Avaliação do potencial probiótico de Lactobacillus casei e Lactobacillus fermentum autóctones e aplicação em leites fermentados com diferentes matrizes
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Data
2018-02-02
Autores
Souza, Bruna Maria Salotti de
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Universidade Estadual Paulista (Unesp)
Resumo
O objetivo deste estudo foi avaliar as propriedades probióticas das estirpes Lactobacillus casei e Lactobacillus fermentum, selecionar novas estirpes seguras para o desenvolvimento de produtos fermentados e a partir desta seleção definir uma matriz apropriada para ser fermentada e avaliar o efeito do leite fermentado pelas cepas selecionadas na translocação bacteriana, análises fecais e morfologia intestinal de camundongos BALB/c. Os testes in vitro realizados foram auto-agregação, co-agregação, hidrofobicidade, produção de β-galactosidase, sobrevivência ao trato gastrointestinal (TGI) e suscetibilidade a antibióticos. As cepas selecionadas foram adicionalmente testadas pela presença de genes que codificam adesão, agregação e colonização, fatores de virulência, resistência a antibióticos e produção de aminas biogênicas, seguido da avaliação de parâmetros cinéticos acidificantes no leite e sobrevivência das cepas sob condições de GIT simuladas durante refrigeração. A maioria das cepas de ambas as espécies apresentou alta auto-agregação, algumas cepas apresentaram capacidade de co-agregação com outras bactérias ácido láticas (BAL) e/ou patógenos, e ambas as espécies apresentaram valores de hidrofobicidade baixos. Sete cepas de L. casei e seis cepas de L. fermentum foram produtoras da enzima β-galactosidase e dez cepas sobreviveram bem a simulação de condições estressantes do TGI. Todas as cepas foram resistentes a vancomicina e quase todas as cepas eram resistentes à kanamicina. L. casei SJRP38 e L. fermentum SJRP43 foram distinguidos entre as outras cepas de BAL pelo seu maior potencial probióticos. Três matrizes foram fermentadas por L. casei SJRP38 e L. fermentum SJRP43 e os parâmetros cinéticos de acidificação ao longo da fermentação foram realizados. Os animais foram divididos em quatro grupos: controle água (CW), controle do leite (CM), leite fermentado por L. casei SJRP38 (FMLC) e leite fermentado por L. fermentum SJRP43 (FMLF), ambas fermentações com co-cultura com a cepa comercial de S. thermophilus TA040 ao longo do tratamento. Após 14 dias de alimentação, os camundongos BALB/c foram contidos manualmente e individualmente, e a eutanásia foi realizada e colhido sangue, fígado, baço, intestino delgado, intestino grosso e conteúdo intestinal foram removidos para estudos de translocação, morfologia intestinal e microbiologia intestinal. Durante o protocolo experimental, nenhuma atividade notável ou alterações comportamentais foram observadas nos grupos de camundongos. Não houve variação estatística do peso de camundongos BALB/c entre os quatro grupos experimentais, em todos os períodos avaliados. Em geral, a administração de leite fermentado resultou em alterações microbiológicas na composição fecal, levando a uma redução no Clostridium spp. e aumento de Streptococcus spp., Lactobacillus spp. e Bifidobacterium spp. A população de Lactobacillus spp. e Bifidobacterium spp. no intestino delgado e no intestino grosso foram afetados pelo grupo de tratamento. O grupo FMLF apresentou menor população de Lactobacillus spp. no intestino delgado e Bifidobacterium spp. no intestino grosso. Não houve diferenças significativas entre os outros grupos. Os resultados de incidência de translocação de bactérias intestinais para diferentes tecidos e sangue mostraram que nenhuma bacteremia foi observada nos diferentes grupos experimentais. Não houve diferença estatística quanto à espessura do epitélio duodeno entre os quatro grupos; no entanto, no grupo CW do grupo do jejuno-íleo apresentou a maior altura epitelial, quando comparado aos outros grupos CM, FMLC e FMLF. A altura do 10 epitélio do cólon também foi maior no grupo CW do que os demais grupos CM, FMLC e FMLF. Quanto à profundidade da cripta, no duodeno houve diferença significativa entre o grupo CM e os demais grupos, que apresentaram maior profundidade de cripta. Na porção jejuno-íleo, a maior profundidade de cripta foi mostrada no grupo CW do que nos outros grupos. Na parte do cólon, não houve variações significativas entre os grupos. A morfometria das vilosidades do intestino delgado mostrou que um menor tamanho das vilosidades do duodeno foi observado no grupo CW, quando comparado aos outros grupos, no entanto, na porção jejuno-íleo não foram observadas diferenças significativas entre os grupos. A avaliação morfométrica das microvilosidades intestinais mostrou que no duodeno, o grupo CM apresentou tamanho de microvilosidades estatisticamente maior do que os outros grupos. Na parcela do jejuno-íleo, apenas os grupos FMLC e FMLF apresentaram diferenças significativas entre si em tamanho de microvilosidades, enquanto na parte do cólon não houve diferenças no tamanho de microvilosidades entre os grupos CW e FMLC e entre os grupos CM e FMLF. Houve uma frequência estatisticamente maior de células caliciformes no duodeno nos grupos CM, FMLC e FMLF, quando comparado ao grupo CW, no entanto, na parcela do jejuno-íleo, a diferença foi restrita entre os grupos CW e CM e a frequência das células caliciformes nos outros grupos foi superior a 40%. Em conclusão, o consumo de leite fermentado com cepas de L. casei SJRP38 e L. fermentum SJRP43 promoveu benefícios ao intestino do hospedeiro.
The objective of this study was to evaluate the probiotic properties of the strains Lactobacillus casei and Lactobacillus fermentum, to select new safe strains for the development of fermented products and from this selection to define an appropriate matrix to be fermented and to evaluate the effect of the fermented milk by the strains selected in the bacterial translocation, fecal analyzes and intestinal morphology of BALB/c mice. The in vitro tests performed were self-aggregation, co-aggregation, hydrophobicity, β-galactosidase production, gastrointestinal tract survival (GIT) and antibiotic susceptibility. The selected strains were additionally tested for the presence of genes encoding adhesion, aggregation and colonization, virulence factors, antibiotic resistance and biogenic amine production, followed by the evaluation of kinetic parameters in the milk and survival of the strains under simulated GIT conditions during cooling. Most of the strains of both species presented high self-aggregation, some strains presented co-aggregation capacity with other lactic acid bacteria (BAL) and / or pathogens, and both species showed low hydrophobicity values. Seven strains of L. casei and six strains of L. fermentum were β-galactosidase enzyme producers and ten strains survived the simulation of stressful conditions of TGI. All strains were resistant to vancomycin and almost all strains were resistant to kanamycin. L. casei SJRP38 and L. fermentum SJRP43 were distinguished among the other strains of BAL by their greater potential probiotics. Three matrices were fermented by L. casei SJRP38 and L. fermentum SJRP43 and the kinetic parameters of acidification along the fermentation were performed. The animals were divided into four groups: water control (CW), milk control (CM), milk fermented by L. casei SJRP38 (FMLC) and milk fermented by L. fermentum SJRP43 (FMLF), both fermentations with coculture with the commercial strain of S. thermophilus TA040 throughout the treatment. After 14 days of feeding, the BALB / c mice were contained manually and individually, and euthanasia was performed and harvested blood, liver, spleen, small intestine, large intestine and intestinal contents were removed for translocation studies, intestinal morphology and intestinal microbiology. During the experimental protocol, no remarkable activity or behavioral changes were observed in the groups of mice. There was no statistical variation in the weight of BALB/c mice among the four experimental groups, in all periods evaluated. In general, administration of fermented milk resulted in microbiological changes in fecal composition, leading to a reduction in Clostridium spp. and increased Streptococcus spp., Lactobacillus sp. and Bifidobacterium spp. The population of Lactobacillus spp. and Bifidobacterium spp. in the small intestine and large intestine were affected by the treatment group. The FMLF group presented lower population of Lactobacillus spp. in small intestine and Bifidobacterium spp. in the large intestine. There were not significative differences among the other groups. Results of incidence of translocation of gut bacteria to different tissues and blood showed that no bacteremia was observed in the different experimental groups. There was no statistical difference regarding the thickness of duodenum epithelium among the four groups; however, in the jejunum-ileum portion CW group presented the highest epithelial height, when compared to the other groups CM, FMLC and FMLF. The height of epithelial colon was also higher in CW group than the other groups CM, FMLC and FMLF. Regarding the crypt depth, in the duodenum there was a significant difference between the CM group and the other groups, which presented greater crypt depth. In the 12 jejunum-ileum portion the greater crypt depth was shown in the CW group than in the other groups. In the colon portion, there were no significant variations among groups. The morphometry of the small intestinal villi showed that a smaller size of the duodenum villi was observed in the CW group, when compared to the other groups, however, in the jejunumileum portion no significant differences among the groups were observed. The morphometrically evaluation of intestinal microvilli showed that in the duodenum, the CM group presented statistically larger microvilli size than the other groups. In the jejunum-ileum portion, only the FMLC and FMLF groups presented significant differences between each other in microvilli size, while in the colon portion, there were no differences in microvilli size between the CW and FMLC groups, and between the CM and FMLF groups. There was a statistically higher frequency of goblet cells in the duodenum in the CM, FMLC and FMLF groups, when compared to the CW group; however, in the jejunum-ileum portion the difference was restricted between the CW and the CM groups and the frequency of goblet cells in the other groups was higher than 40%. In conclusion, the consumption of fermented milk with L. casei strains SJRP38 and L. fermentum SJRP43 promoted health benefits to the host.
The objective of this study was to evaluate the probiotic properties of the strains Lactobacillus casei and Lactobacillus fermentum, to select new safe strains for the development of fermented products and from this selection to define an appropriate matrix to be fermented and to evaluate the effect of the fermented milk by the strains selected in the bacterial translocation, fecal analyzes and intestinal morphology of BALB/c mice. The in vitro tests performed were self-aggregation, co-aggregation, hydrophobicity, β-galactosidase production, gastrointestinal tract survival (GIT) and antibiotic susceptibility. The selected strains were additionally tested for the presence of genes encoding adhesion, aggregation and colonization, virulence factors, antibiotic resistance and biogenic amine production, followed by the evaluation of kinetic parameters in the milk and survival of the strains under simulated GIT conditions during cooling. Most of the strains of both species presented high self-aggregation, some strains presented co-aggregation capacity with other lactic acid bacteria (BAL) and / or pathogens, and both species showed low hydrophobicity values. Seven strains of L. casei and six strains of L. fermentum were β-galactosidase enzyme producers and ten strains survived the simulation of stressful conditions of TGI. All strains were resistant to vancomycin and almost all strains were resistant to kanamycin. L. casei SJRP38 and L. fermentum SJRP43 were distinguished among the other strains of BAL by their greater potential probiotics. Three matrices were fermented by L. casei SJRP38 and L. fermentum SJRP43 and the kinetic parameters of acidification along the fermentation were performed. The animals were divided into four groups: water control (CW), milk control (CM), milk fermented by L. casei SJRP38 (FMLC) and milk fermented by L. fermentum SJRP43 (FMLF), both fermentations with coculture with the commercial strain of S. thermophilus TA040 throughout the treatment. After 14 days of feeding, the BALB / c mice were contained manually and individually, and euthanasia was performed and harvested blood, liver, spleen, small intestine, large intestine and intestinal contents were removed for translocation studies, intestinal morphology and intestinal microbiology. During the experimental protocol, no remarkable activity or behavioral changes were observed in the groups of mice. There was no statistical variation in the weight of BALB/c mice among the four experimental groups, in all periods evaluated. In general, administration of fermented milk resulted in microbiological changes in fecal composition, leading to a reduction in Clostridium spp. and increased Streptococcus spp., Lactobacillus sp. and Bifidobacterium spp. The population of Lactobacillus spp. and Bifidobacterium spp. in the small intestine and large intestine were affected by the treatment group. The FMLF group presented lower population of Lactobacillus spp. in small intestine and Bifidobacterium spp. in the large intestine. There were not significative differences among the other groups. Results of incidence of translocation of gut bacteria to different tissues and blood showed that no bacteremia was observed in the different experimental groups. There was no statistical difference regarding the thickness of duodenum epithelium among the four groups; however, in the jejunum-ileum portion CW group presented the highest epithelial height, when compared to the other groups CM, FMLC and FMLF. The height of epithelial colon was also higher in CW group than the other groups CM, FMLC and FMLF. Regarding the crypt depth, in the duodenum there was a significant difference between the CM group and the other groups, which presented greater crypt depth. In the 12 jejunum-ileum portion the greater crypt depth was shown in the CW group than in the other groups. In the colon portion, there were no significant variations among groups. The morphometry of the small intestinal villi showed that a smaller size of the duodenum villi was observed in the CW group, when compared to the other groups, however, in the jejunumileum portion no significant differences among the groups were observed. The morphometrically evaluation of intestinal microvilli showed that in the duodenum, the CM group presented statistically larger microvilli size than the other groups. In the jejunum-ileum portion, only the FMLC and FMLF groups presented significant differences between each other in microvilli size, while in the colon portion, there were no differences in microvilli size between the CW and FMLC groups, and between the CM and FMLF groups. There was a statistically higher frequency of goblet cells in the duodenum in the CM, FMLC and FMLF groups, when compared to the CW group; however, in the jejunum-ileum portion the difference was restricted between the CW and the CM groups and the frequency of goblet cells in the other groups was higher than 40%. In conclusion, the consumption of fermented milk with L. casei strains SJRP38 and L. fermentum SJRP43 promoted health benefits to the host.
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Palavras-chave
Histologia, Morfometria, Probióticos, Trato gastrointestinal, Gastrointestinal tract, Histology, Morphometry, Probiotics