Influence of the addition of coriander and annatto in the formation of cholesterol oxides during thermal treatment and storage of white hake

Orientador: Neura BragagnoloDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: Pescados são alimentos ricos em ácidos graxos poli-insaturados, os quais são extremamente suscetíveis à oxidação, originando principalmente radicais livres e hidroperóxidos que aceleram o processo oxidativo do colesterol, ocasionando a formação de óxidos de colesterol. Estes óxidos são mais prejudiciais à saúde do que o próprio colesterol, pois aumentam o risco do aparecimento de doenças cardiovasculares e ainda favorecem uma série de efeitos biológicos indesejáveis tais como inflamações, artrite reumatóide e citotoxidade. O objetivo principal do presente trabalho foi avaliar o efeito protetor de antioxidantes naturais na oxidação do colesterol em pescada branca (Cynoscion spp). O filé de pescada adquirido no comércio local de Campinas foi moído em processador doméstico até a obtenção de uma massa homogênea, a qual foi pesada e dividida em quatro partes. Uma parte foi considerada controle sem adição de antioxidantes e as outras três foram adicionadas de 0,1% de urucum, 0,5% de coentro e 0,1% de urucum mais 0,5% de coentro. Foram moldadas almôndegas de 50 ± 2 g, embaladas em filmes de polietileno de baixa densidade (6 µm de espessura) e congeladas em freezer a -18 °C por 120 dias e analisadas nos dias zero, 15, 30, 45, 60, 90 e 120 de armazenamento. Uma parte das amostras foi analisada crua e outra após o cozimento em água fervente a 96 ± 2°C por 30 minutos, até que a temperatura interna atingisse 91 ± 2. As análises de óxidos de colesterol e de colesterol durante o armazenamento e após o tratamento térmico foram realizadas por cromatografia líquida de alta eficiência (HPLC) com detectores de UV e índice de refração. A identificação dos óxidos de colesterol foi feita por (HPLC) e a confirmação desses óxidos realizada por detector de espectrometria de massas (HPLC-MS/MS). Foi realizada a análise dos teores de lipídios totais no tempo zero para caracterizar a amostra e de umidade em todos os tempos para expressar os resultados em base seca. Três óxidos de colesterol, 7-cetocolesterol, 7B-hidroxicolesterol e 7alfa-hidroxicolesterol foram identificados e quantificados durante o armazenamento e tratamento térmico, além disso foram identificados os epóxidos 5,6alfa- e 5,6 B-epoxicolesterol pelo HPLCMS/ MS. O tratamento térmico não alterou os teores de óxidos de colesterol. Durante o armazenamento, as amostras adicionadas de condimentos e controle apresentaram um aumento no teor de óxidos de colesterol. No entanto, não houve diferença significativa entre as amostras controle e tratadas, indicando que a oxidação foi equivalente em todas as amostrasAbstract: Fish meat is a good source of polyunsaturated fatty acids, which are compounds extremely susceptible to oxidation. This process originates free radicals and peroxides, which can accelerate the oxidative process of cholesterol, causing formation of cholesterol oxides. These compounds are more harmful than cholesterol regarding heart diseases, and are also related to some biological effects like inflammation, rheumatoid arthritis and cytotoxicity. The main goal of the present study was the evaluation of the protective effect of natural antioxidants on cholesterol oxidation in white fish (Cynoscion spp). Fish fillet was acquired in a local store in Campinas and was grinded in a domestic food processor to obtain a homogeneous mass. The fish was then weighted and divided in four portions: control without any spice, spiced with annatto (0.1%), spiced with coriander (0.5%) and spiced with both [annatto (0.1%) plus coriander (0.5%)]. Fish meatballs of 50 ± 2 g were molded, packed in low density polyethylene film (thickness 6 µm) and stored frozen at ¿ 18 ºC for 120 days. Samples were analysed at 0, 15, 30, 45, 60, 90, and 120 days of storage. Part of the samples was analyzed raw and another part after cooking in boiling water at 96 ± 2°C for 30 minutes, until the core temperature reached 91 ± 2 ºC. Cholesterol oxides analysis during storage and after heat treatment was carried out by high-performance liquid chromatography (HPLC) coupled to UV and refractive index detectors. Cholesterol oxides identification was performed by HPLC and the confirmation of these oxides was analyzed by high-performance liquid chromatography with a mass spectrometer detector (HPLC-MS/MS). Total lipids were determined at day zero in order to characterize the sample, and moisture content was assayed at all times to express the results in dry weight. Three cholesterol oxides, 7- cetocholesterol, 7B-hydroxycolesterol and 7alpha-hydroxycolesterol were identified and quantified during storage and heat treatment, and also, epoxides 5,6alpha- and 5,6B- epoxicholesterol were identified by HPLC-MS/MS. Heat treatment did not change cholesterol oxides content. During storage, spicy and control samples have presented an increase of cholesterol oxides content. However there was no statistical significant difference among control and treated groups, indicating that all samples have oxidized equallyMestradoMestre em Ciência de Alimento

Chemical inhibition of the contaminant lactobacillus fermentum from distilleries producing fuel bioethanol

The purpose of this study was to determine the Minimum Inhibitory Concentration (MIC) of pure or mixed chemicals for Saccharomyces cerevisiae and Lactobacillus fermentum in the samples isolated from distilleries with serious bacterial contamination problems. The biocides, which showed the best results were: 3,4,4' trichlorocarbanilide (TCC), tested at pH 4.0 (MIC = 3.12 mg/l), TCC with benzethonium chloride (CBe) at pH 6.0 (MIC = 3.12 mg/l) and TCC mixed with benzalkonium chloride (CBa) at pH 6.0 (MIC = 1.53 mg /l). If CBa was used in sugar cane milling in 1:1 ratio with TCC, a 8 times reduction of CBa was possible. This formulation also should be tested in fermentation steps since it was more difficult for the bacterium to develop resistance to biocide. There was no inhibition of S. cerevisiae and there were only antibiotics as an option to bacterial control of fuel ethanol fermentation by S. cerevisiae573441447FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPSem informaçã

Study of biotransformation of rye flour by enzymatic treatment and evaluation of the bioaccessibility of phenolic acids by model of digestion 'in vitro' and of absorption by Caco-2 intestinal cells

Orientador: Gabriela Alves MacedoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: Os ácidos fenólicos possuem propriedades biológicas que desempenham ação antioxidante, anti-inflamatória, antiproliferativa e antimicrobiana. Porém, esses fenólicos de cereais integrais podem ser difíceis de serem absorvidos totalmente pelo organismo, pois estão ligados a outros componentes da parede celular do vegetal ou a um antinutriente (taninos). O zinco, ferro e fósforo de grãos integrais são importantes para o desenvolvimento e manutenção do organismo. No entanto, os cereais ricos em fitato podem apresentar esses minerais complexados ao antinutriente e se tornarem indisponíveis para a absorção. Uma alternativa para melhorar a disponibilidade de ácidos fenólicos e minerais de cereais integrais é o tratamento enzimático. A tanase, tanino acil hidrolase (EC 3.1.1.20), é uma enzima com habilidade de atuar em ligações éster e depsídicas de taninos hidrolisáveis e já foi descrita como capaz de hidrolisar outros fenólicos como ácido tânico, ácido clorogênico, epigalocatequina, dentre outros. A fitase microbiana atua na desfitinização de cereais ricos em fitatos e sua ação melhora a bioacessibilidade de ferro e zinco na formulação infantil. A bioacessibilidade é uma alternativa aos ensaios 'in vivo' que apresentam alto custo de experimental, e pode ser medida pelo modelo de digestão 'in vitro'/absorção pelas células Caco-2. O objetivo deste trabalho foi verificar o efeito de tanase de 'Paecilomyces variotii' e fitase comercial, adicionadas à farinha de centeio, quanto ao teor de fenólicos totais e à identificação e quantificação por CLAE-DAD dos ácidos fenólicos; à capacidade antioxidante (DPPH, ORAC e FRAP); à disponibilidade de minerais (zinco e ferro); ao teor de antinutrientes (tanino hidrolisável e fitato); e à bioacessibilidade dos ácidos fenólicos da farinha. A tanase adicionada à farinha foi capaz de reduzir o teor de tanino hidrolisável, aumentar os fenólicos totais e potencializar a capacidade antioxidante. A farinha tratada com fitase apresentou menor teor de fitato e aumento no conteúdo de fósforo inorgânico proveniente da degradação enzimática. A disponibilidade de zinco e ferro, medida pela razão molar entre os minerais e o fitato foi melhorada após o tratamento da farinha com fitase. Foram identificados os ácidos ferúlico, sinápico e vanílico da farinha de centeio, que após o tratamento com a tanase apresentou aumento no teor de ácido ferúlico em cinco vezes em relação à farinha não tratada. Esse resultado evidencia a ação inédita da tanase de 'P. variotii' como possível feruloil esterase. A farinha biotransformada com tanase apresentou maior teor dos ácidos vanílico, ferúlico e sinápico, porém ao se avaliar a bioacessibilidade houve menor eficiência de transporte desses ácidos quando comparada à farinha controle. Provavelmente, a farinha biotransformada apresentou saturação desses fenólicos sobre a porção apical das células Caco-2 que reduziu a capacidade do transportador de ácidos monocarboxílicos (MCT1) nas duas horas de incubação. Isso corrobora com os resultados obtidos da exposição das células Caco-2 aos padrões de ácidos ferúlico e vanílico na concentração de 50 µl.ml-1 que apresentaram inibição da expressão gênica do MCT1. Contudo, o uso de enzimas como fitase e tanase na farinha de centeio é interessante, pois potencializa sua ação antioxidante, aumenta o teor de fenólicos, aumenta a disponibilidade de zinco e ferro, aumenta o conteúdo de fósforo e reduz os antinutrientes. Para trabalhos futuros seria relevante estudar o tempo de residência de ácidos fenólicos da farinha de centeio no ensaio de transporte celular, pois duas horas de incubação pode ter sido insuficiente para verificar todo o potencial da farinha tratada com tanaseAbstract: Phenolic acids have biological properties that perform antioxidant, anti-inflammatory, antiproliferative and antimicrobial. However, these phenolic of whole grains may be difficult to be completely absorbed by the body because they are connected to other cell wall components of plant or an antinutrient (tannins). Zinc, iron and phosphorus of whole grains are important for the development and maintenance of the organism. However, cereals rich in phytate may have these minerals complexed to antinutrient and become unavailable for absorption. An alternative to improve the availability of phenolic acids and minerals of whole grains is the enzymatic treatment. Tannase, tannin acyl hydrolase (EC 3.1.1.20) is an enzyme with the ability to act on ester bonds and depside bonds of hydrolysable tannins and has been described as capable of hydrolyzing tannic acid, chlorogenic acid, epigallocatechin, among others. Microbial phytase acts on dephytinization of cereals rich in phytate and its action improves the bioaccessibility of iron and zinc in infant formulation. Bioaccessibility is an alternative to in vivo tests that has expensive experimental cost, and can be measured by 'in vitro' model of digestion / absorption by the Caco-2 cells. The aim of this study was to investigate the effect of tannase Paecilomyces variotii and of commercial phytase added to rye flour, as to the total phenolic content and the identification and quantification by HPLC-DAD of phenolic acids; to the antioxidant capacity (DPPH, ORAC and FRAP); to the availability of minerals (zinc and iron); to the antinutrients content (phytate and hydrolyzable tannin); and as to bioaccessibility of phenolic acids of the flour. Tannase added to flour was able to reduce the hydrolyzable tannin content, increase total phenolic and enhance the antioxidant capacity. Flour rye treated with phytase decreased phytate content and increased content of inorganic phosphorus from enzymatic degradation. Availability of zinc and iron of the flour after treatment with phytase was improved, it was measured by the molar ratio between the mineral and phytate. Acids ferulic, sinapic and vanillic were identified and quantified of the rye flour, which after treatment with tannase showed an increase in ferulic acid content in five times bigger that the untreated flour. This result shows the tanase being an original action of 'P. variotii' as possible feruloyl esterase. Biotransformed flour rye with tannase showed a higher content of vanillic acid, ferulic and sinapic, however, when assessing the bioaccessibility was lower transport efficiency of these acids when compared to the control flour. Probably the biotransformed flour showed saturation of these phenolics about the apical portion of Caco-2 cells, they reduced the capacity of the monocarboxylic acids transporter (MCT1) within two hours of incubation. This corroborates the results of exposure of Caco-2 cells to standards of ferulic acid and vanillic at concentration of 50 ?l.mL-1, that showed inhibition of gene expression MCT1. However, the use of enzymes such as phytase and tannase in rye flour is interesting because it enhances antioxidant capacity, it phenolic content is incremented, availability of zinc and iron is bettered, increases the phosphorus content and reduces antinutrients. For further work, would be relevant the study of the residence time of the phenolic acids of rye flour on the cell transport assay, since two hours of incubation may have been insufficient to verify the full potential of flour treated with tannaseDoutoradoCiência de AlimentosDoutora em Ciência de Alimento

Effect of Annatto Seed and Coriander Leaves as Natural Antioxidants in Fish Meatballs during Frozen Storage

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The effects of annatto (0.1 g/100g) and coriander (0.5 g/100g) were assessed against lipid oxidation in white hake meatballs cooked in boiling water (95 +/- 1 degrees C) for 30 min and stored at -18 degrees C for 120 d. The fatty acids (FA) and the nutritional quality, cholesterol, cholesterol oxides, thiobarbituric acid reactive substances (TBARS) values, and conjugated dienes were analyzed to follow the course of oxidation. Annatto and coriander were efficient in the control of lipid oxidation, also preserving the essential FA. At 120 d of storage, the eicosapentaenoic acid (EPA) concentration decreased respectively by 43%, 32%, 12%, and 9% in the control, coriander, annatto, and annatto + coriander patties. For docosahexaenoic acid (DHA), these concentrations decreased, respectively, 44%, 30%, 11%, and 7%, revealing a probable synergistic effect among the antioxidant compounds present in both spices. On the other hand, annatto and coriander were not able to act protecting the meatballs against lipid oxidation when they were cooked, also not exerted any effect in the cholesterol oxidation.766C838C845Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Effect of annatto seed and coriander leaves as natural antioxidants in fish meatballs during frozen storage

The effects of annatto (0.1 g/100g) and coriander (0.5 g/100g) were assessed against lipid oxidation in white hake meatballs cooked in boiling water (95 ± 1 °C) for 30 min and stored at −18 °C for 120 d. The fatty acids (FA) and the nutritional quality, cholesterol, cholesterol oxides, thiobarbituric acid reactive substances (TBARS) values, and conjugated dienes were analyzed to follow the course of oxidation. Annatto and coriander were efficient in the control of lipid oxidation, also preserving the essential FA. At 120 d of storage, the eicosapentaenoic acid (EPA) concentration decreased respectively by 43%, 32%, 12%, and 9% in the control, coriander, annatto, and annatto + coriander patties. For docosahexaenoic acid (DHA), these concentrations decreased, respectively, 44%, 30%, 11%, and 7%, revealing a probable synergistic effect among the antioxidant compounds present in both spices. On the other hand, annatto and coriander were not able to act protecting the meatballs against lipid oxidation when they were cooked, also not exerted any effect in the cholesterol oxidation766C838C845CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPSem informaçãoSem informaçã

Influence of rye flour enzymatic biotransformation on the antioxidant capacity and transepithelial transport of phenolic acids

Phenolic acids have been reported to play a role on the antioxidant activity and other important biological activities. However, as most polyphenolics in food products are either bound to cellular matrices or present as free polymeric forms, the way they are absorbed has not been totally clear until now. Hydrolytic enzymes may act to increase functionalities in polyphenolic-rich foods, enhancing the bioaccessibility of phenolic compounds and minerals from whole grains. The aim of this study was to evaluate the action of tannin acyl hydrolase (tannase) on the total phenols, phenolic acid profile, antioxidant capacity and in vitro bioaccessibility of phenolic acids found in whole rye flour (RF). Besides increasing total phenols and the antioxidant capacity, tannase treatment increased the amounts of ferulic, sinapic and vanillic acids identified in RF, evidencing a new type of feruloyl esterase catalytic action of tannase. Vanillic and sinapic acids in tannase-treated whole rye flour (RFT) were higher than RF after in vitro gastrointestinal digestion, and higher amounts of transported vanillic acid through the Caco-2 monolayer were detected in RFT. However, the bioaccessibility and the transport efficiency of RF phenolic acids were higher than RFT. Underutilized crops like rye and rye-derived products may be an important source of phenolic acids. The tannase biotransformation, even influencing the total phenolics and antioxidant capacity of RF, did not increase the bioaccessibility of phenolic acids under the experimental conditions of this study918891898COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPSem informaçãoSem informaçã

Chemical inhibition of the contaminant Lactobacillus fermentum from distilleries producing fuel bioethanol

The purpose of this study was to determine the Minimum Inhibitory Concentration (MIC) of pure or mixed chemicals for Saccharomyces cerevisiae and Lactobacillus fermentum in the samples isolated from distilleries with serious bacterial contamination problems. The biocides, which showed the best results were: 3,4,4' trichlorocarbanilide (TCC), tested at pH 4.0 (MIC = 3.12 mg/l), TCC with benzethonium chloride (CBe) at pH 6.0 (MIC = 3.12 mg/l) and TCC mixed with benzalkonium chloride (CBa) at pH 6.0 (MIC = 1.53 mg /l). If CBa was used in sugar cane milling in 1:1 ratio with TCC, a 8 times reduction of CBa was possible. This formulation also should be tested in fermentation steps since it was more difficult for the bacterium to develop resistance to biocide. There was no inhibition of S. cerevisiae and there were only antibiotics as an option to bacterial control of fuel ethanol fermentation by S. cerevisiae