A distinct clade of Bifidobacterium longum in the gut of Bangladeshi children thrives during weaning.

The gut microbiome has an important role in infant health and development. We characterized the fecal microbiome and metabolome of 222 young children in Dhaka, Bangladesh during the first two years of life. A distinct Bifidobacterium longum clade expanded with introduction of solid foods and harbored enzymes for utilizing both breast milk and solid food substrates. The clade was highly prevalent in Bangladesh, present globally (at lower prevalence), and correlated with many other gut taxa and metabolites, indicating an important role in gut ecology. We also found that the B. longum clades and associated metabolites were implicated in childhood diarrhea and early growth, including positive associations between growth measures and B. longum subsp. infantis, indolelactate and N-acetylglutamate. Our data demonstrate geographic, cultural, seasonal, and ecological heterogeneity that should be accounted for when identifying microbiome factors implicated in and potentially benefiting infant development.

Mechanism of high-mannose N-glycan breakdown and metabolism by Bifidobacterium longum.

Bifidobacteria are early colonizers of the human gut and play central roles in human health and metabolism. To thrive in this competitive niche, these bacteria evolved the capacity to use complex carbohydrates, including mammalian N-glycans. Herein, we elucidated pivotal biochemical steps involved in high-mannose N-glycan utilization by Bifidobacterium longum. After N-glycan release by an endo-ß-N-acetylglucosaminidase, the mannosyl arms are trimmed by the cooperative action of three functionally distinct glycoside hydrolase 38 (GH38) α-mannosidases and a specific GH125 α-1,6-mannosidase. High-resolution cryo-electron microscopy structures revealed that bifidobacterial GH38 α-mannosidases form homotetramers, with the N-terminal jelly roll domain contributing to substrate selectivity. Additionally, an α-glucosidase enables the processing of monoglucosylated N-glycans. Notably, the main degradation product, mannose, is isomerized into fructose before phosphorylation, an unconventional metabolic route connecting it to the bifid shunt pathway. These findings shed light on key molecular mechanisms used by bifidobacteria to use high-mannose N-glycans, a perennial carbon and energy source in the intestinal lumen.

In vitro conversion of ellagic acid to urolithin A by different gut microbiota of urolithin metabotype A.

The metabolite urolithin A, a metabolite of the dietary polyphenol ellagic acid (EA), has significant health benefits for humans. However, studies on the gut microbiota involved in ellagic acid metabolism are limited. In this study, we conducted in vitro fermentation of EA using human intestinal microbiome combined with antibiotics (vancomycin, polymyxin B sulfate, and amphotericin B). Liquid chromatography-mass spectrometry (LC-MS/MS) analysis demonstrated that the production capacity of urolithin A by gut microbiota co-treated with polymyxin B sulfate and amphotericin B (22.39 µM) was similar to that of untreated gut microbiota (24.26 µM). Macrogenomics (high-throughput sequencing) was used to analyze the composition and structure of the gut microbiota. The results showed that the abundance of Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium bifidum in the gut microbiota without antibiotic treatment or co-treated with polymyxin B sulfate and amphotericin B during EA fermentation was higher than that in other antibiotic treatment gut microbiota. Therefore, B. longum, B. adolescentis, and B. bifidum may be new genera involved in the conversion of EA to urolithin A. In conclusion, the study revealed unique interactions between polyphenols and gut microbiota, deepening our understanding of the relationship between phenolic compounds like EA and the gut microbiota. These findings may contribute to the development of gut bacteria as potential probiotics for further development. KEY POINTS: • Intestinal microbiome involved in ellagic acid metabolism. • Gram-positive bacteria in the intestinal microbiome are crucial for ellagic acid metabolism. • Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium bifidum participate in ellagic acid metabolism.

Anti-Melanogenic and Antioxidant Activity of Bifidobacterium longum Strain ZJ1 Extracts, Isolated from a Chinese Centenarian.

Melanin produced by melanocytes protects our skin against ultraviolet (UV) radiation-induced cell damage and oxidative stress. Melanin overproduction by hyperactivated melanocytes is the direct cause of skin hyperpigmentary disorders, such as freckles and melasma. Exploring natural whitening agents without the concern of toxicity has been highly desired. In this study, we focused on a Bifidobacterium longum strain, ZJ1, isolated from a Chinese centenarian, and we evaluated the anti-melanogenic activity of the distinctive extracts of ZJ1. Our results demonstrated that whole lysate (WL) and bacterial lysate (BL) of ZJ1 ferments efficiently reduce α-melanocyte-stimulating hormone (α-MSH)-induced melanin production in B16-F10 cells as well as the melanin content in zebrafish embryos. BL and WL downregulate melanogenesis-related gene expression and indirectly inhibit intracellular tyrosinase activity. Furthermore, they both showed antioxidant activity in a menadione-induced zebrafish embryo model. Our results suggest that ZJ1 fermentation lysates have application potential as therapeutic reagents for hyperpigmentary disorders and whitening agents for cosmetics.

Mechanism of Cooperative Degradation of Gum Arabic Arabinogalactan Protein by Bifidobacterium longum Surface Enzymes.

Gum arabic is an arabinogalactan protein (AGP) that is effective as a prebiotic for the growth of bifidobacteria in the human intestine. We recently identified a key enzyme in the glycoside hydrolase (GH) family 39, 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase), for the assimilation of gum arabic AGP in Bifidobacterium longum subsp. longum. The enzyme released α-d-Galp-(1→3)-l-Ara and ß-l-Arap-(1→3)-l-Ara from gum arabic AGP and facilitated the action of other enzymes for degrading the AGP backbone and modified sugar. In this study, we identified an α-l-arabinofuranosidase (BlArafE; encoded by BLLJ_1850), a multidomain enzyme with both GH43_22 and GH43_34 catalytic domains, as a critical enzyme for the degradation of modified α-l-arabinofuranosides in gum arabic AGP. Site-directed mutagenesis approaches revealed that the α1,3/α1,4-Araf double-substituted gum arabic AGP side chain was initially degraded by the GH43_22 domain and subsequently cleaved by the GH43_34 domain to release α1,3-Araf and α1,4-Araf residues, respectively. Furthermore, we revealed that a tetrasaccharide, α-l-Rhap-(1→4)-ß-d-GlcpA-(1→6)-ß-d-Galp-(1→6)-d-Gal, was a limited degradative oligosaccharide in the gum arabic AGP fermentation of B. longum subsp. longum JCM7052. The oligosaccharide was produced from gum arabic AGP by the cooperative action of the three cell surface-anchoring enzymes, GAfase, exo-ß1,3-galactanase (Bl1,3Gal), and BlArafE, on B. longum subsp. longum JCM7052. Furthermore, the tetrasaccharide was utilized by the commensal bacteria. IMPORTANCE Terminal galactose residues of the side chain of gum arabic arabinogalactan protein (AGP) are mainly substituted by α1,3/α1,4-linked Araf and ß1,6-linked α-l-Rhap-(1→4)-ß-d-GlcpA residues. This study found a multidomain BlArafE with GH43_22 and GH43_34 catalytic domains showing cooperative action for degrading α1,3/α1,4-linked Araf of the side chain of gum arabic AGP. In particular, the GH43_34 domain of BlArafE was a novel α-l-arabinofuranosidase for cleaving the α1,4-Araf linkage of terminal galactose. α-l-Rhap-(1→4)-ß-d-GlcpA-(1→6)-ß-d-Galp-(1→6)-d-Gal tetrasaccharide was released from gum arabic AGP by the cooperative action of GAfase, GH43_24 exo-ß-1,3-galactanase (Bl1,3Gal), and BlArafE and remained after B. longum subsp. longum JCM7052 culture. Furthermore, in vitro assimilation test of the remaining oligosaccharide using Bacteroides species revealed that cross-feeding may occur from bifidobacteria to other taxonomic groups in the gut.

Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Galß1-3GalNAc reactivity on Fetuin.

Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum (EngBF) belongs to the glycoside hydrolase family GH101 and has a strict preference towards the mucin type glycan, Galß1-3GalNAc, which is O-linked to serine or threonine residues on glycopeptides and -proteins. While other enzymes of the GH101 family exhibit a wider substrate spectrum, no GH101 member has until recently been reported to process the α2-3 sialidated mucin glycan, Neu5Acα2-3Galß1-3GalNAc. However, work published by others (ACS Chem Biol 2021, 16, 2004-2015) during the preparation of the present manuscript demonstrated that the enzymes from several bacteria are able to hydrolyze this glycan from the fluorophore, methylumbelliferyl. Based on molecular docking using the EngBF homolog, EngSP from Streptococcus pneumoniae, substitution of active site amino acid residues with the potential to allow for accommodation of Neu5Acα2-3Galß1-3GalNAc were identified. Based on this analysis, the mutant EngBF variants W750A, Q894A, K1199A, E1294A and D1295A were prepared and tested, for activity towards the Neu5Acα2-3Galß1-3GalNAc O-linked glycan present on bovine fetuin. Among the mutant EngBF variants listed above, only E1294A was shown to release Neu5Acα2-3Galß1-3GalNAc from fetuin, which subsequently was also demonstrated for the substitutions: E1294 M, E1294H and E1294K. In addition, the kcat/KM of the EngBF variants for cleavage of the Neu5Acα2-3Galß1-3GalNAc glycan increased between 5 and 70 times from pH 4.5 to pH 6.0.

Gut Microbiota-Derived Short-Chain Fatty Acids Promote Poststroke Recovery in Aged Mice.

RATIONALE: The elderly experience profound systemic responses after stroke, which contribute to higher mortality and more severe long-term disability. Recent studies have revealed that stroke outcomes can be influenced by the composition of gut microbiome. However, the potential benefits of manipulating the gut microbiome after injury is unknown. OBJECTIVE: To determine if restoring youthful gut microbiota after stroke aids in recovery in aged subjects, we altered the gut microbiome through young fecal transplant gavage in aged mice after experimental stroke. Further, the effect of direct enrichment of selective bacteria producing short-chain fatty acids (SCFAs) was tested as a more targeted and refined microbiome therapy. METHODS AND RESULTS: Aged male mice (18-20 months) were subjected to ischemic stroke by middle cerebral artery occlusion. We performed fecal transplant gavage 3 days after middle cerebral artery occlusion using young donor biome (2-3 months) or aged biome (18-20 months). At day 14 after stroke, aged stroke mice receiving young fecal transplant gavage had less behavioral impairment, and reduced brain and gut inflammation. Based on data from microbial sequencing and metabolomics analysis demonstrating that young fecal transplants contained much higher SCFA levels and related bacterial strains, we selected 4 SCFA-producers (Bifidobacterium longum, Clostridium symbiosum, Faecalibacterium prausnitzii, and Lactobacillus fermentum) for transplantation. These SCFA-producers alleviated poststroke neurological deficits and inflammation, and elevated gut, brain and plasma SCFA concentrations in aged stroke mice. CONCLUSIONS: This is the first study suggesting that the poor stroke recovery in aged mice can be reversed via poststroke bacteriotherapy following the replenishment of youthful gut microbiome via modulation of immunologic, microbial, and metabolomic profiles in the host.

Dietary of Lactobacillus paracasei and Bifidobacterium longum improve nonspecific immune responses, growth performance, and resistance against Vibrio parahaemolyticus in Penaeus vannamei.

This study investigated the effects of two probiotics, namely Lactobacillus paracasei and Bifidobacterium longum, as feed additives on growth performance, nonspecific immunity, immune-related gene expression, and disease resistance against Vibrio parahaemolyticus in Penaeus vannamei. The experimental diets were prepared using L. paracasei and B. longum at concentrations of 105 and 107 CFU/g; these diets were referred to as P5, P7, B5, and B7. After 8 weeks of the diets, regarding growth performance, the B7 group showed the highest weight gain rate (890.34 ± 103.65%), special growth rate (4.08 ± 0.19%), and feed conversion rate (1.52 ± 0.19%) compared with the other groups. Moreover, the total hemocyte counts were significantly increased (p < 0.05) in the P7 groups on day 14 during the 28-day feeding trial. The phagocytosis rate in all experimental groups was increased on day 14 and was persistently significantly activated to day 21, especially in the P7 and B5 group. The phagocytic index of the P7 group showed a significant increase on day 14 and persistent activation to day 21. In the analysis of respiratory burst activity and phenoloxidase activity, the P7 and B5 groups showed a significant increase on day 7 and persistent activation to day 21. The expression level of the immune-related genes of superoxide dismutase, clotting protein, Penaeidin2, Penaeidin3, Penaeidin4, anti-LPS factor, crustin, and lysozyme was significantly increased in the experimental groups, especially in the P7 group. Furthermore, the optimum conditions of feed additives were determined in challenge trials conducted using P7 and B5. Shrimps fed P7 and B5 showed an increased survival rate (72.73% and 66.67%) after the V. parahaemolyticus challenge. In sum, the results revealed that B. longum, as a feed additive at 107 CFU/g, enhanced growth performance. L. paracasei at 107 CFU/g and B. longum at 105 CFU/g can enhance nonspecific immune responses and immune-related gene expression, and 107 CFU/g L. paracasei has the highest resistance ability for V. parahaemolyticus. Thus, dietary supplementation with L. paracasei and B. longum may be a valuable approach in white shrimp aquaculture.

Identification of a Catalytic Nucleophile-Activating Network in the endo-α-N-Acetylgalactosaminidase of Family GH101.

Bifidobacterium longum endo-α-N-acetylgalactosaminidase (GH101), EngBF, is highly specific toward the mucin Core 1 glycan, Galß1-3GalNAc. Apart from the side chains involved in the retaining mechanism of EngBF, Asp-682 is important for the activity. In the crystal structures of both EngBF and EngSP (from Streptococcus pneumoniae), we identified a conserved water molecule in proximity to Asp-682 and the homologue residue in EngSP. The water molecule also coordinates the catalytic nucleophile and three other residues conserved in GH101 enzymes; in EngBF, these residues are His-685, His-718, and Asn-720. With casein-glycomacropeptide as the substrate, the importance of Asp-682 was confirmed by the lack of a detectable activity for the D682N enzyme. The enzyme variants, H685A, H718A, H685Q, and H718Q, all displayed only a modestly reduction in kcat of up to 15 fold for the H718A variant. However, the double-substituted variants, H685A/H718A and H685Q/H718Q, had a greatly reduced kcat value by about 200 fold compared to that of wild-type EngBF. With the synthetic substrate, Galß(1-3)GalNAcα1-para-nitrophenol, kcat of the double-substituted variants was only up to 30-fold reduced and was found to increase with pH. Compared to the pre-steady-state kinetics of wild-type EngBF, a burst of about the size of the enzyme concentration was absent with the double-substituted EngBF variants, indicating that the nucleophilic attack had become at least as slow as the hydrolysis of the enzyme intermediate. Together, the results indicate that not only Asp-682 but also the entire conserved network of His-685, His-718, and what we suggest is a catalytic water molecule is important in the activation of the catalytic nucleophile.

Extracellular Vesicles Produced by Bifidobacterium longum Export Mucin-Binding Proteins.

Extracellular proteins are important factors in host-microbe interactions; however, the specific factors that enable bifidobacterial adhesion and survival in the gastrointestinal (GI) tract are not fully characterized. Here, we discovered that Bifidobacterium longum NCC2705 cultured in bacterium-free supernatants of human fecal fermentation broth released a myriad of particles into the extracellular environment. The aim of this study was to characterize the physiological properties of these extracellular particles. The particles, approximately 50 to 80 nm in diameter, had high protein and double-stranded DNA contents, suggesting that they were extracellular vesicles (EVs). A proteomic analysis showed that the EVs primarily consisted of cytoplasmic proteins with crucial functions in essential cellular processes. We identified several mucin-binding proteins by performing a biomolecular interaction analysis of phosphoketolase, GroEL, elongation factor Tu (EF-Tu), phosphoglycerate kinase, transaldolase (Tal), and heat shock protein 20 (Hsp20). The recombinant GroEL and Tal proteins showed high binding affinities to mucin. Furthermore, the immobilization of these proteins on microbeads affected the permanence of the microbeads in the murine GI tract. These results suggest that bifidobacterial exposure conditions that mimic the intestine stimulate B. longum EV production. The resulting EVs exported several cytoplasmic proteins that may have promoted B. longum adhesion. This study improved our understanding of the Bifidobacterium colonization strategy in the intestinal microbiome.IMPORTANCEBifidobacterium is a natural inhabitant of the human gastrointestinal (GI) tract. Morphological observations revealed that extracellular appendages of bifidobacteria in complex microbial communities are important for understanding its adaptations to the GI tract environment. We identified dynamic extracellular vesicle (EV) production by Bifidobacterium longum in bacterium-free fecal fermentation broth that was strongly suggestive of differing bifidobacterial extracellular appendages in the GI tract. In addition, export of the adhesive moonlighting proteins mediated by EVs may promote bifidobacterial colonization. This study provides new insight into the roles of EVs in bifidobacterial colonization processes as these bacteria adapt to the GI environment.

Indole-3-lactic acid, a metabolite of tryptophan, secreted by Bifidobacterium longum subspecies infantis is anti-inflammatory in the immature intestine.

BACKGROUND: Necrotizing enterocolitis (NEC), a necrotic inflammation of the intestine, represents a major health problem in the very premature infant. Although prevention is difficult, the combination of ingestion of maternal-expressed breastmilk in conjunction with a probiotic provides the best protection. In this study, we establish a mechanism for breastmilk/probiotic protection. METHODS: Ultra-high-performance liquid chromatography-tandem mass spectrometry of Bifidobacterium longum subsp. infantis (B. infantis) secretions was used to identify an anti-inflammatory molecule. Indole-3-lactic acid (ILA) was then tested in an established human immature small intestinal cell line, necrotizing colitis enterocytes, and other immature human enteroids for anti-inflammatory effects and to establish developmental function. ILA was also examined in immature and mature enterocytes. RESULTS: We have identified ILA, a metabolite of breastmilk tryptophan, as the anti-inflammatory molecule. This molecule is developmentally functional in immature but not mature intestinal enterocytes; ILA reduces the interleukin-8 (IL-8) response after IL-1ß stimulus. It interacts with the transcription factor aryl hydrocarbon receptor (AHR) and prevents transcription of the inflammatory cytokine IL-8. CONCLUSIONS: This molecule produced by B. infantis (ATCC No. 15697) interaction with ingested breastmilk functions in a complementary manner and could become useful in the treatment of all at-risk premature infants for NEC if safety and clinical studies are performed.

Characterization of fructooligosaccharide-degrading enzymes in human commensal Bifidobacterium longum and Anaerostipes caccae.

Kestose and nystose are short chain fructooligosaccharides (scFOSs) with degrees of polymerization of 3 and 4, respectively. A previous study revealed that these scFOSs have different growth stimulation properties against two human commensals, i.e. Bifidobacterium longum subsp. longum and butyrogenic Anaerostipes caccae. The present study characterized genes involved in FOS metabolism in these organisms. A. caccae possesses a single gene cluster consisting of four genes, including a gene encoding the putative FOS degradation enzyme sucrose-6-phosphate hydrolase (S6PH). B. longum possesses two gene clusters consisting of three genes each, including genes encoding ß-fructofuranosidase (CscA) and sucrose phosphorylase (ScrP). In A. caccae, the genes were highly transcribed in cells cultured with sucrose or kestose but poorly in cells cultured with glucose or nystose. Heterologously expressed S6PH degraded sucrose and kestose but not nystose. In B. longum, transcription of the genes was high in cells cultured with sucrose or kestose but was poor or not detected in cells cultured with glucose or nystose. Heterologously expressed CscA degraded sucrose, kestose and nystose, but ScrP degraded only sucrose. These data suggested that the different growth stimulation activities of kestose and nystose are due to different substrate specificities of FOS degradation enzymes in the organisms and/or induction activity of the genes in the two scFOSs. This is the first study characterizing the FOS metabolism at the transcriptional level and substrate-specificity of the degradation enzyme in butyrogenic human gut anaerobes.

The MarR Family Regulator BmrR Is Involved in Bile Tolerance of Bifidobacterium longum BBMN68 via Controlling the Expression of an ABC Transporter.

In order to colonize the human gastrointestinal tract and exert their beneficial effects, bifidobacteria must effectively cope with toxic bile salts in the intestine; however, the molecular mechanism underlying bile tolerance is poorly understood. In this study, heterologous expression of a MarR family transcriptional regulator, BmrR, significantly reduced the ox bile resistance of Lactococcus lactis NZ9000, suggesting that BmrR might play a role in the bile stress response. In silico analysis combined with reverse transcription-PCR assays demonstrated that bmrR was cotranscribed with bmrA and bmrB, which encoded multidrug resistance (MDR) ABC transporters. Promoter prediction and electrophoretic mobility shift assays revealed that BmrR could autoregulate the bmrRAB operon by binding to the bmr box (ATTGTTG-6nt-CAACAAT) in the promoter region. Moreover, heterologous expression of bmrA and bmrB in L. lactis yielded 20.77-fold higher tolerance to 0.10% ox bile, compared to the wild-type strain. In addition, ox bile could disrupt the DNA binding activity of BmrR as a ligand. Taken together, our findings indicate that the bmrRAB operon is autoregulated by the transcriptional regulator BmrR and ox bile serves as an inducer to activate the bile efflux transporter BmrAB in response to bile stress in Bifidobacterium longum BBMN68.IMPORTANCE Bifidobacteria are natural inhabitants of the human intestinal tract. Some bifidobacterial strains are used as probiotics in fermented dairy production because of their health-promoting effects. Following consumption, bifidobacteria colonize the lower intestinal tract, where the concentrations of bile salts remain nearly 0.05% to 2.0%. Bile salts, as detergent-like antimicrobial compounds, can cause cellular membrane disruption, protein misfolding, and DNA damage. Therefore, tolerance to physiological bile stress is indeed essential for bifidobacteria to survive and to exert probiotic effects in the gastrointestinal tract. In B. longum BBMN68, the MarR-type regulator BmrR was involved in the bile stress response by autoregulating the bmrRAB operon, and ox bile as an inducer could increase the expression of the BmrAB transporter to enhance the bile tolerance of BBMN68. Our study represents a functional analysis of the bmrRAB operon in the bile stress response, which will provide new insights into bile tolerance mechanisms in Bifidobacterium and other bacteria.

Efficient chemoenzymatic synthesis of UDP-α-6-N3-glucose.

A novel chemo-enzymatic synthetic method for UDP-α-6-N3-glucose was developed by combining the versatility of chemical synthesis and natural enzyme stereo-selectivity of Bifidobacterium longum (BLUSP). This flexible and efficient platform expanded the substrate scope for UDP-sugars on an improved scale, particularly for UDP-sugar substrates containing bioorthogonal functional groups.

Degradative enzymes for type II arabinogalactan side chains in Bifidobacterium longum subsp. longum.

Type II arabinogalactan (AG) is a soluble prebiotic fiber stimulating the proliferation of bifidobacteria in the human gut. Larch AG, which is comprised of type II AG, is known to be utilized as an energy source for Bifidobacterium longum subsp. longum (B. longum). We have previously characterized GH43_24 exo-ß-1,3-galactanase (Bl1,3Gal) for the degradation of type II AG main chains in B. longum JCM1217. In this study, we characterized GH30_5 exo-ß-1,6-galactobiohydrolase (Bl1,6Gal) and GH43_22 α-L-arabinofuranosidase (BlArafA), which are degradative enzymes for type II AG side chains in cooperation with exo-ß-1,3-galactanase. The recombinant exo-ß-1,6-galactobiohydrolase specifically released ß-1,6-galactobiose (ß-1,6-Gal2) from the nonreducing terminal of ß-1,6-galactooligosaccharides, and the recombinant α-L-arabinofuranosidase released arabinofuranose (Araf) from α-1,3-Araf-substituted ß-1,6-galactooligosaccharides. ß-1,6-Gal2 was additively released from larch AG by the combined use of type II AG degradative enzymes, including Bl1,3Gal, Bl1,6Gal, and BlArafA. The gene cluster encoding the type II AG degradative enzymes is conserved in all B. longum strains, but not in other bifidobacterial species. The degradative enzymes for type II AG side chains are thought to be important for the acquisition of type II AG in B. longum.

Impact of combining acerola by-product with a probiotic strain on a gut microbiome model.

In this study, we first investigated the survival of three probiotic strains, individually and combined with acerola by-product during simulated gastrointestinal conditions. Next, we investigated the effects of acerola by-product combined with Bifidobacterium longum BB-46 on a gut microbiota model (SHIME®). Chemical composition, total phenolic compounds, antioxidant activity of the acerola by-product and microbial counts, denaturing gradient gel electrophoresis (DGGE), ammonium ions ( NH4+ ) and short-chain fatty acids (SCFAs) analysis of the SHIME® samples were performed. Acerola by-product revealed high protein and fibre, reduced lipid contents, and showed to be an excellent source of total phenolic compounds with high in vitro antioxidant activity. A decreased amount of NH4+ in the ascending colon and an increase (p < .05) in SCFAs were observed in the three regions of colon during treatment with BB-46 and acerola by-product. BB-46 combined with acerola by-product showed positive effects on the gut microbiota metabolism in SHIME® model.

Gene-trait matching across the Bifidobacterium longum pan-genome reveals considerable diversity in carbohydrate catabolism among human infant strains.

BACKGROUND: Bifidobacterium longum is a common member of the human gut microbiota and is frequently present at high numbers in the gut microbiota of humans throughout life, thus indicative of a close symbiotic host-microbe relationship. Different mechanisms may be responsible for the high competitiveness of this taxon in its human host to allow stable establishment in the complex and dynamic intestinal microbiota environment. The objective of this study was to assess the genetic and metabolic diversity in a set of 20 B. longum strains, most of which had previously been isolated from infants, by performing whole genome sequencing and comparative analysis, and to analyse their carbohydrate utilization abilities using a gene-trait matching approach. RESULTS: We analysed their pan-genome and their phylogenetic relatedness. All strains clustered in the B. longum ssp. longum phylogenetic subgroup, except for one individual strain which was found to cluster in the B. longum ssp. suis phylogenetic group. The examined strains exhibit genomic diversity, while they also varied in their sugar utilization profiles. This allowed us to perform a gene-trait matching exercise enabling the identification of five gene clusters involved in the utilization of xylo-oligosaccharides, arabinan, arabinoxylan, galactan and fucosyllactose, the latter of which is an abundant human milk oligosaccharide (HMO). CONCLUSIONS: The results showed high diversity in terms of genes and predicted glycosyl-hydrolases, as well as the ability to metabolize a large range of sugars. Moreover, we corroborate the capability of B. longum ssp. longum to metabolise HMOs. Ultimately, their intraspecific genomic diversity and the ability to consume a wide assortment of carbohydrates, ranging from plant-derived carbohydrates to HMOs, may provide an explanation for the competitive advantage and persistence of B. longum in the human gut microbiome.

Reconstruction of the Bifidobacterial Pan-Secretome Reveals the Network of Extracellular Interactions between Bifidobacteria and the Infant Gut.

The repertoire of secreted proteins decoded by a microorganism represents proteins released from or associated with the cell surface. In gut commensals, such as bifidobacteria, these proteins are perceived to be functionally relevant, as they regulate the interaction with the gut environment. In the current study, we screened the predicted proteome of over 300 bifidobacterial strains among the currently recognized bifidobacterial species to generate a comprehensive database encompassing bifidobacterial extracellular proteins. A glycobiome analysis of this predicted bifidobacterial secretome revealed that a correlation exists between particular bifidobacterial species and their capability to hydrolyze human milk oligosaccharides (HMOs) and intestinal glycoconjugates, such as mucin. Furthermore, an exploration of metatranscriptomic data sets of the infant gut microbiota allowed the evaluation of the expression of bifidobacterial genes encoding extracellular proteins, represented by ABC transporter substrate-binding proteins and glycoside hydrolases enzymes involved in the degradation of human milk oligosaccharides and mucin. Overall, this study provides insights into how bifidobacteria interact with their natural yet highly complex environment, the infant gut.IMPORTANCE The ecological success of bifidobacteria relies on the activity of extracellular proteins that are involved in the metabolism of nutrients and the interaction with the environment. To date, information on secreted proteins encoded by bifidobacteria is incomplete and just related to few species. In this study, we reconstructed the bifidobacterial pan-secretome, revealing extracellular proteins that modulate the interaction of bifidobacteria with their natural environment. Furthermore, a survey of the secretion systems between bifidobacterial genomes allowed the identification of a conserved Sec-dependent secretion machinery in all the analyzed genomes and the Tat protein translocation system in the chromosomes of 23 strains belonging to Bifidobacterium longum subsp. longum and Bifidobacterium aesculapii.

tuf Gene Sequence Variation in Bifidobacterium longum subsp. infantis Detected in the Fecal Microbiota of Chinese Infants.

Members of the bacterial genus Bifidobacterium generally dominate the fecal microbiota of infants. The species Bifidobacterium longum is prevalent, but the B. longum subsp. longum and B. longum subsp. infantis strains that are known to colonize the infant bowel are not usually differentiated in microbiota investigations. These subspecies differ in their capacities to metabolize human milk oligosaccharides (HMO) and may have different ecological and symbiotic roles in humans. Quantitative PCR provides a quick analytical method by which to accurately ascertain the abundances of target species in microbiotas and microcosms. However, amplification targets in DNA extracted from samples need to be dependably differential. We evaluated the tuf gene sequence as a molecular target for quantitative PCR measurements of the abundances of B. longum subsp. infantis and B. longum subsp. longum in fecal microbiotas. This approach resulted in the detection of a tuf gene variant (operational taxonomic unit 49 [OTU49]) in Chinese infants that has sequence similarities to both B. longum subsp. infantis and B. longum subsp. longum We compared the genome sequence and growth and transcriptional characteristics of an OTU49 isolate cultured in HMO medium to those of other B. longum subsp. infantis cultures. We concluded from these studies that OTU49 belongs to B. longum subsp. infantis, that dependable quantitative PCR (qPCR) differentiation between the B. longum subspecies cannot be achieved by targeting tuf gene sequences, and that functional genes involved in carbohydrate metabolism might be better targets because they delineate ecological functions.IMPORTANCE High-throughput DNA sequencing methods and advanced bioinformatics analysis have revealed the composition and biochemical capacities of microbial communities (microbiota and microbiome), including those that inhabit the gut of human infants. However, the microbiology and function of natural ecosystems have received little attention in recent decades, so an appreciation of the dynamics of gut microbiota interactions is lacking. With respect to infants, rapid methodologies, such as quantitative PCR, are needed to determine the prevalences and proportions of different bifidobacterial species in observational and microcosm studies in order to obtain a better understanding of the dynamics of bifidobacterial nutrition and syntrophy, knowledge that might be used to manipulate the microbiota and perhaps ensure the better health of infants.

Catabolism of citrus flavanones by the probiotics Bifidobacterium longum and Lactobacillus rhamnosus.

PURPOSE: Orange juice (OJ) flavanones undergo limited absorption in the upper gastrointestinal tract and reach the colon where they are transformed by the microbiota prior to absorption. This study investigated the ability of two probiotic bacteria, Bifidobacterium longum R0175 and Lactobacillus rhamnosus subsp. Rhamnosus NCTC 10302 to catabolise OJ flavanones. METHODS: The bacteria were incubated with hesperetin-7-O-rutinoside, naringenin-7-O-rutinoside, hesperetin and naringenin, and the culture medium and intracellular cell extracts were collected at intervals over a 48 h of incubation period. The flavanones and their phenolic acid catabolites were identified and quantified by HPLC-HR-MS. RESULTS: Both probiotics were able to subject hesperetin to ring fission yielding 3-(3'-hydroxy-4'-methoxyphenyl)propionic acid which was subsequently demethylated producing 3-(3',4'-dihydroxyphenyl)propionic acid and then via successive dehydroxylations converted to 3-(3'-hydroxyphenyl)propionic acid and 3-(phenyl)propionic acid. Incubation of both bacteria with naringenin resulted in its conversion to 3-(4'-hydroxyphenyl)propionic acid which underwent dehydroxylation yielding 3-(phenyl)propionic acid. In addition, only L. rhamnosus exhibited rhamnosidase and glucosidase activity and unlike B. longum, which was able to convert hesperetin-7-O-rutinoside and naringenin-7-O-rutinoside to their respective aglycones. The aglycones were then subjected to ring fission and further catabolised in a similar manner to that described above. The flavanones and their catabolites were found in the culture medium but not accumulated in the bacterial cells. CONCLUSIONS: These findings demonstrate the enzymatic potential of single strains of bifidobacterium and lactobacillus which may be involved in the colonic catabolism of OJ flavanones in vivo.