UNIVERSIDAD DE MURCIA...La presente Tesis Doctoral ha sido realizada para optar al grado de Doctor...

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.

Lorena Martínez Zamora PhD Thesis, 2019

i

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


ii

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


iii

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


iv


v

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


vi

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


vii

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


viii


ix

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


x

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


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


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


xi

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


xii


xiii

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.


xiv


xv

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


xvi

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


1

1. Introduction


2


3

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


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.


5

2. Animal origin products


6


7

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).


8

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


9

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-


10

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).


11

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).


12

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).


13

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.).


14

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.).


15

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


16

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.


17

3. Oxidative deterioration in

animal origin products


18


19

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:


20

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


21

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).


22

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.


23

4. Use of antioxidant and

antimicrobial compounds to

preserve animal origin products


24


25

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


26

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


27

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


28

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.


29

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


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,


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,


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.


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.


34


35

5. Development of Clean label

animal origin products


36


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.


38

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.


39

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.


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.


41

6. Justification and Objectives


42


43

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


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.


45

7. Experimental design


46


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.


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


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.


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


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

).


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.


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.


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).


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.


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


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.).


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


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.


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.


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.


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

D T

hesis, 2

019


63

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)



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


Shelf life study of chorizo for 150 days:

- pH

- Colour (CIELab)

- Lipid oxidation: Volatile

compounds (GC-MS)

VII

63

Lo

rena M

artín

ez Za

mo

ra

Ph

D T

hesis, 2

019


64


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


Shelf life study of fish patties for 14 days:

- pH

- Colour (CIELab)



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

Lo

rena M

artín

ez Za

mo

ra

Ph

D T

hesis, 2

019

64


65

8. Results and Discussion


66


67

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.


68

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


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.


69

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


• 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.


70

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


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


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


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


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.


71

Table 8.4. Se retention, transport and cellular uptake (M ± SD) in enriched chicken emulsions. Experimental Treatments


• Se concentration in


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


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


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


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.


72

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%).


73

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


74

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




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.


75

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




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.


76

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).


77

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.


78

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


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

(%

)


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).


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


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).


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.


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.


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


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.


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).


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.


89

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


90

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


91

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).


92

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


93

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.


95

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.


96

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


97

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.


98

C t A c R C o n tro l

0

21 0 0 7

41 0 0 7

61 0 0 7

81 0 0 7

CF

U

A

a

bb c

c

c d

dd

ef

P A P R IK A G A R L IC O R E G A N O C o n tro l

0

21 0 0 7

41 0 0 7

61 0 0 7

81 0 0 7

CF

U

a

b

cccc d c dc d

dd

B

B L A S C h C e W C o nt ro l

0

21 0 0 7

41 0 0 7

61 0 0 7

81 0 0 7

CF

U

C

a

b

d e

cc d

ef

d

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5 0 0 p p m

2 5 0 p p m

d ee e

ef efef ef efeff f f f

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.


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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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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


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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.

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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


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


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.

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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).


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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.


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


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.).


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


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


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.


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.


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”


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.


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.


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.


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)


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


117

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


118

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


119

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.


120

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


121

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.


122

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


123

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 %,


124

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


125

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.


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.


127

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.


128

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.


129

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.


130

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).


131

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


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.


133

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.


134

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


135

9. Conclusions


136


137

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


138

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.


139

10. Perspective for further

research activities


140


141

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.


142


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12. Scientific production


162


163

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.


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.


165

13. Annexes


166


167

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

https://www.mdpi.com/2072-6643/10/8/969


168

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

https://onlinelibrary.wiley.com/doi/abs/10.1002/ejlt.201600518


169

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

https://onlinelibrary.wiley.com/doi/abs/10.1002/jsfa.8240


170

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.




171

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.



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