CONICET | Buscador de Institutos y Recursos Humanos

Lactic acid bacteria (BAL) play an important role in winemaking by undertaking the malolactic fermentation (MLF), that decarboxylates L-malic acid to L-lactic acid and carbon dioxide. Oenococcus oeni is the most tolerant BAL species in unfavourable wine conditions, being mainly responsible for driving the MLF. As a result of MLF, the quality of the wine improves, sine acidity decreases, the sensory characteristics are enhanced with additional flavours, and the microbiological stability of wine increases. Nevertheless, little information is available on other aspects of their physiology, such as their enzymatic activities related with wine aroma and flavour. Many wine volatile compounds such as terpenes can be released from their flavourless glycoconjugate precursors by either acid or enzymatic hydrolysis. Some O. oeni strains well adapted to do MLF could be a good source of glycosidases such as β-D-glucosidase, increasing the fruity aroma in wine. The main objective of the present study has been to study and compare the β-glucosidase (BG) activity in several commercial and other well adapted strains of O. oeni isolated from argentinian and catalan wines, and to look for the cell location of the enzymatic activity. The influence of usual cations on the activity has also been studied. Cells were inoculated in MRS medium supplemented with L-malic acid (2g/l) adjusted to pH 5.0 at 27ºC. At the end of exponential phase (O.D. 0.8-1.0), cells were harvested (9,000 x g for 10 min) and resuspended in Tris buffer. After sonication for 20 min, BG activity was determined in this total cells extract. Then, those extracts were centrifuged at 6,500 x g for 30 min and the resuspended pellet was assayed for BG activity, corresponding to cell wall. After centrifugation of the supernant fraction at 32,000 x g for 30 min, the BG activities of the pellet and the supernatant were analyzed, corresponding to the membrane cell and the cytosol respectively. The β-glucosidase activity was measured by the hydrolysis of the substrate pNPG (4-nitrophenyl-β-D-glucopyranoside) in acetate buffer, pH 5.5, at 30ºC for 30 min, and after stopping the reaction with Na2CO3. Absorbance was measured at 420 nm. One unit of BG activity has been defined as the amount of enzyme releasing 1 μmol of substrate per min. The specific β-glucosidase activity found in total cell extracts has been from 2 to 10 U/g of proteins, depending on strain, being the maximum for some commercial strains. For all strains the maximum activity has been found in cell wall fraction, around 90%. Activity found in membrane and cytosol fractions has been between 5 and 10% in all cases. So, this enzyme would be located near the cell wall. The effect of the most usual cations (10 mM of Mg++, Na+, Mn++, K+ and Ca++) on the β-glucosidase activity of the total cell extract has also been studied in those strains, but neither significant positive nor negative effect has been detected for any strain. As conclusion, this work suggests that β-glucosidase is located near the cell wall in O. oeni. Besides that and the need for following more deeply this study, the variability of this activity among strains makes convenient to consider this characteristic as a point of strain selection to obtain starters wich will give better sensory qualities to wines.