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Bifidobacterium longum subsp. longum BBMN68, isolated from centenarians in Guangxi, China, has been proved to be a promising probiotic strain for its health benefits. In this study, the biodistribution of this strain in the rat gut was first investigated using the quantitative realtime PCR assay and propidium monoazide. Strain-specific primers were originally designed based on the BBMN68 genome sequence. Healthy rats were orally inoculated with either a single dose of BBMN68 (10(10) colony-forming units/kg), or with one dose per day for 7 days and bacterial concentrations were analyzed in detail from the intestinal contents and feces of four different gut locations, including stomach, small intestine, colon, and rectum. Results indicated that strain BBMN68 could overcome the rigors of passage through the upper gastrointestinal tract and transiently accumulate in the colon, even though survival in the stomach and small intestine was not high. A good level of BBMN8 could stay in vivo for 72 h following a 7-day oral administration, and a daily administration is suggested for a considerable and continuous population of BBMN68 to be maintained in the host intestine.

Bifidobacterium animalis subsp. lactis A6 (BAA6) (CGMCC No. 9273) was a probiotic strain isolated from the feces of a centenarian. Previous study showed that BAA6 had high acid resistance to low pH which is a critical factor influencing its healthy benefits. Elaborating the stress resistant mechanisms of bifidobacteria is important to extensively exploit this probiotic. Here, we reported the complete genome sequence of BAA6 that contains 1,958,651 bp encoding 1622 CDSs, 16 rRNA genes, 52 tRNA genes., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Bifidobacterium adolesentis BBMN23 (CGMCC No. 2264) was a probiotic strain originated from the feces of a centenarian. It is an excellent model for the study of the adaptation of genus bifidobacteria to adult human gut, which is a key factor in bifidobacterial strains that allows them to persist in gut and become useful in the food and medical industries. In the present study the complete genome sequence of BBMN23 is presented to provide insight into this strain., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Bifidobacteria are key commensals in human gut, and their abundance is associated with the health of their hosts. Although they are dominant in infant gut, their number becomes lower in adult gut. The changes of the diet are considered to be main reason for this difference. Large amounts of whole-genomic sequence data of bifidobacteria make it possible to elucidate the genetic interpretation of their adaptation to the nutrient environment. Among the nutrients in human gut, starch is a highly fermentable substrate and can exert beneficial effects by increasing bifidobacteria and/or being fermented to short chain fatty acids., Results: In order to determine the potential substrate preference of bifidobacteria, we compared the glycoside hydrolase (GH) profiles of a pooled-bifidobacterial genome (PBG) with a representative microbiome (RM) of the human gut. In bifidobacterial genomes, only 15% of GHs contained signal peptides, suggesting their weakness in utilization of complex carbohydrate, such as plant cell wall polysaccharides. However, compared with other intestinal bacteria, bifidobacteiral genomes encoded more GH genes for degrading starch and starch hydrolysates, indicating that they have genetic advantages in utilizing these substrates. Bifidobacterium longum subsp. longum BBMN68 isolated from centenarian's faeces was used as a model strain to further investigate the carbohydrate utilization. The pathway for degrading starch and starch hydrolysates was the only complete pathway for complex carbohydrates in human gut. It is noteworthy that all of the GH genes for degrading starch and starch hydrolysates in the BBMN68 genome were conserved in all studied bifidobacterial strains. The in silico analyses of BBMN68 were further confirmed by growth experiments, proteomic and real-time quantitative PCR (RT-PCR) analyses., Conclusions: Our results demonstrated that starch and starch hydrolysates were the most universal and favorable carbon sources for bifidobacteria. The low amount of these carbon sources in adult intestine was speculated to contribute to the low relative abundance of bifidobacteria.

Weak acid resistance limits the application of Bifidobacteria as a probiotic in food. The acid tolerance response (ATR), caused by pre-stressing cells at a sublethal pH, could improve the acid resistance of Bifidobacteria to subsequent acid stress. In this study, we used Bifidobacterium longum sub. longum BBMN68 to investigate the effect of the ATR on the acid stress response (ASR), and compared the difference between the ATR and the ASR by analyzing the two-dimensional-PAGE protein profiles and performing physiological tests. The results revealed that a greater abundance of proteins involved in carbohydrate metabolism and protein protection was present after the ASR than after the ATR in Bifidobacterium. Pre-stressing cells increased the abundance of proteins involved in energy production, amino acid metabolism, and peptidoglycan synthesis during the ASR of Bifidobacterium. Moreover, after the ASR, the content of ATP, NH3, thiols, and peptidoglycan, the activity of H+-ATPase, and the maintenance of the intracellular pH in the pre-stressed Bifidobacterium cells was significantly higher than in the uninduced cells. These results provide the first explanation as to why the resistance of Bifidobacterium to acid stress improved after pre-stressing.

Bifidobacteria are normal inhabitants of the human gut, and members of which are generally considered to be probiotic. Before exerting their beneficial properties, they must survive and persist in the physiological concentrations (0.05-2%) of bile in the gut. In this work, the functional role of tlyC1 encoding a hemolysin-like protein from Bifidobacterium longum BBMN68 in bile tolerance was tested. Analysis using the program TMHMM and homologous alignment indicated that TlyC1 is a nontransporter membrane protein and is conserved in many bifidobacteria. Heterologous expression of tlyC1 in Lactococcus lactis NZ9000 was shown to confer 45-fold higher tolerance to 0.15% ox-bile. Notably, the recombinant strains showed threefold higher survival when exposed to sublethal concentration of TCA and TDCA, while no significant change was observed when exposed to GCA and GDCA. Furthermore, real-time quantitative PCR demonstrated that the transcription of tlyC1 was up-regulated c. 2.5- and 2.7-fold in B. longum BBMN68 exposed to sublethal concentration of TCA and TDCA, while no significant change was observed with GCA and GDCA challenges. This study indicated that tlyC1 was specifically induced by tauroconjugates, which provided enhanced resistance to sodium taurocholate and sodium taurodeoxycholate., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Bifidobacteria are natural inhabitants of the human gastrointestinal tract and well known for their health-promoting effects. Tolerance to bile stress is crucial for bifidobacteria to survive in the colon and to exert their beneficial actions. In this work, RNA-Seq transcriptomic analysis complemented with proteomic analysis was used to investigate the cellular response to bile in Bifidobacterium longum BBMN68. The transcript levels of 236 genes were significantly changed (≥ threefold, p < 0.001) and 44 proteins were differentially abundant (≥1.6-fold, p < 0.01) in B. longum BBMN68 when exposed to 0.75 g l(-1) ox-bile. The hemolysin-like protein and bile efflux systems were significantly over produced, which might prevent bile adsorption and exclude bile, respectively. The cell membrane composition was modified probably by an increase of cyclopropane fatty acid and a decrease of transmembrane proteins, resulting in a cell membrane more impermeable to bile salts. Our hypothesis was later confirmed by surface hydrophobicity assay. The transcription of genes related to xylose utilization and bifid shunt were up-regulated, which increased the production of ATP and reducing equivalents to cope with bile-induced damages in a xylan-rich colon environment. Bile salts signal the B. longum BBMN68 to gut entrance and enhance the expression of esterase and sortase associated with adhesion and colonization in intestinal tract, which was supported by a fivefold increased adhesion ability to HT-29 cells by BBMN68 upon bile exposure. Notably, bacterial one-hybrid and EMSA assay revealed that the two-component system senX3-regX3 controlled the expression of pstS in bifidobacteria and the role of this target gene in bile resistance was further verified by heterologous expression in Lactococcus lactis. Taken altogether, this study established a model for global response mechanisms in B. longum to bile., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

To analyze the mechanism of the acid tolerance response (ATR) in Bifidobacterium longum subsp. longum BBMN68, we optimized the acid-adaptation condition to stimulate ATR effectively and analyzed the change of gene expression profile after acid-adaptation using high-throughput RNA-Seq. After acid-adaptation at pH 4.5 for 2 hours, the survival rate of BBMN68 at lethal pH 3.5 for 120 min was increased by 70 fold and the expression of 293 genes were upregulated by more than 2 fold, and 245 genes were downregulated by more than 2 fold. Gene expression profiling of ATR in BBMN68 suggested that, when the bacteria faced acid stress, the cells strengthened the integrity of cell wall and changed the permeability of membrane to keep the H(+) from entering. Once the H(+) entered the cytoplasm, the cells showed four main responses: First, the F(0)F(1)-ATPase system was initiated to discharge H(+). Second, the ability to produce NH(3) by cysteine-cystathionine-cycle was strengthened to neutralize excess H(+). Third, the cells started NER-UVR and NER-VSR systems to minimize the damage to DNA and upregulated HtpX, IbpA, and γ-glutamylcysteine production to protect proteins against damage. Fourth, the cells initiated global response signals ((p)ppGpp, polyP, and Sec-SRP) to bring the whole cell into a state of response to the stress. The cells also secreted the quorum sensing signal (AI-2) to communicate between intraspecies cells by the cellular signal system, such as two-component systems, to improve the overall survival rate. Besides, the cells varied the pathways of producing energy by shifting to BCAA metabolism and enhanced the ability to utilize sugar to supply sufficient energy for the operation of the mechanism mentioned above. Based on these reults, it was inferred that, during industrial applications, the acid resistance of bifidobacteria could be improved by adding BCAA, γ-glutamylcysteine, cysteine, and cystathionine into the acid-stress environment.

Bifidobacterium longum subsp. longum BBMN68, an anaerobic probiotic isolated from healthy centenarian faeces, shows low oxygen (3 %, v/v) tolerance. To understand the effects of oxidative stress and the mechanisms protecting against it in this strain, a proteomic approach was taken to analyse changes in the cellular protein profiles of BBMN68 under the following oxygen-stress conditions. Mid-exponential phase BBMN68 cells grown in MRS broth at 37 °C were exposed to 3 % O(2) for 1 h (I) or 9 h (II), and stationary phase cells were subjected to 3 % O(2) for 1 h (III). Respective controls were grown under identical conditions but were not exposed to O(2). A total of 51 spots with significant changes after exposure to oxygen were identified, including the oxidative stress-protective proteins alkyl hydroperoxide reductase C22 (AhpC) and pyridine nucleotide-disulfide reductase (PNDR), and the DNA oxidative damage-protective proteins DNA-binding ferritin-like protein (Dps), ribonucleotide reductase (NrdA) and nucleotide triphosphate (NTP) pyrophosphohydrolases (MutT1). Changes in polynucleotide phosphorylase (PNPase) plus enolase, which may play important roles in scavenging oxidatively damaged RNA, were also found. Following validation at the transcriptional level of differentially expressed proteins, the physiological and biochemical functions of BBMN68 Dps were further proven by in vitro and in vivo tests under oxidative stress. Our results reveal the key oxidative stress-protective proteins and DNA oxidative damage-protective proteins involved in the defence strategy of BBMN68 against oxygen, and provide the first proteomic information toward understanding the responses of Bifidobacterium and other anaerobes to oxygen stress.

Bifidobacterium longum subsp. longum BBMN68 was isolated from the feces of a healthy centenarian living in an area of BaMa, Guangxi, China, known for longevity. Here we report the main genome features of B. longum strain BBMN68 and the identification of several predicted proteins associated with the ecological niche of longevity.

The acid adaption is commonly used as a strategy to enhance the acid tolerance of bifidobacteria. However, the acid tolerance response (ATR) mechanism elicited by this method is unclear. Real-time relative-quantitative PCR was applied to analyze the changes in the expressions of ffh, uvrA, groES, and dnaK involved in the ATR after acid-adaptation in Bifidobacterium longum BBMN68 in different growth phases. BBMN68 was cultured at a constant neutral pH during the whole growth phase. Without acid-adaptation, the survival ratios at the lethal pH 3.0 were 0.25% and 17% in the exponential and stationary phases, respectively. The genes ffh, uvrA, groES, and dnaK were significantly higher in the stationary phase than in the exponential phase. The results indicated that although there was no acid stress, the acid tolerance of cells was elevated from the exponential phase into stationary phase. After acid-adaptation at pH 5.0 for 120 min, the survival ratios of BBMN68 in the exponential and stationary phases were increased to 2.5 and 31%, respectively. In the exponential phase, ffh, uvrA groES, and dnaK were significantly decreased after acid-adaptation. In the stationary phase, after acid-adaptation for 15, 60, and 120 min, the genes uvrA, groES, and dnaK were significantly decreased, whereas, ffh was significantly up-regulated at 15 min, and then suppressed at 60 and 120 min after acid-adaptation. The results represented that the ATR in B. longum was different from other bacteria, and ffh may be the transient acid gene.

We studied the effects of two bifidobacteria strains isolated from healthy centenarians on intestinal function in mice. Bifidobacterium adolescentis BBMN23 and Bifidobacterium longum BBMN68 were orally administrated to specific pathogen-free BALB/c mice at different doses (2 x 10(11), 2 x 10(9), or 2 x 10(7) CFU/kg body weight) each day for 4 weeks. Villus height, crypt depth, villus width, and villus/crypt ratio (V/C) were determined. The content of duodenal secreted immunoglobulin A (sIgA) was also evaluated. There were clear increases in height and width of duodenal villi in both treated groups. Crypt depths were deeper in animals treated with BBMN23 than in controls, while depths were reduced in animals receiving BBMN68. The V/C ratio was increased after feeding with BBMN68, while BBMN23 had no significant effect. Both strains improved the sIgA content of the duodenum. These results suggest that BBMN23 and BBMN68 may improve intestinal digestion and ability and enhance immune barrier function in the intestine.

The low survival rate in harsh stomach conditions and short retention in intestine of probiotics greatly limit their health benefits. To solve this problem, thiolated oxidized konjac glucomannan (sOKGM) microspheres is designed with pH responsive and mucoadhesive properties. First, an increased survival rate of probiotics by sOKGM microspheres encapsulation in simulated gastric fluid (SGF) is discovered in contrast to the zero‐survival rate of naked probiotics. sOKGM/probiotics even show a higher survival rate in SGF compared with commercial Bb12 formulation. Further, an enhanced mucoadhesion of probiotics to intestinal mucus by mediated interactions with sOKGM is confirmed by isotherm titration calorimetry, rheology, and tensile measurements. The in vivo intestinal transition experiment indicates a prolonged retention of probiotics at intestine by sOKGM encapsulation. Moreover, in vivo evaluation of enhanced colonization and proliferation by sOKGM/probiotics is demonstrated by the fecal and intestinal bacteria copy number via quantitative polymerase chain reaction (qPCR) detection. Further investigation of the alleviation of constipation by sOKGM containing Bifidobacterium animalis subsp. lactis A6 suggests that sOKGM increases the abundance of Bifidobacterium, balanced intestinal flora, and alleviated constipation in mice compared with other formulations. sOKGM with both enhanced gastric acid resistance and adhesion colonization at intestine can effectively improve the function of probiotics. [ABSTRACT FROM AUTHOR]

The underlying mechanisms imparting the growth phase-dependent acid tolerance have not been extensively investigated. In this study, we compared the acid resistance of the Bifidobacterium longum strain BBMN68 from different growth phases at lethal pH values (pH 2.5, 3.0, and 3.5), and analyzed the activity of H-ATPase, the composition of fatty acids, and the mRNA abundance of ffh, uvrA, recA, lexA, groES, and dnaK in cells from different growth phases. The results indicated that the survival rates of cells from early stationary (ES) and late stationary (LS) growth phases at lethal pH values were significantly higher than those of exponential growth phase cells. Our findings indicated that by inducing a continuously auto-acidizing environment during cell growth, the acid resistance of ES and LS cells was strengthened. The higher activity of H-ATPase, the decrease in unsaturated fatty acids, and the increased expression of genes involved in DNA repair and protein protection in the cells in stationary growth phase were all implicated in the significantly increased acid resistance of ES and LS cells compared with exponential growth phase cells of the B. longum strain BBMN68. [ABSTRACT FROM AUTHOR]

Adhesion to gastrointestinal tract is crucial for bifidobacteria to exert their probiotic effects. Our previous work found that bile salts significantly enhance the adhesion ability of Bifidobacterium longum BBMN68 to HT-29 cells. In this study, trypsin-shaving and LC-MS/MS-based surface proteomics were employed to identify surface proteins involved in bile stress response. Among the 829 differentially expressed proteins, 56 up-regulated proteins with a fold change >1.5 were subjected to further analysis. Notably, the minor pilin subunit FimB was 4.98-fold up-regulated in response to bile stress. In silico analysis and RT-PCR confirmed that gene fimB , fimA and srtC were co-transcribed and contributed to the biosynthesis of sortase-dependent pili Pil1. Moreover, scanning electron microscopy and immunogold electron microscopy assays showed increased abundance and length of Pil1 on BBMN68 under bile stress. As the major pilin subunit FimA serves as adhesion component of Pil1, an inhibition assay using anti-FimA antibodies further confirmed the critical role of Pil1 in mediating the adhesion of BBMN68 to HT-29 cells under bile stress. Our findings suggest that the up-regulation of Pil1 in response to bile stress enhances the adhesion of BBMN68 to intestinal epithelial cells, highlighting a novel mechanism of gut persistence in B. longum strains. • Bile stress enhanced the adhesion of Bifidobacterium longum BBMN68 to HT-29 cells. • A total of 56 proteins were identified by surface proteomics in the bile stress response. • The minor pilin subunit FimB was 4.98-fold up-regulated in response to bile stress. • Pil1 in response to bile stress enhances the adhesion of BBMN68 to HT-29 cells. [ABSTRACT FROM AUTHOR]