The production of preconditioned freeze-dried Oenococcus oeni primes its metabolism to withstand environmental stresses encountered upon inoculation into wine.

Oenococcus oeni is the most resistant lactic acid bacteria species to the environmental stresses encountered in wine, particularly the acidity, presence of ethanol and phenolic compounds. Indigenous strains develop spontaneously following the yeast-driven alcoholic fermentation and may perform the malolactic fermentation whereby improving taste, aroma, and the microbial stability of wine. However, spontaneous fermentation is sometimes delayed, prolonged or incomplete. In order to better control its timing and quality, O. oeni strains are selected and developed to be used as malolactic starters. They are prepared under proprietary manufacturing processes to survive direct inoculation and are predominantly provided as freeze-dried preparations. In this study, we have investigated the physiological and molecular alterations occurring in O. oeni cells prepared by an industrial process that consists of preconditioning protocols and freeze-drying, and compared them to the same strain grown in a grape juice medium. We found that compared to cultured cells, the industrial production process improved survival under extreme conditions, i. e. at low pH or high tannin concentrations. In contrast, cultured cells resumed active growth more quickly and strongly than freeze-dried preparations in standard pH wines. A proteomic analysis showed that during the industrial production most non-essential metabolic processes are shut down and components of the general and the stringent stress response are upregulated. The presence of major components of the stress response facilitates protein homeostasis and physiological changes that further ensure the integrity of cells.

Cloning, sequence analysis and expression of the F1F0-ATPase beta-subunit from wine lactic acid bacteria.

The nucleotide sequences of the genes encoding the F1F0-ATPase beta-subunit from Oenococcus oeni, Leuconostoc mesenteroides subsp. mesenteroides, Pediococcus damnosus, Pediococcus parvulus, Lactobacillus brevis and Lactobacillus hilgardii were determined. Their deduced amino acid sequences showed homology values of 79-98%. Data from the alignment and ATPase tree indicated that O. oeni and L. mesenteroides subsp. mesenteroides formed a group well-separated from P. damnosus and P. parvulus and from the group comprises L. brevis and L. hilgardii. The N-terminus of the F1F0-ATPase beta-subunit of O. oeni contains a stretch of additional 38 amino acid residues. The catalytic site of the ATPase beta-subunit of the investigated strains is characterized by the two conserved motifs GGAGVGKT and GERTRE. The amplified atpD coding sequences were inserted into the pCRT7/CT-TOPO vector using TA-cloning strategy and transformed in Escherichia coli. SDS-PAGE and Western blot analyses confirmed that O. oeni has an ATPase beta-subunit protein which is larger in size than the corresponding molecules from the investigated strains.

Oenococcus oeni preference for peptides: qualitative and quantitative analysis of nitrogen assimilation.

Optimization of malolactic fermentation in wine depends mainly on better understanding of nitrogen nutritional requirements of Oenococcus oeni. Four widely used starter strains and the reference ATCC BAA-1163 strain were grown in media containing different N sources: free amino acids, oligopeptides (0.5-10 kDa) or polypeptides (> 10 kDa). Amino acid auxotrophies were determined by the single omission technique. The tested strains were indifferent to only two to four amino acids and two of the starter strains appeared to be particularly demanding. Nitrogen consumption was investigated and a significant level of nitrogen was consumed by O. oeni only in the free amino acid medium. In media containing complex nitrogen sources, a global balance above 5 mg N l(-1) was enough to ensure biomass formation of all tested strains. Moreover, for all strains, bacterial growth yield was higher in the presence of nitrogen from peptides than that from free amino acids. However, no direct relationship between the bacterial growth level and the amount of nitrogen metabolized could be established. These findings were discussed in relation to the physiology of wine malolactic bacteria.