MarceloEdvanDosSantosSilva Dissert
MarceloEdvanDosSantosSilva Dissert
MarceloEdvanDosSantosSilva Dissert
CENTRO DE TECNOLOGIA
TECNOLOGIA DE ALIMENTOS
EXTRAÇÃO E MICROENCAPSULAÇÃO DE
BOVINO
EXTRAÇÃO E MICROENCAPSULAÇÃO DE
BOVINO
EXTRAÇÃO E MICROENCAPSULAÇÃO DE
BOVINO
Dissertação de Mestrado
apresentado ao Programa de Pós-
graduação em Ciência e Tecnologia
de Alimentos, do Centro de
Tecnologia, da Universidade Federal
da Paraíba em cumprimento aos
requisitos para obtenção do título de
Mestre em Ciência e Tecnologia de
Alimentos.
BANCA EXAMINADORA
Dedico.
AGRADECIMENTOS
Ao meu marido, por acreditar e se sacrificar junto comigo. Sem ele, eu não teria
suportado os momentos mais difíceis.
À minha irmã (Melissa Karoly da Silva). Servir de exemplo pra você, me faz
querer ser uma pessoa melhor a cada dia.
Aos meus amigos Ciro Silvestre e Letícia Melo por me encorajarem e vibrar
comigo a cada pequena vitória.
À Professora Dra Tatiana Souza Porto e ao Dr. Rodrigo Lira de Oliveira pela
disponibilidade dos laboratórios e por toda ajuda e acolhimento, sem os quais, seria ainda
mais difícil realizar as pesquisas nesse momento de pandemia.
Agradeço a Professora Dra Suzana Pedroza da Silva, por segurar minha mão desde
a iniciação científica, me orientar no trabalho de conclusão de curso, e agora continuar
como co-orientadora e amiga durante o mestrado. Sou eternamente grato pelos seus
ensinamentos.
Agradeço em especial a Professora Rita Queiroga (in memorian), por suas aulas
inspiradoras e pelas palavras de conforto e motivação.
Agradeço a empresa Polpa de Frutas Canaã por ceder o bagaço da uva, essencial
para o desenvolvimento desta pesquisa.
Figura 1: Formas de uso das uvas (Elaborado a partir de informações obtidas de OIV,
International Organization of vine and wine, 2019 e Venkitasamy et al., 2019).
.........................................................................................................................................45
Figura 2: Encapsulação de compostos fenólicos e, incorporação em embalagens ativas.
(Elaborada a partir de informações obtidas de Estévez e Lorenzo, 2019 e Goméz et al.,
2018). ..............................................................................................................................46
MATERIAL E MÉTODOS
Figura 1: Bagaço obtido após a prensagem da uva (Vitis labrusca L. var. Isabel) para
produção de polpa de fruta congelada. .............................................................................48
Artigo II
Figura 1: Micrografia do extrato do bagaço da uva microencapsulado com maltodextrina
pela técnica de liofilização. (A) Ampliação de 1000x; (B) Ampliação de 500x; (C)
Ampliação de 300x; (D) Ampliação de 146x. ..................................................................87
Figura 3: Concentração dos níveis de malonaldeído nos diferentes tratamentos (SEB, LEB
e MEB) dos hambúrgueres crús e pré-cozidos durante o período de
armazenamento................................................................................................................89
ARTIGO I
Artigo II
Aa Atividade de Água
DE Dextrose Equivalente
MDA Malonaldeído
ME Eficiência de Microencapsulação
UR Umidade Relativa
SUMÁRIO
1 INTRODUÇÃO ...................................................................................................................... 14
2 REFERENCIAL TEÓRICO ................................................................................................. 17
Artigo I: Potencial tecnológico do resíduo agroindustrial do despolpamento da uva para aplicação
em produtos cárneos: uma revisão. ............................................................................................. 17
3 MATERIAL E MÉTODOS ................................................................................................... 48
3.1 MATERIAL .......................................................................................................................... 48
3.2 DELINEAMENTO EXPERIMENTAL ................................................................................ 48
3.3 PREPARO DA AMOSTRA: SECAGEM E TRITURAÇÃO .............................................. 50
3.4 EXTRAÇÃO DOS COMPOSTOS FENÓLICOS A PARTIR DO BAGAÇO DA UVA. ... 50
3.5 DETERMINAÇÃO DO TEOR DE FENÓLICOS NO EXTRATO LIOFILIZADO ........... 51
3.6 RENDIMENTO DA EXTRAÇÃO ....................................................................................... 51
3.7 MICROENCPSULAÇÃO DOS EXTRATOS PELA TÉCNICA DE LIOFILIZAÇÃO ...... 51
3.8 CARACTERIZAÇÃO DAS MICROCÁPSULAS ............................................................... 52
3.8.1 Determinação dos teores de compostos fenólicos nas microcápsulas .......................... 52
3.8.2 Antocianinas monoméricas.............................................................................................. 52
3.8.3 Eficiência de microencapsulação .................................................................................... 53
3.8.4 Determinação da umidade ............................................................................................... 53
3.8.5 Higroscopicidade .............................................................................................................. 53
3.8.6 Atividade de água (Aa) .................................................................................................... 54
3.8.7 Cor instrumental .............................................................................................................. 54
3.8.8 Microscopia eletrônica de varredura (MEV) ................................................................ 54
3.9 AVALIAÇÃO DA CAPACIDADE ANTIOXIDANTE DAS MICROCÁPSULAS ........... 54
3.9.1 Ensaio DPPH .................................................................................................................... 54
3.9.2 Ensaio ABTS ..................................................................................................................... 55
3.9.3 Ensaio FRAP..................................................................................................................... 55
3.10 PROCESSAMENTO DOS HAMBÚRGUERES ............................................................... 55
3.11 AVALIAÇÃO DA COR INSTRUMENTAL DOS HAMBÚRGUERES .......................... 56
3.12 AVALIAÇÃO DA ESTABILIDADE OXIDATIVA DOS HAMBÚRGUERES .............. 56
3.12.1 Teor de fenólicos totais .................................................................................................. 56
3.12.2 TBARS ............................................................................................................................ 57
3.12.3 Compostos carbonílicos totais ....................................................................................... 57
3.13 ANÁLISE ESTATÍSTICA ................................................................................................. 57
4. RESULTADOS E DISCUSSÃO .......................................................................................... 61
Artigo II: Evaluation of oxidative stability of bovine burgers added to lyophilized and
microencapsulated extract from grape pomace (Vitis labrusca. Isabella Var.) used in the
production of fruit pulp. .............................................................................................................. 61
5. CONCLUSÕES GERAIS ..................................................................................................... 93
14
1 INTRODUÇÃO
O efeito adverso a saúde pelo uso de antioxidantes sintéticos tem causado bastante
preocupação entre os consumidores, fato que impulsiona as pesquisas voltadas a
aplicação de antioxidantes naturais na indústria de alimentos (NIRMALA et al., 2018).
Os polifenóis são compostos originários do metabolismo secundário das plantas, que
apresentam capacidade antioxidante além de outras atividades biológicas como anti-
inflamatória e antitumoral. São moléculas que retardam os processos oxidativos e
configuram uma alternativa ao uso de antioxidantes sintéticos (CAROCCHO;
MORALES; FERREIRA, 2018).
2 REFERENCIAL TEÓRICO
Marcelo Edvan dos Santos Silva1, Cristiani Viegas Brandão Grisi2, Suzana Pedroza da
Silva3, Fábio Anderson Pereira da Silva1*
Resumo: Essa revisão teve como objetivo investigar o potencial tecnológico do bagaço
da uva, analisando seu potencial para aplicação em produtos cárneos, abordando os
compostos de interesse e apresentando os desafios da sua aplicação em produtos cárneos.
A viticultura produz anualmente mais de 70 milhões de toneladas de uvas direcionadas
majoritariamente ao processamento. Em consequência são geradas grandes quantidades
de bagaço com elevado potencial tecnológico para aplicação na indústria da carne. O
bagaço da uva contém substâncias bioativas capazes de garantir uma maior estabilidade
oxidativa aos produtos cárneos e inibir o desenvolvimento microbiano, uma vez que estes
são responsáveis pela perda dos atributos de qualidade dos produtos cárneos. A
disponibilidade de nutrientes e de moléculas suscetíveis a oxidação confere uma baixa
estabilidade a carne e seus derivados, indicando a necessidade da aplicação de aditivos
capazes de garantir uma vida de prateleira mais prolongada. Nesse sentido, a aplicação
de compostos extraídos do bagaço da uva em substituição aos aditivos sintéticos usados
em produtos cárneos, atende a atual demanda por produtos naturais e conferem maior
estabilidade. Apesar dos desafios encontrados na aplicação de compostos fenólicos em
produtos cárneos, diversos estudos evidenciam que a aplicação de tecnologias como a
microencapsulação e produção de biofilmes permitem a aplicação dos extratos do bagaço
da uva sem que haja comprometimento da qualidade sensorial. Assim, o
reaproveitamento do bagaço da uva permite a obtenção de moléculas de alto valor
19
agregado a partir de um material de baixo custo, reduz os danos ocasionados pelo descarte
inadequado deste material e se insere nos objetivos do desenvolvimento sustentável
estabelecidos pela ONU.
1. Introdução
A uva (Vitis spp.) é uma das frutas mais consumidas no mundo, seja in natura ou
na forma de produtos derivados. Devido as suas características de perecibilidade, o
processamento é indispensável para a redução de perdas pós-colheita (Beres et al., 2017).
Estima-se que, em 2019, a área mundial de videiras foi de 7,4 milhões de hectares,
ultrapassando a marca de 70 milhões de toneladas de uvas produzidas (OIV, 2020). As
estimativas são de que aproximadamente 50% da produção total de uvas seja destinada
ao processamento, resultando em grandes quantidades de resíduos (Bordiga et al., 2019).
Os resíduos gerados pelo processamento da uva, em especial o bagaço apresenta grande
20
estimativas da FAO (2019), atualmente são perdidos cerca de 14% da produção total de
alimentos, considerando apenas as etapas referentes a cadeia produtiva, dos quais
resíduos de frutas e vegetais representam 21,6% desse montante. Quando somadas as
etapas de varejo e consumo, estima-se que cerca de 1/3 de todo o alimento produzido no
mundo seja desperdiçado, o que equivale a 1,3 bilhão de toneladas. Visando reduzir o
volume de perdas, a indústria processadora de frutas e hortaliças vem buscando minimizar
a geração de resíduos através do uso eficiente dos recursos, valorização dos subprodutos
e consequente redução do impacto ambiental (Leal et al., 2020).
As uvas são consumidas das mais variadas formas, a depender do estado físico e
das interferências político-religiosas de cada país, como a proibição do consumo de
bebidas alcoólicas, por exemplo. As características de perecibilidade impulsionam o
processamento da uva para a obtenção de diferentes produtos derivados, diminuindo o
volume de perdas nas etapas de armazenamento e distribuição (Venkitasamy et al., 2019).
22
O suco de uva é uma bebida não fermentada e rica em substâncias benéficas a saúde,
obtido a partir de um processo tecnológico adequado, sendo considerado um produto de
alto valor comercial (Oliveira et al., 2019). O consumo de suco de uva vem sendo
impulsionado em virtude da crescente demanda por produtos naturais, e tem conquistado
espaço no mercado mundial devido as suas características sensoriais, nutricionais e
nutracêuticas (Mesquita et al., 2020). Nesse sentido, a produção de sucos de uva é
realizada em grandes escalas, consequentemente gerando quantidades expressivas de
bagaço de uva com potencial para o reaproveitamento.
23
Em função do seu valor nutricional, as uvas secas estão entre as frutas desidratadas
mais importantes do mundo. Devido a versatilidade de utilização, podendo ser consumida
diretamente ou aplicada em diversas formulações gastronômicas, as uvas secas
apresentam uma tendência crescente de consumo (Mendonça et al., 2016). A produção de
uvas secas objetiva promover a conservação das uvas, reduzindo as perdas pós-colheita e
permitindo a obtenção de um produto final ou intermediário com aplicação na indústria
de alimentos (Cornejo et al., 2018).
Em relação aos resíduos gerados pela fabricação de uva secas, os dados na literatura
são escassos. No entanto, 1,3 milhões de toneladas de uva foram destinadas a produção
de uva passa em 2018 no mundo. A Turquia direcionou 40,7% da safra (381 mil
toneladas) para a elaboração de uva passa, seguida dos Estados Unidos e da China com
263 e 190 mil toneladas, respectivamente (OIV, 2019). Um estudo de caso publicado em
2004 por uma empresa norte americana relata que o resíduo gerado na produção de uvas
passas é basicamente a água de lavagem que é utilizada para remover a poeira proveniente
do solo. No entanto, certa quantidade de açúcares é dissolvida nessa água durante a
lavagem das uvas, o que inviabiliza a utilização desse rejeito devido à alta demanda
biológica de oxigênio (Elsevier, 2004).
restos de polpa, cascas e caules (constituída principalmente por fibras) e sementes (ricas
em óleo) retêm quantidades consideráveis de compostos bioativos como antocianinas,
flavonóis, ácidos graxos insaturados, proteínas e minerais (Tabela 1). A presença de fibras
alimentares no bagaço da uva também é bastante expressiva, sendo composta
majoritariamente por lignina e polissacarídeos da parede celular e representam cerca de
40% da composição das sementes, podendo compor até 75% do bagaço em base seca. As
cascas presentes no bagaço também são fontes de fibras, sendo constituídas por um
complexo lignocelulósico e apresentam quantidades consideráveis de açúcares
hemicelulósicos que, após a hidrólise, liberam os monossacarídeos glicose, xilose,
arabinose e galactose (Bordiga et al., 2019).
As cascas das uvas são ricas em estilbenos, flavanóis e antocianinas com grande
potencial em eliminar radicais livres, intimamente ligados à sua estrutura. Compostos
fenólicos e fibras que não são extraídas durante o processamento favorecem a introdução
das cascas da uva como fonte de metabólitos funcionais em uma diversa gama de
alimentos (Crupi et al., 2018). A aplicação de extratos da casca da uva em produtos
cárneos promove uma redução na descoloração ao longo do armazenamento.
Antocianinas e outros compostos fenólicos na casca da uva protegem o pigmento da carne
auxiliando na manutenção da cor dos produtos (Andrés et al., 2017).
O engaço é formado pelo caule e pelas ramificações que dão sustentação aos cachos
de uva. Em termos quantitativos, esse subproduto pode representar até 25% do peso total
do bagaço da uva. É rico em compostos fenólicos e fibra alimentar com potencial benéfico
a saúde e apresenta comprovada ação antimicrobiana (Gouvinhas et al., 2020). A
composição fenólica do engaço é semelhante às outras frações do bagaço da uva,
apresentando quantidades consideráveis de flavonóis, antocianinas, ácidos fenólicos,
estilbenos e procianidinas (Leal et al., 2020).
destas alterações pode ser bastante pronunciada em produtos cárneos (Delfanian e Sahari,
2020).
9. Conclusões
aplicação destes compostos. Assim, devem ser mantidos os esforços na busca por
condições aprimoradas para a incorporação de extratos fenólicos em produtos cárneos.
37
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Food and Agricultural By-Products. Academic Press, 2019.
Xiong, Y.; Chen, M.; Warner, R. D.; Fang, Z. Incorporating nisin and grape seed extracts
in chitosan-gelatine adible coating and its effect on cold storage of fresh pork. Food
Control, v. 110, p. 107018, 2020.
Zhang, R.; Zhou, L.; Li, J.; Oliveira, H.; Yang, N.; Jin, W.; Zhu, Z.;Li, Shuyi.; He, J.
Microencapsulation of antocianins extracted from grape skin by emulsification/internal
43
Zhu, M.; Huang, Y.; Wang, Y.; Shi, T.; Zhang, L.; Chen, Y.; Xie, M.; Comparison of
(poly)phenolic compounds and antioxidant properties of pomace extracts from kiwi and
grape juice. Food Chemistry, v. 271, p. 425-432, 2019.
44
Figura 1: Formas de uso das uvas (Elaborado a partir de informações obtidas de OIV,
International Organization of vine and wine, 2019 e Venkitasamy et al., 2019).
Figura 1
Figura 2
Extratos fenólicos
Extratos fenólicos
microencapsulados Extrato fenólico inserido em
embalagem ativa
Tabela 1: Resumo dos compostos nutricionais e bioativos disponíveis no subproduto do processamento da uva de acordo com vários autores.
Fibras Lipídios Açúcares Aminoácidos Minerais Fenólicos Autores
Lignina insolúvel e Ácidos linoleico, oleico _ _ _ Resveratrol, flavonóis, Venkitasamy et al., 2019
polissacarídeos da e palmítico catequinas, taninos
parede celular condensados e
antocianinas,
Lignina insolúvel e α-tocoferol, ß-sitosterol, Ramnose, arabinose, Ácido glutâmico, Potássio, fosforo, Antocianinas, ácidos Bordiga et al., 2019
polissacarídeos da ácidos linoleico, oleico e xilose, manose, aspártico e triptofano. enxofre e magnésio hidroxibenzóico e
parede celular palmítico. galactose, glicose e ácido hidroxicinâmico,
galacturonico. flavonóis e estilbenos.
Lignina insolúvel em Ácidos linoleico, oleico _ Ácido glutâmico, Sais (bitartarato e Antocianinas, García-Lomillo e
ácido e fibras contidas de e palmítico aspártico e triptofano, tartarato) de potássio, flavonoides, ácido Gozález-SanJosé, 2016.
proteínas e taninos. alanina e lisina. fosforo, enxofre, hidroxicinâmico, ésteres
magnésio, cálcio tartáricos, ácido gálico e
ácido protocatecuico.
Lignina insolúvel e Ácidos linoleico, oleico, Ramnose, arabinose, _ _ Ácidos hidroxibenzóico Beres et al.,2017
polissacarídeos da palmítico, esteárico, α- xilose, manose, e hidroxicinâmico,
parede celular tocoferol e ß-sitosterol. galactose, glicose e ácido flavonóis, taninos e
galacturonico. stilbenos.
48
3 MATERIAL E MÉTODOS
3.1 MATERIAL
O bagaço de uva (Vitis labrusca. Var. Isabel) utilizado nos experimentos foi
cedido pela indústria Canaã Polpas de Frutas, localizada na cidade de Goiana – PE. O
bagaço composto por sementes, cascas, caules e polpa residual (Figura 1) foi coletado
após a etapa de prensagem das uvas para obtenção do suco. Posteriormente, a amostra foi
congelada e transportada até o laboratório de apoio localizado na Universidade Federal
da Paraíba - UFPB, onde foi armazenada sob congelamento (-18 °C) até o momento das
análises.
Figura 1: Bagaço obtido após a prensagem da uva (Vitis labrusca L. var. Isabel) para
produção de polpa de fruta congelada.
Para o processo de extração foi adotada a metodologia utilizada por Caldas et al.
(2018). A extração foi realizada de acordo com o esquema apresentado na Figura 3:
Agitação mecânica
Filtração
Rotaevaporação
(60 °C)
Remoção do solvente
Congelamento
Liofilização
(-57 °C; 72h)
51
𝑚𝑔 𝐴 ∗ 𝑀𝑊 ∗ 𝐷𝐹 ∗ 103
𝐴𝑛𝑡𝑜𝑐𝑖𝑎𝑛𝑖𝑛𝑎 (𝑐𝑖𝑎𝑛𝑖𝑑𝑖𝑛𝑎 − 3 − 𝑔𝑙𝑖𝑐𝑜𝑠í𝑑𝑒𝑜, )= (1)
𝐿 ɛ∗1
Onde:
DF = Fator de diluição
10³ = fator de conversão de g para mg
ɛ = 26900, coeficiente molar, para ciadinina-3-glicosídeo
1 = caminho óptico percorrido (cubeta do espectrofotômetro)
TPC - SPC
EE = * (100%) (2)
TPC
3.8.5 Higroscopicidade
salina com a umidade relativa desejada, a mesma foi acondicionada em dessecador, para
evitar variações nos valores de umidade relativa. Em seguida, aproximadamente 1 grama
da amostra foi adicionada em placa de Petri previamente pesada. A placa contendo a
amostra foi acondicionada e mantida por uma semana no dessecador contendo a solução
salina (NaCl: 75,3% UR). Posteriormente, a amostra foi pesada e a higroscopicidade foi
calculada por meio da massa de água adsorvida pela amostra e expressa em g água
adsorvida por 100 g de matéria seca.
mantendo-se sob temperatura ambiente (25 °C) e protegido de luz durante 30 minutos.
Posteriormente, as leituras foram realizadas a 515 nm. Uma curva padrão foi preparada
com uma solução Trolox e os resultados foram expressos em μmol TEAC.100g-1.
3.12.2 TBARS
REFERÊNCIAS
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is increasingly reaching its maturity – a review. International Journal of Food Science
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C. A.; Borguini, R. G.; Godoy, R. L. O.; Cabral, L. M. C.; Tonon, R. V. Phenolic
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Estévez, M.; Cava, R. Lipid and protein oxidation, release of iron from heme molecule
and colour deterioration during refrigerated storage of liver pâté. Meat Science, v. 68, n.
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Meini, M, R.; Cabezudo, I.; Boschetti, C, E.; Romanini, D.; Recovery of phenolic
antioxidants from Syrah grape pomace through the optimization of na anzymatic process.
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changes in oxidized proteins. Journal of Biological Chemistry, v. 262, n. 12, p. 5488-
5491, 1987.
Re R.; Pellegrini N.; Proteggente A.; Pannala A.; Yang M, Rice-Evans C. Antioxidant
activity applying an improved ABTS radical cation decolorization assay. Free Radical
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Ribeiro, J, S.; Santos, M, J, M.; Silva, L, K, R.; Pereira, L, C, L.; Santos, I, A.; Lannes,
S, C, S.; Silva, M, V. Natural antioxidants used in meat products: A brief review. Meat
Science v. 148, p. 181-188, 2019.
Rufino, M. S. M.; Alves, R. E.; Brito, E. S.; Morais, S. M.; Sampaio, C. G.; Pérez-
Jiménes, J.; Saura-Calixto, F. D. Metodologia científica: Determinação da atividade
antioxidante total em frutas pela captura do radical livre DPPH. Comunicado
Técnico Embrapa, ISSN 1679-6535, Fortaleza - CE, julho de 2007.
Yen GC, Chen HY. Antioxidant activity of various tea extracts in relation to their
antimutagenicity. Agricultural and Food Chemistry. v, 43. p, 27–32, 1995.
61
4. RESULTADOS E DISCUSSÃO
Artigo II: Evaluation of oxidative stability of bovine burgers added to lyophilized and
microencapsulated extract from grape pomace (Vitis labrusca. Isabella Var.) used in the
Marcelo Edvan dos Santos Silva1, Rodrigo Lira de Oliveira2, Thamyres Cesar de
Albuquerque Sousa4, Cristiani Viegas Brandão Grisi4, Valquíria Cardoso da Silva
Ferreira4, Tatiana Souza Porto2, Marta Suely Madruga1, Suzana Pedroza da Silva3, Fábio
Anderson Pereira da Silva1*
Abstract: The objective of this work was to obtain and microencapsulate phenolic extract
from grape pomace obtained from the production of juice and frozen pulps and to evaluate
its antioxidant potential in relation to lipid and protein oxidation processes that occur in
precooked bovine hamburgers. The microcapsules were characterized and evaluated for
their morphology and antioxidant potential. Three hamburger formulations containing
lyophilized extract (LEB), microencapsulated extract (MEB) and synthetic sodium
erythrobate antioxidant (SEB) were elaborated. The hamburgers were precooked (72 ºC)
and evaluated for oxidation levels and changes in color. Microencapsulation, with 95.0%
efficiency, provided a material with crystalline structure, rich in phenolics and with high
antioxidant potential. The lyophilized and microencapsulated grape extracts promoted a
greater stability of the red color of hamburgers, indicating a protection of myoglobin
against oxidative processes. The LEB and MEB samples showed lower values of
malonaldehydes and carbonyl compounds, compared to the SEB samples throughout
storage, especially hamburgers that received the microencapsulated extract. Thus,
lyophilized and microencapsulated grape extracts showed superior results when
compared to the synthetic antioxidant, increasing the oxidative stability of hamburgers
and avoiding significant changes in color during the refrigerated storage period.
1 Introduction
The production of grapes to obtain derived products, such as wines and juices,
makes viniculture one of the most important agro-industrial activities in the world, in
economic terms and in harvest volume (Gulcu et al., 2018). According to data from the
International Organization of Vine and Wine (OIV, 2019), in 2018, 77.8 million tons of
grape were produced worldwide, with a planted area of 7.4 million hectares.
Several studies aimed at the reuse of pomace from wine making, however, the
production of frozen grape juices and pulps generates residues in similar proportions,
representing up to 20% of the initial weight of grapes (Zuh et al., 2019). The reuse of
grape pomace allows the obtaining of molecules of high added value and with great
potential for application in the food industry, from a low-cost source (Monteiro et al.,
2021). After processing, approximately 70% of phenolic compounds are retained in grape
pomace. The antioxidant potential of these compounds can protect food systems against
oxidative processes, constituting themselves as the components of greatest interest on the
part of the food industry (Pozo et al., 2021).
Oxidative processes are strongly related to the composition of the food matrix, since
high fat levels, the presence of salts, metal content and water activity increase the
susceptibility of food to oxidative processes. In addition, the processing steps can cause
the loss of the structure and release of pro-oxidant agents (Garcia-Lomillo and Gonzalez-
SanJosé, 2017). In meat and meat products, which have a rich composition of unsaturated
fatty acids, proteins, pigments and vitamins, lipid oxidation is one of the main causes of
loss of sensory and nutritional quality (Pateiro et al., 2018).
delay the discoloration processes of meat products and increase the oxidative stability of
lipids and proteins (Andrés et al., 2017). However, the antioxidant characteristic
associated with the presence of unsaturated bonds in phenolic compounds increases the
sensitivity of these compounds to variations in light, temperature and pH (Tolun et al.,
2020). In addition, the use of phenolic extracts in foods can promote taste changes such
as bitterness and astringency. In this sense, the use of microencapsulation technology
promotes greater stability of microencapsulated extracts and masks sensory changes
(Zhang et al., 2020).
solution. The mixture was stirred for 15 minutes in a magnetic stirrer, frozen, lyophilized
and stored in an amber bottle at -18 °C.
TPC - SPC
EE = * (100%) (1)
TPC
The color parameters of the microcapsules were measured using a portable CM-
2500c colorimeter (Konica Minolta Inc.) calibrated for CIELab color coordinates. In this
system, L* denotes luminosity on a scale from 0 to 100 (black to white); +a* (red color
intensity); -a* (green color intensity); +b* (yellow color intensity), and -b* (blue color
intensity).
For the antioxidant activity test involving the elimination of radical cation ABTS+•
generated from the oxidation of 2,2'-azinobis-3-ethylbenzothiazolin-6-sulfonic acid
(ABTS) 7 mM with potassium persulfate 2.45 mM, the reactionary mixture was pre-
incubated in the dark for 16 hours before use. The ABTS+• solution was adjusted for
absorbance between 0.700 and 0.800 to 734 nm in spectrophotometer, by dilution, being
performed according to the methodology described by Re et al. (1999). Aliquots of 100
to 400 μL of the diluted sample were mixed at 1 mL of the diluted ABTS solution (Radical
ABTS). The reaction mixture was incubated for 6 minutes at room temperature (23 °C).
The absorbance reading was performed at 734 nm and the elimination activity of the
ABTS radical was expressed in μmol of Trolox equivalent (TEAC) for every 100 g of the
sample, from the calibration curve.
2.7.2 DPPH
2.7.3 FRAP
In the formulation of hamburgers, 80% of beef (rear palette) (800 g/kg), 18% of
cold water (180 g/kg) and 2% of sodium chloride (20 g/kg) were used. The mixture was
70
divided into three portions, and the following treatments were applied: SEB – formulation
with the addition of synthetic antioxidant (sodium erythorbate 0.1 g/kg); LEB -
formulation with the addition of lyophilized phenolic extract (1.0 g/kg); MEB -
formulation with the addition of microencapsulated phenolic extract (6.7 g/kg). The
amounts of phenolic extracts, lyophilized and microencapsulated, were calculated
according to the phenolic content obtained in each extract, considering the amount of
sodium erythrobate used, in order to maintain the equivalent proportions. The processing
was carried out in three batches. After mixing each meat mixture and their corresponding
antioxidant, the hamburgers were molded and submitted to pre-cooking in an industrial
oven, with the internal temperature of the burger reaching 72 °C, being measured with
the aid of a digital skewer thermometer. After cooking, the hamburgers were packed in a
polystyrene tray (150x150x18 mm), covered with vinyl polychloride film (PVC) and
stored under refrigeration at an average temperature of 4 °C for 15 days. The samples
were analyzed at 0, 5, 10 and 15 days of storage. To evaluate the effect of the cooking
process on phenolic compound contents, oxidative stability and hamburger color, a batch
of raw hamburgers was produced in the three different treatments (SEB, LEB and MEB).
The instrumental color analysis was performed at 5 random points of the hamburger
surface using a digital colorimeter (MinoltaCo., CR-10, Osaka, Japan). Parameters L
(luminosity), a* (red/green intensity) and b* (yellow/blue color intensity) were evaluated.
2.11 TBARS
Estévez and Cava (2004). A standard tetraethoxypropane curve (TEP) was used to
determine the malonaldehyde (MDA) content. The result was expressed in mg of
MDA/kg sample.
Protein oxidation was evaluated by means of the total carbonyl compounds present
in the samples by the DNPH method (2,4-dinitrophenylhydrazine), by the methodology
adapted by Oliver et al (1987). The samples (1 g) were homogenized with phosphate
buffer 20 mM and NaCl 0.6 M pH 6.5 (1:10, p / v). After homogenization, 150 μL of the
mixture was used to determine protein concentration and carbonyl compound content. In
both cases, the proteins were precipitated with 1 mL of trichloroacetic acid at 10% (TCA)
after cold centrifugation (4 ° C) for 5 minutes at 2400 g. For the determination of total
carbonyl compounds, 1 mL of HCl 2 N + DNPH (0.2%) was added, and for protein
concentration, 1 mL of HCl 2 N was added, and the material was centrifuged at 9000 g
for 10 minutes. After two washes with 1 mL of ethanol / ethyl acetate (1: 1, v / v) followed
by centrifugation, the solvent was evaporated with nitrogen gas. The precipitated proteins
were dissolved in 1.5 mL of phosphate buffer 20 mM (pH 6.5) with Guanidine 6 M
hydrochloride. To determine the total carbonyl compounds and protein concentration,
absorbance readings were performed at 370 and 280 nm, respectively.
The data were processed by two-way analysis of variance (ANOVA). The means
were compared by the Tukey test, considering the significance level of 95% (P < 0.05),
using the statistical program SPSS (v. 20.0).
3 Results
3.1 Extraction yield
The extraction process allowed the obtaining of 147.1 ± 0.07 mg of lyophilized
extract per g of dried pomace. The calculation of phenolic extraction yield was performed
considering the relationship between the phenolic compound content of the extract and
the mass of the dry pomace sample used. The results indicated a yield of 15.21 ± 0.4 mg
GAE.g-1 of dry pomace.
The data obtained in the microencapsulation yield and in the characterization of the
microcapsules are summarized in Table 1. The results obtained for the contents of
phenolic compounds in microcapsules and lyophilized extract were 14.7 ± 0.04 and 103.4
± 0.04 mg GAE.g-1, respectively, with microencapsulation efficiency of 95.0 ± 0.05 %.
The concentration of monomeric anthocyanins in microcapsules was 94.84 ± 0.02 mg
cyanidine-3-glycoside.100g-1 of microencapsulated extract.
The microencapsulated material presented as a fine and dry powder, with low
moisture content (7.06 ± 0.4%), water activity of 0.465 ± 0.02 and hygroscopicity of
2.152 ± 0.2 g of water adsorbed per 100 g of sample. The microcapsules presented a dark
and pink coloration, with 33.6 of L*, 17.7 of a* and b* equal to -27.9. The evaluation of
the morphology of the microcapsules (Figure 1) indicated that the microencapsulation by
lyophilization provided a material with crystalline structure, where the microcapsules
presented a smooth and flake-shaped surface.
The colorimetric parameters obtained after cooking and over the 15 days of
refrigerated storage are presented in Table 2. The pre-cooking process caused statistically
significant changes (P < 0.05) in all color parameters (L*, a*, b*) for all treatments
evaluated (SEB, LEB and MEB). After cooking, the hamburgers presented a lighter
coloration, with an increase in luminosity values. A reduction in a* values was observed,
indicating a loss of red intensity, with SEB treatment being the most affected. For
parameter b*, increases were observed after cooking the samples, showing a tendency of
hamburgers to a yellowish color.
Storage time caused significant changes (P < 0.05) in the instrumental color of all
samples. For the L* (luminosity) coordinate, the SEB treatment showed an increase in
luminosity at the 5th day (P> 0.05). After the 5th day, it did not present changes in the
luminosity values (P< 0.05), but still presented the highest L* values at the end of the
experiment when compared with other treatments. The LEB treatment maintained
73
constant levels of luminosity until the tenth day of storage, presenting a loss of luminosity
at the end of storage. Regarding the MEB treatment, an increase in the values of
luminosity was observed at the 5th day of storage, remaining constant from then on and
not statistically different (P >0.05) when comparing the first and last day of storage.
At the beginning of storage, the precooked samples added from the lyophilized and
microencapsulated extract showed lower intensity of red color (a*). At times 0 and 10
(days), SEB treatment differed significantly (P < 0.05) from LEB and MEB treatments.
At the end of the 15 days, the LEB and MEB samples had the highest values of a*, not
differing from each other. Decay of a* levels was observed throughout storage for all
samples evaluated. However, the microcapsules treatment (MEB) had the lowest
percentage of red color reduction between 0 and 15 days (7.8%) compared to LEB
samples (18.5%) and SEB (38.7%).
There was no difference (P > 0.05) in the b* coordinate (yellow color intensity) of
the samples until the fifth day of storage. However, all treatments showed an increase (P
< 0.05) of b* only from the tenth day on, remaining constant until the end of storage.
3.4 Effect of the pre-cooking and storage process on phenolic compound content and
oxidative stability of hamburgers.
The pre-cooking process significantly increased (P < 0.05) the phenolic compound
levels of hamburgers (Figure 2). The increase in the contents of total phenolic compounds
after cooking ranged from 102.2 to 233.1 mg GAE.g-1 in the LEB treatment and from
109.6 to 208.0 mg GAE.g-1 in the MEB treatment.
In the evaluation of lipid oxidation levels by the TBARS assay, it was observed that
the cooking process did not significantly influence (P > 0.05) the malonaldehyde contents
of the samples (Figure 3A). The treatments SEB, LEB and MEB presented TBARS values
ranging from 0.43 to 0.48 mg MDA.kg-1, 0.27 to 0.37 mg MDA.kg-1, and from 0.29 to
0.42 mg MDA.kg-1, respectively.
The analysis of TBARS along the refrigerated storage showed that, for the
treatments SEB and LEB, increases in MDA levels were observed during the storage
period (Figure 4A). However, for the MEB treatment, no statistically significant
difference was observed in malonaldehyde values, ranging from 0.49 ± 0.02 mg MDA.kg-
1
at the beginning of the experiment to 1.39 ± 0.5 mg MDA.kg-1 at the end of the
74
experiment. The LEB treatment ranged from 0.42 ± 0.07 mg MDA.kg-1 to 2.87 ± 0.3 mg
MDA.kg-1, and the highest malonaldehyde values were found in the SEB treatment
samples ranging from 0.38 ± 0.1 mg MDA.kg-1 to 7.09 ± 0.9 mg MDA.kg-1 between the
first and last day of analysis. It was observed that the SEB treatment showed statistically
significant increases (P < 0.05) in all days of analysis. The LEB treatment showed stable
levels of malonaldehyde up to the tenth day of analysis, presenting a statistically
significant increase (P < 0.05) at the end of the experiment.
According to the results obtained, there was an effect of cooking on the increase of
protein carbonylation for the treatments SEB and LEB (Figure 3B). The exposure of the
hamburgers added with the microencapsulated extract (MEB) to the heat did not alter the
amount of total carbonyl compounds of the samples evaluated.
The total carbonyl contents of the SEB and MEB samples were influenced by
storage time, antioxidant type and the interaction of the two factors (P < 0.05). The SEB
sample showed a significant increase on the 10th day (114.6%), followed by a statistically
significant reduction (P < 0.05) at the end of storage. The MEB sample showed a
significant increase (P < 0.05) in the levels of carbonyl compounds on the 5th day and
then remained constant with the lowest carbonyl values. The LEB treatment maintained
constant levels of carbonyl compounds (P > 0.05) throughout the entire refrigerated
storage.
4 Discussion
4.1 Total phenolic compounds and extraction yield
of the phenolic compounds present in the pomace. Evaluating the concentration of the
extractive solution in the recovery of phenolic compounds of grape pomace, Caldas et al.
(2018) observed that the best yields were obtained in intermediate ethanol concentrations,
corroborating the results obtained in this study.
and 8.06%. These results indicate that the mean of moisture found in the microcapsules
of the present study are characteristic of the microcapsules obtained by lyophilization.
The microcapsules presented a dark color with luminosity of 33.6. Despite the light
coloration of maltodextrin, which can influence the increase in luminosity (Kuck and
Norenañ, 2016) the color intensity promoted by anthocyanins was predominant, resulting
in pink and dark microcapsules. The mean values of coordinates a* (17.7) and b* (-29.9)
reflect a tendency to red and blue, results that can also be attributed to the presence of
anthocyanins in the extract.
The microcapsules presented a characteristic morphology of the materials
microencapsulated by lyophilization. Crystalline structures were observed, with smooth
surface. According to Passos et al. (2015), the crystalline morphology of microcapsules
may be influenced by the dispersion of phenolic compounds during lyophilization. The
non-shrinkage characteristic observed in microcapsule structures is one of the quality
attributes of lyophilization-encapsulated materials. The frozen surface and the lack of
water in the liquid state during sublimation are responsible for the structural stiffness
observed in microcapsules (Kuck and Norenañ, 2016).
The FRAP method is an alternative for evaluating antioxidant activity based on iron
reduction capacity and has a positive correlation with the presence of phenolic
compounds in the sample (Embrapa, 2006). The microcapsules showed a high antioxidant
power of iron reduction (1185.0 μmol TEAC.100g-1). Evaluating the antioxidant capacity
of extracts obtained from different varieties of viniferous grapes, Leal et al. (2020) found
values between 350 μmol TEAC.100g-1 and 1030 μmol TEAC.100g-1 of FRAP. These
results are close to those found in our study. The grape variety and the type of processing
that gave rise to pomace have a significant impact on antioxidant power.
After cooking, a higher luminosity of the samples and loss of red color intensity
were observed, which may be associated with the effect of cooking temperature, causing
degradation of the myoglobin responsible for the red pigmentation of the meat, as well as
the partial loss of anthocyanins present in lyophilized and microencapsulated extracts.
In the evaluation of color parameters during the storage period, it was observed that
the freeze-dried and microencapsulated extracts promoted a stability in the luminosity of
the samples, not differing significantly (P > 0.05) between the first and last day of storage.
On the other hand, the SEB treatment presented higher luminosity values, differing
78
significantly from the other treatments. The SEB and LEB treatments showed significant
decreases (P < 0.05) in red color intensity throughout refrigerated storage. The reduction
of red color is associated with the oxidation of myoglobin, with the appearance of a brown
color on the surface of hamburgers, due to the accumulation of metamyoglobin
(Bouarabe-Chibane et al., 2017). However, no significant change in red color was
perceived in the MEB (P> 0.05) treatment during storage, indicating that
microencapsulation protected anthocyanins, avoiding changes in the red color of
hamburgers. In addition, the microencapsulation of phenolic compounds allows an
effective action of these compounds in the protection of myoglobin against oxidative
processes. The significant increase for the b* parameter in the SEB treatment indicates a
greater tendency to yellowing of the samples, while the treatments LEB and MEB
promoted a greater stability of the color of the hamburgers for parameter b* during the
storage period. Turan and Simsek (2021) evaluated the effects of the addition of
lyophilized phenolic extracts in hamburgers and observed a tendency of discoloration
from red to yellow in the control group samples (without addition of extract). In this sense,
a higher intensity of yellow is related to the loss of red color by oxidation of myoglobin.
The results obtained in the TBARS assay showed that lyophilized (LEB) and
microencapsulated (MEB) extracts of grape pomace promoted a greater inhibition of lipid
oxidation processes in the bovine hamburger during refrigerated storage. The lower
malonaldehyde content observed in the MEB treatment samples indicates that the
79
5 Conclusions
The extraction process using grape pomace from the production of frozen juices
and pulps allows obtaining an extract rich in phenolic compounds. The
microencapsulation process using the freeze-drying technique showed high efficiency,
80
providing microcapsules with crystalline morphology, low moisture and water activity,
showing the stability of the final product. Microcapsules containing phenolic compounds
presented high antioxidant potential, which promoted the inhibition of oxidative
processes in pre-cooked hamburgers. The antioxidant character of freeze-dried and
microencapsulated grape pomace extracts showed greater potential to ensure the stability
of hamburgers during storage compared to the synthetic antioxidant with emphasis on the
microencapsulated extract. The results of this work indicate that phenolic compounds
extracted from grape pomace have the potential to meet a market demand for products
formulated with natural ingredients to preserve meat products.
Acknowledgements
The authors thank the “Canaã” fruit pulp industry for the donation of grape pomace and
the Federal University of Paraiba for its support in carrying out this research.
Funding
The authors declare that the present study was funded in part by the Coordination for the
Improvement of Higher Education Personnel — Brazil (CAPES) for the scholarship
awarded to the first author - Finance Code 001, and “Fundação de Apoio à Pesquisa do
Estado da Paraíba” (FAPESQ-PB) for the financial support through project 005/2019.
81
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FIGURE CAPTIONS
Figure 1- Micrograph of grape pomace extract microencapsulated with maltodextrin by
lyophilization technique. A: magnified 1000x; B: magnified 500x; C: magnified 300x; D:
magnified 146x.
Figure 2 - Phenolic compounds that remained in the LEB and MEB samples after the cooking
process.
Footnote: Different lowercase letters on top of bars denote significant differences (p < 0.05)
between treatments. Different capital letters at the top of the bars indicate significant differences
due to the cooking process.
Figure 3 - Malonaldehyde concentration in different treatments (SEB, LEB and MEB) of raw and
precooked hamburgers during storage.
Footnote: Different lowercase letters on top of bars denote significant differences (p < 0.05)
between treatments. Different capital letters on top of bars denote significant differences due to
storage.
Figure 4 - Concentration of carbonyl compounds in different treatments (SEB, LEB and MEB) of
raw and precooked hamburgers throughout storage.
Footnote: Different lowercase letters on top of bars denote significant differences (p < 0.05)
between treatments. Different capital letters on top of bars denote significant differences due to
storage.
87
Figure 1
88
Figure 2
89
Figure 3
90
Figure 4
91
Means values ± standard deviation. *TPC: Total phenolic compounds (mg GAE.g-1); **TAC:
Total anthocyanin content (mg cyanidine-3-glicoside.100g-1); *** Expressed as a percentage.
92
a* SEB 10.6 ± 0.87aX 7.5 ± 0.30aY 5.4 ± 0.45aZ 4.9 ± 0.43bZ 4.6 ± 0.35bZ
LEB 11.1 ± 0.91aX 6.5 ± 0.15bY 6.0 ± 0.34aYZ 5.8 ± 0.34aYZ 5.3 ± 0.55abZ
MEB 10.0 ± 0.30aX 6.4 ± 0.34bY 5.5 ± 0.17aY 5.8 ± 0.20aY 5.9 ± 0.66aY
b* SEB 12.8 ± 0.45aZ 15.8 ± 0.23aY 16.5 ± 0.58aY 17.8 ± 0.70aX 18.2 ± 0.65aX
LEB 13.1 ± 0.70aZ 15.6 ± 0.20aY 15.9 ± 0.58aY 17.3 ± 0.50aX 16.3 ± 0.10bXY
MEB 12.3 ± 0.20aZ 15.3 ± 0.70aY 16.1 ± 0.56aXY 16.8 ± 0.55aX 16.3 ± 0.56bXY
SEB: hamburger with the addition of sodium erythorbate; LEB: hamburger with the addition of
lyophilized phenolic extract; MEB: hamburger with the addition of microencapsulated phenolic
extract. Different lowercase letters in the same column denote significant differences (p < 0.05)
between treatments. Different capital letters on the same line denote significant differences due
to the cooking process and storage time.
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5. CONCLUSÕES GERAIS
Diante dos resultados obtidos ao longo do período de observações, pode-se concluir que: