Bhawani Chamlagain

Microbial metabolic networks at the mucus layer lead to diet-independent butyrate and vitamin B 12 production by intestinal symbionts

ABSTRACT Akkermansia muciniphila has evolved to specialize in the degradation and utilization of host mucus, which it may use as the sole source of carbon and nitrogen. Mucus degradation and fermentation by A. muciniphila are known to result in the liberation of oligosaccharides and subsequent production of acetate, which becomes directly available to microorganisms in the vicinity of the intestinal mucosa. Coculturing experiments of A. muciniphila with non-mucus-degrading butyrate-producing bacteria Anaerostipes caccae, Eubacterium hallii, and Faecalibacterium prausnitzii resulted in syntrophic growth and production of butyrate. In addition, we demonstrate that the production of pseudovitamin B12 by E. hallii results in production of propionate by A. muciniphila, which suggests that this syntrophy is indeed bidirectional. These data are proof of concept for syntrophic and other symbiotic microbe-microbe interactions at the intestinal mucosal interface. The observed metabolic interactions between A. muciniphila and butyrogenic bacterial taxa support the existence of colonic vitamin and butyrate production pathways that are dependent on host glycan production and independent of dietary carbohydrates. We infer that the intestinal symbiont A. muciniphila can indirectly stimulate intestinal butyrate levels in the vicinity of the intestinal epithelial cells with potential health benefits to the host. IMPORTANCE The intestinal microbiota is said to be a stable ecosystem where many networks between microorganisms are formed. Here we present a proof of principle study of microbial interaction at the intestinal mucus layer. We show that indigestible oligosaccharide chains within mucus become available for a broad range of intestinal microbes after degradation and liberation of sugars by the species Akkermansia muciniphila. This leads to the microbial synthesis of vitamin B12, 1,2-propanediol, propionate, and butyrate, which are beneficial to the microbial ecosystem and host epithelial cells. IMPORTANCE The intestinal microbiota is said to be a stable ecosystem where many networks between microorganisms are formed. Here we present a proof of principle study of microbial interaction at the intestinal mucus layer. We show that indigestible oligosaccharide chains within mucus become available for a broad range of intestinal microbes after degradation and liberation of sugars by the species Akkermansia muciniphila. This leads to the microbial synthesis of vitamin B12, 1,2-propanediol, propionate, and butyrate, which are beneficial to the microbial ecosystem and host epithelial cells.

BluB/CobT2 fusion enzyme activity reveals mechanisms responsible for production of active form of vitamin B12 by Propionibacterium freudenreichii

BackgroundPropionibacterium freudenreichii is a food grade bacterium that has gained attention as a producer of appreciable amounts of cobalamin, a cobamide with activity of vitamin B12. Production of active form of vitamin is a prerequisite for attempts to naturally fortify foods with B12 by microbial fermentation. Active vitamin B12 is distinguished from the pseudovitamin by the presence of 5,6-dimethylbenzimidazole (DMBI) as the lower ligand. Genomic data indicate that P. freudenreichii possesses a fusion gene, bluB/cobT2, coding for a predicted phosphoribosyltransferase/nitroreductase, which is presumably involved in production of vitamin B12. Understanding the mechanisms affecting the synthesis of different vitamin forms is useful for rational strain selection and essential for engineering of strains with improved B12 production properties.ResultsHere, we investigated the activity of heterologously expressed and purified fusion enzyme BluB/CobT2. Our results show that BluB/CoBT2 is responsible for the biosynthesis of the DMBI base and its activation into α-ribazole phosphate, preparing it for attachment as the lower ligand of cobalamin. The fusion enzyme was found to be efficient in metabolite channeling and the enzymes’ inability to react with adenine, a lower ligand present in the pseudovitamin, revealed a mechanism favoring the production of the active form of the vitamin. P. freudenreichii did not produce cobalamin under strictly anaerobic conditions, confirming the requirement of oxygen for DMBI synthesis. In vivo experiments also revealed a clear preference for incorporating DMBI over adenine into cobamide under both microaerobic and anaerobic conditions.ConclusionsThe herein described BluB/CobT2 is responsible for the production and activation of DMBI. Fusing those two activities results in high pressure towards production of the true vitamin B12 by efficiently activating DMBI formed within the same enzymatic complex. This indicates that BluB/CobT2 is the crucial enzyme in the B12 biosynthetic pathway of P. freudenreichii. The GRAS organism status and the preference for synthesizing active vitamin form make P. freudenreichii a unique candidate for the in situ production of vitamin B12 within food products.

Ultra-high performance liquid chromatographic and mass spectrometric analysis of active vitamin B12 in cells of Propionibacterium and fermented cereal matrices

A sensitive and selective method is needed to analyse in situ produced vitamin B12 in plant-based materials, potential new dietary sources of vitamin B12. A UHPLC/UV method was developed and validated for the determination of human active vitamin B12 in cell extracts of Propionibacterium freudenreichii subsp. shermanii and after immunoaffinity purification in extracts of cereal matrices fermented by P. freudenreichii. An Acquity HSS T3 C18 column resulted in a baseline separation, a calibration curve of excellent linearity and a low limit of detection (0.075 ng/5 μL injection). As confirmed by UHPLC-MS, the active vitamin B12 could be separated from pseudovitamin B12. The recovery of vitamin B12 from purified spiked cereal matrices was good (>90%; RSD<5%). A nutritionally relevant amount of active vitamin B12 was produced by P. freudenreichii in cereal malt matrices (up to 1.9 μg/100 g) in 24h at 28 °C.

Comparative genomics and physiology of the butyrate-producing bacterium Intestinimonas butyriciproducens

Intestinimonas is a newly described bacterial genus with representative strains present in the intestinal tract of human and other animals. Despite unique metabolic features including the production of butyrate from both sugars and amino acids, there is to date no data on their diversity, ecology, and physiology. Using a comprehensive phylogenetic approach, Intestinimomas was found to include at least three species that colonize primarily the human and mouse intestine. We focused on the most common and cultivable species of the genus, Intestinimonas butyriciproducens, and performed detailed genomic and physiological comparison of strains SRB521T and AF211, isolated from the mouse and human gut respectively. The complete 3.3-Mb genomic sequences of both strains were highly similar with 98.8% average nucleotide identity, testifying to their assignment to one single species. However, thorough analysis revealed significant genomic rearrangements, variations in phage-derived sequences, and the presence of new CRISPR sequences in both strains. Moreover, strain AF211 appeared to be more efficient than strain SRB521T in the conversion of the sugars arabinose and galactose. In conclusion, this study provides genomic and physiological insight into Intestinimonas butyriciproducens, a prevalent butyrate-producing species, differentiating strains that originate from the mouse and human gut.

Trends of Antibiotic Resistance in Mesophilic and Psychrotrophic Bacterial Populations during Cold Storage of Raw Milk.

Psychrotrophic bacteria in raw milk are most well known for their spoilage potential and cause significant economic losses in the dairy industry. Despite their ability to produce several exoenzyme types at low temperatures, psychrotrophs that dominate the microflora at the time of spoilage are generally considered benign bacteria. It was recently reported that raw milk-spoiling Gram-negative-psychrotrophs frequently carried antibiotic resistance (AR) features. The present study evaluated AR to four antibiotics (ABs) (gentamicin, ceftazidime, levofloxacin, and trimethoprim-sulfamethoxazole) in mesophilic and psychrotrophic bacterial populations recovered from 18 raw milk samples, after four days storage at 4°C or 6°C. Robust analysis of variance and non parametric statistics (e.g., REGW and NPS) revealed that AR prevalence among psychrotrophs, for milk samples stored at 4°C, often equalled the initial levels and equalled or increased during the cold storage at 6°C, depending on the AB. The study performed at 4°C with an intermediate sampling point at day 2 suggested that (1) different psychrotrophic communities with varying AR levels dominate over time and (2) that AR (determined from relative amounts) was most prevalent, transiently, after 2-day storage in psychrotrophic or mesophilic populations, most importantly at a stage where total counts were below or around 105 CFU/mL, at levels at which the milk is acceptable for industrial dairy industrial processes.

Food-Like Growth Conditions Support Production of Active Vitamin B12 by Propionibacterium freudenreichii 2067 without DMBI, the Lower Ligand Base, or Cobalt Supplementation

Propionibacterium freudenreichii is a traditional dairy bacterium and a producer of short chain fatty acids (propionic and acetic acids) as well as vitamin B12. In food applications, it is a promising organism for in situ fortification with B12 vitamin since it is generally recognized as safe (GRAS) and it is able to synthesize biologically active form of the vitamin. In the present study, vitamin B12 and pseudovitamin biosynthesis by P. freudenreichii was monitored by UHPLC as a function of growth in food-like conditions using a medium mimicking cheese environment, without cobalt or 5,6-dimethylbenzimidazole (DMBI) supplementation. Parallel growth experiments were performed in industrial-type medium known to support the biosynthesis of vitamin B12. The production of other key metabolites in the two media were determined by HPLC, while the global protein production was compared by gel-based proteomics to assess the effect of growth conditions on the physiological status of the strain and on the synthesis of different forms of vitamin. The results revealed distinct protein and metabolite production, which reflected the growth conditions and the potential of P. freudenreichii for synthesizing nutritionally relevant amounts of active vitamin B12 regardless of the metabolic state of the cells.

Stability of added and in situ-produced vitamin B12 in breadmaking.

Vitamin B12 exists naturally in foods of animal origin and is synthesised only by certain bacteria. New food sources are needed to ensure vitamin B12 intake in risk groups. This study aimed to investigate the stability of added cyanocobalamin (CNCbl, chemically modified form) and hydroxocobalamin (OHCbl, natural form) and in situ-synthesised vitamin B12 in breadmaking. Samples were analysed both with a microbiological (MBA) and a liquid chromatographic (UHPLC) method to test applicability of these two methods. Proofing did not affect CNCbl and OHCbl levels. By contrast, 21% and 31% of OHCbl was lost in oven-baking steps in straight- and sponge-dough processes, respectively, whereas CNCbl remained almost stable. In sourdough baking, 23% of CNCbl and 44% of OHCbl were lost. In situ-produced vitamin B12 was almost as stable as added CNCbl and more stable than OHCbl. The UHPLC method showed its superiority to the MBA in determining the active vitamin B12.

In situ production of active vitamin B12 in cereal matrices using Propionibacterium freudenreichii

Abstract The in situ production of active vitamin B12 was investigated in aqueous cereal‐based matrices with three strains of food‐grade Propionibacterium freudenreichii. Matrices prepared from malted barley flour (33% w/v; BM), barley flour (6%; BF), and wheat aleurone (15%; AM) were fermented. The effect of cobalt and the lower ligand 5,6‐dimethylbenzimidazole (DMBI) or its natural precursors (riboflavin and nicotinamide) on active B12 production was evaluated. Active B12 production was confirmed by UHPLC–UV–MS analysis. A B12 content of 12–37 μg·kg−1 was produced in BM; this content increased 10‐fold with cobalt and reached 940–1,480 μg·kg−1 with both cobalt and DMBI. With riboflavin and nicotinamide, B12 production in cobalt‐supplemented BM increased to 712 μg·kg−1. Approximately, 10 μg·kg−1 was achieved in BF and AM and was increased to 80 μg·kg−1 in BF and 260 μg·kg−1 in AM with cobalt and DMBI. The UHPLC and microbiological assay (MBA) results agreed when both cobalt and DMBI or riboflavin and nicotinamide were supplemented. However, MBA gave ca. 20%–40% higher results in BM and AM supplemented with cobalt, indicating the presence of human inactive analogues, such as pseudovitamin B12. This study demonstrates that cereal products can be naturally fortified with active B12 to a nutritionally relevant level by fermenting with P. freudenreichii.

Biofortification of riboflavin and folate in idli batter, based on fermented cereal and pulse, by Lactococcus lactis N8 and Saccharomyces boulardii SAA655

Lactococcus lactis N8 and Saccharomyces boulardii SAA655 were investigated for their ability to synthesize B‐vitamins (riboflavin and folate) and their functional role as microbial starters in idli fermentation.