Biogenic amines are indesirable compounds found in fermented products like wine. Lactic acid bacteria from wine, including Oenococcus oeni, the main actor of malolactic fermentation, are able to produce these molecules from nitrogenous precursors. In order to limited biogenic amines accumulation, it is necessary to understand the role of this production by strains responsible for the synthesis of these metabolites in food. That is why the European BiamFood project was put in place. Along my thesis, molecular tools were developed in order to mutate O. oeni genes (encoding decarboxylases), and to express genes of interest. Genetic clusters hdc from L. higardii and odc from O. oeni, responsible for histamine and putrescine synthesis respectively, were cloned into the pGID052 vector and transferred into the laboratory strain O. oeni ATCC BAA-1163, which is not able to produce any biogenic amines. However no biogenic amines production was observed. My work thesis was then redirected in the in vitro biochemical characterization of the ODC from O. oeni. The odc from O. oeni BR14/97 was overproduced in E. coli BL21 via the pET system. Optima pH and temperature were determined with the purified enzyme obtained. Kinetic constants Km = 1 mM and Vmax = 0,57 U.mg-1 were also determined by measuring putrescine production by HPLC (Gomez-Alonso et al, 2007). Furthermore, ODC from O. oeni BR14/97 is specific for the L-ornithine and can not decarboxylated the L-lysine into cadaverine. In a second part of the BiamFood project, the impact of nitrogen sources, especially peptides compared to free amino acids, was studied in a Lactobacillus plantarum strain, isolated from wine and able to produce tyramine. Peptides containing tyrosine residues are used by this strain to produce tyramine. Tyramine production increases during the growth and reaches a maximum at stationary phase. The production is correlated with tyrP expression. The tyrosine measured in culture media shows that peptides may be hydrolyzed in the extracellular medium in L. plantarum.
Study of biogenic amines metabolism in wine lactic acid bacteria