Non- Saccharomyces Yeasts from Organic Vineyards as Spontaneous Fermentation Agents

Abstract

Currently, non-Saccharomyces yeasts are the subject of interest, among other things, for their contribution to the aromatic complexity of wines. In this study, the characterisation of non-Saccharomyces yeasts was addressed by their isolation during spontaneous fermentations of organic Verdejo grapes, obtaining a total of 484 isolates, of which 11% were identified by molecular techniques as non-Saccharomyces yeasts. Fermentative isolates belonging to the species Hanseniaspora meyeri, Hanseniaspora osmophila, Pichia guilliermondii, Pichia kudriavzevii, Torulaspora delbrueckii, and Wickerhamomyces anomalus were analysed. Significant differences were found in the yeast populations established at the different fermentation stages. Interestingly, W. anomalus stood up as a widely distributed species in vineyards, vintages, and fermentation stages. Several of the strains studied stood out for their biotechnological potential in the production of Verdejo wine, showing the presence of relevant enzymatic activity for the release of varietal aromas and the technological improvement of the winemaking process. Three enzymatic activities were found in an important number of isolates, β-glucosidase, protease, and β-lyase, implicated in the positive aromatic impact on this style of white wine. In that sense, all the isolates of W. anomalus presented those activities. T. delbrueckii isolates were highlighted for their significant β-lyase activity. In addition, T. delbrueckii was outlined because of its potential to achieve an elevated fermenting power, as well as the lack of lag phase. The results obtained highlight the importance of maintaining the microbial diversity that contributes to the production of wines with unique and distinctive characteristics of the production region.

Keywords: Verdejo wine; enzymatic activity; microbial diversity; wine quality; yeast ecology.

Figure 1

Identification of non-Saccharomyces isolates. (A) Molecular patterns obtained for each different yeast species by PCR-RFLP of the 5.8S-ITS region using ITS1 and ITS4 primers and restriction enzymes: HaeIII; HinfI; and CfoI. (B) Non-Saccharomyces yeast species and number of isolates (abundance) of each species from the initial collection of Saccharomyces and non-Saccharomyces yeasts were identified. Hanseniaspora meyeri (Hm), Hanseniaspora osmophila (Ho), Papiliotrema laurentii (Pl), Papiliotrema terrestris (Pt), Pichia guilliermondii (Pg), Torulaspora delbrueckii (Td), Rhodotorula mucilaginosa (Rm), Naganishia globosa (Ng), Pichia kudriavzevii (Pk), and Wickerhamomyces anomalus (Wa).

Figure 2

Distribution across vineyard, vintage, and fermentation stages. (A) Distribution of non-Saccharomyces across the vineyards (V1, V2, V3) and the first and second vintage (1st, 2nd). (B) Distribution of non-Saccharomyces across the fermentation stages (freshly crushed grape must, CM; racked must, RM; start of fermentation, SF; tumultuous fermentation, TF; end of fermentation, EF).

Figure 3

Diversity of yeast populations. (A) Yeast community α-diversity based on Shannon index according to fermentation stages. Different letters indicate significant differences among different stages of winemaking process. (B) Yeast community dissimilarity (β-diversity) represented by non-metric multidimensional scaling (NMDS). The groups of strains representative of each vineyard population were defined by different symbols: circles (V1), rhombuses (V2), or rectangles (V3), and they are delimitated by rectangles (V1), circles (V2), and squares (V3). The vintage populations (first and second) were distinguished for the colour of the symbols: white with black border (first); and fully black (second).

Figure 4

Determination of the enzymatic activities found in each of the non-Saccharomyces isolates represented by a heat map (positive and negative activity). The relationship of the isolates according to enzyme activities was represented by a dendrogram.

Figure 5

Quantification of β-lyase activity of the isolated yeasts determined as growth after 48 h (Log₁₀CFU/mL). Different letters indicate significant differences in β-lyase activity among yeast isolates analysed.

Figure 6

CATPCA biplot of first two principal components relating the oenological isolates of interest to ecological factors, fermentation characteristics, and enzymatic activities.