non-Saccharomyces Yeast Species Research Articles

Maximizing Wine Antioxidants: Yeast's Contribution to Melatonin Formation.

Melatonin is commonly found in various fruits, juices, and some fermented beverages. Its concentration in wine is influenced by soil properties, climatic factors, and yeast activity. Even if it is found in fermented beverages in relatively low proportions, melatonin still holds significant nutritional value, giving anti-aging properties, anti-inflammatory actions, and antidepressant effects. In this context, this article focuses on evaluating the impact of different Saccharomyces and non-Saccharomyces yeast species on the formation of melatonin and its contribution to wines' total antioxidant capacity. Considering that the antioxidant activity of wine is usually related to the content of phenolic compounds, ten such compounds were analyzed. The evaluation of bioactive compounds was performed using high-performance liquid chromatography (HPLC) coupled with mass spectrometry. The total antioxidant capacity of wine samples was evaluated by the ABTS+ method. The administration of bâtonnage products increased the efficiency of non-Saccharomyces yeasts. The mixtures of Saccharomyces and non-Saccharomyces yeasts generated higher values for melatonin. The results confirm a significant impact from the grape variety and the specific yeast strains on the melatonin concentration. Also, a strong dependence between antioxidant activity and melatonin levels was observed. Given the limited existing studies on the presence of melatonin in wines, new perspectives are needed for future exploration and understanding.

Sequential Fermentation in Red Wine cv. Babić Production: The Influence of Torulaspora delbrueckii and Lachancea thermotolerans Yeasts on the Aromatic and Sensory Profile.

This research aimed to analyze the impact of two different non-Saccharomyces yeast species on the aromatic profile of red wines made from the cv. Babić (Vitis vinifera L.) red grape variety. The grapes were obtained from two positions in the Middle and South of Dalmatia. This study compared a control treatment with the Saccharomyces cerevisiae (Sc) strain as a type of sequential inoculation treatment with Lachancea thermotolerans (Lt x Sc) and Torulaspora delbrueckii (Td x Sc). The focus was on the basic wine parameters and volatile aromatic compound concentrations determined using the SPME-Arrow-GC/MS method. The results revealed significant differences in cis-linalool oxide, geraniol, neric acid, and nerol, which contribute to the sensory profile with floral and rose-like aromas; some ethyl esters, such as ethyl furoate, ethyl hexanoate, ethyl lactate, ethyl 2-hydroxy-3-methylbutanoate, ethyl 3-hydroxy butanoate, diethyl glutarate, and diethyl succinate, contribute to the aromatic profile with fruity, buttery, overripe, or aging aromas. A sensory evaluation of wines confirmed that Td x Sc treatments exhibited particularly positive aromatic properties together with a more intense fullness, harmony, aftertaste, and overall impression.

Impact of non-Saccharomyces yeasts derived from traditional fermented foods on beer aroma: Analysis based on HS-SPME-GC/MS combined with chemometrics

In recent years, numerous studies have demonstrated the significant potential of non-Saccharomyces yeasts in aroma generation during fermentation. In this study, 134 strains of yeast were isolated from traditional fermented foods. Subsequently, through primary and tertiary screening, 28 strains of aroma-producing non-Saccharomyces yeast were selected for beer brewing. Headspace-solid phase microextraction (HS-SPME) combined with gas chromatography–mass spectrometry (GC–MS) and chemometrics were employed to analyze the volatile flavor substances in beer samples fermented using these strains. Chemometric analysis revealed that distinct species of non-Saccharomyces yeast had a unique influence on beer aroma, with strains from the same genus producing more similar flavor profiles. Accordingly, 2,6-nonadienal, 1-pentanol, phenyl ethanol, isoamyl acetate, ethyl caprate, butyl butyrate, ethyl propionate, furfuryl alcohol, phenethyl acetate, ethyl butyrate, ethyl laurate, acetic acid, and 3-methyl-4 heptanone were identified as the key aroma compounds for distinguishing among different non-Saccharomyces yeast species. This work provides useful insights into the aroma-producing characteristics of different non-Saccharomyces yeasts to reference the targeted improvement of beer aroma.

Genetic diversity of non-Saccharomyces yeasts associated with spontaneous fermentation of Cabernet Sauvignon wines from Ningxia, China.

The organoleptic profile and quality of wine are affected by the presence of different non-Saccharomyces species and strains. Therefore, the identification and characterization of non-Saccharomyces yeasts are the first step to understand their function, and to develop a better strain selection program for winemaking. This study investigated the biodiversity of non-Saccharomyces yeasts associated with spontaneous fermentation of Cabernet Sauvignon wines from five sub-regions (Shi Zuishan, Yinchuan, Yu Quanying, Qing Tongxia and Hong Sibu) in Ningxia, China. Yeast species were identified by sequencing the 26S rRNA D1/D2 region, and strains at the subspecies level were discriminated using tandem repeat-tRNA (TRtRNA) PCR analysis. A total of 524 yeast colonies were isolated, and 19 non-Saccharomyces yeast species belonging to 10 genera were identified, including Aureobasidium pullulans, Cryptococcus albidus, Cryptococcus sp., C. flavescens, C. terrestris, C. magnus, Cystofilobasidium ferigula, Candida zemplinina, Filobasidium magnum, Filobasidium sp., F. elegans, Hanseniaspora uvarum, Metschnikowia pimensis, M. pulcherrima, Naganishia albida, Pichia kluyveri, P. kudriavzevii, Rhodotorula glutinis and R. graminis. Hanseniaspora uvarum, C. zemplinina, and M. pulcherrima were the three most dominated species, while other non-Saccharomyces species were only present in the early stage of spontaneous fermentations at different levels. Further, for the yeast discrimination at strain level, 34 profiles were obtained by amplification with primer pairs TtRNASC/5CAG, while 40 profiles were obtained with primer pairs TtRNASC/ISSR-MB. This study explored the diversity of non-Saccharomyces species in Ningxia, China, and made an important contribution of genetic resources for further strain development.

Diversity of dung beetle-associated yeasts from pristine environments of Botswana.

Yeast-insect interactions are increasingly becoming an attractive source of discovery for previously unknown, unique, diverse, and industrially relevant yeast species. Despite a wealth of studies that have recently focused on yeasts in symbiotic association with Hymenopteran insects, yeasts associated with Coleopteran insects, such as lignocellulosic-rich dung-dependent beetles, remain poorly studied. Trends in yeast discovery suggest that species richness and diversity can be attributed to the ecological niche of the insect. Here, we considered the potential of dung beetles inhabiting the extreme environments of Botswana, characterized by desert-like conditions (semi-arid to arid and hot) as well as protected pristine environments, as possible attribute niches that can shape the extremophilic and diverse life history strategies of yeasts. We obtained a total of 97 phylogenetically diverse yeast isolates from six species of dung beetles from Botswana's unexplored environments, representing 19 species belonging to 11 genera. The findings suggest that the guts of dung beetles are a rich niche for non-Saccharomyces yeast species. Meyerozyma and Pichia were the most dominant genera associated with dung beetles, representing 55% (53 out of 97) of the yeast isolates in our study. Trichosporon and Cutaneotrichosporon genera represented 32% (31 out of 97) of the isolates. The remaining isolates belonged to Apiotrichum, Candida, Diutina, Naganishia, Rhodotorula, and Wickerhamiella genera (12 out of 97). We found out that about 62% (60 out of 97) of the isolates were potentially new species because of their low internal transcribed spacer (ITS) sequence similarity when compared to the most recent optimal species delineation threshold. A single isolate was unidentifiable using the ITS sequences. Using an in silico polymerase chain reaction-restriction fragment length polymorphism approach, we revealed that there was genetic diversity within isolates of the same species. Our results contribute to the knowledge and understanding of the diversity of dung beetle-associated yeasts.

Characterization of Indigenous Non-Saccharomyces Yeast Strains with Potential Use in Winemaking.

The vineyard is a great reservoir of autochthonous yeast strains whose composition is defined by different regional (edaphology, orography or climatology) and anthropological factors (cultivation systems or cultural practices). Most of this yeast diversity corresponds to non-Saccharomyces strains, some of which have potential use in winemaking. The oenological potential of 29 different native non-Saccharomyces strains belonging to 4 species (Lachancea thermotolerans, Torulaspora delbrueckii, Starmerella bacillaris and Metschnikowia spp.) was evaluated, using the autochthonous Saccharomyces cerevisiae XG3 strain as a control. Microfermentations with pure culture of each strain were performed in duplicate and the basic parameters and major volatiles of wines were analysed following official methodology. The best strain within each species was selected using a quantification matrix including the relevant oenological characteristics. The fermentative ability of non-Saccharomyces was lower than S. cerevisiae in all cases, but with differences among species. L. thermotolerans and T. delbrueckii showed higher fermentation rates than Starm. bacillaris, whereas Metschnikowia spp. presented a low fermentative power. At chemical level all non-Saccharomyces strains reduced the alcoholic content, the higher alcohols and the volatile acidity of wines and increased the content of glycerol, with differences among strains within a given species. T. delbrueckii and L. thermotolerans increased the total acidity of wines. The latter and Metschnikowia spp. strains produced lactic acid, which decreased the wine pH in the case of L. thermotolerans. According to their oenological traits the best rated strains of each species were Lt93, Td315, Mf278 and Sb474. In addition, the data obtained in pure fermentations were correlated to those chemical and aromatic compounds obtained with these non-Saccharomyces strains in sequential fermentations. Autochthonous strains of non-Saccharomyces yeast species contribute distinctive chemical characteristics to the wines. The correlations observed between wines fermented with the different non-Saccharomyces indigenous strains in pure and sequential fermentations suggest that their contribution to wine properties remains stable regardless of must composition or winemaking techniques.

Evaluation of Different Molecular Markers for Genotyping Non-Saccharomyces Wine Yeast Species

Wine quality is determined by the particular yeast strains prevailing at various stages of fermentation. Therefore, the ability to make an easy, fast, and unambiguous discrimination of yeasts at the strain level is of great importance. Here, the tandem repeat-tRNA (TRtRNA) method with the 5GAC or ISSR-MB primer sets and random amplified polymorphic DNA (RAPD) analysis with (GTG)3, R5, and RF2 oligonucleotides were tested on various non-Saccharomyces wine yeast species. The TRtRNA-PCR employing ISSR-MB showed the highest capacity in discriminating Lachancea thermotolerans and Metschnikowia pulcherrima isolates. RAPD with RF2 was the most efficient method in resolving Starmerella bacillaris isolates, although it produced few polymorphic bands. RAPD with R5 showed the highest capacity to discriminate among the Issatchenkia orientalis, Hanseniaspora guilliermondii, and Pichia anomala isolates. RAPD with either R5 or RF2 exhibited the highest ability to discriminate among the Torulaspora delbrueckii isolates. RAPD with (GTG)3 was the most discriminating method for the H. uvarum isolates. Here we concluded that both TRtRNA-PCR and RAPD-PCR offer rapid means for typing non-Saccharomyces species. However, each method performs better for a given species when paired with a particular primer set. The present results can be useful in wine research for the fast fingerprinting of non-Saccharomyces yeasts.

Biodiversity and Oenological Property Analysis of Non-Saccharomyces Yeasts Isolated from Korla Fragrant Pears (Pyrus sinkiangensis Yu)

Oenological yeasts play a critical role in the winemaking process. In this study, the biodiversity of the non-Saccharomyces yeast was analyzed and monitored using high-throughput sequencing and culture-dependent approaches. Oenological and fermentation characteristics of these native yeasts were further investigated. A total of 241 fungus species and 5 species of culturable non-Saccharomyces yeasts were detected using high-throughput sequencing and culture-dependent approaches, respectively. Five strains of aroma-producing yeasts (K4, K14, K19, K21, and K26) were isolated, and their growth characteristics, carbon source utilization, hydrogen sulfide production performance, and β-glucosidase activity were different. The oenological condition tolerances of most strains were lower than that of commercial S. cerevisiae X16. The co-inoculum of these strains and S. cerevisiae X16 regulated the volatile aroma characteristics of the fermented Korla fragrant pear (KFP) fruit wine, enriching and complicating the aroma flavor. Thus, the combined inoculation of these indigenous wine yeasts and S. cerevisiae has some application potential in the production of KFP wine.

Exploring Yeast Diversity to Produce Lipid-Based Biofuels from Agro-Forestry and Industrial Organic Residues.

Exploration of yeast diversity for the sustainable production of biofuels, in particular biodiesel, is gaining momentum in recent years. However, sustainable, and economically viable bioprocesses require yeast strains exhibiting: (i) high tolerance to multiple bioprocess-related stresses, including the various chemical inhibitors present in hydrolysates from lignocellulosic biomass and residues; (ii) the ability to efficiently consume all the major carbon sources present; (iii) the capacity to produce lipids with adequate composition in high yields. More than 160 non-conventional (non-Saccharomyces) yeast species are described as oleaginous, but only a smaller group are relatively well characterised, including Lipomyces starkeyi, Yarrowia lipolytica, Rhodotorula toruloides, Rhodotorula glutinis, Cutaneotrichosporon oleaginosus and Cutaneotrichosporon cutaneum. This article provides an overview of lipid production by oleaginous yeasts focusing on yeast diversity, metabolism, and other microbiological issues related to the toxicity and tolerance to multiple challenging stresses limiting bioprocess performance. This is essential knowledge to better understand and guide the rational improvement of yeast performance either by genetic manipulation or by exploring yeast physiology and optimal process conditions. Examples gathered from the literature showing the potential of different oleaginous yeasts/process conditions to produce oils for biodiesel from agro-forestry and industrial organic residues are provided.

Impact of Torulaspora delbrueckii During Fermentation on Aromatic Profile of Vidal Blanc Icewine.

Non-Saccharomyces yeasts usually have a positive effect on improving the diversity of wine aroma and increasing the differentiation of wine products. Among these non-Saccharomyces yeast species, Torulaspora delbrueckii is often studied and used in winemaking in recent years, but its application in icewine has not been reported yet. In this study, indigenous T. delbrueckii strains (TD1 and TD2) and Saccharomyces cerevisiae strains (commercial yeast SC1 and indigenous icewine yeast SC2) were sequentially inoculated for icewine fermentations; meanwhile, pure S. cerevisiae (SC1 and SC2) fermentations were used as the control; TD1, TD2, and SC2 strains used were screened from spontaneous fermentations of Vidal blanc icewine. The aim was to investigate the effect of T. delbrueckii on the aroma complexity of icewine, which is of great significance to the application of T. delbrueckii in icewine production. The results showed that T. delbrueckii was completely replaced by S. cerevisiae at the middle and later fermentative stages in mixed culture fermentations. Compared with the icewine fermented with pure S. cerevisiae, mixed culture fermented icewines contained lower acetic acid and ethanol, and higher glycerol. The inoculation of T. delbrueckii greatly impacted the levels of several important volatile compounds, and more 2-phenylethyl alcohol, isoamyl acetate, linalool, D-limonene, p-cymene and cineole were produced, and the fruity, flowery, and sweet characteristic was intensified. Moreover, the relevance of strain-specificity within T. delbrueckii to aroma compound differences was shown. To our knowledge, this study is the first to investigate the application of T. delbrueckii in Vidal blanc icewine fermentation, and volatile aroma compounds in the icewine fermented by T. delbrueckii and S. cerevisiae.

Whole-Genome Sequencing and Annotation of the Yeast Clavispora santaluciae Reveals Important Insights about Its Adaptation to the Vineyard Environment.

Clavispora santaluciae was recently described as a novel non-Saccharomyces yeast species, isolated from grapes of Azores vineyards, a Portuguese archipelago with particular environmental conditions, and from Italian grapes infected with Drosophila suzukii. In the present work, the genome of five Clavispora santaluciae strains was sequenced, assembled, and annotated for the first time, using robust pipelines, and a combination of both long- and short-read sequencing platforms. Genome comparisons revealed specific differences between strains of Clavispora santaluciae reflecting their isolation in two separate ecological niches—Azorean and Italian vineyards—as well as mechanisms of adaptation to the intricate and arduous environmental features of the geographical location from which they were isolated. In particular, relevant differences were detected in the number of coding genes (shared and unique) and transposable elements, the amount and diversity of non-coding RNAs, and the enzymatic potential of each strain through the analysis of their CAZyome. A comparative study was also conducted between the Clavispora santaluciae genome and those of the remaining species of the Metschnikowiaceae family. Our phylogenetic and genomic analysis, comprising 126 yeast strains (alignment of 2362 common proteins) allowed the establishment of a robust phylogram of Metschnikowiaceae and detailed incongruencies to be clarified in the future.

Investigation of Non-Saccharomyces Yeast Strains for Their Suitability for the Production of Non-Alcoholic Beers with Novel Flavor Profiles

Non-alcoholic beers have seen steady growth in recent years and are attracting customer interest. At the same time, consumer demand for non-alcoholic beers is rising. While wort-like flavors are often criticized in beer and also regarded as off-flavors, there is growing demand for beer flavor diversification. The latest research results prove that certain non-Saccharomyces yeast species can produce distinctive fruity flavor profiles during wort fermentation. In this study, 15 different yeast strains were evaluated for their suitability to produce novel flavor profiles during fermentation in wort from malt extract while being tested for their ability to produce less than 0.5% (v/v) ethanol. The yeast strains were therefore analyzed for their sugar utilization and were compared with the maltose-negative reference yeast strain Saccharomycodes ludwigii TUM SL17. Following the fermentation experiments, the beers were analyzed for secondary metabolites and ethanol content before being tasted by trained assessors. The results reveal that all 15 yeasts are suitable for the production of non-alcoholic beers based on their sugar utilization. Particularly promising flavors were produced by three yeast strains of the species Cyberlindnera saturnus, which were reminiscent of cool mint sweets, pear, and banana. Two further yeasts of the species Kluyveromyces marxianus and Saccharomycopsis fibuligera also produced a wide range of pleasant fruity flavors. Although the secondary metabolites were mostly below the odor thresholds based on regular alcoholic beers, this study reveals that the thresholds in non-alcoholic beers can be classified as significantly lower. Supplemental data for this article is available online at https://doi.org/10.1080/03610470.2021.2012747 .

Strain-Specific Responses by Saccharomyces cerevisiae to Competition by Non-Saccharomyces Yeasts

The use of non-Saccharomyces yeast species generally involves sequential or co-inoculation of a Saccharomyces cerevisiae strain to complete fermentation. While most studies have focused on characterising the impact that S. cerevisiae has on the growth and metabolic activity of these non-Saccharomyces species, microbial interactions work reciprocally. Antagonism or competition of non-Saccharomyces species against S. cerevisiae has been shown to impact subsequent fermentation performance. To date, it remains unclear whether these negative interactions are strain specific. Hence, characterisation of strain-specific responses to co-inoculation would enable the identification of specific S. cerevisiae strain/non-Saccharomyces combinations that minimise the negative impacts of sequential fermentation on fermentation performance. The competitive fitness response of 93 S. cerevisiae strains to several non-Saccharomyces species was simultaneously investigated using a barcoded library to address this knowledge gap. Strain-specific fitness differences were observed across non-Saccharomyces treatments. Results obtained from experiments using selected S. cerevisiae strains sequentially inoculated after Metschnikowia pulcherrima and Torulaspora delbrueckii were consistent with the competitive barcoded library observations. The results presented in this study indicate that strain selection will influence fermentation performance when using non-Saccharomyces species, therefore, appropriate strain/yeast combinations are required to optimise fermentation.

Understanding Wine through Yeast Interactions.

Wine is a product of microbial activities and microbe–microbe interactions. Yeasts are the principal microorganisms responsible for the evolution and fulfillment of alcoholic fermentation. Several species and strains coexist and interact with their environment and with each other during the fermentation course. Yeast–yeast interactions occur even from the early stages of fermentation, determining yeast community structure and dynamics during the process. Different types of microbial interactions (e.g., mutualism and commensalism or competition and amensalism) may exert positive or negative effects, respectively, on yeast populations. Interactions are intimately linked to yeast metabolic activities that influence the wine analytical profile and shape the wine character. In this context, much attention has been given during the last years to the interactions between Saccharomyces cerevisiae (SC) and non-Saccharomyces (NS) yeast species with respect to their metabolic contribution to wine quality. Yet, there is still a significant lack of knowledge on the interaction mechanisms modulating yeast behavior during mixed culture fermentation, while much less is known about the interactions between the various NS species or between SC and Saccharomyces non-cerevisiae (SNC) yeasts. There is still much to learn about their metabolic footprints and the genetic mechanisms that alter yeast community equilibrium in favor of one species or another. Gaining deeper insights on yeast interactions in the grape–wine ecosystem sets the grounds for understanding the rules underlying the function of the wine microbial system and provides means to better control and improve oenological practices.

Influence of Microencapsulation on Fermentative Behavior of Hanseniaspora osmophila in Wine Mixed Starter Fermentation

In recent years, as a consequence of the re-evaluation of the role of non-Saccharomyces yeasts, several studies have been conducted on the use of controlled mixed fermentations with Saccharomyces and different non-Saccharomyces yeast species from the winemaking environment. To benefit from the metabolic particularities of some non-Saccharomyces yeasts, the management of a non-Saccharomyces strain in mixed fermentation is a crucial step, in particular the use of procedures addressed to increase the persistence of non-Saccharomyces strains during the fermentative process. The use of microencapsulation for cell immobilization might represent a strategy for enhancing the competitiveness of non-Saccharomyces yeasts during mixed fermentation. This study was aimed to assess the fermentative performance of a mixed starter culture, composed by a wild Hanseniaspora osmophila strain (ND1) and a commercial Saccharomyces cerevisiae strain (EC1118). For this purpose, free and microencapsulated cells of ND1 strain were tested in co-culture with EC1118 during mixed fermentations in order to evaluate the effect of the microencapsulation on fermentative behavior of mixed starter and final wine composition. The data have shown that H. osmophila cell formulation affects the persistence of both ND1 and EC1118 strains during fermentations and microencapsulation resulted in a suitable system to increase the fermentative efficiency of ND1 strain during mixed starter fermentation.

Genetic, Physiological, and Industrial Aspects of the Fructophilic Non-Saccharomyces Yeast Species, Starmerella bacillaris

Starmerella bacillaris (synonym Candida zemplinina) is a non-Saccharomyces yeast species, frequently found in enological ecosystems. Peculiar aspects of the genetics and metabolism of this yeast species, as well as potential industrial applications of isolated indigenous S. bacillaris strains worldwide, have recently been explored. In this review, we summarize relevant observations from studies conducted on standard laboratory and indigenous isolated S. bacillaris strains.

Comparative uptake of exogenous thiamine and subsequent metabolic footprint in Saccharomyces cerevisiae and Kluyveromyces marxianus under simulated oenological conditions

Managed inoculation of non-Saccharomyces yeast species is regarded as a practical way to introduce new characteristics to wine. However, these yeasts struggle to survive until fermentation is complete. Kluyveromyces marxianus IWBT Y885 is one such yeast. Although it displays interesting oenological properties, a longer persistence during alcoholic fermentation would warranty a stronger impact on wine composition. A key factor for survival, growth and sustained metabolic activity of all yeasts is their nutrient requirements. Thus, identifying nutrients that are essential for maximising fermentation performance, and subsequently ensuring adequate levels of nutrients, is a means to ensure significant contribution of yeasts to wine properties. This study aimed to identify essential nutrients, other than previously studied sugars and nitrogen, for maximum impact of K. marxianus Y885, as well as to characterise the outcomes of their utilisation.A multifactorial experimental design was employed to investigate the impact of nutrient concentrations on fermentation performance with K. marxianus Y885 in synthetic must. B-complex vitamins most significantly impacted fermentation performance of K. marxianus Y885 compared to other nutrient groups investigated. Considering the well-established role of the vitamin, thiamine, for maximum fermentation performance during winemaking and the fact that it may be supplemented to wine fermentations legally, the responses to specifically exogenous thiamine concentration for K. marxianus Y885 and Saccharomyces cerevisiae EC1118 were compared in terms of population viability, fermentation rate, total sugars utilised, thiamine assimilation kinetics, and final wine composition. A saturation effect for initial thiamine concentration of K. marxianus Y885 fermentations was characterised, with a maximum fermentation rate and over 90% of available sugars utilisation obtained at 0.25 mg/L. An appreciably larger comparative increase in exponential cell growth rate, maximum population, fermentation rate and total CO2 production for K. marxianus Y885 compared to S. cerevisiae EC1118 revealed a greater necessity for thiamine to ensure maximum fermentation performance. A delayed uptake of thiamine at higher concentrations for K. marxianus Y885 suggested differential regulation of thiamine uptake compared to S. cerevisiae EC1118. In addition, different trends in metabolites produced between species suggest that thiamine concentration impacts the carbon metabolic flux differently in these two yeasts, potentially impacting final wine properties.

Sterol uptake analysis in Saccharomyces and non-Saccharomyces wine yeast species.

Sterols are essential components of the yeast membrane and their synthesis requires oxygen. Yet, Saccharomyces cerevisiae has developed the ability to take up sterols from the medium under anaerobiosis. Here we investigated sterol uptake efficiency and the expression of genes related to sterol import in Saccharomyces and non-Saccharomyces wine yeast species fermenting under anaerobic conditions. The sterol uptake efficiency of 39 strains was evaluated by flow cytometry (with 25-NBD Cholesterol, a fluorescent cholesterol probe introduced in the medium) and we found an important discrepancy between Saccharomyces and non-Saccharomyces wine yeast species that we correlated to a lower final cell population and a lower fermentation rate. A high uptake of sterol was observed in the various Saccharomyces strains. Spot tests performed on 13 of these strains confirmed the differences between Saccharomyces and non-Saccharomyces strains, suggesting that the presence of the sterol uptake transporters AUS1 and PDR11 could cause these discrepancies. Indeed, we could not find any homologue to these genes in the genome of Hanseniaspora uvarum, H. guillermondii, Lachancea thermotolerans, Torulaspora delbreueckii, Metschnikowia pulcherrima, or Starmarella bacillaris species. The specialization of sterol import function for post genome-duplication species may have favored growth under anaerobiosis.

Learning from 80 years of studies: a comprehensive catalogue of non-Saccharomyces yeasts associated with viticulture and winemaking.

Non-Saccharomyces yeast species are nowadays recognized for their impact on wine´s chemical composition and sensorial properties. In addition, new interest has been given to the commercial exploitation of non-Saccharomyces starter cultures in the wine sector. However, over many years, these yeast species were considered sources of contamination in wine production and conservation, mainly due to the high levels of volatile acidity obtained. The present manuscript systematizes 80 years of literature describing non-Saccharomyces yeast species isolated from grapes and/or grape musts. A link between each reference, the accepted taxonomic name of each species and their geographical occurrence is presented, compiling information for 293 species, in a total of 231 citations. One major focus of this work relates to the isolation of non-Saccharomyces yeasts from grapevines usually ignored in most sampling studies, also as isolation from damaged grapes. These particular niches are sources of specific yeast species, which are not identified in most other explored environments. These yeasts have high potential to be explored for important and diversified biotechnological applications.

Biodiversity of non-Saccharomyces yeasts associated with spontaneous fermentation of Cabernet Sauvignon wines from Shangri-La wine region, China

Shangri-La is a wine region that has the highest altitude vineyards in China. This is the first study investigated the biodiversity of non-Saccharomyces yeasts associated with spontaneous fermentation of Cabernet Sauvignon wines produced from two sub-regions (Lancang River and Jinsha River) of Shangri-La. The culturable yeasts were preliminarily classified based on their colonial morphology on the Wallerstein Laboratory nutrient agar plates. Yeast species were identified by the sequencing of the 26S rRNA D1/D2 region and the 5.8S rRNA ITS region. Twenty-five non-Saccharomyces yeast species belonging to sixteen genera were isolated and identified in Shangri-La wine region. Candida, Hanseniaspora, Pichia, and Starmerella were found in both sub-regions, but the Lancang River showed more diverse yeast species than the Jinsha River. Shangri-La not only exhibited high diversity of non-Saccharomyces yeasts, and furthermore, seven species of non-Saccharomyces yeasts were exclusively found in this region, including B. bruxellensis, D. hansenii, M. guilliermondii, S. vini, S. diversa, T. delbrueckii and W. anomalus, which might play an important role in distinctive regional wine characteristics. This study provide a relatively comprehensive analysis of indigenous non-Saccharomyces yeasts associated with Cabernet Sauvignon from Shangri-La, and has significance for exploring ‘microbial terroir’ of wine regions in China.