Non-Saccharomyces Yeasts: Biotechnological Role for Wine Production

Non-Saccharomyces yeasts play a substantial role in the early stages of wine fermentation. With the increase in alcohol concentration, indigenous or commercial strains of Saccharomyces cerevisiae take over and complete the transformation of the grape must sugars into ethanol, CO2, and other secondary metabolites. The presence of non-Saccharomyces during the fermentation has an impact on the wine composition, and consequently, their contribution during the fermentation process cannot be ignored. The new challenges to enhance the appeal and value of wine elaborated by traditional technology are being achieved by selecting and using autochthonous non-Saccharomyces and Saccharomyces strains that may enhance regional identity of wines. Greater understanding of yeast biochemistry and physiology is enabling the selection and development of yeast strains that have defined specific influences on process efficiency and wine quality. The aim of this chapter was to show the different aspects of non-Saccharomyces species that may play a positive incidence in the biotechnological process to conduct to wine elaboration

The role of non-Saccharomyces yeasts in industrial winemaking

The fermentation of grape juice into wine is a complex microbiological process, in which yeasts play a central role. Traditionally, identification and characterization of yeast species have been based on morphological and physiological characteristics. However, the application of molecular biology techniques represents an alternative to the traditional methods of yeast identification and are becoming an important tool in solving industrial problems. Although Saccharomyces cerevisiae is responsible for the alcoholic fermentation, the presence of non-Saccharomyces species could be important since they produce secondary metabolites, which can contribute to the final taste and flavor of wines

Microbial quality and yeast population dynamics in cracked green table olives' fermentations

Cracked green table olives, from the Manzanilla variety, are a fermented food produced and consumed in Portugal. The objective of the present work was to study the microbiological characteristics and yeast population evolution during the fermentation of cracked green olives. The predominant microorganisms were yeasts while lactic acid bacteria were not detected and a clear decrease of the mould population was observed. At the end of the fermentations, no viable counts of Enterobacteriaceae were found. Yeast isolates were identified by the 5.8S rRNA-ITS region restriction analysis and by sequencing the D1/D2 region of the 26S rRNA gene. During the initial phases of the fermentations a great diversity of yeasts was observed. However, as the processes evolved the biodiversity decreased with the fermentative yeasts Citeromyces matritensis, Zygotorulaspora mrakii and Saccharomyces cerevisiae becoming the dominant species. The presence of these fermentative yeasts at the end of the production process is associated to a risk of spoilage. The results obtained represent a first attempt towards the comprehension of the microbiota of this type of “Natural olives” that constitute an important component of the Mediterranean diet

Use of fortified pied de cuve as an innovative method to start spontaneous alcoholic fermentation for red winemaking

Background and Aims: Some wineries, in order to promote the growth of yeasts able to ferment grape musts, traditionally produce wines using the ‘pied de cuve’ method. The aim of the present work was to study the performance of fortified pied de cuve (FPdC) prepared by addition of wine. Method and Results: Two FPdCs were prepared with the addition of wine at 1.5 and 3% (v/v) of ethanol to the musts and allowed to spontaneously ferment. The FPdCs were then added to fresh bulk musts in order to accelerate the spontaneous alcoholic fermentation (AF). Interestingly, several Saccharomyces cerevisiae strains isolated during the pied de cuve preparation were detected at the highest concentration throughout AF. The chemical composition of the wines conformed to commercial regulations. The volatile organic compounds (VOCs) were mainly represented by diethyl succinate and ethyl lactate, as well as by hexanol and isoamyl alcohol. Principal component analysis of the chemical parameters, VOCs and sensory data showed that the use of experimental pied de cuve influenced positively the composition of the final wines. Conclusion: The FPdC had no negative impact on the microbiological and chemical composition of wines, but it affected the diversity of the species S. cerevisiae present during AF and the sensory profile of the final wine. Significance of the Study: This study provided evidence that FPdC accelerates spontaneous AF and influences the sensory profile of red wines

Bioinformatic approaches for the genetic and phenotypic characterization of a Saccharomyces cerevisiae wine yeast collection

The objective of the present study was to compare genetic and phenotypic variation of 103 Saccharomyces cerevisiae strains isolated from winemaking environments. We used bioinformatics approaches to identify genetically similary strains with specific phenotypes and to estimate a strain's biotechnological potential. 
A S. cerevisiae collection, comprising 440 strains that were obtained from winemaking environments in Portugal has been constituted during the last years. All strains were genetically characterized by a set of eleven highly polymorphic microsatellites and showed unique allelic combinations. Using neural networks, a subset of 103 genetically most diverse strains was chosen for phenotypic analysis, that included growth in synthetic must media at various temperatures, utilization of carbon sources (glucose, ribose, arabinose, xylose, saccharose, galactose, rafinose, maltose, glycerol, potassium acetate and pyruvic acid), growth in ethanol containing media, evaluation of osmotic and oxidative stress resistance, H2S production and utilization of different nitrogen sources. Using supervised data mining approaches we have found that genotype represented with presence/absence of eleven microsatellites relates well with geographical location (performance evaluation using leave-out-out technique resulted in high performance scores; e.g., area under ROC curve was above 0.8 for a number of standard machine learning approaches tested). To find relations between phenotypes and genotypes, we used a two-step approach which first hierarchically clusters the strains according to their phenotype, and then tests if the resulting sub-clusters are identifiable using strain’s genetic data. Several groups of strains with similar phenotype profiles and common features in genotype were identified this way, and they are subject to further investigations. 

Financially supported by the programs POCI 2010 (FEDER/FCT, POCTI/AGR/56102/2004) and AGRO (ENOSAFE, Nº 762).
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The geographic distribution of saccharomyces cerevisiae isolates within three Italian neighboring winemaking regions reveals strong differences in yeast abundance, genetic diversity and industrial strain dissemination

In recent years the interest for natural fermentations has been re-evaluated in terms of increasing the wine terroir and managing more sustainable winemaking practices. Therefore, the level of yeast genetic variability and the abundance of Saccharomyces cerevisiae native populations in vineyard are becoming more and more crucial at both ecological and technological level. Among the factors that can influence the strain diversity, the commercial starter release that accidentally occur in the environment around the winery, has to be considered. In this study we led a wide scale investigation of S. cerevisiae genetic diversity and population structure in the vineyards of three neighboring winemaking regions of Protected Appellation of Origin, in North-East of Italy. Combining mtDNA RFLP and microsatellite markers analyses we evaluated 634 grape samples collected over 3 years. We could detect major differences in the presence of S. cerevisiae yeasts, according to the winemaking region. The population structures revealed specificities of yeast microbiota at vineyard scale, with a relative Appellation of Origin area homogeneity, and transition zones suggesting a geographic differentiation. Surprisingly, we found a widespread industrial yeast dissemination that was very high in the areas where the native yeast abundance was low. Although geographical distance is a key element involved in strain distribution, the high presence of industrial strains in vineyard reduced the differences between populations. This finding indicates that industrial yeast diffusion it is a real emergency and their presence strongly interferes with the natural yeast microbiota

Isolation, identification and oenological characterization of non-Saccharomyces yeasts in a Mediterranean island

We isolated, identified and characterized yeast strains from grapes, and their fermented musts, sampled in the small island of Linosa, where there are no wineries and therefore the possibility of territory contamination by industrial strains is minimal. By traditional culture-dependent methods, we isolated 3805 colonies, distinguished by molecular methods in 17 different species. 544 isolates were analysed for the main oenological characteristics such as fermentative vigor with and without sulphites, sugar consumption, and production of alcohol, volatile acidity, hydrogen sulphide, glycerol and β-glucosidase. This analysis identified Kluyveromyces marxianus (seldomly used in winemaking) as the most interesting candidate yeast for the production of innovative wines. This article is protected by copyright. All rights reserved

Potential Role of Yeast Strains Isolated from Grapes in the Production of Taurasi DOCG

Twelve samples of Aglianico grapes, collected in different locations of the Taurasi DOCG (Appellation of Controlled and Guaranteed Origin) production area were naturally fermented in sterile containers at room temperature. A total of 70 yeast cultures were isolated from countable WL agar plates: 52 in the middle of the fermentation and 18 at the end. On the basis of ITS-RFLP analysis and ITS sequencing, all cultures collected at the end of fermentations were identified as Saccharomyces (S.) cerevisiae; while, the 52 isolates, collected after one week, could be referred to the following species: Metschnikowia (M.) pulcherrima; Starmerella (Star.) bacillaris; Pichia (P.) kudriavzevii; Lachancea (L.) thermotolerans; Hanseniaspora (H.) uvarum; Pseudozyma (Pseud.) aphidis; S. cerevisiae. By means of Interdelta analysis, 18 different biotypes of S. cerevisiae were retrieved. All strains were characterized for ethanol production, SO2 resistance, H2S development, β-glucosidasic, esterasic and antagonistic activities. Fermentation abilities of selected strains were evaluated in micro-fermentations on Aglianico must. Within non-Saccharomyces species, some cultures showed features of technological interest. Antagonistic activity was expressed by some strains of M. pulcherrima, L. thermotolerans, P. kudriavzevii and S. cerevisiae. Strains of M. pulcherrima showed the highest β-glucosidase activity and proved to be able to produce high concentrations of succinic acid. L. thermotolerans produced both succinic and lactic acids. The lowest amount of acetic acid was produced by M. pulcherrima and L. thermotolerans; while the highest content was recorded for H. uvarum. The strain of Star. bacillaris produced the highest amount of glycerol and was able to metabolize all fructose and malic acid. Strains of M. pulcherrima and H. uvarum showed a low fermentation power (about 4%), while, L. thermotolerans, Star. bacillaris and P. kudriavzevii of about 10%. Significant differences were even detected for S. cerevisiae biotypes with respect to H2S production, antagonistic activity and β-glucosidase activity as well as for the production of acetic acid, glycerol and ethanol in micro-vinification experiments