Browsing by Author "Ververis, Ermolaos"

Cobalamin (vitamin B12) occurs naturally in some animal-derived foods and is produced exclusively by microorganisms. An optimised protocol was used for extraction of cobalamin from cheese matrixes. No pseudocobalamin was detected in any of the examined samples. Cobalamin levels (mg/100g FW) detected in commercial emmental cheeses of three ripening stages did not alter significantly (P>0.05). Similar results were observed during the ripening of experimental semi-hard cheeses with or without propionibacteria. Existence of propionibacteria as adjunct culture in experimental cheeses did not alter significantly contribution on cobalamin levels of the cheese (P>0.05). The findings indicate that in the studied cheese matrixes the presence of propionibacteria did not affect the amount of cobalamin. The conditions to which propionibacteria are subjected during cheese manufacture and ripening and the presence of adenosyl-cobalamin in milk may be factors that repress cobalamin synthesis in Swiss- type cheeses. To date, the only known food grade microorganism that can produce cobalamin is Propionibacterium freudenreichii. This microorganism can also produce small amounts of pseudocobalamin, a compound structurally similar to cobalamin. BluB/CobT2 fusion gene is the factor that differentiates the two compounds upon their biosynthesis, by synthesizing and binding 5,6-dimethylbenzimidazole (DMBI) to the final molecule of cobalamin. In the present study, attempts to inactivate this gene were performed in order to investigate the existence of an alternative enzyme, capable of activating adenine for attachment as a lower ligand in pseudocobalamin, instead of DMBI. An electroporation protocol was implemented in order to transform plasmids containing bluB or cobT2 fragments and gene encoding erythromycin resistance in propionibacteria. Following transformation plasmid carrying bacteria were selected by cultivation in medium containing erythromycin. Homologous recombination of the bacterial genome and the non-replicative plasmid was expected to occur, leading to insertional mutagenesis. Colonies appeared after 7 and 11 days and were identified as propionibacteria but the disruption of bluB/cobT2 gene could not be verified. Inefficient transformation protocol, satellite colonies, low transformation efficiency, or choice essentiality of the bluB/cobT2 are among the possible explanations for the outcome of the experiment. Electroporation conditions should be optimized towards a more efficient P. freudenreichii transformation.