Kefir fermentation as a bioprocess to improve lentils antioxidant properties : is it worthwhile?

Kefir, a consortium of acetic and lactic bacteria and yeasts, is versatile for biotransformation of vegetable substrates and may improve their bioactive properties. Lentil is a legume rich in vitamins, minerals, complex carbohydrates and proteins, making it an excellent substrate for fermentation and obtaining bioactive molecules. We report the effect of different process variables on antioxidant properties of lentil extracts obtained using kefir as a biotransformation agent and experimental design. The results showed that the factors agitation (rpm) and incubation temperature exerted significant effects. Incubation temperature of 28 °C and agitation of 75 rpm were the most suitable conditions for kefir fermentation and maximizing the antioxidant properties of the fermented lentils. The highest antioxidant activities for fermented products were 4128.39, 35.87 and 23.20 μmol of Trolox equivalents per gram of sample (μmoL TE g-1) measured by Total Antioxidant Capacity, FRAP and DPPH-radical scavenging methods, respectively. Additionally, we detected that the initial fermentation times (in the first six hours) were enough to positively affect the antioxidant properties of the lentil extracts, reaching a percentage increase of up to 270% in the antioxidant activity of the fermented products in relation to those non-fermented samples233FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2017/02000-

Aplicação da ferramenta de planejamento experimental de misturas como estratégia para a produção de proteases por Aspergillus niger e obtenção de hidrolisados proteicos com múltiplas propriedades funcionais e biológicas

Orientador: Hélia Harumi SatoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: O presente trabalho teve como objetivo utilizar a técnica de delineamento experimental de misturas como estratégia para a produção de proteases por Aspergillus niger LBA02 em fermentação semissólida utilizando formulações contendo diferentes resíduos agroindustriais e produção de hidrolisados proteicos com atividades biológicas e funcionais utilizando a hidrólise enzimática simultânea de proteínas de diferentes fontes. Efeitos sinérgicos e significativos entre as misturas quaternárias de farelo de trigo, farelo de soja, farelo de algodão e casca de laranja foram observados durante a fermentação de A. niger LBA02, atingindo aumentos de 33,7, 7,6, 30,8 e 581,7%, respectivamente, na produção de proteases em comparação com os substratos utilizados de forma isolada. O estudo das características bioquímicas das preparações enzimáticas mostrou que a linhagem de A. niger LBA02 foi capaz de secretar diferentes tipos de proteases em resposta a cada substrato. De um modo geral, as proteases apresentaram atividade ótima a 50 °C e na faixa de pH de 3 a 4. As maiores diferenças entre as preparações de proteases foram observadas para os parâmetros cinéticos e termodinâmicos de ativação e inativação térmica. Na hidrólise enzimática de misturas contendo proteína isolada de soja, proteínas do soro de leite e da clara de ovo utilizando a preparação comercial Flavourzyme® 500L foram observados efeitos sinérgicos entre as formulações contendo misturas binárias ou ternárias, para vários parâmetros. Para atividade antioxidante determinada pelo método DPPH, a mistura contendo proteínas do soro de leite e proteínas da clara de ovo apresentaram aumentos de 45,1 e 37,3% na atividade, quando comparada aos hidrolisados obtidos com as duas fontes de forma isolada, respectivamente. Entre as propriedades funcionais, a capacidade emulsificante foi a que apresentou maior efeito sinérgico, onde os hidrolisados contendo a mistura ternária de proteína isolada de soja, proteínas do soro de leite e da clara de ovo, alcançaram valores 2 a 12 vezes superiores, em relação aos hidrolisados obtidos de forma isolada. A determinação da atividade anti-adipogênica dos hidrolisados revelou que o tratamento de células pré-adipócitas 3T3-L1 com a mistura binária de proteínas do soro de leite e da clara de ovo na concentração de 1.200 ppm reduziu o acúmulo relativo de lipídeos nas células em até 47,9%. Em relação à atividade antimicrobiana, a linhagem de Staphylococcus aureus ATCC 6538 foi a única que apresentou inibição do crescimento quando cultivada em meio suplementado com uma mistura binária de proteína isolada de soja e proteínas da clara de ovo não hidrolisadas, resultando em inibição de 16,82%. Os hidrolisados obtidos com misturas binárias de proteínas do soro de leite e da clara de ovo ou proteína isolada de soja e soro de leite estimularam o crescimento de bactérias lácticas e probióticas, resultando em aumentos de 29,4 a 100% comparados aos meios não suplementados. A utilização de composições contendo diferentes preparações comerciais de proteases para hidrólise de proteína isolada de soja e estudo da atividade antioxidante mostrou diferentes resultados para cada método utilizado. Para inibição dos radicais DPPH, os hidrolisados obtidos com Flavourzyme® 500L combinada com Alcalase® 2.4L mostraram o maior efeito sinérgico, com aumentos de 10,9 e 13,2% da atividade antioxidante, em comparação aos hidrolisados produzidos com as enzimas isoladas. Os hidrolisados obtidos utilizando a mistura ternária de Flavourzyme® 500L, Alcalase® 2.4L e YeastMax® A apresentaram o maior poder de inibição da auto-oxidação do ácido linoleicoAbstract: This study aimed to use the mixture experimental design technique as a strategy to produce proteases by Aspergillus niger LBA02 under solid state fermentation, using formulations containing different agroindustrial wastes. It also aimed to produce protein hydrolysates with biological and functional activities using the simultaneous enzymatic hydrolysis of proteins from the different sources. Synergistic and significant effects between the quaternary mixtures of wheat bran, soybean meal, cottonseed meal and orange peel were observed during fermentation by A. niger LBA02, reaching increases of 33.7, 7.6, 30.8 and 581.7%, respectively, for the production of proteases as compared to the isolated substrates. The study of the biochemical properties of the enzyme preparations showed that the strain of A. niger LBA02 was able to secrete different types of proteases in response to each substrate. In general, the proteases showed optimal activity at 50 °C in the pH range from 3 to 4. The major differences between the protease preparations were observed for the kinetic and thermodynamic parameters of thermal activation and inactivation. In the enzymatic hydrolysis of mixtures containing soy protein isolate, bovine whey protein and egg white protein using the commercial preparation FlavourzymeTM 500L, synergistic effects were observed for various parameters between formulations containing binary or ternary mixtures,. For antioxidant activity as determined by the DPPH assay, the mixture containing bovine whey protein and egg white protein showed increases of 45.1 and 37.3% in their activities as compared to hydrolysates obtained with the isolated proteins, respectively. Of the functional properties, the emulsifying capacity showed the greatest synergistic effect, the hydrolysates containing the ternary mixture of soy protein isolate, bovine whey protein and egg white protein, showing increases ranging from 2 to 12-fold as compared to the hydrolysates obtained using isolated substrates. The determination of the anti-adipogenic activity of the hydrolysates indicated that the treatment of 3T3-L1 preadipocyte cells with 1200 ppm of the mixture containing bovine whey protein and egg white protein reduced the relative lipid accumulation to 47.9%. With respect to antimicrobial activity, the strain of Staphylococcus aureus ATCC 6538 was the only one which showed growth inhibition when cultivated in a medium supplemented with a non-hydrolyzed binary mixture of soy protein isolate and egg white protein, resulting in inhibition of 16.82%. The hydrolysates obtained with binary mixtures of bovine whey protein and egg white protein or soy protein isolate and bovine whey protein stimulated the growth of probiotic and lactic acid bacteria, reaching increases from 29.4 to 100% when compared to non-supplemented media. The use of formulations containing various commercial preparations of proteases for the hydrolysis of soy protein isolate and the study of antioxidant activities showed different results for each method. For DPPH radical scavenging, the hydrolysates obtained with FlavourzymeTM 500L combined with AlcalaseTM 2.4L showed greater synergistic effects, with increases of 10.9 and 13.2% in antioxidant activity as compared to the hydrolysates produced with individual enzymes. The hydrolysates obtained from ternary mixtures of FlavourzymeTM 500L, AlcalaseTM 2.4L and YeastMaxTM A showed the greatest power of inhibition of linoleic acid autoxidationDoutoradoCiência de AlimentosDoutor em Ciência de Alimento

Meddelelse: Medvirkende ved dette nummer

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Bioactive peptides can be defined as specific protein fractions with amino acid sequence that promotes a positive impact on several biologic activities. The hydrolysis of egg white protein might result in releasing or enhancing their biological activity. The aim of this study was to evaluate the effects of the enzymatic hydrolysis of egg white proteins on their antioxidant properties. The hydrolysates were prepared with protease from Aspergillus cuyzae LBA 01 (AO) produced under solid-state fermentation and two commercial proteases: protease from A. oryzae (CAD), and Bacillus lichenifortnis (CBL). The enzymatic hydrolysis with microbial proteases increased the antioxidant properties of egg white proteins. The egg white protein hydrolysates prepared with commercial protease CAD, presented higher antioxidant activity than the hydrolysates obtained with the other proteases. According CCRD analysis, the hydrolysates prepared with 3.0% of egg white proteins and 20.0 U of protease per mL of reaction mixture, presented high radical-scavenging effect, with an ORAC value of 119312 +/- 84.62 and MTH value of 19.05 +/- 0.81 Trolox EQ mu mol g(-1) In ORAC assay, the maximum antioxidant activities were observed in the hydrolysates presenting DH 50.0% obtained after 120 min of hydrolysis. In contrast, for DRPH assay the highest values of antioxidant activity were detected in the hytholysates obtained in the first 30 min, where the degree of hydrolysis was greater than 60.0%. (C) 2014 Elsevier Ltd. All rights reserved.415562Fundação 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)FAPESP [2011/10429-9

Biologically Active Peptides: Processes For Their Generation, Purification And Identification And Applications As Natural Additives In The Food And Pharmaceutical Industries

Recent technological advances have created great interest in the use of biologically active peptides. Bioactive peptides can be defined as specific portions of proteins with 2 to 20 amino acids that have desirable biological activities, including antioxidant, anti-hypertensive, antithrombotic, anti-adipogenic, antimicrobial and anti-inflammatory effects. Specific characteristics, including low toxicity and high specificity, make these molecules of particular interest to the food and pharmaceutical industries. This review focuses on the production of bioactive peptides, with special emphasis on fermentation and enzymatic hydrolysis. The combination of different technologies and the use of auxiliary processes are also addressed. A survey of isolation, purification and peptide characterization methods was conducted to identify the major techniques used to determine the structures of bioactive peptides. Finally, the antioxidant, antimicrobial, anti-hypertensive, anti-adipogenic activities and probiotic-bacterial growth-promoting aspects of various peptides are discussed. (C) 2015 Elsevier Ltd. All rights reserved.7418519

Simultaneous Hydrolysis Of Proteins From Different Sources To Enhance Their Antibacterial Properties Through The Synergistic Action Of Bioactive Peptides

Antimicrobial peptides have been identified as possible substitutes for chemicals used as drugs or food preservatives, owing to their high specificity, easy degradability in the environment, and low toxicity. In the current study, three different protein sources (viz., soy protein isolate [SPI], bovine whey protein [BWP], and egg white protein [EWP]) were hydrolyzed using Flavourzyme 500L, individually or in binary/ternary mixtures, to verify the antibacterial activities of the hydrolysates obtained. The most adequate formulations to inhibit the tested bacterial strains were 1) the ternary mixture of SPI, BWP, and EWP in equal proportions for Escherichia coli ATCC 11229, 2) all binary mixtures of the protein hydrolysates for Salmonella enterica serotype Choleraesuis ATCC 14028, and 3) the binary mixture of SPI and BWP for Staphylococcus aureus ATCC 6538, which decreased the bacterial growth by >= 19%, >= 15%, and >= 48%, respectively. The enzymatic process proposed in our study proved to be a promising alternative for producing protein hydrolysates with high potential bioactivity, by simultaneously hydrolyzing mixtures of different protein sources to obtain peptides that could act synergistically to inhibit pathogenic bacteria.8209212Department of Food Science, School of Food Engineering, University of Campina

Simultaneous hydrolysis of proteins from different sources to enhance their antibacterial properties through the synergistic action of bioactive peptides

Antimicrobial peptides have been identified as possible substitutes for chemicals used as drugs or food preservatives, owing to their high specificity, easy degradability in the environment, and low toxicity. In the current study, three different protein sources (viz., soy protein isolate [SPI], bovine whey protein [BWP], and egg white protein [EWP]) were hydrolyzed using Flavourzyme® 500L, individually or in binary/ternary mixtures, to verify the antibacterial activities of the hydrolysates obtained. The most adequate formulations to inhibit the tested bacterial strains were 1) the ternary mixture of SPI, BWP, and EWP in equal proportions for Escherichia coli ATCC 11229, 2) all binary mixtures of the protein hydrolysates for Salmonella enterica serotype Choleraesuis ATCC 14028, and 3) the binary mixture of SPI and BWP for Staphylococcus aureus ATCC 6538, which decreased the bacterial growth by ≥19%, ≥15%, and ≥48%, respectively. The enzymatic process proposed in our study proved to be a promising alternative for producing protein hydrolysates with high potential bioactivity, by simultaneously hydrolyzing mixtures of different protein sources to obtain peptides that could act synergistically to inhibit pathogenic bacteria.820921

Development of a novel probiotic milk product with enhanced antioxidant properties using mango peel as a fermentation substrate

Mango peel is an agroindustrial waste with excellent nutritional value and a considerable content of bioactive compounds that can be used as a source of antioxidants. Additionally, biotransformation processes by kefir fermentation increase the interest in obtaining products from agroindustrial wastes with good biological properties. In this context, this work aimed to evaluate the effects of milk supplementation with mango peels on the growth rates of kefir microorganisms during fermentation as well as the antioxidant properties of the obtained fermented products. The results were comparatively evaluated between fermented samples in the presence of mango peels (KMP) and in their absence (KC). Significant increases (p ≤ 0.05) in antioxidant activities for KMP were observed for all methods, as follows: 113% for DPPH-radical scavenging (10 h of fermentation), 153% for the Ferric Reducing Antioxidant Power (FRAP) assay (8 h of fermentation), 21% for ABTS-radical scavenging (6 h of fermentation) and 43% for Total Reducing Capacity (Folin-Ciocalteu method) (8 h of fermentation) compared to those of KC fermented samples. According to microbiological analyses, the KMP samples allowed microbial growth of up to 3-fold higher than the growth of KC samples, reaching counts of up to 108 CFU/mL for lactic acid bacteria. According to the results, the products obtained from the fermentation process using milk combined with mango peels as the substrate presented improved antioxidant properties and a potential probiotic property.24CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQSem informaçã

Germinação de grãos: uma revisão sistemática de como os processos bioquímicos envolvidos afetam o conteúdo e o perfil de compostos fenólicos e suas propriedades antioxidantes

Germinação é um processo simples e econômico que melhora a qualidade nutricional e funcional dos grãos, como resultado de transformações complexas. As condições ambientais em que os grãos são expostos influenciam diretamente na composição final do broto e, consequentemente na sua bioatividade. É possível perceber uma correlação positiva entre o processo de germinação e o teor de compostos fenólicos presentes nos grãos, apesar da explicação para isso ser peculiar para cada processo de germinação. De maneira geral, sabe-se que os compostos fenólicos podem estar presentes nas formas solúvel e insolúvel, sendo que a primeira está normalmente armazenada no vacúolo da planta, e a segunda ligada a macromoléculas, constituindo a estrutura dos grãos. Durante o processo de germinação e consequente atuação das enzimas ativadas pela absorção de água, essas duas frações se transformam simultaneamente. A fração solúvel pode se tornar insolúvel devido ao crescimento da radícula e necessidade de formação estrutural e a insolúvel pode ser transformada à solúvel, devido a hidrólise das ligações com as macromoléculas de armazenamento da planta. Dessa forma, a compreensão das diversas vias e consequentes resultados se tornam alvo de estudo da literatura. Dentro desse contexto, o objetivo central dessa revisão é apresentar estudos recentes que investigam as alterações promovidas pelo processo de germinação e sua relação com as propriedades antioxidantes de diferentes grãos. Algumas vias bioquímicas para formação de compostos fenólicos já elucidadas foram abordadas, assim como tendências futuras para uso de germinação como um processo para melhoria de propriedades biológicas dos grãos.31287300Germination is a simple and economical process that improves the nutritional and functional quality of the grains as a result of complex transformations. The environmental conditions in which the grains are exposed directly influence the final composition of the sprout and, consequently, its bioactivity. It is possible to notice a positive correlation between the germination process and the content of phenolic compounds present in the grains, although the explanation for this is peculiar to each germination process. In general, it is known that phenolic compounds can be present in soluble and insoluble forms, the first being normally stored in the plant’s vacuole, and the second linked to macromolecules, constituting the grain structure. During the germination process and the consequent action of enzymes activa-ted by water absorption, these two fractions are transformed simultaneously. The soluble fraction can become insoluble due to root growth and the need for structural formation and the insoluble fraction can be transformed into soluble, due to the hydrolysis of the bonds with the plant’s macromolecules. In this way, the understanding of the different pathways and the consequent results become a target for studying the literature. Within this context, the main objective of this review is to present recent studies that investigate the changes promoted by the germination process and its relationship with the antioxidant properties of different grains. Some biochemical pathways for the formation of phenolic compounds already elucidated have been addressed, as well as future trends for the use of germination as a process to impro-ve the biological properties of grains

Biocatalytic action of proteases in ionic liquids: improvements on their enzymatic activity, thermal stability and kinetic parameters

This study evaluated the effect of the addition of the following ionic liquids (IL): choline chloride (CC), tetramethylammonium bromide (TB) and 1‑ethyl‑3‑methylimidazolium bromide (EM), on some biochemical properties including enzymatic activity and different kinetic parameters of commercial proteases. The enzyme-IL combinations that showed the highest increases in enzyme activities were as follows: CC (0.5 mM) and Neutrase® 0.8 L; CC (5 mM) and Flavourzyme® 500 L; TB (2000 mM) and Alcalase® 2.4 L, with relative increases of 20, 15 and 150% in protease activities, respectively, compared to the control assays. The combination TB and Alcalase® 2.4 L showed a reduction of 50% of the activation energy (Ea), an increase of the relation Vmax/Km of 35% and a 16-fold rise in the values of t1/2, and D. Neutrase® 0.8 L combined with CC showed an increase of 20% in the relation Vmax/Km. The combination Flavourzyme® 500 L and CC presented a 20% higher value of the relation Vmax/Km and a 2-fold increase in the values of t1/2 and D compared to the control assay. In summary, the most positive effects observed in this study included proteases with improved activity and stability properties and a greater affinity for the substrate.11412412