Seleção de novas linhagens de bactérias ácido-láticas probióticas e aplicação de E. faecium em leite
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Data
2017-04-28
Autores
Nascimento, Liane Caroline Sousa [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A busca por alimentos que proporcionem melhorias na saúde e bem estar dos consumidores é uma tendência mundial. Os produtos lácteos fermentados estão incluídos entre os alimentos que atendem a esta demanda. O objetivo desta pesquisa foi selecionar novas linhagens de bactérias ácido-láticas (BAL) potencialmente probióticas e seguras, bem como avaliar a aplicação das cepas selecionadas em leite. Na primeira etapa, o potencial probiótico (capacidade de hidrolise de sais biliares [BSH], capacidade de autoagregação, coagregação com Lactobacillus spp. e Listeria spp. e hidrofobicidade e tolerância em diferentes valores de pH e concentrações de NaCl) e a segurança (susceptibilidade a antibióticos e degradação da mucina) foram avaliados em vinte cepas de BAL, previamente isoladas de queijo muçarela de búfala e identificadas como Lactobacillus helveticus (SJRP56 e SJRP191), Lactobacillus delbrueckii subsp. bulgaricus (SJRP49), Lactobacillus fermentum (SJRP42), Enterococcus durans (SJRP14, SJRP17, SJRP25, SJRP26 e SJRP68), Enterococcus faecium (SJRP20, SJRP28, SJRP65, SJRP69) e Enterococcus sp. (SJRP04, SJRP11, SJRP16, SJRP101, SJRP120, SJRP122 e SJRP125). Seis cepas apresentaram atividade de BSH e a maioria delas apresentou elevada capacidade de autoagregação e hidrofobicidade. Algumas cepas apresentaram capacidade de coagregação com outras BAL e patógenos. A maioria das cepas cresceu bem em pH neutro e a tolerância ao NaCl foi dependente da temperatura. Nenhuma das BAL degradou a mucina; entretanto, algumas apresentaram resistência aos antibióticos testados. Na segunda etapa, considerando as características probióticas e de segurança, as cepas E. faecium SJRP20 e E. faecium SJRP65 foram selecionadas para testes complementares, incluindo tolerância às condições simuladas do trato gastrointestinal (TGI), adesão as células Caco-2, produção da enzima ß- galactosidase e presença de genes associados à virulência e produção de aminas biogênicas. Ambas as cepas resistiram bem às condições do TGI, apresentaram baixa propriedade de adesão, são produtoras de ß-galactosidase e não apresentaram genes relacionados à adesão, agregação e colonização. E. faecium SJRP65 apresentou poucos genes relacionados a resistência a antibióticos e fatores de virulência. Em função disso, foi considerado com boas propriedades funcionais e com características interessantes para aplicação em produtos lácteos. Na terceira etapa, foi avaliada a aplicação de E. faecium SJRP20 e SJRP65 potencialmente probióticos em leite: cinética de acidificação, . pós-acidificação, resistência às condições simuladas do TGI e capacidade de sequestro de radicais livres. E. faecium SJRP20 em leite apresentou maior velocidade máxima de acidificação (Vmáx = 7,11x10-3upH/min), enquanto que E. faecium SJRP65 apresentou menor taxa de acidificação. Não houve diferença na composição centesimal das amostras de leite fermentado inoculado com as cepas de E. faecium. Durante a estocagem refrigerada houve redução do pH e aumento da acidez em todas as amostras de leite fermentado. Os produtos fermentados por ambas as cepas resistiram bem às condições simuladas do TGI, com elevada viabilidade ao longo do período de estocagem. O leite fermentado por E. faecium SJRP20 e SJRP65 apresentou elevada capacidade de sequestro de radicais livres. Com o aumento da concentração de produto e do tempo de estocagem, foi maior a capacidade antioxidante. A fermentação do leite por E. faecium potencialmente probióticos apresentou bons resultados, e a cepa E. faecium SJRP20 reuniu as melhores características tecnológicas para aplicação em produtos lácteos fermentados funcionais: apresentou maior velocidade de acidificação, menor tempo para atingir a velocidade máxima, maior estabilidade às condições do TGI durante a estocagem refrigerada e alta capacidade de sequestro de radicais livres.
The search for food which improves consumer health and well-being is a worldwide trend. The fermented dairy products are included among food which satisfies this demand. The aim of this research was to select new strains of lactic acid bacteria (LAB) potentially probiotic and safe, as well as to evaluate the application of selected strains in milk. In the first stage, the probiotic potential (bile salt hydrolase capacity [BSH], capacity of auto-aggregation, co-aggregation with Lactobacillus spp. and Listeria spp., hydrophobicity, tolerance in different pH values and concentrations of NaCl) and safety (susceptibility to antibiotics and mucin degradation) were evaluated in twenty LAB strains, previously isolated of water- buffalo mozzarella cheese and identified as Lactobacillus helveticus (SJRP56 and SJRP191), Lactobacillus delbrueckii subsp. bulgaricus (SJRP49), Lactobacillus fermentum (SJRP42), Enterococcus durans (SJRP14, SJRP17, SJRP25, SJRP26 and SJRP68), Enterococcus faecium (SJRP20, SJRP28, SJRP65, SJRP69) and Enterococcus sp. (SJRP04, SJRP11, SJRP16, SJRP101, SJRP120, SJRP122 and SJRP125). Six strains presented BSH activity and most of them presented high capacity of auto-aggregation and hydrophobicity. Some strains presented capacity of co-aggregation with other LAB and pathogens. The majority of strains grew well in neutral pH and the tolerance to NaCl was dependent on the temperature. None of the LAB degraded the mucin; however, some of them presented resistance to the tested antibiotics. In the second stage, considering the probiotic features and safety, the strains E. faecium SJRP20 and E. faecium SJRP65 were selected for complementary tests, including tolerance to the simulated GIT conditions, adhesion to Caco-2 cells, production of ß-galactosidase enzyme and presence of genes associated to the virulence factors and production of biogenic amines. Both strains resisted well to the GIT conditions, presented low property of adhesion, ß-galactosidase activity and they did not present genes related to adhesion, aggregation and colonization. E. faecium SJRP65 presented few genes related to the resistance to antibiotics and virulence factors. According to this, it was considered with good functional properties and with interesting characteristics for application in dairy products. In the third stage, the application of potentially probiotic E. faecium SJRP20 and SJRP65 in milk was evaluated: acidification kinetics, post acidification, resistance to the simulated GIT conditions .(GIT) and free radicals scavenging capacity. E. faecium SJRP20 in milk presented higher maximum speed (Vmáx = 7,11x10-3upH/min), while the E. faecium SJRP65 presented lower acidification rate. There was not difference in the gross composition of the samples of fermented milk inoculated with E. faecium strains. During the refrigerated storage, there was a pH reduction and an increase in acidity for all fermented samples. The products fermented by both strains resisted well to the simulated GIT conditions, with high viability during the storage period. The milk fermented by E. faecium SJRP20 and SJRP65 presented high free radicals scavenging capacity. With the increase of the product concentration and the storage time, the antioxidant capacity was higher. The milk fermentation by potentially probiotic E. faecium presented good results, and the E. faecium SJRP20 strain combined the best technological characteristics for application in functional fermented dairy products: presented higher speed of acidification, lower time to reach the maximum speed, higher stability to the GIT conditions during the refrigerated storage and high free radicals scavenging capacity.
The search for food which improves consumer health and well-being is a worldwide trend. The fermented dairy products are included among food which satisfies this demand. The aim of this research was to select new strains of lactic acid bacteria (LAB) potentially probiotic and safe, as well as to evaluate the application of selected strains in milk. In the first stage, the probiotic potential (bile salt hydrolase capacity [BSH], capacity of auto-aggregation, co-aggregation with Lactobacillus spp. and Listeria spp., hydrophobicity, tolerance in different pH values and concentrations of NaCl) and safety (susceptibility to antibiotics and mucin degradation) were evaluated in twenty LAB strains, previously isolated of water- buffalo mozzarella cheese and identified as Lactobacillus helveticus (SJRP56 and SJRP191), Lactobacillus delbrueckii subsp. bulgaricus (SJRP49), Lactobacillus fermentum (SJRP42), Enterococcus durans (SJRP14, SJRP17, SJRP25, SJRP26 and SJRP68), Enterococcus faecium (SJRP20, SJRP28, SJRP65, SJRP69) and Enterococcus sp. (SJRP04, SJRP11, SJRP16, SJRP101, SJRP120, SJRP122 and SJRP125). Six strains presented BSH activity and most of them presented high capacity of auto-aggregation and hydrophobicity. Some strains presented capacity of co-aggregation with other LAB and pathogens. The majority of strains grew well in neutral pH and the tolerance to NaCl was dependent on the temperature. None of the LAB degraded the mucin; however, some of them presented resistance to the tested antibiotics. In the second stage, considering the probiotic features and safety, the strains E. faecium SJRP20 and E. faecium SJRP65 were selected for complementary tests, including tolerance to the simulated GIT conditions, adhesion to Caco-2 cells, production of ß-galactosidase enzyme and presence of genes associated to the virulence factors and production of biogenic amines. Both strains resisted well to the GIT conditions, presented low property of adhesion, ß-galactosidase activity and they did not present genes related to adhesion, aggregation and colonization. E. faecium SJRP65 presented few genes related to the resistance to antibiotics and virulence factors. According to this, it was considered with good functional properties and with interesting characteristics for application in dairy products. In the third stage, the application of potentially probiotic E. faecium SJRP20 and SJRP65 in milk was evaluated: acidification kinetics, post acidification, resistance to the simulated GIT conditions .(GIT) and free radicals scavenging capacity. E. faecium SJRP20 in milk presented higher maximum speed (Vmáx = 7,11x10-3upH/min), while the E. faecium SJRP65 presented lower acidification rate. There was not difference in the gross composition of the samples of fermented milk inoculated with E. faecium strains. During the refrigerated storage, there was a pH reduction and an increase in acidity for all fermented samples. The products fermented by both strains resisted well to the simulated GIT conditions, with high viability during the storage period. The milk fermented by E. faecium SJRP20 and SJRP65 presented high free radicals scavenging capacity. With the increase of the product concentration and the storage time, the antioxidant capacity was higher. The milk fermentation by potentially probiotic E. faecium presented good results, and the E. faecium SJRP20 strain combined the best technological characteristics for application in functional fermented dairy products: presented higher speed of acidification, lower time to reach the maximum speed, higher stability to the GIT conditions during the refrigerated storage and high free radicals scavenging capacity.
Descrição
Palavras-chave
Alimentos funcionais, Fermentação, Viabilidade, Fatores de virulência, Produtos lácteos