Effect of the lower ligand precursors on vitamin B12 production by food-grade Propionibacteria (2023)

Accurate determination of vitamin B₁₂ (VB₁₂) in food matrices remains a big challenge due to the instability of VB₁₂ and extreme complexity of the various matrices. Both the matrix effect and recovery are the main parameters, which are needed to be evaluated meticulously in the measurement. An ultra-high performance liquid chromatography coupled to quadrupole Q-Exactive Plus orbitrap mass spectrometry (UHPLC-QE-Orbitrap-MS) has been developed to detect the VB₁₂ in infant formulas. Besides, four calibration methodologies, including standard addition, standard addition-isotope dilution mass spectrometry, single point isotope dilution mass spectrometry (SP-IDMS), and standard calibration curve isotope dilution mass spectrometry were studied in detail to evaluate the accuracy and precision of the quantification values. The matrix effects of mass spectrometry could be compensated well via a facile SP-IDMS calibration method, which was validated by an F-test and the correction factor. UHPLC-QE-Orbitrap-MS with SP-IDMS calibration method could efficiently determine the VB₁₂ in different infant formula matrices obtained from the supermarket. The precision with reproducibility less than 2.83%, and accuracy with a spiked recovery larger than 97.4% were obtained. The detection and quantification limits of the method were 0.05 and 0.20μg/kg, respectively.

The present study investigated the in situ production of vitamin B12 in eleven cereal, pseudo-cereal and legume materials by fermentation with Propionibacterium freudenreichii DSM 20271 and Levilactobacillus brevis (formerly Lactobacillus brevis) ATCC 14869. P. freudenreichii was used as the vitamin producer and L. brevis was selected to improve the consistency and microbial safety of the process. The study showed that more than 300ng/g dw of vitamin B12 (daily requirement: 2.4μg) were produced during fermentation in most of the studied brans and legumes. The highest vitamin B12 production was observed in the fermentation of the rice bran (ca. 742ng/g dw), followed by the fermentation of buckwheat bran (ca. 631ng/g dw). Furthermore, partial least squares (PLS) regression analysis suggested that the production of vitamin B12 was greatly influenced by the nutrient composition of the fermented raw materials. Meanwhile, L. brevis was found to effectively inhibit the growth of Enterobacteriaceae during fermentation. These results demonstrated that fermentation of cereal, pseudo-cereal and legume materials with P. freudenreichii and L. brevis is effective in fortifying plant-based food with vitamin B12.

The bioaccessibility of vitamin B12 (B12) in plant-based products fortified using wheat bran extract fermented with B12-producing food-grade Propionibacterium freudenreichii was studied by applying a standard static in vitro model. At first, a culture of P. freudenreichii, fresh or heat-treated, was subjected to in vitro assays. Then, food ingredients or products were evaluated for their in vitro bioaccessibility: spray-dried wheat bran extract powder, pasta made with an extruder using fermented bran extract and breads made with spray-dried powder or with added cyanocobalamin. B12 bioaccessibility from the fresh P. freudenreichii culture was only ca. 53%, which, when heated, increased to 73%. The bioaccessibility of B12 from the food products varied from 75% (spray-dried powder) to 95% (breads). B12 from the fortified bread was as bioaccessible as from the bread made with added cyanocobalamin (99%). The in vitro results suggest that B12 synthesized by P. freudenreichii, when fortified in the studied cereal-based products, is largely bioaccessible and could be available for absorption. Plant-based products fortified using fermentation with P. freudenreichii could thus be considered excellent sources of bioaccessible B12.

Propionibacterium freudenreichii has been successfully applied to industrial anaerobic production of vitamin B₁₂. In the traditional fed-batch fermentation process, 5,6-dimethylbenzimidazole (DMB) is used as a precursor. However, DMB can inhibit the synthesis of vitamin B₁₂ metabolic intermediates, which is not conducive to semi-continuous fermentation of P. freudenreichii. To improve the fermentation efficiency, vitamin B₁₂ ex-situ biosynthesis was proposed and a membrane separation coupling fermentation technology was designed with adenosylcobinamide (Ado-cbi) as the indicator signal. The results showed that P. freudenreichii cells should be partly separated online at 84 h with 0.22-μm spiral membrane. The fourfold concentrated suspension was suitable for ex-situ biosynthesis of vitamin B₁₂ with the addition of 3.60 mg L⁻¹ DMB at 30 ℃ and pH 7.0. In the semi-continuous coupling fermentation process, vitamin B₁₂ concentration reached 56.76 ± 3.86 mg L⁻¹, an improvement of 161.6 %, while reutilizing fermentation wastewater and improving the anaerobic fermentation efficiency of P. freudenreichii.

Vitamin B12 contributes many substantial metabolic cycles in the living organisms. Since human beings cannot produce such co-factor by their metabolism, they have to receive this vitamin from foods and supplements. Dimethylbenzimidazole (DMBI) distinguishes the active form of vitamin B12 from pseudo-vitamin B12. De Novo total biosynthesis of vitamin B12 in the bacteria should include DMBI biosynthesis through riboflavin pathway. Propionibacterium freudenreichii can produce vitamin B12 through anaerobic biosynthesis pathway. As vitamin B12 production by P. freudenreichii is the growth-associated phenomena, the effect of different carbon sources (rice bran oil, argan oil), nutrients (DMBI) and amino acids (L-Serin, L-Tryptophan, l-cysteine, l-Methionine) on the growth of Propionibacterium freudenreichii PTCC1674 (pfre) were investigated. Through the statistical analysis of vitamin B12 production, rice bran oil (RBO) was selected as the sole carbon source. By applying Plackett-Burman method, significant parameters of vitamin B12 production were extracted and optimized based on Box-Behnken design of experiments. RBO, DMBI and CaCl₂.2H₂O concentrations and temperature were the four main effective parameters of vitamin B12 production. Via implemented response to surface methodology (RSM), the response was optimized to 2.94mg/L, while 14% increase of vitamin B12 (cyanocobalamin) production was obtained at RBO concentration of 8.648% V/V, temperature of 38.3 (°C), DMBI concentration of 55.758 (mg/L) and elemental solution concentration of 2 (mg/L). It was concluded that pfre can grow on rice bran oil as a new carbon source while the changing of culture media composition alters the growth profile. Box-Behnken design effectively optimized parameters achieved from Plackett-Burman screening method.

International Journal of Food Microbiology, Volume 244, 2017, pp. 103-110

Aquaculture, Volume 434, 2014, pp. 179-183

An 8-week feeding trial was conducted to investigate the optimum dietary vitamin B₁₂ requirement of juvenile Chinese mitten crab, Eriocheir sinensis, and its effect on non-specific immunity responses and resistance against Aeromonas hydrophila. Vitamin B₁₂ was supplemented to the basal diet at 0, 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6mg/kg to formulate seven semi-purified diets. A diet free of vitamin B₁₂ and folic acid was also included as a control. Each diet was fed to E. sinensis (0.051±0.001g) in four replicates, followed by a challenge study for 2weeks. Relative growth rate and specific growth rate were best in crabs fed 0.2 and 0.4mgvitaminB₁₂/kg diets, followed by the 0.1, 0.8, 0, 0.05 and 1.6mg/kg groups and lowest in the control group (vitamin B₁₂ and folic acid free). Feed efficiency and survival rate peaked at the supplementation of 0.2mg/kg. The whole body crude protein of juvenile crabs was significantly higher when crabs fed 0.2–0.8mgvitaminB₁₂/kg than those fed the diets without vitamin B₁₂ supplementation. Total hemocyte count was significantly higher in crabs fed 0.2 and 0.4mgvitaminB₁₂/kg than those in the other feeding groups, and crabs fed the 0.2mg/kg diet had the highest phenoloxidase, lysozyme, acid phosphatase and alkaline phosphatase activities but the lowest cumulative mortality. Hepatopancreatic vitamin B₁₂ concentration increased as the level of vitamin B₁₂ supplementation increased. The optimum dietary vitamin B₁₂ requirement by E. sinensis is recommended at 0.20–0.22mg/kg diet.

International Journal of Food Microbiology, Volume 176, 2014, pp. 38-48

Food Chemistry, Volume 216, 2017, pp. 301-308

Based on increased demands of strict vegetarians, an investigation of vitamin B₁₂ content in plant sources, was carried out. The vitamin B₁₂ concentration was determined by RP-HPLC with UV detection, after prior matrix isolation by immunoaffinity chromatography (IAC). Vitamin B₁₂ was extracted in the presence of sodium cyanide, to transform all forms of cobalamin into cyanocobalamin. Diode array detector was used to monitor vitamin B₁₂, after its chromatographic separation under gradient elution with a mobile phase consisting of acetonitrile and trifluoroacetic acid 0.025% (w/v). The method demonstrated excellent linearity with a limit of detection 0.004μg/ml. The method precision was evaluated for plant samples and it was below 0.7% (n=6). Significant amounts of vitamin B₁₂ in plants were detected in Hippophae rhamnoides (37μg/100g dry weight), in Elymus (26μg/100g dry weight) and in Inula helenium (11μg/100g dry weight).

New Biotechnology, Volume 31, Issue 6, 2014, pp. 553-561

Bacillus megaterium is a bacterium that has been used in the past for the industrial production of vitamin B₁₂ (cobalamin), the anti-pernicious anaemia factor. Cobalamin is a modified tetrapyrrole with a cobalt ion coordinated within its macrocycle. More recently, B. megaterium has been developed as a host for the high-yield production of recombinant proteins using a xylose inducible promoter system. Herein, we revisit cobalamin production in B. megaterium DSM319. We have investigated the importance of cobalt for optimum growth and cobalamin production. The cobaltochelatase (CbiX^L) is encoded within a 14-gene cobalamin biosynthetic (cbi) operon, whose gene-products oversee the transformation of uroporphyrinogen III into adenosylcobyrinic acid a,c-diamide, a key precursor of cobalamin synthesis. The production of CbiX^L in response to exogenous cobalt was monitored. The metal was found to stimulate cobalamin biosynthesis and decrease the levels of CbiX^L. From this we were able to show that the entire cbi operon is transcriptionally regulated by a B₁₂-riboswitch, with a switch-off point at approximately 5nM cobalamin. To bypass the effects of the B₁₂-riboswitch the cbi operon was cloned without these regulatory elements. Growth of these strains on minimal media supplemented with glycerol as a carbon source resulted in significant increases in cobalamin production (up to 200μgL⁻¹). In addition, a range of partially amidated intermediates up to adenosylcobyric acid was detected. These findings outline a potential way to develop B. megaterium as a cell factory for cobalamin production using cheap raw materials.

Bioresource Technology, Volume 319, 2021, Article 124218

The necessity of costly co-enzyme B₁₂ for the activity of glycerol dehydratase (GDHt) is considered as a major bottleneck in sustainable bioproduction of 1,3-propanediol (1,3-PD) from glycerol. Here, an E. coil Rosetta-dhaB1-dhaB2 strain was constructed by overexpressing a B₁₂-independent GDHt (dhaB1) and its activating factor (dhaB2) from Clostridium butyricum. Subsequently, it was used in designing a co-culture with E. coli BL21-dhaT that overexpressed 1,3-PD oxidoreductase (dhaT), to produce 1,3-PD during co-fermentation of glycerol and glucose. The optimum initial ratio of BL21-dhaT to Rosetta-dhaB1-dhaB2 strains in the co-culture was 1.5. Compared to the fermentation of glycerol alone, co-fermentation approach provided 1.3-folds higher 1,3-PD. Finally, co-fermentation was done in a 10 L bioreactor that produced 41.65g/L 1,3-PD, which corresponded to 0.69g/L/h productivity and 0.67mol/mol yield of 1,3-PD. Hence, the developed co-culture could produce 1,3-PD cost-effectively without requiring vitamin B₁₂.