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We investigated extracellular vitamin B12-producing Lactobacillus strains and their characteristics in tolerance to environmental stresses, gastric acid and bile salts. Two isolates, Lactobacillus plantarum LZ95 and CY2, showed high extracellular B12 production, 98±15μg/L and 60±9μg/L respectively. Extracellular B12 from LZ95 were identified as adenosylcobalamin and methylcobalamin using a combination of solid phase extraction and reverse-phase HPLC, while that from CY2 was adenosylcobalamin. Both strains grew under environmental stresses, and LZ95 exhibited better tolerance to low temperature and high ethanol concentration. LZ95 also showed good viability when exposed to gastric acid (pH 2.0 and 3.0) and bile salts (0.3%) as well as good adhesion to Caco-2 cells. The viability of CY2 was significantly reduced under low pH and exposure to bile salt. Together, extracellular B12 producer LZ95 with good probiotic properties might be a candidate for in situ B12 fortification in the food industry.

Vitamin B12 dietary supplement can be critical to the alleviation strategies against micronutrient malnutrition and food insecurity. An HPLC-DAD method has been developed and validated, per AOAC SMPR 2016.017 (Standard Method Performance Requirements), for the quantitation of four bioactive forms of vitamin B12 (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, methylcobalamin) from dietary ingredients and supplements. The method achieves chromatographic baseline resolution of vitamin B12 forms on a modern column platform without the expensive requirement of an ultra-high pressure liquid chromatography and/or mass spectrometry. The method has a wide analytical range (0.0005%w/w–85%w/w), high precision (reproducibility relative standard deviations ranged from 1.43% to 4.67%), and high accuracy (>96% spike recovery rate for 11 out of 12 accuracy testing data points). The method detection and quantification limits are less than 0.16 and 0.52 µg/mL, respectively. To our best knowledge, it is simpler, less time-consuming, and more economical than other published methods for its intended uses.

Vitamin B12 is among the most essential biomolecules required for crucial metabolic processes in humans. Vitamin B12 was extracted from Chlorella vulgaris biomass under aqueous conditions, partially purified by passing the extract through amberlite XAD-2, Sep-Pak columns, and further purified by HPLC. The target peak eluent was subjected to characterisation by tandem mass spectrometry (MS/MS), selected ion recording (SIR) and multiple reaction monitoring (MRM) and identified as methylcobalamin (Me-Cbl). Quantification of Me-Cbl was carried out by microbiological and chemiluminescence methods, and found to be 29.87±2 μg/100 g and 26.84±2 μg/100 g dry weight, respectively. The presence of Me-Cbl was further substantiated using gold nanoparticle (AuNPs) based aptamer analysis, and found to be 28.02±2 μg/100 g dry weight. Good similarity was observed among all the methods. Methylcobalamin, a form of vitamin B12 was identified in C. vulgaris and this finding enhances its use as a nutritional supplement.

The forms of vitamin B12 that have been found in foods are cyanocobalamin, hydroxocobalamin, sulphitocobalamin, adenosylcobalamin and methylcobalamin. These cobalamins, with the exception of sulphitocobalamin, are also known to be present in serum and plasma. The addition of sodium cyanide, potassium cyanide, sodium metabisulphite or sodium nitrite in the extraction procedure employed for the determination of vitamin B12, however, can convert these cobalamins into derivatives which include dicyanocobalamin and nitritocobalamin. It was observed that cyanocobalamin has a binding affinity for hog intrinsic factor equal to that of methylcobalamin, dicyanocobalamin and nitritocobalamin, but not to that of hydroxocobalamin, sulphitocobalamin and adenosylcobalamin. Thus, these latter three cobalamins cannot be measured accurately using a competitive binding assay which employs hog intrinsic factor as its binding protein and cyanocobalamin as the calibration standards unless they are converted to cyanocobalamin, methylcobalamin, dicyanocobalamin or nitritocobalamin before the assay.