Vitamin B12 Production Research Articles

Enhancing vitamin B12 content in co-fermented soy-milk via a Lotka Volterra model

Abstract Soybean products are popular because of its taste, digestibility, and health benefits. However, soybean lacks vitamin, mainly the low water-soluble vitamin B12. This study investigated the effects of fermentation conditions on the synthesis of vitamin B12, production of metabolites, and growth of Lactobacillus reuteri and Propionibacterium shermainii in fermented soy-milk. A Lotka Volterra model was successfully employed to describe the competition relationship between the two microorganisms under various fermentation conditions. A quadratic function between the ratio of interaction coefficients and vitamin B12 content was found. Higher vitamin B12 in soy-milk can be produced when the ratio of interaction coefficients approach to one. Compared with other fermented soybean products, fermented soy-milk contains more acetate, ethanol, and propionic acid. This study successfully demonstrated a mathematical model to enhance soy-milk vitamin B12 production.

Characterization and high-throughput sequencing of a trichlorophenol-dechlorinating microbial community acclimated from sewage sludge

Abstract This study reports the dechlorination characteristics of acclimated biomass, high-throughput sequencing of the 16Sribosomal RNA (rRNA) gene harbored by the microorganisms, and analysis of the relation between the dechlorination function and community structure. Anaerobic 2,4,6-trichlorophenol (TCP)-dechlorinating biomass was acclimated from active sludge, and it showed a high TCP-dechlorination rate. It dechlorinated TCP only at the ortho-position, with 4-chlorophenol as the terminal product. Illumina Hiseq sequencing analyses of a 16SrRNA gene amplicon indicated that the abundance of the phyla Bacteroidetes and Firmicutes doubled in the acclimation process. Compared with that in raw sludge, Betaproteobacteria disappeared from acclimated biomass, and Deltaproteobacteria was replaced by sulfate-reducing bacteria (SRB) including Desulfobulbus, Desulfovibrio, Desulfomicrobium, and Syntrophus. Syntrophomonas of the phylum Firmicutes predominated (abundance 6.7%), and it was responsible for the high efficacy of dechlorination. Dechlorinators included Dechloromonas, Clostridium, SRB and Geobacter. The emergence of SRB provided microbiological evidence of the sulfate-reducing ability of the dechlorinating biomass. Propionicimonas was discovered during this study, and it is known to produce vitamin B12, which is an essential factor for reductive dehalogenase enzymes. The core microbiome resulted in the high dechlorination rate.

In situ fortification of vitamin B12 in wheat flour and wheat bran by fermentation with Propionibacterium freudenreichii

Vitamin B12 is a micronutrient naturally existing in animal products. A growing interest and need to replace animal protein with plant protein sources have resulted in increased attention to developing vitamin B12-fortified plant-based food. Natural fortification by Propionibacterium freudenreichii is a promising alternative to chemical fortification, as P. freudenreichii can synthesize active vitamin B12. In this work, we studied vitamin B12 production in non-sterile matrices prepared from three raw materials of wheat: durum flour, wholewheat flour and wheat bran. Viable cell counts, pH, total titratable acidity and concentration of acids were determined. After seven days of fermentation, vitamin B12 levels reached 33 ± 4, 87 ± 10 and 155 ± 17 ng/g dry weight in durum flour, wholewheat flour, and wheat bran, respectively. While durum flour supported the growth of P. freudenreichii to higher cell densities and more efficient propionic acid production compared with the other two matrices, wholewheat flour and wheat bran were found to be the most promising of the three matrices for in situ production of vitamin B12.

Transient plasma cobalamin elevation in patients with pneumonia – two case reports

We report two cases of transient significantly elevated plasma cobalamin (B12) in geriatric patients acutely admitted with fever, increased C-reactive protein and X-ray verified pneumonia. Extensive diagnostic workup did not reveal kidney or liver disease, neither any signs of cancer. Furthermore, none of the patients had received therapeutic B12 supplementation prior to admission. In both cases, plasma B12 normalized at an out-patient control few months later. We were not able to identify the reason for the initial B12 elevation in any of the patients, since none of the usually recognized causes were evident. Since both patients had an infection and temporarily elevated B12, we suggest a possible inflammatory response or a vitamin B12 production by the infectious agents as the cause. Both suggestions, however, need further exploration.

Production of vitamin B12 and folate using a potent mutant strain of Klebsiella pneumonia

Abstract The production of vitamin B12 and folate under solid state fermentation was investigated by using different mutants of klebsiella penumonia. The results showed that the isolate no ( EM33TE) produce the maximum B12 and folate yields ( 77.45 and 88.22 ug/ml) respectively. The supplementation of the fermentation medium by different carbon sources (glucose, fructose, mannose, sucrose, maltose, lactose ,starch and dextrin) indicated that the best carbon source was glucose and the maximum B12 yield (356.9ug/ml) was obtained by the sequential addition of glucose(10g ) to the fermentation medium . PCR studies revealed that clear differences at bands number and size between the original strain and its mutants using RAPD analysis by three different random primers, these differences in RAPD profiles confirmed the evidence of genetic variations of mutants and K.pneumonia genome after EMS-mutagenesis

Growth engineering of Propionibacterium freudenreichii shermanii for organic acids and other value-added products formation

ABSTRACTPropionic acid production from glucose was studied using Propionibacterium freudenreichii shermanii. Conditions were optimized for high yields of propionic acid and total organic acids by sequential optimization of parameters like pH, inoculum age, inoculum volume and substrate concentration. Near-theoretical yield (0.54 ± 0.023 g/g) was achieved for propionic acid with fermentation of 1% glucose using 20% (v/v) of 48 hr old P. shermanii at 30°C, pH maintained at 5.5. Total organic acid yield under these conditions was 0.74 ± 0.06 g/g. The study resulted in achieving 98% and 95% theoretical yields of propionic acid and total organic acids, respectively. Under optimized conditions, along with organic acids, P. shermanii also produced vitamin B12 and trehalose intracellularly, showing its potential to be used as a cell factory.

Quantification of Vitamin B12-Related Proteins in Marine Microbial Systems Using Selected Reaction Monitoring Mass Spectrometry.

Mass spectrometry-based proteomic approaches to studying microbial systems enable assessment of taxonomically resolved functional capacity. A subset of these proteomic approaches are absolutely quantitative, enabling comparisons of protein expression patterns between different studies and across environments. This chapter outlines a method for applying quantitative assays in marine microbial communities, using proteins involved in vitamin B12 (cobalamin) utilization and production as specific examples. This approach involves identifying important protein targets, determining taxonomic resolution of the required assays, identifying suitable peptides, developing and optimizing liquid chromatography-selected reaction monitoring mass spectrometry assays (LC-SRM-MS), and processing the resulting data. Implementing the method outlined here results in measurements (fmol diagnostic peptide per μg of total bulk protein) that, in this case, define the nutritional status of microbial community members with respect to vitamin B12, and are comparable across and between marine microbial systems.

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

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.

Effects of brewer's yeast hydrolysate on the growth performance and the intestinal bacterial diversity of largemouth bass (Micropterus salmoides)

To investigate the effects of brewer's yeast hydrolysate (BYH) on the growth and intestinal bacteria diversity of largemouth bass (Micropterus salmoides)sthe fish were fed with basal diet supplemented with or without BYH (0, 0.1%, 0.2%) for 8weeks. The results showed that the weight gain rate, survival rate, hepatosomatic index, organ coefficient and condition factor were not significantly different among the groups, except intraperitoneal fat ratio was significantly reduced in 0.2% BYH group. Totally 25 phyla, 56 classes, 103 orders, 155 families, 216 genera and 1051 operational taxonomic units (OTUs) of bacteria were identified from the 2,057,871 sequences obtained by Illumina MiSeq 16S rRNA sequencing. Among them, Fusobacteria, Firmicutes and Proteobacteria were the most three dominant phyla in the intestines regardless of diets. Furthermore, the richness of Fusobacteria, Cyanobacteria, Tenericutes and Actinobacteria in 0.2% BYH group was significantly lower than those of the control group. Interestingly, the ratio of Bacteroides/Firmicutes was decreased in the groups fed with BYH, this might be related to the modulations of energy absorption and storage, resulting in the decrease of fat deposition in fish body. At the genus level, several bacterial genera (Plesiomonas, Mycoplasmas, Synechococcus and Peptostreptococcus) which were potential pathogens of fish were significantly decreased in the groups fed with BYH. By contrast, a significant increase of Cetobacterium which could ferment peptides and carbohydrates to produce vitamin B12 was observed in fish fed with BYH, indicating that dietary BYH was helpful in propagation of some probiotic bacteria. Taken together, the overall intestinal bacterial diversity was decreased in fish fed with BYH which might promote the growth of some probiotics and inhibit some pathogenic bacteria, suggesting that BYH may serve as feed additive in modulation of the gut bacterial community of largemouth bass to decrease the potential pathogens.

Enhanced vitamin B12 production in an innovative lupin tempeh is due to synergic effects of Rhizopus and Propionibacterium in cofermentation

Fermentation represents a valuable and cost-effective approach for food stabilisation and nutritional improvement. Tempeh is an example of soybean solid-state fermentation. In this work, we investigated the possibility of producing a tempeh analogue containing high amounts of vitamin B12 using seeds of three different species of the legume lupin, namely Lupinus albus, L. angustifolius and L. mutabilis, with Rhizopus oligosporus and Propionibacterium freudenreichii cofermentation. Synergic effects of Rhizopus and Propionibacterium in increasing vitamin B12 up to 1230 ng/g dw was observed. These findings indicate that this cofermentation can improve lupin nutritional quality and safety to provide a tempeh analogue with added value for vegan and vegetarian communities and low-income populations. The level of potentially toxic lupin alkaloids was also monitored during the tempeh preparation.

De novo assembly of genomes from long sequence reads reveals uncharted territories of Propionibacterium freudenreichii

BackgroundPropionibacterium freudenreichii is an industrially important bacterium granted the Generally Recognized as Safe (the GRAS) status, due to its long safe use in food bioprocesses. Despite the recognized role in the food industry and in the production of vitamin B12, as well as its documented health-promoting potential, P. freudenreichii remained poorly characterised at the genomic level. At present, only three complete genome sequences are available for the species.ResultsWe used the PacBio RS II sequencing platform to generate complete genomes of 20 P. freudenreichii strains and compared them in detail. Comparative analyses revealed both sequence conservation and genome organisational diversity among the strains. Assembly from long reads resulted in the discovery of additional circular elements: two putative conjugative plasmids and three active, lysogenic bacteriophages. It also permitted characterisation of the CRISPR-Cas systems. The use of the PacBio sequencing platform allowed identification of DNA modifications, which in turn allowed characterisation of the restriction-modification systems together with their recognition motifs. The observed genomic differences suggested strain variation in surface piliation and specific mucus binding, which were validated by experimental studies. The phenotypic characterisation displayed large diversity between the strains in ability to utilise a range of carbohydrates, to grow at unfavourable conditions and to form a biofilm.ConclusionThe complete genome sequencing allowed detailed characterisation of the industrially important species, P. freudenreichii by facilitating the discovery of previously unknown features. The results presented here lay a solid foundation for future genetic and functional genomic investigations of this actinobacterial species.

Applicability of ribosome engineering to vitamin B12 production by Propionibacterium shermanii.

Ribosome engineering has been widely utilized for strain improvement, especially for the activation of bacterial secondary metabolism. This study assessed ribosome engineering technology to modulate primary metabolism, taking vitamin B12 production as a representative example. The introduction into Propionibacterium shermanii of mutations conferring resistance to rifampicin, gentamicin, and erythromycin, respectively, increased per cell production (μg/L/OD600) of vitamin B12 5.2-fold, although net production (μg/L) was unchanged, as the cell mass of the mutants was reduced. Real-time qPCR analysis demonstrated that the genes involved in vitamin B12 fermentation by P. shermanii were activated at the transcriptional level in the drug-resistant mutants, providing a mechanism for the higher yields of vitamin B12 by the mutants. These results demonstrate the efficacy of ribosome engineering for the production of not only secondary metabolites but of industrially important primary metabolites.

Highly Effective Dual Transition Metal Macrocycle Based Electrocatalyst with Macro-/Mesoporous Structures for Oxygen Reduction Reaction

Metal macrocycle based non-noble metal electrocatalysts (NNMEs) with highly efficient oxygen reduction reaction (ORR) activity, good stability, and excellent resistance to the methanol cross-over effect have been regarded as one of the most important alternatives for Pt or Pt based alloys, which are widely used in fuel cells. However, the expensive price of most metal macrocycles hinder further investigation of such a family of NNMEs in large production for practical applications. Here, we introduce a simple strategy to synthesize metal macrocycle based porous carbon (MMPC) material with low cost and easy production of metal macrocycles (hemin (Hm) and vitamin B12 (VB12)) as raw materials by using a hard template of MgO. The pyrolysis of MMPC under the optimal temperature at 900 °C shows comparative ORR performance relative to commercial Pt/C, which could be attributed to the large surface area, macro-/mesoporous structure, the carbon layer encapsulating transition metal based oxides, as well as N-doped carbon species. In addition, MMPC (900) displays a better electrochemical property than 20 wt % Pt/C in terms of durability and tolerance to methanol in O2-saturated 0.1 M KOH media.

Coenzyme B12 synthesis as a baseline to study metabolite contribution of animal microbiota.

SummaryMicrobial communities thrive in a number of environments. Exploration of their microbiomes – their global genome – may reveal metabolic features that contribute to the development and welfare of their hosts, or chemical cleansing of environments. Yet we often lack final demonstration of their causal role in features of interest. The reason is that we do not have proper baselines that we could use to monitor how microbiota cope with key metabolites in the hosting environment. Here, focusing on animal gut microbiota, we describe the fate of cobalamins – metabolites of the B12 coenzyme family – that are essential for animals but synthesized only by prokaryotes. Microbiota produce the vitamin used in a variety of animals (and in algae). Coprophagy plays a role in its management. For coprophobic man, preliminary observations suggest that the gut microbial production of vitamin B12 plays only a limited role. By contrast, the vitamin is key for structuring microbiota. This implies that it is freely available in the environment. This can only result from lysis of the microbes that make it. A consequence for biotechnology applications is that, if valuable for their host, B12‐producing microbes should be sensitive to bacteriophages and colicins, or make spores.

An overview of biotechnological production of propionic acid: From upstream to downstream processes

Abstract The increasing demand for propionic acid (PA) production and its wide applications in several industries, especially the food industry (as a preservative and satiety inducer), have led to studies on the low-cost biosynthesis of this acid. This paper gives an overview of the biotechnological aspects of PA production and introduces Propionibacterium as the most popular organism for PA production. Moreover, all process variables influencing the production yield, different simple and complex carbon sources, the metabolic pathway of production, engineered mutants with increased productivity, and modified tolerance against high concentrations of acid have been described. Furthermore, possible methods of extraction and analysis of this organic acid, several applied bioreactors, and different culture systems and substrates are introduced. It can be concluded that maximum biomass and PA production may be achieved using metabolically engineered microorganisms and analyzing the most significant factors influencing yield. To date, the maximum reported yield for PA production is 0.973 g·g-1, obtained from Propionibacterium acidipropionici in a three-electrode amperometric culture system in medium containing 0.4 mM cobalt sepulchrate. In addition, the best promising substrate for PA bioproduction may be achieved using glycerol as a carbon source in an extractive continuous fermentation. Simultaneous production of PA and vitamin B12 is suggested, and finally, the limitations of and strategies for competitive microbial production with respect to chemical process from an economical point of view are proposed and presented. Finally, some future trends for bioproduction of PA are suggested.

Take Your Pick: Vitamins and Microbiota Facilitate Pathogen Clearance

The gut microbiota plays a chief role in vitamin production. Changes in its membership impact the gut metabolome and host susceptibility to pathogens. In this issue of Cell Host & Microbe, Miki etal. (2017) show that RegIIIβ impacts microbiota composition, decreasing vitamin B6 production and promoting sustained colonization by Salmonella.

Purification and characterization of S-adenosyl-L-methionine:uroporphyrinogen Ⅲ methyltransferase from Rhodobacter capsulatus SB1003

Biosynthesis of vitamin B₁₂ (VB₁₂) requires the methylation at positions C-2 and C-7 of the precursor uroporphyrinogen Ⅲ (urogen Ⅲ) to precorrin-2 by S-adenosyl-L-methionine uroporphyrinogen Ⅲ methyltransferase (SUMT), which is a potential bottleneck step. Most of SUMTs are inhibited by urogen Ⅲ and by-product S-adenosyl-L-homocysteine (SAH). In order to mine an SUMT that lacks such an inhibitory property to drive greater flux through the VB₁₂ biosynthetic pathway, we cloned two SUMT genes (RCcobA1, RCcobA2) from Rhodobacter capsulatus SB1003 and expressed them in Escherichia coli BL21 (DE3). Thereafter, the two enzymes were purified and their specific activity of 27.3 U/mg, 68.9 U/mg were determined respectively. The latter was 2.4 times higher than PDcobA (27.9 U/mg) from Pseudomonas denitrifican. Additionally, RCcobA2 could tolerate over 70 μmol/L urogen Ⅲ, which has never been reported before. Hence, RCcobA2 can be used as an efficient enzyme to regulate the VB₁₂ metabolic pathway and enhance VB₁₂ production in industrial strains.

Adaptation of Propionibacterium freudenreichii to long-term survival under gradual nutritional shortage.

Background Propionibacterium freudenreichii is an Actinobacterium widely used in the dairy industry as a ripening culture for Swiss-type cheeses, for vitamin B12 production and some strains display probiotic properties. It is reportedly a hardy bacterium, able to survive the cheese-making process and digestive stresses.ResultsDuring this study, P. freudenreichii CIRM-BIA 138 (alias ITG P9), which has a generation time of five hours in Yeast Extract Lactate medium at 30 °C under microaerophilic conditions, was incubated for 11 days (9 days after entry into stationary phase) in a culture medium, without any adjunct during the incubation. The carbon and free amino acids sources available in the medium, and the organic acids produced by the strain, were monitored throughout growth and survival. Although lactate (the preferred carbon source for P. freudenreichii) was exhausted three days after inoculation, the strain sustained a high population level of 9.3 log10 CFU/mL. Its physiological adaptation was investigated by RNA-seq analysis and revealed a complete disruption of metabolism at the entry into stationary phase as compared to exponential phase.Conclusions P. freudenreichii adapts its metabolism during entry into stationary phase by down-regulating oxidative phosphorylation, glycolysis, and the Wood-Werkman cycle by exploiting new nitrogen (glutamate, glycine, alanine) sources, by down-regulating the transcription, translation and secretion of protein. Utilization of polyphosphates was suggested.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3367-x) contains supplementary material, which is available to authorized users.

Some improved estimation strategies in high-dimensional semiparametric regression models with application to riboflavin production data

Due to advances in technologies, modern statistical studies often encounter linear models with high-dimension, where the number of explanatory variables is larger than the sample size. Estimation in these high-dimensional problems with deterministic covariates or designs is very different from those in the case of random covariates, due to the identifiability of the high-dimensional semiparametric regression parameters. In this paper, we consider ridge estimators and propose preliminary test, shrinkage and its positive rule ridge estimators in the restricted semiparametric regression model when the errors are dependent under a multicollinear setting, in high-dimension. The asymptotic risk expressions in addition to biases are exactly derived for the estimators under study. For our proposal, a real data analysis about production of vitamin B2 and a Monte–Carlo simulation study are considered to illustrate the efficiency of the proposed estimators. In this regard, kernel smoothing and cross-validation methods for estimating the optimum ridge parameter and nonparametric function are used.

Cobalamin production by Lactobacillus coryniformis: biochemical identification of the synthetized corrinoid and genomic analysis of the biosynthetic cluster.

BackgroundDespite the fact that most vitamins are present in a variety of foods, malnutrition, unbalanced diets or insufficient intake of foods are still the cause of vitamin deficiencies in humans in some countries. Vitamin B12 (Cobalamin) is a complex compound that is only naturally produced by bacteria and archea. It has been reported that certain strains belonging to lactic acid bacteria group are capable of synthesized water-soluble vitamins such as those included in the B-group, as vitamin B12. In this context, the goal of the present paper was to evaluate and characterize the production of vitamin B12 in Lactobacillus coryniformis CRL 1001, a heterofermentative strain isolated from silage.ResultsCell extract of L. coryniformis CRL 1001, isolated from silage, is able to correct the coenzyme B12 requirement of Salmonella enterica serovar Typhimurium AR 2680 in minimal medium. The chemical characterization of the corrinoid-like molecule isolated from CRL 1001 cell extract using HPLC and mass spectrometry is reported. The majority of the corrinoid produced by this strain has adenine like Coα-ligand instead 5,6-dimethylbenzimidazole. Genomic studies revealed the presence of the complete machinery of the anaerobic biosynthesis pathway of coenzyme B12. The detected genes encode all proteins for the corrin ring biosynthesis and for the binding of upper (β) and lower (α) ligands in one continuous stretch of the chromosome.ConclusionsThe results here described show for the first time that L. coryniformis subsp. coryniformis CRL 1001 is able to produce pseudocobalamin containing adenine instead of 5,6-dimethlbenzimidazole in the Coα-ligand. Genomic analysis allowed the identification and characterization of the complete de novo biosynthetic pathway of the corrinoid produced by the CRL 1001 strain.