Enological Traits Research Articles

Exploring wine yeast natural biodiversity to select strains with enological traits adapted to climate change

Wine is widely consumed throughout the world and represents a significant financial market, but production faces increasing challenges. While consumers progressively value more complex flavor profiles, regional authenticity, and decreased use of additives, winemakers strive for consistency among climate change, characterized by rising environmental temperatures and sun burn events. This often leads to grapes reaching phenolic maturity with higher sugar levels, and increased microbial spoilage risk.Herein, we addressed these dual concerns by investigating the use of autochthonous Saccharomyces cerevisiae strains for fermentations of grape musts resulting from these altered conditions. We characterized underexplored repositories of naturally-occurring strains isolated from different environments and geographical regions, regarding adequate enological properties (e.g., high cell growth, reduced production of H2S, ethanol and acetic acid, increased SO2 resistance, killer activity), and other less frequently investigated properties (resistance to osmotic stress, potassium and aluminium silicates and fungicides). The phenotypic data were organized in a biobank, and bioinformatic analysis grouped the strains according to their characteristics. Furthermore, we analyzed the potential of four Portuguese isolates to be used in fermentations of grape musts with high sugar levels, uncovering promising candidates. This research therefore contributes to ongoing efforts to increase sustainability and quality of wine production.

Yeast Diversity in Wine Grapes from Japanese Vineyards and Enological Traits of Indigenous Saccharomyces cerevisiae Strains.

Japan has numerous vineyards with distinct geographical and climatic conditions. To the best of our knowledge, there is no comprehensive analysis of the diversity of yeasts associated with wine grapes from Japan. This study aimed to determine yeast diversity in wine grapes from four wine-producing regions in Japan and to evaluate the physicochemical characteristics of wines produced with indigenous Saccharomyces cerevisiae strains isolated from two regions. A total of 2648 strains were isolated from nine wine grape samples. MALDI-TOF MS and 26S rDNA sequence analyses revealed that the strains belonged to 21 non-Saccharomyces yeasts and 1 Saccharomyces yeast (S. cerevisiae). Non-Saccharomyces yeasts were found in high quantities and were highly distributed among the wine grape samples. Differences in the distribution of the identified yeast species were noted among the different wine grape varieties and regions. Indigenous S. cerevisiae strains of different genotypes from different regions exhibit distinct physiological traits. Our findings are expected to enhance our understanding of the local yeasts associated with Japanese vineyards and contribute to obtaining cultures that can provide region-specific organoleptic characteristics to local wines produced in Japan.

How to modulate mechanical winter pruning according to the productive and enological traits of four Vitis vinifera L. varieties cultivated in the north of Italy

How to modulate mechanical winter pruning according to the productive and enological traits of four <i>Vitis vinifera</i> L. varieties cultivated in the north of Italy

Selection of Native Non-Saccharomyces Yeasts with Biocontrol Activity against Spoilage Yeasts in Order to Produce Healthy Regional Wines

Two major spoilage yeasts in the wine industry, Brettanomyces bruxellensis and Zygosaccharomyces rouxii, produce off-flavors and gas, causing considerable economic losses. Traditionally, SO2 has been used in winemaking to prevent spoilage, but strict regulations are in place regarding its use due to its toxic and allergenic effects. To reduce its usage researchers have been searching for alternative techniques. One alternative is biocontrol, which can be used either independently or in a complementary way to chemical control (SO2). The present study analyzed 122 native non-Saccharomyces yeasts for their biocontrol activity and their ability to be employed under fermentation conditions, as well as certain enological traits. After the native non-Saccharomyces yeasts were assayed for their biocontrol activity, 10 biocontroller yeasts were selected and assayed for their ability to prevail in the fermentation medium, as well as with respect to their corresponding positive/negative contribution to the wine. Two yeasts that satisfy these characteristics were Wickerhamomyces anomalus BWa156 and Metschnikowia pulcherrima BMp29, which were selected for further research in application to mixed fermentations.

Selection of non-Saccharomyces yeasts for orange wine fermentation based on their enological traits and volatile compounds formation.

In order to select the non-Saccharomyces yeasts for orange wine fermentation, the enological traits and volatile compounds formation of ten non-Saccharomyces yeast strains were evaluated through physicochemical methods and solid-phase microextraction coupled to GC-MS, respectively. The results indicated that non-Saccharomyces yeast fermentation had lower maximum populations (7.8-8.0Logcfu/mL), longer fermentation period (7-10days), lower ethanol (4.13-7.79%), lower total acids (7.48-8.51g/L) and higher volatile acids concentrations (0.08-0.23g/L) when compared with those of Saccharomyces cerevisiae fermentation. Hanseniaspora uvarum, Hanseniaspora opuntiae, Hanseniaspora occidentalis, Pichia kudriavzevii and Torulaspora delbrueckii were selected as candidates for orange wine fermentation with higher volatile compounds concentration, odor active values and sensory evaluation scores. This study will provide a valuable selection method of non-Saccharomyces yeasts for orange wine fermentation, and an approach to improve the flavor of orange wine or other fruit wine.

Enological Qualities and Interactions Between Native Yeast and Lactic Acid Bacteria from Queretaro, Mexico

An alternative scheme is proposed for the joint selection of yeast and lactic acid bacteria strains, which allows us to foresee the interactions that may occur between them during winemaking. The kinetic parameters, turbidimetrically measured and analyzed by multivariate methods, simplify the detection of outstanding selectable microorganisms. This methodology can be implemented at any cellar or even any fermentative industry that aims to select compatible yeast and lactic acid bacteria.

Biodiversity and Enological Potential of Non-Saccharomyces Yeasts from Nemean Vineyards

Vineyards in Nemea, the most important viticultural zone in Greece, were surveyed for indigenous non-Saccharomyces (NS) yeasts of enological potential. NS populations were isolated from the final stage of alcoholic fermentation and identified by a range of molecular methods. The enological profiles of Hanseniaspora guilliermondii, H. osmophila, Lachancea thermotolerans, Starmerella bacillaris and Torulaspora delbrueckii strains were evaluated. Significant interspecies variation was observed in fermentation kinetics. H. osmophila and T. delbrueckii showed the highest capacity for prompt initiation of fermentation, while S. bacillaris achieved a higher fermentation rate in the second half of the process. Significant differences were also observed in the chemical parameters of NS strains. S. bacillaris SbS42 and T. delbrueckii TdS45 were further evaluated in mixed-culture fermentations with Saccharomyces cerevisiae. NS strains achieved lower population densities than S. cerevisiae. SbS42 exhibited a higher death rate than TdS45. The chemical profiles of different ferments were separated by principal component analysis (PCA). Both NS strains were associated with lower levels of ethanol, when compared to single S. cerevisiae inoculation. TdS45 increased the ethyl acetate levels, while SbS42 caused a different production pattern of higher alcohols. This is the first report to explore the enological potential of NS wine yeast populations from Nemea. Based on prominent enological traits identified, the selected S. bacillaris and T. delbrueckii strains may be further exploited as co-culture starters for improving the quality and enhancing the regional character of local wines.

Molecular and Phenotypic Characterization of Metschnikowia pulcherrima Strains from Douro Wine Region

Some non-Saccharomyces yeasts, including Metschnikowia pulcherrima, have been proposed as selected starters due to their contribution for the overall aroma and chemical profiles of wines. In this work, we aimed to evaluate the genetic and phenotypic diversity of Metschnikowia pulcherrima strains isolated from different locations of Douro Wine Region, and to explore their potential as co-adjuncts of S. cerevisiae in alcoholic fermentation. For that purpose, a set of 64 M. pulcherrima isolates were used. Polymerase chain reaction (PCR) fingerprinting with M13 primers demonstrated to be an efficient tool in intraspecific discrimination of M. pulcherrima strains. No significant associations were found between genotypic profiles and either geographical origin or winery. The isolates were screened for their stress resistance ability (ethanol, SO2, chitosan, copper, H2O2, and Grape Juice Medium), aroma-related activities (resistance to 5, 5′, 5′′-trifluor-d, l-leucine and cerulenin and β-glycosidase, β-lyase and sulfite-reductase activities) as well as other relevant technological proprieties (protease activity and biogenic amines production). M. pulcherrima response to the different enological traits evaluated was greatly strain-dependent. The most discriminant features were the ability of the strains to grow in Grape-Juice Medium (GJM) and sulfite-reductase, and their β-lyase and protease activities. The enological potential of a selected M. pulcherrima strain in mixed-culture with S. cerevisiae was also assessed in natural grape-juice of a local variety, under two nitrogen regimes. M. pulcherrima proved to be promising for future industrial application as a co-starter, lowering ethanol, acetic acid and, reported here for the first time, lowering hydrogen sulfide levels in the wines.

Detection system for Saccharomyces cerevisiae with phenyl acrylic acid decarboxylase gene (PAD1) and sulphur efflux gene (SSU1) by multiplex PCR

Production of wine with selected yeast strains is a common enological practice followed for the production of wine with desirable organoleptic properties and to guarantee the homogeneity of successive vintages. Sulphur dioxide tolerance and phenyl acrylic acid resistance are the enological traits essential for the survivability of the yeast during fermentation. The present study describes the detection of S. cerevisiae with enological traits, such as phenyl acrylic acid resistance and sulphur dioxide tolerance in a single test. Phenyl acrylic acid decarboxylase (PAD1) and sulphite efflux genes (SSU1) were detected by multiplex PCR, thus confirming the specificity of primers. A single cocktail of all reagents required for the simultaneous detection of both these genes was designed. The ready-to-use formulation optimized was stable at 4 and - 20°C for 6months. The amplification of phenyl acrylic acid decarboxylase and sulphite efflux genes, validated the suitability of the ready-to-use formulation for the detection of S. cerevisiae in food samples. The ready-to-use formulation optimized, minimizes the end user requirements for the detection of S. cerevisiae. Thus, the method was suitable for the identification of S. cerevisiae strains from a mixture of yeast prior to the sequencing analysis, thereby reducing the cost and time of screening.

Modern technology homogenizes enological traits of indigenous Saccharomyces cerevisiae strains associated with Msalais, a traditional wine in China.

In this study, we performed a pilot-scale evaluation of the enological characteristics of indigenous Saccharomyces cerevisiae strains associated with Msalais, a traditional Chinese wine produced by a unique technology of boiling grape juice prior to spontaneous fermentation. Technical and sensory characteristics of top ten indigenous strains previously identified by us by screening a collection of 436 indigenous S. cerevisiae strains (Zhu et al. 2016) were assayed in a traditional craft workshop (TCW) and a modern plant (MP). The use of these strains reduced the spontaneous fermentation (Spo F) period by 6-15days, and resulted in higher sugar and lower alcohol content in TCW Msalais than in MP Msalais. Sensory scores of Msalais fermented by the ten strains were higher than those of wine produced with a commercial strain cy3079, varying in TCW fermentations and significantly different from Spo F, but homogenous for all MP fermentations. Four strains were extensively screened for use in industrial Msalais production. We conclude that modern technology homogenizes enological traits of indigenous strains while traditional craftsmanship maintains their enological diversity. Some strains domesticated in the course of both processes are suitable for industrial Msalais production.

The genetic architecture of low-temperature adaptation in the wine yeast Saccharomyces cerevisiae

BackgroundLow-temperature growth and fermentation of wine yeast can enhance wine aroma and make them highly desirable traits for the industry. Elucidating response to cold in Saccharomyces cerevisiae is, therefore, of paramount importance to select or genetically improve new wine strains. As most enological traits of industrial importance in yeasts, adaptation to low temperature is a polygenic trait regulated by many interacting loci.ResultsIn order to unravel the genetic determinants of low-temperature fermentation, we mapped quantitative trait loci (QTLs) by bulk segregant analyses in the F13 offspring of two Saccharomyces cerevisiae industrial strains with divergent performance at low temperature. We detected four genomic regions involved in the adaptation at low temperature, three of them located in the subtelomeric regions (chromosomes XIII, XV and XVI) and one in the chromosome XIV. The QTL analysis revealed that subtelomeric regions play a key role in defining individual variation, which emphasizes the importance of these regions’ adaptive nature.ConclusionsThe reciprocal hemizygosity analysis (RHA), run to validate the genes involved in low-temperature fermentation, showed that genetic variation in mitochondrial proteins, maintenance of correct asymmetry and distribution of phospholipid in the plasma membrane are key determinants of low-temperature adaptation.

Biocontrol Ability and Action Mechanism of Starmerella bacillaris (Synonym Candida zemplinina) Isolated from Wine Musts against Gray Mold Disease Agent Botrytis cinerea on Grape and Their Effects on Alcoholic Fermentation.

Gray mold is one of the most important diseases of grapevine in temperate climates. This plant pathogen affects plant growth and reduces wine quality. The use of yeasts as biocontrol agents to apply in the vineyard have been investigated in recent years as an alternative to agrochemicals. In this work, fermenting musts obtained from overripe grape berries, therefore more susceptible to infection by fungal pathogens such as Botrytis cinerea, were considered for the selection of yeasts carrying antifungal activity. Thirty-six isolates were identified as Starmerella bacillaris, a species recently proven to be of enological interest. Among them 14 different strains were studied and antifungal activity against B. cinerea was demonstrated, for the first time, to be present in S. bacillaris species. The production of volatile organic compounds (VOCs), tested in vitro, was found to be the main responsible of S. bacillaris antifungal effects. All the strains were able to reduce B. cinerea decay on wounded grape berries artificially inoculated with gray mold. The colonization level of wound was very high reaching, after 5 days, a concentration of 106 cells per ml of grape juice obtained after berry crushing. At this cell concentration S. bacillaris strains were used to ferment synthetic and natural musts. The sequential yeast inoculation, performed by adding S. cerevisiae 48 h after S. bacillaris, was needed to complete sugar consumption and determined a significant increase in glicerol content and a reduction of ethanol and acetic acid concentrations. The high wound colonization ability, found in this work, together with the propensity to colonize grape berry and the interesting enological traits possessed by the selected S. bacillaris strains allow the use of this yeast as biocontrol agent on vine and grape berries with possible positive effects on must fermentation, although the presence of S. cerevisiae is needed to complete the fermentation process. This work introduces new possibilities in wine yeast selection programs in order to identify innovative wine yeasts that are simultaneously antifungal agents in vineyards and alternative wine starters for grape must fermentation and open new perspective to a more integrated strategy for increasing wine quality.

The contribution of indigenous non-Saccharomyces wine yeast to improved aromatic quality of Cabernet Sauvignon wines by spontaneous fermentation

In this study, the aromatic profiles of Cabernet Sauvignon wine and the diversity of non-Saccharomyces yeast in industrial-scale spontaneous fermentation (SF) and inoculated fermentation (IF) were investigated. The wine obtained from SF had distinct aromatic profiles compared with IF wine. SF wine was characterized by a higher amount of medium-chain fatty acids, esters, terpenes (trans-rose oxide) and C13-norisoprenoids (β-damascenone), whereas IF wine featured more aldehydes and higher alcohols. Five non-Saccharomyces yeast species, including Hanseniaspora uvarum, Candida stella, Pichia fermentans, Issatchenkia orientalis and Metschnikowia pulcherrima, were isolated from SF, and their enological traits were examined in mixed fermentation with Saccharomyces cerevisiae, respectively. The mixed culture of P. fermentans and C. stella with S. cerevisiae could generate higher levels of esters, fatty acids and norisoprenoids compared to the monoculture of S. cerevisiae, and a co-culture of M. pulcherrima, I. orientalis and H. uvarum could accumulate higher amounts of higher alcohols. Our results suggested that non-Saccharomyces yeast strains greatly contributed to the fruity aroma quality of SF wine, and the mixed culture of Saccharomyces yeasts with non-Saccharomyces yeasts such as P. fermentans, C. stella or M. pulcherrima is a valuable tool to modulate the volatile profiles and improve the aromatic complexity of wine.

Next Generation Mapping of Enological Traits in an F2 Interspecific Grapevine Hybrid Family.

In winegrapes (Vitis spp.), fruit quality traits such as berry color, total soluble solids content (SS), malic acid content (MA), and yeast assimilable nitrogen (YAN) affect fermentation or wine quality, and are important traits in selecting new hybrid winegrape cultivars. Given the high genetic diversity and heterozygosity of Vitis species and their tendency to exhibit inbreeding depression, linkage map construction and quantitative trait locus (QTL) mapping has relied on F1 families with the use of simple sequence repeat (SSR) and other markers. This study presents the construction of a genetic map by single nucleotide polymorphisms identified through genotyping-by-sequencing (GBS) technology in an F2 mapping family of 424 progeny derived from a cross between the wild species V. riparia Michx. and the interspecific hybrid winegrape cultivar, ‘Seyval’. The resulting map has 1449 markers spanning 2424 cM in genetic length across 19 linkage groups, covering 95% of the genome with an average distance between markers of 1.67 cM. Compared to an SSR map previously developed for this F2 family, these results represent an improved map covering a greater portion of the genome with higher marker density. The accuracy of the map was validated using the well-studied trait berry color. QTL affecting YAN, MA and SS related traits were detected. A joint MA and SS QTL spans a region with candidate genes involved in the malate metabolism pathway. We present an analytical pipeline for calling intercross GBS markers and a high-density linkage map for a large F2 family of the highly heterozygous Vitis genus. This study serves as a model for further genetic investigations of the molecular basis of additional unique characters of North American hybrid wine cultivars and to enhance the breeding process by marker-assisted selection. The GBS protocols for identifying intercross markers developed in this study can be adapted for other heterozygous species.

Segregation and associations of enological and agronomic traits in Graciano × Tempranillo wine grape progeny (Vitis vinifera L.)

The main objective of this research was the evaluation of the variability present in a segregating wine grape population derived from a cross between Graciano × Tempranillo, two Spanish varieties, in order to select improved genotypes with potential for producing high-quality wines in a climate change scenario. For that purpose, the phenotypic segregation of 16 agronomic traits related to production and phenology and 11 enological traits related to technical and phenolic maturity was studied in the progeny for three consecutive years. All traits presented transgressive segregation and continuous variation. Year effect was significant for all traits except total, extractable and skin anthocyanins content. However, a high level of genotype consistency for enological traits was revealed by repeatabilities and correlations between years. Significant correlations among traits were observed but most associations were weak. Furthermore, the CAPS (Cleaved Amplified Polymorphic Sequence) marker for the VvmybA genotype was tested to determine whether it would be useful in indirect selection for berry anthocyanins content. The results showed that the number of homozygous and heterozygous genotypes for the functional colour allele adjusted to a 1:1 segregation ratio, and that homozygous genotypes had significantly higher anthocyanins content. Principal component analysis found eight variables that contributed up to 80 % of the phenotypic variability present in the population. Seven groups of hybrids were distinguished based on ripening time, cluster weight, berry weight and anthocyanins content by cluster analysis; and fourteen genotypes were pre-selected for further research.

Gene expression of specific enological traits in wine fermentation

Background: Wine fermentation is a dynamic process and yeast have a precise genic regulation system that allow them to coordinate the gene transcription levels. However, the fermentation stage at which there is a great variation in the genic expression associated to a particular enological trait is often unknown and there are no simple experimental approaches to define it. Results: To identify the most adequate stage in which to evaluate the expression of the genes associated to specific enological traits we identified three stages of fermentation using the industrial strain EC1118. These stages, called early (0-16.4 hrs), middle (16.4-88.7 hrs) and late (88.7 hrs onwards), were characterized according to changes in the speed of CO 2 production. The greatest nitrogen consumption velocity (1.016 gL/h) was achieved in the early stage. The most significant changes in the consumption of sugar and ethanol production acceleration occurred in the middle stage, and the greatest velocity of ethanol production (0.043% /h) and fructose consumption (0.338 g/Lh) occurred in the late stage. The expression profiles for a set of genes characteristic of these processes were clearly defined during the stages of fermentation: identifying a peak for the genes related to the consumption of nitrogen during the early stage, followed by an increase in the expression of genes related to the consumption of sugars in the middle stage. Finally, during the late stage there is a decrease in the expression of genes involved in the consumption of sugar, except for HXT 3 the levels of which remain high. Conclusions: The kinetic and transcriptional profiles of the enological traits under study are clearly distinct and each is associated to a particular stage of the fermentation. Our results confirm that CO 2 production is a simple parameter to estimate the stages of the fermentation. Therefore, using the kinetics of CO 2 loss it is possible to select the most adequate moment to study the expression of genes associated to the main enological traits: sugar consumption and production of nitrogen and ethanol.

Pilot-scale evaluation the enological traits of a novel, aromatic wine yeast strain obtained by adaptive evolution

In the competitive context of the wine market, there is a growing interest for novel wine yeast strains that have an overall good fermentation capacity and that contribute favorably to the organoleptic quality of wine. Using an adaptive evolution strategy based on growth on gluconate as sole carbon source, we recently obtained wine yeasts with improved characteristics in laboratory-scale fermentations. The characteristics included enhanced fermentation rate, decreased formation of acetate and greater production of fermentative aroma. We report an evaluation of the potential value of the evolved strain ECA5™ for winemaking, by comparing its fermentation performance and metabolite production to those of the parental strain in pilot-scale fermentation trials, with various grape cultivars and winemaking conditions. We show that the evolved strain has outstanding attributes relative to the parental wine yeast strain, and in particular the production of less volatile acidity and greater production of desirable volatile esters, important for the fruity/flowery character of wines. This study highlights the potential of evolutionary engineering for the generation of strains with a broad range of novel properties, appropriate for rapid application in the wine industry.

Experimental approach for target selection of wild wine yeasts from spontaneous fermentation of “Inzolia” grapes

The aim of this research was the study of indigenous yeasts isolated from spontaneous fermentation of Inzolia grapes, one of the most widespread native white grapes in Sicily (Italy). The use of selective medium for the isolation and the screening for sulphur dioxide tolerance were useful for the first selection among 640 isolates. The yeasts characterized by high SO2 tolerance were identified at species level by restriction analysis of ITS region; although the majority of isolates were identified as S. cerevisiae, some non-Saccharomyces yeasts were found. Forty-seven selected yeasts, both S. cerevisiae and non-Saccharomyces yeasts, were characterized for genetic and technological diversity. The genetic polymorphism was evaluated by RAPD-PCR analysis, whereas the technological diversity was analyzed by determining the main secondary compounds in the experimental wines obtained by inoculating these yeasts. Both the molecular and metabolic profiles of selected yeasts were able to clearly discriminate S. cerevisiae from non-Saccharomyces yeasts. This research was useful for the constitution of a collection of selected indigenous yeast strains, including S. cerevisiae and non-Saccharomyces species possessing interesting enological traits. This collection represents a source of wild yeasts, among of which it is possible to select indigenous starters able to maintain the specific organoleptic characteristics of Inzolia wine.

Rational selection of non- Saccharomyces wine yeasts for mixed starters based on ester formation and enological traits

Thirty-eight yeast strains belonging to the genera Candida, Hanseniaspora, Pichia, Torulaspora and Zygosaccharomyces were screened for ester formation on synthetic microbiological medium. The genera Hanseniaspora and Pichia stood out as the best acetate ester producers. Based on the ester profile Hanseniaspora guilliermondii 11027 and 11102, Hanseniaspora osmophila 1471 and Pichia membranifaciens 10113 and 10550 were selected for further characterization of enological traits. When growing on must H. osmophila 1471, which displayed a glucophilic nature and was able to consume more than 90% of initial must sugars, produced levels of acetic acid, medium chain fatty acids and ethyl acetate, within the ranges described for wine. On the other hand, it was found to be a strong producer of 2-phenylethyl acetate. Our data suggest that H. osmophila 1471 is a good candidate for mixed starters, although the possible interactions with S. cerevisiae deserve further research.

SSR-based assessment of genetic diversity in South American Vitis vinifera varieties

Six SSR loci, previously developed for grapevine, were analyzed to evaluate the genetic variability and cultivar relatedness in a collection of 25 autochthonous Vitis vinifera varieties from Perú and Argentina. The number of alleles per locus ranged from 6 to 13, while the number of microsatellites genotypes varied between 9 and 16. The expected heterozygosity varied between 0.71 and 0.89 and the polymorphism information content ranged from 0.70 to 0.88 indicating that the SSRs were highly informative. It was possible to identify 76 different genotypes, with all accessions showing-at least one-specific combination of alleles. Triallelic loci were observed with some SSR. Sequence analysis revealed that variation in the number of repeats and insertion/deletions (InDels) accounted for the polymorphisms observed. Clustering analysis resulted in four separate groups of varieties sharing at least 75% of the markers. A few cases of synonymies were found within the Peruvian accessions. Varieties were clustered following a general pattern of shared morphological and enological traits, rather than geographical origin.