Vitamin B12 (later B12) intake is insufficient in developing countries, and globally, vegetarians and vegans are also at risk of B12 deficiency. Occurring naturally only in foods of animal origin, new affordable and sustainable dietary sources of B12 are needed to ensure sufficient intake. The only known food-grade producers of active B12, Propionibacterium freudenreichii strains, however, are yet to be exploited to enrich plant-based foods with B12. The B12 production capacity of P. freudenreichii depends on the strain, and the availability of the B12 lower ligand (5,6-dimethylbenzimidazole, DMBI) is a key factor for the production of active B12. Bread can be considered as a potential food for B12 fortification; yet the stability of in situ-produced B12 incorporated during breadmaking processes is not known. Current analytical methods such as the microbiological assay (MBA) lack the required specificity and the existing high-performance liquid chromatography (HPLC) methods are only capable of measuring higher B12 levels in fortified foods and supplements. The determination of active B12 in fermented foods, however, needs sensitive and selective methods. An ultra-HPLC (UHPLC) method was developed and validated to measure the active B12 contents. The identity of the B12 form was confirmed with an ion-trap or quadrupole time-of-flight mass spectrometry (MS). The B12 production capacity of 27 P. freudenreichii and 3 Propionibacterium acidipropionici strains was first studied in whey-based medium (WBM), and three of these P. freudenreichii strains were chosen to study B12 production in three aqueous cereal matrices prepared from malted barley (BM; 33% w/v), barley flour (BF; 6% w/v) and wheat aleurone (AM). Riboflavin (RF) and nicotinamide (NAM) as food-grade replacements for DMBI were investigated in WBM and cereal matrices. The stability of in situ-produced B12 and added cyanocobalamin (CNCbl) and hydroxocobalamin (OHCbl) during straight-dough, sponge-dough and sourdough breadmaking was studied. The developed UHPLC method employing an Acquity high-strength silica (HSS) T3 column showed excellent separation of active B12 from its analogues. A low limit of detection (0.075 ng/inj) enabled the measurement of the B12 levels in cell extracts directly and following immunoaffinity purification in extracts of fermented cereal matrices and B12-fortified baking samples. Analysis with UHPLC MS confirmed the production of active B12 by all 27 P. freudenreichii strains in WBM and 3 P. freudenreichii strains in cereal matrices. P. acidipropionici strains, however, produced an inactive form (pseudovitamin B12), thus making them unsuitable for active B12 fortification in foods. The level of B12 production in WBM varied considerably between the strains (0.45‒3.35 µg/mL), which increased up to 4-fold in 12 of the 27 P. freudenreichii strains following supplementation with RF and NAM. In many of these strains, the B12 yield was higher with RF and NAM co-supplementation than with DMBI. In cereal matrices without supplementation, the produced levels of active B12 (9‒37 ng/g) with P. freudenreichii strains were nutritionally significant. The B12 production increased many-fold, reaching up to 430 ng/g in BM, 39 ng/g in BF and 114 ng/g by adding cobalt (Co) and reached 712 ng/g in BM and 180 ng/g in AM with RF and NAM co-supplementation with Co. The incorporated in situ-produced B12 was retained during straight-dough breadmaking and the loss of 29% during sourdough baking was similar to the losses observed for relatively stable CNCbl. However, the added OHCbl decreased by 21%, 31% and 44% respectively in straight-dough, sponge-dough and sourdough breadmaking. These results showed that B12 produced in situ and incorporated during breadmaking was well retained in the bread prepared by the conventional breadmaking processes. This thesis shows that UHPLC combined with MS allows for the accurate identification and quantitation of low levels of active B12 in fermented food matrices (≥1 ng/g). P. freudenreichii strains could be utilised for in situ production of active B12 in cereal matrices and WBM. The availability of RF and NAM could considerably improve B12 production. The produced levels could easily fulfil the recommended dietary allowance set for B12 (e.g. 2‒2.4 µg/day for adults), and could be well retained in bread in the commonly used breadmaking processes.
