THESE Maryse Bonnin Jusserand - Université de Bourgogne

ABSTRACT
Abstract
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.
Key words: biogenic amines, wine, lactic acid bacteria, Oenococcus oeni, heterologous
expression, suicide vectors, peptides.
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