María G. Merín

Characterization of wines produced by mixed culture of autochthonous yeasts and Oenococcus oeni from the northwest region of Argentina

Two autochthonous yeasts from the northwest region of Argentina, Kloeckera apiculata mc1 and Saccharomyces cerevisiae mc2, were used as pure or mixed starter cultures in microvinification trials conducted in Malbec red must. Also, the effect of Oenococcus oeni X2L was evaluated. S. cerevisiae mc2 showed adequate growth and fermentative activity in single and composite fermentations, producing standard concentration of ethanol. The amount of esters was higher in fermentations conducted using mixed yeast starters. Independent of the timing of inoculation of O. oeni, this malolactic bacterium completely depleted malic acid. Sensory evaluation indicated that young wines fermented with mixed yeast cultures and sequential inoculation of O. oeni were preferred, achieving the highest scores for positive descriptors and they allowed better control of the sensory quality. Consequently, this study proposes inclusion of autochthonous K. apiculata mc1 as an adjunct culture to S. cerevisiae mc2 during Malbec must fermentation to improve the organoleptic properties of red wines. Furthermore, sequential inoculation of O. oeni X2L should be carried out after completion of the alcoholic fermentation to enhance sensory characteristics.

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.

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

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.

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.

Highly cold‐active pectinases under wine‐like conditions from non‐Saccharomyces yeasts for enzymatic production during winemaking

The influence of oenological factors on cold‐active pectinases from 15 preselected indigenous yeasts belonging to Aureobasidium pullulans, Filobasidium capsuligenum, Rhodotorula dairenensis, Cryptococcus saitoi and Saccharomyces cerevisiae was investigated. Pectinolytic enzymes were constitutive or partially constitutive; and high glucose concentration (200 g l−1) did not affect or increased pectinase production at 12°C and pH 3·5 (up to 113·9 U mg−1) only in A. pullulans strains. SO2 (120 mg l−1) slightly affected the growth of A. pullulans strains but did not affect pectinase production levels. Ethanol (15%) barely affected pectinase activity of A. pullulans strains but diminished relative activity to 12–79% of basidiomycetous yeasts. Moreover, non‐Saccharomyces strains showed promising properties of oenological interest. This study demonstrates that cold‐active pectinases from some A. pullulans strains were able to remain active at glucose, ethanol and SO2 concentrations usually found in vinification, and suggests their potential use as processing aids for low‐temperature winemaking.

Natural occurrence and production of tenuazonic acid in wine grapes in Argentina

Abstract A survey was carried out to determine natural occurrence of tenuazonic acid (TA) in healthy and rotten wine grapes samples from different varieties (n = 37) collected during 2016 vintage in the region of DOC San Rafael (Argentina). In addition, inoculation experiments with three Alternaria alternata strains in wine grapes were done to elucidate TA production and its major influencing factors. The 16.2% (6/37) of total wine grape samples showed TA contamination with 4% (1/25) of incidence in healthy samples (77 μg·kg−1) and 42% (5/12) in rotten samples (10–778 μg·kg−1). Malbec, Cabernet Sauvignon, and Syrah varieties showed TA contamination, whereas Bonarda, Ancelota, Torrontés, Semillón, and Chenin did not. During inoculation experiments in wine grapes, two of three strains were able to produce TA among the evaluated conditions and the highest TA production was observed at 15°C and 25°C after 24 days of incubation. Nutritional composition of grapes results appropriate for A. alternata infection and TA production and, together with the adequate field conditions, favors TA natural occurrence in wine grapes.

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.

Isolation, identification and selection of antagonistic yeast against Alternaria alternata infection and tenuazonic acid production in wine grapes from Argentina

Epiphytic isolates with yeast characteristics from grapes of the Malbec cultivar were obtained in order to find antagonists against Alternaria alternata. From a total of 111 isolates, 82% corresponded to the yeast-like organism Aureobasidium pullulans and the rest to the non-Saccharomyces yeasts Hanseniaspora uvarum (6.3%), Metschnikowia pulcherrima or spp. (5.4%), Cryptoccocus laurentti II (2.7%), Starmerella bacilaris or Candida zemplinina (2.7%) and Rhodotorula spp. (0.9%). The 22.4% (15 out of 67) of epiphytic yeasts and yeast-like organisms evaluated were able to reduce A. alternata infection from 0.0 to 4.4% when applied 2h previous to pathogen inoculation on wounds of grape berries. From these selected strains, 14 out of 15 strains completely prevented A. alternata infection (0.0%), which implies potential for field application. All Metschnikowia (pulcherrima or spp.), S. bacillaris and almost all H. uvarum evaluated strains showed antagonist capability against A. alternata. Meanwhile, none of the lesser nutritional requirement strains belonging to A. pullulans, Cr. laurenti II and Rhodotorula spp. did. All the yeasts with capacity to prevent A. alternata infection also reduced tenuazonic acid (TA) production by 81.2 to 99.8%, finding TA levels similar to negative controls. Therefore, the epiphytic yeasts selected are promising as biological control agents against Alternaria infection and toxin production in grapes for winemaking.

BACTERIA | Classification of the Bacteria: Traditional

This article is a revision of the previous edition article by Vilma Morata de Ambrosini, Carlos Horacio Gusils, Silvia Nelina Gonzalez, Guillermo Oliver, volume 1, pp 173–178,