Skin and seed grape extract as an antioxidant for Mechanically Deboned Chicken Meat, during frozen storage

Skin and seed grape extract as an antioxidant for mechanically deboned chicken meat, during frozen storage Hernán Horacio Tournour Porto, December 2014 Dissertation Thesis Skin and seed grape extract as an antioxidant for mechanically deboned chicken meat, during frozen storage Dissertation presented to Faculty of Nutrition and Food Science to obtain the degree of Doctor of Philosophy (PhD) in Food Consumption and Nutrition Sciences Supervisor: Dr. Luís Miguel Cunha, Associated Professor, FCUP Co-supervisors: Dr. Marcela Alves Segundo, Assistant Professor, FFUP Dr Jorge Bernardo Lacerda de Queiroz, Assistant Professor, FCUP Hernán Horacio Tournour Porto, 2014 Hernán Tournour is grateful for financial support from Erasmus Mundus Action 2 Strand 1 Lot 16, (ET11D0009ER), Argentina Program - Project 2 Eurotango through a PhD Scholarship. The studies presented here were done at REQUIMTE Laboratory for Food Quality and Preservation of Faculty of Sciences of the University of Porto at the Agrarian Campus in Vairão, at REQUIMTE Laboratory of Applied Chemistry of Faculty of Pharmacy of the University of Porto, at the Higher Institute of Engineering of Porto and at SenseTest Lda., Vila Nova de Gaia. vii Author’s Declaration Under the terms of the “Decreto-lei nº 216/92, de 13 de Outubro” is hereby declared the following original articles were prepared in the scope of this thesis. Under the terms of the referred “Decreto-lei”, the author declares that he afforded a major contribution to the conceptual design and technical execution of the work, interpretation of the results and manuscript preparation of the published articles included in the thesis. This dissertation is constituted by the following papers submitted for publications: I. Hernán H. Tournour, Marcela A. Segundo, Luís M. Magalhães, Luísa Barreiros, Jorge Queiroz, Luís M. Cunha. Valorization of grape pomace: extraction of bioactive phenolics with antioxidant properties. (Submitted for publication). II. Hernán H. Tournour, Marcela A. Segundo, Luís M. Magalhães, Jorge Queiroz, M. Beatriz P. P. Oliveira, Luís M. Cunha. Single and successive oxidative stress factors applied to mechanically deboned chicken meat (MDM): protective effect of grape pomace extract. (Submitted for publication). III. Hernán H. Tournour, Marcela A. Segundo, Luís M. Magalhães, Telmo J. R. Fernandes, M. Beatriz P. P. Oliveira, Luís M. Cunha. Influence of Portuguese grape pomace extracts on the oxidative stability, nutritional and color characteristics of mechanically deboned chicken meat. (Submitted for publication). IV. Hernán H. Tournour, Marcela A. Segundo, Luís M. Magalhães, Anabela S. G. Costa, Luís M. Cunha. Effect of “Touriga nacional” grape extract on quality characteristics of mechanically deboned chicken meat kept under frozen storage. (Submitted for publication). V. Hernán H. Tournour, Luís M. Cunha, Luís M. Magalhães, Rui Costa Lima, Marcela A. Segundo. Evaluation of the joint effect of the incorporation of mechanically deboned meat and grape extract on the formulation of chicken nuggets. (Submitted for publication). viii Moreover, the author declares that he has actively participated in the preparation and writing of each paper, being actively engaged in the stages of experimental design, sample preparation and evaluation, data collection, analysis and interpretation of results. ix Acknowledgements Here there is a small tribute to the institutions and all those people who in one or in other way have brought knowledge, guidance, support and why not friendship during this long journey that comprise a PhD course, University of Porto, Faculty of Nutrition and Food Sciences (FCNAUP) and Faculty of Sciences (FCUP) for accepting me as PhD student, Faculty of Sciences-U.Porto (Lab. Of Food Quality and Preservation at the Agrarian Campus in Vairão), Faculty of Pharmacy-U.Porto (Lab. of Applied Chemistry and Lab. of Bromatology) and Instituto Superior de Engenharia-Porto (Lab. "Grupo de Reação e Análises Químicas") for granting me access to laboratories and facilities where this work was developed, Erasmus Mundus Action 2 Strand 1 Lot 16 Argentina Eurotango2 (ET11D0009ER) for the financial support through a PhD scholarship, Prof. Dr. Maria Daniel Vaz de Almeida, who during her functions as FCNAUP director accepted and gave me the possibility to participate in this fruitful experience as a PhD student, Prof. Dr. Luís Cunha for his support, assistance, willingness, motivation, data analysis assistance and his guidance during this journey of learning, Prof. Dr. Marcela Segundo for her able and concise assistance, I really like to thank her endless patience, scientific advices and mentorship too, Prof. Dr. Jorge Queiroz for his supporting help during the samples selection and provision, Prof. Dr. Beatriz Oliveira for having made possible to work with her research team in the Lab of Bromatology-FFUP, Dr. Luís Magalhães for his accessible communication, having advised and guided during all laboratory activities, Dr. Luísa Barreiros for her help and support provided during the laboratorial activities, xvi Resumo O bagaço de uva, composto de grainhas, películas (também conhecidas como peles), talos e polpas, é um valioso subproduto da indústria de vinificação, reconhecido devido ao seu relevante teor de compostos polifenólicos. A carne de frango mecanicamente desossada (“mechanically deboned chicken meat”, MDM) é uma matéria-prima altamente afetada por reações de degradação associadas à oxidação lipídica. Os antioxidantes sintéticos habitualmente utilizados a nível industrial, a fim de minimizar a peroxidação, são atualmente suspeitos de causar efeitos tóxicos que afetam a saúde do consumidor. Neste contexto, foi realizada uma caracterização das propriedades antioxidantes dos extratos de bagaço (“grape pomace extract”, GPE) de diferentes variedades portuguesas de variedades portuguesas (Vitis vinifera L. variedade "Touriga nacional" –TNac-, "Touriga franca" –TF- e "Tinta roriz" –TR- ), através dos ensaios do conteúdo de compostos fenólicos totais (TPC), capacidade de captação do radical DPPH•, capacidade de absorção de radical peroxilo (ORAC) e capacidade quelante do ferro (II). Além disso, estimada a proteção oferecida pelo GPE de TF em face à oxidação lipídica em condições de degradação acelerada utilizando um modelo de carne sob um único fator de stress ou sob uma exposição sucessiva de fatores de stress, e em comparação com o uso de hidroxitolueno butilado (BHT). Foram analisadas as características gerais de qualidade da MDM suplementadas com diferentes GPEs, incluindo composição nutricional, pH, variáveis de cor e estabilidade oxidativa ao longo do tempo de armazenamento. Finalmente, foi conduzida uma avaliação sensorial por consumidores naive quanto à aparência percebida de nuggets de frango elaborados com diferentes quantidades de MDM e GPE de TNac. Os resultados indicaram que os GPE obtidos, utilizando uma mistura de extracção amiga do ambiente (80 % v/v etanol/água), apresentaram uma elevada propriedade antioxidante. O GPE de TNac apresentou valores significativamente mais altos de TPC (142,4 mg GAE g-1 resíduo), de captação do DPPH• (1,12 mmol TE g-1 resíduo) e de ORAC (1579 µmol TE g-1 resíduo), xvii incluindo também os teores mais altos de compostos fenólicos totais avaliados por HPLC, com todos os valores significativamente (p < 0,05) diferentes dos outros GPEs. Os ensaios de FCR, ORAC e ICA foram efetivos para monitorizar a estabilidade oxidativa de amostras de MDM perante fatores de degradação. A eficácia antioxidante foi dependente da combinação dos factor de stress e do antioxidante (GPE de TF ou BHT). Além disso, verificou-se que a exposição sucessiva às condições de stress afetou o desempenho final do antioxidante. As experiências de o armazenamento congelado indicaram que amostras de MDM suplementadas com diferentes GPEs (60 mg/kg) manteve-se estável até 30 dias sob de acordo com os ensaios de FCR, ORAC e ICA. A suplementação com GPE resultou numa mudança significativa (p < 0,05) em relação às variáveis de cor (CIE-L* a* b*). Por outro lado, após 365D de armazenamento congelado, todas as amostras de MDM suplementadas com GPE não foram significativamente (p > 0,05) diferentes do controlo para 1D, relativamente à oxidação de ácidos gordos agregados em n-3, enquanto que, as amostras com BHT - BHA (hidroxitolueno butilado - hidroxianisole butilado, 200 mg/kg) sofreram oxidação. Foi realizada uma avaliação sensorial de nuggets de frango formulados com MDM e GPE, conduzida por 75 avaliadores naive utilizando uma escada hedônica de 9 pontos complementada com comentários abertos. As conclusões da avaliação de aceitação geral indicam que a adição de GPE até 120 mg/kg e de MDM até 15 %, garante um nível satisfatório de aceitação (valores de aceitação > 7,0) relativamente à aparência percebida em nuggets de frango. Analise dos comentários abertos através da analise de correspondência permitiu a construção de um mapeamento perceptual visando o efeito da variação da composição dos nuggets de frango. MDM e GPE podem ser utilizados com sucesso na elaboração de novos produtos, fomentando a exploração de subprodutos da vinificação. xviii Scope and Aims Portugal is the eighteenth grape global producer with an important wine production spread throughout the whole territory of the country. This production is channeled for the creation of high quality wine, generally yielding relevant amounts of seeds, skins, stalks and pulps, conjointly known as grape pomace. There is a vast amount of literature regarding polyphenolic compounds in grape and their healthy impact in human diet. For several fields, including chemical, pharmacological and food industries this fact represents an inexpensive source of bioactive compounds to be used as target in extractive procedures. On the other hand, the existence of a growing interest by consumers regarding wellbeing and health encourages industries to develop new applications concerning properties of polyphenolic compounds in foods. Moreover, studies related to the antioxidant protection in food matrices, including consumer issues associated to sensory impact of supplemented food are also needed, in order to define a comprehensive understanding and assessment. The work depicted in the present dissertation thesis has the following overall aim: To characterize skin and seed grape (pomace) extracts from Portuguese varieties towards the prevention of the lipid oxidation of mechanically deboned chicken meat (MDM), including the assessment of the final physico-chemical characteristics and consumer acceptance of nuggets containing MDM and supplemented with grape extracts. According to the general work chronogram the results belonging to the present thesis are displayed in four sections in accordance to the specific objectives. Section A: To characterize Portuguese grape pomace extracts (GPE) regarding their antioxidant properties. A1. “Valorization of grape pomace: extraction of bioactive phenolics with antioxidant properties”.  To perform a complete study regarding polyphenolic content and antioxidant capacity of Portuguese GPE. xix  To compare the influence of the choice of solvent on the final antioxidant properties of GPE.  To select the most suitable Portuguese grape variety, under study, towards future recovery of bioactives products and application in other industries. Section B: To evaluate the effectiveness of GPE against lipid oxidation of MDM. B1. “Single and successive oxidative stress factors applied to mechanically deboned chicken meat (MDM): protective effect of grape pomace extract”.  To evaluate methodologies to determine the oxidative stability in a meat model.  To evaluate the performance of GPE against the application of a single stress factor, compared with BHT.  To study the performance of GPE against successive exposure to degradation factors, compared with BHT. Section C: To evaluate the effect of GPE on overall characteristics of MDM through a shelf life real-time analysis, under frozen storage conditions. C1. “Influence of Portuguese grape pomace extracts on the oxidative stability, nutritional and color characteristics of mechanically deboned chicken meat”.  To investigate the effect of GPE supplementation on MDM, regarding oxidative stability and nutritional characteristics.  To understand the contribution of GPE on the changes of color on MDM supplemented samples.  To study the protection conferred by GPE against fatty acids oxidation throughout frozen storage. xx C2. “Effect of “Touriga nacional” grape extract on quality characteristics of mechanically deboned chicken meat kept under frozen storage”.  To understand the influence of the initial MDM composition on the antioxidant performance of GPE.  To evaluate the effect of GPE concentration on the proximate composition, pH, and oxidative stability of frozen MDM.  To study the effect of GPE supplementation on color changes, aiming at possible implications on a finished product. Section D: To evaluate the effect of the implementation of GPE on a real finished product. D1. “Evaluation of the joint effect of the incorporation of mechanically deboned meat and grape extract on the formulation of chicken nuggets”.  To optimize nugget formulation concerning GPE concentration and MDM content.  To study the influence of MDM addition on the nutritional composition of chicken nuggets.  To understand the joint contribution of GPE and MDM on consumers acceptance and perceptual description of chicken nuggets. xxi List of Publications in International Peer-Reviewed Journals Hernán H. Tournour, Marcela A. Segundo, Luís M. Magalhães, Luísa Barreiros, Jorge Queiroz, Luís M. Cunha. Valorization of grape pomace: extraction of bioactive phenolics with antioxidant properties. (Submitted for publication). Hernán H. Tournour, Marcela A. Segundo, Luís M. Magalhães, Jorge Queiroz, M. Beatriz P. P. Oliveira, Luís M. Cunha. Single and successive oxidative stress factors applied to mechanically deboned chicken meat (MDM): protective effect of grape pomace extract. (Submitted for publication). Hernán H. Tournour, Marcela A. Segundo, Luís M. Magalhães, Telmo J. R. Fernandes, M. Beatriz P. P. Oliveira, Luís M. Cunha. Influence of Portuguese grape extracts on the oxidative stability, nutritional, and color characteristics of mechanically deboned chicken meat. (Submitted for publication). Hernán H. Tournour, Marcela A. Segundo, Luís M. Magalhães, Anabela S. G. Costa, Luís M. Cunha. Effect of “Touriga nacional” grape extract on quality characteristics of mechanically deboned chicken meat kept under frozen storage. (Submitted for publication). Hernán H. Tournour, Luís M. Cunha, Luís M. Magalhães, Rui Costa Lima, Marcela A. Segundo. Evaluation of the joint effect of the incorporation of mechanically deboned meat and grape extract on the formulation of chicken nuggets. (Submitted for publication). xxii Poster in Conferences Hernán Tournour, Marcela Segundo, Luís M. Magalhães, Jorge Queiroz, Luís M. Cunha, Estudio comparativo de la capacidad antioxidante de extractos del bagazo de uvas tintas portuguesas en un medio de grado alimenticio, CIBIA9 Congreso Iberoamericano de Ingeniería de Alimentos, Universidad Politécnica de Valencia, España (13-16 de Janeiro de 2014). Hernán H. Tournour, Marcela A. Segundo, Luís M. Magalhães, Jorge Queiroz, Luís M. Cunha, Solvent effect on the antioxidant properties in terms of total phenolic content (TPC) and oxygen radical absorbance capacity (ORAC) in grape pomace extracts from a Portuguese red grape cultivar (“Touriga nacional”), 3rd International ISEKI_Food Conference to be held in Athens, Greece (May 21-23, 2014). Hernán H. Tournour, Marcela A. Segundo, Luís M. Magalhães, Anabela S. G. Costa, Jorge Queiroz, Luís M. Cunha, INFLUENCE of grape pomace extract on the quality characteristics of the mechanically deboned chicken meat (MDM): Towards functional foods, 12º ENCONTRO DE QUÍMICA DOS ALIMENTOS, Instituto Superior de Agronomia, Lisboa, Portugal (10-12 de Setembro de 2014). xxiii Abbreviations The abbreviations list includes acronyms from the dissertation thesis, except from the results section. AAPH 2,2′-azobis(2-amidino-propane) dihydrochloride ABTS 2,2′-Azinobis-(3-ethylbenzothiazole-6-sulphonate) ABTS•+ 2,2′-Azinobis-(3-ethylbenzothiazole-6-sulphonate) radical AOAC Association of American Analytical Chemists AUC Area under curve BHA Butylated hydroxyanisole BHT Butylated hydroxytoluene DAD Diode array detection DPPH• 2,2′-Diphenyl-1-picrylhydrazyl radical EC European Commission EFSA European Food Safety Authority FAO Food and Agriculture Organization of the United Nations FCR Total Phenols Assay by Folin-Ciocalteu Reagent GPE Grape pomace extract HIC Haem iron content HPLC High Performance Liquid Chromatography ICA Iron(II) chelating ability INE Instituto Nacional de Estatistica ISO International Organization for Standardization IVDP Instituto dos Vinhos do Douro e Porto MDM Mechanically deboned meat MhL Millions of hectoliter MRM Mechanically recovered meat MS Mass spectrometry MSM Mechanically separated meat MT Millions of tons NCC National Chicken Council OIV Organisation Internationale de la Vigne et du Vin xxiv ORAC Oxygen Radical Absorbance Capacity psi Pound per square inch TBARS Thiobarbituric Acid Reactive Substances TBHQ tert-butylhydroquinone TE Trolox equivalent TEAC Trolox equivalent Antioxidant Capacity TPC Total Phenolic Content UNESCO United Nations, Educational, Scientific and Cultural Organization USA United States of America UV-vis Ultraviolet and visible xxv Table of Contents Acknowledgements……………………………………………………………….…..ix Abstract…………………………………………………………………………….….xii Resumen……………………………………………………………………………...xiv Resumo……………………………………………………………………………....xvi Scope and Aims…………………………………………………………………….xviii List of Publications in International Peer-Reviewed Journals…………………...xxi Abbreviations………………………………………………………………………..xxiii Table of contents……………………………………………………………………xxv List of tables………………………………………………………………………...xxvii List of figures……………………………………………………………………….xxviii 1. Introduction……………………………………………………………………..1 1.1. Wine production: global and Douro wine region……………………..…3 1.2. Grape pomace: Applications………………………………………………………………...7 1.3. Polyphenolic compounds: antioxidant properties………………….………………………………………………..9 1.4. Extraction of Polyphenols from grape pomace…..………………………………………………………………..17 1.5. Polyphenolic compounds from grape pomace: separation, in vitro characterization and quantification methodologies.……………………………………………………………19 1.6. Poultry meat production: global and in Portugal….………………………………………………………………..23 1.7. Mechanically chicken meat: different uses………………………………………………………………………..28 2. General materials and methods……………………...………………………………………………..45 3. Results and discussion………………………………………………………….………....53 3.1. Paper I: “Valorization of grape pomace: extraction of bioactive phenolics with antioxidant properties”………………………………….55 3.2. Paper II: “Single and successive oxidative stress factors applied to 1. Introduction 4 particular year cannot be the same for the next one. On one hand, the international scene until 2012 for countries such as China, Chile, Australia and South Africa was greatly positive once they have experimented an increasing (from 41 % for China to over 88 % for Chile) in the total amount of wine produced between 2000 and 2012 (OIV, 2012). On the other hand, countries which were commonly recognized as references because of the high quality of their wines and the quantities produced annually, seems to have decreasing perspectives for the future. This is the case of France, Italy and Spain, which have experimented a declining tendency (28, 22, and 27 %, respectively), considering the same period of time (200-2012) in terms of wine production (OIV, 2012). The OIV also shared the ranking for the countries which actively participate in the global wine production (Table 1). Table 1 Wine production (1000 hL excluding juice and musts) a Country 2010 2011 2012 2013 2014 Ranking 2014 France 44,381 50,757 41,548 42,004 46,151 1 Italy 48,525 42,772 45,616 52,429 44,424 2 Spain 35,353 33,397 31,123 45,650 37,000 3 United States 20,890 19,140 21,740 23,500 22,500 4 Argentina 16,250 15,473 11,780 14,984 15,200 5 Australia 11,420 11,180 12,260 12,310 12,560 6 China 13,000 13,200 13,810 11,780 11,178 7 South Africa 9,327 9,725 10,568 10,980 11,420 8 Chile 8,844 10,646 12,554 12,846 10,029 9 Germany 6,906 9,132 9,012 8,409 9,725 10 Portugal 7,148 5,622 6,327 6,238 5,886 11 Romania 3,287 4,058 3,311 5,113 4,093 12 New Zeeland 1,900 2,350 1,940 2,480 3,200 13 Greece 2,950 2,750 3,115 3,343 2,900 14 Brazil 2,459 3,460 2,967 2,710 2,810 15 a Adapted from OIV (2014) 1. Introduction 5 Portugal is the country with the highest variety of wine vinery. It has also the highest global vine biodiversity, including 342 grape varieties (OIV, 2014) and 258 grape varieties of Portuguese origin. Besides, Portugal presents the highest world genetic heritage and varietal density per sq. km (2.7 grape varieties / 1000 km2). Hence, thanks to these characteristics, Portugal reached 5.9 MhL of wine production in 2014 (Table 1). This fact positioned Portugal as the eleventh in the ranking of the world's largest wine producers. Different demarcated regions are displayed for the winemaking in the Portuguese territory. The top five of Portuguese wine productive areas includes Douro, Alentejo, Beiras, Lisboa and Minho, participating with 25, 18, 14, 14 and 13 %, respectively, based on the total wine production. The general characteristics of their vines vary according to the localization, region and also climatic conditions. Regarding to the Douro region, the vineyards were settled down in 1756 thanks to Marquês de Pombal and it was declared a World Heritage region by United Nations, Educational, Scientific and Cultural Organization (UNESCO) in 2001. It is located in Northeast of Portugal, within the Douro River basin, surrounded by craggy mountains that give it very particular soil and climacteric characteristics. This region spreads over a total area of approximately 250,000 hectares and is divided into three sub-regions that differ greatly from each other not only regarding weather aspects but also for socio-economical reasons. The three sub-regions include Lower Corgo, Upper Corgo and Upper Douro, and they differ in terms of area under vines, number of farmers, chemical composition of the soils and climatic characteristics, namely rainfall and temperature. 1. Introduction 6 Located in deep valleys, protected by mountains, the climate in the region is characterized by very cold winters and hot, dry summers. Thus, Douro region has been worldwide recognized as a reference for the high quality of its wines. Different ways to vinify wine "Douro" were developed through time. The traditional prepared in mill using large shallow rock containers, generated wines which exhibited the highest extraction of color and tannins, that gives them a good aging potential. On the other hand, there is a more modern production system, which has recorded a substantial increase, and uses stainless steel tanks with temperature control, improving the enological characteristics in terms of aromas and colors. It has also been an evolution in the maturation of the wine before it is bottled. Large wooden casks were used traditionally as containers that have been gradually being replaced by new oak barrels with lower volume, or by stainless steel vats (IVDP, 2014). Farmers in the Douro region, exhibit a wide range of products, having red, white, rosé and special wines. Red wines are elaborated from indigenous grape varieties such as “Touriga nacional”, “Touriga franca”, “Tinta roriz” (Aragonez), “Tinta barroca” and “Tinto cão”. Basically, several grape varieties are blended with each other in order to increase the richness and complexity of the Douro profile wines. Nevertheless, there are still mono-varietal wines; this means wines produced with only one variety, especially the first three listed before. Figure 1. Demarcated Douro river region. Source: IVDP (2014). 1. Introduction 7 Moreover, Douro region presents strong white wines, because of their dryness resulting by blending several grape varieties such as “Malvasia fina”, “Viosinho”, “Gouveio” and “Rabigato”. Finally, rosé wines are produced in response to global trends in wine consumption, especially among young people. They are elaborated through specific changes during the winemaking processes, reducing the maceration time, which gives the final characteristic pink color (IVDP, 2014). Figure 2. Portuguese grape varieties from Douro, región: a) “Touriga nacional”; b) “Touriga franca” and c) “Touriga roriz”. Source: Eiras-Dias et al. (2011). 1.2 Grape pomace: Applications During winemaking steps, significant amounts of waste, in their majority solids are also generated. Residues of the wine industry, including seeds, peels or skins, stalks and pulps are denominated in its whole as grape pomace. Based on a traditional winemaking process it is estimated that per six liters of wine is generated one kg of solid waste. Thus, taking in consideration a global wine production around 271 MhL (OIV, 2014) over 4.5 million of tons of solid waste would be generated worldwide. Environmental concerns about the production and accumulation of waste worldwide are increasing. Meanwhile the European Commission, early in 2006, issued a series of regulations towards a sustainable European wine sector aimed the inclusion of minimum environmental requirements for the wine sector covering the main pressures a b c 1. Introduction 8 from the sector (notably, soil erosion and contamination, the use of plant protection products, and waste management). The current destinies for the grape pomace comprise the disposal or fertilization/compost (Laufenberg, Kunz, & Nystroem, 2003; Reeve, Carpenter- Boggs, Reganold, York, & Brinton, 2010), cattle feeding (Lu & Yeap Foo, 1999), landfills, fermentation/distillation industry either for the extraction of food natural colorants and bioactive compounds (Lapornik, Prošek, & Golc Wondra, 2005; Mendes, Prozil, Evtuguin, & Lopes, 2013). In certain cases, grape pomace (mainly the seeds) are used in wood adhesives extractive processes (Ping, Pizzi, Guo, & Brosse, 2011). Some authors refer disadvantages in using grape pomace without any pre-treatment cattle feeding or in post winemaking fermentation/distillation process, due to its high polyphenols content with implications in animal nutrition and inhibition of yeasts germination (Mendes et al., 2013; Ping et al., 2011). The most recent and innovate application is associated to a new pesticide, namely “phytosanitary bioproducts” used for the control of the incidence of diseases in some crops (Benouaret et al., 2014; Goupil et al., 2012). The composition of the residue of the grape has significant variations depending on grape variety and technology applied during the winemaking steps. Generally, it consists largely in seeds and skins (or peels), and the rest is represented by stems or stalks. After fermentation step, considerable contents of polyphenols (over 10% on dry bases) are retain in grape pomace, depending on the type of grape (white or red), the part of the tissue (skins, seeds, etc.), as well as the processing conditions (e.g., contact time between skins and must) (Guendez, Kallithraka, Makris, & Kefalas, 2005; Makris, Boskou, & Andrikopoulos, 2007). Regarding to its chemical composition, lignans, cellulose and tannins have been assessed previously by several authors, providing indication for content range as shown in Table 2 (Mendes et al., 2013; Prozil, Evtuguin, & Lopes, 2012; Yu & Ahmedna, 2013). 1. Introduction 9 Table 2 Chemical composition of grape pomace a Components % dry weight Ashes 7.0 – 7.8 Extractives Dichloromethane 1.0 – 5.5 Water 23.7 – 26.4 Proteins 6.1 – 18.8 Tannins 13.8 – 15.9 Cellulose 20.8 – 30.3 Hemicelluloses 12.5 – 21.0 a Data from Mendes et al. (2013); Prozil et al. (2012); Yu and Ahmedna (2013) Many authors highlight the importance in research and development of emerging recovering technologies in order to get advantages from the bioactives compounds in by-products. In this context, numerous systems have been developed in order to extract such bioactives compounds, which as described above have important properties that can be exploited in fields of pharmaceutical, and food industries and also nutrition sciences, cosmetic and medicine (Fontana, Antoniolli, & Bottini, 2013). 1.3. Polyphenolic compounds: antioxidant properties Phenolic compounds (or just polyphenols) represent a wide family of compounds, including various groups of molecules classified as plant secondary metabolites. Phenolics have been considered the most important, numerous and ubiquitous groups of compounds in the plant kingdom (Naczk & Shahidi, 2004). More than 8,000 different compounds have been identified and the number is still growing (Ignat, Volf, & Popa, 2011), including complex chemical structures which exert diverse biological functions. 1. Introduction 10 In order to simplify the understanding, a first classification can be made based on their solubility. The water-soluble polyphenols comprise compounds such as phenolic acids, phenylpropanoids, flavonoids and quinones; whilst those which are water-insoluble include: condensed tannins, lignins and cell-wall bound hydroxycinnamic acids (Haminiuk, Maciel, Plata-Oviedo, & Peralta, 2012). Vermerris & Nocholson classified these bioactive compounds according to the number of phenol rings they contain in: phenolic acids, stilbenes, flavonoids, lignins and tannins (Vermerris & Nicholson, 2006). All these groups present one or more hydroxyl groups directly attached to an aromatic ring, conferring the phenolic characteristics. Flavonoids, the most important single group of polyphenols, include 13 subclasses with more than 5,000 different compounds present mainly in fruits and plants (Bravo, 1998; Haminiuk et al., 2012). Among these subclasses, compounds namely, chalcones, dihydrochalcones, aurones, flavones, flavonols, dihydroflavonol, flavanones, flavanols, flavandiol, anthocyanidins, isoflavoinids, bioflavonoids, and proanthocyanidins or condensed tannins, can be found in food sources. The flavonoids basic structure consists in a common diphenylpropanes (C6-C3-C6) skeleton with an essential structure consisting in two aromatic rings, A and B joined by a 3-carbons bridge, usually in the form of an oxygenated heterocyclic ring C, as shown in Figure 3. Variations in the substituent groups in the ring C give the major flavonoid aforementioned subclasses. Moreover, flavonoids can be found in a non-glycosylated form (aglycone) as occasionally occur in plants, or most commonly attached to a sugar molecule (glycoside) (Bravo, 1998; Ignat et al., 2011). 1. Introduction 11 Considering the importance of flavonoids compounds, a brief description, highlighting the most remarkable characteristics of main subclasses, is given bellow. Flavonols, the most ubiquitous flavonoids in foods, include as main representative compounds, kaempferol and quercetin. They present strong antioxidant properties, mainly quercetin, through the free radical scavenging activity. Quercetin (Figure 4) presents the three fundamental criteria to be considered a strong free radical scavenger as follow:  The O-dihydroxy structure in the B ring, which confers higher stability to the radical form and participates in electron delocalization;  The 2,3 double bond in conjugation with 4-oxo function in the C ring is responsible for the electron delocalization from the B ring, in other words, the antioxidant tendency is associated to this structure regarding the resonance effect of the aromatic nucleus;  The 3- and 5-OH groups with 4-oxo function in A and C rings are required for maximum radical scavenging potential (Rice-Evans et al., 1996). Figure 3. Basic structure and numbering of the flavonoid nucleus. Source: Bravo (1998). 1. Introduction 12 Good flavonols sources are onions, curly lake, blueberries, passion fruit, pomegranate and broccoli. Red wine and tea also contain up to 45 mg flavonols per portion (El Gharras, 2009; Haminiuk et al., 2012). Flavones are much less common than flavonols in fruit and vegetables. Significant quantities are found in the polymethoxylated form as tangeretin, nobiletin and sinensetin in the skin of fruit citrus (essential oil of mandarin, for example). The only important edible sources of flavones identified till these days are parsley and celery. These polymethoxylated flavones are the most hydrophobic flavonoids (Manach, Scalbert, Morand, Rémésy, & Jiménez, 2004). Flavanones are found in tomatoes and certain aromatic plants such as mint, but they are present in high concentrations only in citrus fruit. They can appear in the aglycone form like naringenin in grapefruits, hesperidin in oranges, and eriodictyol in lemons. Nevertheless, the most common forms appear generally as glycosylated (O- or C-glycosides) by a disaccharide in certain cases or by a rutinose in others. High flavanones concentrations are found in the solid parts of citrus fruit, particularly the albedo (the white spongy portion) and the membranes separating the segments (Bravo, 1998; El Gharras, 2009). Isoflavones, such as daidzein and genistein, with ring B of the flavone molecule attached to the carbon 3 of the heterocycle, especially occur in legumes (Bravo, 1998). According to El Gharras isoflavones are provided only by soybeanderived products. They can be present as aglycones or glycosides, depending on the soy preparation. Soya and its processed products are the main source of isoflavones in the human diet (El Gharras, 2009). The interest in this group of Figure 4. Chemical structure of quercetin. Source: Rice-Evans, Miller, and Paganga (1996). 1. Introduction 13 compounds lies in the fact that certain physiological effects are attributed to their similar structure to estrogens like ß-estradiols. Additionally, for this reason they are sometimes described in the literature as "phytoestrogens" (Ignat et al., 2011). Flavanols comprise two types of associations that may exist between compounds. On one hand, it may exist in the form of monomers like catechins and On the other hand, it is also possible to find them in the polymer form reveling a more complex structure (proanthocyanidins). Some sources for catechins are many types of fruits like apricots and sweet cherry, some beverages such as red wine, although green tea and chocolate are the richest sources (Manach et al., 2004). Catechin, epicatechin and gallocatechin are the monomeric constituents of the condensed tannins, although they are also commonly found as free monomers (Bravo, 1998). Catechin and epicatechin are the main flavanols in fruit, whereas gallocatechin, epigallocatechin, and epigallocatechin gallate are found in certain seeds of leguminous plants, in grapes, and more importantly in tea (Manach et al., 2004). Likewise, most of the polyphenolic compounds, catechins and their esters, particularly epigallatocatechin gallate present in the green tea, have shown anticarcinogenic actions in human and animal tissues (Rice-Evans et al., 1996). According to a research work by Arts et al. (2002) the intake of catechin originating from fruits, but not from tea, was associated to a lower risk of cancer of the upper-digestive tract (Arts, Jacobs Jr, Gross, Harnack, & Folsom, 2002). Finally the last groups of compounds belonging to flavonoids are the watersoluble vacuolar pigments that may appear as red, purple, or blue depending on the pH, are the anthocyanins (Ignat et al., 2011). The term anthocyanin refers to the glycoside of anthocyanidins (Bravo, 1998). The anthocyanidins consist of an aromatic ring A bonded to an heterocyclic ring C that contains oxygen, which is also linked by carbon-carbon bond to a third aromatic ring B (Konczak & Zhang, 2004). Up to now there are reports of more than 500 different anthocyanins and 23 anthocyanidins (Castañeda-Ovando, Pacheco-Hernández, Páez-Hernández, Rodríguez, & Galán-Vidal, 2009), although the most frequently reported in the plant kingdom are the following six 1. Introduction 20 Generally speaking, the quantification of the polyphenols in grape pomace starts by evaluating the Total Phenolic Content (TPC), with forward steps consisting in an evaluation of the antioxidant capacity (through more than one single methodology) and a complementary identification and quantification of the individual phenols. The simplest method for a fast estimation of TPC is the measurement of absorption at 280 nm (in a suitably diluted sample). The second method most commonly used for TPC assessment is the Folin−Ciocalteu assay (Fontana et al., 2013) also named Folin-Ciocalteu reducing assay (FCR). The FCR actually measures the sample’s reducing capacity, but this is not reflected in the name “total phenolic assay” (Huang et al., 2005). It has been strongly recommended to use at least two methods for the assessment of antioxidant properties when working with complex matrixes (Schlesier, Harwat, Böhm, & Bitsch, 2002). It is also advantageous to select methods that are commonly accepted, validated and standardized, with a large body of comparable data available in the literature (Magalhães et al., 2008). According to Prior et al., there are certain requirements or criteria which must be followed in order to select and accurately standardize an “ideal” methodology for the antioxidant capacity assessment: (i) measures chemistry actually occurring in potential application(s); (ii) utilizes a biologically relevant radical source; (iii) simple; (iv) uses a method with a defined endpoint and chemical mechanism; (v) instrumentation is readily available; (vi) good within-run and between-day reproducibility; (vii) adaptable for assay of both hydrophilic and lipophilic antioxidants and use of different radical sources; (viii) adaptable to “high-throughput” analysis for routine quality control analyses (Prior et al., 2005). Particularly when working with grape pomace many spectrophotometric assays have been proposed: DPPH• (2,2′-diphenyl-1-picrylhydrazyl) assay, ORAC (Oxygen Reactive Absorbance Capacity) assay, TEAC (Trolox Equivalent Antioxidant Capacity) and TBARS (Thiobarbituric Acid Reactive Substances) assay. Besides, as some polyphenols are also effective as chelators of transition metal ions (which may induce Fenton-type oxidation reactions in their 1. Introduction 21 free states (Rice-Evans et al., 1996)), assays based on this antioxidant property like iron(II) chelating ability (ICA) assay, have been applied. In DPPH• assay, the purple chromogen radical 2,2′-diphenyl-1-picrylhydrazyl (DPPH•) is reduced by antioxidant/reducing compounds to the corresponding pale yellow hydrazine, following the decrease of absorption at 517 nm. Results are typically expressed in Trolox equivalents (TE). In ORAC assay a peroxyl radical is thermally generated in situ from AAPH (2,2′- azobis(2-amidino-propane) dihydrochloride) which reacts later with a fluorescent probe (typically fluorescein or phycoerythrin). The antioxidant presence avoids the fluorescent probe degradation, prolonging its emission upon time. The quantification is performed measuring the area under curve (AUC) that represents the oxidation of the probe along time. The protective effect of antioxidants is evaluated from the net integrated area under the fluorescence decay curves (AUCsample – AUCblank) and results are expressed as µM of TE (Magalhães et al., 2008). TEAC assay, also called ABTS•+ (2,2′-azinobis-(3-ethylbenzothiazole-6- sulphonate) cation radical assay, have been used for antioxidant quantification of grape pomace extracts (González-Paramás, Esteban-Ruano, Santos-Buelga, de Pascual-Teresa, & Rivas-Gonzalo, 2004). It consists in the measurement of the capacity of a given antioxidant to reduce the stable ABTS•+ radical cation (green/blue specie) into the non-radical and colorless species (ABTS), generally in an aqueous media. Separation and analysis of phenols from grape pomace HPLC (High Performance Liquid Chromatography) techniques are broadly used for the polyphenolic separation and later quantification due to their polar nature. Grape pomace dry powders are accurately dissolved in solvent and filtered before analysis. The columns are almost exclusively of the reverse phase type, with C18 as stationary phase (Lorrain, Ky, Pechamat, & Teissedre, 2013). Combination of columns, solvent systems, and conditions has been successfully applied for the separation of families of phenolics such as 1. Introduction 22 anthocyanins, procyanidins, flavanols, isoflavones, flavonols, phenolic acids, flavanones, and stilbenes (Fontana et al., 2013). Among the detection methods, UV-vis (ultra violet visible), photodiode array detector (DAD), fluorescence and mass detectors were implemented, although UV detection remains the most commonly applied. Improvements in the structural information and the possibility of analysis of high complexity matrixes were brought by to more efficient techniques based on mass spectrometry (MS), such as coupling to liquid chromatography or multiple quadruple MS detectors. 1. Introduction 23 1.6. Poultry meat: global and Portuguese production Poultry is defined as any type of domesticated fowl raised for meat and/or eggs according to National Chicken Council (NCC, 2007), including mainly chicken and turkey. Poultry has been and still is a major animal product in diets. Poultry meat and products are consumed broadly around the world due to different reasons, with no intake restriction associated to religion compared with other kind of meat (pork and beef). There are specialized slaughtering industries with Kosher and Halal slaughter procedures (poultry products with certification on meeting Jewish or Islamic dietary laws and standards regarding slaughter and processing, respectively). Additionally, poultry is recognized as a healthy meat because when consumed skinless, the muscle of birds rarely has higher values than 1 % fat and even less saturated fat than in beef. Its proteins are easily digestible and assimilable. Nutritionally, people eat poultry meat for its high content of high-quality protein with all the essential amino acids. Besides, poultry meat has a great potential to be industrialized offering a wide range of food choices for consumers. In addition, with the advances in preservation techniques for fresh poultry and processed products, consumer preferences for poultry and poultry products are higher than ever (Guerrero-Legarreta, 2010). In terms of global production, five countries concentrate the production, ranking as follows: United States of America (USA) (18 %), China (14 %), Brazil (12 %), Russian Federation (4 %) and Mexico (3 %) with a total world production of 92,730,419 MT (millions of tons) of chicken meat, according to FAOSTAT (FAOSTAT, 2012). In Figure 5 is presented the ranking for the ten major producers of poultry meat. 1. Introduction 24 European countries represent almost 17 % of the total world production (15,435,698 MT). The leading countries in poultry meat production are France, closely followed by UK, Spain, Germany and Poland. These five member states account for 60 % of total EU production of poultry meat (EUROSTAT, 2012). Globally speaking, according to Henchion et al., it is sharply established an increasing trend in meat consumption in kg per capita between 1990 and 2009 (Henchion, McCarthy, Resconi, & Troy, 2014), accompanied by a downward Figure 6. European production of poultry meat. Source: EUROSTAT (2012). Other EU-27 30% FR 14% UK 13% ES 11% DE 11% PL 11% IT 10% Figure 5. World production (MT) of chicken meat. Source: FAOSTAT (2012). 0 10,000,000 20,000,000 USA China Brazil Russian Federation Mexico India Iran Indonesia Turkey Argentina 1. Introduction 25 trend for red meat and an important upward trend for white ones, mainly in poultry meat (see Table 3). Possible relative prices of different types of meat can be highlighted as the main reason, as the real price of beef is higher than poultry and pig meat in most countries (Guerrero-Legarreta, 2010). Table 3 Global meat consumption, 1990-2009, kg/capita, adapted from Henchion et al. (2014) 1990 2009 %change Bovine meat 10.4 9.6 -7.7 Mutton and goat meat 1.7 1.9 11.8 Pig meat 13.2 15.8 19.7 Poultry meat 7.7 13.6 76.6 Other meats 0.7 0.9 28.6 Aggregate 33.7 41.9 24.3 According to the perspective established by the European Commission, poultry meat is expected to overtake pig meat as the most consumed meat in the world by 2022. In the other side, a similar analysis was assumed by Kearney from Dublin Institute of Technology, who projected that by 2050 the consumption of meat will increase moderately, and this will largely mirror increases in pork and particularly in poultry (Kearney, 2010). In the European scenario, the trends in meat consumption are exactly the same where white meat is projected to replace the red meat in Europe as well as globally (European-Commission, 2012). Particularly, in Portugal the chicken meat production is ranked in the sixth place of the total commodities produced in this country ahead of the meat pig 1. Introduction 26 production (millions of tons, MT) (283,999 and 282,951 MT, respectively) (FAOSTAT, 2012) (see Figure 7). In terms of consumption in Portugal among 2008 and 2013, a downward trend for cow and pig meat (-14.3% and - 9.1% respectively) was registered by Instituto Nacional de Estatistica (INE), whilst an upward trend (+ 8%) was observed for poultry meat by the same period (INE, 2013). Figure 7. Portuguese production (MT) for main commodities. Source: FAOSTAT (2012). 0 500,000 1,000,000 1,500,000 2,000,000 2,500,000 Milk (cow) Tomatoes Grapes Vegetables Olives Meat (chicken) Meat (pig) Apples Eggs Meat (cattle) 1. Introduction 27 Once perspectives are exposed, it can be deduced that both locally (Portugal), at European and even at worldwide level, projections regarding poultry sector are highly favorable leading due to changes in relative meat prices, to concepts like “nutrition transition” (Hawkesworth et al., 2010) associated to dietary patterns and lifestyle trends, positioning the chicken in a place of privileged. Figure 8. Evolution of consumption for different types of meat A) pork; B) beef and C) poultry (chicken), in Portugal. Source: INE (2013). 40 42 44 46 48 50 2008 2009 2010 2011 2012 2013 kg/capita 15 16 17 18 19 20 2008 2009 2010 2011 2012 2013 kg/capita 32 33 34 35 36 37 2008 2009 2010 2011 2012 2013 kg/capita B) A) C) 1. Introduction 28 1.7. Mechanically chicken meat: different uses According to Point 1(14) of Annex I to Regulation (EC) Nº 853/2004 and Article 3 (1) (n) to Regulation (EC) Nº 999/2001 “mechanically separated meat or MSM means the product obtained by removing meat from flesh-bearing bones after boning or from poultry carcasses, using mechanical means resulting in the loss or modification of muscle fibre structure”. It is noteworthy that MSM from bovine, caprine and ovine animals are currently prohibited by the European regulations. Mechanically deboned meat (MDM), mechanically recovered meat (MRM) or MSM are synonyms used to designate the same product (Püssa, Pällin, Raudsepp, Soidla, & Rei, 2008). Increasing amount of poultry pieces resulted from the industrialization of chicken, turkey and poultry in general, are generated in processing industries these days. Meat attached to the soft bones can be manually or mechanically separated. Although the original aim of MDM technology application was to reduce the rate of repetitive strain injury of workers caused by short cyclic boning work in cutting rooms of meat operations, the procedure became into a profitable way to generate low-price raw material. Thus, mechanical recovery of poultry from necks, backs and other bones with attached flesh started in the late 1950s. Removal of beef and pork from irregularly shaped bones began in the 1970s (Field, 2004). The texture of the resulting meat product is a finely ground material that has a paste-like consistency in which the myofibrils are heavily fragmented (Barbut, 2002). The overall characteristics and therefore the latter destiny of the MDM depend on the specific part of the animal used, conditions (temperature, aeration, pressure, and contact with metal) during the extraction. Although its use in meat sector represents a low-cost source of animal protein with satisfactory binding capacity, strict regulations concerning to risks associated to the use of MDM are mandatory. 1. Introduction 29 In term of proximate composition (calcium content, moisture, protein, ashes and fat) the values can vary broadly depending on the type of machine, anatomical location of bones, animal species, temperature, and amounts of lean meat (Field, 1988). Depending on the pressure applied (or equipment) during the extractive steps, the final product can be classified into low and high pressure MDM, according to European Legislation. The final characteristics of MDM, in terms of overall appearance, consistency, nutritional composition, and even microbiological loading content, depend on the raw materials used and on the strict procedures followed during the extraction itself. In general, the low pressure MDM or also called “Baader meat”, “3 mm meat” or ”desinewed meat” in the meat sector and it has a similar consistency and appearance to a ground meat, whilst the high pressure MDM consists in a fine-consistency product that even macroscopically can clearly distinguishable from the low pressure MDM as a product with a characteristic and particularly pasty texture resulting from the loss or modification of the muscle fibre structure. The mechanical process of removing meat from the bones causes cell breakage, protein changes and increases fat and haem contents, thus, the final product is subjected to strict regulations concerning its use in food preparations. Currently there are three methodologies for the MDM process: 1) belt-drum system, 2) auger type, and 3) hydraulically powered presses (Barbut, 2002). 1. Introduction 36 Table 7 Hygiene requirements of MDM after production. European-Commission (2010) Low pressure MDM High pressure MDM Storage if not immediately used Wrapped and packaged, chilling at max 2 °C or frozen at an internal T of < - 18 °C Wrapped and packaged, chilling at max 2 °C if processed within 1 to 24 h; if not, frozen within 12 h after production, reaching at an internal T of < - 18 °C within 6 h. Maximal storage of frozen MDM of 3 months at < - 18 °C Use In meat preparations which are clearly not intended to be consumed without heat treatment; In meat products Only for heat-treated meat products produced in approved establishments Applications of mechanically deboned meat (MDM) As mentioned in previous sections, there is a global trend regarding to meat consumption consisting in the replacement of the red meat (pig or beef) for “healthier white meat”, mainly poultry (Henchion et al., 2014). Therefore, the use of MDM, principally poultry meat, has increased in the food industry based on this consumption trend in industrialized countries and also due its lower price compared with other kinds of meat (Daros, Lucia Masson, & Amico, 2005). The main applications of MDM are in products which do not require a fibrous texture but demand emulsion, stability, natural color, and relatively low cost (Barbut, 2002). If all the legal requirements described above for the production of MDM are properly complied and controlled, this product can be used as a satisfactory agent for binding structure in product prepared with minced meat and comminuted meat products namely, frankfurter sausages, meatballs, nuggets, and meat emulsions, including “chicken pate”. 1. Introduction 37 The incorporation of MDM into emulsified meat product (10-35 %) and in lower proportions into nonemulsified meat product (1-20 %) has opened up additional markets for this type of meat (Mielnik, Aaby, Rolfsen, Ellekjær, & Nilsson, 2002). Most of these meat products, formulated primarily to suit the local palate, not only target the changing needs of consumers in terms of convenience, nutrition, quality and variety, but also allow a broad marketing of new alternatives (Guerrero-Legarreta, 2010). Therefore, many exotic recipes and ready-to-cook marinated products are presented as dietary convenient options not only for households holding a single individual but also for large family nucleus. Overall aim Once established the state-of-art concerning necessary fundamentals which indicate that implementation of GPE in order to reduce lipid oxidation of MDM still have not been explored, it is pretended as overall aim of the thesis: To characterize skin and seed grape (pomace) extracts from Portuguese varieties towards the prevention of the lipid oxidation of mechanically deboned chicken meat (MDM), including the assessment of the final physico-chemical characteristics and consumer acceptance of nuggets containing MDM and supplemented with grape extracts. 1. Introduction 38 References Arts, I. C., Jacobs Jr, D. R., Gross, M., Harnack, L. J., & Folsom, A. R. (2002). Dietary catechins and cancer incidence among postmenopausal women: the Iowa Women's Health Study (United States). Cancer Causes & Control, 13(4), 373-382. Atanacković, M., Petrović, A., Jović, S., Bukarica, L. G., Bursać, M., & Cvejić, J. (2012). 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Cunha1,* 5 6 1 REQUIMTE/DGAOT Faculdade de Ciências, Universidade do Porto, Rua 7 Padre Armando Quintas, 7, 4486-661 Vairão, Portugal 8 2Faculdade de Ciências da Nutrição e Alimentação, Universidade do Porto, Rua 9 Dr. Roberto Frias, 4200-465 Porto, Portugal 10 3 UCIBIO, REQUIMTE, Departamento de Ciências Químicas, Faculdade de 11 Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 12 Porto, Portugal 13 *Corresponding author: [email protected] 14 Tel.: (+351)252660400 / Fax: (+(351)252661780 15 16 ABSTRACT: 17 Grape pomace can be regarded as an excellent and affordable source of 18 polyphenolic compounds. Hence, the main objective of this work was to conduct 19 a comparative study of different Portuguese grape varieties, using an extraction 20 methodology with possible applications in sustainable agriculture and pest 21 management. Scavenging capacity against DPPH•, oxygen radical absorbance 22 capacity (ORAC), iron(II) chelating ability (ICA) and Folin-Ciocalteu assays were 23 performed in order to evaluate the antioxidant capacity profile and total phenolic 24 content (TPC) in ethanol/water extracts and aqueous grape pomace 25 suspensions. Strong significant correlation between TPC and DPPH• (R = 26 0.944), and a low correlation between ORAC and the other assays was 27 obtained (R ≤ 0.632). ICA was not correlated with any of the other assays (R ≤ 28 0.263). All grape pomace extracts have presented high antioxidant properties 29 (ORAC) and chelating ability, ranging from 906 to 2337 µmol TE g-1 residue and 30 from 55 to 104 % inhib. mg-1 residue, respectively. Results from HPLC analysis 31 showed the presence of gallic acid, caffeic acid, syringic acid, (+)-catechin and 32 (-)-epicatechin being syringic acid and (+)-catechin the major compounds. 33 Although further studies are required, “Touriga Nacional” was the most 34 promising grape variety regarding its highest values for TPC (142.4 ± 1.1 mg 35 58 GAE g-1 dry residue), DPPH• (1.12 ± 0.04 mmol TE g-1 dry residue) and ORAC 36 (1579 ± 244 µmol TE g-1 dry residue) assays. Since Portugal is a major wine 37 producer, utilization of pomace generated during the wine elaboration steps 38 opens a new trend towards compounds extraction with high antioxidant activity 39 in order to contribute to emerging industrial applications and sustainable 40 agriculture. 41 42 Keywords: antioxidant capacity, food products, Portuguese varieties, red grape 43 pomace, environmental-friendly extraction. 44 45 Research highlights 46  Grape pomace from Portuguese varieties was targeted as source of 47 polyphenolic compounds. 48  An environmentally friendly extract with polyphenols was obtained. 49  Extracts showed bioactive properties in radical scavenging and iron(II) 50 chelating assays. 51  “Touriga Nacional” grape variety showed the highest potential for 52 industrial applications. 53 54 59 1. Introduction 55 56 Portugal is now the eighteenth largest global grape producer, with a production 57 close to 839,000 million tons (FAOSTAT, 2012). Winemaking process 58 generates significant amount of wastes (steams, seeds, skins and marcs). It is 59 estimated that for each 6 liters of wine, 1 kg of grape pomace is produced which 60 is mainly destined to animal feed and for compost elaboration (Mendes, Prozil, 61 Evtuguin, & Lopes, 2013). Nevertheless, large amounts of the residual 62 quantities of bioactive substances are maintained into the vegetable tissues 63 (Lapornik, Prošek, & Golc Wondra, 2005). Due to the chemical composition in 64 the final grape waste (high content of sugars, tannins, polyphenols, 65 polyalcohols, pectins and lipids), effluent treatment considerably increase in the 66 chemical oxygen demand (COD) and the biochemical oxygen demand (BOD5). 67 Considering the above, for the industrial sector this situation represents a low 68 cost source of usable polyphenolic compounds. Furthermore, considering the 69 current governmental and legislative pressures, this fact tends to eliminate or 70 reduce the costs associated with effluent treatments. 71 In this context, phenolic compounds present in grape pomace represent 72 bioactive substances with many applications related to healthy benefits: 73 scavenging activity against free radicals, anti-inflammatory properties (Terra et 74 al., 2007), anti-proliferation and cancer therapy (Nandakumar, Singh, & Katiyar, 75 2008). A literature search revealed that the number of publications on the key 76 words: “antioxidant” and “grape pomace” has strongly increased in the last ten 77 years emphasizing the growing interest in the topic. Currently the available 78 information on the polyphenolic compounds from Portuguese grape pomace is 79 limited. Most of the published work are focused on the composition and 80 antioxidant activity of Portuguese grape (whole or parts of the grape) (Cosme, 81 Ricardo-Da-Silva, & Laureano, 2009; Dopico-García et al., 2008; Matias et al., 82 2010; Paixao, Perestrelo, Marques, & Camara, 2007) others have centered 83 their work on the pomace chemical composition (Mendes et al., 2013; Prozil, 84 Evtuguin, & Lopes, 2012), but mainly connected to wines (Baptista, Tavares, & 85 Carvalho, 2001; Jordao et al., 2010; María Monagas, Gómez-Cordovés, 86 Bartolomé, Laureano, & Ricardo da Silva, 2003). 87 60 Grape pomace extracts represent widespread uses in pharmaceutical, 88 cosmetic, and the most recent is linked to the new class of “phytosanitary 89 bioproducts” able to control the incidence of diseases in some crops (Benouaret 90 et al., 2014), Thus, data concerning the polyphenolic content and antioxidant 91 capacity from Portuguese grape pomaces aiming its valorization, are still 92 scarce. Hence, the present study aims to determine the antioxidant profile and 93 the total phenolic content of Portuguese grape pomace extracts. When dealing 94 with complex matrixes coming from vegetables tissues, in order to appropriately 95 assess the antioxidant capacity, experiments should be done through more than 96 one assay, and at least two methods have been recommended (De Nisco et al., 97 2013). Considering this, two antioxidant assays (DPPH• and ORAC) were 98 established. Moreover, the ability of grape pomace extracts to chelate iron(II) 99 was also performed. Since, extraction is a primordial step because it influences 100 the further application of the phenolic compounds, ethanol was chosen as 101 extractive liquid. Firstly, it is the natural solvent present in wines (Spigno, 102 Tramelli, & De Faveri, 2007), it is safe (Shi et al., 2005), due to its relatively low 103 boiling point (volatile) which facilities the elimination and recovering steps, and 104 finally it has an environmentally friendly behavior (Corrales, García, Butz, & 105 Tauscher, 2009), compared to other organic solvents, namely methanol. We 106 also compared the final antioxidant properties in ethanol/water extracts and 107 aqueous re-suspensions of their dry residue in order to enhance the approach 108 towards the grape pomace emerging applications (sustainable pest 109 management, biopesticides and controlled animal diet). It is also intended to 110 select the Portuguese grape variety under study that would be most suitable for 111 future polyphenolic extraction processing towards bioactive products recovery. 112 To our knowledge, this paper represents one of the few attempts to assess the 113 polyphenolic content and the antioxidant profile of the pomace coming from the 114 most representative red grape varieties in Douro, Portugal with perspectives 115 towards a sustainable agriculture and an environmentally friendly pest 116 management approach. 117 118 2. Material and methods 119 2.1. Chemicals 120 61 All chemicals used were of analytical reagent grade. 2,2-diphenyl-1- 121 picrylhydrazyl (DPPH•), 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4,4-disulfonic 122 acid sodium salt (ferrozine) and 2,2-azobis(2-methylpropionamide) 123 dihydrochloride (AAPH) were purchased for Aldrich (Milwaukee, WI). Folin- 124 Ciocalteu (F-C) reagent and fluorescein sodium salt were obtained from Sigma 125 (St. Louis, MO), while iron(II) chloride tetrahydrate, gallic acid, and (±)-6- 126 hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) were obtained 127 from Fluka (Buchs, Switzerland). HPLC standards (Gallic, p-hydroxybenzoic, 128 caffeic, syringic, p-coumaric and o-coumaric, sinapic, ferulic acids employed for 129 Phenolic Acids (PA method), whilst (+)-Catechin, (-)-Epicatechin, (-)-Epicatechin 130 gallate, trans-resveratrol, quercetin, kaempferol and chlorogenic acid, used for 131 Anthoxanthins and Stilbenes (AX method), were purchased from Sigma. Water 132 from Sartorius AG system (resistivity > 18 MΩ cm) (Göttingen, Deutschland) 133 and absolute ethanol p. a. (Panreac Química, Spain) were used in the 134 preparation of all solutions. 135 136 137 138 2.2. Solutions 139 For assessment of total phenolic content (TPC), the commercial F-C reagent 140 was diluted 3:10 (v/v) in water. A solution of Na2CO3∙10 H2O 24.3% (w/v) was 141 prepared, corresponding to 9% (w/v) of sodium carbonate, and also gallic acid 142 standard solutions (1.0 - 15.0 mg L-1) for calibration purposes. For the DPPH• 143 assay, a stock solution of DPPH• in ethanol (600 µM) was prepared and kept in 144 dark at room temperature. Three dilutions from the stock DPPH• solution 145 (between 75 and 225 µM) were prepared in ethanolic solution 50% (v/v) in order 146 to determine the dilution factor needed to provide an absorbance value of 0.900 147 ± 0.020 at 517 nm, after dilution in the microplate well. For DPPH• assay, all 148 Trolox standard solutions (5.0 - 50.0 µM) were prepared in ethanolic solution 149 50% (v/v). For iron(II) chelating ability (ICA) assay, all iron(II) solutions were 150 freshly prepared including the stock solution (6 mM) at pH 3.0 and the iron(II) 151 solution (0.12 mM) added to microplate. The ferrozine solution (0.6 mM) and a 152 solution of acetate buffer (50 mM) were also prepared. For oxygen radical 153 absorbance capacity (ORAC) assay, AAPH (40 mM) and fluorescein stock 154 68 ANOVA models were applied for each assay. Moreover multiple comparisons 353 between varieties were performed using Tukey test. Except if referred, all tests 354 were applied with a 95% confidence level. Statistical data analysis was 355 performed with IBM SPSS Statistics for Windows version 21.0 (IBM SPSS 356 Statistics, New York) 357 358 3. Results and Discussion 359 Different assays were performed in order to evaluate the total phenolic content 360 (TPC), scavenging capacity (DPPH• and ORAC) and iron(II) chelating ability 361 through ICA assay in the ethanol/water extract obtained from the grape pomace 362 and in the aqueous suspension obtained from the dry residue of the previous 363 extract, according to the extraction scheme depicted in Figure 1. 364 365 69 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 Figure 1. Schematic representation of the extraction of polyphenolic 390 compounds present in samples of “Tinta Roriz”, “Touriga Franca”, “Touriga 391 Nacional” and Mix red grape pomace. The whole procedure was performed in 392 duplicate for each grape variety. DPPH•: 2,2-diphenyl-1-picrylhydrazyl radical 393 scavenging; ORAC: oxygen radical absorbance capacity; ICA: iron(II) chelating 394 ability. 395 396 The Folin-Ciocalteu assay is a method commonly used for the total phenolic 397 content measurement and it is based on the ability of certain compounds 398 (phenolic and nonphenolic) in alkaline medium to reduce the phos-399 Grape pomace (20 g) Filtration (Assisted by vacuum, 45 µm membrane) Filtrate c oncentration (Rotavapor, 65 ºC, N 2 flow, vacuum) Folin-Ciocalteu DPPH • ORAC ICA assays Extraction (100 ml ethanol/water 80% v/v, under orbital agitation at 300 rpm 48 h) Ethanol/water extracts (Discarded solid) Dry residue weighing (*) Re - suspension (50 ml water) Folin-Ciocalteu DPPH • ORAC ICA assays Aqueous suspensions Volume adjusted to 100 ml (*) Basis for mass calculation 70 phomolybdic/phosphotungstic acid reagent to complexes, which is 400 spectrophotometrically detected (Luís M. Magalhães, Segundo, Reis, & Lima, 401 2008). In the DPPH• assay, DPPH• radical is reduced due to the presence of 402 antioxidant compounds causing decrease in the absorbance values at 515 nm. 403 Upon reduction, the color of the solution fades (D. Huang, Boxin, & Prior, 2005). 404 The principle for ORAC assay is based on the intensity of fluorescence 405 decrease of the target/probe along time under constant flux of peroxyl radicals 406 (due the thermal decomposition of AAPH) in aqueous buffer. When a sample is 407 analyzed due to the presence of chain-breaking antioxidants the decay of 408 fluorescence is inhibited (Luís M. Magalhães et al., 2008). 409 In the ICA assay, the presence of chelating compounds in the sample, disrupt 410 the complex formed between ion(II) and ferrozine. The colour decreasing on the 411 ion(II)-ferrozine complex monitored at 562 nm is taken as an estimation of the 412 chelating activity. 413 Firstly an extract was obtained from an ethanol/water (80% v/v) solvent, after 414 filtration step. Aqueous suspensions were obtained once the previous solvent 415 was evaporated and the obtained dry extract was re-suspended in water. 416 Before the re-suspension, dry residues obtained were weighted and the 417 percentage of residue recovered per g of initial dry pomace (% extraction yield) 418 was calculated. The percentages for extraction yield were: 3.8 % (“Touriga 419 Nacional”); 6.5 % (“Touriga Franca”); 3.8 % (“Tinta Roriz”); and 6.1 % (Mix) in % 420 g of dry residue per g of dry pomace. Due to these differences and in order to 421 carry an appropriate assessment, results of all analyses were expressed per 422 gram of dry residue (Table 1). 423 71 424 Table Nº1 TPC, antioxidant capacity determined by DPPH • and ORAC assays and iron(II) chelating ability for ethanol/water extracts and aqueous suspensions. Grape varieties TPC†,§ (mg GAE g-1 residue) DPPH•†,§ (mmol TE g-1 residue) ORAC‡,§ (µmol TE g-1 residue) ICA†,§ (%inhibition mg-1 residue) Ethanol/water extract Aqueous suspension Ethanol/water extract Aqueous suspension Ethanol/water extract Aqueous suspension Ethanol/water extract Aqueous suspension TR 69.3 c ± 1.9 75.8 d ± 4.0 0.52 c ± 0.15 0.59 d ± 0.02 1054 c ± 199 1230 b ± 91 76 b ± 5 45 c ± 6 TF 100.1 b ± 7.4 106.1 b ± 1.9 0.87 b ± 0.04 0.90 b ± 0.02 1343 c ± 102 1325 b ± 147 63 c ± 10 55 b,c ± 14 TNac 131.7 a ± 8.1 142.4 a ± 1.1 1.09 a ± 0.13 1.12 a ± 0.04 2337 a ± 368 1579 a ± 244 70 b,c ± 12 66 a,b ± 9 Mix 104.1 b ± 5.5 102.5 c ± 1.8 0.81 b ± 0.09 0.86 c ± 0.02 1649 b ± 164 906 c ± 66 109 a ± 17 73 a ± 10 † Values represent means of quadruplicate ± S.D. ‡ and triplicate ± S.D. § Data were analyzed by MANOVA and within each column different letters indicate statistically differences according to the Tukey multiple comparison test at 95% confidence level (TPC: total phenolic content; DPPH•: 2,2-diphenyl-1-picrylhydrazyl radical assay; ORAC: oxygen radical absorbance capacity; ICA: iron(II) chelating ability; GAE: gallic acid equivalents; TE: Trolox equivalents). 72 3.1. Total phenolic content and antioxidant capacity 425 For ethanol/water extracts, TPC assay values for all samples from single 426 varieties have shown significant differences (P = 0.05) as presented in Table 1. 427 Values for TNac sample were 1.9-fold higher (131.7 ± 8.1 mg GAE g-1 residue) 428 than the average for TR (69.3 ± 1.9 mg GAE g-1 residue). The same result was 429 observed for anti-radical activity assessed through the DPPH• assay, as 1.09 ± 430 0.13 mmol TE g-1 residue and 0.52 ± 0.15 mmol TE g-1 residue were the highest 431 and the lowest values obtained for the same samples. For ORAC assay, TNac 432 sample exhibited the strongest peroxyl scavenging capacity with an average of 433 2337 ± 368 µmol TE g-1 residue, while values for TR and TF were 1054 ± 199 434 µmol TE g-1 residue and 1343 ± 102 µmol TE g-1 residue, respectively. Metal 435 chelating ability represents an important aspect of the antioxidant properties of 436 the polyphenolic compounds. Most of the main strategies to avoid reactive 437 oxygen species (ROS) formation involves ion chelation (Ebrahimzadeh, 438 Pourmorad, & Bekhradnia, 2008). Any compound which exhibits the ability of 439 complexing ions, avoiding or reducing damages caused due to the pro-oxidant 440 effect of transition metals, can be recognized as a potential antioxidant. All the 441 samples have presented important metal binding capabilities, measured 442 through ICA assay. Nevertheless, related to the chelating ability to iron(II), 443 samples did not follow the previous trend. In the ICA assay, Mix sample 444 quelated all available iron (109 ± 17 % inhibition mg-1 residue) and on the other 445 hand, values for TF and TNac samples were the lowest (63 ± 10 %inhibition mg- 446 1 residue and 70 ± 12 %inhibition mg-1 residue). The chelating potential is 447 strongly dependent on the arrangement of hydroxyls and carbonyl group around 448 the molecules (Gülçin, 2012), therefore it depends on specific polyphenolic 449 compounds present in the extract. In this case, there seems to be a synergetic 450 combination of the compounds within Mix sample which increased its ICA 451 values in comparison with those from single variety extracts. 452 When aqueous suspensions are compared, results are similar to those obtained 453 for ethanol/water extracts for TPC and DPPH• assays, where TNac and TR 454 samples showed the highest (142.4 ± 1.1 mg GAE g-1 residue; 1.12 ± 0.04 455 mmol TE g-1 residue) and the lowest (75.8 ± 4.0 mg GAE g-1 residue; 0.59 ± 456 0.02 mmol TE g-1 residue) results, respectively. On the other hand, when 457 evaluating ORAC values among the samples analyzed TNac exhibited the 458 73 strongest peroxyl scavenging capacity (1579 ± 244 µmol TE g-1 residue), and 459 Mix sample corresponded to the lowest value (906 ± 66 µmol TE g-1 residue) in 460 terms of ORAC. Concerning to chelating ability, TR exhibited the lowest ICA 461 values, whilst Mix with 73 ± 9.7 %inhibition mg-1 residue, was 1.6-folds higher 462 than TR regarding to ICA values. Data presented suggests that Mix interfered 463 better than other extracts evaluated, in the iron(II)-ferrozine complex formation 464 by chelating more iron(II) before ferrozine addition. 465 Published data related to extracts from the grape varieties present in this study 466 has not been found in the literature for comparison purposes. Nevertheless, 467 Negro et al. (Negro, Tommasi, & Miceli, 2003) has obtained similar results, 1.40 468 g GAE L-1 extract for TPC values, our results ranged from 1.17 to 2.79 g GAE L- 469 1 extract, (see Table S1 and S2 supplementary data), having worked with marc 470 pomace extract from “Negro amaro” variety under comparable extraction 471 conditions (80% v/v ethanol/water). Jordão et al. (Jordao, Simoes, Correia, & 472 Goncalves, 2012) have recently presented data about wines from TR and TNac 473 winemaking process. Their TPC values were in average 2771 ± 32 mg GAE L-1 474 wine and 3216 ± 105 mg GAE L-1 wine for TR and TNac, respectively. It is 475 important to note that during the traditional winemaking process (maceration), 476 due to the contact between must-seeds and skins and the mass transfer 477 phenomena, once this step is completed, seeds and skins will contain less 478 quantity of polyphenolic compounds. Additionally, Lapornik et al. (Lapornik et 479 al., 2005) have compared extracts prepared from plant by-products using 480 different conditions (solvents and extraction time) and extracted 5790 mg GAE 481 L-1 from grape pomace. Different extraction condition may be the origin of the 482 variance between the values. Cristino et al. (Cristino, Costa, Cosme, & Jordao, 483 2013) have recently published lower values related to total antioxidant capacity 484 for red wines from two Portuguese Appellations of Origin measured by DPPH• 485 with values ranging between 8.0 ± 1.7 and 23.3 ± 0.5 TE mM having worked 486 with 0.1 mL sample. On the other hand, concerning the solvent used in the 487 extraction step, Rockenbach et al. (Rockenbach et al., 2011) have worked with 488 pomace ( skins or seeds) from different Brazilian red grapes extracted by 489 contact with an acidified mixture using methanol instead of ethanol. Their values 490 were in average 2076 and 8517 µmol TE 100 g-1 of dry pomace for skins or 491 seed extracts, respectively. Our results (ranged 2188 - 5688 µmol TE 100 g-1 of 492 74 dry pomace, see Table S2 supplementary data) are comparable having worked 493 with different extractive mixture, and moreover under extraction conditions safer 494 and environmentally friendly as methanol is replaced by ethanol in our case. 495 Regarding ORAC results, our values (906 - 1579 μmol TE g-1 residue, see 496 Table S2 supplementary data) are 3.3-folds lower than those presented by 497 Hogan et al. (Hogan, Canning, Sun, Sun, & Zhou, 2010). Hogan et al. have 498 worked with Norton GPE and they obtained 4133 ± 94 μmol TE g-1 GPE using 499 ethanol 80% (v/v) as extractive solvent under overnight shaking. Yilmaz and 500 Toledo (Yilmaz & Toledo, 2006) have worked with grape/wine industry 501 byproducts and studied the antioxidant properties of the grape and seed 502 extracts through ORAC assay. Their ORAC values ranged from 311 to 638 503 µmol TE g-1 dry seed, and 70 to 103 µmol TE g-1 dry skin. It is important to note, 504 when comparisons were carried on, most of the published literature have 505 presented the results having evaluated parts of the grape separately, not whole 506 grape or pomace. 507 According to previous work published by Ebrahimzadeh et al. our findings are 508 comparable in terms of iron(II) chelating ability. They have worked in the 509 assessment of the iron chelating activity of some medicinal plants from Iran and 510 published values 18.20% inhib. (Feijoa sellowiana, aqueous extract) and 20.8 511 %inhibition (Sambucus ebulus, aqueous extract), both extracts at 3.2 mg ml-1 512 (Ebrahimzadeh et al., 2008). 513 514 3.2. Evaluation of solvent change 515 Results for TPC, DPPH•, ORAC and ICA assays for ethanol/water extracts and 516 aqueous suspensions were shown in Figure 2. 517 518 75 519 Figure 2. (a) Folin: total phenolic content (TPC) (mg GAE g-1 residue); (b) 520 DPPH•: scavenging capacity against DPPH• (mmol TE g-1 residue); (c) ORAC: 521 0 30 60 90 120 150TNAC TR TF MIX (a) Folin(*) 0 25 50 75 100 125TNAC TR TF MIX (d)ICA(*) 0 750 1500 2250 3000TNAC TR TF MIX (c) ORAC(*) (b) DPPH• 0.00 0.30 0.60 0.90 1.20TNAC TR TF MIX (b) DPPH• 76 oxygen radical absorbance capacity (µmol TE g-1 residue) and (d) ICA: iron(II) 522 chelating ability (%inhib. mg-1 residue) for TR (“Tinta Roriz”), TF (“Touriga 523 Franca”), TNac (“Touriga Nacional”) and Mix. (Ethanol/water extracts (solid grey 524 line), aqueous suspensions (dotted line), GAE: gallic acid equivalents; TE: 525 Trolox equivalents). Homogenous groups according to the Tukey multiple 526 comparison test at 95% confidence level. 527 528 Therefore, comparing results for ethanol/water extracts and aqueous 529 suspensions for all assays, and according to a two-factor MANOVA for variety 530 and solvent, with solvent nested into variety, significant differences (P < 0.05; 531 Lambda de Wilks < 0.01) were found for all methods for grape varieties and 532 significant differences were also found for TPC, ORAC and ICA assays (P < 533 0.01; Lambda de Wilks < 0.01), excepting for DPPH• assay results. 534 As the same assay protocol was applied to all samples, the differences found 535 must be ascribed to the solvent change or other factors (temperature, light, and 536 oxygen exposure) that took place during the concentration and ethanol/water 537 solvent removal. In fact, TPC average values significantly (P < 0.01) increased 538 and for ORAC and ICA assay values decreased in average for aqueous 539 suspensions compared with ethanol/water extract. Concerning DPPH• values, 540 there was not any significant difference (P = 0.35) between ethanol/water 541 extract and aqueous suspensions. 542 Published data suggests that temperature has an important effect on structure 543 and biological properties of the polyphenolic compounds. The main 544 consequence is the increase of the polymerization degree which turns one 545 monomer into a compound with higher possibilities of radical stabilization in the 546 aromatic ring. It has been described that this improvement reaches its 547 maximum with four monomers as larger molecules may not be so efficient due 548 to steric interaction between functional groups (Pinelo, Rubilar, Jerez, Sineiro, & 549 Nunez, 2005). Since no significant differences were obtained for DPPH• assay, 550 it seems that the combination time-temperature (1 h at 65 ºC) applied during the 551 concentration step was not significant enough to change the polymerization 552 degree and consequently, enhance the extract scavenging capacity against 553 DPPH• radical. It was reported that at higher temperature (1 h at 150 ºC) 554 77 increased four times DPPH• values for aqueous extracts citrus skins (Jeong et 555 al., 2004). 556 Results for ORAC and ICA assays were significantly (P = 0.01) lower after 557 concentration and solvent change (16% and 20%, respectively). The key for 558 binding ion ability is in the chemical structure (functional groups and their 559 number). It was reported that compounds containing C-OH and C=O functional 560 groups can chelate metal ions (Gülçin, 2012). While the temperature effect 561 tends to increase the number of polymers in the final compounds and 562 consequently improved the chelating ability to iron(II), therefore a reduction in 563 the values for ICA may be due to the influence of solvent change rather than 564 chemical differences arisen from temperature exposition. 565 Published data related to the chemical behavior of antioxidant species in ORAC 566 assay is contradictory. Some authors state that as ORAC is based on hydrogen 567 atom transfer (HAT) it should be solvent and pH independent (Gülçin, 2012). 568 Nevertheless, it has been reported that when changes in solvent take place, 569 changes on oxygen radical absorbance capacity were observed. In this context 570 extracts obtained by aqueous solvents exhibited lower antioxidant capacity in 571 comparison to ethanolic mixtures, according to Pérez-Jimenez et al. (Pérez- 572 Jiménez & Saura-Calixto, 2006). Moreover, regarding the solvent effect on 573 DPPH• values, the same authors observed that, among the different antioxidant 574 capacity assays evaluated for the effect of the solvent, DPPH• was the assay in 575 which the influence of the solvent was weakest in comparison with ORAC, 576 ABTS•+ and FRAP (Ferric Reducing Ability of Plasma) (Pérez-Jiménez & Saura- 577 Calixto, 2006). 578 In order to assess the possible interactions from having mixed TNac, TR and TF 579 the contribution of each grape variety was calculated to the final Mix sample 580 (Mix estimated, Table 2). Standard deviations were estimated according to the 581 equation of propagation of the errors. Finally, Mix experimental and Mix 582 estimated values were compared in a one sample t-test (P = 0.05). Our findings 583 suggest that when grape varieties are mixed their potential antioxidant 584 properties in terms of TPC and ORAC values were significantly reduced (P = 585 0.05). 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Anal., 19(1), 41-48. 831 832 833 87 Supplementary data 834 835 Valorization of grape pomace: extraction of bioactive phenolics with 836 antioxidant properties 837 838 839 Hernán H. Tournour1,2, Marcela A. Segundo3, Luís M. Magalhães3, Luisa 840 Barreiros3, Jorge Queiroz1, Luís M. Cunha1,* 841 842 1 REQUIMTE/DGAOT Faculdade de Ciências, Universidade do Porto, Rua 843 Padre Armando Quintas, 7, 4486-661 Vairão, Portugal 844 845 2 Faculdade de Ciências da Nutrição e Alimentação, Universidade do Porto, 846 Rua Dr. Roberto Frias, 4200-465 Porto, Portugal 847 3 UCIBIO, REQUIMTE, Departamento de Ciências Químicas, Faculdade de 848 Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 849 Porto, Portugal 850 *Corresponding author: lm[email protected].pt 851 Tel.: (+351)252660400 / Fax: (+(351)252661780 852 88 853 Table S1 TPC, antioxidant capacity determined by DPPH• and ORAC assays and iron(II) chelating ability (ICA) for ethanol/water extracts. Grape varieties TPC †,§ (mg GAE L-1 extract) DPPH•†,§ ORAC‡ ,§ ICA†,§ (%inhibition ( g L-1 extract)-1) (mmol TE L-1 extract) (mmol TE 100g- 1 dry pomace) (µmol TE g-1 residue) (µmol TE g-1 dry pomace) TR 534d ± 16 4.1c ± 1.2 1.9c ± 0.6 1054c ± 199 40c ± 7.4 22.8b ± 1.5 TF 1313a ± 80 11.9a ± 1.5 5.9a ± 0.7 1343c ± 102 88a ± 8.2 18.4c ± 2.3 TNac 1000c ± 22 8.4b ± 1.5 4.2b ± 0.7 2337a ± 368 88a ± 12.2 21.0b ± 3.6 Mix 1272b ± 71 9.9b ± 1.4 5.0b ± 0.7 1649b ± 164 101a ± 11.6 32.8a ± 1.3 † Values represent means of quadruplicate ± S.D. ‡ and triplicate ± S.D. § Data were analyzed by MANOVA and within each column different letters indicate statistically differences according to the Tukey multiple comparison test at 95% confidence level (TPC: total phenolic content; DPPH•: 2,2-diphenyl-1-picrylhydrazyl radical assay; ORAC: oxygen radical absorbance capacity; ICA: iron(II) chelating ability; GAE: gallic acid equivalents; TE: Trolox equivalents). 89 854 Table S2 TPC, antioxidant capacity determined by DPPH• and ORAC assays and iron(II) chelating ability (ICA) for aqueous suspensions. Grape varieties TPC †,§ (mg GAE L-1 extract) DPPH•†,§ ORAC‡ ,§ ICA†,§ (%inhibition ( g L-1 extract)-1) (mmol TE L-1 extract) (mmol TE 100g- 1 dry pomace) (µmol TE g-1 residue) (µmol TE g-1 dry pomace) TR 1166d ± 65 9.0d ± 0.3 2.2d ± 0.1 1230b ± 91 46c ± 3.3 13.5c ± 1.9 TF 2790a ± 133 22.9a ± 1.6 5.7a ± 0.4 1325b ± 147 88a ± 11.5 16.7b,c ± 4.3 TNac 2170c ± 144 17.0c ± 1.6 4.3c ± 0.4 1579a ± 244 60b ± 12.3 19.6a,b ± 2.8 Mix 2504b ± 122 20.9b ± 1.1 5.2b ± 0.3 906c ± 66 55b ± 5.2 21.7a ± 3.0 † Values represent means of quadruplicate ± S.D. ‡ and triplicate ± S.D. § Data were analyzed by MANOVA and within each column different letters indicate statistically differences according to the Tukey multiple comparison test at 95% confidence level (TPC: total phenolic content; DPPH•: 2,2-diphenyl-1-picrylhydrazyl radical assay; ORAC: oxygen radical absorbance capacity; ICA: iron(II) chelating ability; GAE: gallic acid equivalents; TE: Trolox equivalents). TPC (mg GAE L-1 extract): (A sample -intercept) / slope (mg GAE L-1).DF DPPH• (mmol TE L-1 extract): (A sample -intercept) / slope (µM TE).DF. (0.05 L g-1 residue) DPPH• (mmol TE 100g-1 dry pomace): [(A sample -intercept) / slope (µM TE).DF. (0.05 L g-1 residue). (g residue g-1 dry pomace)].100 ORAC (µmol TE g-1 residue): (relative AUC - intercept) / slope (µM TE). DF. (0.05 L g-1 residue) ORAC (µmol TE g-1 dry pomace): (relative AUC - intercept) / slope (µM TE). DF. (0.05 L g-1 residue) . (g residue g-1 dry pomace) ICA (%inh. (g L-1 extract) -1); Where: %ICA = [(A1-A2)/A1]. 100; A1= A (Fe2+-Ferrozine); A2= A (sample) - A(sample blank), then %ICA= [%ICA/ (g residue / 0.05 L). DF] / 3 (3 because it were used 100 µl each solution in the well) Paper II Hernán H. Tournour, Marcela A. Segundo, Luís M. Magalhães, Jorge Queiroz, M. Beatriz P. P. Oliveira, Luís M. Cunha. Single and successive oxidative stress factors applied to mechanically deboned chicken meat: protective effect of grape pomace extract. [Submitted for publication]. 93 Single and successive oxidative stress factors applied to mechanically deboned 1 chicken meat: protective effect of grape pomace extract 2 3 Hernán H. Tournour1,2, Marcela A. Segundo3, Luís M. Magalhães3, Jorge 4 Queiroz1, M. Beatriz P.P. Oliveira4, Luís M. Cunha1,*. 5 6 1 LAQV,REQUIMTE/DGAOT, Faculty of Sciences, University of Porto, Porto, 7 Portugal. 8 2 Faculty of Nutrition and Food Sciences, University of Porto, Porto, Portugal. 9 3 UCIBIO,REQUIMTE, Faculty of Pharmacy, Department of Chemistry, University of 10 Porto, Porto, Portugal. 11 4 LAQV,REQUIMTE, Department of Chemistry, Faculty of Pharmacy, University of 12 Porto, Porto, Portugal. 13 *Corresponding author: [email protected] 14 Campus Agrário de Vairão 15 Rua Padre Armando Quintas, 7 16 4486-661 Vairão 17 Portugal 18 Tel.: (+351)252660400 / Fax: (+351)252661780 19 20 21 ABSTRACT 22 The protective effect of grape pomace extract (GPE) added to mechanically deboned 23 chicken meat (MDM) subjected to different oxidative factors: iron(II); UV-C ; modified 24 atmosphere packaging and temperature, was assessed following single and successive 25 exposure. Experimental design followed a four-factor nested ANOVA and compared 26 with the effect of butylated hydroxytoluene (BHT). The effectiveness of Folin-Ciocalteu 27 reducing (FCR), scavenging capacity against 2,2-diphenyl-1-picrylhydrazyl (DPPH•), 28 oxygen radical absorbance capacity (ORAC) and iron(II) chelating ability (ICA) assays 29 to evaluate changes in MDM was examined. All assays evaluated meat oxidation, 30 exception for DPPH•, due to meat pigments interference at λ=517 nm. GPE (150 mg/kg) 31 had a consistent protective effect, although lower than BHT (100 mg/kg). The 32 antioxidant protection depended on the stress factor applied. Successive stress exposure 33 affected antioxidants performance with similar behavior concerning ORAC. FCR, 34 93 2.3. Meat extracts for analysis 156 Extracts for FCR, DPPH•, ORAC and ICA assays were prepared according to Qwele et 157 al. 2013 (Qwele et al., 2013) with some modifications. One gram of each sample was 158 homogenized with 10 mL of 0.05 M KH2PO4 phosphate buffer (pH 7). The extraction 159 step was carried out alternating ultrasound (30 s, 3 times) and vortex cycles (2 min, 3 160 times at 3000 rpm). Before the last cycle, meat samples were left for 10 min to stand in 161 order to assist in tissue hydration and improve the extraction. Finally, extracted samples 162 were centrifuged at 5,580 x g for 30 min at 4 ºC. Supernatant aliquots (1 mL) were 163 disposed in Eppendorf tubes. Extracts were frozen at -80 ºC until analysis by FCR, 164 DPPH•, ORAC and ICA assays. Before performing each assay procedure, analyses were 165 started mixing 200 µL of each extract and 200 µL absolute ethanol p. a. in order to 166 guarantee the total dissolution of polyphenolic compounds. 167 168 2.3.1. Meat extracts 169 FCR reducing capacity (FCR) 170 The FCR was assessed employing a 96-well microplate Folin-Ciocalteu procedure (L. 171 M. Magalhães, Santos, Segundo, Reis, & Lima, 2010; Singleton, Orthofer, & Lamuela- 172 Raventos, 1999). Hence, 150 µL of gallic acid standard solution (1.0 - 15.0 mg/L) or 173 diluted meat extracts (1:50 - 1:100, v/v) and 50 µL of F-C reagent (3:10, v/v) were 174 placed in each well. After that, 100 µL of carbonate solution (9 g/100mL) was added 175 and the reaction was monitored at 760 nm during 120 min. The FCR was expressed as 176 mg of gallic acid equivalents per gram of meat (Abs760 nm = 0.0504×[gallic acid, (mg/L)] 177 + 0.058, R>0.9997, n = 6). 178 Antioxidant capacity assessment 179 DPPH• assay 180 For microplate DPPH• methodology (Brand-Williams, Cuvelier, & Berset, 1995; L. M. 181 Magalhães, Barreiros, Maia, Reis, & Segundo, 2012), 150 µL of Trolox standard 182 solution (5.0 - 50.0 µmol/L) or diluted meat extracts (1:20 – 1:40, v/v) and 150 µL of 183 DPPH• ethanolic solution (50 mL/100mL) were placed in each well. The DPPH• 184 scavenging capacity was monitored at 517 nm during 120 min. The results were 185 94 expressed as mmol of Trolox equivalent (TE) per gram of meat (Abs517 nm = 186 7.20×[Trolox, (mmol/L)] -0.056, R>0.9926, n = 5). 187 188 ORAC assay 189 For ORAC assay (Huang, Ou, Hampsch-Woodill, Flanagan, & Prior, 2002; Wang, 190 Jónsdóttir, & Ólafsdóttir, 2009), 100 µL of Trolox standard solution (1.0 – 7.5 µmol/L) 191 or diluted meat extracts (1:125 – 1:250, v/v) and 100 µL of fluorescein (117 nmol/L) 192 were placed in each well, the microplate was brought to under preincubation for 15 min 193 at 37 ⁰C. Followed, 100 µL of AAPH solution (40 mmol/L) was added by rapidly using 194 multichannel pipet and fluorescence intensity (λexc 485 nm, λem 520 nm) was monitored 195 every minute during 240 min. Reaction carried out in 75 mmol/L phosphate buffer (pH 196 7.4) at 37 ⁰C. The area under the curve (AUC) was calculated for each sample by 197 integrating the relative fluorescence curve over the reaction time. The net AUC of the 198 sample was calculated subtracting the AUC of the control. The regression equation 199 between net AUC and Trolox concentration was determined, and the results were 200 expressed as µmol of Trolox equivalents (TE) per gram of meat by interpolation (Net 201 AUC (%) = 15.1×[Trolox, (µmol/L)] + 21.3, R>0.9983, n = 8). 202 203 Iron(II) chelating ability assay (ICA) 204 For iron(II) chelating ability assay (ICA) (Wang et al., 2009), 100 µL of diluted meat 205 extracts (1:5 - 1:10, v/v) in acetate buffer (50 mmol/L, pH 4.6) were mixed with 100 µL 206 Fe(II) solution (120 µmol/L) and placed in each well. After 5 min, 100 µL of ferrozine 207 solution (600 µmol/L) was added to each well. Solutions were left standing 10 min at 208 room temperature. Thereafter, the absorbance was monitored at 562 nm. The percentage 209 of inhibition of ferrozine-iron(II) complex formation of each sample was calculated 210 according to: ICA (%) = [A0 – (A1 – A2)] / A0 x 100; where A0, A1 and A2 correspond to 211 absorbance of the control, absorbance of sample and blank of the sample, respectively. 212 In A0 the intrinsic absorbance of iron(II) was subtracted from control absorbance. 213 Results were expressed as % inhibition obtained per mg of meat. 214 2.4. Statistical analysis 215 Values for all possible combinations (4 factors at 2 levels each) for Control, GPE and 216 BHT experiments (48 samples in total, 16 for each added antioxidant) were reported as 217 95 mean ± standard deviation (S.D.) for each antioxidant assay. Data regarding the effects 218 of the different oxidative stress factors were assessed using a fully Nested (Hierarchical) 219 ANOVA (Zar, 1999) with four factors organized as follows: iron(II), UV-C radiation 220 (nested within iron(II)), MAP (nested within UV-C radiation), and temperature (nested 221 within MAP). Statistical data analysis was performed with STATISTICA for Windows 222 version 12.0 (STATISTICA 12 Software, StatSoft, Tulsa, OK). Moreover, multiple 223 comparisons were performed using Tukey test at 95% confidence level, in order to 224 identify significant differences between experiments. 225 226 3. Result and Discussion 227 3.1. GPE characterization 228 Information available in literature about pomace extracts from the grape cultivar used in 229 this study is scarce. Nevertheless, our results (Table 2) are in agreement with the total 230 phenolic content (TPC) and antioxidant properties of Portuguese wines (Cristino, Costa, 231 Cosme, & Jordao, 2013; Jordao, Simoes, Correia, & Goncalves, 2012). 232 Table 2 Antioxidant capacity for GPE from “Touriga franca” (vintage 2012) Portuguese cultivar, used during the experiments Antioxidant capacity Mean ± S.D. TPC (mg GAE g extract -1 ) 106.1 ± 1.9 DPPH . (mmol TE g extract -1 ) 0.90 ± 0.02 ORAC (µmol TE g extract -1 ) 1325 ± 147 ICA (%Inhib. mg extract -1 ) 55 ± 14 Values represent means ± standard deviation (S.D.) of triplicate (n = 3). TPC: Total phenolic content; DPPH .: 2,2-diphenyl-1-picrylhydrazyl; ORAC: oxygen reactive absorbance capacity; ICA: Iron(II) chelating ability; GAE: Gallic acid equivalents; TE: trolox equivalents. 233 234 96 3.2. Nutritional and fatty acids composition for MDM raw material 235 The composition of meat is dependent on many factors, namely, type, sex, age, diet of 236 the animals, animal part -with/without skin-for the deboning process and also on 237 operations settings (Hald & Baggesen, 2013; Püssa, Pällin, Raudsepp, Soidla, & Rei, 238 2008). Nutritional and fatty acids composition of MDM is shown in Table S1. The 239 MDM proximate composition indicated 61.2%, 13.4%, 1.4% and 24.8% for moisture, 240 protein, ashes and total fat, respectively and in agreement to previous reports (Henckel, 241 Vyberg, Thode, & Hermansen, 2004; Püssa et al., 2009). Fatty acids profile is shown in 242 Table S1. Palmitic acid was the main saturated fatty acid (SFA) in MDM raw material 243 (24.0 ± 0.1%). In the case of monounsaturated fatty acids (MUFAs) oleic acid was 244 detected in the highest concentration (41.37 ± 0.02%). Linoleic acid was the most 245 predominant polyunsaturated fatty acid (PUFA) with a concentration of 15.2 ± 0.2%. 246 Our results are in agreement to those by Trindade et al. (Trindade, Felício, & Castillo, 247 2004). 248 249 3.3. Evaluation of methodologies to determine the oxidative stability in a meat 250 model 251 Data were obtained from different antioxidant assays (FCR, DPPH•, ORAC and ICA 252 assays) for all possible combinations of meat and oxidative stress factors. Univariate 253 tests of significance, effect sizes, and powers are presented in Table S2. 254 255 97 256 257 258 259 According to results obtained through the DPPH• assay, it was not possible to evaluate 260 the oxidative stability once this assay was not able to distinguish possible differences 261 among experiments (p = 0.59, Table S2). Pigments present in MDM, mainly myoglobin 262 (Mb), can exist in any of the four redox states, namely: deoxymyoglobin (DeoxyMb), 263 oxymyoglobin (OxyMb), carboxymyoglobin (COMb), and metmyoglobin (MetMb), 264 depending on the ligands bound to haem iron and also on the iron redox state 265 (ferrous/Fe2+ or ferric/Fe3+). Additionally, as MDM is not a fresh meat, DoexyMb is not 266 expectable in the samples. These pigments have an absorbance spectrum ranging from 267 500 to 600 nm. OxyMb has double peaks at 542 and 582 nm, whilst MetMb exhibits its 268 maximum peak at 503 nm (Tang, Faustman, & Hoagland, 2004). We hypothesize that 269 the lack of accuracy of DPPH• assay to distinguish differences among experiments may 270 be due to the existence of a spectra/absorption overlapping in the signal registered by 271 the method, once DPPH• assay is spectrophotometrically monitored at 517 nm, as well 272 A) B) D)C) Figure 2 : Boxplots for A) FCR; B) DPPH; C) ORAC and D) ICA assays. FCR: Folin-Ciocalteu reducing content; DPPH•: 2,2-diphenyl-1-picrylhydrazyl radical assay, ORAC: oxygen radical absorbance capacity and ICA: iron(II) chelating ability. MDM: mechanically deboned chicken meat; GPE: grape pomace extract. 98 as, the absorbance spectrum of the own colored phenolic compounds from GPE itself. 273 Tang et al. observed an absorbance for Mb species (namely, DeoMb, MetMb and 274 OxyMb) over 0.60 at 517 nm for a concetration of 0.11 mmol/L (Tang et al., 2004). 275 Additionally, DPPH• assay involves reactions based in a electron transfer mechanism, 276 then, is highly influenced by solvent and pH of the reaction.The DPPH• assay was 277 performed in a 50mL/100mL ethanolic media which may lead to micro protein 278 precipitations with latter interferences in the signal as previously described for samples 279 like plasma by Magalhães et al. (L. M. Magalhães, Segundo, Reis, & Lima, 2008). The 280 Folin-Ciocalteu assay is a method used for the measurement of the total phenolic 281 content and it is based on the ability of certain compounds in alkaline medium to reduce 282 the phosphomolybdic/phosphotungstic acid reagent to complexes, which are 283 spectrophotometrically detected. Despite its non-specificity, it is operationally simple, 284 reproducible, a rather standardized procedure and the absorption of the reaction product 285 at a long-wavelength minimizes interferences from most sample matrixes (L. M. 286 Magalhães et al., 2008). The ORAC assay is based on the intensity of fluorescence 287 decrease of the target/probe along time under constant flux of peroxyl radicals in 288 aqueous buffer. When a sample is analyzed in presence of chain-breaking antioxidants, 289 the decay of fluorescence is inhibited (L. M. Magalhães et al., 2008). In the ICA assay, 290 chelating compounds in the sample disrupt the complex formed between iron(II) and 291 ferrozine. Therefore, the color decreasing on the iron(II)-ferrozine complex monitored at 292 562 nm is taken as an estimation of the chelating activity. FCR, ORAC and ICA assays 293 showed a similar behavior as depicted in Fig. 2, registering lower protection conferred 294 by GPE face to BHT, but consistently present in the assays. Moreover, based in these 295 findings and with supporting statistical analysis about the effectivesness of the FCR, 296 ORAC and ICA assays to identify significant differences among experiments (p < 0.01); 297 three methodologies were applied to monitor the antioxidant performance upon 298 exposure of MDM to stress factors, as showed in the following section. 299 3.4. Evaluation of GPE and BHT performance for protection of MDM against 300 stress factors 301 Results presented in Table 3 allow the comparison upon the addition of GPE and BHT 302 against a single stress factor. No significant differences (p > 0.05) were obtained against 303 the addition of iron(II) as stress factor in ORAC values, whereas FCR and ICA values 304 were significantly different. Iron and copper, are both considered as an important 305 99 catalyst for lipid oxidation reactions. As such, substances capable of chelating ions can 306 act as fat antioxidants (Hogan, Zhang, Li, Wang, & Zhou, 2009). Although less 307 pronounced than for Control samples, both antioxidants were affected by the presence 308 of iron, in FCR values. Chen & Ahn, reported trends of inhibition with increasing final 309 phenolic concentrations in an iron(II)-lipid oxidation (LO) system working with six 310 different polyphenols (Chen & Ahn, 1998). They also showed that caffeic acid (present 311 in our GPE, Table S3) was effective inhibitor of the iron(II)-LO. Additionally, they 312 experimented with BHT showing that the ability of polyphenolic compounds in 313 chelating Fe was not the most critical for phenolics to inhibit iron(II)-LO, as BHT (not 314 chelating agent) had low IC50 (TEAC) (Chen & Ahn, 1998). Concerning UV-C, 315 significant differences were obtained for all methodologies when samples were exposed 316 to UV-C as stress factor. Based on the results, we hypothesize that UV-C originate 317 reactive species with unsaturated fatty acids as target, although in the presence of 318 phenolic compounds from GPE. These bioactive compounds are significantly reduced, 319 affecting the protection they confer to the fluorescent probe oxidation in ORAC assay. 320 321 322 100 Table 3 Influence of a single oxidative stress factor on MDM experiments, measurement through FCR, ORAC and ICA assays Factor Experiment FCR (mg GAE g meat-1) ORAC (µmol TE g meat-1) ICA (% inhibition mg meat-1) Mean SD Mean SD Mean SD Fe Control 1.50d 0.03 21.9a 1.2 14c 2 Control+Fe 1.31e 0.02 18.9a 2.2 33b 6 GPE 1.59c 0.01 23.9a 3.8 37b 4 GPE+Fe 1.48 d 0.02 25.7 a 2.2 58 a 3 BHT 2.21 a 0.04 26.9 a 1.8 62 a 13 BHT+Fe 1.80b 0.03 23.3a 5.9 29b,c 6 UV Control 1.50c 0.03 21.9a,b 1.2 14c 2 Control+UV 1.43d 0.02 12.8c 1.9 32b 3 GPE 1.59b 0.01 23.9a,b 3.8 37b 4 GPE+UV 1.61 b 0.03 15.6 b,c 3.0 56 a 4 BHT 2.21a 0.04 26.9a 1.8 62a 13 BHT+UV 1.49c,d 0.01 22.7a,b 5.3 31b 6 MAP Control 1.50c 0.03 21.9a,b 1.2 14c 2 Control+MAP 1.39d 0.02 20.7a,b 2.9 34b 3 GPE 1.59b 0.01 23.9a,b 3.8 37b 4 GPE+ MAP 1.46c 0.03 13.7c 1.1 35b 2 BHT 2.21a 0.04 26.9a 1.8 62a 13 BHT+ MAP 1.38d 0.01 17.8b,c 1.4 34b 6 Temperature Control 1.50d 0.03 21.9a,b 1.2 14 c 2 Control+T 1.64c 0.04 26.2a 1.2 17 c 4 GPE 1.59c 0.01 23.9a,b 3.8 37 b 4 GPE+T 1.48d 0.03 18.9b,c 2.5 41b 4 BHT 2.21a 0.04 26.9a 1.8 62a 13 BHT+T 1.77b 0.03 15.1c 0.8 36b 4 Data were analyzed using a fully nested ANOVA with four factors; within each column for each factor tested, different letters indicate statistically significant differences according to the Tukey multiple comparison test at 95% confidence level (FCR: Folin-Ciocalteu reducing content; ORAC: oxygen radical absorbance capacity; ICA: iron(II) chelating ability; GAE: gallic acid equivalents). Mean ± S.D. (n = 8). 323 101 Therefore, under advanced scission and degradation processes, proteins from MDM 324 may be exposed to further spoilage, generating smaller peptides with chelating 325 properties (Storcksdieck, Bonsmann, & Hurrell, 2007), as it was verified by the increase 326 of ICA values for Control (14 to 32 % inhibition mg meat-1) and GPE (37 to 56 % 327 inhibition mg meat-1) added samples. MAP is a common operation applied to meat in 328 order to protect its pigments from the discoloration, although it increases the oxidative 329 processes (Faustman, Sun, Mancini, & Suman, 2010). Significant differences were 330 observed for all assays, with the Control exhibiting a similar behavior when UV-C was 331 applied, regarding ICA assay. GPE and BHT added samples showed a stronger 332 reduction when exposed to MAP, in comparison with UV-C exposure results, registered 333 by FCR and ORAC values. Additionally, GPE reduction (1.59 to 1.46 mg GAE g meat- 334 1), although significant when compared to the Control, was less strong than BHT (2.21 335 to 1.38 mg GAE g meat-1) for the previous factor. GPE is composed by anthocyanins, 336 flavonoids, phenolic acids and resveratrol (Teixeira et al., 2014). The antioxidant 337 efficiency of phenols is structure-dependent on their hydrogen-donating ability, which is 338 directly related to the number of phenolic hydroxyl moieties present. Intermediate 339 radicals generated during the lipid oxidation can be stabilized by the resonance 340 delocalization of the electron within the aromatic ring(s) (Gheldof & Engeseth, 2002; 341 Rice-Evans, Miller, & Paganga, 1996). This fact explains the differences between GPE 342 and BHT which has only one aromatic ring not providing the stabilization effects 343 accomplished by more complex structures as presented by polyphenols. Temperature 344 increase leads to the acceleration of reactions, as radical-mediated chain reaction of 345 lipid oxidation was reported to progress in a temperature-dependent fashion (Gatellier, 346 Sante-Lhoutellier, Portanguen, & Kondjoyan, 2009). ICA assay registered significant 347 strong reduction for BHT (62 to 36% inhibition mg meat-1), whilst no significant 348 differences observed for both Control and GPE. Stronger reductions were registered for 349 BHT added samples, compared to GPE, in FCR and ORAC values. Temperature acts 350 differently on polyphenolic compounds depending on their concentrations with other 351 components of studied system (Brewer, 2011). 352 Regarding other assays, FCR indicated presence of reducing species, lower for GPE 353 when compared to BHT (Fig. 2 A). ORAC methodology showed that protection was 354 conferred by both GPE and BHT against ROO• oxidation, but it was lower for GPE 355 compared with BHT. The ratio between pro-oxidant ions (iron(II)) and antioxidant 356 102 concentrations may explain the differences of BHT and GPE performances. 357 Additionally, during the chain reactions taking place upon lipid oxidation, the presence 358 of metal ions can participate as strong pro-oxidant agent, generating radical species 359 from Fenton-type reaction, attacking the fluorescent probe used in ORAC assay or 360 degrading polyphenolic compounds. So, the ability of a compound to inhibit fluorescein 361 oxidation could be influenced by its interactions with pro-oxidants or other antioxidants 362 (Nkhili & Brat, 2011). 363 364 109 Shah, M. A., Bosco, S. J. D., & Mir, S. A. (2014). Plant extracts as natural antioxidants 572 in meat and meat products. Meat Science, 98(1), 21-33. 573 Sharma, G., Srivastava, A. K., Gupta, C., & Prakash, D. IN201102860-I1. 574 Shirahigue, L. D., Plata-Oviedo, M., de Alencar, S. M., d'Arce, M. A. B. R., de Souza 575 Vieira, T. M. F., Oldoni, T. L. C., et al. 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Biostatistical analysis (4th ed.): Pearson Education India. 604 110 Supplementary information 1 Single and successive oxidative stress factors applied to mechanically deboned 2 chicken meat: protective effect of grape pomace extract 3 Hernán H. Tournour1,2, Marcela A. Segundo3, Luís M. Magalhães3, Jorge 4 Queiroz1, M. Beatriz P.P. Oliveira4, Luís M. Cunha1,*. 5 6 1LAQV,REQUIMTE/DGAOT, Faculty of Sciences, University of Porto, Porto, 7 Portugal. 8 2Faculty of Nutrition and Food Sciences, University of Porto, Porto, Portugal. 9 3UCIBIO,REQUIMTE, Faculty of Pharmacy, Department of Chemistry, University of 10 Porto, Porto, Portugal. 11 4LAQV,REQUIMTE, Department of Chemistry, Faculty of Pharmacy, University of 12 Porto, Porto, Portugal. 13 14 Number of pages: 5 15 Number of Tables: 3 16 17 18 19 20 21 22 23 24 25 26 *Corresponding author: lm[email protected]. Tel.: (+351)252660400 27 111 Reagent and solutions 28 29 Stock standard solutions for HPLC analysis, were prepared accurately weighing each 30 compound and by dissolving them in an appropriate solvent (ethanol or water) to a final 31 concentration of 1000 mg/L. Working solutions were prepared from stock standard 32 solutions in mobile phase (5 and 2.5 mg/L; 15 standards mixture). HPLC grade acetic 33 acid and acetonitrile (Aldrich, Milwaukee, WI) were used. All solutions were filtered 34 through a 0.45 µm membrane and degassed in ultrasound. Fatty acid methyl esters 35 (FAME) mixture 37 patterns (Supelco, Bellefonte, PA) were used for the fatty acids 36 profile determination. Boron trifluoride (BF3), n-heptane (C7H16) and anhydrous sodium 37 sulfate (Na2SO4) were purchased in Sigma Aldrich (St. Louis, MO, USA). Water from 38 Sartorius (Goettingen, Germany) (resistivity > 18 MΩ cm) and absolute ethanol p. a. 39 (Panreac Química, Spain) were used in the preparation of all solutions. 40 Chemical assays 41 Characterization of MDM raw material 42 Nutritional components were determined as percentages of moisture, protein, ashes and 43 total fat according to Association of Official Analytical Chemists methodologies 44 (AOAC methods: 985.14; 928.08; 920.153; 991.36, respectively) (AOAC, 1990) in 45 MDM raw material. 46 The fatty acids proportion and the level of un-saturations are determinant for the 47 oxidation degree in fat. Therefore, the assessment of the fatty acids composition was 48 carried out using gas chromatography with flame ionization detector (GC/FID). The 49 fatty fraction was firstly extracted according to Folch method (Folch, Lees, & Sloane- 50 Stanley, 1957), with dichloromethane instead of chloroform. For this, sample was 51 weighed (1 g) into a Falcon tube, followed by addition of 10 mL dichloromethane: 52 methanol (2:1) solution. After homogenization in vortex, the sample was placed in an 53 ultrasound bath for 10 min and centrifuged (3000 rpm, 5 min). Supernatant was 54 displayed in a second Falcon tube where 1 g/100mL NaCl aqueous solution was added 55 in a 1:5 proportion related to the supernatant volume. Sample was homogenized in a 56 vortex and centrifuged again (3000 rpm, 5 min), rejecting the upper portion (aqueous 57 phase) in this case. The same procedure was repeated after addition of anhydrous 58 sodium sulfate. The total fat (organic phase) was placed into a vial tube with 100 µL of 59 112 0.01 g/100mL BHT solution. The fatty acids derivatization into fatty acid methyl esters 60 (FAME) was performed according to Shantha & Ackman (Shantha & Ackman, 1990), 61 with few modifications. The organic phase obtained above was mixed with 500 µL of 62 0.5 mol/L KOH in methanol solution. The sample was homogenized and heated (100 63 ºC, 10 min), followed by cooling into ice, and where 2.5 mL of boron trifluoride (14 64 mL/100mL in methanol) was added. Sample was again homogenized, heated (100 ºC, 65 30 min), cooled in ice, and 2 mL n-heptane added by. The last steps of the derivatization 66 consisted in a separation of the upper phase into a new vial tube with subsequent 67 agitation and centrifugation. Excess water was removed with anhydrous sodium sulfate. 68 Results were expressed as mass percentages of each fatty acid of its methyl esters or 69 area under the curve (AUC) due to the AUC obtained are equivalents to FAME mass, 70 based on: % A = [(AUC A x 100)]-1Σ (AUC peaks), A represents a FAME compound. 71 For the data analises Maitre software (JMBS Developments, Grenoble, France) was 72 used. 73 HPLC analysis 74 The phenolic profile for ethanol/water extracts and aqueous suspensions were obtained 75 using an analytical HPLC unit (Jasco, Easton, USA) comprising: pump, automatic 76 injector, DAD, equipped with a Kinetex (250 × 4.6 mm; 5 µm particle size; C18; 100 77 Å) core-shell column, controlled by Chrom-Nav software. The HPLC characterization 78 was performed according to Kammerer et al. (Kammerer, Claus, Carle, & Schieber, 79 2004), as following: 80 Phenolic acid (PA) method: the mobile phase consisted of 2% (v/v) aqueous acetic acid 81 (eluent A) and 0.5% (v/v) aqueous acetic acid and acetonitrile (50:50, v/v; eluent B) 82 using the following gradient program: from 10 to 15% B (10 min), 15% B isocratic (3 83 min), from 15 to 25% B (7 min), from 25 to 55% B (30 min), from 55 to 100% B (1 84 min), 100% B isocratic (5 min), from 100 to 10% B (10 min), with total run time of 67 85 min. 86 Anthoxanthins and Stilbenes (AX) method: the mobile phase consisted of the same 87 eluents as described above using instead the following gradient program: from 10 to 88 24% B (20 min), from 24 to 30% B (20 min), from 30 to 55% B (20 min), from 55 to 89 100% B (15 min), 100% B isocratic (8 min), from 100 to 10% B (2 min), with a total 90 run time of 95 min. For both methods, the injection volume was 10 µL and the 91 113 absorbance was monitored at three monitoring channels (280, 320 and 370 nm). The 92 flow rate was 1.0 mL/min. The peaks detected in the samples were first compared with 93 respect to retention time and the spectral data with those in the standards mixture. 94 Quantification was performed based on the molar absorptivity (ε, L/mol) values for 95 each compound, according to chromatography peak area, molar mass and standard 96 concentrations. Each sample was injected in duplicate. Results were expressed as means 97 in milligrams GAE per gram of residue (mg GAE g residue -1). 98 99 114 100 Table S1 Proximate composition and fatty acids profile of MDM raw material Measurement % (w/w) Moisture 61.2 ± 1.0 Protein 13.4 ± 0.2 Ashes 1.4 ± 0.1 Total fat 24.8 ± 1.3 Nomenclature Fatty acids % Total fatty acid content mg 100g fat-1 Myristic acid C14:0 0.60 ± 0.02 5.96 Palmitic acid C16:0 24.0 ± 0.1 240.22 Cis-7 hexadecenoic acid C16:1 n-9 0.58 ± 0.001 5.79 Palmitoleic acid C16:1 n-7 6.17 ± 0.01 61.73 Steáric acid C18:0 6.38 ± 0.09 63.79 Oleic acid C18:1 n-9 c 41.37 ± 0.02 413.70 Cis-Vaccenic acid C18:1 n-7 2.09 ± 0.003 20.92 Linoleic acid e C18:2 n-6 c 15.2 ± 0.2 151.09 α-Linolenic acid e C18:3 n-3 0.34 ± 0.01 3.42 Cis-11 eicosenoic acid C20:1 n-9 0.66 ± 0.09 6.63 Arachidonic acid C20:4 n-6 0.53 ± 0.06 5.29 SFA† 31.8 ± 0.2 318 MUFA‡ 51.4 ± 0.04 514 PUFA§ 16.7 ± 0.08 167 n-3 0.61 ± 0.02 6.08 n-6 15.1 ± 0.2 152 n-3 / n-6 0.04 ± 0.002 0.40 Values represent means ± S.D. (n = 2). e Essential fatty acid † Saturated fatty acids ‡ Monounsaturated fatty acids § Polyunsaturated fatty acids 115 101 Table S2 Univariate Tests of Significance, Effect Sizes, and Powers for FCR, DPPH., ORAC and ICA assays, Overparameterized model Type III decomposition Effect FCR (mg GAE g meat-1) DPPH. (µg TE g meat-1) ORAC (µmol TE g meat-1) ICA (% inhib. mg meat-1) F-value p F-value p F-value p F-value p Intercept 170859.5 <0.01 7304.097 <0.01 11943.83 <0.01 6123.177 <0.01 Experiment 114.7 <0.01 0.524 0.59 22.16 <0.01 29.374 <0.01 Iron (Experiment ) 207.8 <0.01 17.524 <0.01 22.27 <0.01 16.206 <0.01 UV (Experiment *Iron) 102.2 <0.01 6.916 <0.01 15.53 <0.01 10.629 <0.01 MAP (Experiment *Iron*UV) 72.7 <0.01 14.659 <0.01 24.1 <0.01 10.967 <0.01 Temp (Experiment *Iron*UV*MAP) 67.3 <0.01 14.9 <0.01 21.67 <0.01 11.369 <0.01 Mean ± S.D. (n = 8). Data were analyzed using hierarchical (Nested) ANOVA at 95% confidence level. ( FCR: total reducing content, DPPH . : 2,2 - diphenyl - 1 - picrylhydrazyl; ORAC: oxygen radical absorbance capacity ; ICA: iron quelating ability; GAE: gallic acid equivalents; TE: Trolox equivalents). 116 102 103 104 105 Table S3 Individual phenols by HPLC, for GPE from “Touriga franca” (vintage 2012) Portuguese cultivar, used during the experiments Individual phenolics by HPLCa Mean ± S.D. Gallic acid (mg g extract-1) 0.53 ± 0.04 Syringic acid (mg g extract-1) 1.43 ± 0.02 (-)-Epicatechin (mg g extrac-1) 0.89 ± 0.07 Caffeic acid (mg g extract-1) 0.46 ± 0.0002 Values represent means ± standard deviation (S.D.) of triplicate (n = 3). a : Values for phydroxybenzoic, p-coumaric and o-coumaric, sinapic, ferulic acids and (+)-Catechin, (-)- Epicatechin gallate, trans-resveratrol, quercetin, kaempferol and chlorogenic acid, were below the detection limit. 117 References 106 AOAC. (1990). Food Composition additives, Natural contaminants Official methods of 107 analysis. Washington D.C. (Reprinted from: 15th Edition). 108 Folch, J., Lees, M., & Sloane-Stanley, G. (1957). A simple method for the isolation and 109 purification of total lipids from animal tissues. Journal of Biological Chemistry, 110 226(1), 497-509. 111 Kammerer, D., Claus, A., Carle, R., & Schieber, A. (2004). Polyphenol screening of 112 pomace from red and white grape varieties (Vitis vinifera L.) by HPLC-DAD-113 MS/MS. Journal of Agricultural and Food Chemistry, 52(14), 4360-4367. 114 Shantha, N., & Ackman, R. (1990). Nervonic acid "versus" tricosanoic acid as internal 115 standards in quantitative gas chromatographic analyses of fish oil longer-chain 116 n-3 polyunsaturated fatty acid methyl esters. Journal of Chromatography B: 117 Biomedical Sciences and Applications, 533, 1-10. 118 119 Paper III Hernán H. Tournour, Marcela A. Segundo, Luís M. Magalhães, Telmo J. R. Fernandes, M. Beatriz P. P. Oliveira, Luís M. Cunha. Influence of Portuguese grape extracts on the oxidative stability, nutritional, and color characteristics of mechanically deboned chicken meat. [Submitted for publication]. 201 Symoneaux, R., Galmarini, M., & Mehinagic, E. (2012). Comment analysis of consumer’s likes 543 and dislikes as an alternative tool to preference mapping. A case study on apples. Food 544 Quality and Preference, 24(1), 59‐66. 545 Tournour, H. H. (2014). Skin and seed grape extract as an antioxidant for mechanically 546 deboned chicken meat, during frozen storage. University of Porto, Porto. 547 Trindade, M. A., Felício, P. E. d., & Castillo, C. J. C. (2004). Mechanically separated meat of 548 broiler breeder and white layer spent hens. Scientia Agricola, 61, 234‐239. 549 Varela, P., & Ares, G. (2014). Novel Techniques in Sensory Characterization and Consumer 550 Profiling: CRC Press. 551 Vidal, L., Ares, G., & Giménez, A. (2013). Projective techniques to uncover consumer 552 perception: Application of three methodologies to ready‐to‐eat salads. Food Quality 553 and Preference, 28(1), 1‐7. 554 Viuda‐Martos, M., Ruiz‐Navajas, Y., Fernández‐López, J., & Pérez‐Álvarez, J. (2010). Effect of 555 orange dietary fibre, oregano essential oil and packaging conditions on shelf‐life of 556 bologna sausages. Food Control, 21(4), 436‐443. 557 WHO. (2003). Diet, nutrition and the prevention of chronic diseases: report of a Joint 558 WHO/FAO Expert Consultation. In: World Health Organization. 559 4. Conclusions 4. Conclusions 204 4.1. Main conclusions Data depicted in the present thesis provide valuable information regarding the characterization of certain Portuguese grape varieties residues which are still under-exploited as a source of polyphenolic compounds. Results from Paper I contribute with a characterization of GPE in two solvents (ethanol/water extract and aqueous suspensions) regarding total phenolic content and antioxidants capacity. Findings indicated that Portuguese GPE presented TPC, DPPH• and ORAC values comparable with previous data, giving a positive perspective on the use of a less efficient extractive mixture (80:20 % v/v absolute ethanol: water) having a food grade classification and being environmentally friendly. Hence, considering that Portugal eleventh largest wine producer in the world, and therefore generating large amounts of grape pomace, these results contribute to the valorization of these by-products, highlighting a variety of industrial applications. Different behaviors were observed regarding the effect of solvent in GPE composition as a dependent response on antioxidant assay. ORAC and ICA values significantly (p < 0.01) decreased when ethanolic/water and aqueous suspensions were compared. However, a significant (p < 0.01) increase in TPC values was determined when the effect of solvent was compared as above. DPPH• values were not significantly (p = 0.350) affected. Concerning the experiments presented in Paper I, although the three most representative Portuguese red grape varieties, from Douro´s region were analyzed, a large number of varieties, and their annual variation were not considered. A broader perspective on those would provide a stronger evidence of our observations. Nevertheless, taking into account these considerations, it was observed that extracts from “Touriga nacional” grape pomace yielded the highest TPC and antioxidant capacity according to DPPH• and ORAC assays. Additionally, TNac exhibited the highest total phenolic content according to HPLC analysis, including mainly gallic acid, (+)-catechin, caffeic acid, syringic 4. Conclusions 205 acid and (-)-epicatechin. These results showed a promising scenario for future applications of this grape variety. In what concerns to the antioxidant protection effect of GPE from “Touriga franca” in food systems, such as a meat model (Paper II), FCR, DPPH•, ORAC and ICA assays were proposed and evaluated as indicators of oxidative meat degradation. Results indicated that FCR, ORAC and ICA assays were suitable and consistent methodologies to monitor the consequences of induced degradation using MDM as food model. In contrast, DPPH• assay was not suitable to distinguish significant differences (p = 0.59) among samples with added antioxidant and control ones. Spectra/absorption overlapping from polyphenols and meat system compounds, along with micro precipitations in 50 % v/v ethanolic medium, were depicted as possible reasons for the assay’s lack of specificity. The antioxidant effectiveness of GPE compared to BHT was evaluated against the application of several stress factors, namely presence of iron(II), UV-C radiation, MAP and temperature abuse, in two experimental approaches: single and successive exposure, combined in a single fully hierarchical design of experiments. Nutritional composition and fatty acid profile of MDM revealed high fat content (over 24 %), including MUFA and PUFA, and with oleic acid (41.37 ± 0.02%) and linoleic acid (15.2 ± 0.2%) as main fatty acids found in MDM composition. Concerning to protection conferred by GPE (150 mg/kg) although it was less effective than that the one conferred by BHT (100 mg/kg), results indicated an antioxidant activity of GPE consistently present in all assays. The antioxidant effectiveness was stress-factor and antioxidant dependent, for instance, this can be depicted if we consider that BHT added samples exerted better protection than GPE against UV, as measured with ORAC; whilst protection conferred by GPE added samples was higher than BHT, regarding temperature abuse, as evaluated with ICA values. Additionally, it yields that a successive exposure to stress conditions affects the final antioxidant performance, causing similar behavior in ORAC values under the effect of both GPE (23.9 to 13.2 µmol TE g-1 meat), and BHT (26.9 to 14.6 µmol TE g-1 meat). 4. Conclusions 206 Information regarding influence of GPE from different Portuguese varieties on oxidative stability, nutritional and physical characteristics of MDM under frozen storage, was provided on Paper III and Paper IV. MDM samples were supplemented with BHT-BHA (200 mg/kg) and GPE at two different levels (60 and 120 mg/kg). The effect of MDM initial composition on the protection of GPE was also evaluated. MDM samples even under vacuum packaging and with antioxidant supplementation showed signs of lipid oxidation as evaluated by FCR, ORAC and ICA assays. Significant changes in meat color, measured through CIE - L*a*b*, were observed with GPE supplementation, mainly with extracts from “Tinta roriz” (TR) and “Touriga franca” (TF). Control and BHT-BHA added samples were lighter, redder and yellower than MDM samples supplemented with any GPE. Protection against lipid oxidation of certain groups of fatty acids in MDM samples supplemented with low concentration of GPE (60 mg/kg), from TR and TF grape varieties, was observed throughout storage. On a relevant note, all MDM samples supplemented with GPE exhibited effective protection against oxidation of n-3 aggregated fatty acids , whilst BHT-BHA and control samples showed a significant (p < 0.05) decrease after advanced storage (365D) (Paper III). From a nutritional point of view, this fact is of valuable importance, given that n-3 fatty acids, including α-Linolenic acid (C18:3 n-3) correspond to essential fatty acids for human diet. Regarding the influence of the initial composition of MDM samples (Paper IV) our findings suggested that MDM composition affected its oxidative stability throughout storage, according to ORAC and ICA methodologies. Additionally, different levels of GPE supplementation (60 and 120 mg/kg) showed minor significant alterations, regarding color variables -mainly b* and Hue angle-. Results from Paper III and IV corroborate that Portuguese grape pomace exerted antioxidant protection, with this protective performance dependent on GPE concentration and on MDM initial composition. In this context, implications of supplementation of MDM with higher GPE levels, from the consumer perspective were analyzed in Paper V. Optimum combinations of MDM content and GPE concentrations for nugget elaboration 4. Conclusions 207 and latter sensory evaluation of overall acceptance were obtained. Naïve assessors, regular consumers of poultry meat products, perceived high acceptance scores: for a fixed overall acceptance of 7 or more points, possible formulations could go up to a MDM content of 20 %, with a fixed concentration of 60 mg/kg of GPE, or up to 90 mg/kg of GPE, in the absence of MDM. If aiming from an acceptance limit of 6 points (for instance, if developing a more economic product), possible formulations could go up to a MDM content of 30 %, with a concentration of up to 105 mg/kg of GPE, or up to 140 mg/kg of GPE, in the absence of MDM. Additionally, through application of Correspondence analysis, these findings were confirmed, increasing information regarding positive attributes such as “light colour” (internal appearance descriptor) associated to previous formulations. Hence, it can be concluded that addition of GPE up to 120 mg/kg and MDM up to 15 % did not adversely affect the perceived appearance of chicken nuggets. These results add up to the valorization of Portuguese grape pomace as an affordable and underestimated source of polyphenolic compounds with relevant properties as a food preservation agent. This study focused on recovery strategies and on the valorization of these agro-food by-products, providing valuable information on their potential industrial applications. Based on this, a new variety of functional products can be offered towards the satisfaction of current consumer demands. 4. Conclusions 208 4.2. Perspectives and future trends Findings presented in this thesis highlighted possible interactions undergone by phenolic compounds from GPE, when incorporated in a food matrix, during storage, by exerting protection against its lipid oxidation. Considering that several reports indicate relevant beneficial properties for consumers’ health, associated to intake of polyphenolic compounds, an additional change in the industrial sector, towards natural and functional ingredients and foods, is anticipated to face consumers´ demands. Considering the relevance that nonextractable polyphenols fraction represent from the research perspective, experiments covering the study of extractive conditions and procedures, compatible with food incorporation should be considered in future work. This should cover the analysis and characterization of those polyphenols fractions. In future, in vitro experiments may be conducted with simpler food matrixes in order to isolate the effects of the application of this grape pomace extract, comprising a deeper study of its chemical mechanisms behind as protective agents, understanding the potential impacts of their antioxidant compounds on the final product characteristics, namely on its lipid content and fatty acids profile. From the consumer point of view, it would be interested to improve nugget – or other meat productformulation and proceed with a larger evaluation of the impact of different processing conditions (e.g., frying, baking or microwave heating). With this being evaluated from a joint metabolomics, nutrigenomics, and consumer perception and acceptance points of view. Such study could be complemented with the evaluation of the impact of different claims, regarding the incorporation of grape pomace extract, on consumers’ willingness to pay for such products.