8., as well as, material
and methods for it are explained in Paper VIII and in next chapter.
Figure 7.8. Graphical abstract Assay V. Paper VIII. HPLC: High-performance liquid chromatography; DPPH: 2,2-diphenyl-1-picrylhydrazyl; ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); FRAP: ferric reducing antioxidant power; ORAC: oxygen radical
absorbance capacity; TVC: Total Viable Count; TCC: Total Coliform Count.
7.5.2. Shelf-life study of fish patties enriched in natural extracts
In this study, Clean Label fish patties were elaborated and a shelf-life study of them was
carried out, which incorporated hydroxytyrosol, rosemary and pomegranate extracts in
combination with essential oils rich in ALA and DHA was developed.
Same procedure previously described was followed for the elaboration of fish patties, but in
this case, samples also incorporated essential oils ECOFLAX ® and BIOMEGA TECH ALGAE
®, which were supplied by BTSA (Alcalá de Henares, Madrid, Spain). ECOFLAX ® was made
from linseeds with 45 % α-linolenic (ALA) fatty acid, while BIOMEGA TECH ALGAE ® was
elaborated from algae with 40 % docosahexaenoic (DHA) fatty acid.
Lorena Martínez Zamora PhD Thesis, 2019
61
Table 7.6. Ingredients (g) of fish patties samples elaborated in Assay V, Paper IX. Ingredients C Ct HXT P R
Hake (g) 852 831 831 831 831
Water (ml) 100 100 100 100 100
Commercial mix (g) 48
Salt (g) 19 19 19 19
Fibers (g) 25 25 25 25
Soy albumin 14 14 14 14
Essential oils (ml):
• Ecoflax ®
• Biomega Tech Algae ®
5.7
5.7
5.7
5.7
5.7
5.7
5.7
5.7
Natural extracts (ppm):
• Acerola
• Ct
• HXT
• P
• R
200
200
200
200
200
200
200
200
Commercial mix®: supplied by Catalina Food Solutions, S.L. (El Palmar, Murcia, Spain) and composed by: vegetables fibres,
salt, potato starch, stabiliser (Pocessed euchema seaweed (PES) E-407-a), acidity correctors (sodium citrate E-331 and sodium
acetate E-262), spices, spice extracts and antioxidant (sodium ascorbate E-301). Ct: Citric extract; HXT: Hydroxytyrosol extract obtained from vegetable waters of olive tree; P: Pomegranate extract, R: Rosemary extract.
Experimental design of this study is represented in Figure 7.9., describes in next chapter and
also in the first draft of Paper IX, in annexes of this present thesis dissertation.
Figure 7.9. Graphical abstract Assay V. Paper IX. ALA: α-Linolenic acid; DHA: Docosahexaenoic acid; TMA: trimethylamine; TVB-N: total volatile basic
nitrogen.
Material and methods followed in the present doctoral thesis are presented in a schematic and
summarized form, in Table 7.7.
Lorena Martínez Zamora PhD Thesis, 2019
62
Table 7.7. Summary of material and methods followed in the present thesis dissertation. W
ay
of
enri
chm
en
t
Food
product
Natural
extract/ingredient
used
Concentration and source Samples Methods Paper Assay
En
do
gen
ou
s +
ex
og
eno
us
Chicken
meat
emulsion
Inorganic (C) and
organic forms (SZ)
of Zn and Se in
feeding diet
0.3 ppm of Na2SeO3 and 80 ppm of
ZnO (C), and 0.2 ppm and 50 ppm of Se
and Zn proteinate (SZ)
C
CHXT
CHXTOl
SZ
SZHXT
SZHXTOl
Bioavailability assay of Fe, Zn, and Se in
Caco-2 cell model
HXT detection by HPLC before and after in
vitro digestion
I I
HXT 50 ppm (23 % from vegetation water)
EVOO (Ol) 10 %
Chicken
nuggets
Inorganic (C) and
organic (SZ) forms
of Zn and Se in
feeding diet
0.3 ppm of Na2SeO3 and 80 ppm of
ZnO (C), and 0.2 ppm and 50 ppm of Se
and Zn proteinate (SZ) C
CRH+P
CRL+GS
CHXT+P+H
SZ
SZRH+P
SZRL+GS
SZHXT+P+H
Characterization of natural extracts by HPLC
Proximal composition
Shelf life study under frozen storage for 12
months:
- pH
- Colour (CIELab)
- Lipid oxidation (TBARs)
- Protein oxidation (Thiol
loss)
- Microbiological analysis:
TVC, TCC, E. Coli, L.
monocytogenes, Salmonella
- Sensory analysis
IV III
Rosemary
1000 ppm 8.1 % Rosmarinic acid (RH)
1000 ppm 5.8 % Carnosic – carnosol
(RL)
Pomegranate (P) 1500 ppm 41.38 % punicalagin (P)
Grape seed (GS) 1500 ppm 2.2 % catechin, 2.2 %
epicatechin, and 95.6 % OPCs
HXT 750 ppm 7.2 % from olive leaf
Harpagophyte (H) 500 ppm 3 % Hapargoside
Ex
og
eno
us Chicken
frankfurters
EVOO (Ol) 10 % C
HXT1
HXT2
HXT3
CW
COL
Characterization of HXT extracts by HPLC
Proximate composition
Fatty acids profile by GC
Lipid quality index
Sensory analysis
Correlation between lipid oxidation and
sensory analysis
II II
Walnuts (W) 2.5 %
62
Lo
rena M
artín
ez Za
mo
ra
Ph
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HXT
50 ppm (7 % from vegetation water)
(HXT 1)
OLW
HXT1OLW Characterization of HXT extracts by HPLC
Proximate composition
Cooking losses
Scanning electron microscopy (SEM)
Shelf life study for 21 days:
- Colour (CIELab)
- Lipid oxidation (TBARs)
- Protein oxidation (Thiol
loss)
- Sensory analysis
III 50 ppm (23 % from vegetation water)
(HXT 2)
50 ppm (7 % from olive leaf) (HXT 3)
Dry-cured
Spanish
“chorizo”
Antioxidants: citrus,
rosemary, and
acerola
500 ppm 55 % hesperidin (C)
500 ppm 14.59% carnosic
acid, 5.84% carnosol, and 0.60% 12-O-
methylcarnosic acid (R)
250 ppm 5 % vitamin C in all samples
Control
CLAW CSCe
CChB RLAW
RSCe
RChB
Characterization of natural extracts:
- Total phenolic content
- Total nitrate content
- Antioxidant activity:
FRAP, ORAC, ABTS, DPPH
- Antimicrobial activity:
Antioxidant and antimicrobial capacity in
dry-cured Spanish “chorizo” for 125 days:
- Volatile compounds (GC-
MS)
- Microbiological analysis
(TVC, TCC, Clostridium
perfringens)
V
IV
Traditional
ingredients: paprika,
garlic, and oregano
30000 ppm paprika
3000 ppm garlic
3000 ppm oregano (of each one in all the
samples)
Nitrate sources: beet
(B), lettuce (L),
arugula (A), spinach
(S), chard (Ch),
celery (Ce), and
watercress (W) 3000 ppm of each one
Protein oxidation study in an oxidized pork
meat model system:
- Thiol loss
- ESR
- Nitrate and nitrite dose-
dependence curve
VI
Proximal composition
Shelf life study of chorizo for 150 days:
- pH
- Colour (CIELab)
- Lipid oxidation: Volatile
compounds (GC-MS)
VII
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- Protein oxidation (Thiol
loss)
- Microbiological analysis:
TVC, TCC, E. Coli, L.
monocytogenes, Salmonella
Sensory analysis
Texture analysis
Fish patties
HXT
200 ppm 11.25 % HXT from olive fruit
(HYT-F)
200 ppm 7.3 % HXT from olive leaf
(HYT-L) Control
P
RA
NOS
NOVS
HYT-L
HYT-F
Characterization of natural extracts:
- Total phenolic content
- HPLC
- Antioxidant activity:
FRAP, ORAC, ABTS, DPPH
- Antimicrobial activity: L.
monocytogenes, S. aureus, E. Coli
Antioxidant and antimicrobial capacity in
fish patties for 11 days:
- Volatile compounds (GC-
MS)
- Microbiological analysis
(TVC, TCC, E. Coli, L.
monocytogenes)
VIII
V
Rosemary
200 ppm 8.1 % Rosmarinic acid (RA)
200 ppm 5.8 % Carnosic – carnosol
(NOS)
200 ppm 5.8 % Carnosic – carnosol +
lecithin (NOVS)
Pomegranate 200 ppm 41.4 % Punicalagin (P)
Citrus 200 ppm 55 % hesperidin (C)
Control
C
HXT
P
R
Proximal composition
Shelf life study of fish patties for 14 days:
- pH
- Colour (CIELab)
- Lipid oxidation (TBARs)
- Protein oxidation (Thiol
loss)
- TMA
- TVB-N
- NH3 content
Sensory analysis
IX
HXT 200 ppm (HXT) 7.3 % from olive
vegetation waters
Rosemary 200 ppm (R) 5.8 % diterpenes
Pomegranate 200 ppm (P) 41.4 % punicalagin
Acerola 200 ppm of 17 % vit C in all samples
Essential oils ALA
and DHA
5.7 % of 45 % ALA and 5.7 % of 40 %
DHA and in all samples OPCs: Oligomers of Proanthocyanidins
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8. Results and Discussion
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Results of each chapter properly discussed are described in the present chapter, as well as, in
attached papers of annexes.
8.1. Assay I:
Obtained results of endogenous enrichment of meat products through
animal diet
In this study, the Caco-2 cell line used to do the bioavailability assay was checked by a
mycoplasma test to ensure that it was free of contamination, which would have affected the results
(Figure 8.1.). The phenol red test was used to check the monolayer permeability. That confirmed
the integrity of the cell membrane for mineral absorption experiments to be carried out (between
days 8 and 21 of subculture). The data obtained were correlated directly with TEER
(transepithelial electrical resistance) when the values were over 1000 Ω cm2, indicating that the
monolayer was full. The results of the MTT assay with different extracts added to the cell
monolayer showed that the percentage of viability did not fall by more than 10%, so these
solutions were not toxic to the cell line.
A
B
Figure 8.1. Negative mycoplasma test in Caco-2 cell line (A: Caco-2 cells and B: cell
nucleuses stained with Hoechst dye.
Otherwise, in Figure 8.2., Caco-2 cell development from day 0 to 20 of seeding can be
appreciated. In this way, it can be observed that bioavailability experiments could be carried out
from the 7th to the 20th, when de cell monolayer was completely formed and differentiation is
started. However, experiments were made at day 13th from seeding to ensure the properly
development in membrane filters.
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Figure 8.2. Caco-2 cell development for 20th days of seeding (A: day 0; B: day 3; C: day 5; D:
day 7; E: day 12; F: day 20).
Firstly, to understand how HXT is degraded after in vitro digestion and to do an estimation of
the amount of this phenolic compound that enterocytes can absorb, Table 8.1. shows the
concentration of HXT (M ± SD) in digested meat emulsions (soluble fraction added to Caco-2
cells), as measured by HPLC. Although HXT decomposition after digestion was very low, there
were significant differences between samples (p < 0.05). For example, HXT degradation was
9.14% in CHXT and 14.86% in SZHXT, both higher than CHXTOl and SZHXTOl, where losses of 3 and
1.04%, respectively, were recorded. This demonstrates that the total hydroxytyrosol content of
this extract does not decrease when it is consumed in food products, as previously mentioned
(Ramírez-Anaya et al., 2015). In this way, results show that HXT becomes more available when
it is combined with EVOO, which is not surprising because both of the compounds share a
common origin: the olive tree.
Table 8.1. HXT concentration in emulsions (soluble fraction added to Caco-2 cells) (M ± SD)
measured by HPLC. Experimental Treatments
C CHXT CHXTOl SZ SZHXT SZHXTOl
HXT added to meat
(ppm) 0.0 ± 0.0 b 50.0 ± 0.0 a 50.0 ± 0.0 a 0.0 ± 0.0 b 50.0 ± 0.0 a 50.0 ± 0.0 a
Digested HXT
concentration (ppm) 0.0 ± 0.0 e 45.4 ± 0.0 c 48.5 ± 0.0 b 0.0 ± 0.0 e 42.6 ± 0.00 d 49.5 ± 0.0 a
% Decomposition 0.0 ± 0.0 e 9.1 ± 0.0 b 3.0 ± 0.0 c 0.0 ± 0.0 e 14.9 ± 0.00 a 1.0 ± 0.0 d
M ± SD: Mean ± standard deviation; HXT: Hydroxytyrosol (23% extract from vegetation waste water). EVOO: Extra Virgin Olive
Oil. C: Control; CHXT: 50 ppm HTX; CHXTOl: 50 ppm HXT + 10% EVOO; SZ: Control fortified with Zn and Se meat; SZHXT: SZ + 50
ppm HXT; SZHXTOl: SZ + 50 ppm HXT + 10% EVOO.
Similar results were obtained in other studies, which showed that the combination of HXT and
EVOO maintained the antioxidant activity of phenolic compounds during cooking (Ramírez-
Anaya et al. 2015). Similarly, Rubio et al. (2014) demonstrated that HXT bioavailability in Caco-
2/HepG2 cells was enhanced when it was combined with other extracts that are rich in phenolic
compounds, such as thyme.
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However, in the SZ samples, the decomposition degree was greater than in C, in which HXT
was not combined with EVOO. This could be due to interference between the organic forms of
Zn and Se and phenolic compounds from the HXT extract. However, no information regarding
this possible effect is available to compare the results of this study. The affinity of HXT for certain
minerals has been reported previously. For example, Ca absorption increases with HXT and
EVOO consumption in osteoporosis patients, preventing the bone loss (Hagiwara et al., 2011).
On the other hand, HXT is bound to Fe (II) in black olives (Wang et al., 2013), so this compound
can be associated with another mineral forms, such as Zn or Se.
8.1.1. Study of mineral bioavailability
To assess Fe bioaccessibility, Table 8.2. shows the results of Fe retention, transport and uptake
(M ± SD) in Caco-2 cells after adding the soluble fraction from chicken emulsions.
Table 8.2. Fe retention, transport and cellular uptake (M ± SD) in enriched chicken emulsions. Experimental Treatments
C CHXT CHXTOl SZ SZHXT SZHXTOl
• Fe concentration in
the soluble fraction
added (mg/ml)
0.3 ± 0.0 d 0.3 ± 0.0 c 0.3 ± 0.0 d 0.4 ± 0.0 b 0.4 ± 0.0 b 0.5 ± 0.0 a
• Mineral added
monolayer (μg) 4.3 ± 0.0 d 4.5 ± 0.0 c 4.6 ± 0.1 b 4.3 ± 0.0 d 4.9 ± 0.0 b 5.0 ± 0.1 a
• Mineral retained in
apical chamber (μg) 1.1 ± 0.1 b 1.2 ± 0.0 ab 1.2 ± 0.1 ab 1.1 ± 0.0 b 1.2 ± 0.0 a 1.2 ± 0.1 ab
• Retention % 26.2 ± 0.4 a 25.5 ± 0.3 b 25.4 ± 0.4 b 26.1 ± 0.6 a 24.8 ± 0.4 c 23.2 ± 0.6 c
• Mineral transported
to basolateral
chamber (μg)
1.1 ± 0.0 c 1.3 ± 0.0 b 1.4 ± 0.0 b 1.2 ± 0.0 c 1.5 ± 0.1 a 1.5 ± 0.0 a
• Transport % 24.5 ± 0.4 f 29.3 ± 0.2 d 29.5 ± 0.1 c 27.2 ± 0.0 e 31.2 ± 0.4 a 31.1 ± 0.2 b
• Mineral uptake by
cells (μg) 2.1 ± 0.0 bc 2.0 ± 0.0 d 2.1 ± 0.1 c 2.0 ± 0.1 de 2.2 ± 0.1 b 2.3 ± 0.1 a
• Uptake % 49.3 ± 2.0 a 45.0 ± 3.1 cd 45.0 ± 4.4 cd 46.7 ± 4.0 b 44.1 ± 4.3 d 45.7 ± 2.1 c
o TE 6.4 ± 1.9 d 7.5 ± 0.1 bc 7.5 ± 0.4 b 7.1 ± 0.2 c 7.7 ± 0.3 a 7.2 ± 0.2 cd
o UE 12.9 ± 1.2 a 11.5 ± 3.0 b 11.5 ± 2.3 b 12.2 ± 0.2 ab 10.9 ± 2.1 c 10.6 ± 1.1 c
M ± SD: Mean ± standard deviation; TE: Transport efficiency; UE: Uptake efficiency. HXT: Hydroxytyrosol (23% extract from vegetation waste water). EVOO: Extra Virgin Olive Oil. C: Control; CHXT: 50 ppm HTX; CHXTOl: 50 ppm HXT + 10% EVOO; SZ:
Control fortified with Zn and Se meat; SZHXT: SZ + 50 ppm HXT; SZHXTOl: SZ + 50 ppm HXT + 10% EVOO.
There were no significant differences between the Fe absorption values in the samples.
However, there were significant differences in basal Fe concentrations and the retained and
transported mineral between the different samples. Moreover, C and SZ had a higher percent of
mineral uptake (p < 0.05), while CHXT, CHXTOl, SZHXT and SZHXTOl showed higher percentages of
mineral transport (p < 0.05). In the same way, the transport and uptake efficiencies behaved
similarly, being higher than 7.5% in CHXT and SZHXT and more than 12% in CHXTOl and SZHXTOl.
This may be because HXT increased Fe transport from the apical to the basolateral chamber,
which may be due to the great affinity of HXT bind to Fe, as was observed by Wang et al. (2013).
In this research, it was observed how HXT binds with gluconate Fe (II) in black olives, which
catalyses the oxidation of this mineral. Moreover, similar results concerning Fe availability were
obtained by Soresen & Bukhave (2010) and Pachón et al. (2008) with enriched pork and chicken
meat, respectively, in Caco-2 cells.
Otherwise, Table 8.3. shows the results that were obtained for Zn retention, transport and
uptake (M ± SD) in Caco-2 cells after addition of the soluble fraction to the Caco-2 cell
monolayer.
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Table 8.3. Zn retention, transport and cellular uptake (M ± SD) in enriched chicken emulsions. Experimental Treatments
C CHXT CHXTOl
SZ SZHXT SZHXTOl
• Zn concentration in
the soluble fraction
added (mg/ml)
0.1 ± 0.0 e 0.2 ± 0.0 de 0.2 ± 0.0 cd 0.2 ± 0.0 bc 0.2 ± 0.0 b 0.5 ± 0.0 a
• Mineral added
monolayer (μg) 3.8 ± 0.0 e 4.5 ± 0.1 c 6.0 ± 0.3 b 4.1 ± 0.1 d 4.6 ± 0.1 c 5.0 ± 0.1 a
• Mineral retained in
apical chamber (μg) 1.7 ± 0.0 c 2.2 ± 0.1 bc 2.7 ± 0.3 ab 1.8 ± 0.1 c 2.4 ± 0.1 cd 1.2 ± 0.1 ab
• Retention % 44.9 ± 0.2 d 49.7 ± 0.0 b 45.2 ± 0.2 c 44.2 ± 0.2 d 51.8 ± 0.6 a 23.2 ± 0.6 c
• Mineral transported
to basolateral
chamber (μg)
0.9 ± 0.0 c 1.3 ± 0.1 bc 2.2 ± 0.2 a 1.2 ± 0.1 bc 1.4 ± 0.1 b 1.5 ± 0.0 a
• Transport % 24.8 ± 0.1 e 28.7 ± 0.0 d 36.9 ± 0.0 a 30.2 ± 0.0 c 30.2 ± 0.0 c 31.1 ± 0.2 b
• Mineral uptake by
cells (μg) 1.2 ± 0.1 ab 1.0 ± 0.1 bc 1.1 ± 0.1 ab 1.0 ± 0.0 abc 0.8 ± 0.0 c 2.3 ± 0.1 a
• Uptake % 30.3 ± 4.2 a 21.6 ± 1.3 c 17.9 ± 3.9 d 25.6 ± 1.3 b 17.9 ± 0.9 d 45.7 ± 2.1 c
o TE 11.1 ± 0.0 e 14.3 ± 0.0 c 16.7 ± 0.2 a 13.4 ± 0.3 d 15.7 ± 0.1 b 7.2 ± 0.2 cd
o UE 13.6 ± 1.9 a 10.7 ± 3.3 c 8.1 ± 6.5 e 11.3 ± 2.4 b 9.3 ± 1.0 d 10.6 ± 1.1 c
M ± SD: Mean ± standard deviation; TE: Transport efficiency; UE: Uptake efficiency; HXT: Hydroxytyrosol (23% extract from
vegetation waste water). EVOO: Extra Virgin Olive Oil; C: Control; CHXT: 50 ppm HTX; CHXTOl: 50 ppm HXT + 10% EVOO; SZ: Control fortified with Zn and Se meat; SZHXT: SZ + 50 ppm HXT; SZHXTOl: SZ + 50 ppm HXT + 10% EVOO
As expected, SZ samples showed a higher basal concentration of Zn than C samples (p < 0.05).
On the other hand, CHXT, CHXTOl, SZHXT and SZHXTOl showed greater Zn bioavailability than C and
SZ (p < 0.05). Then, the mineral retention percent in the apical chamber was higher in C and SZ
samples, reaching 51 % in the SZ batch. However, the mineral uptake by Caco-2 cells was
significantly lower in CHXT and SZHXT, reaching 17 % and 25–30 % in C and SZ, respectively.
For its part, the transported mineral percent was constant in SZ batch (about 30%) and higher in
C (24–37 %).
These results disagree to some extent with those of other researchers, such as, Frontela et al.
(2009) or Frontela et al. (2011), who observed an increase in Zn absorption in milk formulas
enriched with Fe, Zn and Ca. No information exists in the literature concerning the bioavailability
of Zn in Caco-2 using enriched meat, making it an interesting topic for further research.
In addition, the RDA of Zn for a healthy adult is among 8 and 12 mg/day (Frontela et al.,
2011), which according to the quantity ingested, the consumption of 100 g of SZHXTOl supposes
5% of this RDA, while 100 g of CHXTOl supposes 2 %. So, it can be concluded that consumption
of this kind of products helps to reach the recommendation, but it is necessary complete the diet
with other products that are rich in Zn, such as oat, mussels, or cockles.
Finally, Table 8.4. shows the results of Se retention, transport and uptake with normal
distribution (M ± SD) in Caco-2 cells after adding digested chicken emulsions. The table also
shows an estimate of its availability and the values of mineral transport and uptake efficiency.
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Table 8.4. Se retention, transport and cellular uptake (M ± SD) in enriched chicken emulsions. Experimental Treatments
C CHXT CHXTOl SZ SZHXT SZHXTOl
• Se concentration in
the soluble fraction
added (mg/ml)
0.01 ± 0.0 b 0.01 ± 0.0 b 0.01 ± 0.0 b 0.02 ± 0.0 a 0.01 ± 0.0 b 0.5 ± 0.01 a
• Mineral added
monolayer (μg) 4.5 ± 0.0 4.5 ± 0.0 4.5 ± 0.0 4.5 ± 0.0 4.5 ± 0.0 5.0 ± 0.07 a
• Mineral retained in
apical chamber (μg) 1.5 ± 0.0 a 1.5 ± 0.0 a 1.5 ± 0.0 a 1.3 ± 0.0 b 1.5 ± 0.0 a 1.2 ± 0.08 ab
• Retention % 33.3 ± 0.0 a 33.3 ± 0.0 a 33.3 ± 0.0 a 29.2 ± 1.0 b 33.3 ± 0.0 a 23.2 ± 0.58 c
• Mineral transported
to basolateral
chamber (μg)
1.5 ± 0.0 a 1.5 ± 0.0 a 1.5 ± 0.0 a 1.3 ± 0.0 b 1.5 ± 0.0 a 1.5 ± 0.03 a
• Transport % 33.3 ± 0.0 a 33.3 ± 0.0 a 33.3 ± 0.0 a 29.2 ± 0.0 b 33.3 ± 0.0 a 31.1 ± 0.2 b
• Mineral uptake by
cells (μg) 1.5 ± 0.0 b 1.5 ± 0.0 b 1.5 ± 0.0 b 1.9 ± 0.0 a 1.5 ± 0.0 b 2.3 ± 0.1 a
• Uptake % 33.3 ± 0.0 b 33.3 ± 0.0 b 33.3 ± 0.0 b 41.6 ± 0.7 a 33.3 ± 0.0 b 45.7 ± 2.13 c
o TE 11.1 ± 0.0 a 11.1 ± 0.0 a 11.1 ± 0.0 a 8.5 ± 0.1 b 11.1 ± 0.0 a 7.2 ± 0.18 cd
o UE 11.1 ± 0.0 b 11.1 ± 0.0 b 11.1 ± 0.0 b 12.2 ± 0.2 a 11.1 ± 0.0 b 10.6 ± 1.13 c
M ± SD: Mean ± standard deviation; TE: Transport efficiency; UE: Uptake efficiency. HXT: Hydroxytyrosol (23% extract from
vegetation waste water). EVOO: Extra Virgin Olive Oil. C: Control; CHXT: 50 ppm HTX; CHXTOl: 50 ppm HXT + 10% EVOO; SZ: Control fortified with Zn and Se meat; SZHXT: SZ + 50 ppm HXT; SZHXTOl: SZ + 50 ppm HXT + 10% EVOO.
In this case, no significant differences were observed when HXT or EVOO were incorporated
to the formulas (CHXT, CHXTOl, SZHXT and SZHXTOl). However, there was a slight increase (p <
0.05) in the Se initial concentration of Se (0.01 mg/ml higher) and Se uptake (8.31 % higher) by
Caco-2 cells in SZ samples made with chicken meat enriched with organic Se.
These results were similar to others concerning Se bioavailability in seafoods in Caco-2 cells
(Calatayud et al., 2012; Moreda-Piñero et al., 2012). The bioavailability of Se in the intestine is
very low and its absorption efficiency does not exceed 10 %. Although no information on Se
bioavailability in enriched meats has been found, the results that were obtained suggest that the
food matrix used is not a dependent factor for its availability, because mineral uptake is also low
in fish and seafoods. In addition, HXT is not an influential factor, because of the retention,
transport and uptake values were not affected by its presence, in the same way as Zn
bioavailability. This observation can be explained by previous research that has shown how HXT
increases Fe and Ca bioavailability (Wang et al., 2013; Ramírez-Anaya et al., 2015) So, if HXT
acts as transporter of Ca and Fe, which are competitors of Zn and Se, it can be concluded that Fe
acts as a competitor for binding with HXT, preventing the absorption of Zn and Se bound with
this phenolic compound. Consequently, as can be appreciated, Fe availability in this study was
higher in the samples with HXT, while the uptake of Zn and Se combined with HXT was lower.
In addition, the RDA of Se for a healthy adult is among 55 and 70 µg/day (Moreda-Piñero et
al., 2013), that according to the quantity ingested, the consumption of 100 g of SZ samples
supposes the 100% of this RDA. So, it can be concluded that consumption of this kind of products
helps to reach the recommendation about this essential mineral.
Although this research was carefully prepared, this study presents some limitations. One of
the main limitations is the scarce number of animals (n = 70). Another limitation derived from
cell culture methods. These vary in their reproducibility and characterization. On the other hand,
the cell culture model is compared with the digestive system of the human body, which is more
complex and may vary significantly. However, through an exhaustive bibliographical research,
the relationships that occurred during the absorption process of minerals and phenolic compounds
have been justified.
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8.2. Assay II:
Obtained results of the exogenous enrichment of cooked meat product
through the addition of natural antioxidant extracts
In the development of this Assay, two papers have been published (Paper II and III), which
are presented in annexes.
Firstly, the retention times and abundance of the main compounds in the HXT extracts are
shown in the Table 8.5. Phenols (hydroxytyrosol and tyrosol), oleuropeosides (oleuropein and
verbascoside), flavones (luteolin-7-glucoside and apigenin-7-glucoside) and flavan-3-ols
(catechin) were the main group of compounds present. The most abundant compound in HXT1,
HXT2 and HXT3 was hydroxytyrosol (precursor of oleuropein), followed by tyrosol, tyrosol dimer
and verbascoside, in the first case, verbascoside, oleuropein, luteolin, apigenin-7-glucoside and
luteolin-7-glucoside, in the second one, while in HXT3 was followed by tyrosol, tyrosol dimer,
verbascoside, oleuropein, catechin and luteolin.
Table 8.5. Retention time and abundance of the main phenolic present in hydroxytyrosol extracts
(HXT1, HXT2 and HXT3).
Phenolics Retention time
(min)
HXT1
% Absolute
HXT2
% Absolute
HXT3
% Absolute
Hydroxytyrosol 5.7 82.8 76.6 72.4
Luteolin-7-glucoside 5.9 - 2.1 -
Catechin 7.9 - - 1.8
Tyrosol 8.5 9.4 - 10.9
Dimer of Hydroxytyrosol 13.3 5.4 - 7.2
Verbascoside 17.8 2.5 7.9 4.3
Apigenin-7-glucóside 22.4 - 2.2 -
Oleuropein 25.3 - 5.8 2.0
Luteolin 26.2 - 2.3 1.5
Other authors have shown that olives (Olea europaea L.) and olive oil contain polyphenols
with antioxidant properties (Briante et al., 2002; Boitia et al., 2001; Obied et al., 2005). The
polyphenolic chemical nature of these compounds is responsible for the different functionalities
as antimicrobial, antioxidant and health promoting agents (Visioli et al., 2002). Therefore, the
antioxidant activity order of the compounds present in olive oil is: tyrosol < caffeic < oleuropein
< hydroxytyrosol.
8.2.1. Proximate composition and fatty acid profile
The moisture, fat, protein and ash contents of the walnut, chicken meat- and olive oil-
containing formulations are summarised in Table 8.6.
Regarding the fatty acid profile, comparing the three ingredients (meat, extra virgin olive oil
and walnut) used in the formulation of the frankfurters, the SFA content (31.82%) was higher in
the chicken meat and consisted mainly of palmitic acids (24.2%) and estearic acids (6.42%). The
MUFA content (66.5%) was higher in the olive oil, with oleic acids accounting for the majority
of MUFAs (64.6%). The PUFA content was higher (55.8%) in the walnut, with the PUFAs mainly
consisting of linoleic acids (37.4%), linolenic acids (4.56%) and docosadienoic acids (11.8%).
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The results are in accordance with those of several previous studies: Librelotto et al. (2008)
reported that fatty acid profile of extra virgin olive oil consisted primarily of palmitic acid
(11.5%), stearic acid (2.2%), oleic acid (72.0%) and linoleic acid (7.9%); Pereira et al. (2008)
showed than walnuts have high content of MUFA as oleic acid and PUFAs as linolenic and
linoleic acids.
Table 8.6. Nutritional composition (%) and fatty acid profile (%) of chicken meat, walnut paste
and olive oil. Chicken meat Walnut Olive oil
Moisture 73.2 ± 0.9 9.3 ± 1.9 -
Ash 0.9 ± 0.1 1.5 ± 0.2 -
Fat 3.1 ± 1.7 62.1 ± 0.0 100
Protein 17.3 ± 1.2 16.6 ± 0.5 -
Energy (Kcal/100 g) 124.5 ± 4.3 674.2 ± 16.9 888
C16:0 palmitic 24.2 ± 0.0 13.3 ± 1.3 14.1 ± 0.1
C18:0 estearic 6.4 ± 0.0 5.4 ± 0.2 2.3 ± 0.0
C18:1 w-9 oleic 16.1 ± 0.0 24.1 ± 2.3 64.6 ± 0.6
C18:2 w-6 linoleic 0.2 ± 0.0 37.4 ± 6.7 9.9 ± 0.2
C18:3 w-6 α-linolenic 0.2 ± 0.0 4.6 ± 1.0 0.6 ± 0.1
C18:3 w-6 γ-linolenic 0.1 ± 0.0 0.2 ± 0.0 0.3 ± 0.0
C22:2 w-6 docosadienoic 1.0 ± 0.0 11.8 ± 0.2 3.3 ± 0.6
SFA 31.8 ± 0.0 24.1 ± 1.3 18.6 ± 0.2
MUFA 49.4 ± 0.0 25.4 ± 2.7 66.5 ± 0.6
PUFA 18.7 ± 0.0 55.8 ± 6.7 14.8 ± 0.5
PUFA: polyunsaturated fatty acid; MUFA: monounsaturated fatty acid; SFA: saturated fatty acid
Otherwise, the moisture, ash, fat, protein and kcal of the cooked meat frankfurters are
summarised in Table 8.7. As can be seen, the fat contents of the samples with walnut and olive
oil added were higher (3.33–11.59%) and the protein contents were lower (12.63–16.74%) than
those of the control samples (2.2 and 16.74 %, respectively). In this same line, Alvarez et al.
(2011) reported a higher amount of fat and lower protein content in frankfurters with added 2.5
g/100 g of walnut paste compared with control frankfurters.
Table 8.7. Chemical composition of cooked chicken frankfurters elaborated with hydroxytyrosol,
walnut and olive oil. Moisture Ash Fat Protein Kcal
C 78.6±0.7a 0.9±0.4 2.2±0.9d 16.7±0.1a 97.7±7.1a
HXTW 78.0±0.4a 1.9±0.2 4.4±0.1c 15.1±0.4a 106.5±1.2a
W 76.4±0.1a 2.3±0.1 3.3±0.4c 16.7±1.6a 106.8±1.6a
OL 71.7±0.1b 2.1±0.1 10.0±1.1b 12.1±2.3b 158.6±4.8b
OLW 70.2±0.5b 2.1±0.1 9.6±0.2b 13.6±0.9b 163.5±3.1b
HXTOLW 71.9±0.9b 1.8±0.1 11.6±0.6a 12.6±0.6b 166.8±6.0b
Control, C, chicken meat backfat; W, chicken meat backfat walnut 2.5 %; OL, chicken meat olive oil (20 g/100 g); OLW, chicken
meat backfat olive oil (20 g/100 g) walnut 2.5 %; HXTw, chicken meat backfat 50 ppm hydroxytyrosol 2.5 % walnut; HXTOLW, chicken
meat backfat olive oil (20 g/100 g) 50 ppm hydroxytyrosol 2.5 % walnut. Different letters in the same row indicate significant differences (p<0.05).
Cooked frankfurters with olive oil (OLW) had the lowest amount of water among all the
samples analysed. Compared with the control samples, the kcal content of the olive oil- containing
samples was significantly higher (p < 0.05), but it did not increase significantly (p > 0.05) with
the addition of the walnut. The addition of the walnut significantly increased (p < 0.05) the ash
Lorena Martínez Zamora PhD Thesis, 2019
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content of the cooked batter. These results are consistent with other findings for meat batter with
different amounts of walnut added (Álvarez et al., 2011; Ayo et al., 2005).
The fat and protein in the control samples were derived from the chicken meat and pork
backfat, whereas approximately 90% of the fat and 15% of the protein in the raw walnut samples
came from the walnuts. According to the results published in this study, Serrano et al. (2006)
reported that the inclusion of walnuts enriched the fat content of the frankfurters and decreased
moisture and protein content.
The mineral content of the control samples is shown in Table 8.8. The control meat was
composed of calcium (25.9 mg/ 100 g), potassium (220.8 mg/100 g), iron (1.9 mg/100 g),
magnesium (24.4 mg/100 g) and zinc (1.9 mg/100 g). The mineral content profile of the chicken
sausage was in line with that of several food composition tables related to chicken (Ayo et al.,
2005). In this sense, according to Ortega et al. (2004) the mineral content of chicken sausages is
Ca (34.54 mg/100 g), K (210 mg/100 g), Fe (1.44 mg/100 g), P (150 mg/100 g) and Zn (2.62
mg/100 g).
Table 8.8. Mineral content (mg/100 g) of chicken frankfurters elaborated with hydroxytyrosol,
walnut and olive oil. Ca K Fe Mg P Mn Zn Ca/P
C 25.9±0.2b 220.8±3.0b 1.9±0.1b 24.4±0.5b 186±3.3b 0.2±0.0 1.9±0.0b 0.13
HXTW 34.6±0.3ab 639.8±0.8a 2.6±0.0a 53.8±0.3a 364±2.0a 0.6±0.0 2.6±0.0a 0.09
W 32.5±0.0ab 542.3±0.2a 2.4±0.0a 44.8±0.3a 319±2.2a 0.5±0.0 2.0±0.0b 0.10
OL 31.8±0.3ab 571.9±9.1a 2.8±0.0a 40.3±0.4a 314±3.2a 0.3±0.0 2.2±0.1b 0.10
OLW 48.2±0.9ab 568.1±4.1a 2.9±0.0a 44.5±0.2a 301±2.2a 0.5±0.0 2.6±0.1a 0.16
HXTOLW 43.7±0.9ab 465.3±2.6ab 2.4±0.0a 35.9±0.1a 239±0.8a 0.4±0.0 2.1±0.0b 0.18
Control, C, chicken meat backfat; W, chicken meat backfat walnut 2.5 %; OL, chicken meat olive oil (20 g/100 g); OLW, chicken
meat backfat olive oil (20 g/100 g) walnut 2.5 %; HXTw, chicken meat backfat 50 ppm hydroxytyrosol 2.5 % walnut; HXTOLW, chicken
meat backfat olive oil (20 g/100 g) 50 ppm hydroxytyrosol 2.5 % walnut. Different letters in the same row indicate significant differences (p<0.05).
The addition of 2.5 g/100 g of walnut and olive oil altered the concentrations of some of the
minerals present in the frankfurters (Table 8.8). The zinc, calcium, magnesium, potassium and
iron contents of the frankfurters with added walnut were higher (p < 0.05) than those of the control
samples. The manganese content was unaffected by the addition of walnut and olive oil (p > 0.05).
The RDA (recommended daily allowance) for iron is 8 mg/dL in general population and 18
mg/dL in premenopausal women. Therefore, the intake of 100 g of chicken frankfurters with
walnut and olive oil contribute to 35% of RDA of iron. Thus, consumption of this meat product
could benefit individuals vulnerable to dietetic iron deficiency.
Otherwise, changes in the fatty acid composition of the frankfurters are shown in Tables 8.9
and 8.10. There were slight differences between the fatty acid profiles of the sausages without
walnut versus the samples containing walnut and olive oil. In the control samples, oleic acid
(C18:1) was the most abundant fatty acid, followed by palmitic (C16:0), linoleic (C18:2) and
stearic (C18:0) acids. In the walnut sausages (W), linoleic acid (C18:2) was the most abundant
fatty acid, followed by oleic (C18:1), palmitic (C16:0), linoleic (C18:2) and stearic (C18:0) acids.
In the sausages with olive oil (OL), oleic acid (C18:1) was the most abundant fatty acid,
representing 55% of the total fatty acid profile, followed by linoleic (C18:2), palmitic (C16:0) and
linoleic (C18:3) acids. As expected, the addition of HXT did not modify the fatty acid profile.
Lorena Martínez Zamora PhD Thesis, 2019
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The incorporation of 2.5% walnut and olive oil produced significant changes in the fatty acid
profiles of the frankfurters.
Table 8.9. Fatty acid profile (% of the most abundant) of chicken frankfurters elaborated with
hydroxytyrosol, walnut and olive oil. Percentage of total
fatty acids C HXTW W OL OLW HXTOLW
C14:0 Miristic 0.6±0.0a 0.5±0.1b 0.4±0.0b 0.2±0.0c 0.1±0.0c 0.1±0.0c
C16:0 Palmitic 24.2±0.0a 17.2±0.b 17.0±0.0b 15.4±1.4b 15.1±0.0b 15.1±0.2b
C18:0 Estearic 6.4±0.0a 4.9±0.0b 4.9±0.2b 4.4±0.8b 0.1±0.0c 3.4±0.0b
C16:1 Palmitoleic 5.6±0.1a 3.8±0.0b 3.9±0.0b 2.0±0.6b 2.2±0.0b 2.2±0.1b
C18:1 Oleic 43.1±0.0b 31.5±1.3c 32.4±0.2c 34.3±1.3c 55.6±0.2ª 51.9±1.0a
C18:2 Linoleic 16.1±0.0c 31.8±1.5a 32.5±0.4a 12.8±0.0c 18.4±0.9b 18.6±0.7b
C18:3 α-Linolenic 0.2±0.0c 6.2±0.3a 6.4±0.1a 1.2±0.1c 2.9±0.0b 2.8±0.2b
C18:3 γ-Linolenic 0.2±0.0 0.1±0.1 0.2±0.0 0.1±0.0 0.1±0.0 0.1±0.0
Control, C, chicken meat backfat; W, chicken meat backfat walnut 2.5 %; OL, chicken meat olive oil (20 g/100 g); OLW, chicken
meat backfat olive oil (20 g/100 g) walnut 2.5 %; HXTw, chicken meat backfat 50 ppm hydroxytyrosol 2.5 % walnut; HXTOLW, chicken
meat backfat olive oil (20 g/100 g) 50 ppm hydroxytyrosol 2.5 % walnut. Different letters in the same row indicate significant differences (p<0.05).
The incorporation of olive oils as fat replacers in cooked sausages, has been studied previously
by López et al. (2009; 2011) These authors reported that the replacement of animal fat by olive
oil resulted in an increase of percentages of MUFAs, mainly oleic acid without significantly
altering the ratio w6/w3 and a reduction in saturated fatty acids in cooked sausages.
The addition of walnut resulted in a reduction (p < 0.05) in the percentages of SFAs and
MUFAs and an increase in PUFAs with respect to the control and OLW samples. Almost 40 % of
the total fatty acids in the W sausages were PUFAs. The lower percentage of SFAs in the W
samples (with respect to the control) was due to the reduction of miristic, palmitic and stearic
acids (Table 8.10.). The latter is thought to be responsible for an increased risk of diseases as
cardiovascular (Papadopoulos & Boskou, 1991).
PUFAs accounted for 18.67 % of the total fatty acid content in C (Table 8.10.). In contrast, in
the W and OL samples, they accounted for 40.35 and 16 %, respectively, of the total fatty acids.
The most obvious difference in fatty acid content between different frankfurters studied was that
linoleic acid (C18:2) in CW that accounted for 32.5 % of the total fatty acids. The addition of
walnut also resulted in a significant increase (p < 0.05) in the linolenic acid (C18:3) content. The
presence of linoleic acid and linolenic acid in frankfurters is related with benefits in health,
because is related with the prevention of cardiovascular disease (Hu et al., 2001). In general, the
benefits of the substitution of saturated by MUFAs derive from the fact that MUFAs reduces low-
density lipoprotein (LDL) cholesterol in plasma, while some SFAs increases LDL levels and the
risk of cardiovascular disease.
In the present study, the total w6 and w3 contents were significantly higher in the walnut
samples (Table 8.10.). These results are consistent with the lipid profile of walnut fat, which is
rich in PUFAs, with linoleic and linolenic acids accounting for 49–62% and 6–13 %, respectively,
of total fatty acids (Ayo et al., 2005; Krauss et al., 2000). A previous study of the addition of 20
% walnut to restructured beefsteak reported similar results (Serrano et al., 2005). In that study,
replacing animal fat with walnut improved the fatty acid profile of the beefsteak. The presence of
large amounts of walnut fat in a product assures that the diet contains significant quantities of
beneficial fatty acids.
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The dietary recommendation to prevent cardiovascular disease is to reduce the ratio w6/w3
PUFAs to less than 4 (Enser, 2001), which requires increasing the consumption of w3 fatty acids
and decreasing the consumption of w6 in the diet. In the present study, there were significant
differences (P > 0.05) in the w6/w3 ratios between the C samples and frankfurters with added
walnut and olive oil, with greater ratios observed in those containing both olive oil and walnut.
The addition of walnut produced an improvement in the w6/w3 ratio. Although this ratio was
higher than the recommended level (i.e., 4), the w6/w3 ratio also improved in the new
formulations. The latter was due to the increase in the w3 linolenic acid content being
proportionately greater (13 times more) than the increase in the w6 content (four times), giving a
value of 5.12 (CW), which is close to the recommended ratio.
As shown in Table 8.10., there were significant differences in the AI and IT of C versus those
of the frankfurters elaborated with walnut and olive oil, with the indices decreasing significantly
with the addition of walnut or olive oil to the meat formulations.
Table 8.10. Evolution of storage time on the fatty acid composition and nutrioncal index of
chicken frankfurters elaborated with hydroxytyrosol, walnut and olive oil stored under
modified atmosphere during 21 days. Total fatty
acids (%) Day SFA MUFA PUFA n-3 n-6 n-6/n-3 AI IT
C 0 31.8±0.0a 49.4±0.0d 18.7±0.0c 1.2±0.0d 17.5±0.0d 15.0±0.0a 0.29 0.57
21 31.4±0.8a 46.8±1.4d 21.2±2.8c 1.4±0.0d 18.4±0.5d 14.2±0.1a 0.28 0.45
HXTW
0 22.9±0.2bc 38.1±2.2e 39.3±1.7b 6.2±0.3ab 33.1±1.4bc 5.3±0.0d 0.22 0.15
21 23.5±0.4bc 34.2±1.1e 40.7±2.3b 6.7±0.4ab 34.0±0.4bc 5.1±0.7d 0.23 0.15
W 0 23.2±0.1bc 36.9±0.3e 40.4±0.6b 6.6±0.1ª 33.7±0.4bc 5.1±0.1d 0.21 0.15
21 23.2±1.3bc 33.2±0.7e 45.8±0.4b 5.9±0.4a 39.8±0.8bc 6.8±0.6d 0.16 0.17
OL 0 20.8±0.3cd 64.0±1.1ab 16.2±0.8c 1.5±0.0d 14.6±0.8d 9.7±0.7b 0.22 0.14
21 21.0±0.2cd 59.8±3.5ab 17.1±0.9c 1.5±0.1d 15.5±0.8d 10.4±0.1b 0.24 0.21
OLW
0 19.0±0.0d 58.3±0.2bc 22.7±0.2c 3.3±0.0c 19.4±0.2d 5.9±0.1cd 0.18 0.16
21 19.2±0.4d 47.9±3.5bc 25.9±1.7c 3.1±0.0c 22.7±1.8d 7.3±0.7c 0.17 0.13
HXTOLW
0 19.9±0.6d 54.6±1.1cd 25.8±0.1c 3.1±0.1c 22.6±0.1cd 7.3±0.2c 0.18 0.17
21 19.5±1.8d 47.4±5.5cd 28.12±0.8c 3.5±0.3c 24.7±0.5cd 7.2±0.6c 0.16 0.13
Control, C, chicken meat backfat; W, chicken meat backfat walnut 2.5 %; OL, chicken meat olive oil (20 g/100 g); OLW, chicken meat backfat olive oil (20 g/100 g) walnut 2.5 %; HXTw, chicken meat backfat 50 ppm hydroxytyrosol 2.5 % walnut; HXTOLW, chicken
meat backfat olive oil (20 g/100 g) 50 ppm hydroxytyrosol 2.5 % walnut. PUFA: polyunsaturated fatty acid; MUFA: monounsaturated
fatty acid; SFA: saturated fatty acid; IT: thrombogenic index; AI; atherogenic index. Different letters in the same row indicate significant differences (p<0.05).
In this same line, Serrano et al. (2005), reported that addition of 20 % of walnut in restructured
beef steak reduced both indices. In addition, Nieto (2013), studied the influence of the
incorporation of by-products of rosemary and thyme in the diet of pregnant ewes on the fatty acid
profile of lamb meat and reported reduced indices (AI and TI) in supplemented meat. In contrast,
Nkukwana et al. (2014) reported no significant treatment differences observed in both AI and TI
of breast meat supplemented with Moringa oleifera leaf meal over a period of refrigeration.
The atherogenicity index and thrombogenicity index of chicken sausages (ranges of 0.16–0.29
and 0.13–0.57, respectively) were lower than those reported for beef (ranges of 0.75–0.79 and
1.60–1.85, respectively) (Badiani et al., 2002), lamb (ranges of 0.84–1.40 and 0.92–1.31,
respectively) (Nieto, 2013) and broiler chicken (ranges of 0.38–0.44 and 0.72–0.81, respectively)
(Nkukwana et al., 2014).
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In addition, during storage, the proportion of SFAs was not significantly different from day 0
in any of the groups after storage for 21 days (P > 0.05). The similarities in the proportion of
SFAs during storage were mainly due to the fact that most abundant fatty acids (C16:0 and C18:0)
did not significantly change during storage (P > 0.05). In a previous study with chicken meat
(Nkukwana et al., 2014) the fatty acid composition from broiler chicken supplemented with
Moringa oleifera leaf over a period of refrigeration were studied. In this study, no significant
effects were observed in MUFA (Day 1 MUFA 35.98%) content of chicken meat as influenced
by days of storage (8 days: MUFA 36.06%). In the same line, no significant effects were reported
in the SFA percentages ranged from 35.71 day 1 to 40.28 on day 8. However, the PUFA
percentages decreased significantly (p < 0.05) ranged from 28.06 day 1 to 23.44 on day 8.
In addition, Camo et al. (2008) reported that SFA content in lamb meat increased throughout
the storage period, but the period in that study was longer (28 days) than in the present study. In
contrast, Alvarez et al. (2009) did not find that the storage time had a significant effect on the
proportions of SFA in lamb meat. Moreover, Díaz et al. (2011) reported changes in the PUFA and
MUFA content of lamb meat after refrigeration storage for 7 days due to enzymatic hydrolysis of
muscle lipids and oxidation changes.
In the current study, the percentages of MUFAs and PUFAs significantly changed during
chilled storage (Pp < 0.05). Furthermore, the storage period significantly affected the proportion
of MUFAs, decreasing the percentages in all the samples. In contrast, PUFAs increased in all the
groups, except the HXT samples, where the storage time did not exert a significant effect on the
PUFA ratio. A previous study indicated that the susceptibility of unsaturated fatty acids to
oxidation was related to the degree of unsaturation, with PUFAs more prone to oxidation than
MUFAs, mainly due to proximity to pro-oxidant systems, heme pigments and location in cell
membranes (Elmore et al., 1999).
Given the aforementioned findings, the frankfurter enrichment with PUFAs should have
resulted in a product with higher oxidation and fewer PUFAs. However, in this study we observed
the opposite behaviour. The reduced oxidation in the PUFA enriched frankfurters may be
explained by the presence of HXT, which prevented oxidation. In this respect, several studies
have shown that hydroxytyrosol (HXT, 3,4-dihydroxyphe- nylethanol), an extract obtained from
the olive plant, is a powerful antioxidant with other functional properties (antiinflammatory,
hypotensive and an ability to inhibit platelet aggregation and to reduce atherosclerosis and
cardiovascular disease) (DeJong & Lanari, 2009). The main groups of compounds present in HXT
extract were phenols (hydroxytyrosol and tyrosol), oleuropeosides (oleuropein and verbascoside),
flavones (luteolin-7-glucoside and apigenin-7-glucoside) and flavan-3-ols (catechin). The most
abundant compound in HXT extract was hydroxytyrosol (is a precursor of oleuropein), followed
by Tyrosol, a tyrosol dimer and verbascoside. The antioxidative activity of HXT extracts is due
mainly to the to be scavengers of superoxide anions as well as inhibitors of hypochloric acid-
derived radicals (Gordon et al., 2001). The compounds responsible of the radical scavenging are
simple phenols (hydroxytyrosol) and secoiridoids as oleuropein (Visioli et al., 2002). There is a
great scope and potential for the combination of olive oil, walnut and HXT as natural antioxidant
in the development of new functional meat products.
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8.2.2. Shelf life study of chicken frankfurters
Table 8.11. shows that the colour parameters differed significantly between all the groups
studied. This was to be expected as a result of the different fats, vegetable oils and extracts
(Hydroxytyrosol) used in the sausages. In general, the colour of sausages was modified by the
different fat sources, protein percentages and the presence of walnut. Therefore, it was observed
that, the natural pigments of the meat emulsion ingredients influence the final color of
frankfurters.
Table 8.11. Effects of olive oil, hydroxytyrosol, extracts and walnut on colour (L∗ = lightness,
a∗ = redness, b∗ = yellowness) in frankfurters stored in modified atmosphere
packaging (MAP: 70% O2/20% CO2/10%N2) at day 0 of storage. Sample L* SEM P a* SEM P b* SEM P
C 83.3a 0.12 ** 1.0b 0.30 *** 12.0b 0.25 ***
CW 78.4b 3.0a 12.6b
COL 79.5b 3.2a 14.7a
OLW 82.1a 1.3b 14.1a
HXT1 78.6b 2.9a 12.3b
HXT2 78.5b 2.8a 12.4b
HXT3 79.6b 3.2a 12.1b
HTX1OLW 81.8b 2.0b 14.6a
C: Control; CW: Control walnut 2.5%; COL: control olive oil; OLW: olive oil + walnut; HXT1OLW: 50 ppm Hydroxytyrosol + 2.5% walnut+ olive oil. HXT1: 50 ppm Hydroxytyrosol 1 + 2.5% walnut; HXT2: 50 ppm Hydroxytyrosol 2 + 2.5%
walnut; HXT3: 50 ppm Hydroxytyrosol 3 + 2.5% walnut. Mean and SEM. a,b,c Different letters in the same column (effect
of addition of the Hydroxytyrosol extract or olive oil or walnut) indicate significant differences (P<0.05). P: probability; significance levels: ***p < 0.001; **p < 0.01; *p < 0.05; ns: p>0.05.
As regards the colour in greater detail: lightness (L*) was affected by the fat content and
presence of olive oil. The sausages with HXT and olive oil as fat presented lower values of L*,
b* and higher value of a* compared with the control. The chemical interaction of HXT and fat
and protein particles with the pigments of meat (myoglobin) in the fat/protein matrix could be
responsible for these changes in colour. In this sense, too, Estévez et al. (2005) observed that the
color of sausages was modified by the addition of rosemary extract.
In contrast, in samples OLW no changes were detected in L* when walnut was added, although
a* and b* increased, both to a statistically significant extent (p < 0.05). Similarly, Ayo et al. (2007)
reported no change in L* and increased in a* following the addition of walnut, while another
study by Ayo et al. (2005) reported that the value of b* in cooked sausages increased with walnut.
Such changes in CIELab coordinates would be related to brown colour of walnut.
According to other authors, samples with olive oil presented higher a* and b*values. For
example, Muguerza et al. (2002) observed a decrease (P<0.05) in L* and b* when 20% of pork
fat was replaced by olive oil, the main reason for this difference being we used chicken meat
while most previous studies used pork or beef: Alvarez et al. (2011) observed that the values of
a* in a meat emulsion elaborated with olive oil was lower than in a control emulsion (without
addition of olive oil). Kim et al. (2009) observed significantly higher values of a* and b*
coordinates in patties containing 1 % olive oil and tomato powder.
The main differences observed after replacing animal fat by vegetable oils are significantly (p
< 0.05) lower L* values and higher a* or b* values. Previous studies showed that the colour of
sausages made with vegetable oil (canola oil 25%) were modified in the same line as our results
show (Kim et al., 2009). This colour alteration in olive oil sausages would be due to the structure
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79
modifications that occur during the chopping process, when the oil phase is distributed within the
actomyosin matrix, causing an increase in the surface of the fat particles (Ambrosiadis et al.,
1996).
Regarding to cooking loses analysis, Figure 8.3. shows the influence of olive oil and extract
(hydroxytyrosol) or walnut on the average cooking loss (CLoss) for the different meat emulsion
groups studied. The addition of olive oil and walnut in meat emulsions led to a significant (p <
0.05) decrease in CLoss, while the CLoss values for emulsions formulated with walnut (CW) were
not significantly (p < 0.05) from those obtained with the control emulsion.
These results are consistent with those obtained by Ambrosiadis et al. (1996) who observed
that the cooking loss values for batters containing vegetable oils were significantly (p < 0.05)
lower than those recorded for controls containing pork backfat. The high CLoss values detected for
emulsions made with HXT extract suggest possible interactions between HXT and fat-protein
binders during the emulsification process, which would lead to a decrease in exudates during
cooking usages. Regarding walnut, according to Serrano et al. (2007) observed that the addition
of walnut (20g/100 g) improved the texture and yield of cooked restructured beef steaks, while
Saledja et al. (2016) showed that the addition of walnut decreased weight loss. However, these
authors showed that when a higher amount of walnut was used, cooking loss increased. In this
case, the decrease in exudates might be related to the form of the added walnut preparation, i.e.
dried powder, paste etc.
Figure 8.3. Effect of addition of olive oil, walnut or hydroxytyrosol on cooking losses of cooked
frankfurters. a, b, c: Different letters between rows indicate significant differences
(p<0.05). C: Control; CW: Control walnut 2.5%; COL: control olive oil; OLW: olive oil + walnut; HXT1OLW:
50 ppm Hydroxytyrosol + 2.5% walnut+ olive oil. HXT1: 50 ppm Hydroxytyrosol 1 + 2.5% walnut; HXT2: 50
ppm Hydroxytyrosol 2 + 2.5% walnut; HXT3: 50 ppm Hydroxytyrosol 3 + 2.5% walnut. a,b,c Different letters in the same column (effect of addition of the Hydroxytyrosol extract or olive oil or walnut) indicate significant
differences (P<0.05).
Other authors have observed an improvement in the water holding capacity of meat emulsions
as a result of the addition of protein of different origin (e.g., soy, canola, whey) (Feng et al., 2003).
In our experiment, it was observed that CLoss is related with the protein and fat content of the
sausages. On the other hand, CLoss were observed to be significantly (P<0.05) different in the
samples containing higher levels of fat compared with emulsions containing low fat
a
b b
b
b
cc
c
0
2
4
6
8
10
12
C HXT1 HXT2 HXT3 CW COL OLW HXT1OLW
Co
ok
ing
lo
sses
(%
)
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80
concentrations: in the Control, HXT1, HXT2 and HXT3 samples (where the cooking loss was
significantly higher) the fat content was in the range 2.2-4.43 %, whereas in sausages made with
olive oil the fat content increased to percentages of 9.54-11.6 % and the cooking loss was
significantly lower. CLoss values were 26% lower in samples with olive oil (high fat content) than
in HXT samples (low fat content). These results are consistent with those observed by Serdaroglu
(2006) in beef patties and Estevez et al. (2005) in patés, who observed that reducing the fat content
caused a significant increase in cooking loss. This effect in CLoss of sausages with different
percentages of fat can be explained by the increased protein content and hence in the extracted
proteins, which would increase the number of locations in the polypeptide chains capable of
interacting during heating. As a result, a much more stable gel matrix is formed which leads to a
lower release of water and fat, thus improving binding properties of meat emulsions (Carballo et
al., 1995).
An inadequate extraction of soluble protein and the modification of ratio fat-protein may occur
in sausages where a modification of nature of fat (as this study where animal fat is replaced by
olive oil) and nature of protein (with the addition of protein from walnut) is produced. This
adverse effect reduces the stability of final emulsion and the CLoss will increase. It is therefore
very important to investigate how new ingredients affect cooking losses and any economic
consequences.
Taking into account that the estimated economic losses in the meat industry due to a lack of
stability (high CLoss) are between 0.2 and 1.65 billion dollars, improving the formulation of
sausages to improve stability will be welcome. The addition of walnut, olive oil and HXT to
sausages resulted in cooking losses decreasing from 11% (Control) to 3% in HTX1OLW, a decrease
that would be of great economic importance for the meat industry.
Otherwise, the influence of the experimental factors, olive oil, walnut and hydroxytyrosol, on
lipid oxidation in frankfurters during 21 days of storage is shown in Table 8.12. As expected,
TBARS values increased during the storage period, which agrees with the results of Estevez et al.
(2007), who showed that frankfurters underwent intense oxidative deterioration (measured as
TBARS) during refrigeration.
Regarding the components of the formulations, each extract behaved differently: olive oil
showed higher (p <0.05) TBARS values during storage, while the values of the three HXT extracts
(HXT1, HXT2, or HXT3) decreased significantly, an effect that remained constant during the 21
days of storage. The oxidation of lipids clearly increased compared with Control samples
following the replacement of pork fact by olive oil (higher levels of polyunsaturated fatty acid,
33.6 % and lower levels of saturated fatty acid) and in the presence of walnut (sausages enriched
in n-3 fatty acid and more susceptible to lipid oxidation) and diminished with the addition of HXT
extracts. In samples containing both olive oil and HXT, the TBARS values were 71 % lower than
in Control olive oil samples, confirming the need for the addition of a natural antioxidant such as
hydroxytyrosol in reformulated sausages in order to increase oxidative stability. The nature of the
sausage matrix is very important in the oxidation mechanism: the presence of PUFA,
phospholipids, free iron, hydrophilic and hydrophobic components, animal fat, protein, vegetable
oils, walnut, percentage of salt, etc (Jacobsen et al., 2008).
Lorena Martínez Zamora PhD Thesis, 2019
81
In the same line that our results, Kim et al. (2009) showed that the combination of 1% olive
oil and tomato powder produces inhibition of lipid oxidation. In contrast, Alvarez et al. (2011)
reported higher oxidative stability with the addition of combination of olive oil and canola oil (20
g/100 g).
Tab
le 8
.12.
Eff
ects
of
oli
ve
oil
, hy
dro
xy
tyro
sol
extr
acts
an
d w
aln
ut
on
thio
bar
bit
uri
c ac
id-r
eact
ive
subst
ance
s (T
BA
Rs,
mg M
DA
/kg
pro
du
ct)
in f
rankfu
rter
s st
ore
d i
n m
odif
ied a
tmosp
her
e pac
kag
ing (
MA
P:
70%
O2
/20
% C
O2
/10
%N
2)
duri
ng
21
day
s.
SE
M
F
.
0
.46
C
**
*
2
.07
aB
**
*
3
.12
bA
2
.18
bB
CW
0
.50
B
1
.05
bA
1.9
5cA
1
.20
cA
CO
L
0.5
5C
1
.74
aB
5.0
8aA
4
.27
aA
OL
W
0.7
9B
1
.00
bB
1.1
2cB
1
.62
cA
HX
T1
0.4
6B
0
.38
cB
1.2
1cA
0
.78
dB
HX
T2
0.6
5cB
0
.61
dB
0
.50
d
HX
T3
0.2
9B
0
.42
cB
0.8
0dB
1.5
2cA
HT
X1
OL
W
0.5
1B
0.7
3cB
0
.85
dB
1.2
2cA
Lorena Martínez Zamora PhD Thesis, 2019
82
The antioxidative activity of HXT extracts is due mainly to the metal ion chelation and the
radical scavenging activity. The compounds responsible of the radical scavenging are simple
phenols (hydroxytyrosol) and secoiridoids as oleuropein (not present in HXT1), both phenolic
compounds present in the HXT2 and HXT3.
In order to study the protein oxidation, Table 8.13. shows that the sulfhydryl content decreased
during the 21 days of storage that may be due to the higher protein oxidation which is believed to
result in protein fragmentation and degradation of structural protein because sulfhydryl groups
are converted into disulphides during protein oxidation. This behaviour has been observed
previously by Soyer et al. (2010) in chicken meat, Batifoulier et al. (2002) in turkey meat and by
Nieto et al. (2013) in pork patties, who reported the proteins lose thiols up to 9 days and the
formation of cross-linked myosin disulphide after 12 days. The mean content of the thiol groups
fell from 58.58 to 39.05 gmol/mg protein (39.6 % loss) in C and from 14.75 to10.37 nmol/mg
protein (29.7 % loss) in HTX1OLW during 21 days of storage. Cw showed a loss of thiols of 49.1
%, COL 48.7 %, HXT2 35 %, OLw 27 %, HXT1 21 % and HXT3 31 %. Therefore, the greatest loss
of thiols groups was observed in Cw while the losses were less pronounced in the sausages
elaborated with hydroxytyrosol extracts.
The prevention of protein oxidation of meat by natural antioxidants have been reported by
several studies: Ganhão et al. (2010) noticed an inhibition of protein oxidation in cooked burger
patties after the addition of elm-leaf blackberry, arbutus berry, dog rose and hawthorn. In addition,
Jia et al. (2012) showed a significant prevention of loss of protein sulfhydryl’s after the addition
of black currant extract into pork patties.
According to Papuc et al. (2017), the oxidation of the thiol group occurs by 2 main pathways:
the first one is the formation of a non-radical RS forms with thiol group sulfur-containing acids
and the second one is the oxidation of SH groups by free radicals to generate thiyl radicals that
can react with oxygen to produce thiyl peroxi radical or can form disulphide form after its reaction
with other thiols groups.
In addition, there were significant differences in the initial thiol concentration between control
samples and samples containing HXT: C, Cw, COL was found to be ~60 nmol per mg protein and
OLw, HXT1, was ~30 nmol and HXT2, HXT3, HXT1OLW was ~20 nmol per mg protein at day 0.
Jongberg et al. (2011) found initial values of 58.5 ± 0.71 nmol/mg protein in beef, which is similar
to the values of the control sausages in our study (C, Cw and COL).
From the beginning of storage, HXT extracts significantly reduced the thiol concentration
compared with the values of the control elaborated with pork fat (C), control with walnut (Cw)
and control (C) with walnut and olive oil (OLw). The phenolic compounds present in HXT form
covalent bonds between phenols and protein thiols, including the adducts formed by thiol and
quinone from day 0. This binding between the protein from meat and the phenols of the extracts
reduces the thiol concentration from day 0, so the low levels reported in sausages, which include
HXT, could indicate an extreme prooxidative effect of Hydroxytyrosol extract. However, this is
not clear, because contradictory results have been obtained concerning the protection against thiol
oxidation afforded by phenols due to the formation of protein-phenol interactions.
Previous studies with beef showed similar low levels of thiol when grape extract was added to
beef patties (Jongberg et al., 2011) or the model phenol 4-methyl catechol was added to minced
beef stored in modified atmosphere, the authors of both studies mentioning that these low levels
are not due to thiol oxidation, but to protein-phenol covalent interactions (Jongberg et al., 2013).
Lorena Martínez Zamora PhD Thesis, 2019
83
Also, Tang et al. (2015) reported the formation of adducts between thiols of peptides of a myosin
and quinones from rosmarinic acid using MALDI- TOF/TOF MS.
Ta
ble
8.1
3.
Eff
ects
of
oli
ve
oil
, h
yd
roxyty
roso
l ex
tract
s an
d w
aln
ut
on
con
cen
tra
tio
n o
f p
rote
in t
hio
ls i
n f
ran
kfu
rter
s st
ore
d
in m
od
ifie
d a
tmo
sph
ere
pack
agin
g (
MA
P:
70%
O2/2
0%
CO
2/1
0%
N2)
du
rin
g 2
1 d
ay
s.
0
7
14
21
P
ST
5
8.6
aA
5
1.9
aA
4
6.8
aB
3
5.0
aC
**
*
CW
6
7.4
aA
5
4.5
aA
4
4.2
aB
34
.3aC
CO
L
59
.7aA
46
.7aB
45
.6aB
3
0.7
aC
OL
W
30
.9b
2
6.2
b
2
3.7
b
2
2.3
b
ns
HX
T1
24
.6b
2
4.7
b
2
0.4
b
1
9.3
b
ns
HX
T2
19
.7c
1
8.1
c
17
.6b
1
2.8
c
n
s
HX
T3
20
.1c
2
0.7
c
20
.4b
1
6.9
b
ns
HT
X1
OL
W
14
.7c
1
4.1
c
13
.1c
1
0.4
c
n
s
C:
Con
tro
l; C
W:
Co
ntr
ol
wal
nu
t 2
.5%
; C
OL
: co
ntr
ol o
liv
e o
il;
OL
W:
oli
ve
oil
+ w
alnu
t; H
XT
1O
LW
: 50
pp
m H
yd
rox
yty
roso
l +
2.5
% w
aln
ut+
oli
ve
oil
. H
XT
1: 5
0 p
pm
Hyd
roxy
tyro
sol
1 +
2.5
% w
aln
ut;
HX
T2:
50
pp
m H
yd
rox
yty
roso
l 2
+ 2
.5%
wal
nu
t; H
XT
3:
50
pp
m H
yd
rox
yty
roso
l 3
+ 2
.5%
wal
nut.
P:
p -
val
ues
. F
: p
val
ues
of
sausa
ges
fo
rmu
lati
on
wit
h d
iffe
rent
HX
T e
xtr
acts
or
oli
ve
oil
or
wal
nu
t. S
.T:
p v
alues
of
sto
rag
e ti
me.
Mea
n a
nd
SE
M. a,
b,c
Dif
fere
nt
lett
ers
in th
e sa
me
colu
mn
(ef
fect
of
add
itio
n o
f th
e H
yd
rox
yty
roso
l ex
trac
t o
r o
live
oil
or
wal
nut:
a,
b,
c) i
nd
icat
e si
gn
ific
ant
dif
fere
nce
s (P
<0
.05
). A
,B,C
Dif
fere
nt
lett
ers
in t
he
sam
e ro
w (
effe
ct o
f st
ora
ge
day
) in
dic
ate
sign
ific
ant
dif
fere
nce
s (P
<0.0
5).
P:
pro
bab
ilit
y;
sign
ific
ance
lev
els:
***P
< 0
.00
1;
**P
< 0
.01;
*P
< 0
.05;
ns:
P>
0.0
5.
Lorena Martínez Zamora PhD Thesis, 2019
84
Finally, Table 8.14. shows the effect of 21 days of storage on the sensory attributes (odour,
flavour, rancid odour, rancid flavour and acceptability) of sausages. Odour and flavour scores
were maximal at day 0 in fresh sausages and decreased during the 21 days of storage, the decrease
being particularly intense from day 7. Regarding rancid flavour and rancid odour were not
perceptible at day 0, but were detectable from day 7, moderate on day 14 and intense on day 21.
The low scores for flavour and odour are due to the oxidation process (pigment, protein and lipid
oxidation) causing rancid odour, fat darkening, lean browning and loss of metallic blood odour.
In contrast, sausages with olive oil OLw obtained the highest value for rancid odour between all
the samples studied (in the same line as the TBARS results) and the highest score of acceptability
at day 21. These results confirm that HXT extracts delay lipid oxidation, rancidity and general
off-flavours; and olive oil increases rancidity in sausages. In the same line, Fernández- Lopez et
al., (2005) reported that the addition of rosemary extracts delayed the development of rancidity
in beef meat.
In contrast, the application of olive oil and evaluation of sensory evaluation was studied by
Muguerza et al. (2002) in fermented sausages elaborated with 20 % of olive oil. These authors
showed that the higher score for taste and odour of sausages was for sausages elaborated with
olive oil. In our study the OLw presented higher scores of rancid flavours (in the same line that
TBARS results) and the highest acceptability at day 21.
The chicken sausages prepared with walnut and olive oil scored higher for acceptability than
the control and HXT samples. Otherwise, sausages manufactured with HXT2 and HXT3 presented
the lowest acceptability score because the panellists were able to detect the hydroxytyrosol
flavour. For that reason, only the extract HXT1 was used to make sausages with walnut and olive
oil (HXT1OLW) and study the possibility of synergism effect of the different ingredients. The
sample with the highest acceptability score on day 21 was OLW.
This lower degree of acceptability of samples containing HXT might be related to the
characteristic flavour that the extracts imparted to the sausages. In contrast, the panellists were
also able to evaluate flavour of walnut and olive oil in the sausages, but these flavours were noted
positively. To our knowledge, there are not studies in the literature that include a sensory analysis
of meat products elaborated with hydroxytyrosol, an aspect that is very important if these extracts
are to be used in functional meat products. A previous study involving HXT in meat did not make
a sensory evaluation of the resulting product (Cofrades et al., 2011) and only technological and
nutritional properties of the meat product with extracts were evaluated.
Lorena Martínez Zamora PhD Thesis, 2019
85
Table 8.14. Effect of storage time on the odour, flavour, acceptability of frankfurters
stored in modified atmosphere CO2/10 % N2) during 21 days
Sam
ple
s
Ran
cid
flav
ou
r
0
.12
ns
0
.10
ns
0
.15
ns
0
.14
ns
0
.11
ns
0
.09
ns
0
.13
ns
0
.13
ns
0
.12
ns
0
.17
ns
0
.17
ns
3.5
0a
2
.50
a
0
.21
ns
0
.10
ns
0
.25
ns
4.5
0a
3
.25
a
1.7
5b
CW
5.0
0 a
1.0
0c
1
.00
c
2.0
0b
1
.75
b
1
.25
b
CO
L
5
.00
a
2.0
0b
1
.00
c
2.0
0 b
3.2
5b
1
.75
b
1
.25
b
OL
W
5
.00
a
1.0
0b
2
.00
b
2.2
5ns
1
.25
c
2.2
5b
2
.00
a
HX
T1
5
.00
a
1.0
0b
1
.00
c
1.0
0c
1
.25
c
1.7
5b
HX
T2
3
.00
b
1.2
5b
1.0
0c
1
.00
c
HX
T3
3
.00
b
1.0
0b
1
.25
c
1.0
0c
1
.25
c
1.0
0b
HX
T1O
LW
1.2
5b
1.2
5c
1
.50
c
1.2
5b
C:
Co
ntr
ol;
CW
: C
ontr
ol
wal
nut
2.5
%;
CO
L:
con
tro
l oli
ve
oil
; O
LW
: oli
ve
oil
+ w
alnu
t; H
XT
1O
LW
: 50
pp
m H
yd
roxy
tyro
sol
+ 2
.5%
wal
nut+
oli
ve
oil
. H
XT
1:
50
pp
m H
yd
roxyty
roso
l 1
+ 2
.5%
wal
nut;
HX
T2:
50 p
pm
Hy
dro
xyty
roso
l 2
+ 2
.5%
wal
nu
t; H
XT
3:
50
pp
m H
yd
roxyty
roso
l 3 +
2.5
% w
aln
ut.
P:
p -
val
ues
of
sau
sag
es f
orm
ula
tio
n w
ith
dif
fere
nt
HX
T e
xtr
acts
or
oli
ve
oil
or
wal
nut.
Mea
n a
nd
SE
M.
a,b,cD
iffe
ren
t le
tter
s in
the
sam
e c
olu
mn (
effe
ct o
f ad
dit
ion
of
the
Hyd
roxy
tyro
sol
extr
act
or
oli
ve
oil
or
wal
nut:
a,
b,
c) i
nd
icat
e si
gnif
ican
t dif
fere
nce
s (P
<0
.05
). P
: p
rob
abil
ity;
sign
ific
ance
lev
els:
***p
< 0
.00
1;
**p
< 0
.01;
*p
< 0
.05
; n
s: p
> 0
.05
Lorena Martínez Zamora PhD Thesis, 2019
86
According to Booren and Mandigo (1987) the microstructure of a meat emulsion is composed
of different structures within the protein network, the most important factors being: particle size,
surface area availability of myofibrillar proteins for covering fat globules by proteins and forming
a stable network protein matrix that allows more fat globules covered by proteins, which also
allows for a more stable protein matrix after cooking.
Figure 8.4. shows the microstructure of chicken, control (pork fat), HXT1 (HXT and walnut)
and HXT1OLW (HXT, olive oil and walnut) sausages. Different structures were observed for
different ingredients. The matrix network for the chicken sausages was based on the type of fat
(animal fat or olive oil), which affected the properties of the protein and fat globule matrix and,
subsequently, the textural and viscoelastic characteristics of the meat emulsions. Control sausages
had porous structures and cavities mixed with fat globules and meat aggregates. Sausages with
walnut and olive oil (HXT1OLW) had altered aggregation patterns in myofibrillar proteins, giving
a more interactive network and appeared compact and less porous with much smaller fat globules
compared to C, as well as high elasticity and the formation of a stable protein matrix.
Figure 8.4. Scanning electron micrographs (magnification, 500×) of backfat (Control) (A),
hydroxytyrosol extract 1+ 2.5% walnut (HXT1) (B) or hydroxytyrosl 1 + 2.5%
walnut+ 20 g/100 g olive oil (HXT1OLW) (C).
A B
C
Sausages incorporating HXT1 showed different structures than control samples or sausages
with olive oil, related to the composition of the emulsion, void spaces and small HXT globules in
the network protein structure. Walnut and HXT were incorporated into the protein matrix, causing
more fat globules to be covered by walnut protein and increased separation of meat particles in a
more dispersed and less continuous network structure. This microstructure appeared denser and
less spongy than back fat emulsions (control). The microstructures of meat emulsion containing
HXT extracts and combinations with olive oil and walnut have not been previously studied;
therefore, it is difficult to compare the microstructures observed in this study. However, similar
microstructures were described in sausages incorporating walnut and soy proteins by Ayo et al.
Lorena Martínez Zamora PhD Thesis, 2019
87
(2005) and Feng et al. (2003), respectively. In this same line Jiménez-Colmenero et al. (2003)
showed that the addition of walnuts significantly affected morphology of sausages (with an
interference with the formation of protein network structures).
8.3. Assay III:
Obtained results of endogenous and exogenous enrichment of frozen
pre-cooked meat products, through the incorporation of Zn and Se to
animal feed and natural antioxidant extracts during the elaboration of
chicken nuggets
The antioxidant and antimicrobial capacities of studied extracts in the present assay depend on
the concentrations of the phenolic compounds they contain. The extracts obtained from
Rosmarinus officinalis L. had 8.10 % of rosmarinic acid (RH) and 5.76 % of diterpenes (RL), such
as, carnosol, isorosmanol, rosmadial, rosmaridiphenol, picrosalvin and rosmariquinone. The
Harpagophytum procumbens extract (H) had 3.05 % of harpagoside, as bioactive compound.
Grape (Vitis vinífera) seed extract (GS) contained 95.6 % of oligomeric proanthocyanidins
(OPCs), 2.2 % catechin and also 2.2 % epicatechin. Pomegranate (Punica granatum) (P) had
41.38 % punicalagin as the principal bioactive compound. Finally, the hydroxytyrosol extract
(HYT) obtained from olive leaf during olive oil production contained 7.26 % of this compound.
The proximate composition (moisture, ash, protein and lipid contents (%)) of the frozen
chicken nuggets enriched with natural extracts from fuits, seeds and herbs is shown in Table 8.15.
Thomas et al. (2016) and Thomas et al. (2014) showed comparable results in pork nuggets
enriched with kordoi (Averrhoa carambola) fruit and bamboo (Bambusa polymorpha) shoot.
Recently, Carvalho et al. (2018) published similar results in chicken nuggets enriched with
Omega-3 and fibre by chia (Salvia hispanica L.) flour, although they obtained higher values for
the lipid (25-28 g/100g) and ash (4 g/100 g) content due to the incorporation of flour and Omega-
3 in the formula.
Table 8.15. Proximal composition (M ± SD) of chicken frozen nuggets enriched in Zn, Se and
phenolic compounds from natural extracts. Proximate composition (M ± SD)
Treatment Samples Moisture
(%)
Ash
(%)
Protein
(%)
Lipid
(%)
Se
(mg/100g)
Zn
(mg/100g)
Enriched with
inorganic
forms of Zn
and Se
C 66.6 ± 0.1 1.4 ± 0.0 10.8 ± 0.0 3.9 ± 0.0 3.10*10-3 c 0.44b
CRH+P 63.0 ± 0.3 1.4 ± 0.0 11.3 ± 0.0 5.4 ± 0.0 4.20*10-3 bc 0.53b
CRL+GS 67.0 ± 0.9 1.3 ± 0.1 11.0 ± 0.0 4.8 ± 0.0 4.00*10-3 bc 0.51b
CHYT+P+
H
66.8 ± 0.2 1.2 ± 0.1 12.2 ± 0.1 5.1 ± 0.0 3.50*10-3 c 0.49b
Enriched with
organic forms
of Zn and Se
SZ 64.4 ± 0.5 1.6 ± 0.1 11.3 ± 0.0 4.5 ± 0.0 6.70*10-3 a 0.58ab
SZRH+P 64.4 ± 0.2 1.6 ± 0.1 11.2 ± 0.1 5.4 ± 0.0 5.70*10-3 ab 0.74a
SZRL+GS 64.8 ± 0.9 1.2 ± 0.1 11.2 ± 0.0 4.6 ± 0.0 4.20*10-3 bc 0.77a
SZHYT+P
+H
65.6 ± 0.7 1.4 ± 0.1 10.7 ± 0.0 5.1 ± 0.0 4.60*10-3 b 0.72a
C: Control; CRH+P: 1000 ppm Rosemary extract + 1500 ppm Pomegranate extract; CRL+GS: 1000 ppm Nutrox OS + 1500 ppm Grape
seed extract; CHYT+P+H: 1500 ppm Pomegranate extract + 750 ppm Hydroxytyrosol + 500 ppm Harpagophytum; SZ: Control fortified with Zn and Se meat; SZRH+P: 1000 ppm Rosemary extract + 1500 ppm Pomegranate extract; SZRL+GS: 1000 ppm Nutrox OS + 1500
ppm Grape seed extract; SZHYT+P+H: 1500 ppm Pomegranate extract + 750 ppm Hydroxytyrosol + 500 ppm Harpagophytum. a, b, c:
different letters among data in the same column indicate significant differences between samples (p<0.05).
Lorena Martínez Zamora PhD Thesis, 2019
88
However, there were observed significant difference (p < 0.05) between samples in terms of
the Se and Zn contents (mg/100 g). Samples elaborated with meat from chicken broilers fed with
organic forms of Zn and Se showed higher concentrations of Zn and Se than the samples made
from meat enriched with inorganic Zn and Se. This fact can be related with the findings of our
previous research, included in Assay I (Paper I: Martínez, Ros & Nieto, 2018). The highest
content of Se was observed in SZ with no natural extracts, while the highest concentrations of Zn
were found in SZRL+GS, SZRH+P and SZHYT+P+H. Therefore, it seems that phenolic compounds from
Rosmarinus officinalis, grape (Vitis vinifera) seed, Punica granatum, hydroxytyrosol and
Harpagophytum procumbens are rich in Zn, but not in Se, because their incorporation increases
the Zn content, but decreases Se concentration.
The daily consumption of 100 g of chicken nuggets enriched in organic forms of Zn and Se
(SZRH+P, SZRL+GS, SZHYT+P+H) would represent 6.4–9.6 % of the RDA for Zn for a healthy adult
(8–12 mg/day) and 9–10 % of the RDA of Se (55–70 µg/day). It can therefore be claimed that
consumption of this kind of product contributes to the recommended levels of these essential
minerals, as would a diet containing other products rich in Se and Zn, such as oat, mussels,
mushrooms, beer yeast or cockles.
8.3.1. Shelf-life study of frozen chicken nuggets
Variations in pH are associated with food deterioration due to the fact that pH values are an
indicator of food stability associated with microbial growth and chemical reactions. Table 8.16.
shows the changes in pH during the twelve months of frozen storage. As it can be seen, there were
no significant differences among samples at the same day of analysis, but there were differences
between months (p < 0.05). The pH values of chicken nuggets formulated with combinations of
natural extracts ranged from 6.10 to 6.64, due to the fact that natural sources of phenolic
compounds prevent meat oxidation and, therefore, a decrease of pH, while frozen storage reduces
water activity and prevents microbiological growth.
Similarly, Teruel et al. (2015) obtained different results in chicken nuggets. During 9 months
of frozen storage they observed no significant differences (p < 0.05) in pH values, although the
initial pH values were similar. In their research rosemary extracts were incorporated in the chicken
nuggets formula, but they did not combine different sources of phenolic compound, as the present
study does. Verma et al. (2010) and Hwang et al. (2013) also obtained different results after
incorporating apple pulp and Artemisa prínceps Pamp., respectively. However, Verma et al.
(2010) did not carry out a shelf life study while Hwang et al. (2013) did so for 15 days of
refrigerated storage.
Table 8.16. also shows obtained results for CIELab measurements in all the samples during
the twelve months of frozen storage. L* (lightness), a* (redness) and b* (yellowness) showed
significant differences (p < 0.05) between the months of storage (0, 3, 6, 9 and 12), but there were
no differences between samples at these times.
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Table 8.16. Results of pH values and colour CIELab (M ± SD) in chicken frozen nuggets for
twelve months under frozen storage. CIELab Storage time (months)
Samples 0 3 6 9 12
pH
C 6.46 ± 0.03b 6.55 ± 0.08a 6.26 ± 0.05c 6.14 ± 0.08d 6.45 ± 0.09c
CRH+P 6.48 ± 0.05b 6.54 ± 0.04a 6.36 ± 0.01c 6.15 ± 0.02d 6.42 ± 0.07c
CRL+GS 6.50 ± 0.10b 6.64 ± 0.02a 6.55 ± 0.05c 6.17 ± 0.03d 6.40 ± 0.20c
CHYT+P+H 6.49 ± 0.08b 6.62 ± 0.05a 6.43 ± 0.08c 6.20 ± 0.05d 6.46 ± 0.16c
SZ 6.41 ± 0.06b 6.56 ± 0.07a 6.39 ± 0.05c 6.10 ± 0.10d 6.42 ± 0.04c
SZRH+P 6.38 ± 0.04b 6.61 ± 0.05a 6.33 ± 0.11c 6.11 ± 0.06d 6.41 ± 0.02c
SZRL+GS 6.54 ± 0.05b 6.63 ± 0.09a 6.48 ± 0.18c 6.24 ± 0.10d 6.45 ± 0.15c
SZHYT+P+H 6.44 ± 0.02b 6.62 ± 0.11a 6.40 ± 0.04c 6.19 ± 0.15d 6.38 ± 0.06c
L* (lightness)
C 75.64 ± 2.06c 83.62 ± 2.01a 81.93 ± 1.98a 84.23 ± 1.25a 74.22 ± 1.14b
CRH+P 66.83 ± 1.88c 80.59 ± 2.15a 82.77 ± 1.85a 85.27 ± 1.36a 69.84 ± 0.87b
CRL+GS 62.51 ± 1.25c 80.10 ± 1.84a 79.86 ± 3.01a 81.36 ± 2.05a 65.57 ± 1.54b
CHYT+P+H 64.64 ± 1.54c 81.19 ± 1.91a 78.94 ± 2.54a 80.65 ± 2.47a 69.86 ± 1.99b
SZ 78.50 ± 1.78c 82.24 ± 1.35a 83.92 ± 1.25a 84.74 ± 1.88a 75.68 ± 2.30b
SZRH+P 66.23 ± 2.31c 77.47 ± 1.88a 80.08 ± 1.86a 80.05 ± 1.79a 75.18 ± 3.14b
SZRL+GS 64.49 ± 3.01c 77.70 ± 2.22a 82.47 ± 1.88a 82.07 ± 0.98a 73.23 ± 2.11b
SZHYT+P+H 65.56 ± 2.89c 76.03 ± 2.54a 79.78 ± 1.96a 79.12 ± 1.30a 70.50 ± 1.85b
a* (redness)
C 1.64 ± 0.03a 0.41 ± 0.01bc 0.55 ± 0.05bc 0.47 ± 0.03c 1.13 ± 0.47b
CRH+P 6.31 ± 1.04a 1.77 ± 0.17bc 2.02 ± 0.85bc 0.86 ± 0.11c 2.82 ± 1.15b
CRL+GS 7.4 ± 1.07a 1.69 ± 0.94bc 1.44 ± 0.34bc 0.16 ± 0.02c 4.41 ± 1.99b
CHYT+P+H 6.85 ± 1.17a 1.59 ± 0.69bc 2.63 ± 1.02bc 1.91 ± 0.91c 2.43 ± 0.87b
SZ 2.98 ± 0.05a 0.30 ± 0.01bc 0.73 ± 0.08bc 0.04 ± 0.00c 1.03 ± 0.55b
SZRH+P 6.29 ± 0.01a 3.23 ± 1.25bc 2.96 ± 1.15bc 2.17 ± 0.88c 3.03 ± 1.02b
SZRL+GS 6.35 ± 0.15a 1.12 ± 0.05bc 0.71 ± 0.22bc 0.93 ± 0.15c 2.27 ± 0.77b
SZHYT+P+H 5.27 ± 1.24a 2.11 ± 0.24bc 2.69 ± 0.88bc 1.75 ± 0.79c 2.64 ± 0.97b
b* (yellowness)
C 28.80 ± 1.15a 21.01 ± 3.00c 22.03 ± 1.42bc 20.12 ± 1.25b 10.80 ± 0.54d
CRH+P 33.90 ± 1.26a 21.92 ± 1.87c 21.60 ± 1.87bc 24.78 ± 0.86b 15.51 ± 0.32d
CRL+GS 28.89 ± 1.98a 20.82 ± 2.03c 22.01 ± 1.24bc 22.48 ± 2.74b 14.61 ± 0.01d
CHYT+P+H 33.45 ± 2.41a 21.93 ± 1.96c 21.88 ± 2.56bc 24.15 ± 2.81b 14.12 ± 2.14d
SZ 33.77 ± 1.47a 20.09 ± 2.01c 23.22 ± 3.05bc 22.44 ± 3.05b 13.63 ± 1.24d
SZRH+P 32.83 ± 1.88a 19.25 ± 1.44c 22.46 ± 2.87bc 25.91 ± 1.78b 15.78 ± 1.01d
SZRL+GS 23.49 ± 1.99a 17.50 ± 1.87c 20.45 ± 1.85bc 17.48 ± 1.45b 12.64 ± 0.83d
SZHYT+P+H 29.88 ± 2.54a 19.15 ± 1.25c 19.48 ± 0.76bc 20.92 ± 1.58b 13.45 ± 1.02d
C: Control; CRH+P: 1000 ppm Rosemary extract + 1500 ppm Pomegranate extract; CRL+GS : 1000 ppm Nutrox OS + 1500 ppm Grape seed extract; CHYT+P+H : 1500 ppm Pomegranate extract + 750 ppm Hydroxytyrosol + 500 ppm Harpagophytum; SZ: Control fortified
with Zn and Se meat; SZRH+P : 1000 ppm Rosemary extract + 1500 ppm Pomegranate extract; SZRL+GS : 1000 ppm Nutrox OS + 1500
ppm Grape seed extract; SZHYT+P+H: 1500 ppm Pomegranate extract + 750 ppm Hydroxytyrosol + 500 ppm Harpagophytum. a, b, c: different letters among data in the same row indicate significant differences between month of analysis (p<0.05).
An analysis of these results points to no significant differences between samples (p < 0.05),
although it can be observed that the samples with the lowest variations in CIELab colour were the
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nuggets enriched in organic forms of Zn and Se, especially the sample that incorporated rosemary
and pomegranate, SZRH+P, followed by SZRL+GS and SZHYT+P+H. The least stable samples in this
respect were C and SZ. So, although incorporating organic forms of Zn and Se helps to maintain
the colour, it is also necessary to incorporate sources of phenolic compounds, such as rosemary,
pomegranate or hydroxytyrosol.
Other studies with chicken nuggets obtained similar results concerning colour (Carvalho et al.,
2018; Teruel et al., 2015; Teruel et al., 2014; Hwang et al., 2013) by incorporating chia, rosemary
and even ascorbic acid with ganghwayakssuk. However, these studies were shorter than the
present research, while no studies that combine feed sources of organic minerals and the addition
of extracts rich in phenolic compounds have been found.
In the same way, the malondialdehyde (MDA) content of the frozen nuggets is shown in Figure
8.5. (A). The TBARs values represent the aldehydes and carbonyls as secondary lipid oxidation
products that alter the flavour of meat. As can be appreciated, lipid oxidation increased
significantly (p < 0.05) up to 1.5 mg MDA/kg at month 9 of frozen storage in the C, SZ and CRL+GS
samples. However, when organic forms of Zn and Se were combined with rosemary and
pomegranate in SZRH+P, this sample resisted lipid oxidation and showed 47 % lower TBARs
values than C or SZ after 12 months of storage (p < 0.05). The decrease in lipid oxidation recorded
at this time might be caused by losses in the oxidation products formed or the reaction of MDA
with proteins (Maqsood & Benjakul, 2010).
This antioxidant effect observed in samples enriched exogenously with natural extracts
obtained as food industrial by-products would be due to their high phenolic content. For example,
the sample with the lowest MDA level combined RH, with 8.10 % rosmarinic acid and P with
41.38 % punicalagin. In addition, the incorporation of HYT (7.16 %), diterpenes form RL (5.8 %)
and catechins from GS (4.4 %) also reduced the TBARs levels by a 25–35 % compared with the
values recorded in C and SZ.
0 3 6 9 1 2
0 .0
0 .5
1 .0
1 .5
2 .0
T im e (m o n th s )
TB
AR
S
(mg
MD
A/k
g n
ug
ge
t)
A
0 3 6 9 1 2
0
1 0
2 0
3 0
4 0
T im e (m o n th s )
nm
ol
th
iol/
mg
pr
ote
in
C
C R H + P
C R L + G S
C H Y T + P + H
S Z
S Z R H + P
S Z R L + G S
S Z H Y T + P + H
B
Figure 8.5. Results of lipid oxidation, TBARs (mg MDA/kg) (A); protein oxidation, thiol groups
(nmol thiol/mg protein) (B) of chicken frozen nuggets for twelve months of storage. C: Control; CRH+P: 1000 ppm Rosemary extract + 1500 ppm Pomegranate extract; CRL+GS : 1000 ppm Nutrox OS + 1500 ppm Grape
seed extract; CHYT+P+H : 1500 ppm Pomegranate extract + 750 ppm Hydroxytyrosol + 500 ppm Harpagophytum; SZ: Control fortified with Zn and Se meat; SZRH+P : 1000 ppm Rosemary extract + 1500 ppm Pomegranate extract; SZRL+GS : 1000 ppm Nutrox OS + 1500
ppm Grape seed extract; SZHYT+P+H: 1500 ppm Pomegranate extract + 750 ppm Hydroxytyrosol + 500 ppm Harpagophytum.
Similar trends in TBARs values were observed by Hwang et al. (2013) in chicken nuggets that
incorporated ganghwayakssuk (Artemisia prínceps Pamp.) in combination with ascorbic acid to
increase the shelf life (15 days refrigerated storage at 4ºC). While no similar results have been
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identified in this kind of product, Nieto et al. (2017) (Assay II) observed the same trend in chicken
sausages, which were enriched with hydroxytyrosol extracts, walnuts and extra virgin olive oil
and analysed during 21 days of refrigerated storage.
On the other hand, protein oxidation was determined by reference to the thiol groups (see
Figure 8.5. (B) in which it can be seen that their concentration slightly decreased during frozen
storage). Nevertheless, samples that incorporated Nutrox OS4 and grape seed extract (CNOS+GS
and SZNOS+GS) showed much higher concentrations of thiol groups than C and SZ. In the same
way, the incorporation of organic forms of Zn and Se in SZ decreased protein oxidation compared
with C, due to the antioxidant capacity of the minerals. So, it can be said that the combination of
phenolic compound sources with Zn and Se protects against the loss of thiol groups for up to 1
year of frozen storage. Even though no similar studies have been obrerved, our group, Nieto et al.
(2013) observed a similar protective effect against the loss of thiol groups during 9 days of chilled
storage in pork patties containing sources of phenolic compounds (in this case, the essential oils
of oregano, rosemary or garlic). Jongberg et al. (2018) observed a reduction in protein oxidation
in brine-injected pork loins containing ascorbate and green tea or mate extracts during chilled
storage. It is clear, then, that antioxidant compounds can reduce the concentration of thiol groups,
acting as an indicator of protein oxidation. Similarly, high quantities of polyphenols can reduce
the amount of thiol groups, leading to the formation of protein cross-links, the smallest phenolic
compounds, such as diterpenes from rosemary, penetrating inter-fibrillar regions of proteins
forming crosslink peptide chains (Mulaudzi et al., 2012). This might explain why CRH+P, SZRH+P
and SZRL+GS had lower levels of thiol groups at month 9 and 12 than the rest of the samples, which
were also rich in phenolic compounds, but had a higher molecular weight, preventing crosslinking
with the protein chain.
The results of the microbiological analyses (cfu/g) made in frozen chicken nuggets over the
twelve months are shown in Table 8.17. As can be seen, all the results comply with the legal limits
(EC 2073/2005 for Europe; RD 474/2014 for Spain). All the samples showed <10 cfu/g of E. Coli
and S. Aureus and no L. Monocytogenes and Salmonella in 25 g at all sampling times. However,
significant differences were obtained for the total viable counts (cfu/g) among different samples
and months of frozen storage.
Table 8.17. Results of microbiological analysis (M ± SD cfu/g) in chicken frozen nuggets for 12
months under frozen storage. Storage time (months)
Microorganism Samples 0 3 6 9 12
TVC C 550±40c z 2400±120a yz 6500±250a xy 8950±425a wx 10000±500a w
CRH+P 725±50b z 975±45bc yz 1000±60cd xy 2350±115bc x 4500±425bc w
CRL+GS 665±46bc z 780±50c yz 1200±50c wxy 1500±80c wx 2500±180c w
CHYT+P+H 615±34bc z 900±70bc yz 1450±25c xy 1850±95c x 4700±200bc w
SZ 1100±90a z 1200±80b yz 3150±210b xy 4500±120b x 8500±350b w
SZRH+P 725±67b z 1200±90b yz 1500±200c xy 3200±320bc wx 4500±495bc w
SZRL+GS 220±40c z 400±50c yz 700±80d xy 1000±60c wx 2500±120c w
SZHYT+P+H 1020±98a z 1475±115b yz 2300±90bc xy 3350±250bc wx 5000±290bc w
E. Coli <10
S. Aureus <10
L. Monocytogenes Absence in 25 g
Salmonella Absence in 25 g
TVC: Total Viable Count. C: Control; CRH+P: 1000 ppm Rosemary extract + 1500 ppm Pomegranate extract; CRL+GS : 1000 ppm
Nutrox OS + 1500 ppm Grape seed extract; CHYT+P+H : 1500 ppm Pomegranate extract + 750 ppm Hydroxytyrosol + 500 ppm
Harpagophytum; SZ: Control fortified with Zn and Se meat; SZRH+P : 1000 ppm Rosemary extract + 1500 ppm Pomegranate extract;
SZRL+GS : 1000 ppm Nutrox OS + 1500 ppm Grape seed extract; SZHYT+P+H: 1500 ppm Pomegranate extract + 750 ppm Hydroxytyrosol
+ 500 ppm Harpagophytum. a, b, c, d: different letters among data in the same row indicate significant differences between samples (p<0.05). w, x, y, z: different letters among data in the same line indicate significant differences between month of analysis (p<0.05).
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It can be appreciated how the control samples, C and SZ, with inorganic and organic forms of
Zn and Se, respectively, had higher TVC values than the rest of the samples that included natural
extracts. Moreover, samples that incorporated RL and GS extract (CRL+GS, SZRL+GS) obtained the
best results for microbiological growth (75 % and 70 %, respectively less than C and SZ),
followed by samples containing RH and P (CRH+P, SZRH+P), with 55 % and 47 % less, respectively.
Samples that combined HYT, P and H (CHYT+P+H, SZHYT+P+H) showed a 53 % and 41 % lower TVC
than the controls (C and SZ). This demostrates that, although the final counts were lower in
samples incorporating organic Zn and Se, the results could be improved if phenolic compound
sources are added.
Similar TVC results were obtained by Hwang et al. (2013) in chicken nuggets enriched with
ganghwayakssuk and by Thomas et al. (2014 and 2016) in pork nuggets with kordoi fruit juice
and bamboo shoot extract on day 0.
Finally, sensory analysis of chicken nuggets was carried out at 0 and 12 months of frozen
storage. The results are shown in Figure 8.6. (A) and (B).
Figure 8.6. Results of sensory evaluation (A: at time 0 and B: at month 12) of chicken frozen
nuggets for twelve months of storage.
0
1
2
3
4
5Own Odor
Rancid Odor
Extract Odor
Own Colour*
Brown Colour*
Extract Colour*
Own Flavour
Rancid Flavour
Extract Flavour
Acceptability
Month 0
0
1
2
3
4
5Own Odor
Rancid Odor
Extract Odor
Own Colour*
Brown Colour*
Extract Colour*
Own Flavour
Rancid Flavour
Extract Flavour
Acceptability
Month 12
C
CRH+P
CRL+GS
CHYT+P+H
SZ
SZRH+P
SZRL+GS
SZHYT+P+H
A
B
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As regards the colour values, there were significant differences (p < 0.05) between the “Own
Colour”, “Brown Colour” and “Extract Colour” results, all related with the CIELab results. The
C and SZ samples obtained the highest score for “Own Colour, at 0 and 12 months, while the rest
of samples were valued as browner (“Brown Colour”). On the other hand, rancid odour and
flavour were not appreciated at either time, which may be related with the TBARs values which
did not exceed 2 mg MDA/kg (Gray & Pearson, 1987). Therefore, “Own Odor” and “Own
Flavour” were valued positively, while “Extract Flavour” was highly scored in samples with
natural extracts at month 0 of analysis, although this attribute has disappeared by month 12.
However, no previous research results have been detected to compare this effect. It is possible
that the compounds responsible for strong flavours, HYT, RH or GS, are degraded during lengthy
frozen storage, because phenolic compounds react with the molecules produced by lipid and
protein oxidation. This effect needs further investigation. The data regarding to textural attributes
are not presented because there were no significant differences between the samples and controls
(C and SZ). Finally, “Acceptability” was positively valued in all the samples at month 0 as 12, so
the incorporation of phenolic compounds exogenously and the minerals Zn and Se endogenously
had little effect on the sensory quality compared with control samples (C and SZ).
These results can be compared with those of previous research. For example, Banerjee et al.
(2012) showed that the incorporation of broccoli extract did not affect goat meat nuggets stored
refrigerated for 16 days. However, using chicken meat with its stronger flavour than goat meat,
Radha et al. (2014) observed that Syzygium aromaticum, Cinnamomum cassia, Origanum vulgare
and Brassica nigra extracts negatively affected the sensory quality. Similarly, the addition of
rosemary extracts at 300–900 ppm to chicken nuggets had the same effect, decreasing the sensory
quality of the product (Teruel et al., 2015). In contrast, Carvalho et al. (2018) obtained chicken
nuggets with good sensory quality after incorporating chia (Salvia hispánica L.) flour, although
no herbs or spices with strong flavour were added.
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8.4. Assay IV:
Obtained results of exogenous enrichment of dry-cured meat
products through the addition of natural antioxidant and nitrate source
extracts
8.4.1. Characterization of natural extracts and application in
Spanish “chorizo”
Obtained results from phenolic and nitrate content are shown in Table 8.18. Anlyzed extract
with the highest concentration of phenolic compounds was R, obtained from Rosmarinus
officinalis, with 1913 mg GAE/100 g, followed by paprika, C (Citrus cinensis) and oregano, with
1707, 1683 and 1439.7 mg GAE/100 g, respectively. The natural extracts and ingredients of W,
A, Ch, S and B reported values from 334.7 to 215.3 mg GAE/100 g, followed by L, garlic, Ce
and Ac, with the lowest quantity of phenolic compounds. Otherwise, regarding the nitrate content,
significant differences (p < 0.05) were obtained among the tradicional ingredients from Spanish
cuisine and green leaf vegetable extracts (Table 8.18.), whereas natural extracts obtained from
citrics, acerola and rosemary (Ct, Ac and R) did not report significant results. As can be observed,
leafy green vegetables presented the highest results of nitrates (p < 0.05): B, Ch, A, S, Ce, L and
W, followed by oregano, garlic and paprika, in this order.
Table 8.18. Total phenolic content (TPC) (mg GAE/100 g) and total nitrate content (TNC) (ppm
NO3-) in natural extracts (M ± SD). Samples TPC TNC
mg GAE 100 g−1 ppm NO3−
Ct 1683.70 ± 8.6 c Nd
Ac 57.67 ± 1.5 i Nd
R 1913 ± 29 a Nd
Paprika 1707 ± 20.1 b 21.8 ± 0.5 i
Garlic 87.3 ± 2.5 hi 50.2 ± 0.7 h
Oregano 1439.7 ± 7.5 d 51.5 ± 0.3 h
B 215.3 ± 9.6 ef 1384.1 ± 1.2 a
L 145.3 ± 5.1fg 736.4 ± 0.9 f
A 296.3 ± 5.7 ef 1160.5 ± 1.0 c
S 255 ± 6 ef 948.8 ± 0.8 d
Ch 278 ± 37 ef 1213.4 ± 1.5 b
Ce 80 ± 1 hi 921.3 ± 1.1 e
W 334.7 ± 4 e 472.9 ± 0.8 g
Ct: Citric; R: Rosemary; Ac: Acerola; L: Lettuce; A: Arugula; S: Spinach; Ch: Chard; Ce: Celery; W: Watercress. Superscript letters
indicate significant differences (p < 0.05) between samples. M ± SD: Mean ± standard deviation.
Actually, all these extracts are obtained from natural foods, fruits, vegetables and herbs, known
to be excellent sources of phenolic compounds. For example, R is a natural rosemary (Rosmarinus
officinalis) extract, with hydrophobic powder containing 14.6% carnosic acid and 5.8% carnosol,
which justifies the TPC result shown. In the same way, C, the citric extract, contained 55.11%
flavonoids as hesperidin measured by HPLC. On the other hand, paprika was shown to have a
high concentration of capsaicin, a phenolic compound responsible for its characteristic colour and
flavour (Gougoulias et al., 2017). For example, Škrovánková et al. (2017) announced similar
results for the TPC in different paprika spices, from 1467 to 2878 mg GAE/100 g.
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Similarly, Kruma et al. (2008) obtained from 72.12 to 52.15 mg phenolic compounds per 100
g of oregano using different solvents for the extraction, with methanol or ethanol. Considering
that different phenolic compounds have been identified in this herb, such as phenolic acids and
its derivatives (caffeic, rosmarinic acid and their dimmers), flavons (apigenin and luteolin) and
flavonols (eriodictyol or naringenin), the obtained result can also be justified (Santos et al., 2012).
Regarding the green leafy vegetables analysed, W, A, Ch, S, L, B and Ce, previous studies
showed comparative TPC values. For example, Zeb (2015) reported 290 mg phenolic compounds
per 100 g of water-soluble extract of watercress roots. Corleto et al. (2018) showed 600 µg
GAE/ml beetroot juice and 780 µg GAE/ml arugula juice. Alarcón-Flores et al. (2014) described
70 mg phenolic compounds per kg of spinach; Pyo et al. (2004) reported 290 mg GAE/100 g in
chard. Pérez-López et al. (2018) obtained 100 mg GAE/100 g in lettuce, while Yao et al. (2010)
reported lower values in celery, from 3.48 to 5.02 mg GAE/100 g. This fact can be explained by
the concentration of flavonoids, such as catechins, myricetin, quercetin and kaempferol, or
phenolic acids, such as gallic, p-hydroxybenzoic, protocatechuic, syringic, vanilic, chlorogenic,
caffeic, p-coumaric, or ferulic acid, which have been described in all references previously cited.
Finally, garlic and Ac were reported to have lower TPC values due to these extracts containing
higher quantities of allicin or vitamin C, respectively. However, garlic has also shown phenol
structures in its formula, such as phenolic acids (caffeic and ferulic acid) and flavonoids (apigenin
and quercetin) (Alarcón-Flores et al., 2014). While Vendramini and Trugo (2004) reported that
the content of anthocyanins or ripe acerola skin was estimated as 37.5 mg per 100 g.
The antioxidant activity of all extracts was measured by four methods and two of them showed
the chelating activity percentages against ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-
sulphonic acid) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical cation, in a hydrophilic and
lipophilic system, respectively (Table 8.19.). The other two methods showed the efficiency to
reduce Fe3+ to Fe2+ (FRAP) and the hydrophilic antioxidant capacity obtained by measuring the
oxygen radical absorbance (ORAC), both expressed in µM Trolox equivalents (TE)/100 g (Table
8.19.).
Table 8.19. Antioxidant activity of natural extracts by measuring their ABTS and DPPH radical
scavenging activity, together with their ORAC and FRAP (µM TE/100 g) (M ± SD). Samples Chelating Activity Percent (%) Antioxidant Activity (µM TE/100 g± SD)
ABTS DPPH ORAC FRAP
Ct 15.4 ± 0.2 h 8.45 ± 0.3 k 4828.5 ± 19.9 d 6004.7 ± 29.6 c
Ac 46.5 ± 0.3 c 78.3 ± 0.5 b 16,80.7 ± 19.3 g 1925.7 ± 28.7 f
R 70.2 ± 0.1 b 76.7 ± 1.7 c 19,909.0 ± 59.8 a 17,790 ± 53.3 a
Paprika 21.1 ± 1.6 f 48.7 ± 0.2 ef 5746.0 ± 21.7 c 2491.3 ± 17.1 e
Garlic 25.4 ± 0.8 e 51.5 ± 0.3 d 1919.3 ± 23.4 g 1915.7 ± 52.5 f
Oregano 15.6 ± 0.5 h 41.3 ± 0.2 j 11,436.7 ± 27.5 b 9355.3 ± 46.4 b
B 85.7 ± 1.1 a 90.2 ± 0.6 a 3509.0 ± 26.3 e 3690 ± 58.8 d
L 14.6 ± 1.1 i 49.9 ± 0.1 e 1723.3 ± 35.1 g 1998 ± 18.9 f
A 25.9 ± 3.1 e 49.2 ± 1.2 e 2881.3 ± 28.4 f 2071 ± 16.3 ef
S 20.1 ± 0.1 g 43.6 ± 3.6 i 1491.3 ± 22.1 gh 1995.3 ± 9.6 f
Ch 19.7 ± 0.0 g 47.4 ± 0.6 g 2150.7 ± 35.0 fg 2216.7 ± 19.4 e
Ce 12.0 ± 0.5 j 48.7 ± 0.4 ef 993.7 ± 18.5 i 804.7 ± 33.6 g
W 33.4 ± 2.6 d 46.5 ± 0.1 h 1200.7 ± 15.0 h 2510.3 ± 39.4 e
Ct: Citric; R: Rosemary; Ac: Acerola; L: Lettuce; A: Arugula; S: Spinach; Ch: Chard; Ce: Celery; W: Watercress. Superscript letters
indicate significant differences (p < 0.05) between natural extracts. M ± SD: Mean ± standard deviation; TE: Trolox equivalents.
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Firstly, it can be reported that beet, acerola and rosemary showed the highest chelating activity
against DPPH and ABTS radical cations. Garlic powder obtained a 51.5% scavenging activity
against DPPH, while the lowest value was presented by Ct with 8.45% of quelation power.
On the other hand, the scavenging activity against the hydrophilic radical, ABTS, is generally
lower than against DPPH. For this reason, the scavenging activity against ABTS followed the
next hierarchy: W, garlic, A, paprika, S, Ch, oregano, Ct, Le and Ce, with values from 33.4% to
12%, after B, R and Ac, which presented values of 85.7%, 70.2% and 46.5%, respectively.
Secondly, in Table 8.19., we can also observe a similar behaviour regarding the efficiency of
each extract to reduce Fe3+ to Fe2+ by comparing the hydrophilic antioxidant capacity measured
by their oxygen radical absorbance. In this case, applying the FRAP method, natural extracts rich
in phenolic compounds are the first on the list: R from rosemary, oregano, Ct from citrics, B, W
and paprika followed by Ch, A, L, S, Ac, garlic and Ce, the last one with 804.7 µM TE/100 g,
50% less than garlic with 1915.7 or 95 % less than R with 17,790 µM TE/100 g.
It can be interpreted that the scavenging power of different extracts lies in their composition
and the molecular structure of bioactive substances, such as the presence of catechol and gallate
groups in phenol groups, their polymerization and conjugation, or the combination with other
substances, such as nitrates, pigments and/or vitamins.
In this way, R is a natural extract obtained from Rosmarinus officinalis L. with 14.59%
carnosic acid, 5.84% carnosol and 0.60% 12-O-methylcarnosic acid, while C obtained from Citrus
sinensis L. contains 55.11% hesperidin as has been described previously. Considering this, it can
be understood why the highest values in the FRAP and ORAC analysis were obtained by R.
However, it must be noted that the antioxidant behaviour of flavanones (C) varies according to
the oxidant radical used. For example, Gardner et al. (2000) reported that the antioxidant power
of flavanones obtained by DPPH* was much lower than that using ABTS*, which was also proven
in the present study.
Otherwise, the Ac extract from Malpighia emarginata, with 5% vitamin C, is also rich in
phenolic compounds, such as anthocyanins, anthocyanidin, phenolic acids (p-coumaric, caffeic
and ferulic acid), flavonols (quercetin and kaempferol) and catechins (Franco-Vega et al., 2016;
Pérez-López et al., 2018). In addition, it contains β-carotene and minerals (Gardner et al., 2000),
which make it a functional fruit and justifies the results obtained from the different analyses
carried out in the present study.
The traditional ingredients from Spanish cuisine also had higher values in the antioxidant
assays but behaved differently according to the method used for assessment. For instance, the
antioxidant activity of oregano, paprika and garlic was higher when measured by the FRAP
method and ORAC method. The antioxidant activity of oregano is mainly due to the concentration
of phenol and catechol groups in the molecular structure of its principal phenolic compounds,
such as oreganoside. On the other hand, paprika is a source of important compounds for its
antioxidant capacity, such as carotenoids, capsaicinoids and vitamins C and E (α and γ-tocopherol
from pepper seeds) (Kim et al., 2016). However, the concentration of this kind of compound
varies due to several reasons, like the crop, the degree of ripeness, or the temperature used to air-
dry the peppers (Kim et al., 2016).
Additionally, garlic is an excellent scavenger of hydroxyl radicals due to its content of
flavonoids (quercetin and kaempferol) and organosulphurs (allyl-cysteine, dialyl sulphide and
dialyl trisulphide) (Brewer, 2011). Thiosulphonated compounds, such as allicin, provide the
characteristic odour to garlic, however, this compound is related to its anti-inflammatory activity
Lorena Martínez Zamora PhD Thesis, 2019
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but not with its antioxidant effect (Kim et al., 2011), which also can explain the obtained results
in the present study.
Finally, the obtained results regarding leafy green vegetables can also be related to their
concentrations in phenolic compounds. For example, B, with the highest scavenging power
against DPPH and ABTS radicals, is the principal source of nitrates, as was commented
previously and it is also rich in phenolic compounds, with 215.3 mg GAE/100 g, like
anthocyanidins. In addition, the natural purple colour of beet is due to the presence of betanin,
also known for its antioxidant power, being a derivative from betalamic acid, which is obtained
from the L-DOPA molecule. Moreover, different authors have obtained comparative results, such
as Saani and Lawrence (Saani & Lawrence, 2017), who showed a 50% scavenging DPPH radical
activity, or Ou et al. (2002), who obtained a higher antioxidant capacity using ORAC and FRAP
assays in beet of 11500 and 8600 µM TE/100 g, respectively.
The remaining leafy green vegetables obtained similar results in the different antioxidant
assays, which can be associated with the fact that they also share the same bioactive compounds:
Phenolic acids (gallic, ferulic, caffeic and p-coumaric acids), flavonoids (quercetin, kaempferol
and apigenin) and chlorophyll as the principal pigment responsible for their green colour. In the
same way, it can also be appreciated that celery had a lower antioxidant capacity than other
vegetables.
Figure 8.7. shows the antimicrobial capacity against Clostridium perfringens growth in the
presence of all studied extracts, species and vegetables. In these graphics, it can be appreciated
that all extracts reported antimicrobial activity by inhibiting growth or causing bacterial death of
Clostridium perfringens.
Taking into account that the control sample represents the total bacterial growth (100%
bacteria), it can be observed that B only reduced 65% of bacterial growth, while acerola and C
decreased by 85% using 1000 ppm of each extract. Moreover, the rest of the ingredients reduced
the bacterial growth between 90% and 100% compared to the control. Similarly, it can be said
that the concentration of each ingredient applied directly influences their antimicrobial capacity,
at least from 250 to 1000 ppm, because it may be possible that a higher concentration causes a
loss of this effect due to saturation of the system.
On the other hand, the extracts that reported the highest antimicrobial activity (p < 0.05) were
R (100% at 1000 ppm), followed by garlic, paprika, oregano and the rest of leafy green vegetables
rich in nitrates (Ce, L, S, Ch, A and W, from 98% to 90%, in this order, at 1000 ppm).
Consequently, it can be affirmed that the antimicrobial power of the extracts studied against
Clostridium perfringens growth is related to the total phenolic and nitrate content. Actually, the
bacterial growth (CFU) of Clostridium perfringens has been directly related (p < 0.05) to the
concentration of nitrates, which was already described by Hasan and Hall (1975).
In addition, phenolic compounds from R (Rosmarinus officinalis) and allicin from garlic have
been described as antimicrobial agents acting in different ways: Affecting the cytoplasmic
membrane structure, blocking protein synthesis and affecting any of the phases of this process
(activation, initiation, binding of the tRNA amino acid complex to ribosomes, or elongation),
affecting the metabolism of nucleic acids and/or blocking any bacterial metabolic pathways.
Lorena Martínez Zamora PhD Thesis, 2019
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C t A c R C o n tro l
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Figure 8.7. Antimicrobial activity of natural extracts expressed by bacterial growth (cfu) at
different concentrations in Clostridium perfringens NCTC 8237 CECT 376 after 48 h
incubation at 37 °C under anaerobic conditions. (A) obtained results for Ct: Citric; R:
Rosemary; Ac: Acerola; (B) obtained results for Paprika, Garlic and Oregano; (C)
obtained results for L: Lettuce; A: Arugula; S: Spinach; Ch: Chard; Ce: Celery; W:
Watercress. Superscript letters indicate significant differences (p < 0.05) between
samples. Control sample represents the normal bacterial growth without any extract.
Once the antioxidant and antimicrobial capacities of each ingredient were measured in vitro,
they were incorporated as preservative agents to delay the lipid oxidation and the microbiological
growth in a cured meat product. The obtained results of volatile fatty acids analysis by GS-MS
are shown in Table 8.20.
Volatile compounds from lipid oxidation (propan-2-ol, hexanal and nonanal) were
significantly affected (p < 0.05) by the ripening time and addition of antioxidants (Table 8.20.).
In contrast, octen-2-ol was not affected by the ripening time or addition of antioxidants. In
general, 2-propanol increased from 0.45 to 1.75 mg/g meat during 125 days area units to 316 ×
106 area units during the first 4 days. In contrast, the increase in samples with natural extracts
was less pronounced, especially RLAW with a value of 0.85 mg/g at day 125. The production of
octen-2-ol was not detected in any of the samples, suggesting good product sensory quality
because these compounds have a low threshold off-odour.
Lorena Martínez Zamora PhD Thesis, 2019
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Table 8.20. Average values and standard deviations of volatile compounds /mg/g meat) in
chorizo for 0, 25, 50 and 125 days, under retail conditions. Volatile
Compounds Sample Day 0 Day 25 Day 50 Day 125
propan-2-ol
Control 0.45 ± 0.02 0.54 ± 0.02 1.02 ± 0.01 a 1.75 ± 0.03 a
RLAW 0.37 ± 0.01 0.46 ± 0.01 0.37 ± 0.02 b 0.85 ± 0.04 c
RSCe 0.38 ± 0.03 0.44 ± 0.02 0.92 ± 0.01 b 1.10 ± 0.05 b
RChB 0.58 ± 0.02 0.65 ± 0.01 0.66 ± 0.02 b 1.27 ± 0.01 b
CLAW 0.65 ± 0.01 0.70 ± 0.01 0.89 ± 0.03 b 1.20 ± 0.02 b
CSCe 0.61 ± 0.03 0.69 ± 0.03 0.59 ± 0.01 b 1.33 ± 0.00 b
CChB 0.38 ± 0.02 0.50 ± 0.02 0.55 ± 0.01 b 1.08 ± 0.01 b
octen-2-ol
Control 0.11 ± 0.01 0.10 ± 0.00 0.15 ± 0.01 0.10 ± 0.01
RLAW 0.10 ± 0.02 0.18 ± 0.02 0.15 ± 0.02 0.15 ± 0.02
RSCe 0.14 ± 0.02 0.18 ± 0.01 0.12 ± 0.01 0.11 ± 0.01
RChB 0.14 ± 0.01 0.13 ± 0.01 0.16 ± 0.02 0.25 ± 0.02
CLAW 0.12 ± 0.01 0.12 ± 0.02 0.14 ± 0.03 0.19 ± 0.01
CSCe 0.16 ± 0.00 0.15 ± 0.01 0.15 ± 0.01 0.16 ± 0.02
CChB 0.10 ± 0.01 0.13 ± 0.01 0.13 ± 0.01 0.11 ± 0.01
Hexanal
Control 0.11 ± 0.01 0.14 ± 0.02 0.21 ± 0.02 a 0.44 ± 0.03 a
RLAW 0.12 ± 0.01 0.14 ± 0.01 0.08 ± 0.01 b 0.18 ± 0.01 b
RSCe 0.10 ± 0.02 0.12 ± 0.01 0.12 ± 0.03 b 0.18 ± 0.02 b
RChB 0.13 ± 0.01 0.16 ± 0.00 0.15 ± 0.02 b 0.20 ± 0.01 b
CLAW 0.11 ± 0.02 0.14 ± 0.02 0.09 ± 0.00 b 0.19 ± 0.02 b
CSCe 0.12 ± 0.01 0.14 ± 0.01 0.18 ± 0.01 b 0.21 ± 0.01 b
CChB 0.13 ± 0.03 0.12 ± 0.01 0.19 ± 0.01 b 0.25 ± 0.02 b
Nonanal
Control 0.18 ± 0.01 0.39 ± 0.04 0.45 ± 0.02 a 0.58 ± 0.01 a
RLAW 0.17 ± 0.01 0.27 ± 0.03 0.32 ± 0.01 b 0.41 ± 0.03 b
RSCe 0.22 ± 0.01 0.16 ± 0.01 0.27 ± 0.02 b 0.27 ± 0.02 b
RChB 0.14 ± 0.01 0.18 ± 0.01 0.35 ± 0.01 b 0.30 ± 0.02 b
CLAW 0.15 ± 0.02 0.18 ± 0.02 0.20 ± 0.03 b 0.23 ± 0.01 b
CSCe 0.18 ± 0.03 0.15 ± 0.01 0.27 ± 0.02 b 0.24 ± 0.02 b
CChB 0.17 ± 0.02 0.10 ± 0.01 0.19 ± 0.01 b 0.25 ± 0.01 b
RLAW: 500 ppm rosemary extract + 250 ppm acerola + 3000 ppm lettuce, arugula and watercress; RSCe: 500 ppm rosemary extract
+ 250 ppm acerola + 3000 ppm spinach and celery; RChB: 500 ppm rosemary extract + 250 ppm acerola + 3000 ppm chard and beet;
CLAW: 500 ppm citric extract + 250 ppm acerola + 3000 ppm lettuce, arugula and watercress; CSCe: 500 ppm citric extract + 250 ppm
acerola + 3000 ppm spinach and celery; CChB: 500 ppm citric extract + 250 ppm acerola + 3000 ppm chard and beet. Superscript letters
indicate significant differences (p < 0.05) between natural extracts.
The behaviour of nonanal and hexanal was quite similar, with both increasing during storage
and showing significant differences between the control and samples with extracts from day 50
onwards. Nonanal is associated with waxy and painty descriptors, while 1-octen-3-ol is amongst
the compounds responsible for rancid odours and it is an autoxidation indicator of linoleic and
arachidonic acids. In addition, hexanal is an aldehyde that can be generated from arachidonic
acids, oleic acid and through the degradation of deca-2,4-dienal (Kerler and Grosch, 1997).
Volatile alcohols, such as heptanol, are formed from oleic acid (Forss, 1973), whereas pentanol
and 1-octen-3-ol are by-products of the autoxidation of linoleic and arachidonic acids. Hexanal
concentration ranging from 2 to 7 g kg−1 was reported in cooked pork (Forss, 1973) cooked
turkey (Meynier et al., 1999) and cooked ground beef (Tikk et al., 2008).
The addition of antioxidants decreased the total volatile compounds from lipid oxidation (2-
propanol, hexanal and nonanal). At the end of process, hexanal contents were found in the
following order: C, RLAW, RSCe, CLAW, RChB, CSCe and CChB. These results indicated that the
addition of R and Ct improved the control of lipid oxidation compared to the control sample.
These results are consistent with the polyphenol content and the in vitro evaluation of the
antioxidant activity of the extracts (Table 8.20.).
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100
According to Kerler and Grosch (1997), all volatile compounds analysed (hexanal, heptanal,
octen-2-ol and propan-2-ol) are components that contribute the most to the emergence of
unpleasant notes of flavour due to their high rate of formation and low flavour threshold (Alfawaz
et al., 1994). The loss of acceptance depends to a large extent on odour and flavour deterioration
in meat and meat products (Ahn et al., 2007). In general, the volatile profile of chorizo strongly
depends on the composition. Polyphenols are metal chelating agents and also act on free radicals,
since their benzene rings inhibit chain reactions during lipid oxidation. Previous studies have
demonstrated that a rosemary diet delays lipid oxidation in raw meat from broilers (Ulrich &
Grosch, 1988), pigs (Ramarathnam et al., 1993) and lambs (Basmacioglu et al., 2004).
Regarding the obtained results of oxidative damage of Spanish chorizo for 125 days, acids,
which were practically absent at the start, showed the largest increase among the volatiles during
ripening. Carbohydrate metabolism (Saani & Lawrence, 2017), lipolysis (Ou et al., 2002), amino
acid catabolism (Kandler, 1983) and smoke (Dwidevi & Snell, 1975) might account for the
formation of these acids. The only alcohol present in all the samples of chorizo was
furfurylalcohol. Johansson et al. (1994) reported the presence of this compound as the major
alcohol in a smoked dry fermented sausage. Lipid oxidation (Töth & Potthast, 1984),
carbohydrate metabolism (Saani & Lawrence, 2017) and amino acid catabolism (Kandler, 1983)
could be the most important pathways accounting for the production of volatile alcohols in
fermented dry sausages. These compounds could also come from smoke, like furfurylalcohol,
which is abundant in wood smoke (Dwidevi & Snell, 1975; Johansson, et al., 1994).
A total lack of straight chain aldehydes and ketones, which are typical breakdown products of
the hydroperoxides derived from fatty acids (Töth & Potthast, 1984), was observed in chorizo
unlike other varieties of dry fermented sausage (Frankel, 1991; Maga, 1987; Croizet et al., 1992).
On the other hand, branched and cyclic carbonyls were detected in greater profusion. The bulk of
the carbonyls was formed during ripening and each of the ketones isolated increased during
ripening, whereas the aldehydes did not show a definite evolution. The presence of some cyclo-
pentanones and cyclopentanones as volatile constituents of dry fermented sausages has not been
previously reported. Nonetheless, these substances are typical of wood smoke (Dwidevi & Snell,
1975; Johansson, et al. 1994). The presence of methyl-branched aldehydes may be explained by
amino acid catabolism (Kandler, 1983) and by ketones (such as diacetyl, acetoin) and
hydroxypropanone by carbohydrate metabolism (Saani & Lawrence, 2017). Large amounts of
furfural and 5-methylfuran-2-carbaldehyde, which are characteristic products of the Maillard
reaction, were observed principally in industrial chorizo. Apart from spices and smoke, it is
generally accepted that the formation of volatiles during the ripening of dry fermented sausages
would be due to the occurrence of a set of reactions between the precursors of flavor, such as
carbohydrates, lipids and proteins, with microbial or endogenous enzymes being involved in
many instances. Several low molecular weight compounds isolated from chorizo, i.e., formic,
acetic and propanoic acids, propanol, butan-2,3-diol, diacetyl, 1-hydroxy-2-propanone, acetoin,
ethyl acetate, ethyl propionate, propyl acetate and ethyl butyrate, might derive to a great extent,
whether directly or indirectly, from carbohydrate metabolism (Saani & Lawrence, 2017). There
was approximately twice the quantity of these substances in industrial chorizo with regard to the
traditional ones. Therefore, a more intense fermentation metabolism in industrial chorizo seemed
probable. The production of 2-methylpropanal, 2- and 3-methylbutanal, 2-methylpropanol, 2- and
3-methylbutanol, 2-methylpropanoic and 2- and 3-methylbutanoic acids from valine, leucine and
isoleucine would be explained by amino acid degradation (Kandler, 1983). The larger total
content of these substances in industrial chorizo would imply that major amino acid catabolism
Lorena Martínez Zamora PhD Thesis, 2019
101
developed in this type of chorizo. On the other hand, it appeared that there was a smaller incidence
of amino acid degradation as contrasted with carbohydrate fermentation in chorizo.
Lipid autooxidation accounts for the appearance of numerous volatile compounds in dry
fermented sausage (Maga, 1987; Croizet et al., 1992). However, the absence of key intermediates
of autooxidation in the chorizo analyzed implies that the development of lipid oxidation is
irrelevant, aromatically speaking. This was also suggested by Berger et al. (1990) for another type
of dry sausage. This could be due to the antioxidant effect of paprika and smoke. The addition of
curing agents, which possess a positively recognised antioxidant effect, seemed to produce no
especially marked repercussions on the flavour of chorizo in light of the following two points.
First, it was not possible to impute to the curing agents a restrictive effect on the formation of
volatiles originating from chemical oxidation, since these substances were not observed either in
industrial or in traditional chorizo (Berdagué et al., 1993; Berger et al., 1990)
The antimicrobial capacity of different extracts was studied in cured meat products elaborated
with pork meat. Consequently, the microbiological results of Spanish chorizo after 50 days from
elaboration are shown in Table 8.21.
Table 8.21. Microbiological results (cfu/g) of Spanish chorizo analysis after 50 days under
refrigerated storage Samples Analysis
TVC TCC Clostridium perfringens
Control 6.20 × 104 b 2.77 × 102 10 a
RLAW 5.12 × 105 a 1.28 × 102 Absence in 10 g b
RSCe 4.25 × 105 a 2.01 × 102 Absence in 10 g b
RChB 3.62 × 105 a 1.10 × 102 Absence in 10 g b
CLAW 4.05 × 104 b 1.56 × 102 Absence in 10 g b
CSCe 6.22 × 104 b 1.79 × 102 Absence in 10 g b
CChB 5.98 × 104 b 2.10 × 102 Absence in 10 g b
RLAW: 500 ppm rosemary extract + 250 ppm acerola + 3000 ppm lettuce, arugula and watercress; RSCe: 500 ppm rosemary extract
+ 250 ppm acerola + 3000 ppm spinach and celery; RChB: 500 ppm rosemary extract + 250 ppm acerola + 3000 ppm chard and beet; CLAW: 500 ppm citric extract + 250 ppm acerola + 3000 ppm lettuce, arugula and watercress; CSCe: 500 ppm citric extract + 250 ppm
acerola + 3000 ppm spinach and celery; CChB: 500 ppm citric extract + 250 ppm acerola + 3000 ppm chard and beet. Superscript letters
indicate significant differences (p < 0.05) between natural extracts. TVC: Total viable count; TCC: Total coliform count.
As can be observed, the only sample that presented Clostridium perfringens growth was the
control sample, while the rest of samples enriched with natural extracts (RLAW, RSCe, RChB, CLAW,
CSCe and CChB) presented an absence of this bacteria in the 10 g sample. Similarly, samples that
incorporated R or Ct extracts in their formula decreased from 24% to 60% of the total coliform
count in all the samples compared to the control. This fact could be due to the presence of
monoterpens and rosmarinic acid from the R extract in case of RLAW, RSCe and RChB, or the
presence of hesperidin in the case of CLAW, CSCe and CChB. It is also important to note that the
combination of L, A and W with R was more effective than combined with Ct, while the mix
among S and Ce with Ct presented lower bacterial growth than with R.
Otherwise, the total viable bacteria growth was lowest in samples enriched with citric extract
(CLAW, CSCe and CChB), which demonstrated the synergism between Ct and natural nitrate sources.
This behaviour was not visible after the combination of R and natural nitrate sources. This
synergistic activity could be due to the reaction between the flavonoid, hesperidin, with other
phenolic compounds from L, A, W, S, Ce, Ch and B, such as flavonoids quercetin, kaempferol
and apigenin, or phenolic acids, such as gallic, ferulic, caffeic and p-coumaric acid. In addition,
this reaction could also be produced among nitrates and hesperidin.
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In this way, the obtained results from the antimicrobial in vitro test of each ingredient and
natural extract showed that natural nitrate sources and rosemary especially presented an excellent
antimicrobial activity against Clostridium perfringens. This fact is due to the presence of nitrates
that directly affect bacterial growth, as was also described by Hasan and Hall (1975).
Nevertheless, when these combined extracts were used in cured meat products, such as Spanish
chorizo in the present study, Ct combined with natural nitrate sources showed a synergistic
activity that was not shown by R in the same conditions. This behaviour must be studied in future
research, but now it can be affirmed that the reaction among flavonoids as hesperidin and natural
nitrate sources from green leafy vegetables demonstrates a synergistic effect in the preservation
of cured meat products, which is also elaborated by paprika, oregano, garlic and acerola extract,
which is also rich in Vitamin C. Furthermore, they are able to increase the antioxidant and
antimicrobial activity of the studied ingredients by separation. This increas could be produced by
vitamin C, which acts as a proton donor to the phenolic compounds, whose hydroxyl groups are
responsible for the antioxidant and antimicrobial capacity.
These reactions can explain the antimicrobial activity that can be produced by different
methods. For instance, by affecting the cytoplasmic membrane structure, blocking protein
synthesis, affecting any of the phases of this process (activation, initiation, binding of the tRNA
amino acid complex to ribosomes, or elongation), affecting the metabolism of nucleic acids and/or
blocking any bacterial metabolic pathways.
8.4.2. Obtained results of protein oxidation in pork meat after
application of natural extracts
Results of this research were obtained during the stay abroad in the “Department of Food
Sciences, of the University of Copenhagen, Denmark”, under the direction of the Professor Leif
Skibsted and the supervision of the Associate Professor Sisse Jongberg. During this time, several
tecniques and methods were learnt and applied in order to measure the protein oxidation process
in a meat matrix.
In this way, the future paper that is going to be published in next months (Paper VI) is attached
in annexes. In this study, an oxidized pork meat model system was elaborated to measure the
influence of the application of Mediterranean ingredients to avoid the protein oxidation.
As a result, the concentration of protein thiols in the control pork meat model system (C-
NoOX) was detected to be 48.4 ± 4.0 mmol/mg protein and is comparable to previous results
reported by Jongberg, Tørngren & Skibsted (2018) in brine-injected pork loins. Subjecting the
meat model system to oxidation by the hydrophilic initiation system (OXHydro) or the lipophilic
initiation system (OXLip) resulted in thiol concentrations of 25.5 ± 2.7 mmol/mg protein and 26.8
± 2.5 mmol/mg protein, respectively. The thiol concentration in the oxidized meat model systems
are presented as relative values compared to the C-NoOX, which represents 100 % (Figure 9.8.).
Analysis of the meat model system subjected to the OXHydro or OXLip system resulted in 51.6 %
and 53.3 % thiol groups, respectively.
Electron Spin Resonance (ESR) spectroscopy evaluates the radical formation from the
absorption of electromagnetic energy by radicals/unpaired electrons. Subjecting the liophilized
meat model systems to ESR spectroscopy showed a signal in the magnetic field of 336 mT. The
radical signal intensity determined as the peak height of radical signal is presented in in Figure
8.9. Radicals were generated in the meat model system subjected to oxidation by the OXHydro or
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OXLip systems, which release peroxyl radicals that react rapidly to extract hydrogen atoms from
oxidation substrates in the meat model system. Hence, the radical intensity can be considered as
a measure of initial oxidative modifications. Quantification of radicals represents accordingly a
method for meat oxidation assessment and the scavenging activity of added potential antioxidants
(Jongberg, Tørngren & Skibsted, 2018). C-NoOX showed a radical signal intensity of 80.8 ± 3.9
AU, while the meat model systems subjected to OXHydro or OXLip resulted in 222.6 ± 9.4 and 256.6
± 11.6 AU, respectively, indicating an increase in radicals signal intensity of 175 % and 218 %,
respectively. These increments are directly related to the oxidation status of the meat model
system.
CO
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Figure 8.8. Percentage thiol groups in meat model systems oxidized by AAPH (OXHydro) or
AMVN (OXLip) after addition of phenolic extracts (Citrus (500 ppm), Acerola (250
ppm) and Rosemary (500 ppm)) (A), traditional ingredients (Paprika (30000 ppm),
Garlic (4000 ppm) and Oregano (4000 ppm)) (B), or natural nitrate sources (1500 ppm
Beet, Lettuce, Arugula, Spinach, Celery, Chard or Watercress) (C) relative to a control
meat model system without oxidant (C-NoOX). All data points represent the mean ±
SD of triplicated determinations. Different letters (a-i) indicate significant differences
between samples (p<0.05).
Consequently, evaluation of the effects of the phenolic extracts on thiol oxidation in the two
oxidizing systems, OXHydro or OXLip, showed that Citrus was a more effective antioxidant against
protein thiol loss as compared to Acerola and Rosemary, especially in the OXHydro system (Figure
8.8. (A)). Rosemary was found to be slightly, though significantly, prooxidative in the OXHydro
system (Figure 9.8.A.). Evaluation of the radical scavenging activities of the phenolic extracts
showed that Rosemary efficiently scavenged radicals to a level similar to the non-oxidized control
Lorena Martínez Zamora PhD Thesis, 2019
104
(C-NoOX) (Figure 8.9. (A)). Citrus also showed protective radical scavenging activities being
most effective in the OXHydro system. In contrast, Acerola showed significant prooxidant activities
especially in the OXLip system leading to 7-fold increase in radical signal intensity as compared
to the C-NoOX (Figure 8.9. (A)).
The antioxidant capacity of Citrus may be due to its high concentration of hesperidin (55 %).
Hesperidin is a bioflavonoid glycoside and is a sugar-bound form of the flavonoid hesperetin and
the glycoside rutin, whose antioxidant capacity lies in the high number of hydroxyl groups (Figure
8.12.). The chemical structure may explain the radical scavenging activity of Citrus in the meat
model system subjected to OXHydro or OXLip as evidenced in Figure 8.9., protecting the thiols from
oxidation and maintaining a thiol concentration comparable to C-NoOX (Figure 8.8. (A)). A
recent study by Martínez et al. (2019), which results has been exposed in previous chapter,
demonstrated the potent antioxidative activity of Citrus by several antioxidant assays (ORAC,
FRAP, ABTS and DPPH). Gravador et al. (2014) also showed promising antioxidant effects of
dried citrus pulp, rich in naringin and hesperidin, when incorporated endogenously by the diet in
lamb meat. The present study showed the ability of citrus flavonoids to delay the protein oxidation
by keeping the thiol concentration as well as the protein radical signal at the same leven as the
non-oxidized control (C-NoOX).
Rosemary showed a prooxidant effect on the thiols in the meat model system subjected to
OXHydro resulting in a lower thiol group concentration than the oxidized control model systems
(Figure 8.8. (A)). On the opposite Rosemary was observed to be an efficient scavenger of radicals
in both oxidizing systems as determined by ESR spectroscopy (Figure 8.9. (A)). Rosemary extract
from Rosmarinus officinalis L. herb and contained 14.59 % carnosic acid, 5.84 % carnosol and
0.60 % 12-O-methylcarnosic acid of the total amount of phenolics present in the extract (Martínez
et al., 2019). Wang et al. (2018) showed in a recent study on myofibrillar proteins that thiol groups
were lost by high rosmarinic acid addition (60 or 300 µM/g protein), whereas a low dose of
rosmarinic acid (12 µM/g protein) partially prevented the thiol loss. The same study also
demonstrated cross-linking of myofibrillar proteins due to the multiple reaction sites on
rosmarinic acid, including the two ο-catechol rings. Jongberg et al. (2013) proposed that thiol loss
by addition of phenolic compounds to meat products may result in the formation of covalent bonds
between protein thiol groups and quinones as oxidized ο-catechol. Carnosic acid and carnosol
contain one possible site of reaction and it is likely that protein thiols in the present study may
have reacted with quinones, in effect reducing the thiols in the meat model system. Furthermore,
these reactions may terminate both protein and phenoxyl radicals and hereby explain the low
radical signal intensity in both oxidizing systems. Jongberg et al. (2013) obtained comparable
results in Bologna type sausages prepared from oxidatively stressed pork which was protected
from protein oxidation by Rosemary extract and as was seen again in the present study.
The antioxidative activity of Acerola against thiol loss was more pronounced in the OXLip
system as compared to the OXHydro system. Acerola has been associated with nutritional and
therapeutic properties that are due to the high content of vitamin C, which may vary between 1.2–
1.8 % (Lima et al., 2005). Moreover, high concentrations of carotenoids, group B vitamins and
minerals such as Fe, Ca and P have been found in Acerola (Lima et al., 2005; Muller et al., 2010).
Ascorbic acid degrades to dehydroascorbic acid when oxidized, in effect protecting other
substrates against oxidation, including the thiols. In presence of reducing agents, such as phenolic
compounds, it may be regenerated to ascorbic acid, which again can act as an electron donor
(Becker, Nissen & Skibsted, 2004). The combination of trace metals and ascorbic acid in a system
containing azo-initiators generating peroxyl radicals may facilitate Fenton reactions and, in this
Lorena Martínez Zamora PhD Thesis, 2019
105
sense, Acerola becomes prooxidative as evidenced by the high radical signal intensity (Figure
9.9.A.). As reviewed by Becker, Nissen and Skibsted (2004), ascorbic acid may in combination
with lipid soluble antioxidants also generate synergistic effects. Acerola contains lipid soluble β-
and α-carotene or lutein (Lima et al., 2005; Muller et al., 2010) that are able to reduce radicals
generated by AMVN directly in the lipid phase, orienting the radical species towards the interface
to the aqueous phase, where the radicals may be transferred to ascorbic acid in the aqueous phase
serving as an electron donor to the carotenoid.
The significantly increased radical signal intensity of Acerola in the OXLip system may also be
explained by the formation of ascobyl radicals produced through scavenging of radicals generated
by AMVN in the OXLip system. A similar increase in radical intensity was observed by Tsuchiya
et al. (2002) in a system containing erythrocyte membranes and ascorbic acid oxidized by AMVN
and this increment in radicals was described the accumulation of ascorbyl radicals. However, the
high concentration of ascorbic acid would be expected also to result in a high radical signal in the
OXHydro system as the peroxyl radicals generated would have direct access to the ascorbic acid.
Only a moderate increase was observed, which may be explained by a rapid formation and faster
degradation of the ascorbyl radicals when the peroxyl radicals are generated directly in the
aqueous phase, where other electron donators are present.
CO
NT
RO
L
CIT
RU
S
AC
ER
OL
A
RO
SE
MA
RY
0
2 0 0
4 0 0
6 0 0
8 0 0
Ra
dic
al
sig
na
l in
te
ns
ity
(A
U)
a
bc
e
f
A
d
fff
CO
NT
RO
L
PA
PR
IKA
GA
RL
IC
OR
EG
AN
O
0
2 0 0
4 0 0
6 0 0
8 0 0
Ra
dic
al
sig
na
l in
te
ns
ity
(A
U)
B
a
ec
bc
e dff
CO
NT
RO
L
BE
ET
LE
TT
UC
E
AR
UG
UL
A
SP
INA
CH
CH
AR
D
CE
LE
RY
WA
TE
RC
RE
SS
0
2 0 0
4 0 0
6 0 0
8 0 0
Ra
dic
al
sig
na
l in
ten
sit
y (
AU
)
C
a
bd e
ce
fed
C -N o O X Ox H y d ro Ox L ip
d e
ff ffe
f gg
Figure 8.9. Radical signal intensity in meat model systems oxidized by AAPH (OXHydro) or
AMVN (OXLip) after addition of phenolic extracts (Citrus (500 ppm), Acerola (250
ppm) and Rosemary (500 ppm)) (A), traditional ingredients (Paprika (30000 ppm),
Garlic (4000 ppm) and Oregano (4000 ppm)) (B), or natural nitrate sources (1500 ppm
Beet, Lettuce, Arugula, Spinach, Celery, Chard or Watercress) (C) relative to a control
meat model system without oxidant (C-NoOX). All data points represent the mean ±
SD of triplicated determinations. Different letters (a-g) indicate significant differences
between samples (p<0.05).
Lorena Martínez Zamora PhD Thesis, 2019
106
Otherwise, Paprika, Garlic and Oregano were all able to reduce protein thiol loss and radical
signal intensity in the systems subjected by OXHydro or OXLip, indicating antioxidant properties of
all ingredients (Figure 8.8. (B) and Figure 8.9. (B)). Garlic, Paprika and Oregano were all added
in high concentrations as compared to the phenolic extracts and it is likely that the mere presence
of the ingredients will result in an apparent protective effect. Addition of Paprika and Oregano to
the meat model system in the present study resulted in 512 and 58 ppm gallic acid equivalents
(GAE), respectively. These concentrations exceed the levels of GAE introduced by the phenolic
extracts by far, but the antioxidant activities are not proportionally improved. The protecting
effect observed for Paprika and Oregano may not be a direct antioxidant activity, but perhaps
simply a result of the ingredients being oxidized in preference to other components present in the
model system, acting as “sacrificial compounds” due to their excess concentration (Mathew,
Abraham & Zakaria, 2015). These observations stress the importance of applying efficient
antioxidants in the production of foods.
All the traditional ingredients showed better protection against protein thiol loss when the
radicals were generated in the lipid pase, while all the ingredients were found to be better
scavengers of radicals generated in the aqueous pase. This phenomenon was especially apparent
for Garlic, which however is in contrast to previous reports showing prooxidative activity of
Garlic on thiol oxidation in pork patties (Nieto, Jongberg andersen & Skibsted, 2013). The
mechanism behind this thiol loss was explained by Nagy, Lemma & Ashby (2007), who
demonstrated that allicin, a principal compound in Garlic, reacts with thiols to form a sulfenic
acid and a disulphide from its thiosulfinate ester, hereby reducing the thiol concentration. Allicin
is responsible for numerous beneficial properties by Garlic consumption, but not necessarily for
its antioxidant power (Petropoulos et al., 2018). Garlic powder also contains flavonoids and
phenolic acids, such as quercetin, kaempferol, apigenin, caffeic acid, ferulic acid, vanillic acid,
p-hydroxybenzoic acid and p-coumaric acid (Martins, Petropoulos & Ferreira, 2016), which in
the present study may serve as antioxidants. However, the total phenolic content was calculated
to be 3.5 ppm and may hence not explain the overall antioxidative effect. Okada et al. (2005)
reported the need for a combination of the allyl (-CH2CH=CH2) and -S(O)S- groups for the
antioxidant action of thiosulfinates in Garlic extracts, which may explain this antioxidant
protection. Selenium is another important compound from Garlic that may increase the
antioxidant activity (Gorinstein et al., 2005), a behaviour which was also reported by Nieto,
Skibsted andersen & Ros (2012).
Paprika and Oregano also showed antioxidant activity in both systems (OXHydro and OXLip).
Paprika is an oleoresin and its principal compound is capsaicin (Riquelme & Matiacevich, 2016).
This molecule is bipolar, which means that its catechol ring is hydrophilic while its amide bond
together with its fatty acid chain forms its lipophilic domain (Claudino, Jonsson & Johansson,
2013). Due to the structure capsaicin may be located in the interface between the lipid and aqueous
phase generating a bridge across the interface. Oregano contains 10-11% of lipids, from which
the essential oil is commonly obtained, but it is also rich in phenolic acids and diterpenes, which
are water and lipid soluble, respectively.
A more general conclusion from these studies seems to be that the use of proper concentrations
of natural antioxidants from herbs and spices is important in order to protect thiols as the balance
between pro- and antioxidative effects in strongly depends on concentration. Moreover, the
interaction between lipophilic antioxidants and hydrophilic antioxidants in the interface between
the aqueous and lipid phase may change the effective antioxidant concentration through
regeneration.
Lorena Martínez Zamora PhD Thesis, 2019
107
In the same way, regarding to natural nitrate sources, all vegetables extracts were able to
reduce thiol loss with Lettuce and Spinach being more effective (Figure 8.8. (C)). Additionally,
it was observed for protection against thiol loss, initiation in the aqueous phase and in the lipid
phase by, OXHydro or OXLip, respectively, had similar effect on oxidation, except for addition of
Beet, which showed to be more effective against thiol loss initiated by OXHydro in the aqueous
phase (Figure 8.8. (C)) as compared to initiation by OXLip in the lipid phase. Similarly, when
analysing the radical scavenging activity, all-natural nitrate sources were able to scavenge the
radicals, except for nitrates from Beet in the OXLip system, where a prooxidative activity was
observed (Figure 8.9. (C)). All other natural nitrate sources reduced the radical signal intensity,
especially the radicals generated in the OXHydro system (Figure 8.9. (C)).
Similarly, for the application of the traditional ingredients, relatively high concentrations were
applied to the meat model system, which may induce some degree on sacrificial effect of the
natural nitrate sources. However, the distinct effect of especially Lettuce and Spinach as an
inhibitor of thiol oxidation in the meat model system should be further investigated.
C -N o O X O x H y dr o O x L ip
0
2 5
5 0
7 5
1 0 0
1 2 5
% T
hio
l g
ro
up
s
f
c
d
c
ed
c
b
c c
f
e
b cc
a 0 .0 0 1 p p m
0 .5 p p m
3 7 .5 p p m
3 7 5 p p m
1 5 0 0 p p m
6 0 0 0 p p m
0 p p m
Figure 8.10. Percentage thiol groups in meat model systems oxidized by AAPH (OXHydro) or
AMVN (OXLip) after addition of 0, 0.001, 0.5, 37.5, 375, 1500 and 6000 ppm of
NaNO2. All data points represent the mean ± SD of triplicated determinations.
Different letters (a-e) indicate significant differences (p<0.05) between OXHydro
samples and C-NoOX. Different letters (A-H) indicate significant differences (p<0.05)
between OXLip samples and C-NoOX.
A study of the dose-dependence was carried out for the effect of nitrite in the meat model
system. Nitrate or nitrite is commonly added to meat products for antimicrobial protection. When
nitrate is added it is reduced to nitrite by microbial reductases (Moller, Jongberg, Skibsted, 2015).
Levels of nitrite applied are normally 60-150 ppm, but most of it will be lost immediately after
addition due to reactions with meat components (Alahakoon et al., 2015). In the present study,
nitrite was applied in the concentration range from 0.001-6000 ppm to the meat model system
subjected to both oxidizing systems, OXHydro or OXLip and a clear dose-dependent effect was
observed especially in the OXLip system. Nitrite was found to protect against thiol loss, with
optimal efficiency at 37.5 ppm in the OXLip system. A similar experiment with nitrate was
conducted showing the same tendency, though with less pronounced effects (data not shown).
Addition of nitrite was found to protect against thiol loss, with optimal efficiency at 37.5 ppm in
the OXLip system (Figure 8.10.). The high concentration of nitrite (6000 ppm) was found to have
prooxidative effect in both oxidizing systems, whereas all concentrations applied showed radical
scavenging activities (Figure 8.11.). Evaluation of the radical scavenging indicated a clear dose-
dependent effect especially for the OXHydro system with the same optimum concentration level as
for the protection against thiols (Figure 8.11.).
Lorena Martínez Zamora PhD Thesis, 2019
108
C -N o O X O x H y dr o O x L ip
0
1 0 0
2 0 0
3 0 0
Ra
dic
al
sig
na
l in
ten
sit
y (
AU
)d
a
bFc
c
b
dc
A
BC
DE
GH
0 .0 0 1 p p m
0 .5 p p m
3 7 .5 p p m
3 7 5 p p m
1 5 0 0 p p m
6 0 0 0 p p m
0 p p m
Figure 8.11. Radical signal intensity in meat model systems oxidized by AAPH (OXHydro) or
AMVN (OXLip) after addition of 0, 0.001, 0.5, 37.5, 375, 1500 and 6000 ppm of
NaNO2. All data points represent the mean ± SD of triplicated determinations.
Different letters (a-e) indicate significant differences (p<0.05) between OXHydro
samples and C-NoOX. Different letters (A-H) indicate significant differences (p<0.05)
between OXLip samples and C-NoOX.
The concentration of nitrate (TNC) in the meat model systems was calculated and only little
variation occurred between samples (0.7-2.1 ppm), indicating that the concentration of nitrate
alone may not explain the ability of especially Lettuce or Spinach to inhibit thiol oxidation. The
low concentrations of nitrate are a result of working with a meat model system, where the meat
and all additives have been diluted in the model system buffer. The concentration of nitrate
relative to the content of meat was 0.08–0.23 ppm, approximately. It is remarkable to mention
that Beet extract was observed to have the highest concentration of nitrate, but show minor
antioxidant effect against thiol loss and even to have prooxidative activity in the form of increased
radical signal intensity. This clearly indicates a non-proportional relation between nitrate
concentration and antioxidant capacity, which also is stressed by the significantly higher
protective effect against thiol loss of Lettuce and Spinach. No clear explanation of the
prooxidative activity of Beet has been established. Beet extract is hydrophilic, containing phenolic
compounds, nitrates, pigments and betanins (Kale et al., 2018). The antioxidant effect may not be
ascribed to the nitrate alone, but other bioactive compounds present in the vegetables may add to
the antioxidative effect. Accordingly, it has been demonstrated that natural nitrate sources are
efficient scavengers against DPPH and ABTS radicals (Martínez et al., 2019). The phenolic acids
present in the natural nitrate sources (gallic acid, ferulic acid, caffeic acid and p-coumaric acid),
flavonoids (quercetin, kaempferol and apigenin) (Figure 8.12.) and pigments (betanin from Beet),
will all contribute to the antioxidant effect by their functional groups (catechol, gallate and
hydroxyl). However, as proven by the dose-dependence experiment, nitrite alone clearly protects
meat protein thiols (Figure 8.9.). This discovery may facilitate the application of nitrate-rich
vegetables as ingredients with multiple protective actions in Clean Label meat products. Avoiding
phenolic extracts in the production will reduce possible reactions between phenolic compounds
and proteins, which can affect meat texture and protein functional properties (Jongberg et al.,
2013, Jongberg et al., 2015; Tang et al., 2017; Jia et al., 2017, Ozdal et al., 2013, Wang et al.,
2018). As for the protection against lipid oxidation in meat products, no additional effect was
obtained from combining nitrite phenolic rich extract, as compared to nitrite alone. This indicates
that nitrate-rich vegetables extracts may serve as natural antioxidant and antimicrobial agents in
meat (Martínez et al., 2019). Reports describe nitrosilation of thiol groups and proteins, which
happens when NO is produced by nitrate and nitrite reduction and reacts with free thiols forming
Lorena Martínez Zamora PhD Thesis, 2019
109
nitrosothiol complexes (R-SH-N=O) (Wu et al., 2011; Sullivan & Sebranek, 2012). However, this
effect was not found in the present study as no additional loss of thiols was observed.
Generally, it can be concluded that natural nitrate sources may serve as antioxidants, protecting
against as thiol loss and radical formation. For this reason and combined with previous reports on
the protection of nitrite against lipid oxidation, green leafy vegetables may potentially substitute
addition of synthetic or phenolic antioxidants in meat products obtaining a Clean Label product
and avoiding interactions between phenolic compounds and proteins which have been observed
to disturb structural properties of meat proteins (Jongberg et al., 2018; Cao & Xiong, 2017; Nieto
et al., 2013; Jongberg et al., 2015). However, more studies are needed to verify this protective
effect of natural nitrate sources on the formation of other potential harmful oxidation products in
meat.
Figure 8.12. Relevant bioactive compounds from phenolic extracts, traditional ingredients and
natural nitrate sources.
Lorena Martínez Zamora PhD Thesis, 2019
110
8.4.3. Shelf-life study of Spanish “chorizo” enriched in natural
extracts
In order to complete this assay, a shelf life study of 150 days of a dry-cured meat product has
been carried out. For that, firstly proximal composition of the product is showed in Table 8.22.
All Spanish “chorizo” samples showed similar values of proximate composition, regarding to
water content, dry extract, airing losses, ash, fat, proteins and minerals like Na and K. As it can
be appreciated in Table 8.22., there were not significant differences among treatments on
Tab
le 8
.22.
Pro
xim
ate
com
po
siti
on
(g
/100
g),
air
ing
lo
sses
(%
), n
itra
te (
pp
m)
an
d n
itri
te (
pp
m)
con
ten
t (M
± S
D)
in
Sp
an
ish
“ch
ori
zo” e
nri
ched
wit
h n
atu
ral
extr
act
s.
S
am
ple
s
C
on
tro
l R
LA
W
RS
Ce
RC
hB
C
LA
W
CS
Ce
CC
hB
Mo
istu
re
28
.2 ±
0.4
4
27
.7 ±
0.5
1
29
.1 ±
0.4
9
28
.6 ±
0.6
8
30
.5 ±
0.3
9
31
.1 ±
0.0
2
27
.5 ±
0.3
3
Dry
ex
tra
ct
71
.8 ±
0.4
4
70
.3 ±
0.5
1
70
.9 ±
0.4
9
71
.4 ±
0.6
8
69
.5 ±
0.3
9
68
.9 ±
0.0
2
72
.5 ±
0.3
3
Air
ing
lo
sses
4
5.4
4 ±
1.2
2
45
.09
± 1
.71
4
5.7
6 ±
2.0
2
43
.95
± 1
.05
4
4.9
4 ±
0.9
8
45
.62
± 2
.12
4
3.9
7 ±
0.8
6
Ash
6
.09
± 0
.01
5
.43
± 0
.02
5.2
4 ±
0.2
2
5.2
6 ±
0.0
5
4.8
3 ±
0.0
8
5.6
8 ±
0.0
7
5.7
1 ±
0.0
6
Fa
t 2
6.5
± 0
.75
33
.7 ±
0.3
0
31
.2 ±
0.4
3
31
.8 ±
0.2
6
30
.5 ±
1.1
5
29
.9 ±
1.2
3
29
.6 ±
0.7
6
Pro
tein
s 2
5.5
± 0
.91
2
5.8
± 1
.10
29
.8 ±
0.9
5
27
.5 ±
0.4
3
27
.0 ±
0.5
1
29
.7 ±
1.3
7
29
.1 ±
0.9
1
Na
0
.49
± 0
.01
0
.57
± 0
.01
0.5
8 ±
0.0
0
.54
± 0
.0
0.6
3 ±
0.0
0
.54
± 0
.0
0.5
8 ±
0.0
K
0.5
2 ±
0.0
1
0.3
2 ±
0.0
0
.35
± 0
.01
0.5
1 ±
0.0
0
.36
± 0
.0
0.2
8 ±
0.0
0
.30
± 0
.0
NO
3
12
.25
± 0
.01
a 8
.60
± 0
.0b
5.3
0 ±
0.0
b
7.7
6 ±
0.0
b
9.2
1 ±
0.0
b
8.0
0 ±
0.0
b
8.5
5 ±
0.0
b
NO
2
56
.23
± 0
.04
a 1
3.2
9 ±
0.0
3c
13
.28
± 0
.01
c 2
3.5
6 ±
0.0
3b
21
.35
± 0
.01
b
21
.03
± 0
.03
b
23
.28
± 0
.01
b
RL
AW
: 50
0 p
pm
Ro
sem
ary e
xtr
act
+ 2
50 p
pm
Ace
rola
+ 3
00
0 p
pm
Let
tuce
, A
rugu
la a
nd
Wat
ercr
ess;
RS
Ce:
50
0 p
pm
Ro
sem
ary
ex
trac
t +
25
0 p
pm
Ace
rola
+ 3
000
pp
m
Sp
inac
h a
nd C
eler
y;
RC
hB:
500
pp
m R
ose
mar
y e
xtr
act
+ 2
50
pp
m A
cero
la +
30
00
pp
m C
har
d a
nd B
eet;
CL
AW
: 500
pp
m C
itri
c ex
trac
t +
250
pp
m A
cero
la +
3000
pp
m
Let
tuce
, A
rug
ula
an
d W
ater
cres
s; C
SC
e: 5
00 p
pm
Cit
ric
extr
act
+ 2
50
pp
m A
cero
la +
3000
ppm
Spin
ach
and
Cel
ery
; C
Ch
B:
500
pp
m C
itri
c ex
trac
t +
250
pp
m A
cero
la +
3000
pp
m C
har
d a
nd
Bee
t.
Lorena Martínez Zamora PhD Thesis, 2019
111
chemical composition. However, different results were previously obtained by Perea-Sanz et al.
(2019), who have recently showed the effect of vacuum storage and nitrate reduction of dry
fermented sausages with 30 % of fat, 50 % of protein and 40 % moisture, approximately.
Nevertheless, nitrate and nitrite content were significantly higher (p < 0.05) in Control sample.
This fact is predictable because of Control sample was elaborated with a Commercial mix made
of synthetic nitrate and nitrite, while R and C samples were elaborated with leafy green vegetables
as natural nitrate sources. In addition, it can be observed that the nitrite contain was higher than
the nitrate contains, due to the reduction of nitrate to nitrite by bacteria (Micrococcus and
Staphylococcus) producing the enzyme nitrate-reductase (Polenski, 1981). Once nitrites are
formed from nitrates, the reddish colour is produced by the reaction of nitric oxide (NO) with
meat pigments, myoglobin (Mb). This reaction produces nitrosomyoglobin (NOMb), which is the
reddish pigment that forms the nitrosylhemochrome complex during the cook with the
characteristic pink colour (Skibsted, 1992). The nitrate and nitrite function in dry-cured meat
products are: reddish colour formation, bacteria inhibition growth (Clostridium botulinum and
Clostridium perfringens), development of characteristic flavour and antioxidant activity avoiding
the aparition of rancid flavour and organoleptic alterations. For this reason, the increased
concentration of nitrates and nitrites can be related with other factors as the bacteria content, the
reddish colour or the development of rancid flavour, characteristic of the degradation of meat.
After that, an evaluation of the stability of meat product was carried out during 25 days of
curation process and after that until 125 days under refrigerated storage: 150 days in total.
In general, there was a significant decrease in pH values for all the treatments (P < 0.001)
during the 150 days of analysis (Table 8.23.). However, there were no significant differences
among pH values obtained from different “chorizo” samples at different days of analysis. As it is
widely known, starter cultures composed of lactic acid bacteria (Pediococcus, Staphylococcus
xylosus and Staphylococcus carnosus, in this case) that ferment sugars producing a decreasement
of pH to values close to 5 and generates an inhibition of the growth of pathogenic microorganisms
(Ordoñez & Hoz, 2001). The main function of these cultures is the acidification of the meat
product as a result of their metabolism. However, they also perform other functions such as the
proteolytic activity by which essential amino acids are relased for the development of lactic acid
bacteria, the generation of aromas (Fordyce, Crow & Thomas, 1984), or the production of
bacteriocines that inhibit the growth of other pathogenic microorganisms (De Vuyst & Leroy,
2007).
In this way, initial pH values ranged between 6.18 and 5.82, while since day 2 until day 150,
pH values oscillated from 4.96 to 4.66. This fact shows that the incorporation of rosemary or citric
extracts as antioxidants, either natural nitrate sources obtained from leafy green vegetables did
not affect to pH behaviour during refrigerated storage. On the other hand, Fernándes et al. (2018)
demonstrated a tendency to lower pH values in cured sheep sausages enriched with oregano
extract and stored at room temperature for 135 days. But, in the present study, oregano is used at
the same concentration in all the samples, together with garlic and paprika, for this reason no
change can be appreciated.
Otherwise, unless the moisture was no significantly affected by the incorporation of natural
nitrate sources, this analysis was only carried out until day 50 after samples elaboration. However,
in Table 8.23., evolution of water activity (aw) values are showed from the beginning until the
end of the shelf-life study. As it can be appreciated, there are no appreciable changes in water
activity values at day 0 and 2 from elaboration. The aw decreased from initial values of 0.962–
0.949 to about 0.863–0.807 at the end of ripening (day 25). In this moment, Spanish “chorizo”
Lorena Martínez Zamora PhD Thesis, 2019
112
samples are vacuum packed and stored at 4ºC. Then aw decreased until values around 0.808–
0.740, showed at day 150 of the present study. Nevertheless, aw was significantly affected (p <
0.05) since day 10 by ripening time and addition of natural nitrate sources. This fact demonstrates
how the presence of synthetic nitrate sources can affect negatively to water activity since day 10
until day 150 of the study. Then, the use of natural nitrate sources from leafy green vegetables
delayed the water loss during ripening time. Similar conclusions were also reached by Hospital
et al. (2016) in nitrate and nitrite-reduced dry fermented Spanish sausages (“salchichón” and
“fuet”), but this shelf-life study was only carried out for 28 days. In addition, it must be taken into
account that Spanish “chorizo” samples with natural nitrate sources were also enriched with meat
protein as water retained instead of vegetable fibres that were incorporated to Control sample
(Commercial mix ®). Thus, this increasement in water retention could be due to protein meat or
leafy green vegetable presence in Clean label “chorizo” samples.
Table 8.23. also presents the development of colour parameters (lightness coordinate (L*),
redness coordinate (a*) and yellowness coordinate (b*)), which were measured on the surface of
the “chorizo” slices along the 150 days of ripening and storage. As it can be appreciated during
the shelf-life study, L* has the same tendency in all the samples. In this way, it increases from
day 0 to 2. However, during the ripening and vacuum packaged storage, these values suffer a
decrease since 40.0 to 15.0 after 150 days, approximately. These results indicated normal changes
due to the time. A similar behaviour has been previously observed for 28 days in dry-cured
“chorizo” enriched in tiger nut fibre (Sánchez-Zapata, et al., 2013). Nevertheless, obtained values
by samples enriched with tiger nut fibre showed a slightly increasement regarding to Control
sample, due to the water retention by the fibre, while there were not significant differences
(p<0.05) among samples in the present study due to the fact that used extracts did not present
water retention.
At the same time, a slight increase was observed in a* values. This small change in redness
coordinate during the ripening and storage processes is attributed to the formation of
nitrosomyoglobin in dry-cured products. For this reason, Control sample also presents higher
values of a* than samples enriched with nitrate natural sources, because of Control incorporated
to its formula nitrite, hence the reddish colour was produced before, as it has been explained
previously. However, it must be taken into account that paprika pigments (capsaicin) could mask
the effect of the natural extracts in Spanish “chorizo” samples (Fernández-López et al., 2002).
Apart from that, b* values experimented a decrease from day 25, characterized by the loss of
yellow colour and an increasement of brown tones. Then, there were not significant differences
(p<0.05) among samples at day 0, 2 and 10 from elaboration. These changes during the last days
of dry-curing process and the vacuum packaged, could be due to the oxygen consumption by
microorganisms during their exponential growth phase and at the same time the reduction in
oxymyoglobin content which greatly contributes to the value of b* value, as it was stated by
Sánchez-Zapata et al. (2013), because microorganism produce metabolites that induce the
oxidation of meat and fat present in the product and contribute to the decrease of b* (Pérez-
Álvarez et al., 1999). In addition, this reduction was lower in samples that combined Beet (B)
extract with (C) and (R). This fact could be produced by betanins (pigments of beet) that act as
scavenger of Fe retaining this mineral in its molecular structure resulting in complex with Fe
heme of meat, as it was also described by Wybraniec et al. (2013), who described the effects of
metal cations on betanin stability in aqueous solutions and it can be related with the studies
samples. This complex can be result in brown colours, however, this behaviour has not been
described by other authors, it could be interesting to study in future studies.
Lorena Martínez Zamora PhD Thesis, 2019
113
Table 8.23. Results of pH, water activity (aw) and colour CIELab (M ± SD) in Spanish “chorizo”
enriched with natural extracts for 150 days of refrigerated storage Days of ripening Days of vacuum-packed storage
Sample 0 2 10 25 50 75 100 125 150
pH
Control 6.18±0.01 4.73±0.0 4.79±0.0 4.73±0.01 4.73±0.0 4.95±0.11 4.93±0.12 4.88±0.03 4.91±0.05
RLAW 5.84±0.0 4.90±0.13 4.80±0.0 4.79±0.01 4.83±0.0 4.83±0.02 4.85±0.08 4.86±0.06 4.83±0.05
RSCe 5.82±0.04 4.72±0.0 4.82±0.0 4.80±0.0 4.81±0.0 4.88±0.05 4.96±0.01 4.82±0.05 4.82±0.01
RChB 5.90±0.01 4.80±0.0 4.78±0.0 4.72±0.0 4.73±0.0 4.81±0.03 4.86±0.04 4.71±0.04 4.77±0.02
CLAW 5.82±0.02 4.73±0.0 4.80±0.0 4.87±0.02 4.88±0.0 4.80±0.04 4.76±0.04 4.69±0.07 4.79±0.05
CSCe 5.89±0.01 4.66±0.05 4.76±0.01 4.78±0.01 4.79±0.0 4.79±0.03 4.82±0.02 4.76±0.08 4.80±0.02
CChB 6.13±0.01 4.72±0.01 4.80±0.0 4.72±0.0 4.73±0.01 4.80±0.0 4.82±0.04 4.74±0.06 4.78±0.02
aw
Control 0.962±0.0 0.951±0.0 0.902±0.0b 0.807±0.0b 0.792±0.0b 0.775±0.0b 0.768±0.0b 0.752±0.0b 0.740±0.0b
RLAW 0.962±0.0 0.955±0.0 0.921±0.0a 0.813±0.1b 0.801±0.0a 0.797±0.0a 0.778±0.0a 0.764±0.0a 0.742±0.1b
RSCe 0.961±0.0 0.958±0.0 0.910±0.0a 0.841±0.0a 0.834±0.0a 0.822±0.0a 0.800±0.0a 0.788±0.1a 0.760±0.0a
RChB 0.957±0.0 0.954±0.0 0.921±0.0a 0.861±0.0a 0.874±0.0a 0.867±0.0a 0.847±0.0a 0.821±0.0a 0.802±0.0a
CLAW 0.961±0.0 0.949±0.0 0.925±0.0a 0.863±0.0a 0.882±0.0a 0.865±0.0a 0.844±0.1a 0.828±0.0a 0.808±0.0a
CSCe 0.962±0.0 0.954±0.0 0.912±0.0a 0.847±0.0a 0.832±0.0a 0.823±0.0a 0.810±0.0a 0.795±0.0a 0.777±0.0a
CChB 0.949±0.0 0.942±0.0 0.922±0.0a 0.836±0.0a 0.826±0.0a 0.811±0.0a 0.799±0.0a 0.772±0.0a 0.751±0.0ab
Colour parameters: L*
Control 42.2±0.52 42.2±0.02 40.2±0.28 35.3±0.08 33.1±0.02 25.2±0.03 14.1±0.02 15.1±0.01 11.2±0.09
RLAW 48.5±2.61 49.6±0.10 42.2±0.05 36.0±0.04 34.0±0.11 29.0±0.02 18.9±0.09 18.8±0.08 15.3±0.02
RSCe 43.1±0.04 45.8±0.24 41.1±0.01 33.6±0.03 32.6±0.10 30.1±0.07 27.3±0.04 13.1±0.02 11.8±0.0
RChB 39.5±0.12 43.1±0.12 38.9±0.04 35.0±0.25 34.5±0.04 29.3±0.01 16.1±0.10 12.0±0.03 17.4±0.07
CLAW 44.4±0.02 52.4±0.65 40.8±0.70 37.0±0.16 36.2±0.10 21.8±0.05 14.9±0.01 14.1±0.01 18.1±0.02
CSCe 44.2±0.80 46.9±0.29 42.2±0.08 35.7±0.18 33.9±0.12 20.0±0.0 17.9±0.03 10.4±0.04 11.4±0.08
CChB 41.2±0.23 45.4±0.19 38.5±0.03 32.2±0.02 30.3±0.05 19.1±0.11 8.9±0.04 5.6±0.01 18.2±0.05
a*
Control 29.8±0.42 35.1±0.06 31.0±0.20 25.9±0.08 24.6±0.08 28.5±0.08 38.5±0.04 34.6±0.02 37.0±0.02
RLAW 21.8±1.51 26.0±0.04 22.7±0.07 20.5±0.04 18.9±0.10 29.1±0.30 32.4±0.03 30.6±0.09 31.2±0.02
RSCe 16.9±0.05 21.1±0.15 18.5±0.03 16.6±0.02 16.0±0.03 20.9±0.14 24.4±0.05 24±9±0.08 24.4±0.07
RChB 17.0±0.10 23.1±0.11 21.7±0.04 18.9±0.19 17.2±0.04 21.2±0.06 29.0±0.04 29.0±0.04 31.6±0.07
CLAW 20.5±0.07 26.2±0.32 23.3±0.53 20.3±0.08 18.4±0.04 25.9±0.21 31.3±0.04 30.5±0.06 32.1±0.07
CSce 17.9±0.47 21.3±0.13 19.8±0.05 18.0±0.05 16.5±0.15 23.4±0.01 28.9±0.08 24.4±0.09 24.7±0.03
CChB 16.7±0.12 22.8±0.10 21.2±0.04 17.5±0.03 16.5±0.07 22.1±0.05 28.3±0.08 21.8±0.0 26.3±0.08
b*
Control 28.5±0.40 32.7±0.04 28.7±0.31 20.9±0.09 18.0±0.02 20.1±0.06 20.9±0.09 26.2±0.05 15.9±0.02
RLAW 32.4±0.45 39.0±0.11 30.7±0.06 21.4±0.05 19.1±0.14 19.4±0.07 29.0±0.04 27.7±0.06 22.8±0.04
RSCe 28.1±0.04 34.5±0.27 29.0±0.01 18.8±0.04 18.0±0.07 22.0±0.05 32.4±0.05 18.4±0.02 16.1±0.02
RChB 23.5±0.06 28.9±0.15 28.2±0.06 19.1±0.30 18.8±0.06 20.4±0.15 22.0±0.03 15.6±0.04 26.4±0.04
CLAW 29.5±0.03 39.4±0.56 31.9±0.79 22.8±0.16 19.8±0.08 18.2±0.01 22.1±0.09 20.5±0.09 27.8±0.04
CSCe 30.2±0.80 34.0±0.21 32.4±0.08 21.0±0.05 18.2±0.23 19.0±0.05 27.2±0.08 13.6±0.06 15.8±0.03
CChB 23.5±0.24 30.1±0.16 24.8±0.02 15.8±0.02 15.0±0.09 12.3±0.05 11.0±0.07 15.0±0.09 19.9±0.02
RLAW: 500 ppm Rosemary extract + 250 ppm Acerola + 3000 ppm Lettuce, Arugula and Watercress; RSCe: 500 ppm Rosemary extract + 250 ppm Acerola + 3000 ppm Spinach and Celery; RChB: 500 ppm Rosemary extract + 250 ppm Acerola + 3000 ppm Chard and
Beet; CLAW: 500 ppm Citric extract + 250 ppm Acerola + 3000 ppm Lettuce, Arugula and Watercress; CSCe: 500 ppm Citric extract + 250 ppm Acerola + 3000 ppm Spinach and Celery; CChB: 500 ppm Citric extract + 250 ppm Acerola + 3000 ppm Chard and Beet.
Parallelly, microbiological values obtained at the end of the ripening process are showed in
Table 8.24.
Lorena Martínez Zamora PhD Thesis, 2019
114
Table 8.24. Results of microbiological analysis (cfu/g) in Spanish “chorizo” after 50 days of
refrigerated storage. Analysis
Samples TVC E. Coli Listeria
monocytogenes
Salmonella
Control 6.20 × 104 b < 10 Absence in 10 g Absence in 25 g
RLAW 5.12 × 105 a < 10 Absence in 10 g Absence in 25 g
RSCe 4.25 × 105 a < 10 Absence in 10 g Absence in 25 g
RChB 3.62 × 105 a < 10 Absence in 10 g Absence in 25 g
CLAW 4.05 × 104 b < 10 Absence in 10 g Absence in 25 g
CSCe 6.22 × 104 b < 10 Absence in 10 g Absence in 25 g
CChB 5.98 × 104 b < 10 Absence in 10 g Absence in 25 g
TVC: Total Viable Count. RLAW: 500 ppm Rosemary extract + 250 ppm Acerola + 3000 ppm Lettuce, Arugula and Watercress; RSCe:
500 ppm Rosemary extract + 250 ppm Acerola + 3000 ppm Spinach and Celery; RChB: 500 ppm Rosemary extract + 250 ppm Acerola
+ 3000 ppm Chard and Beet; CLAW: 500 ppm Citric extract + 250 ppm Acerola + 3000 ppm Lettuce, Arugula and Watercress; CSCe: 500 ppm Citric extract + 250 ppm Acerola + 3000 ppm Spinach and Celery; CChB: 500 ppm Citric extract + 250 ppm Acerola + 3000
ppm Chard and Beet.
There were no significant differences among Spanish “chorizo” samples regarding to E. Coli,
Listeria monocytogenes and Salmonella growth. Then, obtained results to each strain were: < 10
ufc / g for E. Coli, absence of Listeria monocytogenes in 10 g sample and absence of Salmonella
in 25 g sample. Nevertheless, regarding to Total Viable Count (TVC) results, there were no
significant differences among Control sample and “chorizo” samples that incorporated citric
extract to their formula (CLAW, CSCe and CChB), while samples enriched with rosemary extract
(RLAW, RSCe and RChB) had high growth rates of mesophilic microorganisms. This fact
demonstrated that the combination of C with acerola and natural nitrate sources obtained from
leafy green vegetables was as effective as synthetic nitrate and nitrite (Control) and more effective
against mesophile growth than R with the same ingredients in Spanish “chorizo”. Others authors
have obtained comparable results in “chorizos” formulated with natural extracts as substitutes of
synthetic additives. For instance, Sánchez-Zapata et al. (2013) presented higher TVC results
incorporating tiger nut fiber to “chorizos” formula, while Pateiro et al. (2015) reached a
decreasement of TVC in samples that incorporated 200 ppm of natural extracts rich in phenolic
compounds such as tea, chestnut, or beer extracts, but not with 200 ppm of grape seed in
comparison with the use of 200 ppm of butylated hydroxytoluene (BHT) as synthetic additive.
This decreasement in mesophile bacteria growth should be due to the antimicrobial activity of
flavonoids (hesperidin, quercetin, kaempferol, apigenin, cyanidin, etc.) and phenolic acids
(carnosic acid, carnosol, gallic acid, ferulic acid, caffeic acid, etc.) which has previously been
reported by Martínez et al. (2019) in a recent research, which is previously in the first part of the
present chapter. This study has showed the antimicrobial capacity of all these extracts against the
bacterial growth of Clostridium perfringens. The antimicrobial power of these natural extracts
resides in the presence of tannins, saponins, phenolic compounds, essential oils and flavonoids,
biologically active compounds with antimicrobial activity. In the present study, it is known that
in case of Citrus sinensis extract (C), with 55 % hesperidin. For instance, Espina et al. (2011)
showed that extracts obtained from peels of Citrus sinensis inhibits celular protein cynthesis due
to the formation of irreversible complexes with proteins rich in proline. This makes it possible to
understand the biological properties of extracts rich in flavonoids or phenolic acids, hence the
antimicrobial activity against the bacteria growth.
Lorena Martínez Zamora PhD Thesis, 2019
115
Results of protein oxidation related with thiol group loss for 150 days of refrigerated storage
are shown in Table 8.25.
The concentration of protein thiols in the Control “chorizo” sample was observed to be 55.2 ±
2.2 mmol/mg protein, which is comparable to previous results previously reported by us in an
oxidized pork meat model system (8.4.2.), or by Jongberg, Tørngren & Skibsted (2018) in brine-
injected pork loins. Then, a gradually decrease in the concentration of thiol groups was observed
in all the Spanish “chorizo” samples. For instance, Control sample suffered a decrease of 83 % of
thiol groups concentration, which is directly related with the protein oxidation. This fact occurs
as a result of the protein oxidation produced when free thiols form bounds among proteins, which
Ta
ble
8.2
5. R
esu
lts
of
pro
tein
oxid
ati
on
rel
ate
d w
ith
th
iol
gro
up
loss
(n
mol
thio
l/m
g p
rote
in),
res
pec
tiv
ely
, fo
r 1
50
days
of
refr
iger
ate
d s
tora
ge
(M ±
SD
)
D
ay
s o
f ri
pen
ing
Da
ys
of
va
cuu
m-p
ack
ed s
tora
ge
Sa
mp
le
0
2
10
25
50
75
10
0
12
5
15
0
Pro
tein
ox
ida
tio
n:
Th
iol
gro
up
s
Co
ntr
ol
55
.2±
2.2
v
46
.8±
1.1
w
37
.4±
2.9
a x
18
.6±
1.2
a y
9.8
±0
.5b z
6
.4±
1.7
b z
1
2.3
±1
.1a
yz
12
.4±
1.1
a yz
9.6
±0
.9b z
RL
AW
5
8.1
±1
.9v
45
.4±
1.4
w
29
.4±
2.1
b x
1
1.8
±0
.6b y
1
1.1
±0
.8b y
9
.4±
0.5
b y
z 1
1.0
±0
.7a
y
7.7
±0
.3b y
z 9
.8±
1.0
b y
z
RS
Ce
51
.4±
4.1
v
42
.1±
2.3
w
25
.5±
2.6
b x
1
9.5
±1
.6a
y
14
.2±
1.1
ab y
z 6
.8±
0.8
b z
3
.46
±0
.1c
z 5
.6±
0.3
bc
z 2
.9±
0.5
c z
RC
hB
5
7.6
±2
.1v
44
.5±
1.8
w
24
.4±
2.2
b x
1
9.9
±1
.2a
y
13
.4±
1.4
ab y
z 8
.9±
0.8
b y
z 8
.6±
0.5
b y
z 9
.5±
0.8
b y
z 1
3.0
±1
.2a
yz
CL
AW
5
5.9
±3
.2v
43
.1±
2.8
w
25
.7±
1.6
b x
1
8.0
±1
.1a
y
17
.1±
1.0
a y
9.0
±0
.7b y
z 1
4.1
±1
.2a
y
8.4
±0
.9b y
z 1
2.9
±0
.5a
yz
CS
Ce
55
.3±
4.0
v
45
.2±
3.5
w
26
.2±
2.0
b x
2
0.2
±1
.5a
xy
19
.2±
1.1
a y
14
.4±
1.5
a y
9.4
±0
.8b y
z 9
.3±
0.8
b y
z 3
.6±
0.3
c z
CC
hB
5
2.1
±3
.9v
46
.9±
2.5
w
27
.1±
1.9
b x
2
1.5
±1
.7a
xy
18
.2±
0.4
a y
7.8
±1
.0b y
z 9
.7±
0.8
b y
z 7
.5±
0.5
b y
z 1
2.7
±1
.1a
y
RL
AW
: 500
ppm
Ro
sem
ary
extr
act
+ 2
50 p
pm
Ace
rola
+ 3
000
pp
m L
ettu
ce,
Aru
gula
an
d W
ater
cres
s; R
SC
e: 5
00
pp
m R
ose
mar
y e
xtr
act
+ 2
50
ppm
Ace
rola
+ 3
00
0 p
pm
Sp
inac
h a
nd
Cel
ery;
RC
hB:
50
0 p
pm
Ro
sem
ary
extr
act
+ 2
50
ppm
Ace
rola
+ 3
000
pp
m C
har
d a
nd
Bee
t; C
LA
W:
500
pp
m C
itri
c ex
trac
t +
25
0 p
pm
Ace
rola
+ 3
000
pp
m
Let
tuce
, A
rug
ula
and
Wat
ercr
ess;
CS
Ce:
500
pp
m C
itri
c ex
trac
t +
25
0 p
pm
Ace
rola
+ 3
00
0 p
pm
Sp
inac
h a
nd
Cel
ery
; C
ChB:
50
0 p
pm
Cit
ric
extr
act
+ 2
50
pp
m A
cero
la +
3000
pp
m C
har
d a
nd
Bee
t. D
iffe
ren
t su
bsc
rip
t le
tter
s in
dic
ate
signif
ican
t d
iffe
ren
ces
(p<
0.0
5)
bet
wee
n s
ample
s (a
, b,
c) a
nd d
ay o
f an
aly
sis
(v,
w, x,
y, z)
Lorena Martínez Zamora PhD Thesis, 2019
116
changes protein structure. Samples that incorporated C to their formula presented significant (p <
0.05) lower values of protein oxidation in comparison with R.
However, it is impossible to associate this variation to the presence of natural nitrate sources
from leafy green vegetables. Samples that combined R or C with SCe (Spinach + Celery) obtained
higher values of protein oxidation (93-94 % thiol loss), while the combination ChB (Chard +
Beet) with R or C, even C combined with LAW (Lettuce + Arugula + Watercress) presented the
lowest values at day 150 after elaboration (76-77 % thiol loss). Actually, this combination of
extracts protected in 7 % the Spanish “chorizo” samples against natural thiol loss, regarding to
the Control sample. A recent study carried out by our research group (8.4.2.) demonstrated in an
oxidized pork meat model system as C acted as antioxidant against thiol loss while R did not
prohibit this behaviour. In addition, in this investigation, R produced an increase of thiol loss,
which might be produced by reactions between phenolic compounds from rosemary and free thiol
groups that may form thiol-quinone adducts. In this way, o-catechol groups from carnosic acid
and carnosol (bioactive compounds of rosemary) than can be oxidized forming quinones, which
may form covalent bonds with free thiols resulting in thiol-quinone complex (Jongberg et al.,
2013). In the same researcher line, Nieto et al. (2013) investigated the addition of essential oil of
rosemary, oregano and garlic in pork patties. Obtained results from this study also showed as
protein disulfide cross-link formation was produced after incorporation of garlic and phenolic
compounds from oregano and rosemary. However, in the last study (8.4.2.) protein oxidation was
not affected by natural sources of nitrate. Then it is possible that protein oxidation was inhibited
by the combination of nitrate, from leafy green vegetables and phenolic compounds, from C and
R.
Lipid oxidation was measured through the analysis of the volatile compounds related with this
alteration and results are shown in Table 8.26.
Table 8.26. Evolution of volatile compounds of Spanish “chorizo” samples for 150 days of
refrigerated storage (M ± SD). Storage day
Volatile
Compounds
Sample 0 50 100 150
Lipid oxidation
1-butoxy-2-
propanol
Control 1.01±0.03 0.50±0.02 0.42±0.01 0.34±0.01
RLAW 0.81±0.04 0.53±0.00 0.46±0.02 0.41±0.03
RSCe 1.10±0.05 0.43±0.04 0.28±0.01
RChB 1.27±0.01 0.43±0.01 0.63±0.01 0.47±0.02
CLAW 1.20±0.02 0.58±0.02 0.76±0.06 0.64±0.04
CSCe 1.33±0.00 0.58±0.01 0.59±0.04 0.48±0.03
CChB 1.08±0.01 0.58±0.03 0.51±0.03 0.43±0.01
2,6-dimethyl-
7-octen-2-ol
Control 0.11±0.01 0.14±0.00 0.09±0.0 0.12±0.0
RLAW 0.16±0.02 0.15±0.02 0.19±0.01
RSCe 0.14±0.02 0.22±0.03 0.15±0.02
RChB 0.14±0.01 0.34±0.01 0.21±0.08
CLAW 0.12±0.01 0.37±0.02 0.19±0.01
CSCe 0.16±0.00 0.13±0.04 0.12±0.01 0.17±0.02
CChB 0.20±0.02 0.11±0.0 0.15±0.0
Heptanal
Control 0.09±0.01
RLAW 0.11±0.01 0.15±0.02
RSCe 0.11±0.01 0.13±0.01
RChB 0.11±0.01 0.26±0.04
CLAW 0.12±0.01 0.14±0.02
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CSCe 0.13±0.02 0.20±0.02
CChB 0.13±0.01 0.15±0.03
2-Heptenal
Control
RLAW 0.44±0.02 0.47±0.03
RSCe 0.61±0.03 0.65±0.05
RChB 0.75±0.04
CLAW 0.74±0.03
CSCe 0.40±0.02
CChB 0.41±0.01
Nonanal
Control 0.18±0.01 0.89±0.02 0.13±0.0 0.31±0.03
RLAW 0.17±0.01 0.55±0.03 0.28±0.0 0.43±0.02
RSCe 0.22±0.01 0.53±0.01 0.35±0.06
RChB 0.14±0.01 0.52±0.03 0.44±0.05
CLAW 0.15±0.02 0.59±0.0 0.24±0.01
CSCe 0.28±0.03 0.25±0.04 0.17±0.01 0.36±0.02
CChB 0.50±0.02 0.25±0.03 0.39±0.04
2-Bornanone
Control 0.10±0.01 0.17±0.02 0.08±0.0 0.07±0.0
RLAW 0.14±0.01 0.11±0.01 0.10±0.0 0.10±0.0
RSCe 0.11±0.01 0.13±0.01 0.08±0.0
RChB 0.13±0.02 0.14±0.02 0.10±0.01
CLAW 0.10±0.01 0.22±0.04
CSCe 0.12±0.01 0.08±0.01 0.08±0.0 0.09±0.01
CChB 0.08±0.0 0.07±0.0
Phenol
Control 0.29±0.02
RLAW 0.57±0.05 0.29±0.02 0.46±0.0
RSCe 0.39±0.02 0.61±0.02 0.52±0.06
RChB 0.31±0.02 0.65±0.03 0.59±0.02
CLAW 0.54±0.04 0.33±0.05
CSCe 0.35±0.06 0.40±0.03
CChB 0.33±0.02 0.65±0.03 0.42±0.03 0.57±0.04
Dodecane
Control 0.40±0.05 0.11±0.0
RLAW 0.20±0.04 0.08±0.01 0.11±0.01
RSCe 0.13±0.01 0.29±0.02 0.12±0.01
RChB 0.37±0.03 0.13±0.0
CLAW 0.08±0.01 0.41±0.0 0.10±0.01
CSCe 0.22±0.01 0.12±0.01
CChB 0.36±0.02 0.08±0.0 0.13±0.03
Microbiological degradation
Acetic acid
Control 1.25±0.05 5.90±0.08 11.66±0.10 12.29±0.22
RLAW 2.33±0.01 13.08±0.04 12.37±0.07
RSCe 3.40±0.03 4.90±0.04 12.73±0.04 12.52±0.11
RChB 3.45±0.02 14.27±0.14 14.09±0.05
CLAW 2.80±0.02 15.72±0.02 14.22±0.21
CSCe 3.45±0.03 8.68±0.05 12.93±0.08
CChB 2.91±0.02 7.94±0.03 12.63±0.04
3-methyl
butanoic acid
Control 0.33±0.01 0.61±0.03 0.86±0.02
RLAW 0.69±0.05 0.69±0.03
RSCe 0.53±0.02 0.87±0.04
RChB 0.74±0.02
CLAW 0.77±0.04
CSCe 0.41±0.01 0.76±0.01
CChB 0.46±0.02 0.84±0.02
2,3-
1butanediol
Control 0.98±0.03 9.93±0.12 12.05±0.04 14.17±0.12
RLAW 8.94±0.05 7.99±0.04 9.17±0.09
RSCe 8.11±0.07 6.19±0.06 9.76±0.04
RChB 12.01±0.09 5.73±0.08 6.18±0.06
CLAW 16.14±0.11 6.70±0.11 7.31±0.19
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CSCe 10.32±0.10 6.96±0.10 11.93±0.31
CChB 10.86±0.04 7.14±0.02 8.55±0.06
RLAW: 500 ppm Rosemary extract + 250 ppm Acerola + 3000 ppm Lettuce, Arugula and Watercress; RSCe: 500 ppm Rosemary
extract + 250 ppm Acerola + 3000 ppm Spinach and Celery; RChB: 500 ppm Rosemary extract + 250 ppm Acerola + 3000 ppm Chard and Beet; CLAW: 500 ppm Citric extract + 250 ppm Acerola + 3000 ppm Lettuce, Arugula and Watercress; CSCe: 500 ppm
Citric extract + 250 ppm Acerola + 3000 ppm Spinach and Celery; CChB: 500 ppm Citric extract + 250 ppm Acerola + 3000
ppm Chard and Beet
Table 8.26. shows the mean quantities of the identified compounds grouped according to their
probable origins as from lipid oxidation, microbial esterification, carbohydrate fermentation,
amino acid catabolism and spices. Although some of these compounds could have different
sources because they are result of secondary reactions between substances derived from different
catabolic routes. Most compounds identified have been reported in Spanish sausages (Andrade et
al., 2010; Purriños et al., 2012).
The compound butanoic acid, 3-methyl has been associated with the characteristic ripened
aroma of cured meat products (Ruiz et al., 1999). Otherwise, diallyl sulfide, β-pinene, d-
limonene, o-cymene, Copaene, Caryophyllene are volatile compounds detected from added
spices, all of them derived from garlic, paprika, rosemary, vegetables, and citric. The highest
concentration of these compounds was found in chorizo elaborated with natural extracts.
Volatile compounds from lipid oxidation (propan-2-ol, nonanal, and heptanal) were affected
(P< 0.05) by addition of antioxidants and by ripening time and (Table 8.26.). In contrast, octen-
2-ol was not affected by these factors.
In general, 2-propanol increased during 150 days of storage. In contrast, the increase in
samples with natural extracts was less pronounced, especially RLAW with a value of 0.85 mg/g
at day 150. Production of octen-2-ol was not detected in all the samples.
The behaviour of nonanal and hexanal was similar; they reported significant differences
between Control and samples with extracts from day 50 until the end of storage. In addition, both
compound increasing during storage that is because lipid oxidation increased trough storage.
Hexanal is associated with rancid odour and nonanal with painty and waxy descriptors. In
addition, hexanal is an aldehyde that is generated from the degradation of deca-2,4-dienal,
arachidonic acids and oleic acid, while octen-2-ol is an indicator of autoxidation of arachidonic
and linoleic acids.
The antioxidant extracts added to the chorizo samples decreased total volatile compounds from
lipid oxidation (2-propanol, hexanal and nonanal). These results indicated that the addition of
Rosemary and Citrus extracts improved the control of lipid oxidation, compared to the Control
sample. These results were already reported in Table 8.18. where results of the polyphenol content
and the in vitro evaluation of antioxidant activity of the extracts were shown (Table 8.19.).
All the volatile compounds analysed (heptanal, octen-2-ol, and propan-2-ol) have a high rate
of formation and a low flavour threshold; therefore, these compounds have an influence on the
formation of unpleasant attributes of flavour.
Generally, the volatile profile of chorizo depends on the ripening time, lipid oxidation and
composition. Therefore, deterioration on flavour and odour causes a loss of acceptance. The
addition of natural extract rich in polyphenols, such as rosemary, act as antioxidant because they
are metal chelating agents and also act on free radicals, since their benzene rings, inhibit chain
reactions during lipid oxidation.
Lipid autooxidation accounts for the appearance of numerous volatile compounds in dry
fermented sausage. However, the absence of key intermediates of autooxidation in the chorizos
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analysed implies that the development of lipid oxidation is irrelevant, aromatically speaking. This
was also suggested by Berger et al. (1990) for another type of dry sausage. This could be due to
the antioxidant effect of paprika and spices. The addition of curing agents, which possess a
positively recognised antioxidant effect, seemed to produce no especially marked repercussions
on the flavour of chorizo in the light of the following two points.
Sensory QDA (Quantitative Descriptive Analysis) of Spanish “chorizo” samples is
represented in Figure 8.13. In colour QDA (Figure 8.13. A), can be observed that Control sample
gave higher value of “Reddish colour”, related with a* values, significant (p < 0.05) higher in
Control samples. In this same sense, samples that included beet into their ingredients (RChB and
CChB) showed higher scores for “Brown colour”, while RLAW, RSCe, CLAW and CSCe gave a more
visible “Orange colour”. This “Brown colour” can be related with lower values of b* coordinate
previously explained. There were no significant differences regarding to visual “Homogeneity”
of the samples, neither with visual “Colour Extract”.
On the other hand, the perceptible odour (Figure 8.13. (B)) of all the samples was also similar
regarding to the “General odour”, “Cured odour”, “Smoked odour” and “Extract odour”.
However, Control sample presented an intense “Rancid odour” at day 50, which was also related
with commented volatile compounds results.
As it can be appreciated in Figure 8.13. (C), “Rancid flavour” was also perceptible in Control
at day 50 after manufacturing. Otherwise, unless citric extract did not affect to the flavour of the
samples, the “chorizos” that incorporated rosemary in their formulas presented a characteristic
“Extract flavour” provided by phenolic compounds from R. This behaviour can be related with
obtained “Acceptability” results (Figure 8.13. (E)). For instance, the lowest value of this
parameter was showed by Control, followed by samples elaborated with R extract and the highest
score was obtained by CLAW, CSCe and CChB. These three samples did not present neither rancid
nor extract flavour, for this reason they were qualified better by panellists.
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Figure 8.13. Results of organoleptic analysis, colour (A), odour (B), flavour (C), texture (D) and
Aceptability of Spanish “chorizo” at 50 days of chilled storage. (F) represents the
hardness in Newton (N) measured by a texturometer TA-XT2i (ANAME, Madid,
Spain). RLAW: 500 ppm Rosemary extract + 250 ppm Acerola + 3000 ppm Lettuce,
Arugula and Watercress; RSCe: 500 ppm Rosemary extract + 250 ppm Acerola + 3000
ppm Spinach and Celery; RChB: 500 ppm Rosemary extract + 250 ppm Acerola +
3000 ppm Chard and Beet; CLAW: 500 ppm Citric extract + 250 ppm Acerola + 3000
ppm Lettuce, Arugula and Watercress; CSCe: 500 ppm Citric extract + 250 ppm
Acerola + 3000 ppm Spinach and Celery; CChB: 500 ppm Citric extract + 250 ppm
Acerola + 3000 ppm Chard and Beet.
0,0
1,0
2,0
3,0
4,0Reddish
Brown
ExtractOrange
Homogeneity
Colour
0,0
1,0
2,0
3,0
4,0General
Cured
SmokedRancid
Extract
Odour
0,0
1,0
2,0
3,0
4,0General
Rancid
Acid
SmokedCured
Spicy
Extract
FlavourC
0,0
1,0
2,0
3,0
4,0Hardness
Cohesiveness
Massicability
Juiciness
Granularity
Fibrosity
TextureD
0
2
4
6
8
10
Hed
on
ic S
cale
AceptabilityE
0
20
40
60
80
100
120
140
Ha
rdn
ess
(N)
Texture
B A
F
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From another point of view, texture was measured both objectively (Figure 8.13. (F)) and
subjectively (Figure 8.13. (D)). Unless no significant differences were appreciated considering
only the overall texture evaluated by the hedonic test, when this parameter was objectively
measured using a texturometer, Control sample presented a significant increase (p < 0.001) of the
hardness, measured in Newton, in comparison with the rest of the samples. However, it seems
that the combination of the meat protein with lettuce, arugula and watercress (LAW) in RLAW and
CLAW was more effective regards to the hardness than the combination with the rest of extracts.
Unfortunately, final conclusions cannot be reached with only these data obtained.
Comparable results regarding to hardness and overall acceptance were also reported by
Fonseca et al. (2013), who studied the effect of autochthonous starter cultures on the sensory
properties of Galician chorizo.
Finally, some appreciable correlations exist among all the studied parameters (Table 8.27.).
For instance, the microbiological growth is conditioned by aw, pH, or nitrate and nitrite content
in samples. It can be appreciated as a negative correlation (p<0.001) exists between nitrate and
nitrite content with mesophilic microorganism growth. Apart from that, all the oxidative
phenomena are closely related. Nonanal is an indicator of the oxidative state of fat in all the
samples, while thiol groups content was an indicator of protein oxidation. Consequently, a lower
value of thiols was indirectly correlated with higher values of nonanal (p<0.01). This fact can be
explained because the same oxidants (reactive species (ROS) or secondary products of oxidative
stress) that induce lipid peroxidation, also produce protein oxidation, characterized by protein
thiol loss and carbonyl formation (Xiong, 2010). In addition, it was observed that thiol loss was
also directly related (p<0.001) with hardening of the product, which can be justified by disulphide
bonds generated between proteins, that form conglomerates that provides consistency.
Nevertheless, hardness is also directly related with pH (p<0.01) and aw (p<0.001). During
ripening of the product pH suffers a decrease caused by fermentations. This acid pH denaturalizes
protein structure increasing the consistency of the product. Parallelly, water loss during this
process also helps to hardening of the product (Hammes & Hertel, 1998).
Because of the production of reddish colour from the incorporation of nitrate and nitrite to the
meat (Skibsted, 1992), it can be explained the correlation among different parameters as the
reddish colour and the nitrite concentration (p<0.05). That is, as it has been previously explained,
the nitric oxide produced from nitrite content reacts with myoglobin producing the
nitrosylmyoglobine (NOMb), with a characteristic reddish colour. or the hardness and the nitrite
concentration.
Table 8.27. Pearson correlations between different measured parameters.
TVC Aw pH Hardness Rancid
Flavour
Nonanal
Nitrate content -0.618***
Nitrite content -0.584***
Thiol groups -0.847*** 0.532*** -0.387*** -0.498**
Aw 0.292NS -0.735*** -0.581*** -0.601*** -0.553***
pH -0.405* -0.799*** -0.306NS -0.429**
Hardness -0.339* 0.760**
Rancid Flavour 0.449*
TVC: Total Viable Count; aw: water activity. Significance levels: NS: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001.
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8.5. Assay V:
Obtained results of exogenously enrichment of processed fish
products through the addition of natural antioxidant extracts
In this last assay, natural extracts rich in antioxidant and antimicrobial bioactive compounds
have been used for the preservation of other kind of animal origin products: fish patties.
8.5.1. Characterization of natural extracts and application in fish
patties
For that, firstly, antioxidant and antimicrobial capacities of chosen natural extracts were
studied both in vitro and applied during the elaboration of fish patties preserved for 14 days.
(a) (b)
(c)
(d) (e)
Figure 8.14. HPLC chromatograms for natural extract. (a) RA: Rosemary extract rich in
Rosmarinic Acid, (b) NOS: Rosemary extract rich in diterpenes and NOVS: Rosemary
extract rich in diterpenes and with lecitin as emulsifier, (c) P: Pomegranate extract, (d)
HYT-F: Hydroxytyrosol extract obtained from olive fruit, (e) HYT-L: Hydroxytyrosol
extract obtained from olive leaf.
The antioxidant and antimicrobial capacities of these extracts depend on the concentration of
the phenolic compounds. Extracts obtained from Rosmarinus officinalis L. contained 8.10% of
rosmarinic acid (RA) and 5.76% of diterpenes (NOS and NOVS), more specifically, carnosic acid
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and carnosol. P had 41.38% of total punicalagins, as principal bioactive compounds. Otherwise,
hydroxytyrosol extracts, obtained from different parts of the olive tree (Olea europaea) during
the manufacture of olive oil, had different concentrations of the bioactive compound: HYT-F had
11.25% hydroxytyrosol, while HYT-L presented 7.26%. HPLC chromatograms for each extract
are presented in Figure 8.14.
Determination of the total phenolic content by means of the Folin–Ciocalteau method allows
a comparative evaluation of the content of this kind of compounds, considering, at molecular
level, the significant structural difference between the various polyphenols present in the extracts
being analysed. The results obtained (mg GAE/g) are shown in Table 8.28.
HYT-F showed the highest quantity of phenolic compounds, with 41.44 mg GAE/g, followed
by P, NOS, RA, HYT-L and NOVS. This last one with 35.95 mg GAE/g, 13.2% less than the first
extract. All the extracts showed more than 35 mg GAE/g. In that way, hydroxytyrosol (HYT-F
and HYT-L), rosmarinic (RA, NOS and NOVS) and pomegranate extracts had similar total
phenolic amounts, between 36 and 41 mg GAE/g.
Firstly, the obtained results of total phenolic content agree with previous findings by other
authors using the Folin-Ciocalteau method or by HPLC (Fuentes et al., 2018; Balasundran et al.,
2006; Presti et al., 2017). Results obtained in the present spectrophotometric determination were
not strictly correlated with the data obtained by HPLC analysis, which is due to the different
response factors of each of the polyphenol structures present in the extracts (punicalagins,
rosmarinic acid, carnosic acid, carnosol and hydroxytyrosol) regarding the pattern used, as the
gallic acid in this case. It is difficult to make a structural interpretation of the results obtained for
the antioxidant capacity measurements using the studied methods, although, clearly, some factors
are related with the molecular structure of the active substances: the presence of phenols, their
conjugation and polymerisation, cathecol and/or gallate groups presence, etc. In both methods, P
shows the best results, probably, due to the presence of some conjugated polyphenol structures
and a significant amount of gallic acid groups (tri-hydroxy phenol structures). Regarding the olive
extracts, HYT-L, with its lower level of hydroxytyrosol than HYT-F, as principal active
compound, showed a higher antioxidant capacity in both models, making it one of the most
powerful extracts. This fact could originate from the presence of flavonoid compounds in
combination with the hydroxytyrosol, providing a synergistic effect in terms of antioxidant
activity. RA is the most structurally similar extract to olive extracts, due to the presence of
rosmarinic acid as an active compound. This substance could be termed “double-hydroxytyrosol,”
only for their structural similarity, perhaps for this reason both extracts showed proximate
chelating activity values. The difference among rosemary extracts was significant. The water-
soluble extract (RA) was more active in the ABTS method, while liposoluble extracts (NOS and
NOVS) showed a higher activity in the DPPH model. This behaviour could be explained by the
different chemical structure of the radical used in each technique and by the different properties
of the molecular structures of phenylpropanoids (rosmarinic acid) and diterpens (carnosic acid
and carnosol). However, both structures have a cathecol group and a carborxylic acid group.
These results can be compared with previous research. For example, Hmid et al. (2017) and
Elfalleh et al. (2009) obtained similar values for pomegranate extracts using the same methods,
as well as hydroxytyrosol (Kouka et al., 2009) and rosemary extracts (Pereira et al., 2017; Erkan
et al., 2008).
The chelating activity percentages obtained using two different methods, are also shown in
Table 8.28. ABTS + radical cation assay and the DPPH free radical scavenging method were
used. The capacity of P, HYT-L and RA to scavenge the ABTS + radical was higher than 80 %,
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while the chelating activity of HYT-F, NOVS and NOS was of 79.62%, 70.61 % and 70.17 %,
respectively. On the other hand, scavenging ability of P was also the highest by measuring the
stability of the DPPH radical, 92.55 %. The chelating activity of HYT-L, NOVS, NOS, RA and
HYT-F ranged from 89 % to 78 %. Thus, the extracts with greatest chelating capacity were those
obtained from pomegranate, hydroxytyrosol and rosemary, which is related with their high
content of phenols, such as, punicalagin, hydroxytyrosol and rosmarinic compounds (carnosic
acid, carnosol and rosmarinic acid), respectively.
The hydrophilic antioxidant capacity of the natural extracts obtained by measuring the oxygen
radical absorbance is shown in Table 8.28. However, in this case, the extract with the greatest
antioxidant activity was HYT-L (147.46 µM TE/g), followed by P (146.39 µM TE/g), HYT-F
(140.54 µM TE/g), RA (123.2 µM TE/g), NOVS (49.16 µM TE/g) and NOS (45.18 µM TE/g).
As it can be appreciated, a great significant difference (p < 0.05) exists among rosmarinic extracts
(NOS and NOVS) rich in diterpenes (carnosic acid and carnosol) and RA rich in rosmarinic acid,
together with P, HYT-L and HYT-F. This fact could be explained by lipophilic activity of NOS
and NOVS, because of this measurement is carried out in a hydrophilic system. In this way, the
rest of extracts are water-soluble, as P, as well as HYT-L, HYT-F and RA present dual affinities,
both to polar and non-polar solvents.
Table 8.28. Total phenolic content (TPC) of natural extracts (mg GAE/g) (M ± SD) and their
antioxidant activity by measuring their ABTS and DPPH radical scavenging activity,
together to their ORACHP and FRAP (µM TE/g) (M ± SD). Samples TPC ABTS DPPH ORACHP FRAP
mg GAE/g % Chelating
Activity
% Chelating
Activity µM TE/g µM TE/g
RA 36.4 ± 0.0 b 81.1a 81.29 b 123.2 ± 0.2 b 73.8 ± 1.5 a
NOS 36.5 ± 0.0 b 70.2b 87.98 ab 45.2 ± 0.2 c 64.2 ± 1.5 b
NOVS 36.0 ± 0.0 b 70.6b 88.76 a 49.2 ± 0.5 c 73.4 ± 2.0 a
P 40.7 ± 0.0 a 83.1a 92.55 a 146.4 ± 0.9 a 61.3 ± 1.0 b
HYT-F 41.4 ± 0.1 a 79.6ab 77.96 b 140.5 ± 1.0 a 71.2 ± 1.9 a
HYT-L 36.3 ± 0.0 b 82.1a 88.95 a 147.5 ± 1.4 a 65.0 ± 2.9 b
GAE: Gallic acid equivalents; SD: Standard deviation; Superscript letters indicate significant differences (p < 0.05) between natural extracts. P: Pomegranate extract, RA: Rosemary extract rich in Rosmarinic Acid; NOS: Rosemary extract rich in diterpenes; NOVS:
Rosemary extract rich in diterpenes and with lecitin as emulsifier; HYT-L: Hydroxytyrosol extract obtained from olive leaf; HYT-F:
Hydroxytyrosol extract obtained from olive fruit.
The efficiency of the natural plant extracts to reduce Fe+++ to Fe++ as an antioxidant power
measured is also presented in Table 8.28. expressed in µM Trolox Equivalents (TE)/g. Obtained
data showed as that all the extracts have a good and similar ferric reducing antioxidant power,
ranging from 73.8 µM TE/g (RA) to 61.3 µM TE/g (P). The order of antioxidant activity using
this method was RA > NOVS > HYT-F > HYT-L > NOS > P. All the extracts had high levels of
reducing power, which indicated the presence of some compounds that are electron donors and
could react with free radicals to convert them into more stable products.
Hydrophilic ORAC is one of the most widely used methods for evaluating antioxidant
capacity, but it is clear that the results may be conditioned not only by the antioxidant capacity of
each compound, but also by the physical and chemical properties, particularly its water solubility.
Pomegranate and olive extracts obtained similar values for their antioxidant activity unrelated to
their origin (leaves, fruit or vegetation water). In this case, the different of cathecol and gallate
groups did not seem to be significant. Despite this, HYT-L again showed a higher activity. The
antioxidant capacity of RA was lower than that of the above (–15%), although it followed the
same order. It seems obvious that the structural similarity goes on establishing a parallelism in
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the antioxidant activity, also in this model. If not, the lower ORAC activity of the fat-soluble
rosemary extracts (diterpens) compared with the hydrosoluble extracts that were already
described. Previous researchers, such as Azaizeh et al. (2012), obtained similar results analysing
hydroxytyrosol in olive (Olea europaea) vegetation waters, while Sueishi et al. (2018) obtained
results that were 50% higher when measuring the seasonal variations of oxygen radical
scavenging ability in rosemary leaf extract using the same method. In research carried out by
Durante et al. (2017), the authors measured the antioxidant activity of diferent extracts from
tomato, grape and pomegranate seeds, obtaining similar results as the last. In the same way,
previous research obtained similar results to that obtained results by the FRAP method regarding
to rosemary (Pereira et al., 2017), pomegranate (Hmid et al., 2017) and hydroxytyrosol (Kouka
et al., 2009).
Data obtained from measuring the antimicrobial capacity of different extracts are presented in
Table 8.29. The results differed according to the bacterial strain used, L. monocytogenes KCTC
3569 CECT 7467 (Gram-positive), S. Aureus ATCC 25923 CECT 435 (Gram-positive) or E. Coli
O157:H7 ATCC 25922 CECT 434 (Gram-negative). All the extracts showed a lower growth
inhibition capacity than cloramphenicol (positive control), a broad-spectrum antibiotic.
HYT-L, followed by HYT-F had the highest antimicrobial capacity values against S. Aureus
ATCC 25923 CECT 435 (gram-positive) growth, because of they presented 28.1 and 25.2 mm of
growth inhibition, respectively. These extracts were followed by RA, P, NOS and NOVS. In this
case, clearly, the most active compound was the hydroxytyrosol, as obtained from olive leaves as
from olive fruits. In second place was RA, which is the most similar in terms of its molecular
structure. Both of them provide an opportunity to study the action mechanism of these compounds
in future research. However, the inhibitory capacity of the rest of the compounds was lower and
does not allow any structure-activity hypothesis to be proposed.
Table 8.29. Antimicrobial activity of natural extracts measured by the disc diffusion method (mm
± SD).
Samples Concentration
(ppm) Dilution (µL)
L.
monocytogenes
KCTC 3569
CECT 7467
(Gram-positive)
S. Aureus
ATCC 25923
CECT 435
(Gram-positive)
E. Coli O157:H7
ATCC 25922
CECT 434
(Gram-negative)
RA 1000
30 8.0 ± 0.0 7.8 ± 0.7 6.9 ± 1.0
60 9.6 ± 0.6 10.5 ± 0.5 12.3 ± 0.6
90 12.3 ± 0.6 16.8 ± 1.8 15.2 ± 0.3
NOS 1000
30 8.0 ± 0.5 7.5 ± 0.0 8.0 ± 0.5
60 10.8 ± 0.7 9.0 ± 0.5 10.5 ± 1.0
90 14.0 ± 0.7 13.1 ± 0.5 20.0 ± 1.0
NOVS 1000
30 - - -
60 7.7 ± 1.0 6.7 ± 0.5 7.2 ± 0.7
90 14.7 ± 1.0 11.9 ± 0.3 18.5 ± 1.0
P 1000
30 10.3 ± 0.6 9.0 ± 0.0 8.0 ± 0.0
60 12.5 ± 0.5 11.2 ± 0.3 10.2 ± 0.3
90 15.3 ± 0.6 13.8 ± 0.8 12.6 ± 0.6
HYT-F 1000
30 6.0 ± 0.6 7.8 ± 0.5 -
60 10.0 ± 0.6 14.5 ± 0.3 -
90 13.0 ± 1.5 25.2 ± 0.5 11.3 ± 0.8
HYT-L 1000
30 10.0 ± 0.5 9.0 ± 0.5 -
60 12.5 ± 0.6 18.5 ± 0.6 8.0 ± 0.5
90 15.0 ± 0.5 28.1 ± 0.5 12.0 ± 0.5
Chloramphenicol 30 µM 34.7 ± 0.6 32.7 ± 0.5 33.7 ± 0.7 Chloramphenicol: positive control; SD: Standard Deviation. P: Pomegranate extract, RA: Rosemary extract rich in Rosmarinic Acid; NOS: Rosemary extract rich in diterpenes; NOVS: Rosemary extract rich in diterpenes and with lecitin as emulsifier; HYT-L:
Hydroxytyrosol extract obtained from olive leaf; HYT-F: Hydroxytyrosol extract obtained from olive fruit.
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126
On the other hand, the gram-positive bacterium L. monocytogenes KCTC 3569 CECT 7467 is
the most resistant strain to phenolic compounds. In this case, P was the most antimicrobial with
15.3 mm growth inhibition, which corresponds with 44.1% of the positive control,
chloramphenicol. This was followed by HYT-L, NOVS, NOS, HYT-F and RA. It can be
considered, then, that all the studied compounds showed similar inhibitory activities against this
microorganism.
Finally, NOS, was the most antimicrobial extract against the gram-negative bacteria E. Coli
O157:H7 ATCC 25922 CECT 434, with 20 mm of growth inhibition, 40.6% less than
chloramphenicol. This phenolic extract was followed by NOVS, RA, P, HYT-L and HYT-F. In
this case, of great interest and significance is the fact that fat-soluble compounds such as
terpenoids had a higher inhibitory capacity against the growth of gram-negative bacteria. This
was followed by the rest of extracts, all of which showed similar values of antimicrobial activity,
making it difficult to offer any considerations on their structure-activity.
Regarding antimicrobial activity, the terpenoid structure did not provide good results, although
this does not mean that this compound has a lower antioxidant capacity. While not significant, it
is interesting to point out that NOVS, which contains lecitin as an emulsionant, shows higher
antioxidant activity than NOS, which does not contain this excipient. This method has been used
in much research to test the antimicrobial capacity of many drugs and natural extracts. For
example, Laincer et al. (2014) measured the antimicrobial activity of several olive phenolics,
including HYT, against E. Coli and S. Aureus, obtaining similar results. Weckesser et al. (2007)
analysed the antimicrobial activity of plant extracts, such as Rosmarinus officinalis L. against
bacteria of dermatological relevance, among them E. Coli and S. Aureus, obtaining similar results
using the diffusion disk method. Regarding the P extract, Kharchoufi et al. (2018) obtained similar
results for pomegranate peel extracts against Pseudomonas putida, Penicillium digitatum and
Saccharomyces cerevisae, but not against any strains used in the present study. Finally, applying
rosemary extracts, Santomauro et al. (2017) obtained similar results (more than 10 mm of
inhibition) in different strains.
Regarding the oxidative and antimicrobial damage of fish products under refrigerated storage
for 11 days, all the natural extracts showed an antioxidative effect against formation of volatile
compounds related to lipid oxidation.
Table 8.30. shows the results obtained from the of GS-MS analysis of the volatile organic
compounds: 1-Penten-3-ol, hexanal, 2-nonanone, 1,6-octadien-3-ol, octanal, pentadecane in fish
patties. In general, all the volatile compounds analysed increased (p < 0.05) from the beginning
of storage for all the treatments. These results point to the degradation that is shown in fish patties,
that is due to oxidation phenomena, as most straight chain aldehydes are derived from the
oxidation of unsaturated fatty acids.
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Table 8.30. Average values and standard deviations of organic compounds (mg/g) in fish patties
stored for 11 days, under retail conditions. Organic
compounds
(mg/g)
Sample Day 0
(M ± SD)
Day 11
(M ± SD)
Organic
compounds
(mg/g)
Day 0
(M ± SD)
Day 11
(M ± SD)
1-Penten-3-ol
Control 0.05 ± 0.01 5.14 ± 0.01 a
1,6-octadien-3-
ol
16.79 ± 0.04 a 22.32 ± 0.21 a
P 0.06 ± 0.05 3.86 ± 0.05 b 2.05 ± 0.04 b 2.32 ± 0.05 b
RA 0.04 ± 0.02 3.54 ± 0.02 b 2.05 ± 0.03 b 2.55 ± 0.03 b
NOS 0.05 ± 0.04 1.95 ± 0.04 c 0.65 ± 0.01 b 0.44 ± 0.01 b
NOVS 0.01 ± 0.03 2.01 ± 0.03 c 0.77 ± 0.02 b 0.77 ± 0.02 b
HYT-F 0.04 ± 0.05 4.24 ± 0.05 b 0.57 ± 0.02 b 0.57 ± 0.02 b
HYT-L 0.07 ± 0.04 4.27 ± 0.04 b 0.87 ± 0.02 b 0.87 ± 0.02 b
Hexanal
Control 0.03 ± 0.00 4.25 ± 0.02 a
Nonanal
0.16 ± 0.01 1.81 ± 0.02
P 0.09 ± 0.01 2.01 ± 0.03 b 0.51 ± 0.01 1.30 ± 0.02
RA 0.04 ± 0.02 2.96 ± 0.04 b 0.37 ± 0.01 1.75 ± 0.02
NOS 0.07 ± 0.01 1.53 ± 0.02 b 0.33 ± 0.01 0.66 ± 0.02
NOVS 0.02 ± 0.05 1.47 ± 0.02 b 0.29 ± 0.01 0.66 ± 0.01
HYT-F 0.03 ± 0.01 2.88 ± 0.05 b 0.47 ± 0.01 1.20 ± 0.03
HYT-L 0.03 ± 0.01 2.96 ± 0.03 b 0.39 ± 0.01 1.57 ± 0.02
2-nonanone
Control 0.16 ± 0.01 0.39 ± 0.02
Pentadecane
1.13 ± 0.02 1.60 ± 0.02
P 0.51 ± 0.01 0.46 ± 0.04 1.17 ± 0.03 1.44 ± 0.02
RA 0.37 ± 0.01 0.22 ± 0.03 0.10 ± 0.0 1.04 ± 0.03
NOS 0.33 ± 0.01 0.16 ± 0.01 0.62 ± 0.01 1.08 ± 0.01
NOVS 0.29 ± 0.01 0.25 ± 0.03 0.57 ± 0.01 1.05 ± 0.03
HYT-F 0.47 ± 0.01 0.27 ± 0.05 0.82 ± 0.03 1.54 ± 0.01
HYT-L 0.39 ± 0.01 0.32 ± 0.01 0.75 ± 0.03 1.27 ± 0.01
Results are expressed as mean ± standard deviation in arbitrary area units (× 106). P: Pomegranate extract, RA: Rosemary extract rich in
Rosmarinic Acid; NOS: Rosemary extract rich in diterpenes; NOVS: Rosemary extract rich in diterpenes and with lecitin as emulsifier; HYT-L: Hydroxytyrosol extract obtained from olive leaf; HYT-F: Hydroxytyrosol extract obtained from olive fruit
Hexanal and 1,6-octadien-3-ol were the dominant aldehyde in the fish patties meat in all the
groups. Hexanal values ranged from 0.1 mg/kg, in day 0, to 5.14 mg/kg after 11 days, in control
samples. These values are in the same line than those reported by Brunton et al. (2000), who found
hexanal values of 4.01 l/g in cooked turkey stored for 6 days at 4 °C.
Differences in the mean hexanal levels between C and patties with natural extracts were
significant (p < 0.05) on day 11. On day 11, NOVS showed lower (39%) hexanal values than C,
meaning that Rosemary extracts improved lipid stability of the fish patties. In this sense, Shahidi,
Yun and Rubin (1987) reported that such an increase in hexanal is a good indicator of lipid
oxidation. Indeed, these authors suggested hexanal as a valid indicator of oxidative stability and
flavour acceptability in cooked ground meat.
The behaviour of nonanal and 1-penten-3-ol was similar to hexanal, both increasing (p < 0.05)
during storage and showing significant differences between C and natural extracts samples on day
11. Nonanal is a waxy flavour and descriptors, while 1-penten-3-ol is amongst the compounds
responsible for the rancid odour in mayonnaise. Note the absence of significant differences in 2-
nonanone and pentadecane on day 11.
Table 8.30. shows that all the volatile compounds analysed are the main components that
contribute the most to the emergence of unpleasant notes of flavour, due to the low flavour
threshold (Kerler & Grosch, 1997). In general, the presence of natural extract (especially NOVS)
in the fish patties delayed the formation of all the volatile lipid-derived compounds. In the same
line, Nieto et al. (2011a and 2011b) reported lower hexanal values, rancid odour and rancid
flavour scores in lamb meat from ewes fed thyme leaves or rosemary by-products, respectively.
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Antimicrobial capacity of different extract was also proved in fish products elaborated from
thawed hake. Consequently, microbiological results of fish patties at days 0, 4, 7 and 11 from
elaboration, are shown in Table 8.31.
Table 8.31. Microbiological results (cfu/g) of fish patties analysis at days 0, 4, 7 and 11 under
refrigerated storage.
Analysis Sample Storage day
0 4 7 11
TVC
Control 2.0 × 103 6.2 × 103 2.8 × 104 5.6 × 107
P 1.5 × 103 5.1 × 103 1.3 × 104 5.4 × 107
RA 9.1 × 102 4.3 × 103 2.0 × 104 7.3 × 107
NOS 7.3 × 102 3.6 × 103 1.1 × 104 6.5 × 107
NOVS 1.0 × 103 4.1 × 103 1.6 × 104 6.9 × 107
HYT-L 1.9 × 103 6.2 × 103 1.8 × 104 3.5 × 107
HYT-F 2.0 × 103 6.0 × 103 2.1 × 104 3.2 × 107
TCC
Control <10 8.4 × 102 4.8 × 103 5.5 × 104
P <10 1.4 × 103 7.8 × 103 6.8 × 103
RA <10 2.0 × 103 6.1 × 103 3.8 × 104
NOS <10 1.3 × 103 5.3 × 103 2.6 × 104
NOVS <10 1.7 × 103 6.0 × 103 3.1 × 104
HYT-L <10 4.4 × 102 3.3 × 103 7.2 × 104
HYT-F <10 5.9 × 102 3.9 × 103 7.8 × 104
E. Coli
Control <10 <10 <10 <10
P 10 <10 <10 <10
RA <10 <10 <10 <10
NOS <10 <10 <10 <10
NOVS <10 <10 <10 <10
HYT-L <10 <10 20 <10
HYT-F <10 <10 <10 <10
L. monocytogenes
Control Absence in 25 g Absence in 25 g Absence in 25 g Absence in 25 g
P Absence in 25 g Absence in 25 g Absence in 25 g Absence in 25 g
RA Absence in 25 g Absence in 25 g Absence in 25 g Absence in 25 g
NOS Absence in 25 g Absence in 25 g Absence in 25 g Absence in 25 g
NOVS Absence in 25 g Absence in 25 g Absence in 25 g Absence in 25 g
HYT-L Absence in 25 g Absence in 25 g Absence in 25 g Absence in 25 g
HYT-F Absence in 25 g Absence in 25 g Absence in 25 g Absence in 25 g
TVC: Total Viable Count; TCC: Total Coliform Count P: Pomegranate extract, RA: Rosemary extract rich in Rosmarinic Acid; NOS:
Rosemary extract rich in diterpenes; NOVS: Rosemary extract rich in diterpenes and with lecitin as emulsifier; HYT-L:
Hydroxytyrosol extract obtained from olive leaf; HYT-F: Hydroxytyrosol extract obtained from olive fruit.
As it can be appreciated, TVC results after 11 days of refrigerated storage were lower in
samples that incorporated HYT-F and HYT-L in their formula, followed by P, control sample,
NOVS, NOS and RA. While, TCC results showed as both pomegranate and rosemary extracts,
diterpens rich extracts (NOS and NOVS) and rosmarinic acid rich extract (RA) obtained the
lowest results in comparison with the control sample, or samples that incorporated HYT to their
formulas. These last results can be related with antimicrobial activity of all the extracts against E.
Coli O157:H7 ATCC 25922 CECT 434. On the other hand, results obtained from analysis of E.
Coli and L. monocytogenes did not present significant differences (p < 0.05) among incorporation
of natural extracts. In addition, it must be taken into account that only samples enriched with
HYT, both from fruit or leaf, did not exceed the limit stablished by European legislation regarding
to TVC in fish products (5,000,000 cfu/g). In parallel, only P and rosemary extracts (RA, NOS
and NOVS) kept fish product samples below legal microbiological safety limits of TCC (50,000
cfu/g). However, all the natural extracts, including the control sample prevented against
microbiological growth of E. Coli and L. monocytogenes.
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In Table 8.31., natural extracts also acted as antimicrobial agents against TVC and TCC
proliferation. This behaviour has been previously observed by other researchers using other
natural extracts. For example, Del Nobile et al. (2009) studied the combined effect of different
gas mix compositions (MAP 30:40:30 O2:CO2:N2, 50:50 O2:CO2 and 5:95 O2:CO2) and three
essential oils (thymol, lemon and grapefruit seed extracts) on fresh blue fish burgers. Results
obtained showed as the combination of 110 ppm of thymol, 100 ppm of grapefruit seed extract,
or 120 ppm of lemon extract with MAP 5:95 O2:CO2 was able to maintain the microbial quality
of fish burgers for 28 days under refrigerated storage. In the same way, the combined effect of
antimicrobial mixtures of chitosan, nisin and sodium lactate with MAP 55:45 CO2:N2 was able
to guarantee the microbial acceptance of hake burgers for 30 days of refrigerated storage
(Schelegueda et al., 2016). On the other hand, Smaldone et al. (2017) have observed that only
MAP 5:60:35 O2:CO2:N2 application can extend the microbiological shelf-life of hake burgers
for 15 days after elaboration. However, in the present study, modified atmosphere package
treatment was not assessed, neither in previous research on fish products using natural extracts
from pomegranate, rosemary or olive tree (Olea europaea). Likewise, with obtained results it can
be concluded that bioactive compounds from studied extracts (P, RA, NOS, NOVS, HYT-L and
HYT-F) act as antimicrobial agents, which has also demonstrated in vitro and it is due to their
high concentration of phenolic compounds (punicalagin, carnosic acid, carnosol, rosmarinic acid
and hydroxytyrosol) with known antimicrobial activity, as it has been exposed in the introduction
of this work. For this reason, it is not surprising that their application avoided L. monocytogenes
or E. Coli growth. Nevertheless, is important to know that samples that incorporated rosemary
extracts presented TVC growth higher than the control sample at day 11, similar to hydroxytyrosol
extracts that showed higher TCC growth than the control. This fact can be explained by the great
amount of antioxidant compounds in combination with spices and spice extracts that the
commercial mix contained and which can produce a synergism between them, increasing the
shelf-life of fish products.
8.5.2. Shelf-life study of fish patties enriched in natural extracts
This last study is the continuation of the previous one, were five new batches of fish patties
were elaborated using the best antioxidant compounds, taking into account obtained results in last
studies (showed in 8.4.1. and 8.5.1. chapters).
Proximal composition of different samples of fish patties is shown in Table 8.32. As it can be
observed, there were no significant differences among all the samples, which were made with
frozen hake, as main ingredient (83–85 %). Hence, samples presented 77.24–79.08 % moisture,
2.72–3.29 % ash, 14.84–15.99 % protein and 1.11–1.78 % fat. Similar results were also obtained
by Martí et al. (2015), Igor et al. (2010) and Izquierdo et al. (1999), who also used more than 80
% fish for the elaboration of fish patties. These results show as the incorporation of both phenolic
rich natural extracts and omega 3 rich essential oils did not affect to the proximal content of fish
patties.
Lorena Martínez Zamora PhD Thesis, 2019
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Table 8.32. Proximal composition ( ± SD) of fish patties samples.
Samples Moisture
(%)
Ash
(%)
Protein
(%)
Fat
(%)
C 79.0±0.0 2.9±0.0 14.8±0.0 1.1±0.0
Ct 79.1±0.0 2.7±0.3 15.2±0.0 1.7±0.1
HXT 77.2±0.2 3.0±0.4 16.0±0.4 1.6±0.0
P 77.5±0.3 3.3±0.1 15.4±0.5 1.7±0.1
R 77.6±0.5 3.1±0.0 15.8±0.3 1.8±0.2
C: Control; Ct: 200 ppm acerola + 200 ppm Citric extract; HXT: 200 ppm acerola + 200 ppm Hydroxytyrosol
extract; P: 200 ppm acerola + 200 ppm Pomegranate extract; R: 200 ppm acerola + 200 ppm Rosemary extract. M ± SD: Mean ± Standard Deviation. Different letters in the same row indicate significant differences between
samples (p<0.05).
Otherwise, table 8.33. shows obtained results of micronutrients analysis in fish patties,
together with the percent of RDA that corresponds if 100 g per day are consumed of this
manufactured fish product.
In a general view, Na was the most concentrated mineral, followed by, K, P, Mg, Fe, Zn and
Se. Additionally, there were significant differences in the mineral content of fish preparations.
Hence, control sample was the most abundant sample in potassium, magnesium and zinc, while
P was the richest one in iron and R in Na and P.
Table 8.33. Mineral content (M ± SD) (mg/100 g) of fish patties and RDA percent that supposes
consumption of 100 g per day. Samples Fe K Mg Na Se Zn P
C 0.7a
5%
534a
15.3%
68a
20%
409e
20.5%
0.03
45%
0.4a
2.6%
214d
30.6%
Ct 0.5ab
3.9%
288e
8.2%
39d
11.5%
651d
32.5%
0.02
28.8%
0.2c
1.6%
209e
29.9%
HXT 0.3b
1.8%
349b
10%
49b
14.4%
751c
37.6%
0.022
35.8%
0.3b
1.7%
281b
40.1%
P 0.7a
5.1%
305c
8.7%
42c
12.4%
818b
41%
0.02
25.6%
0.2e
1.4%
272c
38.9%
R 0.4b
3.5%
300d
8.5%
40cd
11.8%
846a
42%
0.02
30.2%
0.2d
1.5%
292a
41.8%
RDA
(mg/día) 10-18 3500 330-350 <2000
0,055-
0,070 15 700
C: Control; Ct: 200 ppm acerola + 200 ppm Citric extract; HXT: 200 ppm acerola + 200 ppm Hydroxytyrosol extract; P: 200 ppm
acerola + 200 ppm Pomegranate extract; R: 200 ppm acerola + 200 ppm Rosemary extract. RDA; Recommended Dietary Allowance;
M ± SD: Mean ± Standard Deviation. Different letters in the same row indicate significant differences between samples (p<0.05).
If the studied samples are compared with other commercial fish derivatives (croquettes or hake
fingers) we can observe that it contains a higher proportion of Na, K, P and Mg (Planells et al.,
2003).
As far as the daily recommendations are concerned, the consumption of manufactured fish
products studied does not cover the necessary nutritional needs, but it can be said that their
consumption provides 50% more of minerals, as P or Mg, than the consumption of hake fresh,
which has a concentration of 190 and 23 mg/100 g, respectively (Moreiras et al., 2013). Then, the
inclusion of fish products such as this in a correct balanced diet can be beneficial when helping
to achieve the appropriate consumption requirements, especially for population groups with lower
fish consumption, such as young people between 5 and 18 years of age (MAPA, 2019).
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In another way, regarding to obtained data of shelf-life study of fish pattie samples, results of
pH and colour (CIELab) measurements are shown in Table 8.34.
Table 8.34. Obtained results of pH and colour (CIELab) (M ± SD) evolution of fish patties for
14 days under refrigerated storage.
Days refrigerated storage
Sample 0 4 7 11 14
pH
C
Ct
HXT
P
R
6.8±0.0c
6.8±0.0c
6.8±0.0c
6.8±0.0c
6.8±0.1c
7.0±0.0by
7.1±0.0by
7.0±0.1by
6.8±0.1cz
6.8±0.0cz
8.0±0.1x
7.6±0.0ay
7.4±0.0by
6.8±0.0cz
7.0±0.1byz
7.3±0.0by
7.2±0.0by
7.3±0.0by
6.7±0.0cz
7.3±0.0byz
7.1±0.0by
7.2±0.0by
7.2±0.1by
6.6±0.1cz
7.1±0.0by
Colour parameters: L*
C
Ct
HXT
P
R
72.3±0.3
80.0±1.2
87.8±2.3
82.3±0.9
81.9±1.1
65.0± 0.8
73.9 ±1.3
73.6±0.8
70.9±0.5
71.7±0.4
62.8±0.6
72.2±0.4
71.8±0.3
70.4±0.5
70.2±0.4
64.7±0.5
72.3±0.5
72.8±0.7
69.5±0.8
70.3±0.6
64.5±0.2
73.0±0.6
72.5±0.3
69.2±0.4
69.8±0.3
a*
C
Ct
HXT
P
R
3.5±0.0a
2.4±0.1b
1.9±0.1b
0.7±0.1c
2.6±0.0b
3.7±0.1a
2.4±0.1b
2.1±0.0b
1.4±0.1c
2.7±0.0b
4.7±0,1a
3.4±0.1b
3.0±0.1b
1.9±0.1c
3.3±0.0b
4.2±0.1a
3.4±0.8b
2.9±0.0b
2.2±0.2c
3.3±0.1b
3.7±0.1a
2.4±0.1b
2.4±0.1b
1.5±0.0c
2.8±0.0b
b*
C
Ct
HXT
P
R
11.4±0.1c
11.9±0.4b
11.9±0.4c
18.7±0.4a
12.1±0.4c
11.3±0.2c
13.7±0.1b
11.1±0.2c
17.0±0.3a
11.1±0.7c
12.2±0.3c
14.8±0.1b
13.0±0.3c
16.2±0.2a
11.5±0.1c
12.1±0.2c
14.6±0.4b
12.4±0.3c
16.5±0.3a
10.6±0.5c
11.0±0.1c
13.8±0.3b
12.4±0.1c
15.7±0.2a
11.2±0.2c
C: Control; Ct: 200 ppm acerola + 200 ppm Citric extract; HXT: 200 ppm acerola + 200 ppm Hydroxytyrosol extract; P: 200 ppm acerola + 200 ppm Pomegranate extract; R: 200 ppm acerola + 200 ppm Rosemary extract; M ± SD: Mean ± Standard Deviation. a,
b, c: Different letters in the same line indicate significant differences between days of analysis (p<0.05). x, y, z: Different letters in
the same row indicate significant differences between samples (p<0.05).
As it can be appreciated in Table 8.34., there were significant differences (p<0.05) in pH
analysis both on the day of analysis and between samples. Regarding to the day of analysis, it is
observed that all samples had a pH value of 6.8 at day 0 and a maximum peak is reached at day 7
of the study, decreasing again afterwards. As for the samples, it is observed as P sample kept the
pH levels constant on all the days of the study, while the rest of them increased, even reaching
pH 8 in the control sample.
Increases in pH are an indicator of the accumulation of alkaline compounds such as ammonia
and TMA and can also derive from the action of microorganisms, however, the decrease of pH
values from day 7 until 14 is an indicator of lactic acid bacteria growth. Once pH value decreases
due to the bacteria growth, fish flavour turns to acid and sensory acceptance also decreases
(Hebard, Flick & Martin, 1982).
Regarding to colour measurements, also presented in Table 8.34., there are significant
differences (p<0.05) between the a* and b* coordinate results in all samples, indicating that the
addition of extracts causes colour modifications. However, this effect was not appreciated in the
brightness parameter: L*. Otherwise, parameters such as water retention capacity, collagen
content, free water and fat content affect L* coordinate, as does the addition of additives and other
Lorena Martínez Zamora PhD Thesis, 2019
132
technological factors such as cooking (Fuentes-Zaragoza et al., 2009). Therefore, it would be
expected obtaining differences in brightness (L*) between the preparations and control sample,
made without phenolic rich extracts, but it does not during refrigerated storage, because of fish is
a product that retains a large amount of water (Fuentes-Zaragoza et al., 2009).
P sample is the one with the lowest values in the a* coordinate, followed by the HXT, C, R
and Control sample. However, the opposite occurs at coordinate b* where P has the highest
content followed by C, HXT, R and control. These differences could be observed visually, since
the fish preparation with pomegranate extract presented a more intense orange coloration, which
coincides with the values of a* and b*. The red-green component is related to the presence of
pigments, so its presence in foods, such as fish, in this case, will depend on the values of
hemoglobin/myoglobin or punicalagin and carotenoids that may have been incorporated
(Czeczuga & Kylszeiko, 1986).
Nevertheless, obtained results of lipid and protein oxidation studies for 14 days are shown in
Table 8.35., together with fish degradation results along the same period of time under chilled
storage.
In the obtained TBARs values (lipid oxidation analysis), significant differences (p<0.05) have
been observed between the samples throughout the shelf-life study. Unless the values were slowly
decreasing along the study, P sample was less oxidized in comparisson with the rest of samples,
followed by R, HXT, Control and finally C, which is the worst behaving, surpassing the control
sample in lipid oxidation.
It can be said that hake contained low levels of fat (2 % approximately) so it could be expected
that in general the samples did not have high values of lipid oxidation. Also, lower TBARS values
were obtained by Laura Martí et al. (2015) in tuna and seaweed hamburgers, possibly because
they were packaged in a modified atmosphere and vacuum.
In the analysis of thiols, it can be observed that the sample with the lowest protein oxidation
throughout the shelf-life study was P, followed by HXT, C, R and Control. The fact that the C
sample presents such high values of protein oxidation versus lower values of lipid oxidation
contrasts. This may be due to the fact that the present phenolic in this extract (naringin, hesperidin)
are powerful acting on the agents responsible for this protein oxidation (transition metals,
hydrogen peroxide). In addition, these results can be also compared with previous exposed results
in 8.4.2., where the same extract, C, was tested in an oxidized pork meat model system and it
helped to control de protein oxidation in presence of APPH and AMVN agents.
Lorena Martínez Zamora PhD Thesis, 2019
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Table 8.35. Obtained results of lipid oxidation (TBARs), protein oxidation (thiol loss) and fish
degradation (TMA and TVB-N) (M ± SD) of fish patties for 14 days under refrigerated
storage. Days refrigerated storage
Sample 0 4 7 11 14
TBARs (mg MDA/kg)
C
Ct
HXT
P
R
0.7±0.1ayz
0.8±0.0ay
0.7±0.0az
0.6±0.1az
0.6±0.1az
0.6±0.0cyz
0.6±0.1cy
0.4±0.1cz
0.5±0.1cz
0.3±0.0cz
0.6±0.1bcyz
0.7±0.1bcy
0.5±0.0bcz
0.5±0.0bcz
0.4±0.1bcz
0.6±0.0abcyz
0.7±0.0abcy
0.6±0.0abcz
0.5±0.0abcz
0.5±0.0abcz
0.6±0.1abyz
0.7±0.1aby
0.6±0.0abz
0.6±0.0abz
0.6±0.0abz
Thiol loss
C
Ct
HXT
P
R
37.5±0.1by
22.9±0.0byz
18.4±0.1byz
19.0±0.0bz
16.4±0.0byz
32.0±0.0by
21.4±0.0byz
17.1±0.1byz
11.5±0.0bz
27.0±0.0byz
26.7±0.0by
16.4±0.1byz
15.2±0.1byz
10.7±0.1bz
10.1±0.0byz
15.7±0.0by
5.4±0.0byz
8.6±0.0byz
6.7±0.1bz
13.5±0.4byz
92.0±0.0ay
32.9±0,06ayz
34.7±0.1ayz
24.1±0.0az
69.0±0.0ayz
TMA (mg/100 g)
C
Ct
HXT
P
R
1.0±0.0c
0.5±0.0c
0.2±0.1c
0.9±0.0c
0.6±0.1c
4.5±0.0bc
4.7±0.0bc
4.4±0.0bc
1.5±0.2bc
3.5±0.0bc
7.8±0.0b
7.0±0.1b
6.3±0.0b
2.9±0.0b
5.1±0.1b
17.3±0.0a
16.1±0.0a
10.8±0.0a
7.1±0.0a
9.2±0.1a
18.3±0.0a
17.3±0.0a
15.4±0.1a
10.7±0.0a
11.7±0.0a
TVB-N (mg N/100 g)
C
Ct
HXT
P
R
4.0±0.1c
5.2±0.1c
4.6±0.0c
4.4±0.1c
4.8±0.0c
32.2±0.1c
23.9±0.1c
27.9±0.0c
7.5±0.0c
14.2±0.0c
87.9±0.2b
81.8±0.1b
50.6±0.1b
12.4±0.1b
42.2±0.1b
110.0±0.1a
114.4±0.0a
108.1±0.0a
34.8±0.1a
91.1±0.0a
115.2±0.1a
118.4±0.0a
120.6±0.0a
49.7±0.0a
100.9±0.0a
C: Control; Ct: 200 ppm acerola + 200 ppm Citric extract; HXT: 200 ppm acerola + 200 ppm Hydroxytyrosol extract; P: 200 ppm
acerola + 200 ppm Pomegranate extract; R: 200 ppm acerola + 200 ppm Rosemary extract. RDA; Recommended Dietary Allowance; M ± SD: Mean ± Standard Deviation. a, b, c: Different letters in the same line indicate significant differences between samples
(p<0.05). y, z: Different letters in the same row indicate significant differences between samples (p<0.05).
Otherwise, if we focus in autolytic changes that produces in fish meat, in Table 8.35. can be
also appreciated obtained results of TMA and TVB-N. Regarding to them, there were no
significant differences between the samples, there were only differences in the days of storage,
increasing progressively to reach its maximum on day 14. However, if we carefully study the
results, it can be observed as fish patties enriched with P extract showed better results than R,
HXT, C and Control sample, in this order. Hence, stablished quality limit of 14-15 mg/100 g of
fish is exceeded by C and Control sample at day 11, while P and R sample still maintain TMA
values under this level after 14 days. Then, P acted as better preservative agent in fish than HXT
or R, with known antioxidative and protective capacities.
Similarly, the legal limits for TVB-N are stablished from 25 to 35 mg N/100 g. The only
sample that reaches day 11 without exceeding these values was P sample. The increase of TVB-
N and TMA is related to the microbiological activity, which coincides with obtained results in
previous study (8.5.1.), since on day 11 the microbial count increases considerably, being
especially high after 14 days. In a comparative manner, in the study carried out by Laura Martí et
al. (2015), TVB-N and TMA did not exceed 20 mg NBVT/100 g neither 15 mg TMA/100 g, due
to the protective effect of packaging in a modified atmosphere. Nevertheless, in the present study
it was preferred to study the action of antioxidant agents in aerobic conditions.
Lorena Martínez Zamora PhD Thesis, 2019
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Finally, the sensory analysis of fish patties samples was carried out at day 0 from elaboration
in order to know the acceptability of these Clean Label fish products. Obtained results are shown
in Figure 8.15.
Figure 8.15. Obtained results of organoleptic analysis of fish patties enriched in phenolic
compounds and essential fatty acids.
Significant differences (p<0.05) have been obtained in the score of the attribute of the proper
colour. In Control, C, HXT and R a hake colour of its own was observed, while in P it was not.
Similarly, “Extract colour” was appreciable only in P sample, being imperceptible in Control,
HXT, C, or R sample. Actually, this colour changes can also be appreciated in Table 8.34. were
a* values have been presented and it is that P samples presented a yellowish tone different from
the rest of samples.
Otherwise, no significant differences have been observed between the different formulas in
the parameters of proper odour and extract odour. Regarding to flavour, differences have been
observed in the taste of hake. Hence, Control sample presented a spiced flavour, while samples
enriched in C, HXT, P and R reported high scores of hake flavour (5, 5, 4.7, and 4.7, respectively).
In this sense, a higher extract flavour score was obtained by R, followed by the Control, P, C and
HXT. This fact is due to the presence of phenolic and volatile compounds with intense flavour,
such as rosmarinic acid, carnosic and carnosol.
It can be observed that the most accepted hake preparations were those of the C and HXT
samples followed by P, R and Control, which was the worst scored, due to the presence of a lot
of synthetic flavorings in the commercial mix that gave to the sample a flavour completely
different from fish. Comparable results were shown by José Igor et al. (2010) on surimi
preparations. After shelf-life study, it can be said that all samples had a maximum consumption
date of 7 days, except P sample, that reached 14 days under refrigerated storage. In addition, these
sample were better sensory evaluated than Control sample. Hence, P sample was the best qualified
in all the analysis carried out.
0,0
1,0
2,0
3,0
4,0
5,0Own colour
Extract colour
Own odour
Extract odourOwn flavour
Extract flavour
AceptabilityC
Ct
HXT
P
R
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9. Conclusions
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In this thesis dissertation, different possibilities of elaboration of Clean label animal origin
products have been approached. Consequently, the following conclusions have been achieved.
Firstly, organic forms of minerals Zn and Se have demonstrated to be more bioavailable in a
chicken meat emulsion enriched endogenously with those minerals and also exogenously through
the incorporation of EVOO and HXT using an in vitro Caco-2 cell model system. An important
outcome is related with HXT degradation, that was minimum during the “in vitro” digestion, that
lead to the idea for future investigations that at least 90 % HXT consumed can be available at
intestinal level.
As expected, the use of EVOO and nuts as ingredients, improves the fatty acid profile in
chicken meat emulsions, providing a good nutritional profile with higher concentration of MUFA
and PUFA. At the same time, the exogenous use of HXT extract avoids protein and lipid oxidation
for 21 days in frankfurters, while maintaining organoleptic quality in combination with EVOO
and nuts.
The addition of phenolic compounds as natural extracts from seeds, herbs, and fruits, together
with organic forms of Zn and Se, delays the microbial growth (longer LAG phase, bacteria adapt
themselves to growth conditions), reduces protein and lipid oxidation time, and do not modify the
sensory quality, that as an overall conclusion, extends the shelf-life of chicken nuggets during one
year under frozen storage (-18 ºC).
Regarding antioxidant and antimicrobial capacity of natural extracts used as ingredient to
produce Spanish “chorizo”, rosemary showed the most intense antimicrobial activity compound
followed by natural sources of nitrates (beet, lettuce, arugula, spinach, chard, celery, and
watercress) and spices, such as paprika, garlic, and oregano. Among all the natural extracts, citrus
ones (herperidin) was the only that showed the higher antioxidant capacity, at the same time that
the lowest antimicrobial activity. Nevertheless, the combination of citric extract with leafy green
vegetables extracts rich in nitrates showed a higher antimicrobial power. Consequently,
hesperidin and natural nitrate sources showed a synergistic behaviour, but it did not present the
same effectiveness in combination with the monoterpenes from rosemary extracts (carnosic acid
and carnosol). This combination of extracts allows the maintenance of dry-cured Spanish
“chorizo” samples for 150 days at refrigeration storage without modifying their sensory quality.
Citrus, as well as Lettuce and Spinach, almost fully protect against protein thiol loss in the
meat model system, initiated by the hydrophilic initiator, OXHydro and by the lipophilic initiator,
OXLip. The same components showed also efficient radical scavenging activity as determined by
ESR spectroscopy. In addition, natural nitrate sources were found to protect against protein thiol
oxidation and were able to scavenge radicals in the oxidizing meat system. The potential
substitution of synthetic or phenolic antioxidants with natural nitrate sources from green leafy
vegetables in the production of meat products for the protection against oxidation and a
prolongation of shelf-life is pointed with the results achived.
Natural extracts tested (pomegranate, olive tree, rosemary and citrus) are also suitable to
extend fish patties shelf-life up to 11 days, with mechanisms that slow down autolytic phases
(degradation of non-protein nitrogen -NPN- components) as well as spoilage microbiological
growth, and any lipid or protein oxidation, keeping the same sensorial high acceptability for
panellists, and with no detection of abnormal flavours (smell or taste).
As a final remark of the current PhD Thesis, the strategies followed provide an useful tool to
“Clean label” animal origin products (meat or fish based), where synthetic additives with
analogical effect have been substituted by natural extracts produced from traditional
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Mediterranean ingredients rich in bioactive compounds, whose consumption leads to significant
improvements in the health of the human body. In addition, this change did not affect to the
sensory properties of the product, which showed a high acceptance avoiding the oxidative damage
and the microbiological growth.
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10. Perspective for further
research activities
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The present thesis dissertation can be the basis for further research in the development of
animal origin “Clean label” products enriched in Mediterranean ingredients, but also with the
perspective of obtaining the basic mechanistic understanding of the interactions between quality
of animal foods and the incorporation of this kind of products obtained as Food Industry by-
products. Based on the knowledge acquire in the current PhD Thesis, some areas are suggested
for further investigation in order to increase the general understanding of the complex interactions
and mechanisms involved:
- Characterization and study of new antioxidant and antimicrobial substances, or traditional
ingredients that can act as preservative agents in foods. Understanding their content in
bioactive compounds and their biochemical formula to know the possible interactions
between protein or lipid modifications is crucial for prevention of the food deterioration
without altering their normal structure.
- New technologies to extract or apply these extracts with the aim of reducing the amount
of waste produced by Food Industry in order to promote the sustainable development of
this sector.
- New technologies for incorporation and distribution of phenolic antioxidants into meat
of fish pieces should be developed, and the effect hereof on meat tenderness and juiciness
before implementation in the Food Industry. Some of these techniques in the development
of animal origin products could be marinating, injection, formulation, or spraying in order
to produce ready-to-eat products such as sliced or 5th range foods.
- Shelf-life and commercial studies of new products free of synthetic additives, in the same
sense as the present Thesis, in order to satisfy the demands of a health-conscious
consumer who is looking for new ready-to-eat products, both healthy and
environmentally friendly.
- Study of health claims into the human body of both, natural ingredients and also food
elaborated with them. For that, monitored and controlled studies on humans should be
carried out prior to the marketing of any of these products. In them, parameters as useful
for current society as their antioxidant, anti-inflammatory, antitumor, satiating, or even
antidepressant capacities could be evaluated.
Finally, for the development of these research will require a multidisciplinary approach,
composed of a team of nutritionists, doctors, chemists and biochemists, psychologists, engineers,
and food technologists, working hand-in-hand with the Food Industry to the development of a
healthy society in which tasty “Clean label” foods will be included.
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11. References
Lorena Martínez Zamora PhD Thesis, 2019
144
Lorena Martínez Zamora PhD Thesis, 2019
145
AESAN/MARM. (2011). Encuesta Nacional de Ingesta
Dietética (ENIDE). Agencia Española de
Seguridad Alimentaria y Nutrición. Ministerio de
Agricultura, Alimentación y Medio Ambiente.
Ahmad Shah, M., Don Bosco, S.J., Ahmad Mir, S. (2014). Plant extracts as natural antioxidants in meat and
meat products. Meat Science, 98: 21–33.
Ahn, Y.H.; Lee, S.J.; Shin, K.M.; Park, E.J. (2007). The vegetation and flora of village groves in
Paengseong-eup, Pyongtaek city, Gyonggi-Do,
Korea. Journal of Korean Institution of Environment and Ecology, 21: 515–525.
Alahakoon, A.U., Jayasena, D.D., Ramachandra, S., Jo, C.
(2015). Alternatives to nitrite in processed meat: Up to date. Trends Food Science and
Technology;45(1): 37-49.
Alarcón-Flores, M.I.; Romero-González, R.; Martínez-Vidal, J.L.; Garrido-Frenich, A. (2014). Determination
of phenolic compounds in artichoke, garlic and
spinach by ultra-high-performance liquid chromatography coupled to tandem mass
spectrometry. Food Analysis and Methods, 7:
2095–2106.
Albert, C.M., Gaziano, J.M., Willnett, W.C. y Manson, J.E.
(2002). Nut consumption and decreased risk of
sudden cardiac death in the physician health study. Archives of Internal Medicine, 162(12): 382-387.
Alfawaz, M.; Smith, J.S.; Jeon, I.J. (1994). Maillard reaction
products as antioxidants in pre-cooked ground
beef. Food Chemistry, 51: 311–318.
Alu’datt, M.H., Rababah, T., Alhamad, M.N., Gammoh, S.,
Al-Mahasneh, M.A., Tranchant, C.C., Rawshdeh, M. (2018). Chapter 15 – Pharmaceutical
Nutraceutical and Therapeutic properties of
selected wild medicinal plants: thyme, spearmint and Rosemary. In: Therapeutic, Probiotic and
Unventional Food, edited by Grumezescu, A.M.
& Holban, A.M. Academic Press: 275-290. DOI:10.1016/B978-0-12-814625-5.00014-5
Álvarez, I., De La Fuente, J., Cañeque, V., Lauzurica, S., et
a., (2009). Changes in the fatty acid composition of M. longissimus dorsi of lamb during storage in
a high-oxygen modified atmosphere at different
levels of dietary vitamin E supplementation.
Journal of Agriculture and Food Chemistry, 57:
140–146.
Álvarez, D., Delles, R.M., Xiong, Y.L., Castillo, M., Payne, F.A., y Laencina, J. (2011). Influence of canola-
olive oils, rice bran and walnut on functionality
and emulsion stability of frankfurters. LWT- Food Science and Technology, 44: 1435-1442.
Amagase, H. (2006). Clarifying the real bioactive constituents of garlic. Journal of Nutrition, 136:
716S–725S.
Ambrosiadis, J., Vareltzis, K.P., Georgakis, S.A. (1996). Physical, chemical and sensory characteristics of
cooked meat emulsion style products containing
vegetable oils. International Journal of Food Science and Technology, 31: 189- 194.
Andrade, M.J., Córdoba, J.J., Casado, E.M., Córdoba, M.G.,
Rodríguez, M. (2010). Effect of selected strains of
Debaryomyces hansenii on the volatile compound
production of dry fermented sausage "salchichón". Meat Science, 85(2): 256-264.
AOAC. (1990). Procedure 920.39 C. Soxhlet method for
quantitative analysis of fat. Association of analytical communities.
AOAC. (1990). Procedure 955.04. Kjeldahl method for
quantitative analysis of protein. Association of analytical communities.
AOAC Official Method 971.14. Trimethylamine nitrogen in
seafood. In "AOAC Official Methods of Analysis of AOAC International". 2002; volume II, chapter
5, 9.
AOAC (Association of Official Analytical Chemists) Procedure 973.49 Nitrogen (ammonia) in water.
Colorimetric method.
AOAC. (2002). Official Methods of Analysis of AOAC International, 17th Edn., Association of Official
Analyticial Chemistry, Maryland, USA.
Aruoma, O.I. (1999). Antioxidant actions of plant foods, use of oxidative DNA damage as a tool for studying
antioxidant efficacy. Free Radical Research; 30:
419–427.
Ayo, J., Carballo, J., Serrano, J., Olmedilla-Alonso, B., Ruiz-
Capillas, C., Jiménez-Colmenero, F. (2007).
Effect of total replacement of pork backfat with walnut on the nutritional profile of frankfurters.
Meat Science, 77: 173-181.
Ayo, J., Carballo, J., Solas, M.T., Jiménez-Colmenero, F. (2005). High pressure processing of meat batters
with added walnuts. International Journal of Food
Science and Technology, 40: 47-54.
Azaizeh, H., Halahlih, F., Najami, N., Brunner, D., Faulstich,
M., Tafesh, A. (2012). Antioxidant activity of
phenolic fractions in olive mill wastewater. Food Chemistry, 134: 2226–2234.
Badiani, A., Stipa, S., Bitossi, F., Gatta, P.P., Vignola, G.,
Chizzolini, R. (2002). Lipid composition, retention and oxidation in fresh and completely
trimmed beef muscles as affected by common
culinary practices. Meat Science, 60: 169–186.
Babinsky, L., Langout, D.J., Verstegen, M.W.A., Den
Hartog, L.A., Joling, P. (1994). Effect of vitamin
e and fat source in sow’s diets on immune response of suckling and weaned piglets. Journal
of Animal Science, 69: 1833-1842.
Bahadoran, Z., Mirmiran, P., Jeddi, S., Azizi, F., Ghasemi, A., Hadaegh, F. (2016). Nitrate and nitrite content
of vegetables, fruits, grains, legumes, dairy
products, meat and processed meats. Journal of Food Composition and Analysis, 51: 93-105.
Balasundram, N., Sundram, K., Samman, S. (2006). Phenolic compounds in plants and agri-industrial by-
products: Antioxidant activity, occurrence and
potential uses. Food Chemistry, 99: 191–203.
Banerjee, R., Verma, A.K., Das, A.K., Rajkumar, V.,
Shewalkar, A.A., Narkhede, H.P. (2012).
Antioxidant effects of broccoli poder extract in goat meat nuggets. Meat Science, 91: 179-184.
Lorena Martínez Zamora PhD Thesis, 2019
146
Baranauskaite, J., Kubiliene, A., Marksa, M., Petrikaite, V.,
Vitkevičius, K., Baranauskas, A., Bernatoniene, J. (2017). The Influence of Different Oregano
Species on the Antioxidant Activity Determined
Using HPLC Postcolumn DPPH Method and Anticancer Activity of Carvacrol and Rosmarinic
Acid. BioMed Research International: 1–7.
DOI:10.1155/2017/1681392
Barbut, S. (1998). Use of a fiber optic probe to predict meat
emulsion breakdown. Italian Journal of Food
Science, 3: 253–259.
Barbut, S. (2011). Producing battered and breaded meat
products.In: Meat processing technology series
Champaign, IL: American Meat Science Association.
Basmacioglu, H.; Tokusoglu, O.; Ergul, M. (2004). Effect of
oregano and rosemary essential oils or alpha-
tocopheryl acetate on performance and lipid
oxidation of meat enriched with n-3 PUFA’s in
broilers. South African Journal of Animal Science, 34: 197–210.
Batifoulier, F., Mercier, Y., Gatellier, P., Renerre, M. (2002).
Influence of vitamin E on lipid and protein oxidation induced by H2O2-activated MetMb in
microsomal membranes from turkey muscle. Meat
Science, 61: 389–395.
Becker, E.M., Nissen, L.R., Skibsted, L.H. (2004).
Antioxidant evaluation protocols: Food quality or
health effects. European Food Research and Technology, 219: 561-571. DOI 10.1007/s00217-
004-1012-4
Benavente-García, O., Castillo, J., Lorente, J., Ortuño, A., Del Río, J.A. (2000). Antioxidant activity of
phenolics extracted from Olea europea L. leaves.
Food Chemistry, 68: 457-462.
Bendeddouche, M.S., Benhassaini, H., Hazem, Z., Romane,
A. (2011). Essential oil analysis and antibacterial
activity of Rosmarinus tournefortii from Algeria. Natural Production Communitary; 6: 1511–1514
Benzie, I.F.F.; Strain, J.J. (1996) The ferric reducing ability
of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Annals of Biochemistry,
239: 70–76.
Berdagué, J.L.; Monteil, P.; Montel, M.C.; Talon, R. (1993).
Effects of starter cultures on the formation of
flavour compounds in dry sausage. Meat Science,
35: 275–287.
Berger, R.G.; Macku, C.; German, J.B.; Shibamoto, T.
(1990). Isolation and identification of dry salami
volatiles. Journal of Food Science, 55: 1239–1242.
Berian, M.J., Gómez, I., Ibáñez, F.C., Sarriés, M.V., Ordoñez, A.I. (2018). Chapter 1 – Improvement of
the functional and healthy properties of meat
products. In: Food quality: balancing health and disease. Handbook of Food Bioengineering: 1-74.
DOI: 10.1016/B978-0-12-811442-1.00001-8
Blomhoff, R., Carlsen, M.H., Frost Andersen, L., Jacobs, D.
R. Jr. (2006). Health benefits of nuts, potential
role of antioxidants. British Journal Nutrition, 96:
S52-S60.
Boitia, J.M., Ortuño, A., Benavente-Garcia, O., Baidez, A.G.,
Frias, J., Marcos, D., Del Río, J.A. (2001) Modulation of the biosynthesis of some phenolic
compounds in Olea europea L. fruits: Their
influence in olive oil quality. Journal of Agriculture and Food Chemistry, 49: 355–358.
Boletin Oficial del Estado (BOE). (2007). Regulation (ES)
No 32/2007, for the Care of Animals, in their Exploitation, Transportation, Experimentation
and Sacrificie; Boletin Oficial del Estado: Madrid,
Spain: 45914–45920.
Booren, A.M., Mandigo, R.W. (1987). Fundamentals of
production. In: A. M. Pearson, R., T. Dutson
(Eds.), Restructured meat and poultry products, advance in meat research, vol 3. (pp. 351– 382)
New York: Van Nostrand.
Bozin, B., Mimica-Dukic, N., Samojlik, I., Jovin, E. (2007).
Antimicrobial and Antioxidant properties of
Rosemary and Sage (Rosmarinus officinalis L.
and Salvia officinalis L., Laminaceae) essential oils. Journal of Agriculture and Food Chemistry;
55: 7879–7885
Brand-Williams, W.; Cuvelier, M.E.; Berset, C. (1995). Use of a free radical method to evaluate antioxidant
avtivity. LWT - Food Science and Technology, 28:
25–30.
Brewer, M.S. (2011). Natural Antioxidants: Sources,
Compounds, Mechanisms of Action, and Potential
Applications. Comprehensive Reviews in Food Science and Food Safety, 10(4):221–247.
Briante, R., Batumi, M., Terenziani, S., Bismuto, E., Febraio,
F., Nucci, R. (200 2). Olea europea L. leaf extract and derivatives: Antioxidant properties.
Journal of Agriculture and Food Chemistry, 50:
4934–4940.
Brkić, D.; Bošnir, J.; Bevardi, M.; Gross-Bošković, A.;
Miloš, S.; Lasić, D.; Krivohlavek, A.; Racz, A.;
Mojsović-Ćuić, A.; Trstenjak, N.U. (2017). Nitrate in leafy green vegetables and estimated
intake. African Journal of Traditional,
Complementary and Alternative Medicine, 14: 31–41.
Brunton, N.P., Cronin, D.A., Monahan, F.J., Durcan, R.A.
(2000). comparison of solid-phase microextraction (SPME) fibers for measurement
of hexanal and pentanal in cooked turkey. Food
Chemistry, 68: 339–345.
Cabrera, M.C., Ramos, A., Saadoun, A., Brito, G. (2010).
Selenium, copper, zinc, iron and manganese
content of seven meat cuts from Hereford and Braford steers fed pasture in Uruguay. Meat
Science, 84(3): 518-528.
Camo, J., Beltrán, J. A., Roncalés, P. (2008). Extension of the
display life of lamb with an antioxidant active
packaging. Meat Science, 80: 1086–1091.
Capuano, E., Fogliano, V. (2011). Acrylamide and 5-
hydroxymethylfurfural (HMF): A review on
metabolism, toxicity, occurrence in food, and mitigation strategies. LWT-Food Science and
Technology, 44(4); 793-810. DOI:
10.1016/j.lwt.2010.11.002
Lorena Martínez Zamora PhD Thesis, 2019
147
Calatayud, M., Devesa, V., Virseda, J.R., Barberá, R.,
Montoro, R., Vélez, D. (2012) Mercury and selenium in fish and shellfish: Occurrence,
bioaccessibility and uptake by caco-2 cells. Food
Chemistry and Toxicology, 50: 2696–2702.
Cao, Y., Xiong, Y.L. (2017). Interaction of whey proteins
with phenolic derivatives under neutral and acidic
pH conditions. Journal of Food Science, 82(2): 409-419. DOI: 10.1111/1750-3841.13607
Cardoso, P.C.; Tomazini, A.P.B.; Stringheta, P.C.; Ribeiro,
S.M.R.; Pinheiro-Sant’Ana, H.M. (2011). Vitamin C and carotenoids in organic and
conventional fruits grown in Brazil. Food
Chemistry, 126: 411–416.
Carvalho, J., Sichetti, P.E., Alves, M., Rodrigues, I., Slaoui,
O., Da Costa, C.E., Trindade, M.A. (2018).
Omega-3- and fibre-enriched chicken nuggets by
replacement of chicken skin with chia (Salvia
hispánica L.) flour. LWT- Food Science and
Technology, 90: 283-289.
Cataldo, D.A.; Maroon, M.; Schrader, L.E.; Youngs, V.L.
(1975). Rapid colorimetric determination of
nitrate in plant tissue by nitration of salicylic acid. Communication in Soil Science and Plant
Analysis, 6: 71–80.
CEE. Reglamento (CE) nº2074/2005 de la comisión de 5 de diciembre de 2005 por el que se establecen
medidas de aplicación para determinados
productos (…). Sección II, Capítulo I: Valores límite de nitrógeno básico volátil total (NBVT)
para determinadas categorías de productos de la
pesca y métodos de análisis que deberán utilizarse.
Diario Oficial de la Unión Europea. 2005b;
L338,36.
Channon, H.A., Trout, G.R. (2002). Effect of tocopherol concentration on rancidity development during
frozen storage of a cured and an uncured
processed pork product. Meat Science, 62(1): 9-17.
Chang, T.W., Pan, A.Y. (2008). Chapter 2; Cumulative
environmental changes, skewed antigen exposure and the increase of allergy. Advances in
Inmunology, 98: 39-83.
Chanwitheesuk, A., Teerawutgulrag, A., Kilburn, J.D., Rakariyatham, N. (2007). Antimicrobial gallic
acid from Caesalpinia mimosoides Lamk. Food
Chemistry, 100: 1044-1048.
Charlebois, A.; Jacques, M.; Archambault, M. (2014).
Biofilm formation of Clostridium perfringens and
its exposure to low-dose antimicrobials. Frontiers in Microbiology, 5: 183.
Chen, C.H., Pearson, A.M., Gray, J.I. (1992). Effects of synthetic antioxidants (BHA, BHT and PG) on the
mutagenicity of IQ-like compounds. Food
Chemistry; 43: 177–183.
Choi, Y. S., Choi, J. H., Han, D. J., Kim, H. Y., Lee, M. A.,
Kim, H. W., Lee, J. W., Chung, H. J. y Kim, C. J.
(2009). Optimization of replacing pork back fat with grape seed oil and rice bran fiber for reduced-
fat meat emulsion systems. Meat Science, 84: 212-
218.
Chowdhury, R., Warnakula, S., Kunutsor, S., Crowe, F.,
Ward, H.A., Johnson, L., Franco, O.H., Butterworth, A.S., Forouhi, N.G., Thompson, S.
G., Thaw, K. T., Mozaffarian, D., Danesh, J. y Di
Angelantonio, E. (2014). Association of Dietary, Circulating and Supplement Fatty Acids with
Coronary Risk: A Systematic Review a Meta-
analysis. Annals of Internal Medicine, 160(6): 398-406.
Cicerale, S., Lucas, L.J. y Keast, R.S.J. (2009). Chemistry
and health of olive oil phenolics. Critical Reviews in Food Science and Nutrition, 49: 218-236.
Claudino, M., Jonsson, M., Johansson, M. (2013). Thiol-ene
coupling kinetics of D-limonene: a versatile “non-click” free-radical reaction involving a natural
terpene. The Royal Society of Chemistry, 3:
11021-11034. DOI: 10.1039/c3ra40696b
Clough, S.R. (2014). Sodium sulfite. In: Encyclopedia of
Toxicology, 3rd ed.; Elsevier: Amsterdam, The
Netherlands: 341-343.
Cofrades, S., Sandoval, L.S., Delgado-Pando, G., López-
López, I., Ruiz-Capillas, C., Jiménez- Colmenero.
F. (2011). Antioxidant activity of hydroxytyrosol in frankfurters enriched with n-3 polyunsaturated
fatty acids. Food Chemistry, 129(2): 429-436
Comission Directive 2002/63/CE, of July 11th of 2002, about methods for the official control of pesticide
residues in animal and vegetable products, and
which repealed Directive 79/700/EEC.
Corleto, K.A.; Singh, J.; Jayaprakasha, G.K.; Patil, B.S.
(2018). Storage stability of dietary nitrate and
phenolic compounds in beetroot (Beta vulgaris) and arugula (Eruca sativa) juices. Journal of Food
Science, 83: 1237–1248.
Crews, C. (2014). Processing Contaminants: N-Nitrosamines. Encyclopedia of Food Safety, (2):
409-415.
Croizet, F.; Denoyer, C.; Tran, N.; Berdagué, J. (1992). Les composes volatils du saucisson sec. Evolution au
cours de la maturation. Viandes Prod. Carnes, 13:
167–170.
Czeczuga, B., Klyszejko, B. (1986). Carotenoids in fish XL.
Carotenoids in fish from the flaklands region. Acta
Ichthyologica et piscatorial; 16: 73-86.
Daza, A., Salado, S., Gálvez, J.F., Gutiérrez-Barquín, M.
(2000) Efecto de la suplementación con vitamina
E y selenio sobre el sistema inmune, parámetros hematológicos y parámetros productivos de
lechones recién destetados. Investigación
Agrícola: Producción y Sanidad Animal, 15(1-2).
Dalgaard, P., Gram, L., Huss, H.H. (1993). Spoilage and shelf
life of cod fillets packed in vacuum or modified atmospheres. International Journal of Food
Microbiology, 19: 283-294.
De la Torre Carbot, K., Chavez Servin, J. L., Jauregui, O., Castellote, Al., Lamuela Raventos, R. M., Nurmi,
T., Pulsen, H. E., Gaddi, A. V., Kaikkonen, J.,
Zunft, H. F. y colaboradores. (2010). Elevated
circulating LDL phenol levels in men who
consumed virgin rather than refined olive oil are
associated with less oxidation of plasma LDL. Journal of Nutrition, 140: 501-508.
Lorena Martínez Zamora PhD Thesis, 2019
148
De Marco, M., Zoon, M.V., Margetyal, C., Picart, C.,
Ionescu, C. (2017). Dietary administration of glycine complexed trace minerals can improve
performance and slaughter yield in broilers and
reduces mineral excretion. Animal Feed Science and Technology, 232: 182-189.
De Mey, E., De Klerck, K., De Maere, H., Dewulf, L.,
Derdelinckz, G., Peeters, M.C., Fraeye, I., Vander Heyden, Y., Paelinck, H. (2014). The occurrence
of N-nitrosamines, residual nitrite and biogenic
amines in commercial dry fermented sausages and evaluation of their occasional relation. Meat
Science, 96(2-A): 821-828.
De Vuyst, L., Leroy, F. (2007). Bacteriocins from lactic acid bacteria: Production, purification, and food
applications. Journal of Molecular Microbiology
and Biotechnology, 13(4): 194-199.
Deiana, N., Corona, G., Incani, A., Loru, D., Rosa, A., Atzeri,
A., Paola Melis, M. y Assunta Dessi, M. (2010).
Protective effect of simple phenols from extravirgin olive oil against lipid peroxidation in
intestinal caco-2 cells. Food Chemistry and
Toxicology, 48: 3008-3016.
DeJong, S., Lanari, M.C. (2009). Extracts of olive
polyphenols improve lipid stability in cooked beef
and pork: Contribution of individual phenolics to the antioxidant activity of the extract. Food
Chemistry, 116: 892–897.
Del baño, M.J., Lorente, J., Castillo, J., Benavente-Garcia, O., Marín, P., Del Río, J.A., Ortuó, A., Ibarra, I.
(2004). Flavoid distribution during the
development of leaves flowers, stems and roots of
Rosmarinus officinalis postulation of the
Biosynthetic pathway. Journal of Agriculture and
Food Chemistry, 52: 4987–4992.
Del Nobile, M.A., Corbo, M.R., Speranza, B., Sinigaglia, M.,
Conte, A., Caroprese, M. (2009). Combined effect
of MAP and active compounds on fresh blue fish burger. International Journal of Food
Microbiology, 135: 281–287, DOI:
10.1016/j.ijfoodmicro.2009.07.024.
Delles, R.M., Xiong, Y.L., True, A.D. (2011). Mild protein
oxidation enhanced hydration and myofibril
swelling capacity of fresh ground pork muscle packaged in high oxygen oatmosphere. Journal of
Food Science, 76: C760-C767.
Delles, R.M., Xiong, Y.L., True, A.D., Ao, T., Dawson, K.A. (2014). Dietary antioxidant supplementation
enhances lipid and protein oxidative stability of
chicken broiler meat through promotion of antioxidant enzyme activity. Poultry Science,
93(6): 1561-1570. DOI: 10.3382/ps.2013-03682.
Decker, E. A., Faustman, C., López Bote, C. J. (2000).
Antioxidants in muscle foods: nutritional
strategies to improve quality. John Wiley & Sons. Inc. New York: 499.
Díaz, M. T., Cañeque, V., Sánchez, C. I., Lauzurica, S., Pérez,
C., Fernández, C., Álvarez, I., De la Fuente, J. (2011). Nutritional and sensory aspects of light
lamb meat enriched in n-3 fatty acids during
refrigerated storage. Food Chemistry, 124: 147–155.
Dikeman, M., Devine, M. (2014). Encyclopedia of Meat
Sciences, 1 (2nd ed.). Academic Press (Cambridge, MA, USA).
Dobrinas, S.; Soceanu, A.; Popescu, V.; Stanciu, G. (2013).
Nitrate determination in spices. Ovidius Univ. Ann. Chem, 24: 21–23.
Dvorska, J.E., Pappas, A.C., Karadas, F., Speake, B.K., Surai,
P.F. (2007). Protective effect of modified glucomannans and organic selenium against
antioxidant depletion in the chicken liver due to T-
2 toxin- contaminated feed consumption. Comparative Biochemistry and Physiology Part
C: Toxicology & Pharmacology, 145(4): 582-587.
Dwidevi, B.K.; Snell, F.D. (1975). Meat flavor. Critical Review of Food Research, 5(4): 487–535.
Elfalleh, W., Nasri, N., MArzougui, N., Thabti, I., M’Rabet,
A., Yahya, Y., Lachiheb, B., Guasmi, F., Ferchichi, A. (2009). Physico-chemical properties
and DPPH-ABTS scavenging activity of some
local pomegranate (Punica granatum) ecotypes. International Journal of Food Science and
Nutrition, 60: 197-210.
Elgaard, L., Sevier, C.S., Bulleid, N.J. (2017). How are proteins reduced in the endoplasmic reticulum?
Trends in Biochemical Sciences, 1402: 1-12. DOI:
10.1016/j.tibs.2017.10.006.
Ellis, R.P., Vorhies, M.V. (1976) Effect of supplemental
dietary vitamin e on the serologic response of
swine to an echerichia coli bacterin. Journal American of Veterinary Medical Association, 168:
231-232.
Elmore, J.S., Mottram, D.S., Enser, M., Wood, J.D. (1999). Effect of the polyunsaturated fatty acid
composition of beef muscle on the profile of
aroma volatiles. Journal of Agriculture and Food Chemistry, 47: 1619–1625.
Enser, M. (2001). In: Rossell, B. (Ed.), Animal Carcass Fats.
Oils and Fats, Leatherhead Publishing, Leatherhead, Surrey, UK: 77–122.
Erkan, N., Ayranci, G., Ayranci, E. (2008). Antioxidant
activities of rosemary (Rosmarinus Officinalis L.) extract, blackseed (Nigella sativa L.) essential oil,
carnosic acid, rosmarinic acid and sesamol. Food
Chemistry, 110: 76–82.
Erkmen, O., Bozoglu, F. (2016). Spoilage of meat and meat
products. In: Food microbiology: principles into
practice, 2: 285. Chichester (U.K.): John Wiley & Sons.
Espina, L., Somolinos, M., Loran, S., Conchello, P., García,
D., Pagan, R. (2011). Chemical compositions of commercial Citrus fruit essential oils and
evaluation of their antimicrobial activity acting alone or in combined processes. Food Control,
22(6): 896-902.
Estévez, M. (2011). Protein carbonyls in meat systems: A review. Meat Science; 89: 259–279.
Estévez, M., Cava, R. (2005). Effectiveness of rosemary
essential oil as an inhibitor of lipid and protein oxidation: Contradictory effects in different types
of frankfurters. Meat Science, 72: 348-355.
Lorena Martínez Zamora PhD Thesis, 2019
149
European Parliament and of the Council. (2004). Regulation
(EC) No 852/2004 on the Hygiene of Foodstuffs; L 139; Official Journal of the European Union:
Brussels, Belgium; p. 1.
Estevez, M., Ventana, S., Cava, R. (2005). Protein oxidation in frankfurters with different levels of added
Rosemary essential oil: Effect on colour and
texture deterioration. Journal of Food Science, 70: 427–432.
Estevez, M., Ventanas, S., Cava, R. (2007). Oxidation of
lipids and proteins in frankfurters with different fatty acid composition and tocopherols and
phenolics contents. Food Chemistry, 100: 55–63.
European Parliament and of the Council. (2004). Regulation (EC) No 853/2004 Laying Down Specific
Hygiene Rules for on the Hygiene of Foodstuffs;
L 139; Official Journal of the European Union:
Brussels, Belgium; p. 55.
European Parliament and of the Council. (2004). Regulation
(EC) No 854/2004 Laying Down Specific Rules for the Organisation of Official Controls on
Products of Animal Origin intended for Human
Consumption; L 226; Official Journal of the European Union: Brussels, Belgium; p. 88.
Fang, X., Wada, S. (1993). Enhancing the antioxidant effect
of α-tocopherol with rosemary in inhibiting catalyzed oxidation caused by Fe2+ and
hemoprotein. Food Research International; 26:
405–411.
Feng, J., Xiong, Y.L., Mikelr, W.B. (2003). Textural
properties of pork frankfurters containing
thermally/enzymatically modified soy protein. Journal of Food Science, 68: 1220–1224.
Fernandes, R.P.P., Trindade, M.A., Lorenzo, J.M., de Melo,
M.P. (2018). Assessment of the stability of sheep sausages with the addition of different
concentrations of Origanum vulgare extract
during storage. Meat Science, 137: 244-257. DOI: 10.1016/j.meatsci.2017.011.018
Fernández-López, J., Pérez-Alvarez, J.A., Sayas-Barberá, E.,
López-Santoveña, F. (2002). Effect of paprika (Capsicum annum) on color of Spanish-type
sausages during the resting stage. Journal of Food
Science, 67(6): 2410–2414.
Fernández-López, J., Zhi, N., Aleson-Carbonell, L., Pérez-
Álvarez, J.A., Kuri, V. (2005). Antioxidant and
antibacterial activities of natural extracts: application in beef meatballs. Meat Science, 69:
371–380.
Festing, M.F.W.; Altman, D.G. (2002). Guidelines for the design and statistical analysis of experiments
using laboratory animals. ILAR J, 43: 244–258.
Folch, J., Lees, M., Sloane Stanley, G. H. (1957). A simple
method for the isolation and purification of total
lipids from animal tissues. Journal of Biology and Chemistry, 226(1): 497-509.
Fonseca, S., Cachaldora, A., Gómez, M., Franco, I., Carballo,
J. (2013). Effect of different autochthonous starter
cultures on the volatile compounds profile and
sensory properties of Galician chorizo, a
traditional Spanish dry fermented sausage. Food Control, 33: 6-14.
Fordyce, A.M., Crow, V.L., Thomas, T.D. (1984).
Regulation of product formation during glucose or lactose limitation in non-growing cells of
Streptococcus lactis. Application of
Environmental Microbiology. 48(2): 332-337.
Forss, D.A. (1973). Odor and flavor compounds from lipids.
Programme Chemistry of Fats and Other Lipids,
13: 177–258.
Fortin, A., Robertson, W.M. y Tong, A.K.W. (2005). The
eating quality of Canadian pork and its
relationship with intramuscular fat. Meat Science, 69: 297-305.
Franco-Vega, A., Reyes-Jurado, F., Cardoso-Ugarte, G.A.,
Sosa-Morales, M.E., Palou, E., López-Malo, A. (2016). Chapter 89 – Sweet Orange (Citrus
sinensis) oils. Essential Oils in Food Preservation,
Flavor and Safety: 783-790.
Frankel, E.N. (1991). Recent advances in lipid oxidation.
Journal of Food Agriculture, 54: 495–511.
Frontela, C., Peso-Echarri, P., González, C.A., López, R., Martínez, C., Ros, G. (2011). A critical
perspective on cells lines studies in nutrition: The
case of intestinal absorption. In Caco-2 and Their Uses. Nova Science Publishers: Hauppauge, NY,
USA.
Frontela, C., Ros, G., Martínez, C. (2009). Iron and calcium availability from digestion of infant cereals by
Caco-2 cells. Europe Food Researcher and
Technology, 228(5): 789-797.
Fuentes, E., Paucar, F., Tapia, F., Ortiz, J., Jimenez, P.,
Romero, N. (2018). Effect of the composition of
extra virgin olive oils on the differentiation and antioxidant capacities of twelve monovarietals.
Food Chemistry, 243: 285–294.
Fuentes-Zaragoza, E., Pérez-Álvarez, J.A., Sánchez-Zapata, E. (2009). Efecto de la concentración de aditivos
e ingredientes sobre el color de pastas de merluza
(Merluccius australis) tratadas térmicamente. Óptica Pura y Aplicada; 42(1): 9-21.
Gao, M., Feng, L., Jiang, T., Zhu, J., Fu, L., Yuan, D., Li, J.
(2014). The use of rosemary extract in combination with nisin to extend the shelf life of
pompano (Trachinotus ovatus) fillet during
chilled storage. Food Control; 37: 1–8.
Ganhão, R., Morcuende, D., Estévez, M. (2010). Protein
oxidation in emulsified cooked burger patties with
added fruit extracts: Influence on colour and texture deterioration during chill storage. Meat
Science, 85: 402–409.
Gardner, P.T.; White, T.A.; McPhail, D.B.; Duthie, G.G. (2000). The relative contribution of vitamin C,
carotenoids and phenolics to the antioxidant potential of fruit juices. Food Chemistry, 68: 471–
474.
Gaston, B. (1999). Nitric oxide and thiol groups. Biochimica et biophysica acta, 1411 (2-3): 323-333.
Ghaly, A.E., Dave, D., Budge, S., Brooks, M.S. (2010). Fish
Spoilage Mechanisms and Preservation Techniques: Review. American Journal of
Applied Sciences; 7 (7): 859-877.
Lorena Martínez Zamora PhD Thesis, 2019
150
Gordon, M.H. (1990). The mechanism of antioxidant action
in vitro. In Food Antioxidants; Hudson, B.J.F., Ed.; Elsevier Science Publishing: New York, NY,
USA: 1–18
Gordon, M.H., Paiva-Martins, F., Almeida, M. (2001). Antioxidant activity of hydroxytyrosol acetate
compared with that of other olive oil polyphenols.
Journal of Agriculture and Food Chemistry, 49: 2480–2485.
Gorinstein, S., Drzewiecki, J., Leontowicz, H., Leontowicz,
M., Najman, K., Jastrzebski, Z., Zachwieja, Z., Barton, H., Shtabsky, B., Katrich, E.,
Trakhtenberg, S. (2005). Comparison of the
bioactive compounds and antioxidant potentials of fresh and cooked Polish, Ukranian and Israeli
garlic. Journal of Agriculture and Food
Chemistry, 53(7): 2726-2732. DOI: 10.1021/jf0404593
Gougoulias, N.; Wogiatzi, E.; Vagelas, I.; Giurgiulescu, L.;
Gogou, I.; Ntalla, M.N.; Kalfountzos, D. (2017). Comparative study on polyphenols content,
capsaicin and antioxidant activity of different hot
peppers varieties (Capsicum annum, L.) under environmental conditions of Thessaly region,
Greece. Carpathian Journal of Food Science and
Technology, 9: 109–116.
Gravador, R.S., Jongberg, S., Andersen, M.L., Luciano, G.,
Priolo, A., Lund, M.N. (2014). Dietary citrus pulp
improves protein stability in lamb meat stored under aerobic conditions. Meat Science, 97: 231-
236. DOI: 10.1016/j.meatsci.2014.01.016
Gray, J.L., Pearson, A.M. (1987). Rancidity and warmed-
over flavor. In: Pearson AM & Dutson TR (Eds.)
Advances in meat research. New York: Van
Nostrand: 221-269.
Guyon, C., Meynier, A., De Lamballerie, M. (2016). Protein
and lipid oxidation in meat: A review with
emphasis on high-pressure treatments. Trens in Food science & Technology, 50: 131-143.
Ha, J.K.; Lindsay, R.C. (1990). Method for the quantitative
analysis of volatile free and total branched-chain fatty acids in cheese and milk fat. Journal of Dairy
Science, 73: 1988–1999.
Haga, S., Ohashi, T. (1984). Heat-induced gelation of a mixture of myosin B and soybean protein.
Agriculture, Biology, and Chemistry, 48: 1001–
1007.
Hagiwara, K., Goto, T., Araki, M., Miyazaki, H., Hagiwara,
H. (2011). Olive polyphenol hydroxytyrosol
prevents bone loss. European Journal of Pharmacology, 662: 78–84.
Hall, G.M. (2011). Freezing and chilling of fish and fish products. In: Hall, G.M. (Ed). Fish processing-
sustainability and new opportunities. Blackwell
Publishing Ltd: 77-97.
Hammes, W.P., Hertel, C. (1998). New developments in meat
starter cultures. Meat Science, 49(1).
Hasan, S.M.; Hall, J.B. (1975). The physiological function of
nitrate reduction in Clostridium perfringens.
Journal of Genetic and Microbiology, 87: 120–
128.
Haytowitz, D. B., Bhagwat, S. (2010). USDA Database for
the Oxygen Radical Absorbance Capacity (ORAC) of Selected Foods, Release 2. U.S.
Department of Agriculture (USDA), Maryland,
USA.
He, K. (2009). Fish, Long- Chain Omega-3 Polyunsaturated
Fatty Acids and Prevention of Cardiovascular
Disease- Eat Fish or Take Fish Oil Supplement? Progress in Cardiovascular Diseases, 52(2): 95-
114.
Hebard, C.E., Flick, G.J., Martín, R.E. (1982). Occurrence and significance of trimethylamine oxide and its
derivatives in fish and shellfish. In: Martin, R.E.:
Chemistry and biochemistry of marine food products: 149-304. Westport, CT: AVI
publishing.
Herrmann, S.S., Granby, K., Duedahl-Olesen, L. (2015).
Formation and migration of N-nitrosamines in
nitrite preserved cooked sausages. Food
Chemistry, 174: 516-526.
Higgs, J.D. (2000). The changing nature of meat: 20 years of
improving nutritional quality. Trends in Food
Science and Technology, 11(3): 85-95.
Hmid, I., Elothmani, D., Hanine, H., Oukabli, A., Mehinagic,
E. (2017). Comparative study of phenolic
compounds and their antioxidant attributes of eighteen pomegranate (Punica granatum L.)
cultivars grown in Morocco. Arabian Journal of
Chemistry, 10: S2675–S2684.
Hölihan, C.M., Ho, C.T., Chang, S.S. (1984). Elucidation of
the chemical structure of a novel antioxidant,
rosmaridiphenol, isolated from rosemary. Journal of the American Oil Chemists’ Society; 61: 1036–
1039
Hosomi, R., Yoshida, M., Fukunaga, K. (2012). Seafood consumption and components for health. Global
journal of health science, 4(3): 72–86.
doi:10.5539/gjhs.v4n3p72
Hospital, X.F., Hierro, E., Stringer, S., Fernández, M. (2016).
A study on the toxigenesis by Clostridium
botulinum in nitrate and nitrite-reduced dry fermented sausages. International Journal of
Food Microbiology;218: 66–70.
Hwang, K.E., Choi, Y.S., Choi, S.M., Kim, H.W., Choi, J.H.,
Lee, M.A., Kim, C.J. (2013). Antioxidant action
of ganghwayakssuk (Artemisa princeps Pamp.) in
combination with ascorbic acid to increase the shelf life in raw and deep fried chicken nuggets.
Meat Science, 95: 593-602.
Hu, F. B., Manson, J.E., Willett, W.C. (2001). Types of dietary fat and risk of coronary heart disease: A
critical review. Journal of the American College of Nutrition, 20: 5–19.
Igor, J., Gutiérrez, A., Rivera, L.J. (2010). Análisis
microbiológico y sensorial de productos elaborados a partir de surimi de carduma
(Cetengraulis mysticetus) y plumuda
(Opisthonema spp.). Revista BioAgro;8(2).
International commission on microbiological specifications
for foods – ICMSF. (2000). Microorganisms in
Foods I. 2nd Edition.
Lorena Martínez Zamora PhD Thesis, 2019
151
International Life Sciences Institute (ILSI) Europe. (1998).
Functional Food Science in Europe. British Journal of Nutrition, 80(1): S1-S193.
International Standards Organization – ISO (1999). Meat and
meat products – measurements of pH (Reference method), ISO 2917. Geneva, Switzerland.
International Standards Organization – ISO. 2012. Sensory
analysis – general guidance for the selection, training and monitoring of assessors, ISO 8586.
Geneva, Switzerland.
Izquierdo, P., Torres, G., González, E., Barboza-Martínez, Y., Márquez, E., Allara, M. (1999). Composición
de ácidos grasos y contenido de humedad en doce
especies de pescado de importancia comercial en Venezuela. Revista científica FCV-Luz;9(6): 463-
468.
Jackson, A.L., Sullivan, G.A., Kulchaiyawat, C., Sebranek, J.G., Dickson, J.S. (2011). Survival and Growth of
Clostridium perfringens in Commercial No-
Nitrate-or-Nitrite-Added (Natural and Organic) Frankfurters, Hams, and Bacon. Journal of Food
Protein;74(3): 410-416.
Jacobsen, C., Let, M.B., Nielsen, N.S., Meyer, A.S. (2008). Antioxidant strategies for preventing oxidative
flavour deterioration of foods enriched with n-3
polyunsaturated lipids: A comparative evaluation. Trends of Food Science and Technology, 19(2):
76–93.
Jang, C.S., Chen, S.K. (2015). Integrating indicator-based geostatistical estimation and aquifer vulnerability
of nitrate-N for establishing groundwater
protection zones. Journal of Hydrology, 523: 441-451.
Jaskiewicz, T., Sagan, A., Puzio, I. (2014). Effect of the
Camelina sativa oil on the performance, essential fatty acid level in tissues and fat-soluble vitamins
content in the livers of broiler chicken. Livestock
Science, 165: 74-79.
Jessen F, Nielsen J, Larsen E. (2014). Chilling and freezing
of fish. In: Boziaris, I.S. (Ed). Seafood processing:
Seafood Processing: Technology, Quality and Safety: 98-118.
Jiang, Y., Wu, N., Fu, Y.J., Wang, W., Luo, M., Zhao, C.J.,
Zu, Y.G., Liu, X.L. (2011). Chemical composition
and antimicrobial activity of the essential oil of
Rosemary. Environmental Toxicology and
Pharmacology; 32: 63–68.
Jia, N., Kong, B., Liu, Q., Diao, X. Xia, X. (2012).
Antioxidant activity of black currant (Ribes
nigrum L.) extract and its inhibitory effect on lipid and protein oxidation of pork patties during
chilled storage. Meat Science, 91: 533–539.
Jia, N., Wang, L., Shao, J., Liu, D., Kong, B. (2017). Changes
in the structural and gel properties of pork
myofibrillar protein induced by catechin modification. Meat Science, 127: 45-50. DOI:
10.1016/j.meatsci.2017.01.004.
Jiang, J., Xiong, Y.L. (2016). Natural antioxidants as food
and feed additives to promote health benefits and
quality of meat products: A review. Meat Science;
120: 107–117.
Jiménez-Colmenero, F., Carballo, J., Cofrades, S. (2001).
Healthier meat and meat products: their role as functional foods. Meat Science, 59(5): 5-13.
Jiménez-Colmenero, F., Sánchez Muniz, F. J., Olmedilla
Alonso, B., Ayo, J., Carballo, J., Cofrades, S., Ruiz-Capillas, C., Serrano, A. (2010). Design and
development of meat-based functional foods with
walnut: Technological, nutritional and health impact. Food Chemistry, 123: 959-967.
Jiménez-Colmenero, F., Serrano, A., Ayo, J., Solas, M.T.,
Cofrades, S., Carballo, J. (2003). Physicochemical and sensory characteristics of restructured beef
steak with added walnuts. Meat Science, 65:
1391–7.
Johansson, G.; Berdagué, J.L.; Larsson, M.; Tran, N.; Borch,
E. (1994). Lipolysis, proteolysis and formation of
volatile components during ripening of a
fermented sausage with Pediococcus pentosaceus
and Staphylococcus xylosus as starter cultures.
Meat Science, 38(2): 203–218.
Jongberg, S. (2012). Antioxidative protection of protein in
meat and meat systems. PhD Thesis. Copenhagen,
Denmark. ISBN 978-87-7611-473-2.
Jongberg S., Lund, M.N., Waterhouse, A.L., Skibsted, L.H.
(2011). 4-Methyl catechol inhibits protein
oxidation in meat but not disulfide formation. Journal of Agriculture and Food Chemistry, 59:
10329–10335.
Jongberg, S., Terkelsen, L. de S., Miklos, R., Lund, M.N. (2015). Green tea extract impairs meat emulsion
properties by disturbing protein disulfide cross-
linking. Meat Science, 100: 2-9. DOI: 10.1016/j.meatsci.2014.09.003
Jongberg, S., Tørngren, M.A., Gunvig, A., Skibsted, L.H.,
Lund, M.N. (2013). Effect of green tea or rosemary extract on protein oxidation in Bologna
type sausages prepared from oxidatively stressed
pork. Meat Science, 93: 538–546.
Jongberg, S., Torngren, M.A., Skibsted, L.H. (2018). Brine-
injected porl loins added ascorbate or extracts of
green tea or maté during chill-storage in high-oxygen modified atmosphere. Medicines, 5(7).
DOI: 10.3390/medicines5010007.
Kale, R., Sawate, A.R., Kshirsagar, R.B., Patil, B.M., Mane,
R.P. (2018). Studies on evaluation of physical and
chemical composition of beetroot (Beta vulgaris
L.). International Journal of Chemical Studies, 6(2): 2977-2979. P-ISSN: 2349–8528; E-ISSN:
2321–4902.
Kanner, J. (2007). Dietary advanced lipid oxidation end products are risk factors to human health.
Molecular Nutrition & Food Research, 51(9): 1094-1101.
Kanner, J., Lapidot T. (2001). The stomach as a bioreactor:
dietary lipid peroxidation in the gastric fluid and the effects of plan-derived antioxidants. Free
Radical Biology and Medicine, 31(11): 1388-
1395.
Kakhki, R.A.M., Bakhshalinejad, R., Shafiee, M. (2016).
Effect of dietary zinc and α-tocopherol acetate on
broiler performance, immune responses, antioxidant enzyme activities, minerals and
Lorena Martínez Zamora PhD Thesis, 2019
152
vitamin concentration in blood and tissues of
broilers. Animal Feed Science and Technology, 221(A): 12-26.
Kandler, O. (1983). Carbohydrate metabolism in lactic acid
bacteria. Journal of Microbiology, 49: 209–224.
Kerler, J., Grosch, W. (1997). Character impact odorants of
boiled chicken: Changes during refrigerated
storage and reheating. European Food Research Technology, 205: 232–238.
Kharchoufi, S., Licciardello, F., Siracusa, L., Muratore, G.,
Hamdi, M., Restuccia, C. (2018). Antimicrobial and antioxidant features of ‘Gabsi’ pomegranate
peel extracts. Industrial Crops Production, 111:
345–352.
Khwairakpam, A.D., Bordoloi, D., Thakur, K.K., Monisha,
J., Arfuso, F., Sethi, G., Mishra, S., Kumar, A.P.,
and Kunnumakkara, A.B. (2018). Possible use of Punica granatum (Pomegranate) in cancer
therapy. Pharmacologic Research; 133: 53-64.
Kim, J.S., Ahn, J., Lee, S.J., Moon, B., Ha, T.Y., Kim, S. (2011). Phytochemicals and antioxidant activity
of fruit and leaves of paprika (Capsicum Annuum
L., var. special) cultived in Korea. Journal of Food Science, 76: C193–C198.
Kim, J.S., An, C.G., Park, J.S., Lim, Y.P., Kim, S. (2016).
Carotenoid profiling from 27 types of paprika (Capsicum annuum L.) with different colours,
shapes and cultivation methods. Food Chemistry,
201: 64–71.
Kim, I.S., Jin, S.K., Kang, S.N., Hur, I.C., Choi, S.Y. (2009).
Effect of olive-oil prepared tomato powder
(OPTP) and refining lycopene on the physicochemical and sensory characteristics of
seasoned raw pork during storage. Korean Journal
of Food Science and Animal Resources, 29(3): 329–334.
Kontogianni, V.G., Tomic, G., Nikolic, I., Nerantzaki, A.,
Sayyad, A., Stosic-Grujicic, N., Stojanovic, S., Gerothanassis, I.P., Tzakos, A.G. (2013).
Phytochemical profile of Rosmarinus officinalis
and Salvia officinalis extracts and correlation to their antioxidant and anti-proliferative activity.
Food Chemistry; 136: 120–129.
Kouka, P., Priftis, A., Stagos, D., Angelis, A., Stathopoulos,
P., Xinos. N., Skaltsounis, A.L., Mamoulakis, C.,
Tsatsakis, A.M., Spandidos, D.A., Kouretas, D.
(2017). Assessment of the antioxidant activity of an olive oil total polyphenolic fraction and
hydroxytyrosol from a Greek Olea europea variety
in endothelial cells and myoblasts. International Journal Molecular Medicine, 70: 703–712.
Krauss, R.M., Eckel, R.H., Howard, B., Appel, L.J., Daniels, S.R., Deckelbaum, R.J., Erdman, J.W., Kris-
Etherton,P., Goldberg, I.J., Kotchen, T.A.,
Lichtenstein, A.H., Mitch, W.E., Mullis, R., Robinson, K., Wylie-Rosett, J., St Jeor, S., Suttie,
J., Tribble, D.L., Bazarre, T.L. (2000). AHA
scientific statement. AHA dietary guidelines. A statement for healthcare professionals from the
nutrition committee of the American heart
association. Revision. Circulation, 102: 2284–2299.
Kroll, N.G., Rawel, H.M., Rohn, S. (2003). Reactions of plant
phenolics with food proteins and enzymes under special consideration of covalents bonds. Food
Science and Technology Research, 9: 205-218.
Laincer, A., Laribi, R., Tamendjari, A., Arrar, L., Rovellini, P., Venturini, S. (2014). Olive oils from Algeria:
Phenolic compounds, antioxidant and
antibacterial activities. Grasas y Aceites, 65(1).
Lalpanmawia, H., Elangovan, A.V., Sridhar, M., Shet, D.,
Ajith, S., Pal, D.T. (2014). Efficacy of phytase on
growth performance, nutrient utilization and bone mineralization in broiler chicken. Animal Feed
Science and Techonology, 192: 81-89.
Larsen, C.S. (2003). Animal source foods and human health during evolution. The Journal of Nutrition,
133(11): 3893S-3897S.
Lee, B.J., Hendricks, D.G., Conrnforht, D.P. (1998). Effect of sodium phytate, sodium pyrophosphate on
physico-chemical characteristics of restructured
beef. Meat Science, 50: 273-283.
Lee Richards, K. (2014). The Most Powerful Natural
Antioxidant Discovered to Date—
Hydroxytyrosol. Pro-Health, Available online: http://www.prohealth.com/library/print.cfm?libid
=17054 (accessed on 12 January 2018).
Lemonakis, N., Poudyal, H., Halabalaki, M., Brown, L., Tsarbopoulos, A., Skaltsounis, A.L., Gikas, E.
(2017). The LC-MS-based metabolomics of
hydroxytyrosol administration in rats reveals amelioration of the metabolic syndrome. Journal
of Chromatography; 1041: 45–59.
León, H., Shibata, M.C., Sivakumaran, S., Dorgan, M., Chatterley, T., Tsuyuki, R. T. (2008). Effect of
fish oil on arrhytmias an mortality: systematic
review. British Medical Journal, 337.
Li, J., Xie, S., Ahmed, S., Wang, F., Gu, Y., Zhang, C., Chai,
X., Wu, Y., Cai, J., Cheng, G. (2017).
Antimicrobial activity and resistance: Influence factors. Frontiers in Pharmacology, 8(364): 1-11.
Librelotto, J., Bastida, S., Serrano, A., Cofrades, S., Jiménez-
Colmenero, F., Sánchez-Muniz, F.J. (2008). Changes in fatty acids and polar material of
restructured low- fat or walnut-added steaks pan-
friedinolive oil. Meat Science, 80: 431–441.
Lima, E.A. Melo, M.I.S. Maciel, F.G. Prazeres, R.S. Musser,
D.E.S. 2005. Total phenolic and carotenoid
contents in acerola genotypes harvested at three ripening stages, Food Chemistry, 90, 565–568.
DOI: 10.1016/j.foodchem.2004.04.014
Löliger, J. (1991). The use of antioxidants in foods. In Free Radicals and Food Additives; Aruoma, O.I.,
Halliwell, B., Eds.; Taylor & Francis: London, UK: 121–150
López-Caballero, M.E., Gómez-Guillén, M.C., Pérez-
Mateos, M., Montero, P. (2005). A chitosan-gelatin blend as a coating for fish patties. Food
Hydrocolloids, 19(2): 303-311.
López López, I., Cofrades, S., Cañeque, V., Díaz, M.T., López, O., Jiménez-Colmenero, F. (2011). Effect
of cooking on the chemical composition of low-
salt, low-fat wakame/olive oil added beef patties
Lorena Martínez Zamora PhD Thesis, 2019
153
with special reference to fatty acid content. Meat
Science, 89: 27–34.
López López, I., Cofrades, S., Jiménez Colmenero, F. (2009).
Low fat frankfurters enriched with n-3 PUFA and
edible seaweed: Effects of olive oil and chilled storage on physicochemical, sensory and
microbial characteristics. Meat Sciencie, 83: 148-
154.
López Uriarte, P., Nogués, R., Sáez, G., Bulló, M., Romeu,
M., Masana, L., Tormos, C., Casas Agustench, P.
y Salas-Salvadó, J. (2010). Effect of nut consumption on oxidative stress and the
endothelial function in metabolic syndrome.
Clinical Nutrition, 29: 373-380.
Lorenzo, J.M., Bedia, M., Bañón, S. (2013). Relationship
between flavour deterioration and the volatile
compound profile of semi-ripened sausage. Meat
Science, 93: 614-620.
Loru, D., Incani, A., Deiana, M., Corona, G., Atzeri, A.,
Melis, M. P., Rosa, A. y Dessi, M. A. (2009). Protective effect of hydroxytyrosol and tyrosol
against oxidative stress in kidney cells. Toxicology
and Health, 25: 301-310.
Lund, M.N., Lametsch, R., Hviid, M.S., Jensen, O.N.,
Skibsted, L.H. (2007). High-oxygen packaging
atmosphere influences protein oxidation and tenderness of porcine longissimus dorsi during
chill storage. Meat Science, 77: 295-303.
Lund, M.N, Ray, C.A. (2017). Control of Maillard reactions in foods: strategies and chemical mechanisms.
Journal of Agricultural Food and Chemistry,
65(23); 4537-4552. DOI: 10.1021/acs.jafc.7b00882.
Lunn, J., Theobald, H. E. (2006). The health effects of dietary
unsaturated fatty acids. Nutrition Bulletin, 31(3): 178-224.
Machowetz, A., Pulsen, H. E., Gruendel, S., Weimann, A.,
Fito, M., Marrugat, J., De la Torre, R., Salonen, J. T., Nyyssonen, K., Mursu, J. y colaboradores.
(2007). Effect of olive oils on biomarkers of
oxidative DNA stress in Northern and Southern Europeans. The FASEB Journal, 21(1): 45-52.
Maga, J.A. (1987). The flavor chemistry of wood smoke.
Food Review International, 3: 139–183.
Maqsood, S., Benjakul, S. (2010). Synergistic effect of tannic
acid and modified atmospheric packaging on the
prevention of lipid oxidation and quality losses of refrigerated striped catfish slices. Food Chemistry,
121(1), 29–38.
Martí, L., Fuentes, A., Fernández-Segovia, I. (2015). Evaluación de la vida útil de hamburguesas
elaboradas a base de pescado y algas. Universidad Politécnica de Valencia, Comunidad Valenciana,
Spain.
Martín Cerdeño, V.J. (2017). Consumo de pescados y mariscos en España. Un análisis de los perfiles de
la demanda. Distribución y Consumo; 4.
Martín-Juárez, B. (2005). Estudio de las comunidades microbianas de embutidos fermentados
ligeramente acidificados mediante técnicas
moleculares. Estandarización, seguridad y mejora
tecnológica. Available from:
http://www.tdx.cat/handle/10803/7790.
Martínez, L., Bastida, P., Castillo, J., Ros, G., Nieto, G.
(2019). Green alternatives to synthetic
antioxidants, antimicrobials, nitrates, and nitrites in clean label Spanish “chorizo”. Antioxidants,
8(6): 184. DOI: 10.3390/antiox8060184
Martínez, L., Ros, G., Nieto, G. (2018). Fe, Zn and Se bioavailability in chicken meat emulsions
enriched with minerals, Hydroxytyrosol and Extra
Virgin Olive Oil as measured by Caco-2 cell model. Nutrients. 10(8). DOI:
10.3390/nu10080969.
Martins, N., Petropoulos, S., Ferreira, I.C.F.R. (2016). Chemical composition and bioactive compounds
of garlic (Allium sativum L.) as affected by pre-
and post- harvest conditions: A review. Food
Chemistry, 211(15): 41-50. DOI:
10.1016/j.foodchem.2016.05.029.
Mathew, S., Abraham, T.E., Zakaria, Z.A. (2015). Reactivity of phenolic compounds towards free radicals
under in vitro conditions. Journal of food science
and technology, 52(9): 5790-5798. DOI: 10.1007/s13197-014-1704-0.
McWangi, N., Chen, W., Vermaak, I., Viljoen, A.M., and
Gericke, N. (2012). Devil´s claw-A review of the ethnobotany, phytochemistry and biological
activity of Harpagophytum procumbens. Journal
of Ethnopharmacology; 143(3): 755-771.
Merra, E., Calzaretti, G., Bobba, A., Storelli, M.M., Casalino,
E. (2014). Antioxidant role of hydroxytyrosol on
oxidative stress in cadmium-intoxicated rats: Different effect in spleen and testes. Drug and
Chemical Toxicology, 37(4): 420-426.
Meynier, A., Novelli, E., Chizzolini, R., Zanardi, E., Gandemer, G. (1999). Volatile compounds of
commercial Milano salami. Meat Science, 51:
175–183.
Ministerio de Agricultura, Pesca y Alimentación (MAPA).
(2019). Infome de consumo alimentario en España
2018. Ministerio de Agricultura, Pesca y Alimentación, Madrid, Spain.
Molnár, H., Kónya, É., Zalán, Z., Bata-Vidács, I.,
Tömösközi-Farkas, R., Szèkács, A., Adányi, N.
(2018). Chemical characteristics of spice paprika
of different origins. Food Control, 83: 54-60.
Mondal, M.K., Das, T.K., Biswas, P., Samanta, C.C., Bairagi, B. (2007). Influence of dietary inorganic and
organic copper salt and level of soybean oil on
plasma lipids, metabolites and mineral balance of broiler chickens. Animal Feed Science and
Techonology, 139: 212-233.
Moreda-Piñero, J., Moreda-Piñero, A., Romarís-Hortas, V.,
Domínguez-González, R., Alonso-Rodríguez, E.,
López-Mahía, P., Muniategui-Lorenzo, S., Prada-Rodríguez, D., Bermejo-Barrera, P. (2013). In
vitro bioavailability of total selenium and
selenium species from seafood. Food Chemistry, 139: 872–877.
Moreiras, O., Carbajal, A., Cabrera, L., Cuadrado, C. (2013).
Tablas nacionales de composición de alimentos. Ed. Pirámide, (16º ed).
Lorena Martínez Zamora PhD Thesis, 2019
154
Mosmann, T. (1983). Rapid colorimetric assay for cellular
growth and survival: Application to proliferation and citotoxicity assays. Journal Inmunology
Methods, 65: 55-63.
Moura, C.F.H., Oliveira, L. de S., De Souza, K.O., Da Franca, L.G., Ribeiro, L.B., De Souza, P.A., De Miranda,
M.R.A. (2018). Acerola – Malpiguia emarginata.
Exotic Fruits, Reference Guide: 7-14.
Muguerza, E., Fista, G., Ansorena, D., Astiasaran, I.,
Bloukas, J.G. (2002). Effect of fat level and partial
replacement of pork backfat with olive oil on processing and quality characteristics of
fermented sausages. Meat Science, 61(4): 397–
404.
Mulaudzi, R.B., Ndhlala, A.R., Kulkarni, M.G., Staden, J.V.
(2012). Pharmacological properties and protein
binding capacity of phenolic extracts of some
Venda medicinal plants used against cough and
fever. Journal of Ethnopharmacology, 143: 185-
193.
Müller, L., Gnoyke, S., Popken, A.M., Böhm, V. (2010).
Antioxidant capacity and related parameters of
different fruit formulations. LWT – Food Science and Technology, 43, 992–999. DOI:
10.1016/j.lwt.2010.02.004
Nagi, P., Lemma, K., Ashby, M.T. (2007). Reactive sulfur species: kinetics and mechanisms of the reaction
of cysteine thiolsulfinate ester with cysteine to
give cysteine sulfenic acid. Journal of Organic Chemistry, 72: 8838-8846. DOI:
10.1021/jo701813f
Nemec, M., Butler, G., Hidiroglou, M., Farnworth, E.R., Nielsen, K. (1994). Effect of supplementing gilts’
diets with different levels of vitamin e and
differents fats on the humoral and cellular immunity of gilts and their progeny. Journal of
animal science, 72(3): 665-676.
Nieto, G. (2013). Incorporation of by-products of rosemary and thyme in the diet of ewes: Effect on the fatty
acid profile of lamb. European Food Research
and Technology, 236: 379–389.
Nieto, G., Castillo, M., Xiong, Y.L., Álvarez, D., Payne, F.,
Garrido, M.D. (2009). Antioxidant and
emulsifying properties of alacalase-hydrolyzed potato protein in meat emulsions with different fat
concentrations. Meat Science, 90: 24–30.
Nieto, G., Díaz, P., Bañón, S., Garrido, M.D. (2010). Dietary administration of ewe diets with a distillate from
rosemary leaves (Rosmarinus officinalis L.):
Influence on lamb meat quality. Meat Science, 84: 23–29.
Nieto, G., Estrada, M., Jordán, M.J., Garrido, M.D., Bañón, S. (2011). Effects in ewe diet of rosemary by-
product on lipid oxidation and the eating quality
of cooked lamb under retail display conditions. Food Chemistry, 124: 1423–1429.
Nieto, G., Jongberg, S., Andersen, M.L., Skibsted, L.H.
(2013) Thiol oxidation and protein cross-link formation during chill storage of pork patties
added essential oil of oregano, rosemary, or garlic.
Meat Science; 95: 177–184.
Nieto, G., Martínez, L., Castillo, J., Ros, G. (2017b). Effect
of hydroxytyrosol, walnut and olive oil on nutritional profile of Low-Fat Chicken
Frankfurters. European Journal of Lipid Science
and Technology, 119: 1600518.
Nieto, G., Martínez, L., Ros, G. (2017a). Hydroxytyrosol
extracts, olive oil and walnuts as functional
components in chicken sausages. Journal of Science of Food and Agriculture.
Nieto, G., Skibsted, L.H., Andersen, M.L., Ros, G. (2012).
Antioxidant and prooxidant activity of essential oil of garlic by electron spin resonance. Anales de
Veterinaria de Murcia, 28(39): 23-33. ISSN:
0213-5434
Nieto, G., Xiong, Y.L., Payne, F., and Castillo, M. (2014).
Predicting frankfurters quality metrics using light
backscatter. Journal of Food Engineering, 143:
132–138.
Nkukwana, T.T., Muchenje, V., Masika, P.J., Hoffman, L. C.,
Descalzo, A.M. (2014). Fatty acid composition and oxidative stability of breast meat from broiler
chickens supplemented with Moringa oleifera leaf
meal over a period of refrigeration. Food Chemistry, 142: 255–261.
Noseda, B., Vermeulen, A., Ragaert, P., Devlieghere, F.
(2014). Packaging of fish and fishery products. In: Boziaris, I.S. (Ed). Seafood Processing:
Technology, Quality and Safety: 237-261.
Nowshehri, J.A., Bhat, Z.A., Shah, M.Y. (2015). Blessings in disguise: Bio-functional benefits of grape seed
extracts. Food Research International; 77: 333-
348.
Nowzari, F., Shábanpour, B., Ojagh, S.M. (2013).
Comparison of chitosan-gelatin composite and
bilayer coating and film effect on the quality of refrigerated rainbow trout. Food Chemistry,
141(3): 1667-1672.
Obied, H.K., Allen, M.S., Bedgood, D.R., Prenzler, P.D., Robards, K., Stockmann, R. (2005). Bioactivity
and analysis of biophenols recovered from olive
mill waste. Journal of Agricultural and Food Chemistry 53: 823–837.
Okada, Y., Tanaka, K., Fujita, I., Sato, E., Okajima, H.
(2005). Antioxidant activity of thiosulfinates
derived from garlic. Redox Rep, 10(2): 96-102.
DOI: 10.1179/135100005X38851
Olmedo, R.H., Nepote, V., Grosso, N.R. (2013). Preservation of sensory and chemical properties in flavoured
cheese prepared with cream cheese base using
oregano and rosemary essential oils. LWT-Food Science and Technology; 53: 409–417
Ordóñez, J.A., Hierro, E.M., Bruna, J.M., de la Hoz, L. (1999). Changes in the components of dry-
fermented sausages during ripening. Critical
Reviews in Food Science and Nutrition; 39: 329-367.
Ordoñez, J.A., Hoz, L. (2001). Embutidos crudo curados.
Tipos. Fenómenos madurativos. Alteraciones. En:
Enciclopedia de la carne y de los productos
cárnicos, 51(2). Ediciones Martín y Macias,
Plasencia (Cáceres, Spain): 1063-1091.
Lorena Martínez Zamora PhD Thesis, 2019
155
Organización de las Naciones Unidas para la Agricultura y la
Alimentación (FAO). (1998). El pescado fresco: su calidad y cambios de su calidad. Roma: FAO;
348.
Organización de las Naciones Unidas para la Agricultura y la Alimentación (FAO). (2016). El estado mundial
de la pesca y la acuicultura. Contribución a la
seguridad alimentaria y la nutrición para todos. Roma: FAO; 224.
Ou, B., Huang, D., Hampsch-Woodill, M., Flanagan, J.A.,
Deemer, E.K. (2002). Analysis of antioxidant activities of common vegetables employing
oxygen radical absorbance capacity (ORAC) and
ferric reducing antioxidant power (FRAP) assays: A comparative study. Journal of Agriculture and
Food Chemistry, 50: 3122–3128.
Ozdal, T., Capanoglu, Altay, F. (2013). A review on protein-
phenolic interactions and associated changes.
Food Research International, 51: 964-970. DOI:
10.1016/j.foodres.2013.02.009
Özen, H., Kamber, U., Karaman, M., Gül, S., Atakisi, E.,
Özcan, K., Atakisi, O. (2014). Histopathologic,
biochemical and genotoxic investigations on chronic sodium nitrite toxicity in mice.
Experimental and Toxocologic Pathology, 66(8):
367-375.
Pachón, H., Stoltzfus, R.J., Glahn, R.P. (2008). Chicken
thigh, chicken liver, and iron-fortified wheat flour
increase iron uptake in an in vitro digestion/caco-2 cell model. Nutrition Research, 28: 851–858.
Palou, A., Serra, F., Pico, C. (2003). General aspects on the
assessment of functional foods in the European Union. European Journal of Clinical Nutrition;
57(1): 12-17.
Papadopoulos, G., Boskou, D. (1991). Antioxidant effects of natural phenols onolive oil. Journal of American
Oil Chemistry Society, 68: 669–671.
Papuc, C., Goran, G.V., Predescu, C.N., Nicorescu, V. (2017). Mechanisms of oxidative processes in
meat and toxicity induced by postprandial
degradation products: A reviw. Comprehensive Reviews in Food Science and Food Safety, 16(1):
96-123.
Parmeggiani, B., Pimentel-Moura, A., Grings, M., Bumbel,
A.P., De Moura-Alvorcem, L., Tauana Pletsch, J.,
Gonçalves Fernandes, C., Wyse, A., Wajner, M.,
Leipnitz, G. (2015). In vitro evidence that sulfite impairs glutamatergic neurotransmission and
inhibits glutathione metabolism-related enzumes
in rat cerebral cortex. International Journal of Developmental Neuroscience, 42: 68-75.
Pateiro, M., Bermúdez, R., Lorenzo, J.M., Franco, D. (2015). Effect of addition of natural antioxidants on the
shelf-life of “chorizo”, a Spanish dry-cured
sausage. Antioxidants, 4: 42-67.
Pereira, D., Pinheiro, R.S., Heldt, L.F.S., Mour, C., Bianchin,
M., Almedia, J.F., Reis, A.S., Ribeiro, I.S.,
Haminiuk, C.W.I., Carpes, S.T. (2017). Rosemary as natural antioxidant to prevent oxidation in
chicken burgers. Food Science and Technology
Camp., 37: 17–23.
Pereira, J. A., Oliveira, I., Sousa, A., Ferreira, I.C., Beto, A.,
Estevinho, L. (2008). Bioactive properties and chemical composition of six walnut (Juglans
regia L.) cultivars. Food Chemistry and
Toxicology, 46: 2103–2111.
Pérez-Alvarez, J.A., Sayas-Barberá, M.E., Fernández-López,
J., Aranda-Catalá, V. (1999). Physicochemical
characteristics of Spanish-type dry-cured sausage. Food Research International, 32: 599–607.
Pérez-López, U., Sgherri, C., Miranda-Apodaca, J., Micaelli,
F., Lacuesta, M., Mena-Petite, A., Quartacci, M.F., Muñoz-Rueda, A. (2018). Concentration of
phenolic compounds is increased in lettuce grown
under high light intensity and elevated CO2. Plant Physiology and Biochemistry, 123: 233–241.
Petropoulos, S., Fernandes, A., Barros, L., Ciric, A., Sokovic,
M., Ferrerira, I.C.F.R. (2018). Antimicrobial and
antioxidant properties of various Greek garlic
genotypes. Food Chemistry, 245: 7-12.
Planells, E., Baró, L., Mataix, J., Ochoa, J. (2003). Análisis de la composición mineral en alimentos
congelados precocinados de consumo habitual.
Ars Pharmaceutica;44(4).
Polenski E. (1981). Uber den Verlust, welchen das
Rinkfleisch und Nahrwert durch das Pokein
erleidet, sowie uber die Veranderungen salpeterhaltiger Pokellaken. Arb. K. GesundhAmt,
7: 471.
Presti, G., Guarrasi, V., Gulotta, E., Provenzano, F., Provenzano, A., Giulano, S., Monfreda, M.,
Mangione, M.R., Passantino, R., San Biagio, P.L.,
Costa, M.A., Giacomazza, D. (2017). Bioactive compounds from extra virgin olive oils:
Correlation between phenolic content and
oxidative stress cell protection. Biophysics Chemistry, 230: 109–116.
Prior, R.L., Hoang, H., Gu, L., Wu, X., Bacchiocca, M.,
Howard, L., Hampsch-Woodil, M., Huang, D., Ou, B., Jacob, R. (2003). Assays for hydrophilic
and lipophilic antioxidant capacity (oxygen
radical absorbance capacity (ORACFL)) of plasma and other biological and food samples. Journal of
Agricultural and Food Chemistry, 51: 3273–
3279.
Purriños, L., Franco, D., Carballo, J., Lorenzo, J.M. (2012).
Influence of the salting time on volatile
compounds during the manufacture of dry-cured pork shoulder “lacón”. Meat Science, 92(4): 627-
634.
Pyo, Y.H., Lee, T.C., Logendra, L., Rosen, R.T. (2004). Antioxidant activity and phenolic compounds of
Swiss chard (Beta vulgaris subspecies cycla) extracts. Food Chemistry, 85: 19–26.
Radha, K., Babuskin, S., Ashagu, P., Sasikala, M., Sabina, K.,
Archana, G., Sivarajan, M., Sukumar, M. (2014). Antiomicrobial and antioxidant effects of spice
extracts on the shelf life extension of raw chicken
meat. International Journal of Food Microbiology, 171: 32-40.
Ramarathnam, N., Rubin, L.J., Diosady, L.L. (1993). Studied
on meat flavor. A novel method for trapping volatile components from uncured and cured pork.
Lorena Martínez Zamora PhD Thesis, 2019
156
Journal of Agriculture and Food Chemistry, 41:
933–938.
Ramírez-Anaya, J.d.P., Samaniego-Sánchez, C., Castañeda-
Saucedo, M.C., Villalón-Mir, M., de la Serrana,
H.L.-G. (2015). Phenols and the antioxidant capacity of mediterranean vegetables prepared
with extra virgin olive oil using different domestic
cooking techniques. Food Chemistry, 188: 430–438.
Ranguelova, K., Rice, A.B., Lardinois, O.M., Triquigneauz,
M., Steinckwich, N., Deterding, L.J., Garantziotis, S. y Mason, R.P. (2013). Sulfite-mediated
oxidation of myeloperoxidase to a free radical:
Inmuno-spin trapping detection in human neutrophils. Free Radical Biology and Medicine,
60: 98-106.
Riquelme, N., Matiacevich, S. (2016). Characterization and
evaluation of some properties of oleoresin from
Capsicum annuum var. cacho de cabra. CyTA –
Journal of Food, 15(3): 344-351. DOI: 10.1080/19476337.2016.1256913.
Rubió, L., Macià, A., Castell-Auví, A., Pinent, M., Blay,
M.T., Ardévol, A., Romero, M.P., Motilva, M.J. (2014). Effect of the co-occurring olive oil and
thyme extracts on the phenolic bioaccesibility and
bioavailability assessed by in vitro digestion and cell models. Food Chemistry, 149: 277–284.
Rutherfurd, S., Montoya, C.A., Moughan, P.J. (2014). Effect
of the oxidation of dietary proteins with performic acid on true ileal amino acid digestibility as
determined in the growing rat. Journal of
Agricultural and Food Chemistry, 62(3): 699-
707.
Ruiz, J., Ventanas, J., Cava, R., Andrés, A., García, C. (1999).
Volatile compounds of dry-cured Iberian ham as affected by the length of the curing process. Meat
Science, 52(1): 19-27.
Saani, M., Lawrence, R. (2017). Evaluation of pigments as antioxidant and antibacterial agents from Beta
vulgaris linn. International Journal of Current
Pharmacologic Research, 9: 37–41.
Salejda, A.M., Janiewicz, U., Korzeniowska, M., Kolniak-
Ostek, J., Krasnowska, G. (2016). Effect of walnut
green husk addition on some quality properties of cooked sausages. LWT - Food Science and
Technology, 65: 751-757.
Salgado, P.R., López-Caballero, M.E., Gómez-Guillén, M.C., Mauri, A.N., Montero, M.P. (2013).
Sunflower protein films incorporated with clove
essential oil have potential application for the preservation of fish patties. Food Hydrocolloids,
33(1): 74-84.
Sampels, S. (2015). The effects of processing technologies
and preparation on the final quality of fish
products. Trends in Food Science & Tecnology; 39: 1206-1215.
Sánchez-Moreno, C., Larrauri, J.A., Saura-Calixto, F. (1998).
A procedure to measure the antiradical efficiency of polyphenols. Journal of Science and Food
Agriculture, 76: 270–276.
Sánchez-Zapata, E., Zunino, V., Pérez-Alvarez, J.A., López-Fernández, J. (2013). Effect of tiger nut fibre
addition on the quality and safety of a dry-cured
pork sausage (“Chorizo”) during the dry-curing process. Meat Science, 95: 562-568.
Santiago, P. (2017). Impact of high‐pressure processing on
chemical constituents and nutritional properties in aquatic foods: a review, International Journal of
Food Science & Technology;53(4): 873-891.
Santomauro, F., Sacco, C., Donato, R., Bellumori, M., Innocenti, M., Mulinacci, N. (2017). The
antimicrobial effects of three phenolic extracts
from Rosmarinus officinalis L.; Vitis vinifera L. and Polygonum cuspidatum L. on food pathogens.
Natural Products Research.
DOI:10.1080/14786419.2017.1375920.
Santos, R.D., Shetty, K., Cecchini, A.L., da Silva-
Miglioranza, L.H. (2012). Phenolic compounds
and total antioxidant activity determination
rosemary and oregano extracts and its use in
cheese spread. Ciencias Agrárias Londrina, 33:
655–666.
Schelegueda, L.I., Delcarlo, S.B., Gliemmon, M.F., Campos,
C.A. (2016). Effect of antimicrobial mixtures and
modified atmosphere packaging on the quality of Argentine hake (Merluccius hubbsi) burgers.
LWT-Food Science and Technology, 68: 258–264,
doi:10.1016/j.lwt.2015.12.012.
Secci, G., Parisi, G. (2016). From farm to fork: Lipid
oxidation in fish products. A review. Italian
journal of Animal Science; 15(1): 124-136.
Sehgal, H.S., Shahi, M., Sehgal, G.K., Thind, S.S. (2011).
Nutritional, microbial and organoleptic qualities
of fish patties prepared from carp (Cyprinus carpio Linn.) of three weight groups. Journal of food
science and technology, 48(2): 242–245.
doi:10.1007/s13197-010-0118-x
Serdaroglu, M. (2006). The characteristics of beef patties
containing different levels of fat and oat flour.
International Journal of Food Science and Technology, 41: 147–153.
Serrano, N., Cetó, X., Núñez, O., Arago, M., Gámez, A.,
Ariño, C., Díaz-Cruz, J.M. (2018). Characterization and classification of Spanish
paprika (Capsicum annuum L.) by liquid
chromatography coupled to electrochemical detection with screen-printed carbon-based
nanomaterials electrodes. Talanta, 189: 296-301.
Serrano, A., Cofrades, S., Ruiz-Capillas, C., Olmedilla- Alonso, B., Herrero-Barbudo, C., Jiménez-
Colmenero, F. (2005). Nutritional profile of
restructured beef steak with added walnuts. Meat Science, 70: 647–654.
Serrano, A., Librelotto, J., Cofrades, S., Sanchez-Muniz, F.J., Jimenez-Colmenero, F. (2007). Composition and
physicochemical characteristics of restructured
beef steaks containing walnuts as affected by cooking method. Meat Science 77: 304-313.
Shahidi, F., Yun, J., Rubin, L.J. (1987). The hexanal content
as an indicator of oxidative stability and flavour acceptability in cooked ground meat. Canadian
Institute of Food Science and Technology Journal,
20: 104–106.
Lorena Martínez Zamora PhD Thesis, 2019
157
Sienkiewicz, M., Lysakowska, M., Pastuszka, M., Bienias,
W., Kowalczyk, E. (2013). The potential of use Basil and Rosemary essential oils as effective
antibacterial agents. Molecules; 18: 9334–9351
Sindelar, J.J., Milkowski, A.L. (2012). Human safety controversies surrounding nitrate and nitrite in the
diet. Nitric Oxide, 26(4): 259-266.
Singleton, V.L.; Rossi, J.A.Jr. (1965). Colorimetry of total phenolics with phosphomolybdic-
phosphotungstic acid reagents. American Journal
of Enology and Viticulture, 16: 144–158.
Shahidi, F., Amigaipalan, J. (2015). Phenolics and
polyphenolics in foods, beverages and spices:
Antioxidant activity and health effects-A review. Journal of Functional Foods, 18: 820-897.
Skendi, A., Irakli, M., Chatzopoulou, P. (2017). Analysis of
phenolic compounds in greek plants of Lamiaceae family by HPLC. Journal of applied research on
medicinal and aromatic plants, 6: 62-69.
Skibsted L.H. (1992). Cured meat products and their Oxidative Satbility. In: The Chemistry of muscle-
based Foods. Ed: Ledward D.A., Johnston D.E. y
Knight M.K. The Royal Society of Chemistry: 266-287.
Škrovánková, S., Mlček, J., Orsavová, J., Juriková, T.,
Dřímalová, P. (2017). Polyphenols content and antioxidant activity of paprika and pepper spices.
Slovak Journal of Food Sciences, 11(1): 52-57.
Smaldone, G., Marrone, R., Zottola, T., Vollano, L., Grossi,
G., Cortesi, M.L. (2017). Formulation and shelf-
life of fish burgers served to preschool children.
Italian Journal of Food Safety, 6: 63-73. doi:10.4081/ijfs.2017.6373.
Soubra, L., Sarkis, D., Hilan, C., Verger, P.H. (2007). Dietary
exposure of children and teenagers to benzoates, sulphites, butylhydroxyanisol (BHA) and
butylhidroxytoluen (BHT) in Beirut (Lebanon).
Regulatory Toxicology and Pharmacology, 47: 68-77.
Soyer, A., Özalp, B., Dalmısß, Ü., VolkanBilgin, V. (2010).
Effects of freezing temperature and duration of frozen storage on lipid and protein oxidation in
chicken meat. Food Chemistry, 120: 1025-1030.
Sørensen, A.D., Bukhave, K. (2010). Iron uptake by caco-2 cells following in vitro digestion: Effects of heat
treatments of pork meat and PH of the digests.
Journal of Trace Elements and Medical Biology, 24: 230–235.
Stahnke, L.H. (1994). Aroma compounds from dried
sausages fermented with Staphylococcus xylosus. Meat Science, 38: 39–53.
Sueishi, Y., Sue, M., Masamoto, H. (2018). Seasonal variations of oxygen radical scavenging ability in
rosemary leaf extract. Food Chemistry, 245: 270–
274.
Sullivan, G.A., Sebranek, J.G. (2012). Nitrosylation of
myoglobin and nitrosation of cysteine by nitrite in
a model system simulating meat curing. Journal of Agricultural and Food Chemistry, 60: 1748-1754.
DOI: 10.1021/jf204717v
Surette, M.E., Gill, T.A., Leblanc, P.J. (1988). Biochemical
basis of post morten nucleotide catabolism in cod (Gadus morhua) and its relationship to spoilage.
Journal of Agriculture and Food Chemistry. 36:
19-22.
Tai, J., Cheung, S., Wu, M., Hasman, D. (2012).
Antiproliferation effect of Rosemary (Rosmarinus
officinalis) on human ovarian cancer cells in vitro. Phytomedicine; 19: 436–443.
Tang, C.B., Zhang, W.G., Dai, C., Li, H.X., Xu, X.L., Zhou,
G.H. (2015). Identification and quantification of adducts between oxidized rosmarinic acid and
thiol compounds by UHPLC-LTQ-orbitrap and
MALDI-TOF/TOF tandem mass spectrometry. Journal of Agriculture and Food Chemistry, 63:
902–911.
Tang, C.B., Zhang, W.G., Zou, Y.F., Xing, L.J., Zheng, H.B.,
Xu, X.L., Zhou, G.H. (2017). Influence of RosA-
protein adducts formation on myofibrillar protein
gelation properties under oxidative stress. Food Hydrocolloids, 67: 197-205.
Teruel, M.R, García-Segovia, P., Martínez-Monzó, J.,
Linares, M.B., Garrido, M.D. (2014). Use of vaccum-frying in chicken nugget processing.
Innovation in Food Science and Emerging
Technologies, 26: 482-489.
Teruel, M.R., Garrido, M.D., Espinosa, M.C., Linares, M.B.
(2015). Effect of different format-solvent
rosemary extracts (Rosmarinus officinalis L.) on frozen chicken nuggets quality. Food Chemistry,
172: 40-46.
Tesoriere, L., D’Arpa, D., Butera, D., Pintaudi, A.M., Allegra, M., Livrea, M.A. (2002). Exposure to
malondialdehyde induces an early redox
unbalance preceding membrane toxicity in human erythrocytes. Free Radical Research, 36(1): 89-
97.
The International Agency for Research on Cancer (IARC). (2015). Q&A on the carcinogenicity of the
consumption of red meat and processed meat.
Press Release Nº240. Monographs-Q&A, vol 114.
Thomas, R., Jebin, N., Barman, K., Das, A. (2014). Quality
and shelf life evaluation of pork nuggets
incorporated with fermented bamboo shoot (Bambusa polymorpha) mince. Meat Science, 96:
1210-1218.
Thomas, R., Jebin, N., Saha, R., Sarma, D.K. (2016). Antioxidant and antimicrobial effects of kordoi
(Averrhoa carambola) fruit juice and bamboo
(Bambusa polymorpha) shoot extract in pork nuggets. Food Chemistry, 190: 41-49.
Tikk, K.; Haugen, J.E.; Andersen, H.; Aaslying, M. (2008). Monitoring of warmed-over flavor in pork using
the electronic nose-Correlation to sensory
attributes and secondary lipid oxidation products. Meat Science, 80: 1254–1263.
Torabian, S., Haddad, E., Rajaram, S., Banta, J. y Sabaté, J.
(2009). Acute effect of nut consumption on plasma total polyphenols, antioxidant capacity
and lipid peroxidation. Journal of Human
Nutrition and Dietetics, 22: 64-71.
Lorena Martínez Zamora PhD Thesis, 2019
158
Töth, L.; Potthast, K. (1984). Chemical aspects of the
smoking of meat and meat products. Advances in Food Research, 29: 87–158.
Trichopoulou, A., Dilis, V. (2007). Olive oil and longevity.
Molecular Nutrition and Food Research, 51(10): 1275-1278.
Trichopoulou, A., Martínez-González, M. A., Tong, T. Y.,
Forouhi, N. G., Khandelwal, S., Prabhakaran, D., Mozaffarian, de Lorgeril, M. (2014). Definitions
and potential health benefits of the Mediterranean
diet: views from experts around the world. BMC medicine, 12(1): 112. DOI: 10.1186/1741-7015-
12-112
Tsuchiya, M., Asada, A., Kasahara, E., Sato, E.F., Shindo, M., Inoue, M. (2002). Antioxidant protection of
Propofol and its recycling in erythrocyte
membranas. American Journal of Respiratory and
Critical Care Medicine, 165: 54-60. DOI:
10.1164/ajrccm.165.1.2010134
Uchiyama, H., Ehira, S. (1974). Relation between freshness and acid-soluble nucleotides in aseptic cod and
yellowtail muscles during ice storage. Bulletin of
Tokai Regional Fisheries Research Laboratory, 78: 23-31.
Ullrich, F., Grosch, W. (1988). Identification of the most
intense odor compounds formed during autoxidation of methyl linolenate at room
temperature. Journal of American Oil Chemistry
Society, 65: 1313–1317.
UNE- ISO 5580. (1996). Animal and vegetable fats and oils.
analysis by gas chromatography of methyl esters
of fatty acids. International Organization for Standardization Publications.
Uri, N. (1961). Mechanisms of antioxidation. In W.O.
Lundberg (Ed.). Antioxidation and Antioxidants, New York: John Wiley & Sons, Inc.
Vendramini, A.L.A.; Trugo, L.C. (2004). Phenolic
compounds in acerola fruit (Malpighia punicifolia, L.). Journal of the Brazilian
Chemistry Society, 15: 664–668.
Verma, A.K., Sharma, B.D., Banerkee, R. (2010). Effect of sodium chloride replacement and apple pulp
inclusión on the physico-chemical, textural and
sensory properties of low fat chicken nuggets.
LWT-Food Science and Technology, 43: 715-719.
Viadel, M.B. (2002). Biodisponibilidad de calcio, hierro y
cinc en leguminosas mediante ensayos in vitro con cultivos celulares. Doctoral thesis. Facultad de
Farmacia. Universidad de Valencia.
Visioli, F., Poli, A., Gall, C. (2002). Antioxidant and other biological activities of phenols from olives and
olive oil. Medicinal Research Reviews, 22 (1): 65–75.
Visioli F., Wolfram, R., Richard, D., Abdullah, M. I. C. B.,
Crea, R. (2009). Olive phenolics increase glutathione levels in healthy volunteers. Journal
of Agricultural and Food Chemistry, 57(5): 1793-
1796.
Wang, D., Williams, B.A., Ferruzzi, M.G., D’Arcy, B.R.
(2013). Microbial metabolites, but not other
phenolics derived from grape seed phenolic
extract, are transported through differentiated
Caco-2 cell monolayers. Food Chemistry, 138: 1564–1573.
Wang, L.L., Xiong, Y.L. (2005). Inhibition of lipid oxidation
in cooked beef patties by hydrolyzed potato protein is related to tis reducing and radical
scavenging ability. Journal of Agricultural and
Food Chemistry, 53: 9186-9192.
Wang, S., Zhang, Y., Chen, L., Xu, X., Zhou, G., Li, Z., Feng,
X. (2018). Dose-dependence effects of rosmarinic
acid on formation of oxidatively stressed myofibrillar protein emulsion gel at different
NaCl concentrations. Food Chemistry, 243: 50-
57. DOI: 10.1016/j.foodchem.2017.09.114
Waterhouse, A.L., Laurie, V.F. (2006). Oxidation of wine
phenolics: A critical evaluation and hypotheses.
American Journal of Enology and Viticulture, 57:
306-313.
Weber, R.W. (2014). Chapter 32. Adverse reactions to the
antioxidants butylated hydroxyanisole and hydroxytoluene. In: Food Allergy: Adverse
reactions to foods and food additives, 5th Ed. John
Willey & Sons, Ltd: 393-401.
Weckesser, S., Engel, K., Simon-Haarhaus, B., Wittmer, A.,
Pelz, K., Schempp, C.M. (2007). Screening of
plant extract for antimicrobial activity against bacteria and yeast with dermatological relevance.
Phytomedicine, 14: 508–516.
Wu, C., Parrot, A.M., Fu, C., Liu, T., Marino, S.F., Gladyshev, V.N., Jain, M.R., Baykal, A.T., Li, Q.,
Oka, S., Sadoshima, J., Beuve, A., Simmons,
W.J., Li, H. (2011). Thioredoxin 1-mediated post-translational modifications: reduction,
transnitrosylation, denitrosylation and related
proteomics methodologies. Antioxidants & Redox Signaling: 1-40. DOI: 10.1089/ars.2010.3831
Wybraniec, S., Starzak, K., Skopi´nska, A., Szaleniec, M.,
Slupski, J., Mitka, K., Kowalski, P., Michalowski, T. (2013). Effects of metal cations on betanin
stability in aqueous-organic solutions. Food
Science and Biotechnology, 22(2): 353-363.
Xiong, Y.L. (2010). Chapter 4: Protein oxidation and
implications for muscle food quality. In:
Antioxidants in Muscle Foods: Nutritional Strategies to Improve Quality; Decker, E.A.,
Faustman, C., Lopez-Bote, C.J., Eds.; John Wiley
& Sons, Inc.: Hoboken, NJ, USA: 85–112, ISBN 0-471-31454-4.
Xiong, Y.L., Blanchard, S.P., Ooizumi, T., Ma, Y. (2010).
Hydroxyl radical and ferryl-generating systems promote gel network formation of myofibrillar
protein. Journal of Food Science, 75: C215–C221.
Yadav, A.S., Singh, R.P. (2004). Natural preservatives in
poultry meat products. Natural Production
Radiance, 3: 300–303.
Yao, Y., Sang, W., Zhou, M., Ren, G. (2010). Phenolic
composition and antioxidant activities of 11 celery
cultivars. Journal of Food Science, 75: C9–C13.
Yerlikaya, P., Gokoglu, N., Uran, H. (2005). Quality changes
off ish patties produced from anchovy during
refrigerated storage. European Food Research and Technology, 220(3): 287-291.
Lorena Martínez Zamora PhD Thesis, 2019
159
Zakrys-Waliwander, P.I., O’Sullivan, M.G., O’Neill, E.E.,
Kerry, J.P. (2012). The effects of high oxygen modified atmosphere packaging on protein
oxidation of bovine M. longissimus dorsi muscle
during chiller storage. Food Chemistry, 131: 527-532.
Zeb, A. (2015). Phenolic profile and antioxidant potential of
wild watercress (Nasturtium officinale L.). SpringerPlus, 4: 714.
Lorena Martínez Zamora PhD Thesis, 2019
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Lorena Martínez Zamora PhD Thesis, 2019
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12. Scientific production
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12.1. Publications
Nieto, G., Martínez, L., Castillo, J., Ros, G. (2017). Effect of hydroxytyrosol, walnut and
olive oil on nutritional profile of low-fat chicken frankfurters. European Journal of Lipid Science
and Technology, 119: 1600518
Nieto, G. Martínez, L., Castillo, J., Ros, G. (2017). Hydroxytyrosol extracts, olive oil and
walnuts as functional components in chicken sausages. Wiley Online Library. DOI:
10.1002/jsfa.8240
Martínez, L., Ros, G., Nieto. G. (2018). Hydroxytyrosol: Health Benefits and Use as
Functional Ingredient in Meat. Medicines, 5(1): 13.
Martínez, L., Ros, G., Nieto, G. (2018). Fe, Zn and Se bioavailability in chicken meat
emulsions enriched with minerals, hydroxytyrosol and Extra Virgin Olive Oil as measured by
Caco-2 cell model. Nutrients, 10.
Martínez, L., Bastida, P., Castillo, J., Ros, G., Nieto, G. (2019). Green alternatives to synthetic
antioxidants, antimicrobials, nitrates and nitrites in Clean Label Spanish chorizo. Antioxidants,
8(6).
Martínez, L., Castillo, J., Ros, G., Nieto, G. (2019). Antioxidant and antimicrobial activity of
rosemary, hydroxytyrosol and pomegranate natural extracts in fish patties. Antioxidants, 8.
González, C.M., Martínez, L., Ros, G., Nieto, G. (2019). Evaluation of nutritional profile and
total antioxidant capacity of the Mediterranean Diet from the Southern of Spain. Food Science
and Nutrition.
12.2. Book chapters
Martínez, L., Ros, G., Nieto, G. (2019). Oregano: Health benefits and its use as functional
ingredient in meat products.
12.3. Scientific congresses
Martínez, L., Nieto, G., Ros, G. (2016). Fe, Zn and Se availability of a functional chicken
meat producto enriched with olive oil and Hydroxytyrosol in an in vitro gastrointestinal digestion
system and Caco-2 cells. II Jornadas Doctorales de la Universidad de Murcia. Murcia, España,
31 Mayo, 1 y 2 Junio 2016. Oral comunication.
Martínez, L., Nieto, G., Ros, G. (2017). Mejora del perfil lipídico en productos cárnicos tipo
mortadela, a través de la incorporación de nueces y Aceite de Oliva Virgen Extra, como fuente
de Omega 3. III Jornadas Doctorales de la Universidad de Murcia. Murcia, España, 29-30 Mayo
y 1 Junio 2017. Oral comunication.
Nieto, G., Martínez, L., Ros, G. (2017). Total antioxidant capacity of chicken meat from
organic mineral supplementation. 63rd International Congress of Meat Science and Technology
(ICOMST). Cork, Irlanda, 13-18 Agosto 2017. Poster.
Lorena Martínez Zamora PhD Thesis, 2019
164
Martínez, L., Nieto, G., Ros, G. (2018). Antioxidant and antimicrobial capacity of natural
extracts of fruits and leaves from the Region of Murcia. V National and IV International Student
Congress of Food Science and Technology. Valencia, España, 22 y 23 Febrero 2018. Poster.
Martínez, L., Bastida, P., Ros, G., Nieto, G. (2018). Antioxidant capacity and antimicrobial
capacity against Clostridium perfringens of natural extracts obtained from vegetables from the
Region of Murcia. IV Jornadas Doctorales de la Universidad de Murcia. Murcia, España, 29, 30
y 31 Mayo 2018. Poster.
Martínez, L., Ros, G., Nieto, G. (2018). Effect of natural extracts from food industrial by-
products on nutritional and antioxidant quality of chicken nuggets enriched with Zn and Se. II
International Congress of Food of Animal Origin. Hayvansal, Chipre, 8-11 Noviembre 2018.
Poster.
Martínez, L., Serrano, A., Ros, G., Nieto, G. (2018). Antioxidant and antiinflamatory capacity
of frozen chicken nuggets enriched with phenolic compounds from food industrial by-products,
Zn and Se. III Jornadas Científicas del IMIB-Arrixaca. Murcia, España, 19 y 20 Noviembre 2018.
Poster.
Martínez, L., Bastida, P., Ros, G., Nieto, G. (2019). Capacidad antioxidante y antimicrobiana
contra Clostridium perfringens del pimentón, ajo y orégano. X Congreso Nacional CyTA-CESIA.
León, España, 15-17 Mayo 2019. Poster.
Martínez, L., Bastida, P., Ros, G., Nieto, G. (2019). Desarrollo de un chorizo “clean label”,
con la adición de extractos de romero, cítricos, espinaca y apio como ingredientes funcionales. X
Congreso Nacional CyTA-CESIA. León, España, 15-17 Mayo 2019. Poster.
Nieto, G., Lloret, P., Martínez, L., Ros, G. (2019). Efect antioxidant de flavonoids cítricos y
polifenoles del olivo, romero y granada en preparados de pescado funcionales. X Congreso
Nacional CyTA-CESIA. León, España, 15-17 Mayo 2019. Oral comunication.
Martínez, L., Bastida, P., Nieto, G., Ros, G. (2019). Elaboración de chorizo sarta “Clean
label” rico en compuestos fenólicos, vitamina C y nitratos provenientes de frutas y verduras. V
Jornadas Doctorales de la Universidad de Murcia. Murcia, España, 29, 30 y 31 Mayo 2019.
Poster.
Martínez, L., Ros, G., Nieto, G. (2019). Effect of natural extracts on nutritional quality and
protein oxidation of chicken nuggets enriched through diet with organic Zn and Se. 65th
International Congress of Meat Science and Technology (ICOMST). Berlín, Alemania, 4-9
Agosto 2019. Poster.
Martínez, L., Bastida, P., Ros, G., Nieto, G. (2019). Antioxidant activity of rosemary and
citrus extract and natural sources of nitrate in Clean Label Spanish “chorizo”. 65th International
Congress of Meat Science and Technology (ICOMST). Berlín, Alemania, 4-9 Agosto 2019.
Poster.
12.4. Prizes
Nieto, G., Lloret, P., Martínez, L., Ros, G. (2019). Efect antioxidant de flavonoids cítricos y
polifenoles del olivo, romero y granada en preparados de pescado funcionales. X Congreso
Nacional CyTA-CESIA. León, España, 15-17 Mayo 2019.
Lorena Martínez Zamora PhD Thesis, 2019
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13. Annexes
Lorena Martínez Zamora PhD Thesis, 2019
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Assay I
Paper I
Martínez L., Ros, G., Nieto, G. (2018). Fe, Zn and Se bioavailability in chicken meat emulsions
enriched with minerals, Hydroxytyrosol and extra virgin olive oil as measured by Caco-2 cell
model. Nutrients, 10: 969. DOI: 10.3390/nu10080969
Abstract
There is a high demand for functional meat products due to increasing concern about food and
health. In this work, Zn and Se bioavailability was increased in chicken meat emulsions that are
enriched with Hydroxytyrosol (HXT), a phenolic compound obtained from olive leaf. Six
different chicken emulsions were elaborated. Three were made with broiler chicken meat
supplemented with inorganic Zn and Se: control, one with HXT (50 ppm) added and one with
HXT (50 ppm) and Extra Virgin Olive Oil (EVOO) (9.5%) added; and, three were made with
chicken meat from chickens fed a diet that was supplemented with organic Zn and Se: control,
one with HXT (50 ppm) added and one with HXT (50 ppm) and EVOO (9.5%) added. The
samples were digested in vitro and the percent decomposition of phenolic compounds was
measured by HPLC. Mineral availability (Fe, Zn and Se) was measured by cell culture of the
Caco-2 cell line and the results were compared with mineral standards (Fe, Zn, and Se). The data
obtained showed that neither HXT resistance to digestion nor Fe availability was affected by the
presence of organic Zn and Se or phenolic compounds. Zn uptake increased in the presence of
HXT, but not when its organic form was used, while Se uptake increased but it was not affected
by the presence of HXT. It was concluded that the enrichment of meat—endogenously with
organic minerals and exogenously with phenolic compounds—could be considered an interesting
strategy for future research and applications in the current meat industry.
URL: https://www.mdpi.com/2072-6643/10/8/969
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Assay II
Paper II
Nieto, G., Martínez L., Castillo, J., Ros, G., Nieto, G. (2017). Effect of Hydroxytyrosol, walnut
and olive oil on nutritional profile of low-fat chicken frankfurters. European Journal of Lipid
Science and Technology, 119: 1600518. DOI: 10.1002/ejt.201600518.
Abstract:
The aim of this study was to evaluate the effect of hydroxytyrosol extract (HXT, 50 ppm), walnut
paste (2.5 g/100 g) and extra olive oil (as substitute of animal fat, 20 g/100 g) on fatty acid profiles,
mineral content and sensory analysis of chicken frankfurters. Low-fat chicken sausages produced
with olive oil as fat replacement, walnut and HXT extract remained stable without a significant
loss of sensory attributes during storage at 4°C for 21 days. The sausages with HXT were found
to decrease rancid odour, and the samples with walnut were darker, compared with control.
Whereas positive correlations were established between rancid odour, saturated fatty acid (SFA)
and monounsaturated fatty acid (MUFA), and negative correlations were found between
polyunsaturated fatty acid (PUFA), rancid odour and thiobarbituric acid-reactive substances
(TBARS); no significant correlations were established between TBARS and MUFA. Sausages
with walnut or olive oil contained significantly larger (P<0.05) percentages of minerals (K, Fe,
Mg, Mn, Ca, P and Zn), MUFAs, and n3 PUFAs, mainly a-linolenic acid, in addition to
significantly lower amounts (P<0.05) of SFAs, mainly miristic, palmitic and stearic acid. They
also contained a significantly lower n6/n3 PUFA ratio, atherogenic index (AI) and
thrombogenicity index (IT) and significantly higher (P<0.05) PUFA ratio. In conclusion, walnut,
HXT and olive oil can be applied in meat products as additives with functional properties.
URL: https://onlinelibrary.wiley.com/doi/abs/10.1002/ejlt.201600518
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Assay II
Paper III
Nieto, G., Martínez L., Castillo, J., Ros, G., Nieto, G. (2017). Hydroxytyrosol extracts, olive oil
and walnuts as functional components in chicken sausages. Journal of Science and Food
Agriculture, 97: 3761-3771. DOI: 10.1002/jsfa.8240.
Abstract:
BACKGROUND: Olive oil, hydroxytyrosol and walnut can be considered ideal Mediterranean
ingredients for their high polyphenolic content and healthy properties. Three extracts of
hydroxytyrosol obtained using different extraction processes (HXT 1, 2, 3) (50 ppm) were
evaluated for use as antioxidants in eight different chicken sausage formulas enriched in
polyunsaturated fatty acids (2.5 g 100 g−1 walnut) or using extra virgin olive oil (20 g 100 g−1)
as fat replacer. Lipid and protein oxidation, colour, emulsion stability, and the microstructure of
the resulting chicken sausages were investigated and a sensory analysis was carried out.
RESULTS: The sausages with HXT extracts were found to decrease lipid oxidation and to lead
to the loss of thiol groups compared with control sausages. Emulsion stability (capacity to hold
water and fat) was greater in the sausages containing olive oil and walnut than in control sausages.
In contrast, the HXT extracts produced high emulsion instability (increasing cooking losses).
Sensory analysis suggested that two of the HXT extracts studied (HXT2 and HXT3) were
unacceptable, while the acceptability of the other was similar to that of the control products.
Sausages incorporating HXT showed different structures than control samples or sausages with
olive oil, related to the composition of the emulsion.
CONCLUSION: These results suggest the possibility of replacing animal fat by olive oil and
walnut in order to produce healthy meat products.
URL: https://onlinelibrary.wiley.com/doi/abs/10.1002/jsfa.8240
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Assay IV
Paper V
Martínez, L., Bastida, P., Castillo, J., Ros, G., Nieto, G. (2019). Green alternatives to synthetic
antioxidants, antimicrobials, nitrates, and nitrites in Clean Label Spanish chorizo. Antioxidants,
8(6): E184. DOI: 10.3390/antiox8060184
Abstract:
Natural extracts obtained from fruit and vegetable processing are important sources of phenolic
compounds and nitrates, with excellent antioxidant and antimicrobial properties. The aim of this
study was to characterize and determine the antioxidant and antimicrobial capacity of several
natural extracts (citric (Ct), acerola (Ac), rosemary (R), paprika, garlic, oregano, beet (B), lettuce
(L), arugula (A), spinach (S), chard (Ch), celery (Ce), and watercress (W)), both in vitro and
applied to a cured meat product (chorizo). For that, the volatile compounds by GC-MS and
microbial growth were determined. The total phenolic and nitrate contents were measured and
related with their antioxidant capacity (measured by DPPH, ABTS, FRAP, and ORAC methods)
and antimicrobial capacity against Clostridium perfringens growth in vitro. In order to study the
antioxidant and antimicrobial activities of the extracts in food, their properties were also measured
in Spanish chorizo enriched with these natural extracts. R and Ct showed the highest antioxidant
capacity, however, natural nitrate sources (B, L, A, S, Ch, Ce, and W) also presented excellent
antimicrobial activity against C. perfringens. The incorporation of these extracts as preservatives
in Spanish chorizo also presented excellent antioxidant and antimicrobial capacities and could be
an excellent strategy in order to produce clean label dry-cured meat products.
URL: https://www.mdpi.com/2076-3921/8/6/184
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Assay V
Paper VIII
Martínez, L., Castillo, J., Ros, G., Nieto, G. (2019). Antioxidant and antimicrobial activity of
rosemary, hydroxytyrosol, and pomegranate natural extracts in fish patties. Antioxidants, 8(4):
86. DOI: 10.3390/antiox8040086
Abstract:
Natural extracts (rich in bioactive compounds) that can be obtained from the leaves, peels and
seeds, such as the studied extracts of Pomegranate (P), Rosemary (RA, Nutrox OS (NOS) and
Nutrox OVS (NOVS)), and olive (Olea europaea) extracts rich in hydroxytyrosol (HYT-F from
olive fruit and HYT-L from olive leaf) can act as antioxidant and antimicrobial agents in food
products to replace synthetic additives. The total phenolic compounds, antioxidant capacity
(measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-Azinobis (3-ethylbenzothiazolin) -6-
sulphonic acid (ABTS), Ferric Reducing Antioxidant Power (FRAP), and Oxygen Radical
Absorbance Capacity (ORACH)) and their antimicrobial power (using the diffusion disk method
with the Escherichia Coli, Lysteria monocytogenes, and Staphilococcus Aureus strains) were
measured. The results showed that all the extracts were good antioxidant and antimicrobial
compounds in vitro. On the other hand, their antioxidant and antimicrobial capacity was also
measured in fish products acting as preservative agents. For that, volatile fatty acid compounds
were analysed by GS-MS at day 0 and 11 from elaboration, together with total vial count (TVC),
total coliform count (TCC), E. Coli, and L. monocytogenes content at day 0, 4, 7 and 11 under
refrigerated storage. The fish patties suffered rapid lipid oxidation and odour and flavour spoilage
associated with slight rancidity. Natural extracts from pomegranate, rosemary, and
hydroxytyrosol delayed the lipid oxidation, measured as volatile compounds, and the
microbiological spoilage in fish patties. Addition of natural extracts to fish products contributed
to extend the shelf life of fish under retail display conditions.
URL: https://www.mdpi.com/2076-3921/8/4/86