SCREENING OF ENZYMATIC ACTIVITIES RELATED TO AROMA IN LACTIC ACID BACTERIA ISOLATED FROM WINERY WASTE

MARÍA ROSA MORALES, LUCIANA RIVERO, FABIANA SAGUIR

Congreso; LVI Reunión Anual SAIB + Reunión Anual SAMIGE; 2020

The aim of this study was to examine the esterase, β-glucosidase, α-rhamnopyranosidase, and α-arabinofuranosidase activities in lactic acid bacteria strains. Such activities are related to the hydrolysis of aroma precursors, involved in enhancing the varietal wine aroma. We examined Oenococcus and Lactobacillus strains isolated from both wine and winery waste and selected in previous studies for their promising enological traits. Enzymatic assays were performed in triplicate in three types cell fractions: cell suspension, supernatant, and cell-free extract. Cells grown in MRS media supplemented with malic acid (4 g/L) and fructose (5 g/L) were harvested by centrifugation (at 8000 rpm and 4°C for 15 min), washed once in Citrate-Phosphate buffer (0.1 M, pH 5.0), and resuspended in the same buffer (25%, w/v). Cells were then disrupted on ice by sonication (10 cycles of 30 s, 80% pressure, 1000 psi). Subsequently, cell suspensions were centrifuged at 8000 rpm and 4°C for 15 min, and the supernatants were reserved for enzyme activity. Protein concentration of enzyme extracts was determined by the Bradford method using bovine serum albumin as a standard protein. The substrates used for the assays were p-nitrophenyl acetate (esterase activity), p-nitrophenyl-β-D-glucopyranoside (βglucosidase activity), p-nitrophenyl-α-L-rhamnopyranoside (αrhamnopyranosidase activity), and p-nitrophenyl-α-L-arabinofuranoside (α-arabinofuranosidase activity). The final 1 mL reaction mixture contained Citrate-Phosphate buffer (pH 5.0), the respective cell fraction (reaching a final a final OD600 of 0.5), and 40 μL substrate solution, yielding a final 1 mM substrate concentration. Control assays contained the same reaction mixture but without cells. After incubation of samples at 37°C for 2 hours, the cells were pelleted by centrifugation (8000 rpm, 15 min. An aliquot of 900 μL of each cell fraction was transferred to a corresponding cuvette, while 100 μL of 0.5 M sodium hydroxide were used to stop the reaction. Absorbance at 410 nm was measured in a spectrophotometer and compared to a p-nitrophenol standard curve. The enzyme activity was expressed as μmoles of p-nitrophenyl released per minute per gram of cell dry weight (U/g). Esterase activity was high in the B18 (Oenococcus oeni, isolated from wine lees), X2L, MS46, and m (Oenococcus oeni, isolated from wine) strains in the cellfree extract. The highest esterase activity was observed in the OT200 strain (Lactobacillus plantarum, isolated from grape pomace) in the supernatant. The β-glucosidase was only present in the cell-free extract of OT200 and B16 (Lactobacillus brevis, isolated from wine lees), while the two remaining activities were found in the m strain (cell-free extract) but in a low level