Culturable yeast community associated with grape must and honey bees sampled from apiaries located in the vineyards.

AIM: In this study, we investigated culturable yeast community, present in grape must sampled from vineyards with apiaries on the borders, and in honey bees collected in these apiaries. METHODS AND RESULTS: To this aim, yeasts isolated from spontaneously fermented grapes randomly collected in two vineyards (P1 and P2) with apiaries on the borders (A1 and A2) were compared to those isolated from spontaneously fermented grapes collected from a vineyard without apiary (P4). At the same time, yeast community was analyzed on bees collected in each apiary placed in the vineyards, in comparison to yeasts isolated from an apiary (A3) located far from the vineyards. The analysis was performed for two consecutive years (2021 and 2022). The isolated yeasts were identified by restriction analysis of amplified ITS region, followed by sequencing of ITS fragment.Our research showed that the presence of apiaries seems to increase yeast counts of grape must, in particular of Saccharomyces cerevisiae; furthermore, the permanence of apiaries in the vineyards allowed the recovering of these yeasts also from bees. CONCLUSIONS: Our findings seem to corroborate the role of bees as vectors and reservoirs of oenologically relevant yeasts, such as a source of non-conventional yeasts with potential biotechnological applications.

Saccharomyces cerevisiae and Hanseniaspora uvarum mixed starter cultures: Influence of microbial/physical interactions on wine characteristics.

The growing trend in the wine industry is the revaluation of the role of non-Saccharomyces yeasts, promoting the use of these yeasts in association with Saccharomyces cerevisiae. Non-Saccharomyces yeasts contribute to improve wine complexity and organoleptic composition. However, the use of mixed starters needs to better understand the effect of the interaction between these species during alcoholic fermentation. The aim of this study is to evaluate the influence of mixed starter cultures, composed by combination of different S. cerevisiae and Hanseniaspora uvarum strains, on wine characteristics and to investigate the role of cell-to-cell contact on the metabolites produced during alcoholic fermentation. In the first step, three H. uvarum and two S. cerevisiae strains, previously selected, were tested during mixed fermentations in natural red grape must in order to evaluate yeast population dynamics during inoculated fermentation and influence of mixed starter cultures on wine quality. One selected mixed starter was tested in a double-compartment fermentor in order to compare mixed inoculations of S. cerevisiae/H. uvarum with and without physical separation. Our results revealed that physical contact between S. cerevisiae and H. uvarum affected the viability of H. uvarum strain, influencing also the metabolic behaviour of the strains. Although different researches are available on the role of cell-to-cell contact-mediated interactions on cell viability of the strains included in the mixed starter, to our knowledge, very few studies have evaluated the influence of cell-to-cell contact on the chemical characteristics of wine.

Identification by phenotypic and genetic approaches of an indigenous Saccharomyces cerevisiae wine strain with high desiccation tolerance.

During active dry yeast (ADY) production process, cells are exposed to multiple stresses, such as thermal, oxidative and hyperosmotic shock. Previously, by analysing cells in exponential growth phase, we selected an indigenous Saccharomyces cerevisiae wine strain, namely CD-6Sc, for its higher tolerance to desiccation and higher expression of specific desiccation stress-related genes in comparison to other yeast strains. In this study, we performed a desiccation treatment on stationary phase cells by comparing the efficacy of two different methods: a 'laboratory dry test' on a small scale (mild stress) and a treatment by spray-drying (severe stress), one of the most appropriate preservation method for yeasts and other micro-organisms. The expression of selected desiccation-related genes has been also assessed in order to validate predictive markers for desiccation tolerance. Our data demonstrate that the 'mild' and the 'severe' desiccation treatments give similar results in terms of cell recovery, but the choice of marker genes strictly depends on the growth phase in which cells undergo desiccation. The indigenous CD-6Sc was ultimately identified as a high dehydration stress-tolerant indigenous strain suitable for ADY production. This study highlights the exploitation of natural yeast biodiversity as a source of hidden technological features and as an alternative approach to strain improvement by genetic modifications. Copyright © 2017 John Wiley & Sons, Ltd.

Comparative study of Saccharomyces cerevisiae wine strains to identify potential marker genes correlated to desiccation stress tolerance.

The most diffused formulation of starter for winemaking is active dry yeast (ADY). ADYs production process is essentially characterized by air-drying stress, a combination of several stresses, including thermal, hyperosmotic and oxidative and cell capacity to counteract such multiple stresses will determine its survival. The molecular mechanisms underlying cell stress response to desiccation have been mostly studied in laboratory and commercial yeast strains, but a growing interest is currently developing for indigenous yeast strains which represent a valuable and alternative source of genetic and molecular biodiversity to be exploited. In this work, a comparative study of different Saccharomyces cerevisiae indigenous wine strains, previously selected for their technological traits, has been carried out to identify potentially relevant genes involved in desiccation stress tolerance. Cell viability was evaluated along desiccation treatment and gene expression was analyzed by real-time PCR before and during the stress. Our data show that the observed differences in individual strain sensitivity to desiccation stress could be associated to specific gene expression over time. In particular, either the basal or the stress-induced mRNA levels of certain genes, such as HSP12, SSA3, TPS1, TPS2, CTT1 and SOD1, result tightly correlated to the strain survival advantage. This study provides a reliable and sensitive method to predict desiccation stress tolerance of indigenous wine yeast strains which could be preliminary to biotechnological applications.

Implementing principles of traditional concentrated grape must fermentation to the production of new generation balsamic vinegars. Starter selection and effectiveness.

In an effort to implement principles of traditional concentrated grape must fermentation to the production of new generation balsamic vinegars (BVs), the specific goals of the study were the isolation and molecular identification of the predominant yeasts in concentrated grape must (cv. Xinomavro), their technological characterization and the evaluation of the fermentative aptitude of the selected strains. Tolerance against 5-hydroxymethyl-furfural (HMF) and furfural, acetic acid and glucose concentration was examined by appropriate methods and tests. The enological characteristics studied were acetic acid and H2S production, foaming and flocculation ability and key enzymatic activity. PCR-RFLP analysis revealed only the presence of Saccharomyces cerevisiae and Hanseniaspora uvarum among the 14 predominant osmophilic yeast isolates. Tolerance to both HMF and furfural was found strain- and dose-dependent and was suggested as a critical factor in the pre-selection of yeast starters. The most tolerant yeasts to these stress factors, a S. cerevisiae and a non-Saccharomyces strains, showed satisfactory growth in the presence of high glucose and acetic acid content (up to 600 g/L and 2 % w/w, respectively) and desirable enological characteristics. Results from the comparative evaluation of the fermentative aptitude of these strains with a commercial wine strain highlighted that the isolates had glucophilic behaviour and ability to produce desirable amounts of ethanol (100-120 g/kg) in short time (~20 d). The key volatiles useful for varietal discrimination and differentiation between the BVs and the traditional ones were also evaluated.

Impact of yeast starter formulations on the production of volatile compounds during wine fermentation.

The most diffused starter formulation in winemaking is actually represented by active dry yeast (ADY). Spray-drying has been reported as an appropriate preservation method for yeast and other micro-organisms. Despite the numerous advantages of this method, the high air temperatures used can negatively affect cell viability and the fermentative performance of dried cells. In the present study, 11 wine S. cerevisiae strains (both indigenous and commercial) were submitted to spray-drying; different process conditions were tested in order to select the conditions allowing the highest strain survival. The strains exhibited high variability for tolerance to spray-drying treatment. Selected strains were tested in fermentation at laboratory scale in different formulations (free fresh cells, free dried cells, immobilized fresh cells and immobilized dried cells), in order to assess the influence of starter formulation on fermentative fitness of strains and aromatic quality of wine. The analysis of volatile fraction in the experimental wines produced by selected strains in different formulations allowed identification of > 50 aromatic compounds (alcohols, esters, ketones, aldehydes and terpenes). The results obtained showed that the starter formulation significantly influenced the content of volatile compounds. In particular, the wines obtained by strains in dried forms (as both free and immobilized cells) contained higher numbers of volatile compounds than wines obtained from fresh cells.

Preliminary investigation on the potential involvement of an ABC-like gene in Halomicronema metazoicum (Cyanobacteria) tolerance to low seawater pH in an ocean acidification scenario.

Decreasing ocean surface pH, called ocean acidification (OA), is among the major risks for marine ecosystems due to human-driven atmospheric pCO2 increase. Understanding the molecular mechanisms of adaptation enabling marine species to tolerate a lowered seawater pH could support predictions of consequences of future OA scenarios for marine life. This study examined whether the ATP-binding cassette (ABC)-like gene slr2019 confers tolerance to the marine cyanobacterium Halomicronema metazoicum to low seawater pH conditions (7.7, 7.2, 6.5) in short- and long-term exposures (7 and 30 d). Photosynthetic pigment content indicated that the species can tolerate all three lowered-pH conditions. At day 7, slr2019 was up-regulated at pH 7.7 while no changes were observed at lower pH. After 30-d exposure, a significant decrease in slr2019 transcript levels was observed in all low-pH treatments. These first results indicate an effect of low pH on the examined transporter expression in H. metazoicum.

Genetic improvement of Saccharomyces cerevisiae wine strains for enhancing cell viability after desiccation stress.

In the last few decades spontaneous grape must fermentations have been replaced by inoculated fermentation with Saccharomyces cerevisiae strains as active dry yeast (ADY). Among the essential genes previously characterized to overcome the cell-drying/rehydration process, six belong to the group of very hydrophilic proteins known as hydrophilins. Among them, only SIP18 has shown early transcriptional response during dehydration stress. In fact, the overexpression in S. cerevisiae of gene SIP18 increases cell viability after the dehydration process. The purpose of this study was to characterize dehydration stress tolerance of three wild and one commercial S. cerevisiae strains of wine origin. The four strains were submitted to transformation by insertion of the gene SIP18. Selected transformants were submitted to the cell-drying-rehydration process and yeast viability was evaluated by both viable cell count and flow cytometry. The antioxidant capacity of SIP18p was illustrated by ROS accumulation reduction after H2 O2 attack. Growth data as cellular duplication times and lag times were calculated to estimate cell vitality after the cell rehydration process. The overexpressing SIP18 strains showed significantly longer time of lag phase despite less time needed to stop the leakage of intracellular compounds during the rehydration process. Subsequently, the transformants were tested in inoculated grape must fermentation at laboratory scale in comparison to untransformed strains. Chemical analyses of the resultant wines indicated that no significant change for the content of secondary compounds was detected. The obtained data showed that the transformation enhances the viability of ADY without affecting fermentation efficiency and metabolic behaviour.

Indigenous yeast population from Georgian aged wines produced by traditional "Kakhetian" method.

The yeast microbiota present in wines produced by the ancient "Kakhetian" method in Georgia (EU) was studied. This technique involves the use of terracotta vessels (amphoras), during spontaneous fermentation, maceration phase and wine ageing. The analysed yeasts were collected from wines after maturation for one year in ten amphoras from a Georgian winery. The 260 isolates were all identified as Saccharomyces cerevisiae, and the majority were classified as flor yeasts by restriction analysis of ITS region. A first technological and molecular screening was used to select 70 strains for further characterization. Both genetic and metabolic characterization discriminated flor from non-flor strains. The combined results obtained by analysis of interdelta region and mtDNA-RFLP yielded 23 different biotypes; no biotype was common to flor and non-flor strains. The wines produced by flor yeasts showed a high content in acetaldehyde, acetic acid, acetoin, whereas the level of other compounds was similar to wines obtained by non-flor strains. This study represents the first report on the composition of yeast microbiota involved in the maturation of this traditional wine. These flor strains represent an interesting yeast population, in possession of peculiar characteristics allowing them to survive during wine ageing, becoming the dominant flora in the final wine.

Role of Yeasts on the Sensory Component of Wines.

The aromatic complexity of a wine is mainly influenced by the interaction between grapes and fermentation agents. This interaction is very complex and affected by numerous factors, such as cultivars, degree of grape ripeness, climate, mashing techniques, must chemical−physical characteristics, yeasts used in the fermentation process and their interactions with the grape endogenous microbiota, process parameters (including new non-thermal technologies), malolactic fermentation (when desired), and phenomena occurring during aging. However, the role of yeasts in the formation of aroma compounds has been universally recognized. In fact, yeasts (as starters or naturally occurring microbiota) can contribute both with the formation of compounds deriving from the primary metabolism, with the synthesis of specific metabolites, and with the modification of molecules present in the must. Among secondary metabolites, key roles are recognized for esters, higher alcohols, volatile phenols, sulfur molecules, and carbonyl compounds. Moreover, some specific enzymatic activities of yeasts, linked above all to non-Saccharomyces species, can contribute to increasing the sensory profile of the wine thanks to the release of volatile terpenes or other molecules. Therefore, this review will highlight the main aroma compounds produced by Saccharomyces cerevisiae and other yeasts of oenological interest in relation to process conditions, new non-thermal technologies, and microbial interactions.

In Vitro Study of Probiotic, Antioxidant and Anti-Inflammatory Activities among Indigenous Saccharomyces cerevisiae Strains.

Nowadays, the interest toward products containing probiotics is growing due to their potential health benefits to the host and the research is focusing on search of new probiotic microorganisms. The present work was focused on the characterization of indigenous Saccharomyces cerevisiae strains, isolated from different food matrixes, with the goal to select strains with probiotic or health-beneficial potential. A preliminary screening performed on fifty S. cerevisiae indigenous strains, in comparison to a commercial probiotic strain, allowed to individuate the most suitable ones for potential probiotic aptitude. Fourteen selected strains were tested for survival ability in the gastrointestinal tract and finally, the strains characterized for the most important probiotic features were analyzed for health-beneficial traits, such as the content of glucan, antioxidant and potential anti-inflammatory activities. Three strains, 4LBI-3, LL-1, TA4-10, showing better attributes compared to the commercial probiotic S.cerevisiae var. boulardii strain, were characterized by interesting health-beneficial traits, such as high content of glucan, high antioxidant and potential anti-inflammatory activities. Our results suggest that some of the tested S. cerevisiae strains have potential as probiotics and candidate for different applications, such as dietary supplements, and starter for the production of functional foods or as probiotic to be used therapeutically.

Screening of Saccharomyces and Non-Saccharomyces Wine Yeasts for Their Decarboxylase Activity of Amino Acids.

The type and quantity of precursor amino acids present in grape must that are used by wine yeasts affect the organoleptic and health properties of wine. The aim of this work was to conduct a preliminary screening among Saccharomyces and non-Saccharomyces indigenous strains, which were previously isolated from different Italian regional grape varieties. This was performed in order to evaluate their decarboxylase activity on certain important amino acids-such as arginine, proline, serine, and tyrosine-that are present in grape must. In particular, a qualitative test on 122 wine yeasts was performed on a decarboxylase medium using arginine, proline, serine, and tyrosine as precursor amino acids. Our results showed a considerable variability among the microbial species tested for this parameter. Indeed, Saccharomyces cerevisiae strains exhibited a high decarboxylase capability of the four amino acids tested; moreover, only 10% of the total (i.e., a total of 81) did not show this trait. A high recovery of decarboxylation ability for at least one amino acid was also found for Zygosaccharomyces bailii and Hanseniaspora spp. These findings can, therefore, promote the inclusion of decarboxylase activity as an additional characteristic in a wine yeast selection program in order to choose starter cultures that possess desirable technological traits; moreover, this also can contribute to the safeguarding of consumer health.

Impact of Starmerella bacillaris and Zygosaccharomyces bailii on ethanol reduction and Saccharomyces cerevisiae metabolism during mixed wine fermentations.

The bulk of grape juice fermentation is carried out by the yeast Saccharomyces cerevisiae, but non-Saccharomyces yeasts can modulate many sensorial aspects of the final products in ways not well understood. In this study, some of such non-conventional yeasts were screened as mixed starter cultures in a defined growth medium in both simultaneous and sequential inoculations. One strain of Starmerella bacillaris and another of Zygosaccharomyces bailii were chosen by their distinct phenotypic footprint and their ability to reduce ethanol levels at the end of fermentation. S. bacillaris losses viability strongly at the end of mixed fermentations, while Z. bailii remains viable. S. cerevisiae viability was unchanged by the presence of the other yeasts. Physiological characterization of both strains indicates that S. bacillaris behavior is overall more different from S. cerevisiae than Z. bailii. In addition, S. cerevisiae transcriptome changes to a bigger degree in the presence of S. bacillaris in comparison to mixed fermentation with Z. bailii. S. bacillaris induces the translation machinery and repress vesicular transport. Both non-Saccharomyces yeasts induce S. cerevisiae glycolytic genes, and that may be related to ethanol lowering, but some aspects of carbon-related mechanisms are specific for each strain. Z. bailii presence increases the stress-related polysaccharides trehalose and glycogen, while S. bacillaris induces gluconeogenesis genes.

Validation of a Standard Protocol to Assess the Fermentative and Chemical Properties of Saccharomyces cerevisiae Wine Strains.

This paper reports on a common experiment performed by 17 Research Units of the Italian Group of Microbiology of Vine and Wine (GMVV), which belongs to the Scientific Society SIMTREA, with the aim to validate a protocol for the characterization of wine strains of Saccharomyces cerevisiae. For this purpose, two commercial S. cerevisiae strains (EC 1118 and AWRI796) were used to carry out inter-laboratory-scale comparative fermentations using both synthetic medium and grape musts and applying the same protocol to obtain reproducible, replicable, and statistically valid results. Ethanol yield, production of acetic acid, glycerol, higher alcohols, and other volatile compounds were assessed. Moreover, the Fourier transform infrared spectroscopy was also applied to define the metabolomic fingerprint of yeast cells from each experimental trial. Data were standardized as unit of compounds or yield per gram of sugar (glucose and fructose) consumed throughout fermentation, and analyzed through parametric and non-parametric tests, and multivariate approaches (cluster analysis, two-way joining, and principal component analysis). The results of experiments carried out by using synthetic must showed that it was possible to gain comparable results from three different laboratories by using the same strains. Then, the use of the standardized protocol on different grape musts allowed pointing out the goodness and the reproducibility of the method; it showed the main traits of the two yeast strains and allowed reducing variability amongst independent batches (biological replicates) to acceptable levels. In conclusion, the findings of this collaborative study contributed to the validation of a protocol in a specific synthetic medium and in grape must and showed how data should be treated to gain reproducible and robust results, which could allow direct comparison of the experimental data obtained during the characterization of wine yeasts carried out by different research laboratories.

Geographical markers for Saccharomyces cerevisiae strains with similar technological origins domesticated for rice-based ethnic fermented beverages production in North East India.

Autochthonous strains of Saccharomyces cerevisiae from traditional starters used for the production of rice-based ethnic fermented beverage in North East India were examined for their genetic polymorphism using mitochondrial DNA-RFLP and electrophoretic karyotyping. Mitochondrial DNA-RFLP analysis of S. cerevisiae strains with similar technological origins from hamei starter of Manipur and marcha starter of Sikkim revealed widely separated clusters based on their geographical origin. Electrophoretic karyotyping showed high polymorphism amongst the hamei strains within similar mitochondrial DNA-RFLP cluster and one unique karyotype of marcha strain was widely distributed in the Sikkim-Himalayan region. We conceptualized the possibility of separate domestication events for hamei strains in Manipur (located in the Indo-Burma biodiversity hotspot) and marcha strains in Sikkim (located in Himalayan biodiversity hotspot), as a consequence of less homogeneity in the genomic structure between these two groups, their clear separation being based on geographical origin, but not on technological origin and low strain level diversity within each group. The molecular markers developed based on HinfI-mtDNA-RFLP profile and the chromosomal doublets in chromosome VIII position of Sikkim-Himalayan strains could be effectively used as geographical markers for authenticating the above starter strains and differentiating them from other commercial strains.

Yeasts isolated from olive mill wastewaters from southern Italy: technological characterization and potential use for phenol removal.

Olive mill wastewater (OMW) samples from two traditional varieties (Peranzana and Ogliarola Garganica) of Apulian region (southern Italy) and produced through continuous and traditional methods were microbiologically and chemically examined; thus, 104 yeasts were isolated and selected for further analyses. The strains were identified as Candida boidinii, Pichia holstii, Pichia membranifaciens, and Saccharomyces cerevisiae and analyzed to assess their suitability to metabolize phenols. Based on phenol metabolism, 27 strains were selected and inoculated into OMW aliquots to determine their ability to reduce phenols in vivo; then, five strains (identified with the codes 682-C. boidinii and 625, 642, 647, and 941-P. holstii) were used as a cocktail in wastewaters for a final validation step. In this last experiment, the effects of the temperature (10-30 degrees C) and (NH(4))(2)SO(4) (0.0-6.0 g l(-1)) were studied through a central composite design approach, and the results highlighted that the cocktail was able to reduce phenols by 40% at 10 degrees C with 6.0 g l(-1) of (NH(4))(2)SO(4) added.

Biotechnological Approach Based on Selected Saccharomyces cerevisiae Starters for Reducing the Use of Sulfur Dioxide in Wine.

Sulfites are considered the main additives in winemaking for their antimicrobial, antioxidant and anti-oxidasic activities. The current concern about the potential negative effects of sulfur dioxide (SO2) on consumer health has focused the interest on replacing or reducing SO2 use. Our work aims to develop a strategy based on the use of selected starter culture, able to perform wine fermentation without SO2 addition. Four selected Saccharomyces cerevisiae indigenous strains were tested as mixed starter cultures in laboratory scale fermentations. The starter culture, characterized by a similar percentage of dominance of both strains composing the mixed starter and able to produce a wine characterized by the best combination of chemical and aromatic characteristics, was chosen. This mixed culture was tested as a starter at pilot scale with and without SO2 addition, by using a higher inoculum level in the vinification without SO2. The selected starter confirmed higher dominance ability in vinification without SO2 addition than in SO2-added fermentation, demonstrating that sulfite addition is not a guarantee to reach an absolute dominance of starter culture on indigenous microflora. The proposed biotechnological tool allowed to produce good quality wines possessing also "functional properties", as NO-SO2 added wines were characterized by high polyphenol content and antioxidant activity.

Corrigendum: Genome Sequencing and Comparative Analysis of Three Hanseniaspora uvarum Indigenous Wine Strains Reveal Remarkable Biotechnological Potential.

[This corrects the article DOI: 10.3389/fmicb.2019.03133.].

SPME-GC method as a tool to differentiate VOC profiles in Saccharomyces cerevisiae wine yeasts.

The aim of this work was to study the variability of 36 Saccharomyces cerevisiae wild strains isolated from different grape varieties and from two very distant zones, located in Northern and Southern Italy. The strains were differentiated on the basis of parameters of technological interest, such as resistance to antimicrobial compounds frequently present in wine, and the production of volatile aromatic compounds (VOC), determined by SPME procedure in the experimental wines obtained by inoculated fermentations. The VOC profile allowed to differentiate the yeasts in function of isolation area: S. cerevisiae isolated from Southern Italy grapes were able to produce more volatile compounds than those from Northern Italy. The compounds synthesized by all the yeasts, besides the ethanol, were 3-methyl-1-butanol and ethyl acetate. The production of acids during the alcoholic fermentation was a characteristic of Southern yeast strains. The screening of S. cerevisiae strains for technological parameters, such as sulphur dioxide, copper and ethanol resistance or hydrogen sulphide production, revealed similar behaviour for sulphur dioxide resistance among Northern and Southern S. cerevisiae strains. Copper resistance and sulphur dioxide production were correlated to isolation area: S. cerevisiae "Northern" strains showed higher copper resistance and lowest hydrogen sulphide production than that exhibited from "Southern" strains.

Genome Sequencing and Comparative Analysis of Three Hanseniaspora uvarum Indigenous Wine Strains Reveal Remarkable Biotechnological Potential.

A current trend in winemaking has highlighted the beneficial contribution of non-Saccharomyces yeasts to wine quality. Hanseniaspora uvarum is one of the more represented non-Saccharomyces species onto grape berries and plays a critical role in influencing the wine sensory profile, in terms of complexity and organoleptic richness. In this work, we analyzed a group of H. uvarum indigenous wine strains as for genetic as for technological traits, such as resistance to SO2 and ß-glucosidase activity. Three strains were selected for genome sequencing, assembly and comparative genomic analyses at species and genus level. Hanseniaspora genomes appeared compact and contained a moderate number of genes, while rarefaction analyses suggested an open accessory genome, reflecting a rather incomplete representation of the Hanseniaspora gene pool in the currently available genomes. The analyses of patterns of functional annotation in the three indigenous H. uvarum strains showed distinct enrichment for several PFAM protein domains. In particular, for certain traits, such as flocculation related protein domains, the genetic prediction correlated well with relative flocculation phenotypes at lab-scale. This feature, together with the enrichment for oligo-peptide transport and lipid and amino acid metabolism domains, reveals a promising potential of these indigenous strains to be applied in fermentation processes and modulation of wine flavor and aroma. This study also contributes to increasing the catalog of publicly available genomes from H. uvarum strains isolated from natural grape samples and provides a good roadmap for unraveling the biodiversity and the biotechnological potential of these non-Saccharomyces yeasts.