Yeast Multi-Enzymatic Systems for Improving Colour Extraction, Technological Parameters and Antioxidant Activity of Wine.

Research background: Wine yeasts are a heterogeneous microbial group with high enzymatic potential that makes them a useful tool in winemaking. With a better understanding of their oenological properties, selection procedures can be optimised to obtain more efficient strains. The present study aims to isolate and select yeasts from wine grape surface by studying their production of enzymes that hydrolyse plant cell wall polymers and by linking them to different technological parameters and antioxidant activity of wines. Experimental approach: Yeasts that are able to produce carbohydrolases and related enzymes of oenological importance were firstly selected on plates and subsequently identified. Then, a secondary selection of yeasts was carried out according to technological effects of their extracellular enzyme extracts on short macerations. In this way, the colour extraction, total polyphenol content, clarification, filterability and antioxidant activity were studied. This approach makes it possible to correlate the microorganism capacity to produce cell wall-depolymerizing enzymes with their technological effects. Results and conclusions: From 366 isolates, 96 strains (26.2%) showed at least one of the polysaccharidase activities and 55 strains (57.3%) of them exhibited activities of multiple enzymes that degrade plant cell wall polymers. Sixteen strains were selected and identified as Aureobasidium, Candida, Debaryomyces, Hanseniaspora, Metschnikowia, Pichia, Saccharomyces and Torulaspora. Pectinolytic enzymes had the highest hydrolytic activity. Aureobasidium pullulans had a broader enzyme blend and higher activity, dominated by pectinases and followed by xylanases and cellulases. Moreover, the Torulaspora delbrueckii m7-2 strain produced high amounts of polysaccharidase and this was strain-dependent. Strains that produced enzyme extracts with a wide range of activities that were also the highest, also had the best chromatic and technological properties. Cluster analysis confirmed that A. pullulans R-22, m11-2, m86-1 and m86-2 and T. delbrueckii m7-2 could be correlated with a better effect on filterability, clarification and extraction of bioactive compounds, encouraging future studies regarding their application in winemaking. Novelty and scientific contribution: The study of yeast multi-enzymatic systems impacting the grape maceration process enables a proper selection criterion for wine yeasts to improve colour extraction, technological parameters and antioxidant activity of Malbec wine. This work shows that A. pullulans and T. delbruekii have a high enzymatic potential for oenological purposes.

Kinetic and metabolic behaviour of the pectinolytic strain Aureobasidium pullulans GM-R-22 during pre-fermentative cold maceration and its effect on red wine quality.

Pectinolytic yeasts can be applied to winemaking with the purpose of improving sensory and technological properties of wine because of their enzymes secreted during vinification. In this work, the autochthonous yeast-like pectinolytic strain from D.O. San Rafael viticulture region, Aureobasidium pullulans GM-R-22, was used in co-culture with Saccharomyces cerevisiae IOC 18-2007 in microvinification trials with Malbec must applying pre-fermentative cold maceration (PCM). A. pullulans remained viable during PCM and S. cerevisiae growth and fermentative kinetics were not affected in mixed culture with respect to pure S. cerevisiae culture. High pectinolytic activity (9.13 U/mg) was detected in mixed A. pullulans vinification during PCM, in which conditions of low temperature (8 °C), low pH (3.8) and high sugar concentration (250.6 g/L) governed. Mixed A. pullulans wine showed enhanced colour compared with pure S. cerevisiae wine, characterised by higher colour index and percentage of red colour, lower tonality and percentage of yellow colour, and negative values of b* and h* indicating more bluish and purplish tonalities. Moreover, filtration time and turbidity diminished by a 40% in mixed A. pullulans wine. The presence of GM-R-22 strain improved the production of desirable volatile compounds, such as esters and norisoprenoids, which displayed the maximum odour activity values (OAVs), whereas this strain reduced the total content of higher alcohols when compared to pure S. cerevisiae fermentation. Sensory analysis indicated that A. pullulans impacted on wine highlighting the violet hue, plum jam aroma, body and equilibrium that are distinctive features of Malbec variety. A. pullulans GM-R-22 seems to be promising for applying to low-temperature red winemaking as an adjunct culture to S. cerevisiae to improve the wine quality and vinification process.

Pectinolytic yeasts from viticultural and enological environments: novel finding of Filobasidium capsuligenum producing pectinases.

In this study indigenous yeasts associated with wineries, grapes and Malbec fermented must from San Rafael viticulture region (Argentina) were isolated to select pectinolytic strains for their potential use in enology. Pectinolytic yeasts were identified by physiological and molecular methods. Among 78 isolates, only nine were able to produce extracellular pectinases. Six isolated from berry surface were identified as Aureobasidium pullulans and the remaining isolates, recovered from wineries, belonged to Saccharomyces cerevisiae and Filobasidium capsuligenum species. Pectinase production was evaluated under vinification-related conditions: pH 3.5, 12 and 28 °C. A. pullulans U-12 produced the highest pectinolytic activity at low temperature (1.16 U ml(-1) ), while F. capsuligenum strains showed good activity at 12 and 28 °C (0.77 and 1.15 U ml(-1) , respectively) being this study the first report on the capacity of this species to produce pectinases. The pectinolytic activity of F. capsuligenum B-13 showed an optimum at pH 4.5 and two peaks at 20 and 50 °C. The enzyme half-life was 2 h at 40 °C and retained 65% of its activity at 40 °C after 1 h of incubation. This pectinolytic system displayed remarkable activity at pH and temperatures found in vinification, suggesting a potential candidate for applying to wine-making.

Isolation and selection of yeasts from wine grape ecosystem secreting cold-active pectinolytic activity.

The present study was undertaken with the purpose of selecting yeasts from wine grapes that are able to produce extracellular cold-active pectinases. After two consecutive selections yeast isolates were identified by pheno- and genotyping, and pectinolytic activity was preliminarily characterised at proximate winemaking conditions. Out of 1023 indigenous microorganisms isolated from grape skins of D.O. San Rafael (Mendoza, Argentina) viticulture region, 565 (55%) showed pectinolytic activity on plates and, among them, 96 (17%) were chosen in a primary selection. Ten isolates were finally selected for exhibiting the greatest activity at low temperature (12 °C) and identified as Aureobasidium pullulans. GM-R-22 strain demonstrated the highest pectinolytic activity (0.751 U/mL) at pH 3.5 and 12 °C. Yeast pectinases were constitutively produced. This study is the first report about strains of A. pullulans producing pectinases which are able to show good activity at low temperature. These pectinolytic strains could be of interest in wine production.