| Bifidobacteria are common inhabitants of the human and animals gastrointestinal tract. Several species in the genus Bifidobacterium are considered probiotics and their presence has been associated with health-promoting effects; therefore, they are commonly applied in probiotic products. It is generally believed that they must survive passage through the gastrointestinal tract and reach the distal part of the intestine in sufficient numbers to function as probiotics. However, various acidic environments(e.g., the low pH of fermented dairy products in which bifidobacteria are added as probiotics, the low pH of the stomach and the low pH of fermented processing due to the productions of lactate and acetate) commonly reduce the survival of bifidobacteria, resulting in less than the recommended sufficient numbers reaching the intestine. Consequently, acid tolerance is recognized as a desirable property of potential probiotic bifidobacteria. However, the acid tolerance of most bifidobacteria have a weak acid tolerance, which limits their application in probiotic products. Hence, improving the acid tolerance of bifidobacteria is critical for exerting their beneficial effects efficiently as probiotic products.To improve the acid tolerance of bifidobacteria, it is necessary to fully understand the acid tolerance mechanisms. So far, studies on acid tolerance of bifidobacteria are still in the initial stage and the mechanisms of acid tolerance are unclear.In this study, we isolated two acid-resistant derivatives from their parental strains(one Bifidobacterium longum strain and one Bifidobacterium breve strain); the role of cell membrane in acid tolerance was explored by comparing the cell membrane of the two acid-resistant derivatives and their corresponding parental strains. To investigate the possible acid tolerance mechanisms of B. breve, a combined RNA-seq, qRT-PCR and physiological approach was used to characterize differences between the acid-resistant derivative of B. breve and its parental strain. Additionally, to explore the role of membrane-associated protein in acid tolerance, a combined SDS-PAGE and LC-MS/MS approach was used to identify the differential membrane-associated protein between the two acid-resistant derivative and their corresponding parental strains. Therefore, the aim of this study was to reveal the acid tolerance mechanisms in bifidobacteria, which would provide a basis for developing new strategies to enhance the acid tolerance of bifidobacterial strains.In this study, the two acid-resistant derivatives were successfully isolated from their corresponding parental strains by the method of prolonged exposure to low pH after repeating the isolation procedures many times. The two acid-resistant derivatives were named B. longum JDY1017 dpH and B. breve BB8 dpH, respectively. The stability of the acid-resistant phenotype of the two acid-resistant derivatives was evaluated by testing their ability to survive in acidic conditions by daily cultivation in medium for 20 consecutive days. The results showed that viable cell counts of the two acid-resistant derivatives were obviously higher than that those of their corresponding parental strains after exposure to acidic conditions at every day, which indicated the two acid-resistant derivatives having a stable acid-resistant phenotype.In this study, the relationship between acid tolerance and cell membrane was investigated by comparing the acid tolerance, cell membrane fatty acid compositions, membrane fluidity, and the expression of cfa gene encoding cyclopropane fatty acid synthase between the two acid-resistant derivatives and their corresponding parental strains when cells were grown in medium with and without Tween 80. The fold increase in acid tolerance of the two acid-resistant derivatives relative to their parental strains was much higher when cells were grown in medium with Tween 80(104-105-fold) than without Tween 80(181- and 245-fold). Moreover, when cells were grown in medium with Tween 80, the two acid-resistant derivatives exhibited more C18:1 and cycC19:0, higher mean fatty acid chain length, lower membrane fluidity, and higher expression of cfa gene than their parental strains. No significant differences in cell membrane were observed between the two acid-resistant derivatives and their corresponding parental strains when cells were grown in medium without Tween 80. These results implied that, when cells were grown in MRSC medium with Tween 80, the significant fold increase in acid tolerance of the two acid-resistant derivatives was mainly ascribed to the pronounced changes in cell membrane compared with their parental strains.To investigate the possible reasons for the higher acid tolerance of B. breve BB8 dpH, as compared with its parental strain, a combined RNA-seq, qRT-PCR and physiological approach was used to characterize differences between the two strains, when cells were grown in standard MRSC medium(containing Tween 80). An analysis of the transcriptome and RNA-seq indicated that the expression of 121 genes was increased by more than 2-fold, while the expression of 146 genes was reduced more than 2-fold, in B. breve BB8 dpH. To validate the results obtained from RNA-seq, the expression of 27 selected differentially expressed genes was measured using qRT-PCR, and the results showed that there was a strong positive correlation(R2 =0.91) between the RNA-seq data and the qRT-PCR data, demonstrating the RNA-seq results highly reliable. The comparison analysis, based on differentially expressed genes, indicated that acid tolerance of B. breve BB8 dp H reduce the expression of genes involved in the synthesis and transport of branched chain amino acid as the precursors of branched chain fatty acids, implying B. breve BB8 dpH might decrease the cell membrane fluidity to withstand acidic environments by reducing the production of branched chain fatty acids. The results supported the lower membrane fluidity in B. breve BB8 dpH, as compared with its parental strain B. breve BB8, when cells were grown in MRSC medium with Tween 80. Moreover, B. breve BB8 dpH might enhanced the ability cell envelope to prevent the entry H~+ into cells by increasing the expression of genes involved in synthesis of peptidoglycan and exopolysaccharide. When the H~+ entered the cytoplasm, the B. breve BB8 dpH could have several strategies to withstand acidic environments by increasing the expression of genes related to carbohydrates transport and metabolism, synthesis and transport of glutamate and glutamine, and histidine synthesis. Furthermore, an analysis of physiological data showed that B. breve BB8 dpH displayed higher production of exopolysaccharide and lower H~+-ATPase activity than B. breve BB8. These data obtained from this study allowed us to propose a mode for the possible reasons for the higher acid tolerance of B. breve BB8 dpH.In this study, we compared the membrane-associated protein profiles between the two acid-resistant derivative and their corresponding parental strains when cells were grown in standard MRSC medium(containing Tween 80). The results indicated one differential membrane-associated protein was identified as aminopeptidase N between B. longum JDY1017 dpH and its parental strain JDY1017. The aminopeptidase N was involved in the glutathione metabolism. Moreover, the differential membrane-associated protein were involved in exopolysaccharide synthesis, glycogen metabolism, protein synthesis, the transport of oligopeptides and glutamine between B. breve BB8 dpH and its parental strain B. breve BB8. The B. longum JDY1017 dpH might increase the production of intracellular glutathione to withstand acidic environments by regulating the expression of aminopeptidase N. The B. breve BB8 dpH might increase the production of exopolysaccharide to withstand acidic environments by regulating the expression of glycosyltransferase. The results supported the higher production of exopolysaccharide in B. breve BB8 dpH, as compared with its parental strain B. breve BB8, when cells were grown in MRSC medium with Tween 80. In addition, the B. breve BB8 dpH might increase the production of intracellular glutamine and glycogen to withstand acidic environments by regulating the expression of membrane-associated proteins related to glycogen metabolism and transport of glutamine. The result further supported the conclusions obtained by RNA-seq. Moreover, the B. breve BB8 dpH might regulate protein synthesis and transport of oligopeptide to withstand acidic environments. |
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