• baking
• bread
• Propionibacterium freudenreichii
• stability
• vitamin B12

Vitamin B12 is an essential macromolecule in mammal metabolism. However, it can be detected in sufficient amount only in animal-derived foods. This fact exposes vegetarians and vegans to vitamin B12 deficiency. Therefore the need to develop plant-based food rich in vitamin B12 is high. However, it is important to know the stability of both supplemented vitamin B12 forms and naturally produced vitamin B12, during the different food processes. Many attempts have been done during the last decades in order to produce vitamin B12- fortified plant-based products, whose main aim was to replace vitamin B12 supplements, in accordance with the increasing demands of natural and healthy products. This study aimed to investigate the stability of vitamin B12 during different bread-making processes, Firstly adding standard solutions, and subsequently mixing the natural vitamin B12 produced by the fermentation of a selected strain of Propionibacterium freudenreichii subsp. freudenreichii. Firstly, the stability of di_erent forms of vitamin B12 (methylcobalamin (Me-Cbl), 5_ -deoxyadenosylcobalamin (Ado-Cbl), cyanocobalamin (CN-Cbl) and hydroxocobalamin (OH-Cbl) after different times of exposure to light was investigated. While Me-Cbl and Ado-Cbl stability was tested only in water, CN-Cbl and OH-Cbl tests were performed at different pH conditions (pH 2.5, 4.5, 7.0, water). Furthermore, the stability of CN-Cbl and OH-Cbl was studied during three different bread-making methods: straight-dough, sponge-dough and sourdough. Finally, a malt extract medium fermented by a selected strain of P. freudenreichii subsp. freudenreichii was used in the straight-dough baking. The vitamin B12 was quantified by a microbiological assay (MBA) and the ultra high performance liquid chromatography (UHPLC). Ado-Cbl and the Me-Cbl were extremely photolable and they converted entirely into OH-Cbl within 30 min, while both CN-Cbl and OH-Cbl showed a greater stability under light depending on the pH conditions: the CN-Cbl was more photo-sensitive at low pHs, while the OH-Cbl was more unstable at higher pHs. In addition, one unidentified peak was observed in the chromatogram originated from OH-Cbl and CN-Cbl due to the exposure to light. The proofing steps had not effect on the loss of supplemented OHCbl and CN-Cbl in straight- and sponge-doughs. Instead, they were lost during the baking in the oven, where the loss of OH-Cbl was higher than CN-Cbl. In straight-dough and sponge-dough processes, the loss of OH-Cbl was 15% and 32% , and the loss of CN-Cbl 6% and 7%, respectively. The sourdough method caused the highest destruction. During the baking, OH-Cbl and CN-Cbl losses were 57% and 42%, respectively. In straight-dough baking with the fermented malt extract, the loss of naturally produced vitamin B12 was on average 29±12% obtained with the MBA. By contrast, results analysed with the UHPLC methods showed no loss, and vitamin B12 content in baked breads was still considerable, on average 106 ng/g dm. This difference between the results may indicate about inactive B12 forms produced by the selected strain of P. freudenreichii subsp. freudenreichii and also high stability of vitamin B12 produced by such bacterium. Generally, the supplementation of OH-Cbl and CN-Cbl did not effect on appearance and taste of breads. However, the specific volume of the breads baked with fermented malt extract was slightly lower than control breads. Producing a vitamin B12-fortified bread using fermented malt extract, as presented in this study, is possible and may represent a great opportunity for vegetarian and vegan people. However, further studies are needed, e.g., to clarify the effects of the compounds in the fermented media on the yeast growth, as well as the determination and the quantification of the inactive corrinoids produced by such bacteria during the fermentation step.