Characterization of pectinase activity for enology from yeasts occurring in Argentine Bonarda grape

Abstract

Pectinolytic enzymes are greatly important in winemaking due to their ability to degrade pectic polymers from grape, contributing to enhance process efficiency and wine quality. This study aimed to analyze the occurrence of pectinolytic yeasts during spontaneous fermentation of Argentine Bonarda grape, to select yeasts that produce extracellular pectinases and to characterize their pectinolytic activity under wine-like conditions. Isolated yeasts were grouped using PCR-DGGE and identified by partial sequencing of 26S rRNA gene. Isolates comprised 7 genera, with Aureobasidium pullulans as the most predominant pectinolytic species, followed by Rhodotorula dairenensis and Cryptococcus saitoi. No pectinolytic activity was detected among ascomycetous yeasts isolated on grapes and during fermentation, suggesting a low occurrence of pectinolytic yeast species in wine fermentation ecosystem. This is the first study reporting R. dairenensis and Cr. saitoi species with pectinolytic activity. R. dairenensis GM-15 produced pectinases that proved to be highly active at grape pH, at 12 °C, and under ethanol and SO2 concentrations usually found in vinifications (pectinase activity around 1.1 U/mL). This strain also produced cellulase activity at 12 °C and pH 3.5, but did not produce β-glucosidase activity under these conditions. The strain showed encouraging enological properties for its potential use in low-temperature winemaking.

Keywords: Aureobasidium pullulans; Cryptococcus saitoi; Rhodotorula dairenensis; pectinolytic activity; winemaking.

Figure 1. DGGE profiles of yeasts isolated from Argentine Bonarda grapes and must during spontaneous fermentation. Correlations between lane designations and colony groups as well as Roman numerals and DGGE patterns are indicated in Table 1. The bands common to all isolates are single stranded DNA artifacts that were not influenced differentially by the gradient (Cocolin et al., 2000)

Figure 2. Occurrence of pectinolytic yeasts among representative yeasts isolated from grapes, fresh must and fermentation (sampling time: days 3 to 12) of cv. Argentine Bonarda. Number of isolates of yeast species (A) and of pectinolytic yeast species (B) found on grapes and during spontaneous fermentation. Aureobasidium pullulans, Hanseniaspora sp., Metschnikowia sp., Rhodotorula dairenensis, Cryptococcus saitoi, Candida zemplinina and Saccharomyces cerevisiae

Figure 3. Relative pectinolytic activity of Cr. saitoiGM-4 and R. dairenensis GM-15 assayed at 12 and 28 °C in presence of 15% (v/v) ethanol or 120 mg/L SO₂ or a combination of both compounds. Reference activity (100% of relative activity): 0.736 ± 0.071 U/mL at 12 °C and 1.349 ± 0.133 U/mL at 28 °C for Cr. saitoi GM-4, and 0.972 ± 0.083 U/mL at 12 °C and 1.483 ± 0.091 U/mL at 28 °C for R. dairenensis GM-15. (*): significant difference, (ns): not significant, according to the LSD test (n = 3, p < 0.05). In all cases SD was lower than 10%