Project description:Two novel phytases have been characterized from Bifidobacterium pseudocatenulatum and Bifidobacterium longum subsp. infantis. The enzymes belong to a new subclass within the histidine acid phytases, are highly specific for the hydrolysis of phytate, and render myo-inositol triphosphate as the final hydrolysis product. They represent the first phytases characterized from this group of probiotic microorganisms, opening the possibilities for their use in the processing of high-phytate-content foods.
Project description:Bifidobacterium longum subsp. infantis ATCC 15697 utilizes several small-mass neutral human milk oligosaccharides (HMOs), several of which are fucosylated. Whereas previous studies focused on endpoint consumption, a temporal glycan consumption profile revealed a time-dependent effect. Specifically, among preferred HMOs, tetraose was favored early in fermentation, with other oligosaccharides consumed slightly later. In order to utilize fucosylated oligosaccharides, ATCC 15697 possesses several fucosidases, implicating GH29 and GH95 α-L-fucosidases in a gene cluster dedicated to HMO metabolism. Evaluation of the biochemical kinetics demonstrated that ATCC 15697 expresses three fucosidases with a high turnover rate. Moreover, several ATCC 15697 fucosidases are active on the linkages inherent to the HMO molecule. Finally, the HMO cluster GH29 α-L-fucosidase possesses a crystal structure that is similar to previously characterized fucosidases.
Project description:Bifidobacterium infantis is associated with the gut microbiota of breast-fed infants. B. infantis promotes intestinal barrier and immune function through several proposed mechanisms, including interactions between their surface polysaccharides, the host, and other gut microorganisms. Dairy foods and ingredients are some of the most conspicuous food-based niches for this species and may provide benefits for their delivery and efficacy in the gut. Milk phospholipid (MPL)-rich ingredients have been increasingly recognized for their versatile benefits to health, including interactions with the gut microbiota and intestinal cells. Therefore, our objective was to investigate the capacity for MPL to promote survival of B. infantis during simulated digestion and to modulate bacterial polysaccharide production. To achieve these aims, B. infantis was incubated with or without 0.5% MPL in de Man, Rogosa, and Sharpe (MRS) media at 37 °C under anaerobiosis. Survival across the oral, gastric, and intestinal phases using in vitro digestion was measured using plate count, along with adhesion to goblet-like intestinal cells. MPL increased B. infantis survival at the end of the intestinal phase by at least 7% and decreased adhesion to intestinal cells. The bacterial surface characteristics, which may contribute to these effects, were assessed by ζ-potential, changes in surface proteins using comparative proteomics, and production of bound polysaccharides. MPL decreased the surface charge of the bifidobacteria from –17 to –24 mV and increased a 50 kDa protein (3-fold) that appears to be involved in protection from stress. The production of bound polysaccharides was measured using FTIR, HPLC, and TEM imaging. These techniques all suggest an increase in bound polysaccharide production at least 1.7-fold in the presence of MPL. Our results show that MPL treatment is positively correlated with increased survival during simulated digestion, a stress resistance surface protein, and bound polysaccharide production of B. infantis, suggesting its use as a functional ingredient to enhance probiotic and postbiotic effects.
Project description:Bifidobacteria are among the most abundant microorganisms inhabiting the intestine of humans and many animals. Within the genus Bifidobacterium, several beneficial effects have been attributed to strains belonging to the subspecies Bifidobacterium longum subsp. longum and Bifidobacterium longum subsp. infantis, which are often found in infants and adults. The increasing numbers of sequenced genomes belonging to these two subspecies, and the availability of novel computational tools focused on predicting glycolytic abilities, with the aim of understanding the capabilities of degrading specific carbohydrates, allowed us to depict the potential glycoside hydrolases (GH) of these bacteria, with a focus on those GH profiles that differ in the two subspecies. We performed an in silico examination of 188 sequenced B. longum genomes and depicted the commonly present and strain-specific GHs and GH families among representatives of this species. Additionally, GH profiling, genome-based and 16S rRNA-based clustering analyses showed that the subspecies assignment of some strains does not properly match with their genetic background. Furthermore, the analysis of the potential GH component allowed the distinction of clear GH patterns. Some of the GH activities, and their link with the two subspecies under study, are further discussed. Overall, our in silico analysis poses some questions about the suitability of considering the GH activities of B. longum subsp. longum and B. longum subsp. infantis to gain insight into the characterization and classification of these two subspecies with probiotic interest.
Project description:Riboflavin or vitamin B2 is the precursor of the essential coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Despite increased interest in microbial synthesis of this water-soluble vitamin, the metabolic pathway for riboflavin biosynthesis has been characterized in just a handful of bacteria. Here, comparative genome analysis identified the genes involved in the de novo biosynthetic pathway of riboflavin in certain bifidobacterial species, including the human gut commensal Bifidobacterium longum subsp. infantis (B. infantis) ATCC 15697. Using comparative genomics and phylogenomic analysis, we investigated the evolutionary acquisition route of the riboflavin biosynthesis or rib gene cluster in Bifidobacterium and the distribution of riboflavin biosynthesis-associated genes across the genus. Using B. infantis ATCC 15697 as model organism for this pathway, we isolated spontaneous riboflavin overproducers, which had lost transcriptional regulation of the genes required for riboflavin biosynthesis. Among them, one mutant was shown to allow riboflavin release into the medium to a concentration of 60.8 ng mL-1. This mutant increased vitamin B2 concentration in a fecal fermentation system, thus providing promising data for application of this isolate as a functional food ingredient.
Project description:Prebiotics are non-digestible substrates that stimulate the growth of beneficial microbial populations in the intestine, especially Bifidobacterium species. Among them, fructo- and galacto-oligosaccharides are commonly used in the food industry, especially as a supplement for infant formulas. Mechanistic details on the enrichment of bifidobacteria by these prebiotics are important to understand the effects of these dietary interventions. In this study the consumption of galactooligosaccharides was studied for 22 isolates of Bifidobacterium longum subsp. infantis, one of the most representative species in the infant gut microbiota. In general all isolates showed a vigorous growth on these oligosaccharides, but consumption of larger galactooligosaccharides was variable. Bifidobacterium infantis ATCC 15697 has five genes encoding ?-galactosidases, and three of them were induced during bacterial growth on commercial galactooligosaccharides. Recombinant ?-galactosidases from B. infantis ATCC 15697 displayed different preferences for ?-galactosides such as 4' and 6'-galactobiose, and four ?-galactosidases in this strain released monosaccharides from galactooligosaccharides. Finally, we determined the amounts of short chain fatty acids produced by strain ATCC 15697 after growth on different prebiotics. We observed that biomass and product yields of substrate were higher for lactose and galactooligosaccharides, but the amount of acids produced per cell was larger after growth on human milk oligosaccharides. These results provide a molecular basis for galactooligosaccharide consumption in B. infantis, and also represent evidence for physiological differences in the metabolism of prebiotics that might have a differential impact on the host.