Propriedades antioxidantes e reológicas de geleia de goiaba com
Transcrição
Propriedades antioxidantes e reológicas de geleia de goiaba com
UNIVERSIDADE ESTADUAL DE MARINGÁ CENTRO DE CIÊNCIAS AGRÁRIAS Programa de Pós-Graduação em Ciência de Alimentos PROPRIEDADES ANTIOXIDANTES E REOLÓGICAS DE GELEIA DE GOIABA COM ADIÇÃO DE SUCO DE UVA CONCENTRADO RÚBIA CARVALHO GOMES CORRÊA Maringá 2012 RÚBIA CARVALHO GOMES CORRÊA PROPRIEDADES ANTIOXIDANTES E REOLÓGICAS DE GELEIA DE GOIABA COM ADIÇÃO DE SUCO DE UVA CONCENTRADO Dissertação apresentada ao Programa de Pós Graduação em Ciência de Universidade Alimentos da Estadual de Maringá para obtenção do grau de Mestre em Ciência de Alimentos. Orientadora: Profa. Dra. Angélica Marquetotti Salcedo Vieira Maringá 2012 Orientadora Profa. Dra. Angélica Marquetotti Salcedo Vieira Co-orientadora Profa. Dra. Rosangela Bergamasco BIOGRAFIA Rúbia Carvalho Gomes Corrêa nasceu em 26 de junho de 1986 na cidade de Maringá - PR. Concluiu o Ensino Fundamental na escola Notre Dame de Maringá e o Ensino Médio no Colégio Marista de Maringá. Em 2004 ingressou no curso de Engenharia de Alimentos da Universidade Estadual de Maringá, graduando-se em 2009. Durante o mesmo ano, realizou estágio curricular obrigatório na COCAMAR Cooperativa Agroindustrial. No ano de 2010, iniciou o mestrado no Programa de Pós-Graduação em Ciência de Alimentos - UEM. Neste mesmo período passou a integrar, como bolsista recém-formada, o projeto de extensão universitária Suporte Técnico à Agroindústria Familiar de Conservas, financiado pela Fundação Araucária do Paraná. Em 2011 concluiu o "Curso de Especialização em Biotecnologia Aplicada à Agroindústria", do Departamento de Biologia Celular e Genética - UEM. Participou de trabalhos nas áreas de Tecnologia de Produtos Vegetais, Aproveitamento de Subprodutos e Tecnologia de Alimentos Dietéticos e Nutricionais, com experiência em análises físico-químicas, microbiológicas e sensoriais em alimentos. DEDICO Aos meus amados pais Susy e Pedro Aos meus irmãos e amigos Susana, Pedro e Raísa AGRADECIMENTOS Primeiramente agradeço a Deus, que sempre esteve a meu lado e colocou em meu caminho pessoas capacitadas para me auxiliarem, mas também queridas que permanecerão para sempre em meu coração. Aos meus pais, por investirem constantemente em minha vida, e por sonharem junto comigo a realização deste mestrado. À professora Angélica, que me acompanha desde a graduação, pelo incentivo, orientação, paciência, generosidade, carinho e presença constante. À professora Rosangela, pelo grande otimismo, carinho e generosidade. Ao professor Charles, que nos recebeu na Universidade Tecnológica Federal do Paraná campus Campo Mourão, pela orientação valiosa, generosidade e essencial ajuda neste trabalho. À querida Gisele, por ter me acolhido em sua casa por diversas vezes, além de ter colaborado em todas as etapas desta pesquisa, sendo acima de tudo uma grande amiga e exemplo. Aos amigos e colegas do mestrado, em especial à Katieli, pela amizade, incentivo, companheirismo e grande ajuda nos momentos mais difíceis. À Universidade Estadual de Maringá, por mais esta oportunidade de formação e crescimento profissional. Aos professores do Programa de Pós-Graduação em Ciência de Alimentos - UEM, por terem contribuído para a minha formação, amadurecimento, e por servirem de inspiração. À Lúcia e à professora Rosane, pela competência e dedicação ao trabalho realizado no PPC. À CAPES e à Fundação Araucária pelo suporte financeiro e incentivo à pesquisa. A todos que contribuíram de alguma forma para a realização deste trabalho. APRESENTAÇÃO Esta dissertação de mestrado está apresentada na forma de artigo científico, que será submetido para a revista Lebensmittel-Wissenschaft & Technologie / LWT - Food Science & Technology, com extrato qualis A2 referente à avaliação trienal 2010 e com fator de impacto 2,292 para o mesmo ano. Rúbia Carvalho Gomes Corrêa, Gisele Teixeira de Souza Sora, Charles Windson Isidoro Haminiuk, Rosangela Bergamasco, Angélica Marquetotti Salcedo Vieira. Antioxidant and rheological properties of guava jam with addition of concentrated grape juice. Autores: Rúbia Carvalho Gomes Corrêa1 ([email protected]) Gisele Teixeira de Souza Sora1 ([email protected]) Charles Windson Isidoro Haminiuk2 ([email protected]) Rosangela Bergamasco3 ([email protected]) Angélica Marquetotti Salcedo Vieira4 ([email protected]) Filiações: 1 Universidade Estadual de Maringá, Programa de Pós-Graduação em Ciência de Alimentos. 2 Universidade Tecnológica Federal do Paraná, campus Campo Mourão. 3 Universidade Estadual de Maringá, Departamento de Engenharia Química. 4 Universidade Estadual de Maringá, Departamento de Engenharia de Alimentos. GENERAL ABSTRACT INTRODUCTION. The oxidative stress originated by reactive oxygen species (ROS) is fundamentally linked to the manifestation of numerous chronic and degenerative diseases. Fortunately, studies have strongly demonstrated that a diet rich in antioxidants can protect from health harms caused by the oxidation process. Guava (Psidium guajava L.) is a popular tropical fruit characterized by excellent nutritional and sensory properties, besides an extraordinary content of carotenoids, especially lycopene. Because fruits constitute the major dietary source of phenolic compounds, it has been suggested that fruit juices have potential to be used as functional ingredients in food industry. As grapes are rich sources of potent antioxidants with proven cardio protective properties, concentrated grape juice could be applied as a potential functional ingredient in food formulations. In this context, guava and concentrated grape juice could figure as great raw materials in jam formulations, thus originating potential functional products with a high sensory appeal. AIMS. The present study aimed to develop production technology of guava jam with addition of concentrated grape juice, prioritizing the antioxidants profile and rheological characterization of this new product. MATERIALS AND METHODS. The following elements were used in the jams processing: pink guava fruits, Paluma cultivar; commercial sucrose; mineral water; high methoxyl pectin; citric acid and potassium sorbate. The concentrated grape juice was obtained by reverse osmosis process from the natural juice of Isabel cultivar grapes. One standard formulation (GP) and one enriched formulation (GS), in which concentrated grape juice was added in the proportion of 29.70%, were elaborated. Guava jams were therefore prepared using different concentrations of sugar (39.52 % and 19.80%) and pectin (0.69 %, and 0.99 %). For the physicochemical characterization of the jams, the following analyses were performed: total soluble solids (°Brix), acidity (% citric acid), moisture and total sugar. In order to investigate the antioxidant potential of the guava jams, fresh samples were subjected to sequential extractions with methanol and acetone, and in the end of the process the two extracts were combined. The amount of phenolic compounds in the extracts was determined by the Folin-Ciocalteu colorimetric method and gallic acid were used as standard. The results were expressed as gallic acid equivalents (GAE). Extracts of the guava jams and the concentrated grape juice used as a enriching ingredient were analysed by High-performance liquid chromatography (HPLC) to quantify different phenolic compounds. Standards of gallic acid, catechin, caffeic acid, p-coumaric acid, ferulic acid, resveratrol, quercetin and kaempferol were used to obtain the standard curve. Two antioxidant systems, which act by distinct mechanisms, were used to evaluate the antioxidant activity of the guava jams. That were the β-carotene-linoleic acid coupled oxidation system and the 1,1Diphenyl-2-picrylhydrazyl radical (DPPH•) method. The stability of the phenolic compounds and the antioxidant activity of the jams were evaluated through 90-day storage at room temperature (on average 25°C). The rheological behaviour of the samples were assessed at temperatures of 25°C, 35°C, 45°C and 55°C using a Brookfield rheometer model DVIII with a SC4-34 spindle. The data of deformation rate and shear stress were adjusted by the Power Law model, while the effect of temperature on apparent viscosity was described by an equation analogous to Arrhenius. RESULTS AND DISCUSSION. The results showed all jam formulations had total soluble solids content compatible with the requirements of the Brazilian legislation, which establishes a minimum of 65 °Brix for total soluble solids in jam. The values obtained for the moisture content in GP and GS, which were 36.34% and 37.16% (wet basis), respectively, were also in accordance with the legislation. The total sugar content found for both guava jam formulations are close to the values related in other references. The acidity (% citric acid) found for GS formulation was higher than the value found for GP formulation, which indicates that the addition of grape juice promoted a increasing in the jam acidity, though GS acidity value still is in accordance to legislation parameters. As expected, was found a greater concentration of phenolic compounds (PC) in the GS formulation than in the GP formulation, and most important, the enriched jam showed a PC content of more than two times the standard formulation content. Moreover, the data obtained over the monitoring period showed a decreasing on the PC content with the days, for both GP and GS formulations. The pair of guava jams presented similar stability over the storage time, since the PC content of GP formulation dropped 32.18% while the PC content of GS formulation dropped 36.21%. The major compound identified in the samples was the flavonoid catechin, followed by gallic acid and quercetin in very minor concentrations. The GS formulation presented greater contents of galic acid, caffeic acid, p-coumaric acid and ferrulic acid than the GP formulation, and even presented superior contents of these compounds than the concentrated grape juice that was added to its formulation. In this last, was found an extraordinary content of catechin in addition to less representative contents of the other compounds, reason why the addition of concentrated grape juice in guava jam formulation provided a significant contribution in terms of phenolic compounds, especially in terms of catechin. Among the phenolic compounds quantified in the guava jams, catechin represented 99.93% and 52.58%, respectively, of the total value of compounds found for GS and GP. In the guava jams extracts were also found low contents of quercetin and kaempferol. The GS formulation showed a content of quercetin 2-fold higher than the GP formulation, while both formulations showed equivalent contents of kaempferol. At the 45th day of storage, the results obtained for the measure of antioxidant activity of the jams by the β-carotene-linoleic acid system were: among the positive controls used in this test, Trolox showed the highest value of antioxidant activity , which were 58.28 ± 2.78% at 100 ppm; GS presented an antioxidant activity of 53.862 ± 0.388%, that were 18.5% higher than the value found for GP; and the antioxidant activity of the positive controls BHT, Trolox and Propyl gallate were not significantly different (p < 0.05).The data of the antioxidant activity monitoring of the guava jams based on the β-carotene method, during 90-day storage, surprisingly revealed a increasing in antioxidant activity with time, for both jams and for the positive controls. So the antioxidant activity of GP at the end of the 90-day storage was 1.7-fold higher than the value found for the 1st day, and GS showed a corresponding increase of 1.5-fold the original value. This results could be explained by a possible transformation in the phenolic compounds with time, where the originated polyphenols presented lower antioxidant activity in the hydrophilic DPPH• system and greater activity in the lipophilic β-carotene system. On the results of the DPPH• assay at the 45th day of storage, the antioxidant activity ranged from 95.86 ± 0.15 % (Propyl gallate) to 63.94 ± 1.03 % (GP). GS extract showed better antioxidant efficiency value than GP extract, with an antioxidant capacity of 67.93 ± 0.81 %. The EC50 values, inversely proportional to the antioxidant activity values, were of 25.45 ± 1.03 µg/mL for GP and 20.40 ± 0.22 µg/mL for GS, which indicates that the enriched jam provided higher protection against oxidation than the standard guava jam. In the rheological behaviour study of both guava jams, the values of consistency coefficient, K, and the flow behaviour index, n, were obtained by curve fitting using least square method. The satisfactory regression coefficients, R2, all superior to 0.984, showed the effectiveness in fitting. The n values, found for the pair of samples, showed that GP and GS exhibited exhibited a non-Newtonian shear-thinning behaviour (n < 1) at all evaluated temperatures. The increasing of temperature showed no significant influence on the pseudoplasticity of both guava jams. Also the phenomena of decreasing on consistence coefficient, K, with temperature, was observed. There were a decreasing in the GP and GS values of apparent viscosity with increasing shear rate and temperature. A close observation on the apparent viscosity results showed that the GS formulation suffered higher drop in its viscosity than the GP formulation, with the temperature increasing. GP and GS values of apparent viscosity dropped 26.78% and 34.67%, respectively, when the temperature increased from 25°C to 55°C. The GS formulation presented higher apparent viscosity values than GP formulation, for all evaluated temperatures. The values of activation energy found for GP and GS were of 8.16 kJ.mol-1 and 12.47 kJ.mol-1, respectively, which indicate that the GP was more sensitive to temperature changes than GS, thus, the enriched formulation presented higher stability than the standard formulation when exposed to temperature variation. CONCLUSIONS. The incorporation of concentrated grape juice in the jam formulation promoted an increase of more than 2-fold in its phenolic compounds content, with catechin representing 99.93% of the total value of compounds found for GS, furthermore, provided an increase in the antioxidant activity of the product in almost 20%. This way, the consumption of the enriched guava jam could potentially deliver health benefits by the supply of natural antioxidants, especially the flavonoid catechin. The enriched guava jam exhibited a non-Newtonian behaviour and behaved as a shear thinning fluid for all evaluated temperatures. The Power Law model described appropriately the rheological behaviour of the elaborated jam. This study revealed that the guava jam enriched through the addition of concentrated grape juice constitute a new product with a notable antioxidant potential, even greater than the potential presented by the standard guava jam, worldwide appreciated delicacy. RESUMO GERAL INTRODUÇÃO. O estresse oxidativo originado por espécies reativas de oxigênio (ROS) é fundamentalmente ligado à manifestação de inúmeras doenças crônicas e degenerativas. Felizmente, estudos têm demonstrado que uma dieta rica em antioxidantes pode proteger dos danos à saúde causados pelo processo oxidativo. A goiaba (Psidium guajava L.) é uma fruta tropical popular caracterizada por excelentes propriedades nutricionais e sensoriais, além de um conteúdo extraordinário de carotenóides, especialmente licopeno. Devido ao fato das frutas constituírem a principal fonte dietética de compostos fenólicos, têm-se sugerido que os sucos de frutas apresentam potencial para serem utilizados como ingredientes funcionais na indústria de alimentos. Como uvas são ricas fontes de potentes antioxidantes com comprovadas propriedades cardioprotetoras, o suco de uva concentrado pode ser aplicado como um potencial ingrediente funcional em formulações de alimentos. Neste contexto, goiaba e suco de uva concentrado poderiam figurar como ótimas matérias-primas na formulação de geleias, originando assim produtos com potencial funcionalidade e grande apelo sensorial. OBJETIVOS. O presente estudo teve como objetivo desenvolver tecnologia de produção de geleia de goiaba com adição de suco de uva concentrado, priorizando o perfil de antioxidantes e caracterização reológica deste novo produto. MATERIAL E MÉTODOS. Os seguintes elementos foram utilizados no processamento das geleias: goiabas vermelhas do cultivar Paluma; sacarose comercial; água mineral; pectina de alto grau de metoxilação; ácido cítrico e sorbato de potássio. O suco de uva concentrado foi obtido por processo de osmose inversa a partir do suco natural de uvas do cultivar Isabel. Uma formulação padrão (GP) e uma formulação enriquecida (GS), na qual o suco de uva concentrado foi adicionado na proporção de 29,70%, foram elaboradas. As geleias de goiaba foram, portanto, preparadas com diferentes concentrações de açúcar (39,52% e 19,80%) e pectina (0,69% e 0,99%). Para a caracterização físico-química das geleias, as seguintes análises foram realizadas: determinação de sólidos solúveis totais (°Brix), acidez (ácido cítrico %), umidade e açúcares totais. A fim de investigar o potencial antioxidante das geleias de goiaba, as amostras frescas foram submetidas a extrações sequenciais com metanol e acetona, e ao final do processo os dois extratos foram combinados. A determinação de compostos fenólicos nos extratos foi feita pelo método colorimétrico de Folin-Ciocalteau e ácido gálico foi utilizado como padrão. Os resultados foram expressos como equivalentes de ácido gálico (GAE). Extratos das geleias de goiaba e do suco de uva concentrado utilizado como ingrediente enriquecedor foram analisados por Cromatografia Líquida de Alta Eficiência (CLAE), para a quantificação de diferentes compostos fenólicos. Padrões de ácido gálico, catequina, ácido caféico, ácido p-cumárico, ácido ferúlico, resveratrol, quercetina e kaempferol foram usados para obter a curva padrão. Dois sistemas antioxidantes, que agem por mecanismos distintos, foram utilizados para avaliar a atividade antioxidante das geleias de goiaba. Estes foram o sistema de cooxidação do β-caroteno/ácido linoléico e o método de sequestro do radical livre 1,1difenil-2-picrilhidrazil radical (DPPH •). A estabilidade dos compostos fenólicos e da atividade antioxidante nas geleias foi avaliada durante 90 dias de estocagem à temperatura ambiente (média de 25°C). O comportamento reológico das amostras foi avaliado em temperaturas de 25°C, 35°C, 45°C e 55°C utilizando um reômetro Brookfield modelo DVIII com spindle SC4-34. Os dados de taxa de deformação e tensão de cisalhamento foram ajustados pelo modelo de Lei da Potência, enquanto que o efeito da temperatura sobre a viscosidade aparente foi descrito por uma equação análoga à de Arrhenius. RESULTADOS E DISCUSSÃO. Os resultados mostraram que todas as formulações de geleia atingiram o teor de sólidos solúveis compatíveis com as exigências da Legislação Brasileira, que estabelece um mínimo de sólidos solúveis totais de 65 °Brix para geleias. Os valores obtidos para o teor de umidade em GP e GS, que foram 36,34% e 37,16% (base úmida), respectivamente, também encontravam-se de acordo com a legislação. O teor de açúcar total encontrado para ambas as formulações de geleia de goiaba estão próximos aos valores relatados em outras referências. A acidez (% de ácido cítrico) encontrada para a formulação GS foi maior do que a acidez encontrada para a formulação GP, o que indica que a adição de suco de uva promoveu um aumento na acidez da geleia, embora o valor de acidez de GS ainda se encontrasse em conformidade com os parâmetros da legislação. Como esperado, foi descoberto uma maior concentração de compostos fenólicos (CF) na formulação GS do que na formulação GP, e mais importante, a geleia enriquecida apresentou um conteúdo de CF equivalente a mais de duas vezes o conteúdo da formulação padrão. Além disso, os dados obtidos ao longo do período de monitoramento mostraram um decréscimo sobre o teor de CF com os dias, tanto para a formulação GP quanto para a formulação GS. Ambas as geleias de goiaba apresentaram estabilidade semelhante ao longo do tempo de armazenamento, uma vez que o conteúdo de CF da formulação GP caiu 32,18%, enquanto que o conteúdo de CF da formulação GS caiu 36,21%. O principal composto identificado nas amostras analisadas foi o flavonoide catequina, seguido pelo ácido gálico e quercetina em concentrações muito menores. A formulação GS apresentou teores maiores de ácido gálico, ácido caféico, ácido p-cumárico e ácido ferrúlico do que a formulação GP, e até mesmo apresentou teores superiores destes compostos quando comparada ao suco de uva concentrado que foi adicionado à sua formulação. Neste último, foi encontrado um conteúdo extraordinário de catequina, além de conteúdos menos representativos dos outros compostos, razão pela qual a adição de suco de uva concentrado na formulação da geleia de goiaba proporcionou uma significativa contribuição em termos de compostos fenólicos, especialmente em relação à catequina. Entre os compostos fenólicos quantificados nas geleias de goiaba, a catequina representou 99,93% e 52,58%, respectivamente, do valor total de compostos encontrados para GS e GP. Nos extratos de geleia de goiaba também foram encontrados baixos teores de quercetina e kaempferol. A formulação GS apresentou um conteúdo de quercetina duas vezes maior do que a formulação GP, enquanto ambas as formulações apresentaram teores equivalentes de kaempferol. No 45o dia de armazenamento, os resultados obtidos para a medida da atividade antioxidante das geleias de goiaba pelo sistema β-caroteno-ácido linoléico foram: entre os controles positivos utilizados no ensaio, Trolox apresentou o valor mais elevado de atividade antioxidante, que foi de 58,28 ± 2,78%, a 100 ppm; GS apresentou uma atividade antioxidante de 53,862 ± 0,388%, a qual foi 18,5% maior do que o valor encontrado para GP; e as atividades antioxidantes dos controles positivos BHT, Trolox e Galato de propila não foram significativamente diferentes (p < 0,05). Os dados de monitoramento da atividade antioxidante das geleias de goiaba baseado no método do β-caroteno, durante 90 dias de armazenamento, surpreendentemente revelaram um crescimento da atividade antioxidante com o tempo, para ambas as geleias e os controles positivos. Assim, a atividade antioxidante de GP ao final do armazenamento de 90 dias foi 1,7 vezes mais elevada do que o valor encontrado para o 1o dia de armazenamento, e GS mostrou um aumento correspondente de 1,5 vezes o valor original de atividade antioxidante. Este resultado pode ser explicado por uma possível transformação nos compostos fenólicos com o tempo, em que os polifenóis originados apresentaram menor atividade antioxidante no sistema hidrofílico do método DPPH• e maior atividade antioxidante no sistema lipofílico do método do β-caroteno. Em relação aos resultados do ensaio com o método DPPH • realizados ao 45o de armazenamento, os valores encontrados para a atividade variaram entre 95,86 ± 0,15% (Galato de propila) e 63,94 ± 1,03% (GP). O extrato de GS apresentou um melhor valor de eficiência antioxidante do que o extrato de GP, com uma capacidade antioxidante de 67,93 ± 0,81%. Os valores de EC50, inversamente proporcionais aos valores de atividade antioxidante, foram de 25,45 ± 1,03 mg / mL para GP e 20,40 ± 0,22 mg / mL para GS, o que indica que a geleia enriquecida proporcionou maior proteção contra a oxidação do que a geleia padrão. No estudo do comportamento reológico de ambas as geleias de goiaba, os valores do coeficiente de consistência, K, e do índice de comportamento de fluxo, n, foram obtidos por ajuste de curva, usando para isto o método dos mínimos quadrados. Os satisfatórios coeficientes de regressão, todos superiores a 0,984, mostraram a eficiência do ajuste dos dados. Os valores de n, encontrados para as duas amostras, indicam que GP e GS exibiram um comportamento não-Newtoniano e se comportaram como fluidos pseudoplásticos (n <1) em todas as temperaturas avaliadas. O aumento da temperatura não apresentou influência significativa sobre a pseudoplasticidade de ambas as geleias de goiaba. Também o fenômeno do decréscimo do coeficiente de consistência, K, com a temperatura, foi observado. Houve uma diminuição nos valores de viscosidade aparente de GP e GS com o aumento da taxa de cisalhamento e da temperatura. Uma observação mais acurada dos resultados de viscosidade aparente mostrou que a formulação GS sofreu maior queda em sua viscosidade do que a formulação GP, com o aumento da temperatura. Os valores de viscosidade aparente de GP e GS caíram 26,78% e 34,67%, respectivamente, quando a temperatura aumentou de 25°C para 55°C. A formulação GS apresentou maiores valores de viscosidade aparente do que a formulação GP, para todas as temperaturas avaliadas. Os valores de energia de ativação encontrados para GP e GS foram de 6,93 kJ.mol-1 e 9,02 kJ.mol-1, respectivamente, o que indica que GP foi mais sensível às mudanças de temperatura do que a formulação GS, assim, a formulação enriquecida apresentou maior estabilidade que a formulação padrão quando exposta à variações de temperatura. CONCLUSÕES. A incorporação do suco de uva concentrado na formulação da geleia promoveu um aumento de mais de duas vezes no seu conteúdo de compostos fenólicos, com catequina representando 99,93% do valor total de compostos encontrados em GS, e ainda proporcionou um aumento na atividade antioxidante do produto em quase 20%. Desta forma, o consumo da geleia enriquecida poderia oferecer benefícios à saúde, através do fornecimento de antioxidantes naturais, especialmente a catequina. A geleia enriquecida exibiu um comportamento não-Newtoniano e se comportou como fluido pseudoplástico para todas as temperaturas avaliadas. O modelo Lei da Potência descreveu adequadamente o comportamento reológico da geleia elaborada. Este estudo revelou que a geleia enriquecida através da adição de suco de uva concentrado constitui um novo produto, com um potencial antioxidante notável, ainda maior do que o potencial apresentado pela geleia de goiaba padrão, iguaria apreciada em todo o mundo. ANTIOXIDANT AND RHEOLOGICAL PROPERTIES OF GUAVA JAM WITH ADDITION OF CONCENTRATED GRAPE JUICE Authors: Rúbia Carvalho Gomes Corrêa1*, Gisele Teixeira de Souza Sora1, Charles Windson Isidoro Haminiuk2, Rosangela Bergamasco3, Angélica Marquetotti Salcedo Vieira1. Affiliations: 1 Universidade Estadual de Maringá, Programa de Pós-Graduação em Ciência de Alimentos, Maringá, PR, Brazil. 2 Universidade Tecnológica Federal do Paraná, Programa de Pós-Graduação em Tecnologia de Alimentos, Campus Campo Mourão, PR, Brazil. 3 Universidade Estadual de Maringá, Departamento de Engenharia Química, Maringá, PR, Brazil. Abstract In this research, guava jam with addition of concentrated grape juice (GS), a new product, was studied for its antioxidant potential and rheological behaviour. Total phenolic content, 1,1diphenyl-2-picrylhydrazyl radical (DPPH•) scavenging activity and β-carotene-linoleic acid couple oxidation assays were used to evaluate the antioxidant properties of the samples during 90-day storage. HPLC analysis was performed to quantify different phenolic compounds (PC). The GS showed a PC content of 1109.23 ± 2.18 mg GAE/100g of jam at production, which dropped 36.21 % through the monitoring period. The flavonoid catechin constituted the major compound found in GS, corresponding to 99.93 % of the total value of compounds. The antioxidant capacity measured by the antioxidant tests increased with time on the β-carotene assay and decreased with time on the DPPH assay. The enriched guava jam exhibited a nonNewtonian shear-thinning behaviour at temperatures ranging from 25 to 55 °C. This study revealed that the enriched jam constitutes a product with notable antioxidant potential, even greater than the potential presented by the standard guava jam, worldwide appreciated delicacy. Keywords: jam, guava, phenolic compounds, rheology 1. Introduction The definition widely accepted by several organizations for “functional foods” is: “foods or ingredients of foods that provide an additional physiological benefit beyond their basic nutrition” (ADA, 2004; ILSI, 1999). Fruits and vegetables constitute the simplest form of this food category, because they are originally rich in bioactive phytochemicals like polyphenols and carotenoids. Due to the popularization of such information, a surge of research and product development in the food industry emerged to meet demand from consumers for healthful food (Day, Seymour, Pitts, Konczak & Lundin, 2009). Guava (Psidium guajava L.) is a tropical fruit characterized by a low content of carbohydrates, fats, proteins, high vitamin C and fibre levels (Gutiérrez, Mitchell & Solis, 2008), besides an extraordinary content of carotenoids, especially lycopene (4–6 mg/100 g of edible portion) (Wilberg & Rodriguez-Amaya, 1995). In addition to its nutritious profile, guava presents notable sensory properties, serving as raw material in manufacturing of marmalades, jellies, jams and juices, also due to its high perishability (Osorio, Forero & Carriazo, 2011). Another fruit with remarkable content in antioxidants, including phenolic compounds, is the grape. Grapes are rich sources of anthocyanins, catechins, proanthocyanidins, flavonols, stilbenes, and other phenolics, all of them potent antioxidants that show cardio protective properties (Zern & Fernandez, 2005), so that consumption of grape juice has been associated with the decrease of platelet aggregation (Keevil, Osman, Reed & Folts, 2000). Studies have strongly demonstrated that a diet rich in antioxidants can protect from health harms caused by the oxidation process. Especially the polyphenols, which present antiinflammatory, anti-carcinogenic, anti-atherogenic and cardio protective effects, besides a protective role towards brain degenerative processes (Brenna, Ceppi & Giovanelli, 2009). The oxidative stress originated by reactive oxygen species (ROS) is fundamentally linked to the manifestation of numerous chronic and degenerative diseases (Azizova, 2002), being associated to the occurrence of atherosclerosis, cancer, diabetes mellitus, cardiovascular and neurodegenerative diseases, besides the ageing process itself (Valko, Leibfritz, Moncol, Cronin, Mazur & Telser, 2007) Since fruits constitute the major dietary source of phenolic compounds, it has been suggested that fruit juices have potential to be used as functional ingredients in food industry (Karaaslan, Ozden, Vardin & Turkoglu, 2011). In this context, concentrated grape juice could be applied as a potential functional ingredient in food formulations, especially jams, also because the combination of fruits tends to add a higher nutritional value and thus add value to the new product. Fruit jam is the delicacy obtained by cooking fruit, sugar and water until producing a gelatinous consistency that, when extracted from its container, is able to remain in semi-solid state. It may undergo the addition of acidulants and pectin, if necessary, to compensate for any deficiency on natural acidity or pectin content of the fruit (Brazil, 2007). It is acknowledged that the quantity of added sugar, proportion and kind of gelling agent, fruit pulp content, besides the process temperature, are factors that directly influence the rheological properties of fruit jams (Basu & Shivhare, 2010). The development of a new product, as a jam with addition of concentrated fruit juice, demands the determination of its rheological parameters, once the use of pre-existing parameters in industry could easily incur process errors. This because of the importance of rheological behaviour to the design of processes such as pumping, stirring and transport in pipelines (Branco & Gaspareto, 2005). Not least, the study of rheological properties works as a great tool in quality control, also permitting the correlation of food texture with sensory attributes (Basu & Shivhare, 2010). Therefore, the present study aimed to develop production technology of guava jam with addition of concentrated grape juice, prioritizing the antioxidants profile and rheological characterization of this new product. 2. Materials and methods 2.1. Materials The following elements were used for the preparation of the guava jams: pink guava fruits (Psidium guajava, Paluma cultivar), provided by Sitio Centenario (Mandaguaçu, PR, Brazil); commercial sucrose; mineral water; high methoxyl pectin (CPKelco, Limeira, SP, Brazil); citric acid and potassium sorbate (Sigma–Aldrich Co., Steinheim, Germany). The concentrated grape juice (Vitis labrusca) was obtained from the natural juice of Isabel cultivar grapes. 2.2. Jams Processing One standard formulation (GP) and one enriched formulation (GS), in which concentrated grape juice was added in the proportion of 29.70 %, were elaborated. Guava jams were therefore prepared using different concentrations of sugar (39.52 % and 19.80 %), pectin (0.69 %, and 0.99 %). The guava fruits were cleaned with a sodium hypochlorite solution, peeled and then its cores (central part which contains all the seeds) were took out and discarded. The guava pulp was processed in blender with enough mineral water to form paste, which was transferred to a cooking pot with controlled heating, being constantly homogenized. After 15 minutes of cooking process, the pectin and sucrose were added to the paste. Exclusively for GS formulation, the addition of concentrated grape juice was made in the final stage of cooking, in order to avoid and minimize the degradation of antioxidants by the heat. Next, the additives were incorporated and the heating proceeded until the ending cooking point at 65°Brix. The guava jams were bottled in sterilized jars. 2.3. Physicochemical characterization All the physical-chemical analysis was performed in triplicate, with results expressed as mean ± standard deviation (SD). The following analysis were performed according to regulations of the (AOAC, 2005): total soluble solids (°Brix), acidity (% citric acid), moisture and total sugar. 2.4. Phenolic compounds extraction Jam samples were weighed (g) in centrifuge tubes and extracted sequentially with 40 ml of methanol/water (50:50, v/v) by agitation in a test tube rotary mixer at room temperature for 1 h. The tubes were centrifuged at 6.000 rpm for 15 min and the supernatant was recovered. Then 40 ml of acetone/water (70:30, v/v) was added to the residue at room temperature, extracted for 60 min and centrifuged. Finally, methanol and acetone extracts were combined, made up to 100 ml with distilled water and used to determine total phenolic compounds and antioxidant capacity (Rufino, Alves, de Brito, Pérez-Jiménez, Saura-Calixto & Mancini-Filho, 2010). Aliquots analyzed by HPLC suffered only methalonic extraction, as described above. The supernatant was filtered in 0.25 µm of Millipore membrane (Millipore, Bedford, MA). 2.5. Phenolic compounds analysis The amount of phenolic compounds in the guava jam extracts was determined by the FolinCiocalteau colorimetric method (Singleton & Rossi, 1965), using gallic acid as a standard. Diluted samples or blanks (100 µL) were mixed with 5 mL of distilled water, Folin–Ciocalteau reagent (500 µL), 1.5 mL of 15 % sodium carbonate (after 3 minutes) and finally, distilled water enough to complete each 10 mL volumetric baloon. The mixture was incubated in the dark at room temperature for 2 h. The absorbance was then measured at 765 nm using an UV⁄ Vis double beam spectrophotometer T-80 (PG Instruments Limited, Beijing, China). The results were expressed as gallic acid equivalents (GAE) using a calibration curve over the range of 5– 250 ppm (Vasco, Ruales & Kamal-Eldin, 2008). High-performance liquid chromatography analysis was used to quantify the presence of individual phenolic compounds. The Dionex Ultimate 3000 HPLC (Dionex, Idstein, Germany) system was used, equipped with an Ultimate 3000 pump, Ultimate 3000 sampler column compartment, Ultimate 3000 photodiode array detector, and Chromeleon software to qualify and quantify the phenolic compounds. The Acclaim ® 120, C18 5 µm 120 A (4.6 mm x 250 mm) reversed-phase column was used to separate the phenolic compounds. The column was kept at 40 °C throughout the analysis, and detection was performed at three wavelengths (280, 306, and 370 nm). The sample injection volume was 10 µL. The mobile phase (A) comprised water acidified with 1% phosphoric acid and phase (B) with methanol. The elution of phenolic compounds was performed by means of a gradient between both mobile phases. After 45 minutes, the column was held at 100% mobile phase B for 5 minutes. Phase (B) was then gradually reduced until it reached the initial injection condition for column conditioning purposes (95% A and 5% B). A flow of 1.0 mL/min was used. Standards of gallic acid, catechin, caffeic acid, p-coumaric acid, ferulic acid, resveratrol, quercetin, kaempferol were used to obtain the standard curve of the phenolic compounds. The tests were replicated twice. 2.6. 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH•) assay The free radical scavenging activity was assessed with the DPPH• method as previously described by (Mensor, Menezes, Leitão, Reis, Santos, Coube et al., 2001). Based on the total phenolic compound values, six different concentrations of the jam extract were used to perform the DPPH• assay. The 0.3 mmol L-1 DPPH• ethanol solution (1 mL) was added to 2.5 mL of sample solutions of different concentrations, and the mixtures were allowed to react at room temperature. After 30 min, the absorbance values were measured at 518 nm, and they were converted into the antioxidant activity percentage (AA%) using the following equation: ⎡ (Abssample − Absblank ) ⎤ AA% = 100 − ⎢ x100⎥ Abscontrol ⎣ ⎦ (1) Where Abssample = sample absorbance The antioxidant capacity was expressed as the concentration of antioxidant required to reduce the original amount of free radicals by 50% (EC50) and values expressed as µg/mL. 2.7. β-Carotene bleaching method The antioxidant capacity of each sample was estimated by the β-carotene bleaching method, following the procedure described by (Emmons, Peterson & Paul, 1999) with modifications proposed by (Prado, 2009). Aliquots of the β-carotene-linoleic acid emulsion (3 mL) were mixed with 50 µL of the jam extract (100 ppm) (Haminiuk, Plata-Oviedo, Guedes, Stafussa, Bona & Carpes, 2011), the mixtures were kept in a water bath at 50 °C. Spectrophotometric readings were made at 470 nm 3 minutes after the mixing and then at 30–120 min intervals. The antioxidant activity was expressed as percent inhibition relative to the control after incubation for 120 min using the following equation: ⎛ DR − DRS AOA = 100x⎜⎜ C DRC ⎝ ⎞ ⎟⎟ ⎠ (2) where AOA is the antioxidant activity, DRC is the degradation rate of the control (ln(a ⁄ b) ⁄ 120), DRS is the degradation rate of the sample (ln(a ⁄ b) ⁄ 120), a is the initial absorbance at time zero, and b is the absorbance at 120 min. Trolox [(±)-6-hydroxyl-2,5,7,8tetramethlychromane-2-carboxylic acid], BHT (buthylated hydroxytoluene) and Propyl gallate were used as standards with a concentration of 100 ppm. The total phenolic content and the antioxidant activity of the guava jams, based on the both DPPH• method and coupled oxidation of β-carotene-linoleic acid system, were evaluated during 90-day storage at room temperature (on average 25 °C). 2.8. Rheological behaviour study This study assessed the rheological behaviour of guava jams at temperatures of 25 °C, 35 °C, 45 °C and 55 °C. Rheological measurements were performed using a Brookfield rheometer model DVIII with a SC4-34 spindle endowed with a small sample amount adapter (Brookfield Engineering Laboratories, Massachusetts, USA). A Tecnal Thermostatised T-184 thermostatic bath (Tecnal, Piracicaba, São Paulo, Brazil) was used to adjust the temperature of jam samples to a temperature range between 25 and 55 °C. The data on apparent viscosity (ηap), shear stress (τ), and shear rate ( γ ) were collected by means of Rheocalc software (version V3.1-1, Brookfield Engineering Laboratories, USA). To perform the rheological analysis, 10 mL samples were used for each measurement (as recommended by the manufacturer), which were kept for 15 minutes before testing for temperature stabilization. In each experimental run, the ascending curve lasted for 2 minutes with shear rates varying between 0,1 and 10 s-1. Two experimental runs were performed on each sample; the resulting shear stress was the average of the two experimental values. The experimental values of shear stress and shear rate were fitted to the rheological models of Ostwald-de-Waele (Power Law). τ = κγ n where τ = shear stress (Pa) K = consistence index (Pa.sn) n = fluid behaviour index (dimensionless) γ = shear rate (s-1) (3) To non-Newtonian fluids, define the term apparent viscosity (ηap) is fundamental, since the ratio between the shear stress (τ) and the rate of deformation (γ) is not a constant, being that for the Power Law model, the apparent viscosity can be defined as (Guedes, Ramos & Diniz, 2010): ηap = Kγ n−1 (4) The apparent viscosity was calculated using the equation 4 and based on a deformation rate of 10 s-1. The effect of temperature on apparent viscosity is described by an equation analogous to Arrhenius (Guedes, Ramos & Diniz, 2010): ⎛ Ea ⎞ ⎟ ⎝ RT ⎠ η ap = η 0 exp⎜ (5) where η ap = apparent viscosity (Pa.s) η 0 = constant (Pa.s) Ea = activation energy for viscous flow (kJ.g.mol-1) R = gas constant (8,314J.g.mol-1.K-1) T = absolute temperature (K) 2.8. Statistical analysis Assays were performed in triplicate for each sample. The results were expressed as the mean values ± SD. Data were evaluated by variance analysis (ANOVA) and the values that showed significant mean difference were then treated by the post-hoc Tukey’s test, at probability (p ≤ 0.05) level of 5%, using Statistica 7.1 software (StatSoft, Tulsa, OK, USA). The data of flow curves for the two guava jam formulations were adjusted by the Power Law model (OstwaldDe-Walle) using the software Origin 7.1 (OriginLab Corporation, MA, USA) in order to obtain the rheological and statistical parameters (n, K and R2). 3. Results and Discussion 3.1. Physicochemical characterization The proximate composition of the principal raw material and formulated guava jams is presented in Table 1. The results showed the pair of jams formulations had total soluble solids content compatible with the requirements of the Brazilian legislation, which establishes a minimum of 65 °Brix for total soluble solids in jam (Brazil, 2007), with no significant difference between the values. The values obtained for the moisture content in GP and GS, which were 36.34 % and 37.16 % (wet basis), respectively, were also in accordance with the legislation. The total sugar content found for both guava jam formulations are close to the values related by (Besbes, Drira, Blecker, Deroanne & Attia, 2009) for date jams. Statistically significant differences were found between the GS and GP values of acidity (% citric acid). The acidity value found for GS was more than 1.4-fold the value found for GP, which indicates that the addition of grape juice promoted a increasing in the jam acidity, though GS acidity value still is in accordance to legislation parameters. Table 1 Composition of the concentrated grape juice, guava (Psidium guajava, var. Paluma) and guava jams (GP and GS) Property Concentrated Guava GP GS grape juice Moisture (%) nd 84.96 ± 0.07a 36.34 ± 0,07 c 37.16 ± 0.09b ° Brix 18.40 ± 0.05b 7.40 ± 0.02c 65.04 ± 0.01a 65.08 ± 0.00a Acidity (% acid citric) 1.20 ± 0.01a 0.63 ± 0.06c 0.87 ± 0.02b 1.24 ± 0.02a Total Sugar A nd 87.14 ± 0.51c 92.90 ± 0.13a 91.73 ± 0.13b GP: standard formulation; GS: enriched formulation. Means in line with different letters are significantly different (p < 0.05). A g /100 g dry matter. nd = not determined 3.1. Phenolic compound content Nowadays, is notable the increasing interest in the possible health-promoting effects of phenolic compounds. In this sense, the characterisation, quantification and identification of these compounds are fundamental to authenticate their potential health benefits in human nutrition (Fischer, Carle & Kammerer, 2011). To that end, the stability of the phenolic compounds and the antioxidant activity of the jam were evaluated through 90-day storage at room temperature (on average 25 °C). Table 2 shows the monitoring of the phenolic compounds (PC) content of the both guava jam formulations during 90-day storage. As expected, it was found a greater concentration of phenolic compounds in the GS formulation than in the GP formulation, and most important, the enriched jam showed a PC content of more than 2-fold the standard formulation content. Moreover, the data obtained over the monitoring period showed a decreasing on the PC content with the days, with significant differences between the values found at the 1st, 45th and 90th day of storage, for both GP and GS formulations. The pair of guava jams presented similar stability over the storage time, since the PC content of GP formulation dropped 32.18 % while the PC content of GS formulation dropped 36.21 %. Table 2 Phenolic compound content of the guava jam formulations in mg GAE/100g during 90-day storage. Jam samples Storage time (days) 1st 45th 90th GP 490.00 ± 0.54a 397.692 ± 1.63b 332. 308 ± 1.09c GS 1109.23 ± 2.18a 870.769 ± 1.09b 759. 231 ± 1.63c GP: standard formulation; GS: enriched formulation. Means in line with different letters are significantly different (p < 0.05). * GAE: galic acid equivalents Extracts of the guava jams and the concentrated grape juice used as an enriching ingredient were analysed by HPLC to quantify different phenolic compounds. Figure 1 shows the chromatograms corresponding to the samples, which were standard formulation (GP), enriched formulation (GS) and concentrated grape juice. The major compound identified in the samples was the flavonoid catechin, followed by gallic acid and quercetin in very minor concentrations. Flavonoids have been reported to exhibit potent antioxidative and free radical scavenging activities, and catechin is one of the most representative flavonoids in the human diet, demonstrating, as other flavonoids, excellent antioxidant potential (Uzun, Demir, Kalender, Bas & Kalender, 2010). Catechin is the basic monomeric unit of grape and wine proanthocyanidins (Riberau-Gayon, Glories, Maujean & Dubourdieu, 2006), that present diverse pharmacological effects, like cardioprotection and diuretic and hypotensive actions, being the main polyphenolic component in tea and fruit (Uzun, Demir, Kalender, Bas & Kalender, 2010). Figure 1 HPLC chromatograms of the standard formulation (GP), enriched formulation (GS) and concentrated grape juice extracts. How can be observed in Figure 1, the GS formulation presented greater contents of galic acid, caffeic acid, p-coumaric acid and ferrulic acid than the GP formulation, and even presented superior contents of these compounds than the concentrated grape juice that was added to its formulation. In this last, was found an extraordinary content of catechin in addition to less representative contents of the other compounds, reason why the addition of concentrated grape juice in guava jam formulation provided a significant contribution in terms of phenolic compounds, especially in terms of catechin. Among the phenolic compounds quantified in the guava jams, catechin represented 99.93 % and 52.58 %, respectively, of the total value of compounds found for GS and GP. In the guava jam extracts were also found low contents of quercetin and kaempferol. The GS formulation showed a content of quercetin 2-fold higher than the GP formulation, while both formulations showed equivalent contents of kaempferol. 3.2. Antioxidant activity assays Considering that the antioxidant capacity of food is defined by a mixture of different antioxidants, with diverse action mechanisms including synergistic interactions, it is fundamental to associate more than one method to properly evaluate the antioxidant capacity of foodstuff (Almeida, de Sousa, Arriaga, do Prado, Magalhães, Maia et al., 2011). For this reason, two antioxidant systems, which act by distinct mechanisms, were used to evaluate the antioxidant activity of the guava jams. That were the β-carotene-linoleic acid coupled oxidation system and the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) methods. While the first method determines the activity of a sample or composed to protect a lipid substrate from oxidation, the method of inhibition of DPPH • radical is based on the transfer of electrons from an antioxidant compound to an oxidant (Duarte-Almeida, Santos, Genovese & Latojo, 2006). On the coupled oxidation of β-carotene and linoleic acid system, antioxidant capacity is defined by the capacity of extracts to inhibit β-carotene bleaching promoted by free radicals generated during linoleic acid peroxidation. Figure 2 (a) presents the antioxidant activity of the guava jam extracts at the 45th day of storage, as measured by the bleaching of the β-carotene-linoleic acid system. Among the positive controls used in this test, Trolox showed the highest value of antioxidant activity (inhibition of the free radical peroxidation of lipids): 58.28 ± 2.78 % at 100 ppm, although antioxidant activity values found for Trolox, BHT and Propyl gallate were not significantly different (p ≤ 0.05). The GS extract presented antioxidant activity only 7.6 % lower than the Trolox, and still 18.5 % higher than the value found for GP formulation. This result indicates the positive effect of the addition of concentrated grape juice in GS formulation, improving its inhibition of lipid peroxidation. Figure 2. (A) Antioxidant activity of the guava jams by coupled oxidation of β-carotene and linoleic acid at the 45th day of storage. (B) Antioxidant activity and EC50 values of guava jams on the DPPH• assay at the 45th day of storage. ND = not determined. Bars with different letters are significantly different (p < 0.05). In the DPPH • method, the reduction of DPPH • radical by radical scavengers is evaluated spectrophotometrically by monitoring the decrease in absorbance, as the DPPH radical is decolorized from deep violet to pale yellow (Al-Dabbas, Al-Ismail, Kitahara, Chishaki, Hashinaga, Suganuma et al., 2007). Namely, the DPPH• radical scavenging capacity assay monitors the percentage of the DPPH• remaining after a given time (Brand-Williams, Cuvelier & Berset, 1995). Antioxidant activity and EC50 values of guava jam extracts on the DPPH• assay at the 45th day of storage are presented in Figure 2 (b). It can be seen that antioxidant activity ranged from 95.86 ± 0.15 % (Propyl gallate) to 63.94 ± 1.03 % (GP). GS extract showed better antioxidant efficiency value than GP extract, with an antioxidant capacity of 67.93 ± 0.81 %. The EC50 values, inversely proportional to the antioxidant activity values, were of 25.45 ± 1.03 µg/mL for GP and 20.40 ± 0.22 µg/mL for GS, which indicates that the enriched jam provided higher protection against oxidation than the standard guava jam. The results of the antioxidant activity monitoring of the guava jams based on the coupled oxidation of β-carotene and linoleic acid system during 90-day storage are presented in Table 3. The data revealed an increasing in antioxidant activity with time, for both jams and for the positive controls. The antioxidant activity of GP at the end of the 90-day storage was 1.7-fold higher than the value found for the 1st day, and GS showed a corresponding increase of 1.5-fold the original value. Table 3 Antioxidant activity (%) of the guava jams based on the coupled oxidation of β-carotene and linoleic acid system, during 90-day storage. Trolox, BHT and propyl gallate were used as standards with a concentration of 100 ppm. Sample Storage time (days) 1st 45th 90th GP 36.664 ± 1.168c 43.904 ± 0.447b 62.181 ± 1.481a GS 44.485 ± 1.169c 53.862 ± 0.388b 66.966 ± 0.042a BHT 38.307 ± 0.476c 46.014 ± 0.548b 69.114 ± 2.493a Trolox 50.130 ± 1.297b 58.283 ± 2.781b 83.985 ± 1.723a Propyl gallate 42.463 ± 1.852b 43.904 ± 0.447b 62.181 ± 1.481a GP: standard formulation; GS: enriched formulation. Means in line with different letters are significantly different (p ≤ 0.05). These results could be explained by a possible transformation in the phenolic compounds with time, where the originated polyphenols presented lower antioxidant activity in the hydrophilic DPPH• system and greater activity in the lipophilic β-carotene system. The phenomenon of changing in the phenolic profile during storage was related by (Baiano, Gambacorta, Terracone, Previtalli, Lamacchia & La Notte, 2009) in their research over changes in phenolic and antioxidant activity of olive oils during store. Corroborating the advantage of using both methods for assessment of the antioxidant activity, (Alén-Ruiz, García-Falcón, Pérez-Lamela, Martínez-Carballo & Simal-Gándara, 2009) highlighted that DPPH• and linoleic acid/β-carotene assays provide complementary information, once the assays measure hydrophilic and lipophilic antioxidants, respectively. The evolution of DPPH • radical scavenging antioxidant activity in the guava jams with time, through 90-day storage, is presented in Table 4. The values of antioxidant activity observed for both guava jam formulations were significant, and as expected, smaller than the values of antioxidant activity found for the positive controls. Over the whole storage period GS formulation showed greater antioxidant activity than GP formulation, so that GS had an antioxidant activity 10 % higher than GP at the first day of storage, advantage which was also observed in the monitoring of antioxidant activity by the bleaching of the β-carotene-linoleic acid system. The values of antioxidant activity of the jams remained rather stable throughout storage, since there was a decreasing of 17.67 % for GP formulation and 18.68 % for GS formulation. (Piljac-Žegarac, Valek, Martinez & Belščak, 2009) also observed a decreasing in antioxidant capacity of dark fruit juices in 29-day refrigerated storage. Table 4 Antioxidant activity (%) of the guava jams based on the DPPH• assay, during 90-day storage. Trolox, BHT and propyl gallate were used as standards with a concentration of 100 ppm. Sample Storage time (days) 1st 45th 90th GP 65.884 ± 0.867a 63.938 ± 1.026a 54.241 ± 0.592b GS 71.195 ± 0.433a 67.927 ± 0.806b 57.906 ± 0.296c BHT 96.425 ± 0.144a 95.492 ± 0.220b 95.550 ± 0.052b Trolox 96.170 ± 0.072a 95.130 ± 0.147b 95.864 ± 0.222a Propyl gallate 96.476 ± 0.072a 95.855 ± 0.147b 95.026 ± 0.074c GP: standard formulation; GS: enriched formulation. Means in line with different letters are significantly different (p ≤ 0.05). 3.4. Rheological behaviour study The rheograms in Fig. 3 and Fig. 4 show plots of shear stress versus shear rate of GP and GS formulations, respectively, measured in a temperature range from 25 to 55 °C. As can be observed in both rheograms, there were a decreasing in the shear stress with the increasing of temperature. Figure 3 GP rheogram for temperatures ranging from 25 to 55 °C. Figure 4 GS rheogram for temperatures ranging from 25 to 55 °C. The values of consistency coefficient, K, and the flow behaviour index, n, for both guava jams, were obtained fitting the experimental data into the Power Law model. The correspondent values are presented in Table 5, showing effectiveness in fitting with satisfactory regres0sion coefficients, R2, all superior to 0.9885. Table 5 Parameters from power law equation fitting τ = Kγn; consistency coefficient, K, and flow behaviour index, n and apparent viscosity, ηap calculated at 10 s-1 of guava jams and temperatures in °C. Sample T(°C) Consistency Flow behaviour Coefficient of Apparent coefficient, K index, n determination, viscosity, ηap n GP GS 2 (Pa.s ) (dimensionless) R 25 344.61 ± 3.25 0.3230 ± 0.0052 0.9934 15.25 ± 0.12 35 347.65 ± 3.81 0.2900 ± 0.0061 0.9885 13.24 ± 0.02 45 308.48 ± 3.34 0.3017 ± 0.0060 0.9898 12.39 ± 0.89 55 285.49 ± 1.87 0.2956 ± 0.0036 0.9960 11.17 ± 0.33 25 401.19 ± 1.79 0.3883 ± 0.0024 0.9990 23.96 ± 0.61 35 390.58 ± 3.16 0.3545 ± 0.0044 0.9961 20.05 ± 1.33 45 334.77 ± 2.82 0.3289 ± 0.0046 0.9950 16.58 ± 1.03 55 329.84 ± 2.36 0.3341 ± 0.0039 0.9964 15.40 ± 2.03 (Pa.s) GP: standard formulation; GS: enriched formulation. The n values obtained for both samples, showed that GP and GS exhibited a non-Newtonian shear-thinning behaviour (n < 1) at all evaluated temperatures. The increasing of temperature showed no significant influence on the pseudoplasticity of both guava jams. Similar results were observed by (Chin, Chan, Yusof, Chuah & Talib, 2009) when analysing pummelo juice at 50 °Brix, fluid with a total soluble solids value close to the approximated 65 °Brix found for our guava jam formulations. The phenomenon of decreasing on consistence coefficient, K, with temperature, was also observed by (Vitali & Rao, 1982) in guava puree and by (Basu & Shivhare, 2010) in mango jam. As presented in Table 5, there were a decreasing in the GP and GS values of apparent viscosity with increasing shear rate and temperature. A close observation on the apparent viscosity results shows that the GS formulation suffered higher drop in its viscosity than the GP formulation, with the temperature increasing. The GP and GS values of apparent viscosity dropped 26.78 % and 34.67 %, respectively, when the temperature increased from 25 °C to 55 °C. It also can be seen in Table 5 that GS formulation presented higher apparent viscosity values than GP formulation, for all evaluated temperatures. The linear relationship of apparent viscosity (η ap ) by temperature (1 / T), for both samples, is presented in Figure 5. The values of activation energy found for GP and GS were of 8.16 kJ.mol-1 and 12.47 kJ.mol-1, respectively, which indicate that the GP was more sensitive to temperature changes than GS, thus, the enriched formulation presented higher stability than the standard formulation when exposed to temperature variation. Figure 5 The linear relationship of apparent viscosity (ηap) by temperature (1 / T) for the both GP and GS formulations. 4. Conclusion The incorporation of concentrated grape juice in the jam formulation promoted an increase of more than 2-fold in its phenolic compounds content, with catechin representing 99.93 % of the total value of compounds found for GS, furthermore, provided an increase in the antioxidant activity of the product in almost 20 %. This way, the consumption of the enriched guava jam could potentially deliver health benefits by the supply of natural antioxidants, especially the flavonoid catechin. The enriched guava jam exhibited a non-Newtonian behaviour and behaved as a shear thinning fluid for all evaluated temperatures. The Power Law model described appropriately the rheological behaviour of the elaborated jam. This study revealed that the guava jam enriched through the addition of concentrated grape juice constitute a new product with a notable antioxidant potential, even greater than the potential presented by the standard guava jam, worldwide appreciated delicacy. Acknowledgements The authors would like to thank the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES), Federal University of Technology – Paraná (Campus Campo Mourão), National Council for Scientific and Technological Development (CNPq; Process Number 501535/2009-8) and Araucaria Foundation for financial support. We would also like to thank CPKelco which donated the pectin used in jams preparation. References ADA. (2004). Position of the American Dietetic Association: Functional Foods. Journal of the American Dietetic Association, 109(4), 735-746. Al-Dabbas, M. M., Al-Ismail, K., Kitahara, K., Chishaki, N., Hashinaga, F., Suganuma, T., & Tadera, K. (2007). The effects of different inorganic salts, buffer systems, and desalting of Varthemia crude water extract on DPPH radical scavenging activity. Food Chemistry, 104(2), 734-739. Almeida, M. M. B., de Sousa, P. H. M., Arriaga, Â. M. C., do Prado, G. M., Magalhães, C. E. d. C., Maia, G. A., & de Lemos, T. 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