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Yeast population dynamics in spontaneous fermentations: Comparison between two different wine-producing areas over a period of three years
Abstract
Yeast ecology, biogeography and biodiversity are important and interesting topics of research. The population dynamics of yeasts in several cellars of two Spanish wine-producing regions was analysed for three consecutive years (1996 to 1998). No yeast starter cultures had been used in these wineries which therefore provided an ideal winemaking environment to investigate the dynamics of grape-related indigenous yeast populations. Non-Saccharomyces yeast species were identified by RFLPs of their rDNA, while Saccharomyces species and strains were identified by RFLPs of their mtDNA. This study confirmed the findings of other reports that non-Saccharomyces species were limited to the early stages of fermentation whilst Saccharomyces dominated towards the end of the alcoholic fermentation. However, significant differences were found with previous studies, such as the survival of non-Saccharomyces species in stages with high alcohol content and a large variability of Saccharomyces strains (a total of 112, all of them identified as Saccharomyces cerevisiae) with no clear predominance of any strain throughout all the fermentation, probably related to the absence of killer phenotype and lack of previous inoculation with commercial strains.
... (Renouf et al., 2007). Cependant Saccharomyces cerevisiae est la plus efficiente pour l'accomplissement de cette étape (Albergaria & Arneborg, 2016;Beltran et al., 2002;Heard & Fleet, 1988;Torija et al., 2001). Les autres espèces ont été testées pour comparer leurs performances respectives, mais aucune autre levure n'est aussi efficace que S. cerevisiae. ...
... Dans la baie de raisin, sa population est largement minoritaire (Mortimer & Polsinelli, 1999) mais à la suite du foulage/pressurage elle sera considérablement favorisée. Cette levure peut se développer rapidement dans des concentrations en sucre supérieures à 200 g/L, mais elle possède aussi une tolérance importante pour réaliser la fermentation dans l'environnement du vin où le pH est acide (3,0 -3,5) et la concentration en azote faible (150-200 mg/L) (Albergaria & Arneborg, 2016;Beltran et al., 2002;Heard & Fleet, 1988;Torija et al., 2001). Il est important de noter que la souche de levure effectuant la fermentation aura un impact sur le niveau des composés aromatiques et sur la perception de certains caractères fruités (Gammacurta et al., 2017). ...
... Une représentation schématique de la réaction de fermentation alcoolique est présentée dans la Molina et al., 2007;Ribéreau-Gayon et al., 2007;Torija et al., 2001). La nutrition des levures peut être contrôlée via la concentration en azote. ...
Il existe un besoin de développement d'outils de caractérisation rapide et fiables de milieux biologiques. Ce travail de thèse a pour but de développer une méthode se basant sur l'acquisition de matrices d'émission et d'excitation de fluorescence (MEEF) couplée avec l'utilisation de la mesure du temps de vie de fluorescence en spectroscopie et microscopie (FLIM). Ces techniques ont un fort potentiel du fait de leur rapidité, du faible volume d’échantillon nécessaire à l’analyse, d’une analyse non-destructrice de l’échantillon et d’un faible coût. Ce projet s’est focalisé sur deux milieux biologiques ayant un fort intérêt dans l’industrie agro-alimentaire : le vin et les spores bactériennes. D’une part, nous avons une boisson représentant un marché mondial de grande envergure, et d’autre part, un contaminant alimentaire responsable de diverses maladies. Le couplage de nos différentes techniques a permis de caractériser des sondes fluorescentes natives aux deux milieux biologiques et d’évaluer leurs caractéristiques en fonction de différents paramètres, tels que la fermentation malolactique ou des changements de pH et d’activité de l’eau. Cet outil s’est concentré sur diverses sondes natives, avec les acides organiques et phénoliques dans notre matrice vin, et l’acide dipicolinique pour la matrice spore bactérienne. Ce projet a permis de bâtir une base de données permettent le suivi in-situ de ces sondes fluorescentes dans leur environnement natif et donne lieu à la mise en place d’une nouvelle méthode de caractérisation des milieux biologiques.
... This genus is commonly found in soil, and some species are responsible for plant growth [25]. The Bacillus species are mostly associated with spoilage in wine fermentation due to the organic acid metabolites that they produce [26,27]. The isolated Bacillus species are reported to produce a wide range of enzymes, such as polygalacturonase and pectin esterase involved in smoothening and liquefying the fermenting juice on the last few days of fermentation [28]. ...
... Bacillus spp. also produces high amounts of lactic acid under aerobic and anaerobic conditions [27,28], which can be more than 3.7 g/L of lactic acid with a glucose conversion yield of more than 49% [29]. When in abundance, as in the instance of cactus pear juice and wine, Bacillus spp. ...
... The non-fermenting yeasts Aureobasidium pullulans, Aurobasidium leucospermi and Starmerella bacillaris were observed earlier in the fermentation process. These were previously reported elsewhere in spontaneous fermentation studies [27]. Starmerella bacillaris is commonly isolated by winemakers from different fruits [29]. ...
The cactus pear (Opuntia ficus-indica) fruit is widely cultivated and grown naturally in arid regions because it is adaptive to a wide range of soil and environments. The pear fruit is inhabited by different micro-organisms and has chemical composition suitable for wine making. Profiling the contributing micro-organisms and evaluating the chemical parameters of cactus pear wine can assist in selecting reliable microbes for use as starter cultures. Spontaneous fermentation was carried out for 13 days and followed by three months of cold storage. Fermenting microbes were isolated, characterised and identified. The chemical parameters, namely, sugar concentration, ethanol concentration, pH and total acidity, were analysed. A total of 22 micro-organisms were identified, among which nine yeast species, two acetic acid bacteria (Gluconobacter spp.) and eight Bacillus spp. were isolated. The simple sugars were used up, and ethanol was produced to a high concentration of 50.9 g/L. The pH ranged between 2.8 and 2.9; hence, a maximum total acidity of ±25 g/100 mL was achieved. At least 78% of the available tannins were used in the early stages of fermentation. Potassium and magnesium were the highest minerals obtained, and zinc was the lowest. The highest ash content obtained was 7.9 g/100 mL. The vitamin C content was retained and gradually increased throughout the fermentation process. The findings indicate that lasting flavoured wine can be developed from cactus pear fruit because of the fermenting microbes and the chemical composition of the fruit.
... The counts obtained for yeasts and bacteria in non-treated must (4.93 ± 1.02 and 3.92 ± 0.89 log10 cfu/m, respectively) were like those obtained from microwave-treated must (5.03 ± 1.77 and 3.96 ± 1.10 log10 cfu/m), and no significant differences were found. The populations of bacteria and yeasts were as expected in fresh must when yeasts increase the viable cells while bacteria decrease them [36]. ...
... Most of the species identified agree with the native species usually reported by other studies in musts [36,42] with no presence of Saccharomcyes spp. As it has been reported above, the total yeast population was not affected by the microwave treatment, but there was a reduction in the number of species identified and their percentages were affected by the treatment. ...
... The species that mainly prevailed after microwave maceration were D. hansenii and K. thermotolerans, which increased their percentages from 20 to 35% and from 20 to 25% respectively. Most of the species identified agree with the native species usually reported by other studies in musts [36,42] with no presence of Saccharomcyes spp. As it has been reported above, the total yeast population was not affected by the microwave treatment, but there was a reduction in the number of species identified and their percentages were affected by the treatment. ...
The objective of this study was to evaluate the effect of microwave treatment of crushed grapes on the yeast population of the must and on the development of alcoholic fermentation, as well as on the extraction of different compounds from the grapes such as polysaccharides and amino acids that can affect the organoleptic quality and stability of the wine. This study demonstrated for the first time the effect of the microwave treatment of grapes on native yeast species and their diversity, producing an increase in fermentation kinetics and a decrease in the lag phase. The microwave treatment produced a positive effect on the extraction of amino acids and polysaccharides from the grapes, resulting in significantly higher amounts of the main amino acids of the must and some major volatile compounds in the treated samples. The polysaccharides most affected by the microwave treatment were the PRAGs, the main polysaccharides liberated from grapes during the maceration.
... Saccharomyces cerevisiae is known to be the major contributor to leading and finishing such natural wine fermentations, as evidenced by the many studies devoted to the identification and dynamics of the natural floras of fermenting grape musts [9][10][11][12][13], and its presence has been confirmed in one of the most ancient known wine lees [14]. It has been reported that many S. cerevisiae strains usually coexist in natural fermentations [15][16][17][18][19][20][21][22][23]. Such complexity is observed even when musts are inoculated with selected starters, though natural equilibrium is affected in such cases [24][25][26]. ...
... Numerous studies have looked into different aspects of Saccharomyces cerevisiae's genetic diversity since the first application of molecular tools, some three decades ago, which made it possible to conduct such explorations, the scale of which grew over time. Many of these studies focusing on oenological S. cerevisiae communities have looked into vineyard floras [17,33,35,43,[75][76][77][78][79] collected to conduct fermentations in the lab, and only a few have explored the fermenting floras in their natural environment, i.e., the wineries [21][22][23], while some have looked at both populations [19,27,41,80]. Even if a constant circulation and exchange of strains is most probable between wineries and vineyards [19,27,43], wineries might also have their own implanted yeast lineages that conduct or participate in the natural fermentations and are different from the surviving ones in the vineyards [27,34,[80][81][82]. ...
... A pronounced biodiversity was revealed within S. cerevisiae fermentative population dispatched across the territory on the basis of both interdelta and microsatellites analysis. Diversity figures from similar surveys concerning wineries in other oenological regions are different from one study to the other (some of the variability factors probably being the use of different methods and different sampling schemes), but all such reports agree on high S. cerevisiae diversities [20][21][22][23]55,83]. ...
A total of 296 isolates of Saccharomyces cerevisiae sampled from naturally fermenting grape musts from various locations in Lebanon were typed by interdelta fingerprinting. Of these, 88 isolates were compared with oenological strains originating from various countries, using microsatellite characterization at six polymorphic loci. These approaches evidenced a large diversity of the natural oenological Lebanese flora over the territory as well as in individual spontaneous fermentations. Several cases of dominance and perenniality of isolates were observed in the same wineries, where fermentations appeared to involve lineages of sibling isolates. Our work thus evidenced a “winery effect” on strains’ relatedness. Similarly, related or identical strains were also detected in vicinal wineries, suggesting strain circulation within small geographical areas and a further “vicinity effect”. Moreover, and despite its diversity, the Lebanese flora seemed interrelated, on the basis of microsatellite loci analysis, in comparison to worldwide communities. We finally tested the ability of 21 indigenous strains to act as potential starters for winemaking. Seven of them passed our pre-selection scheme and two of them at least may be good candidates for use provided pilot-scale assays confirm their suitability.
... The most RFLP used for bacteria is the 16S rRNA gene, which has been denominated Amplified Ribosomal DNA Restriction Analysis (ARDRA). The application to wine species was initiated by Guillamón et al. (1998) and Esteve-Zarzoso et al. (1999) and several studies have used this technique later on (Torija et al. 2001, Beltran et al. 2002. ARDRA has been used to identify LAB (Rodas et al. 2005) and AAB , Ruiz et al. 2000, González et al. 2006a, Gullo et al. 2006. ...
... Then, the restriction pattern DNA with enzymes that target sequences such as GCAT will cut less frequently the mtDNA than the nuclear DNA. So far, this was the most used technique to genotype the strains of S. cerevisiae (Torija et al. 2001, Beltran et al. 2002, although it still has the need to extract the DNA and it needs more time consuming than the direct PCR that can be performed with delta elements. ...
... The number of yeasts on the grape berry and grape must change depending on the geographical situation of the vineyard, climatic conditions, sanitary state of the berries and pesticide treatments of the vineyard (Beltran et al. 2002, Romano et al. 2006). At harvest time, the yeast population is quite complex and the major fermenting yeast, S. cerevisiae, is not very abundant (Beltran et al. 2002, Torija et al. 2001. Therefore, the non-Saccharomyces population is expected to be dominant in the early stages of grape must processing. ...
Alcoholic fermentation and the production of wine has accompanied humanity for more than 10000 years. However, it has been only in the last 50 years when the winemakers have had the tools to manage and control the process. The methodology to analyze and monitor the succession of the microorganisms that participate in the process along with the effective use of antimicrobial compounds (for instance sulfur dioxide), the control of the temperature and, above all, the use of cellar-friendly fermentation starters (mostly as Active Dry Wine Yeast) have provided the appropriate conditions for that control. However, the use of a limited number of commercial presentations of the starters has generated an unwanted uniformity of the wines produced. Furthermore, new tendencies in wine making with limited or no human intervention have considered these tolls as a negative aspect in the wine quality, although most of these concerns are only philosophical, without clear scientific evidence. We present a revision of the present state of the art in these methodologies where our research group has been working for the last 25 years.
... The most RFLP used for bacteria is the 16S rRNA gene, which has been denominated Amplified Ribosomal DNA Restriction Analysis (ARDRA). The application to wine species was initiated by Guillamón et al. (1998) and Esteve-Zarzoso et al. (1999) and several studies used this technique later on (Torija et al., 2001;Beltran et al., 2002;Raspor et al., 2006, etc.). ARDRA has been used to identify LAB (Rodas et al., 2005) and AAB Ruiz et al., 2000;González et al., 2006ª;Gullo et al., 2006;Vegas et al., 2010). ...
... Then, the restriction pattern DNA with enzymes that target sequences, such as GCAT, will cut less frequently the mtDNA than the nuclear DNA. So far, this was the most used technique to genotype the strains of S. cerevisiae (Torija et al., 2001;Beltran et al., 2002;Nikolaou et al., 2007;Maqueda et al., 2010), although it still has the need to extract the DNA and is more time consuming than direct PCR that can be performed with delta elements. ...
... The number of yeasts on the grape berry and grape must change, depending on the geographical situation of the vineyard, climatic conditions, sanitary state of the berries and pesticide treatments of the vineyard (Beltran et al., 2002;Romano et al., 2006;Padilla et al., 2016). At harvest time, the yeast population is quite complex and the major fermenting yeast, S. cerevisiae, is not very abundant (Beltran et al., 2002;Torija et al., 2001). Therefore, the non-Saccharomyces population is expected to be dominant in the early stages of grape must processing. ...
... However, the picture seems to be more complex. Indeed, several studies revealed that there are no strains representative of a winery or an area [20][21][22]. By comparing the S. cerevisiae biodiversity of two regions in Spain (Priorat and Terra Alta) during three consecutive years, Torija et al. (2001) [21] reported that the yeast population changed from year to year. ...
... Indeed, several studies revealed that there are no strains representative of a winery or an area [20][21][22]. By comparing the S. cerevisiae biodiversity of two regions in Spain (Priorat and Terra Alta) during three consecutive years, Torija et al. (2001) [21] reported that the yeast population changed from year to year. Furthermore, they observed that some identical strains present in different cellars in the same area were also present in the cellars of different areas. ...
... Indeed, several studies revealed that there are no strains representative of a winery or an area [20][21][22]. By comparing the S. cerevisiae biodiversity of two regions in Spain (Priorat and Terra Alta) during three consecutive years, Torija et al. (2001) [21] reported that the yeast population changed from year to year. Furthermore, they observed that some identical strains present in different cellars in the same area were also present in the cellars of different areas. ...
Wine terroir is characterized by a specific taste and style influenced by the cultivar of the fermented grapes, geographical factors such as the vineyard, mesoclimate, topoclimate, and microclimate, soil geology and pedology, and the agronomic approach used. These characteristics together define the concept of "terroir". Thus, regional distinctive flavors in wine have been the subject of many studies aimed at better understanding the link between the wine and the vineyard. Indeed, the identification of key environmental elements involved in the regional variation of grape and wine quality characteristics is a critical feature for improving wine production in terms of consumer preference and economic appreciation. Many studies have demonstrated the role of abiotic factors in grape composition and consequently in wine style. Biotic factors are also involved such as grape microbial communities. However, the occurrence and effects of region-specific microbiota in defining wine characteristics are more controversial issues. Indeed, several studies using high throughput sequencing technologies have made it possible to describe microbial communities and revealed a link between grape must and soil microbial communities, and the geography of the territory. Based on these observations, the concept of "microbial terroir" emerged. However, this concept has been subject to contradictory studies. The aim of this opinion article is to take a step back and examine in perspective the concept of microbial terroir, by comparing numerous data from different studies and providing arguments in favor of or against this concept to stimulate discussion and point out that experimental research is still needed to study the contribution of this assembly of microorganisms to the final product and to support or refute the concept.
... However, the picture seems to be more complex. Indeed, several studies revealed that there are no strains representative of a winery or an area [20][21][22]. By comparing the S. cerevisiae biodiversity of two regions in Spain (Priorat and Terra Alta) during three consecutive years, Torija et al. (2001) [21] reported that the yeast population changed from year to year. ...
... Indeed, several studies revealed that there are no strains representative of a winery or an area [20][21][22]. By comparing the S. cerevisiae biodiversity of two regions in Spain (Priorat and Terra Alta) during three consecutive years, Torija et al. (2001) [21] reported that the yeast population changed from year to year. Furthermore, they observed that some identical strains present in different cellars in the same area were also present in the cellars of different areas. ...
... Indeed, several studies revealed that there are no strains representative of a winery or an area [20][21][22]. By comparing the S. cerevisiae biodiversity of two regions in Spain (Priorat and Terra Alta) during three consecutive years, Torija et al. (2001) [21] reported that the yeast population changed from year to year. Furthermore, they observed that some identical strains present in different cellars in the same area were also present in the cellars of different areas. ...
Wine terroir is characterized by a specific taste and style influenced by the cultivar of the fermented grapes, geographical factors such as the vineyard, mesoclimate, topoclimate, and microclimate, soil geology and pedology, and the agronomic approach used. These characteristics together define the concept of "terroir". Thus, regional distinctive flavors in wine have been the subject of many studies aimed at better understanding the link between the wine and the vineyard. Indeed, the identification of key environmental elements involved in the regional variation of grape and wine quality characteristics is a critical feature for improving wine production in terms of consumer preference and economic appreciation. Many studies have demonstrated the role of abiotic factors in grape composition and consequently in wine style. Biotic factors are also involved such as grape microbial communities. However, the occurrence and effects of region-specific microbiota in defining wine characteristics are more controversial issues. Indeed, several studies using high throughput sequencing technologies have made it possible to describe microbial communities and revealed a link between grape must and soil microbial communities, and the geography of the territory. Based on these observations, the concept of "microbial terroir" emerged. However, this concept has been subject to contradictory studies. The aim of this opinion article is to take a step back and examine in perspective the concept of microbial terroir, by comparing numerous data from different studies and providing arguments in favor of or against this concept to stimulate discussion and point out that experimental research is still needed to study the contribution of this assembly of microorganisms to the final product and to support or refute the concept.
... A similar observation was reported that at the initial stages of fermentation (<10 days), LAB population showed a declined trend and after 20 days the population remained stable until the end of their experiments (50 th days) [37]. Likewise, Torija et al. [38], reported that the non-Saccharomyces yeasts were limited to early stages of fermentation of wines, whilst the Saccharomyces yeasts dominated towards the end of alcoholic fermentation. This is due to the fact that Saccharomyces yeast are more tolerant to ethanol and more competitive in a growth medium containing high concentrations of sugar [39]. ...
... In all the samples of our study, high ash content was observed in the longest time of maturity. Ozabor et al. [38] also reported the same characteristics of ash content increment with an increase of maturity time. ...
Tej is an Ethiopian traditional alcoholic beverage with significant social and economic importance. Due to the spontaneous fermentation process of Tej, several issues such as safety, quality, and physicochemical properties of the final products is rquired to be assessed. Thus, this study was aimed to assess the microbial quality, physicochemical, and proximate properties of Tej associated with different maturity time. The microbial, physicochemical and proximate analyses were carried out by standard protocol. Lactic acid bacteria (6.30 log CFU/mL) and yeast (6.22 log CFU/mL) were the dominat microorganisms of all Tej samples at different maturity time, with significant differences (p = 0.001) in mean microbial count among samples. The mean pH, titratable acidity and ethanol content of Tej samples were 3.51, 0.79 and 11.04% (v/v), respectively. There were significant differences (p = 0.001) among the mean pH and titratable acidity values. The mean proximate compositions (%) of Tej samples were as follows: moisture (91.88), ash (0.65), protein (1.38), fat (0.47) and carbohydrate (3.91). Statistically significant differences (p = 0.001) were observed in proximate compositions of Tej samples from different maturity time. Generally, Tej maturity time has a great impact on the improvement of nutrient composition and the increment of the acidic contents which in turn suppress the growth of unwanted microorganisms. Further evaluation of the biological, and chemical safety and development of yeast-LAB starter culture are strongly recommended to improve Tej fermentation in Ethiopia.
... Yeast of several genera and species can convert sugars into ethanol and carbon dioxide as well as other metabolites during alcohol fermentation. While the diversity of microorganisms associated with winemaking is high, Saccharomyces cerevisiae is the dominant yeast species in alcoholic fermentation (Torija et al., 2001). Despite agreement for the role of microorganisms in the winemaking process, no consensus yet exists about the relationships between microbial biodiversity, vinification process and wine quality (Tempère et al., 2018) (Fig. 1). ...
... They found a positive correlation between genetic relatedness of the different strains and aroma profiles in wines which they interpret as evidence for a role of microbial populations to the terroir concept. However, this interpretation is controversial as several studies demonstrated that there are no S. cerevisiae strains representative of a given vineyard and that strains are highly variable between year with no common strains detected between neighbouring vineyards (Alexandre, 2020; Torija et al., 2001;Vigentini et al., 2015). Even if S. cerevisiae is considered as the main actor of wine fermentation, discrepancies in results may come from the fact that other taxa in the microbial community may be involved in fermentation and contribute to any potential terroir effect. ...
... Yeast of several genera and species can convert sugars into ethanol and carbon dioxide as well as other metabolites during alcohol fermentation. While the diversity of microorganisms associated with winemaking is high, Saccharomyces cerevisiae is the dominant yeast species in alcoholic fermentation (Torija et al., 2001). Despite agreement for the role of microorganisms in the winemaking process, no consensus yet exists about the relationships between microbial biodiversity, vinification process and wine quality (Tempère et al., 2018) (Fig. 1). ...
... They found a positive correlation between genetic relatedness of the different strains and aroma profiles in wines which they interpret as evidence for a role of microbial populations to the terroir concept. However, this interpretation is controversial as several studies demonstrated that there are no S. cerevisiae strains representative of a given vineyard and that strains are highly variable between year with no common strains detected between neighbouring vineyards (Alexandre, 2020; Torija et al., 2001;Vigentini et al., 2015). Even if S. cerevisiae is considered as the main actor of wine fermentation, discrepancies in results may come from the fact that other taxa in the microbial community may be involved in fermentation and contribute to any potential terroir effect. ...
For decades, and at global scales, vineyard landscapes have experienced a profound intensification of management. These socio-ecosystems are now facing major environmental, agronomical and economic issues that challenge their future sustainability. In this context, implementing agroecological management of these landscapes is no longer an alternative, and multiple lines of evidence demonstrate the central role of biodiversity and associated ecosystem services for the future functioning of these landscapes. In this paper, we provide a comprehensive overview of this evidence, indicate major gaps in our current knowledge and highlight how biodiversity could become a major asset in the design of multifunctional vineyard landscapes in the context of global change.
... Five mono glucoside anthocyanins, namely delphinidin (3), cyanidin (5), petunidin (6), peonidin (8), and malvidin (9), together with malvidin-3-O-acetylglucoside (15), malvidin-3-O-caffeoylglucoside (17), cyanidin-3-O-(p-coumaroyl)glucoside (17), peonidin-3-O-trans-(p-coumaroyl)glucoside (20), and malvidin-3-O-trans-(p-coumaroyl)glucoside (21) were revealed in all the samples. Whilst other acyl compounds, such as peonidin-3-O-acetylglucoside (14), petunidin-3-O-(p-coumaroyl)glucoside (18), and malvidin-3-Ocis-(p-coumaroyl)glucoside (18), also belonging to the group of free-anthocyanins directly extracted from grape skin [36,37], were not detected in Aleatico wines. ...
... Regarding Primitivo at draining off (Figure 1a), FA18 was characterized by a higher content of vitisin A (10), but also reddish/violet ethylidene-bridged compounds (11, 12, and 19) and bluish pigment (4). On the contrary, SCE16 (and less SCE138) showed greater amounts of free anthocyanins, especially the compounds 6,9,17,20, and 21 together with pyruvic and vinyl derivatives (7 and 16, respectively). Having λ max > 530 nm [31], the relative predominance of the compounds 4, 10, 11, 12, and 19 could partially explain the slightly higher CI in FA18 than SCE wines (Table 1). ...
Mixed fermentation using Starmerella bacillaris and Saccharomyces cerevisiae has gained attention in recent years due to their ability to modulate the qualitative parameters of enological interest, such as the color intensity and stability of wine. In this study, three of the most important red Apulian varieties were fermented through two pure inoculations of Saccharomyces cerevisiae strains or the sequential inoculation of Saccharomyces cerevisiae after 48 h from Starmerella bacillaris. The evolution of anthocyanin profiles and chromatic characteristics were determined in the produced wines at draining off and after 18 months of bottle aging in order to assess the impact of the different fermentation protocols on the potential color stabilization and shelf-life. The chemical composition analysis showed titratable acidity and ethanol content exhibiting marked differences among wines after fermentation and aging. The 48 h inoculation delay produced wines with higher values of color intensity and color stability. This was ascribed to the increased presence of compounds, such as stable A-type vitisins and reddish/violet ethylidene-bridge flavonol-anthocyanin adducts, in the mixed fermentation. Our results proved that the sequential fermentation of Starmerella bacillaris and Saccharomyces cerevisiae could enhance the chromatic profile as well as the stability of the red wines, thus improving their organoleptic quality.
... In Spanish wineries, de Celis et al. described important differences in the S. cerevisiae diversity (from 1 to 151 strains) among wineries related to farming system or the use of commercial or autochthonous yeasts [30]. Other authors using mtDNA-RFLPs found until 38 different profiles in wineries from La Rioja [24]; whereas up to 112 strains were distinguished in two wineries in three vintages in Cataluña using the same methodology [58]. ...
... In addition, we observed differences in the yeast population between red and white must fermentations ( Figure 4). These differences could be expected since both substrates differ in their characteristics and fermentation protocols [25,58,63]. In addition, the interaction of S. cerevisiae and other yeast species in must also influence yeast population dynamics during fermentation. ...
Yeast play an essential role in wine quality. The dynamics of yeast strains during fermentation determine the final chemical and sensory characteristics of wines. This study aims to evaluate the Saccharomyces cerevisiae strains diversity in organic wineries from Galicia (NW Spain). Samples from spontaneous fermentations were taken in five wineries over three consecutive years (2013 to 2015). The samples were transported to the laboratory and processed following standard methodology for yeast isolation. S. cerevisiae strains were differentiated by mDNA-RFLPs. A total of 66 different strains were identified. Some of them presented a wide distribution and appeared in several wineries. However, other strains were typical from a specific winery. Similarity analysis using two different statistical tests showed significant differences in strain diversity among wineries. The results also revealed high biodiversity indexes; however, only some strains showed an important incidence in their distribution and frequency. Our findings confirmed that spontaneous fermentation favored the existence of a high S. cerevisiae strain diversity in organic wineries from Galicia. The presence of different yeasts during fermentation, specially winery-specific strains, contribute to increased wine complexity and differentiation.
... Several ecological surveys, using molecular methods of identification, report a large diversity of genetic patterns among the enological fermentative flora. S. cerevisiae strains seem to be widely distributed in a given viticultural region [16][17][18][19], can be found in consecutive years [20,21] and there are also strains predominant in the fermenting flora [2,22], which support the notion that specific native strains can be associated with a terroir. ...
... The vast majority of the patterns were unique, demonstrating an enormous biodiversity of S. cerevisiae strains in the Vinho Verde Region. Considering the ratio between the number of isolates and the number of patterns as an approximate biodiversity estimative, our results showed similar values to previously published surveys on genetic diversity of autochthonous oenological S. cerevisiae strains in other regions with viticulture traditions such as Bordeaux [2], Charentes [17,45], Campagne and Loire Valley [21], in France; El Penedèz [46], Tarragona [7], Priorato [20,22] and La Rioja [47] in Spain; Germany and Switzerland [41]; Tuscany, Sicily [48] and Collio [49] in Italy; Amyndeon and Santorini [42] in Greece; Western Cape [16,18,43] in South Africa; and Patagonia [19] in Argentina. The present study has been carried out in a viticultural region that has never been characterized before and includes aspects that have not been considered in previous works, such as the appearance of several commercial yeast strains, and the comparison of yeast populations that can be found in grape samples before and after the harvest. ...
One thousand six hundred and twenty yeast isolates were obtained from 54 spontaneous fermentations performed from grapes collected in 18 sampling sites of three vineyards (Vinho Verde Wine Region in northwest Portugal) during the 2001-2003 harvest seasons. All isolates were analyzed by mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) and a pattern profile was verified for each isolate, resulting in a total of 297 different profiles, that all belonged to the species Saccharomyces cerevisiae. The strains corresponding to seventeen profiles showed a wider temporal and geographical distribution, being characterized by a generalized pattern of sporadic presence, absence and reappearance. One strain (ACP10) showed a more regional distribution with a perennial behavior. In different fermentations ACP10 was either dominant or not, showing that the final outcome of fermentation was dependent on the specific composition of the yeast community in the must. Few of the grape samples collected before harvest initiated a spontaneous fermentation, compared to the samples collected after harvest, in a time frame of about 2 weeks. The associated strains were also much more diversified: 267 patterns among 1260 isolates compared to 30 patterns among 360 isolates in the post- and pre-harvest samples, respectively. Fermenting yeast populations have never been characterized before in this region and the present work reports the presence of commercial yeast strains used by the wineries. The present study aims at the development of strategies for the preservation of biodiversity and genetic resources as a basis for further strain development.
... As previously described in literature [58,59], some strains seem specific to a vintage: for example, the strain AF10 was detected in all vats of the 2016 vintage and after AF on floor and walls (T3 2016), and the strains AF15 and AF17 were specific to 2017 vintage ( Figure 3B). The presence of the strains However, three months after the AF (T3), no Saccharomyces cerevisiae strains from the PDC were detected on the WREs: walls, floor and equipment ( Figure 2B). ...
... Some differences were observed between both strains: the surface coverage for the strain P19 was more scattered and the microcolonies structures were less developed compared to the strain P1 (Figure 4). As previously described in literature [58,59], some strains seem specific to a vintage: for example, the strain AF10 was detected in all vats of the 2016 vintage and after AF on floor and walls (T3 2016), and the strains AF15 and AF17 were specific to 2017 vintage ( Figure 3B). The presence of the strains AF15 and AF17on winery equipment at T0 2017, prior to the fermentation, is probably due to must handling before the sampling of the winery equipment. ...
The aim of this work was to study the fungal colonization of a new winery over time, specifically for Saccharomyces cerevisiae. Therefore, we analyzed the flora present before the arrival of the first harvest on the floor, the walls and the equipment of this new winery by Illumina MiSeq. The genus Saccharomyces (≤0.3%) was detected on floor and equipment but the presence of S. cerevisiae species was not reported. Wild S. cerevisiae strains were isolated from a ‘Pied de Cuve’ used during the first vintage to ensure the alcoholic fermentation (AF). Among 25 isolates belonging to this species, 17 different strains were identified highlighting a great intraspecific diversity. S. cerevisiae strains were also isolated from different vats throughout the spontaneous fermentations during the first vintage. The following year, some of these strains were isolated again during AF. Some of them (four) were found in the winery equipment before the arrival of the third harvest suggesting a potential colonization by these strains. To better understand what promotes the yeast colonization of the winery’s environment, the ability to form a biofilm on solid surfaces for eight colonizing or non-colonizing strains was studied. This capacity, different according to the strains, could partly explain the colonization observed for certain strains.
... Wineries displayed distinct and, in some cases, temporally unstable fungal communities, and this is consistent with the observations reported in similar studies (Bokulich et al., 2014;Knight et al., 2015;Liu & Howell, 2021;Torija et al., 2001;Vigentini et al., 2015). Despite this, the effect size of these associations was considerably lower than previously reported, likely due to differences in sampling size, geographic coverage, number of grape varieties, and years of sampling. ...
Over 6 years, we conducted an extensive survey of spontaneous grape fermentations, examining 3105 fungal microbiomes across 14 distinct grape‐growing regions. Our investigation into the biodiversity of these fermentations revealed that a small number of highly abundant genera form the core of the initial grape juice microbiome. Consistent with previous studies, we found that the region of origin had the most significant impact on microbial diversity patterns. We also discovered that certain taxa were consistently associated with specific geographical locations and grape varieties, although these taxa represented only a minor portion of the overall diversity in our dataset. Through unsupervised clustering and dimensionality reduction analysis, we identified three unique community types, each exhibiting variations in the abundance of key genera. When we projected these genera onto global branches, it suggested that microbiomes transition between these three broad community types. We further investigated the microbial community composition throughout the fermentation process. Our observations indicated that the initial microbial community composition could predict the diversity during the early stages of fermentation. Notably, Hanseniaspora uvarum emerged as the primary non‐ Saccharomyces species within this large collection of samples.
... There were 42 genotypes in SF, called SF-1 to SF-42; 45 genotypes in YF, called YF-1 to YF-38, SF-1 to SF-6, and CECA; and 32 genotypes in YDF called YDF-1 to YDF-24, SF-1 to SF-3, YF-1 to YF-4, and CECA (Table 3). The degree of variability (Torija et al., 2001), the percentage of the relative abundance of different strains found among the analyzed colonies, in SF (37.17%, 42/113) was higher than in the other treatments: 27.95% (45/161) in YF and 13.79% (32/232) in YDF. In SF, genotype SF-2 was the dominant strain for the entire fermentation Fig. 2. Yeast community dynamics at species level revealed by (a) culture-dependent and (b) culture-independent methods during three wine fermentation processes. ...
... Therefore, secondary aromas have a greater influence among the main volatile compounds that define the overall quality of wine, for which the yeast inoculum and the fermentation methodology play an essential role [69]. Despite this, the importance of indigenous isolates of S. cerevisiae is also amply demonstrated during the production of wine, reflecting defined territorial characteristics [70,71]. Several studies reported that the use of native S. cerevisiae yeasts leads to wines with higher ester concentrations and sweet/fruity aromas [72]. ...
Wine represents a complex matrix in which microbial interactions can strongly impact the quality of the final product. Numerous studies have focused on optimizing microbial approaches for addressing new challenges to enhance quality, typicity, and food safety. However, few studies have investigated yeasts of different genera as resources for obtaining wines with new, specific traits. Currently, based on the continuous changes in consumer demand, yeast selection within conventional Saccharomyces cerevisiae and unconventional non-Saccharomyces yeasts represents a suitable opportunity. Wine fermentation driven by indigenous yeasts, in the various stages, has achieved promising results in producing wines with desired characteristics, such as a reduced content of ethanol, SO2, and toxins, as well as an increased aromatic complexity. Therefore, the increasing interest in organic, biodynamic, natural, or clean wine represents a new challenge for the wine sector. This review aims at exploring the main features of different oenological yeasts to obtain wines reflecting the needs of current consumers in a sustainability context, providing an overview, and pointing out the role of microorganisms as valuable sources and biological approaches to explore potential and future research opportunities.
... Yeast characterization was undertaken to assess the influence of the yeast strain on the fermentation performance. The results of laboratory-scale fermentations are in accordance with the obser-vations generally reported in the literature for wine yeast, showing that S. cerevisiae strains possess higher fermentative power than non-Saccharomyces strains [6,[38][39][40][41][42]. M. pulcherrima, when isolated, showed low fermentative capacity, probably due to its sensibility to high concentrations of ethanol, making the mixed use with S. cerevisiae to completely ferment the grape must necessary [21,38,43]. ...
The use of indigenous yeasts for the production of wines is a tool to defend the typicity of a particular region. The selection of appropriate indigenous yeasts ensures the maintenance of oenological characteristics by simulating spontaneous alcoholic fermentation (AF) while avoiding the risks of stuck or sluggish fermentations. In this study, autochthonous yeasts from Verdejo grape juice (Appellation of Origin Rueda) were selected, identified, and characterized to exploit the characteristics of the ‘terroir’. The fermentation capacity of seven strains was studied individually at the laboratory scale. The most suitable strains (Saccharomyces cerevisiae: Sacch 1, Sacch 2, Sacch 4, and Sacch 6) and Sacch 6 co-inoculated with Metschnikowia pulcherrima were characterized at the pilot scale. The fermentation kinetics, bioproduct release, volatile composition, and sensory profile of the wines were evaluated. Significant differences were found, especially in the aroma profile. In particular, Sacch 6 and Sacch 6 co-inoculated with M. pulcherrima produced higher amounts of ethyl esters and acetates and lower amounts of higher alcohols than the spontaneous AF. Wines inoculated with indigenous yeasts had higher sensory scores for fruit aromas and overall rating. The selection of indigenous yeasts improved the aroma of Verdejo wines and could contribute to determining the wine typicity of the wine region.
... The use of indigenous strains of Saccharomyces cerevisiae as region-specific wine starters is increasingly attracting the interest of wine researchers and winemakers [1]. These yeasts are well adapted to micro-area conditions of a given region [2][3][4] and could ensure the maintenance of the typical flavor and aroma of wines obtained by grapevine cultivars of a specific geographical area [5][6][7]. Indeed, S. cerevisiae strains collected from ecologically and geographically diverse sources typically show genetic divergences associated with habitat type [8][9][10]. ...
In winemaking, the influence of Saccharomyces cerevisiae strains on the aromatic components of wine is well recognized on a laboratory scale, but few studies deal with the comparison of numerous strains on a pilot scale fermentation. In this scenario, the present work aimed to validate the fermentative behavior of seven wild S. cerevisiae strains on pilot-scale fermentations to evaluate their impact on the aromatic profiles of the resulting wines. The strains, isolated from grapes of different Italian regional varieties, were tested in pilot-scale fermentation trials performed in the cellar in 1 hL of Aglianico grape must. Then, wines were analyzed for their microbiological cell loads, main chemical parameters of enological interest (ethanol, total sugars, fructose, glucose, total and volatile acidity, malic and lactic acids) and volatile aroma profiles by GC/MS/SPME. Seventy-six volatile compounds belonging to six different classes (esters, alcohols, terpenes, aldehydes, acids, and ketones) were identified. The seven strains showed different trends and significant differences, and for each class of compounds, high-producing and low-producing strains were found. Since the present work was performed at a pilot-scale level, mimicking as much as possible real working conditions, the results obtained can be considered as a validation of the screened S. cerevisiae strains and a strategy to discriminate in real closed conditions strains able to impart desired wine sensory features.
... In MIX treatments, the content of ethyl myristate (F1 and F2 sampling times) and ethyl 9-hexadecenoate (F2 sampling time) were significantly lower than in SC, whereas in the case of ethyl octanoate, a similar trend was observed as in LT treatment. The obtained results agree with other Fermentation 2023, 9,29 14 of 18 studies [35] proving the lower amount of ester in L. thermotolerans products. In contrast, Benito et al. [16] reported an increase in the concentration of wine isoamyl alcohol, ethyl octanoate, and isoamyl acetate in mixed fermentations of L. thermotolerans-S. ...
This work presents the attempt to develop a production technology for grape–plum low-alcohol beverages and enhance their chemical composition and flavor complexity through the non-Saccharomyces species. Saccharomyces cerevisiae (SC) pure cultures were used as reference beverages. Pure cultures of Lachancea thermotolerans (LT) and co-inoculated Lachancea thermotolerans with Saccharomyces cerevisiae (MIX) were included for grape–plum must fermentation at a pilot scale. The process involves two steps: a primary alcoholic fermentation in stainless steel tanks (F1) and a secondary fermentation in a bottle after dextrose syrup addition (F2). The chemical compositions of all beverages obtained in F1 and F2 were studied. Compared to SC, must inoculated with L. thermotolerans (LT and MIX) required four more days to complete the fermentation of sugars during F1. SC fermentation tended to have slightly higher pH and titratable acidity values and lower concentrations of total phenols. Final levels of aromatic precursor nitrogen and sulfur amino nitrogen were obtained more in SC than in LT and MIX. SC treatment had higher final levels of histidine, phenylalanine, isoleucine, lysine, methionine, threonine, valine, and cysteine. Related to individual amino acids, SC treatment had higher final levels of histidine, phenylalanine, isoleucine, lysine, methionine, threonine, valine, and cysteine. Analysis of the volatile composition showed that, compared with SC, MIX had the highest percentage of higher alcohols (3-methyl-1-butanol and 2-methyl-1-butanol) and acetates (isoamyl acetate and isobutyl acetate) which are associated with fruity and banana aromas. A decreasing trend in volatile fatty acids was observed in LT and MIX compared to SC. LT application, both in pure and mixed culture, significantly modified the values of the percentage of 5 of the 10 ethyl ester compounds analyzed. Finally, the sensory analysis showed that there were no significant differences, even though the non-Saccharomyces had a higher percentage of volatile metabolites. The results have shown that through this process an innovative and high-quality product was obtained: a low-alcohol beverage made from grapes and plums, which could be developed at an industrial level due to the increasing interest of consumers in this type of product.
... Detection of Saccharomyces in mid fermentation and end fermentation is logical and corresponds to the inoculum of the commercial strain. The high relative abundance of Saccharomyces in must differs, however, from previous observations, describing this genus as generally weakly represented (Hierro et al., 2006;Morrison-Whittle & Goddard, 2018;Torija et al., 2001). ...
High‐throughput sequencing approaches, which target a taxonomically discriminant locus, allow for in‐depth insight into microbial communities’ compositions. Although microorganisms are historically investigated by cultivation on artificial culture media, this method presents strong limitations, since only a limited proportion of microorganisms can be grown in vitro. This pitfall appears even more limiting in enological and winemaking processes, during which a wide range of molds, yeasts, and bacteria are observed at the different stages of the fermentation course. Such an understanding of those dynamic communities and how they impact wine quality therefore stands as a major challenge for the future of enology. As of now, although high‐throughput sequencing has already allowed for the investigation of fungal communities, there is no available comparative study focusing on the performance of microbial deoxyribonucleic acid (DNA) extraction in enological matrixes. This study aims to provide a comparison of five selected extraction methods, assayed on both must and fermenting must, as well as on finished wine. These procedures were evaluated according to their extraction yields, the purity of their extracted DNA, and the robustness of downstream molecular analyses, including polymerase chain reaction and high‐throughput sequencing of fungal communities. Altogether, two out of the five assessed microbial DNA extraction methods (DNeasy PowerSoil Pro Kit and E.Z.N.A.® Food DNA Kit) appeared suitable for robust evaluations of the microbial communities in wine samples. Consequently, this study provides robust tools for facilitated upcoming studies to further investigate microbial communities during winemaking using high‐throughput sequencing.
... Allele sizing of the microsatellite loci discriminated 94 different patterns among the 150 isolated S. cerevisiae strains ( Figure 1). The number of Saccharomyces strains present during spontaneous fermentation is known to be strongly variable, from less than 20 different strains [26,27] to more than 110 reported in two Spanish cellars [28]. Thus, a highly S. cerevisiae diverse population underlies the Aljarafe region ( Figure 1). ...
Natural diversity represents an inexhaustible source of yeasts for the diversification of wines and the improvement of their properties. In this study, we analysed the genetic diversity of autochthonous Saccharomyces cerevisiae wine yeasts in the Aljarafe of Seville, one of the warmest winemaking regions of Spain. Through multiplex-PCR analysis of five microsatellite markers and RT-PCR determination of the killer genotype, we found 94 different patterns among 150 S. cerevisiae yeast strains isolated from spontaneous fermentation of grape must, thereby representing a highly diverse population. Remarkably, 92% of the isolated strains exhibited high sporulation capacity. Tetrad analysis of sporulating strains rendered a microsatellite marker’s combinatory that mimics patterns observed in the native population, suggesting that the high polymorphism of microsatellite markers found in these wild yeasts might result from sexual reproduction in their natural environment. The identification of unconventional M2/L-A-lus totivirus combinations conferring the killer phenotype also supports this suggestion. One idea behind this study is to determine to what extent the vineyards microbiota in areas with warm climates can provide useful natural yeasts to adapt fermentation processes to the needs imposed by global warming. Analysis of traits of oenological interest in regions potentially affected by global climate changes, such as growth tolerance to ethanol and to sugar stress in the analysed strains, indicated that this broad combinatorial diversity of natural S. cerevisiae yeasts provides a wide range of autochthonous strains with desirable profiles for quality winemaking in warm regions. This combinatorial diversity renders strains with diverse oenological performing abilities. Upon microvinification assays and organoleptic attests, a S. cerevisiae strain with interesting oenological properties has been identified. This result can be considered a successful outcome in industry–academia collaboration.
... In addition, the cost of using this technique is lower, and it could work completed faster and better than ITS PCR and fluorescent ITS PCR capillary electrophoresis (Wheeler 2008). High-throughput sequencing technology has been widely used in microbial diversity sequencing, microbial community structure sequencing, and dynamic changes of dominant species, and NGS was employed in the analysis of microbiota diversity in various fermentation processes (Torija et al. 2001;Bao et al. 2021). In addition, the application of this technique in microbial community dynamic analysis has been relatively mature. ...
The ‘Kyoho’ grape (Vitaceae, Plantae) has large ears, plenty of flesh, and rich nutrition and is planted across a large area in China. There are few reports on this variety in winemaking, especially on the dynamic changes of fungi in the wine fermentation broth. In this study, we used the ‘Kyoho’ grapes as raw materials and adopted a high throughput to analyze dynamic changes in fungal species composition of the natural fermentation broth at four time points: day 1 (D1P), day 3 (D3P), day 5 (D5P), and day 15 (D15P). Changes in fungal metabolic pathways and dominant yeasts were also analyzed. A total of 78 families, 110 genera, and 137 species were detected, in the natural fermentation broth samples. Forty-nine families, 60 genera, and 72 species were found in the control check (CK). A total of 66 differential metabolic pathways were enriched; of those, 41 were up-regulated compared to CK, such as CDP-diacylglycerol biosynthesis I (PWY 5667), chitin degradation to ethanol (PWY 7118), and the super pathway of phosphatidate biosynthesis (PWY 7411). Changes in fungal metabolic pathways were in line with the dynamic changes of dominant yeast species in the whole process of fermentation. Pichia kluyveri, P. membranifaciens, and Citeromyces matritensis are the dominant species in the later stages of natural fermentation. These yeast species may play vital roles in the ‘Kyoho’ wine industry in the future.
... Candida stellata is one non-conventional yeast that appears regularly in considerable amounts (between 5% to 12%, or even up to 50% of the total population) in most early spontaneous fermentations (Combina et al., 2005;Torija et al., 2001). High populations are common in overripe or botrytized grape berries. ...
Climate change is generating several problems in wine technology. One of the main ones is lack of acidity and difficulties performing malolactic fermentation to stabilize wines before bottling. Among the different available acidity management technologies, such as direct acid addition, ion exchange resins, electro-membrane treatments, or vineyard management, the microbiological option is reliable and deeply studied. The main approach is the increase in malic acid content because of the metabolism of specific Saccharomyces strains and to increase lactic acid because of the metabolism of Lachancea genus. Other non-Saccharomyces yeasts, such as Starmerella bacillaris or Candida stellata can also acidify significantly because of the production of pyruvic or succinic acid. Wine industry needs the removal of malic acid in most red wines before bottling to achieve wine stability. Oenococus oeni performs the malolactic fermentation of red wines on most conditions because of the metabolization of malic acid into lactic acid. However, modern oenology challenges such as high ethanol concentrations, high pH or low levels of malic acid have made researchers to look for other options to reduce potential risks of deviation. Other wine-related microorganisms able to de-acidify malic acid have appeared as interesting alternatives for specific difficult scenarios. Lactiplantibacillus plantarum and Schizosaccharomyces genus make up nowadays the main studied alternatives.
... This elevated polymorphism in both nonconsecutive vintages indicated a high persistence over the years in this specific organic winery where starter yeasts have never been used. Similar results were reported also by Torija et al. [46] and Granchi et al. [45] that described the persistence of recurrent and predominant S. cerevisiae strains during spontaneous wine fermentations of a specific wineproducing area. In this regard, Granchi et al. [45] found that native S. cerevisiae strains are representative of a specific oenological ecosystem and that some of them termed "dominant" persisted in different fermentations in the same winery from one year to another, and they seemed to be representative of a single winery rather than of an oenological area. ...
The relation between regional yeast biota and the organoleptic characteristics of wines has attracted growing attention among winemakers. In this work, the dynamics of a native Saccharomyces cerevisiae population was investigated in an organic winery. In this regard, the occurrence and the persistence of native S. cerevisiae were evaluated in the vineyard and winery and during spontaneous fermentation of two nonconsecutive vintages. From a total of 98 strains, nine different S. cerevisiae biotypes were identified that were distributed through the whole winemaking process, and five of them persisted in both vintages. The results of the oenological characterization of the dominant biotypes (I and II) show a fermentation behavior comparable to that exhibited by three common commercial starter strains, exhibiting specific aromatic profiles. Biotype I was characterized by some fruity aroma compounds, such as isoamyl acetate and ethyl octanoate, while biotype II was differentiated by ethyl hexanoate, nerol, and β-damascenone production also in relation to the fermentation temperature. These results indicate that the specificity of these resident strains should be used as starter cultures to obtain wines with distinctive aromatic profiles.
... Actually, S. cerevisiae was isolated in musts and grape skins from all regions except Sardinia, demonstrating that this species is found in grape samples. This finding contradicts other studies postulating that this species was found in low proportions or was even absent in grapes [30,66]. However, in this work, it was found in relatively considerable proportions in both grapes and musts, which agrees with other previous studies [25,31]. ...
Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi is recognized as the dioecious parental generation of today’s cultivars. Climatic change and the arrival of pathogens and pests in Europe led it to be included on the International Union for Conservation of Nature (IUCN) Red List of Threatened Species in 1997. The present work focused on the study of culturable yeast occurrence and diversity of grape berries collected from wild vines. Sampling was performed in 29 locations of Azerbaijan, Georgia, Italy, Romania, and Spain. In total, 3431 yeast colonies were isolated and identified as belonging to 49 species, including Saccharomyces cerevisiae, by 26S rDNA D1/D2 domains and ITS region sequencing. Isolates of S. cerevisiae were also analyzed by SSR–PCR obtaining 185 different genotypes. Classical ecology indices were used to obtain the richness (S), the biodiversity (H’), and the dominance (D) of the species studied. This study highlights the biodiversity potential of natural environments that still represent a fascinating source of solutions to common problems in winemaking.
... Vol:. (1234567890) causative organism Schizosaccharomyces pombe was identified (Delfini et al. 1983). S. pombe was detected in wine fermentations in Spain (Torija et al. 2001), in Greece (Jörgensen 1909), South Africa (van der Walt and van Kerken 1958) and Portugal (Couto et al. 2005). In Argentina S. pombe was detected in several samples of concentrated grape juices (Combina et al. 2008). ...
The fission yeast genus Schizosaccharomyces contains important model organisms for biological research. In particular, S. pombe is a widely used model eukaryote. So far little is known about the natural and artificial habitats of species in this genus. Finding out where S. pombe and other fission yeast species occur and how they live in their habitats can promote better understanding of their biology. Here we investigate in which substrates S. pombe, S. octosporus, S. osmophilus and S. japonicus are present. To this end about 2100 samples consisting of soil, tree sap fluxes, fresh fruit, dried fruit, honey, cacao beans, molasses and other substrates were analyzed. Effective isolation methods that allow efficient isolation of the above mentioned species were developed. Based on the frequency of isolating different fission yeast species in various substrates and on extensive literature survey, conclusions are drawn on their ecology. The results suggest that the primary habitat of S. pombe and S. octosporus is honeybee honey. Both species were also frequently detected on certain dried fruit like raisins, mango or pineapple to which they could be brought by the honey bees during ripening or during drying. While S. pombe was regularly isolated from grape mash and from fermented raw cacao beans S. octosporus was never isolated from fresh fruit. The main habitat of S. osmophilus seems to be solitary bee beebread. It was rarely isolated from raisins. S. japonicus was mainly found in forest substrates although it occurs on fruit and in fruit fermentations, too.
... For example, Baiano et al. [13] reported a minor variation of malic acid level obtained during winemaking, tartaric acid content was significantly diminished when using cryomaceration technique compared to traditional technology and citric acid presented upper values. Other authors demonstrated a major influence of temperature, sugar, and pH levels [3] on succinic acid [14]. The formation of acetic acid is significantly affected by the yeast strain [15], sugar level, pH [16,17], or fermentation temperature [3]. ...
Organic acids represent naturally occurring compounds that are found in many types of food and beverages, with important functions in defining products’ final quality. Their proportions in wine are dependent on grape composition and winemaking conditions (temperature, pH levels, oxygen, and carbon dioxide concentration). Therefore, this article studied the influence of different fermentation conditions (200 hL tanks vs. 50 L glass demijohns) and various yeasts on the evolution of the main organic acids during alcoholic fermentation of “Aligoté” wines. The fermentation lasted 22 days and samples were collected daily. Laboratory analyses were quantified according to the International Organization of Vine and Wine recommendations. High-performance liquid chromatography for the identification and quantification of organic acids was used. The data showed the important effect of winemaking conditions on sugar consumption, density or acidity values, and sensory characteristics. Significant differences in organic acid concentrations (especially for tartaric acid) were obtained for all variants, depending on the fermentation conditions, inoculated yeast and the sampling moment. The quantities of most of the identified organic acids were generally significantly increased when glass vessels were used, compared to those fermented in tanks. Most organic acids concentrations were favored by lower pH and showed higher values at lower temperatures.
... A concentration between 5-7 per cent (v/v) of ethanol has been often cited as the maximum tolerance for non-Saccharomyces species (Heard and Fleet, 1988;Gao and Fleet, 1988). However, it's noteworthy that some strains of non-Saccharomyces have been isolated at late stages and even at the end of spontaneous fermentations (Torija et al., 2001;Llauradó et al., 2002;Nurgel et al. 2005;Wang and Liu, 2013;Padilla et al., 2016). Recent studies also found that some non-Saccharomyces strains of H. uvarum, H. guilliermondii, T. delbrueckii and S. bacillaris could tolerate ethanol of 10 per cent (Pina et al., 2004;Pérez-Nevado et al., 2006;Wang et al., 2015), which means that these species may be more resistant to ethanol than it was considered in the past. ...
... These findings reinforce the idea of a microbial component of the terroir (Martínez et al., 2007;Raspor et al., 2006;Valero et al., 2007) which supports the increasing interest on the composition of yeast communities from grape surfaces and its possible effects on wine's quality (Jolly et al., 2014). The concept of microbial terroir also provides support to the selection programs of autochthonous S. cerevisiae strains, that have been performed since long in several wine-producing regions around the world Khan et al., 2000;Lopes et al., 2002;Pramateftaki et al., 2000;Torija et al., 2001), leading to a rediscovery of wines obtained by using indigenous yeasts naturally occurring on grapes (Francesca et al., 2009). ...
Biogeography is the study of the distribution of species over space and time and aims to
understand where, why, and at what abundance organisms live. Revealing the diversity and
distribution pattern of populations and communities at multiple spatial scales is thus a central issue in ecology. Recent biogeographic studies based on genetic technologies revealed that microorganisms are not randomly distributed over space and time, showing that their distribution is systematically heterogeneous and structured, revealing specific patterns of microbial distribution. In this context, the study of microbial communities associated with vineyards points to the existence of patterns of microbial distribution across viticultural areas, suggesting a microbiological component of the terroir concept. In the light of this knowledge, the composition of the yeast flora present on grapes, besides representing a long-term known factor of wine quality tends to be presently seen as a potential factor of wine typicality. To assess grape yeast diversity and to understand the ecological and geographical factors shaping the yeast communities and populations composition is of great importance for modern oenology. In this chapter we present
on overview of the research developed in this scope.
... In recent years, many studies have been conducted to investigate indigenous wine yeasts around the world, including in France, Australia, Spain, Italy, Hungary, the United States, etc. [9,10,[14][15][16][17][18]. In China, researchers also have investigated indigenous wine yeasts. ...
The investigation of indigenous wine yeast can facilitate the production of distinctive Beijing wine by providing wineries with more choices in distinctive wine yeasts. Wine yeasts that were isolated from grape must during spontaneous fermentation and from vineyard soil were preliminarily classified by their colony morphologies when cultured on Wallerstein Laboratory Nutrient (WLN) medium and then were identified by 5.8S ITS sequence analysis. Four selected strains were involved in inoculated fermentation tests. The results showed that five yeast species were isolated from the grape must, and five non-Saccharomyces yeast species were found in vineyard soil. The colony morphology of Pichia occidentalis on the WLN medium was described herein for the first time. An effective way which could be used for isolate and select wine yeasts from soil samples on WLN medium was described. And one of the selected indigenous strains shared over 99% similarity on 5.8S ITS sequence with commercial wine yeast, which indicated that commercial yeast might have influenced the local yeast diversity. The two selected indigenous Saccharomyces cerevisiae strains, which showed good fermentation performances, could be applied in the local wine industry in the future.
... This may also explain why individual strains were found in consecutive years. Similar conclusions have been reached in the case of grape fermentation [53][54][55]. ...
The influence of fruit varieties on yeast ecology during spontaneous plum mash fermentation was investigated. Yeast colonies were isolated from mashes obtained from four plum varieties throughout fermentation in laboratory conditions during two consecutive years. The yeast strains were differentiated by random amplification of polymorphic DNA (RAPD-PCR) and identified by the 26S rDNA D1/D2 sequence analysis. Hanseniaspora uvarum, Metschnikowia spp. and Pichia kudriavzevii were the dominant yeasts during the early stages of plum mash fermentation, while the middle and end phases were dominated by Saccharomyces cerevisiae. The strains of Candida sake, Nakazawaea ernobii, Pichia kluyveri, Rhodotorula mucilaginosa and Wickerhamomyces anomalus were also detected in fermenting plum mashes. Metschnikowia sp. M1, H. uvarum H1 and H2 strains were detected in all samples, irrespective of the tested variety and year. Investigation of the impact of individual yeast strains on the production of volatile compounds showed the potential possibility of using them as starter cultures.
... Vineyard geography, environment and management practices, and harvest, juice/must processing and fermentation conditions can all affect yeast population dynamics during wine fermentation [7][8][9][10][11][12][13]. Of those fermentation conditions that are readily modulated by winemakers, the addition of the antimicrobial sulphur dioxide (SO 2 ) represents the most broadly available intervention practice. ...
Uninoculated wine fermentations are conducted by a consortium of wine yeast and bacteria that establish themselves either from the grape surface or from the winery environment. Of the additives that are commonly used by winemakers, sulphur dioxide (SO2) represents the main antimicrobial preservative and its use can have drastic effects on the microbial composition of the fermentation. To investigate the effect of SO2 on the resident yeast community of uninoculated ferments, Chardonnay grape juice from 2018 and 2019 was treated with a variety of SO2 concentrations ranging up to 100 mg/L and was then allowed to undergo fermentation, with the yeast community structure being assessed via high-throughput meta-barcoding (phylotyping). While the addition of SO2 was shown to select against the presence of many species of non-Saccharomyces yeasts, there was a clear and increasing selection for the species Hanseniaspora osmophila as concentrations of SO2 rose above 40 mg/L in fermentations from both vintages. Chemical analysis of the wines resulting from these treatments showed significant increases in acetate esters, and specifically the desirable aroma compound 2-phenylethyl acetate, that accompanied the increase in abundance of H. osmophila. The ability to modulate the yeast community structure of an uninoculated ferment and the resulting chemical composition of the final wine, as demonstrated in this study, represents an important tool for winemakers to begin to be able to influence the organoleptic profile of uninoculated wines.
... F. Aydın, et al. International Journal of Food Microbiology 325 (2020) 108647 small subunit (SSU) of rDNA comparably separated and identified different yeast species isolated from different food sources such as cheese, yogurt, kefir, wine, and grape (Banjara et al., 2015;Belloch et al., 2002;Caggiaa et al., 2001;Constantı et al., 1998;Dertli and Çon, 2017;Eder et al., 2017;Torija et al., 2001). The sequence comparison in the GenBank database by the BLAST algorithm revealed that all yeast isolates we used have high nucleotide similarity with the corresponding isolates available in GenBank, which confirm that the sequence analysis of the ribosomal DNA region is a reliable tool for the yeast identification. ...
Yeasts are one of the main organisms in the food industry and effective components of many ecosystems. The method for identifying and detecting certain yeast species or strains is a crucial step for the food industry and should be simple, reliable, fast, and inexpensive. In our study, inter-priming binding sites (iPBS) retrotransposon marker system was employed to elucidate the genetic variability at intraspecies and interspecies levels among 112 yeast strains belonging to eight species previously obtained from fermented foods. The molecular identification of yeast strains was firstly confirmed by sequencing the D1/D2 domain of the 26S rRNA. The eight selected retrotransposon-based primers produced 278 bands, all of which were polymorphic with an average of 34.75 polymorphic fragments per primer. The averages of polymorphism information contents and the resolving power values for the iPBS marker system were 0.23 and 10.11, respectively. The genetic parameters within each yeast species obtained from iPBS markers were observed as; the percentage of polymorphic loci for each species ranging from 19.23% to 71.21%, Nei's gene diversity from 0.085 to 0.228, while Shannon's information index values ranging from 0.125 to 0.349. The value of gene flow (0.09) and genetic variation among the populations (0.85) showed higher genetic variation among the species. UPGMA analyses demonstrated considerable genetic variability in the yeast strains, clustered them according to their species, and revealed the intraspecific variation. Each of the selected iPBS primer provided enough species-discrimination. Present evaluations suggest the utility of iPBS marker system to estimate the genetic variation of yeast strains. This study is a preliminary point for further studies on the identification methodology, and population genetics of yeast species having importance in the food industry with iPBS markers.
... Some species that belong to the last group can participate in the beginning of the fermentation process [2]. The non-Saccharomyces species present in the grape juice and, in the first stages of fermentation, are divided into three groups: yeasts that are mostly aerobic (Pichia spp., Rhodotorula spp., or Cryptococcus albidus), yeasts with low fermentation ability (Kloeckera apiculata, K. apis and K. javanica) and yeasts that display fermentative metabolism (Metschnikowia pulcherrima, Kluyveromyces marxiamus and Zygosaccharomyces bailii) [3,4]. Nevertheless, non-Saccharomyces yeasts can also be detected before the fermentation process, i.e., during ripening and harvest processes. ...
There a lot of studies including the use of non-Saccharomyces yeasts in the process of wine fermentation. The attention is focused on the first steps of fermentation. However, the processes and changes that the non-Saccharomyces yeast populations may have suffered during the different stages of grape berry ripening, caused by several environmental factors, including antifungal treatments, have not been considered in depth. In our study, we have monitored the population dynamics of non-Saccharomyces yeasts during the ripening process, both with biochemical identification systems (API 20C AUX and API ID 32C), molecular techniques (RFLP-PCR) and enzymatic analyses. Some unusual non-Saccharomyces yeasts have been identified (Metschnikowia pulcherrima, Aureobasidium pullulans, Cryptococcus sp. and Rhodotorula mucilaginosa). These yeasts could be affected by antifungal treatments used in wineries, and this fact could explain the novelty involved in their isolation and identification. These yeasts can be a novel source for novel biotechnological uses to be explored in future work.
... S. cerevisiae was the fastest yeast to ferment all tested media, due to its well-known good fermentative capacity [13,[48][49][50]. In SM-Mix, T. delbrueckii and L. thermotolerans, which also had good fermentation performance, exhibited a similar nitrogen consumption pattern to that of S. cerevisiae. ...
Non-Saccharomyces yeasts have long been considered spoilage microorganisms. Currently, oenological interest in those species is increasing, mostly due to their positive contribution to wine quality. In this work, the fermentative capacity and nitrogen consumption of several non-Saccharomyces wine yeast (Torulaspora delbrueckii, Lachancea thermotolerans, Starmerella bacillaris, Hanseniaspora uvarum, and Metschnikowia pulcherrima) were analyzed. For this purpose, synthetic must with three different nitrogen compositions was used: a mixture of amino acids and ammonium, only organic or inorganic nitrogen. The fermentation kinetics, nitrogen consumption, and yeast growth were measured over time. Our results showed that the good fermentative strains, T. delbrueckii and L. thermotolerans, had high similarities with Saccharomyces cerevisiae in terms of growth, fermentation profile, and nitrogen assimilation preferences, although L. thermotolerans presented an impaired behavior when only amino acids or ammonia were used, being strain-specific. M. pulcherrima was the non-Saccharomyces strain least affected by the nitrogen composition of the medium. The other two poor fermentative strains, H. uvarum and S. bacillaris, behaved similarly regarding amino acid uptake, which occurred earlier than that of the good fermentative species in the absence of ammonia. The results obtained in single non-Saccharomyces fermentations highlighted the importance of controlling nitrogen requirements of the wine yeasts, mainly in sequential fermentations, in order to manage a proper nitrogen supplementation, when needed.
... 3). It is worth mentioning that musts and white wines are characterized by lower diversity of yeasts compared to red ones, which is associated with their lower pH, creating less favourable conditions for the growth of these microorganisms [17]. Analogous studies were conducted on grape must obtained from red grape varietes of Rondo and Regent [9]. ...
... These microorganisms are found in fresh must (e.g. Saccharomyces cerevisiae; Pichia spp.; Rhodotorula spp.; Candida stellata; Hanseniaspora guilliermondii; Lachancea thermotolerants; Kluyveromyces marxianus; Torulaspora delbrueckii; Dekkera bruxellensis and Zygosaccharomyces bailii) (Combina et al., 2005;Fleet, 2008;Lonvaud-Funel, 1996;Querol et al., 1990;Torija et al., 2001) and, some of them contribute to the sensorial profile of wine, influencing its organoleptic quality and complexity (Bisson and Kunkee, 1991;Ciani and Ferraro, 1998;Clemente-Jimenez et al., 2004;Fleet, 2003Fleet, , 2008Jolly et al., 2013). However, several non-Saccharomyces species, those as ethanol tolerant as S. cerevisiae (Z. ...
... In recent years, a lot of research on the ecology of wine yeasts and the genetic diversity of S. cerevisiae has been published (Cocolin et al. 2001;Cordero-Bueso et al. 2011a;Frezier and Dubourdieu 1992;Izquierdo-Cañas et al. 1997;Schuller et al. 2012;Settani et al. 2012;Tello et al. 2012;Vezinhet et al. 1992). Results have revealed that must microbiota vary from one year to another, not only when comparing different regions but also between different regional harvests (Gutiérrez et al. 1999;Torija et al. 2001). These variations are due to climate conditions, grape variety, maturity at harvest, use of fungicides, physical damage to the grapes, and vinification technology (Cordero-Bueso et al. 2011a;Querol and Ramón 1996;Schütz and Gafner 1993). ...
This paper has studied the success of implantation for 16 commercial active dry yeasts (ADYs) during industrial fermentation (30) and the impact of these yeasts during spontaneous fermentations (19) in 10 wineries from the Denomination of Origin “Vinos de Madrid” over two consecutive years. Yeasts strains were identified by molecular techniques, pulsed field electrophoresis and microsatellite analysis. According to these techniques, all the ADYs were different with the exceptions of two strains, L2056 and Rh, which showed the same karyotype and loci size. The results showed that inoculating fermentations with ADYs did not ensure their dominance throughout the fermentation; the implantation level of ADYs was above 80% in only 9 of the 30 commercial fermentations studied; while in 16 fermentations, the dominance of the inoculated ADYs was below 50%. The type of vinification with the best implantation results overall were those associated with red wine fermentations. ADYs affected spontaneous fermentations, although their impact was observed to decrease in the second year of the study. Therefore, specific adaptation studies are necessary before using commercial yeasts during the fermentation process. At the same time, a study was carried out on the frequency of commercial strains in IMIDRA’s yeast collection, made up of strains isolated from spontaneous fermentations of the different areas and cellars since the beginning of the Denomination of Origin “Vinos de Madrid” in 1990. Six different ADYs were found with a frequency of less than 5%.
Scotch Whisky, a product of high importance to Scotland, has gained global approval for its distinctive qualities derived from the traditional production process which is defined in law. However, ongoing research continuously enhances Scotch Whisky production and is fostering a diversification of flavour profiles. To be classified as Scotch Whisky, the final spirit needs to retain the aroma and taste of “Scotch”. While each production step contributes significantly to whisky flavour—from malt preparation and mashing to fermentation, distillation, and maturation—the impact of yeast during fermentation is crucially important. Not only does the yeast convert the sugar to alcohol, it also produces important volatile compounds, for example esters and higher alcohols, that contribute to the final flavour profile of whisky. The yeast chosen for whisky fermentations can significantly influence whisky flavour, so the yeast strain employed is of high importance. This review explores the role of yeast in Scotch Whisky production and its influence on flavour diversification. Furthermore, an extensive examination of non-conventional yeasts employed in brewing and winemaking is undertaken to assess their potential suitability for adoption as Scotch Whisky yeast strains, followed by a review of methods for evaluating new yeast strains.
Wine production has been performed during most of the recent history of humanity. Yeasts are the primary players involved in the transformation of grape must into wine, and the need for proper control of fermentation requires good microbiological knowledge and appropriate tools to monitor the process. The primary task of microorganisms in nature is to recycle organic matter, including sugars. Among these microorganisms, yeasts have a high capacity to ferment sugars to alcohol whenever fruits are ripe. During the fermentation process, a succession of microorganisms occurs, and the methods available for analyzing microbiological dynamics can be classified as “classical” methods, molecular methods, and next‐generation sequencing methods. Grape must have diverse secondary metabolites that are either volatiles or can be transformed into volatiles and contribute to the aroma. Microbiological control is necessary to produce wines with properties that define the quality and reproducibility of the wines.
Wine making has evolved since its origins in the Caucasus more than 8000 years ago to a modern scientific and technological discipline. Novel methodologies and practices have been implemented continuously in the elaboration of wines. The industry has been normally keen to accept those developments and incorporate them into their protocols. However, the complexity of some of the new developments, the “return” to old practices driven by some influencing wine makers or opinion makers, commercial regulations, and consumer concerns are growing limitations for the incorporation of new methodologies. This chapter is focused on new microbial methodologies that can be applied to modern winemaking to control the process microbiologically and discuss about the possible challenges of their incorporation.
The ‘Kyoho’ grape (Vitaceae, Plantae) has large ears, plenty of flesh, and rich nutrition, and is planted across a large area in China. There are few reports on this variety in winemaking, especially on the dynamic changes of fungi in the wine fermentation broth. In this study, we used the ‘Kyoho’ grapes as raw materials and adopted a high throughput to analyze dynamic changes in fungal species composition of the natural fermentation broth at four time points: day one (D1P), day three (D3P), day five (D5P), and day fifteen (D15P). Changes in fungal metabolic pathways and the dynamic changes of dominant yeast were also analyzed. A total of 78 families, 110 genera, and 137 species were detected, in the natural fermentation broth samples. The control check (CK) had 49 families, 60 genera, and 72 species. A total of 66 differential metabolic pathways were enriched; of those 66, 41 pathways were up-regulated compared to CK, such as pyrimidine deoxyribonucleotides biosynthesis from CTP (PWY 7210), CDP-diacylglycerol biosynthesis I (PWY 5667), and chitin degradation to ethanol (PWY 7118). Changes in fungal metabolic pathways were in line with the dynamic changes of dominant yeast species in the whole process of fermentation. Pichia kluyveri , P. membranifaciens , and Citeromyces matritensis are the dominant species in the later stages of natural fermentation. These yeast species may play vital roles in the ‘Kyoho’ wine industry in the future.
Fermentation of the Longyan grape at a low temperature typically leads to the production of the more aromatic and characteristic Longyan white wine. However, the role of microorganisms during fermentation, particularly their contribution to aroma formation, is poorly understood. The dynamics of volatile compounds and microbial succession during the fermentation process of Longyan wine were monitored at 10 °C, 15 °C, and 20 °C, by using HS-SPME-GC/MS and HST. Fourteen aromas markers and 13 microbial biomarkers were responsible for the differences between fermentation at these temperatures. Although the total volatile compounds content was higher at 20 °C mainly due to the higher contents of ethyl octanoate and ethyl decanoate (P < 0.05), wines fermented at 10 °C had higher complexity of esters and terpenes. Orthogonal partial least squares (variable importance in projection >1), and Spearman's correlation (|ρ| > 0.7) indicated that the unique core microorganisms (Geobacillus, Lactobacillus, and Terribacillus) at 10 °C were positively correlated to aromatic markers ((R)-lavandulyl acetate, β-caryophyllene, isoamyl hexanoate, P < 0.05; diethyl succinate, octyl acetate, decyl acetate, P < 0.01). Reducing the fermentation temperature had a potential application prospect for flavor improvement.
The genus Hanseniaspora is the main yeast group isolated from grapes and musts. In this review, we will show some of the main results obtained in real winemaking conditions for the application of Hanseniaspora vineae, the main species of what was named the “fermentation clade” of the genus. The winemaking potential of H. vineae will be discussed in order to understand how to apply it, taking advantage at the winemaking level of its capacity to produce white wines with increased sensory complexity compared with Saccharomyces cerevisiae conventional wine yeasts.
This species is evaluated in some practical aspects for its fermentation capacity at low assimilable nitrogen levels. Furthermore, it lacks H2S production and has good SO2 resistance. Other desirable characteristics are also studied, such as its ability to increase both flavor complexity with neutral grape varieties (e.g., Trebbiano, Macabeu, or Albillo) and palate due to fast cell autolysis processes. Although H. vineae shows a great capacity to produce differentiated wines, some fine-tuning adjustments are still necessary to improve the end of fermentation in highly mature white grapes by sequential inoculation with S. cerevisiae in the middle of the process. Nutrient management and mixed strain selection of the Saccharomyces pairing should be carefully defined to complete total sugar depletion.
This study focuses on the isolation of a consortium of microorganisms from spontaneously fermenting must that naturally contain lactic acid bacteria, non-saccharomyces yeasts, and saccharomyces yeasts. To collect the greatest diversity of microorganisms, the consortium was taken from the point of micro-sparkling. Based on the growth curves, isolation was performed using individual special nutrient media, and the isolates were subsequently multiplied in the nutrient medium. Individual isolates were then used for fermentation tests to monitor the percentage of fermented sugar and hydrogen sulphide production. The highest fermentation abilities were achieved in the isolates containing Saccharomyces cerevisiae and Zygosaccharomyces bailii. The smallest amount of ethanol was formed from the isolates containing Hanseniaspora uvarum, while Candida sake isolate produced the lowest amount of hydrogen sulphide and Zygosaccharomyces bailii produced the highest. The other isolates produced an average amount. Based on these results, a consortium containing the given isolates in a certain ratio was compiled.
Polysaccharides are biopolymers made up of a large number of monosaccharides joined together by glycosidic bonds. Polysaccharides are widely distributed in nature: Some, such as peptidoglycan and cellulose, are the components that make up the cell walls of bacteria and plants, and some, such as starch and glycogen, are used as carbohydrate storage in plants and animals. Fungi exist in a variety of natural environments and can exploit a wide range of carbon sources. They play a crucial role in the global carbon cycle because of their ability to break down plant biomass, which is composed primarily of cell wall polysaccharides, including cellulose, hemicellulose, and pectin. Fungi produce a variety of enzymes that in combination degrade cell wall polysaccharides into different monosaccharides. Starch, the main component of grain, is also a polysaccharide that can be broken down into monosaccharides by fungi. These monosaccharides can be used for energy or as precursors for the biosynthesis of biomolecules through a series of enzymatic reactions. Industrial fermentation by microbes has been widely used to produce traditional foods, beverages, and biofuels from starch and to a lesser extent plant biomass. This review focuses on the degradation and utilization of plant homopolysaccharides, cellulose and starch; summarizes the activities of the enzymes involved and the regulation of the induction of the enzymes in well-studied filamentous fungi.
Two hundred and thirty killer yeast strains were selectively isolated from Chenin blanc grapes and grape skins collected from six wineries. The killer yeasts were divided into nine groups based on their colony morphology and colour on modified Wallerstein laboratory nutrient agar. All strains fermented Chenin blanc grape must (pH 3,5; 40 mg/ I free SO, and 5% (v/v) ethanol) at l4°C. Existing techniques in which methylene blue are used were evaluated to detect killer yeasts, to determine interactions between different killer phenotypes, and to determine the sensitivity of commercial strains to the killer toxins.
An extensive survey of different methods of yeast strain identification (classical microbiological tests, whole-cell protein electrophoresis, chromosomal patterns, DNA hybridization and mitochondrial DNA restriction analysis) has been carried out in order to differentiate, with industrial purposes, strains present in the Alicante wine ecosystem. Only chromosomal patterns and mitochondrial DNA (mtDNA) restriction analysis show differences between strains. Both techniques are very complex to be used in bio technological industries. For this reason, we have developed a new, simple, unexpensive and rapid method based on mtDNA restriction analysis.
Electrophoretic karyotyping, mitochondrial DNA restriction fragment length polymorphism analysis, and PCR amplification of interspersed repeats were used to study the variability, phylogenetic affinities, and biogeographic distribution of wild Saccharomyces cerevisiae enological yeasts. The survey concentrated on 42 individual wine cellars in the Charentes area (Cognac region, France). A limited number (35) of predominant S. cerevisiae strains responsible for the fermentation process have been identified by the above molecular methods of differentiation. One strain (ACI) was found to be distributed over the entire area surveyed. There seemed to be little correlation between geographic location and genetic affinity.
Several strains of the four sibling species of the genus Saccharomyces (S. bayanus, S. cerevisiae, S. paradoxus, and S. pastorianus) were characterized by using a rapid and simple method of restriction analysis of mitochondrial DNA. Patterns obtained with four-cutter endonucleases (such as AluI, DdeI, HinfI, and RsaI) made it possible to differentiate each species. S. cerevisiae and S. paradoxus presented a greater number of large fragments than S. pastorianus and S. bayanus with all the assay enzymes. With AluI and DdeI, species-specific bands clearly permitted differentiation between S. pastorianus and S. bayanus. To test the resolution of this method, wild Saccharomyces strains were analyzed. The correct assignment of these strains to a known taxon by this rapid method was confirmed by means of electrophoretic karyotyping.
Electrophoretic karyotyping and mitochondrial DNA restriction analysis were used to analyze natural yeast populations from fermenting musts in El Penedès, Spain. Both analyses revealed a considerable degree of polymorphism, indicating heterogeneous natural populations. By specifically designed genetic selection protocols, strains showing potentially interesting phenotypes, such as high tolerance to ethanol and temperature or the ability to grow and to ferment in wine-water-sugar mixtures, were isolated from these natural populations. Genetic analysis showed a strong correlation between the selected phenotypes and mitochondrial DNA polymorphisms. Karyotype analysis revealed several genetically similar yeast lineages in the natural yeast microflora, which we interpret as genetically isolated subpopulations of yeast strains with distinct genetic traits, which may correspond to specific microenvironments. Thus, molecular polymorphism analysis may be useful not only to study the geographical distribution of natural yeast strains but also to identify strains with specific phenotypic properties.
In this study, we identified a total of 33 wine yeast species and strains using the restriction patterns generated from the region spanning the internal transcribed spacers (ITS 1 and 2) and the 5.8S rRNA gene. Polymerase chain reaction (PCR) products of this rDNA region showed a high length variation for the different species. The size of the PCR products and the restriction analyses with three restriction endonucleases (HinfI, CfoI, and HaeIII) yielded a specific restriction pattern for each species with the exception of the corresponding anamorph and teleomorph states, which presented identical patterns. This method was applied to analyze the diversity of wine yeast species during spontaneous wine fermentation.
The identification and classification of yeasts have traditionally been based on morphological, physiological and biochemical traits. Various kits have been developed as rapid systems for yeast identification, but mostly for clinical diagnosis. In recent years, different molecular biology techniques have been developed for yeast identification, but there is no available database to identify a large number of species. In the present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts. In many cases, the size of the PCR products and the restriction patterns obtained with endonucleases CfoI, HaeIII and HinfI yielded a unique profile for each species. Accordingly, the use of this molecular approach is proposed as a new rapid and easy method of routine yeast identification.
The growth of yeasts that occur naturally in grape juice was quantitatively examined during the fermentation of four wines that had been inoculated with Saccharomyces cerevisiae. Although S. cerevisiae dominated the wine fermentations, there was significant growth of the natural species Kloeckera apiculata, Candida stellata, Candida colliculosa, Candida pulcherrima, and Hansenula anomala.
A simple and rapid method of yeast strain characterization based on mitochondrial DNA restriction analysis was applied to the control of wine fermentations conducted by active dry yeast strains. This molecular approach allows us to understand several important aspects of this process, such as the role of the active dry yeast strain and that of the natural Saccharomyces cerevisiae flora during vinification. In this paper, we demonstrate that the inoculated strain is really responsible for the fermentation but does not suppress significant development of natural strains during the first stages. During this early period, natural strains could have important effects on wine flavor.
A study was conducted of the dynamics of Saccharomyces and non-Saccharomyces populations during alcoholic fermentation of Albarino musts from two enological subzones located in Galicia (Northwest Spain). Sixteen microvinifications were carried out (8 in each must, M and E) with five indigenous Saccharomyces cerevisiae strains, two commercial active dry strains, and the corresponding spontaneous fermentation. The volatile compounds in the resulting wines were measured using gas chromatography. The study of different physiological and biochemical characteristics allowed us to follow the evolution of the inoculated S. cerevisiae strains. The different cellular concentrations of these strains in the musts produced different growth rates during fermentation. The growth of non-Saccharomyces flora depended on the initial starter culture concentrations and on their growth rate during winemaking. Statistical analysis (factorial and cluster) of data obtained by gas chromatography created statistical relationships between the production of some components of wine aroma and the evolution of yeast flora during alcoholic fermentation.
We identify and characterize 31 Saccharomyces strains from different wine regions, deposited at the Spanish Type Culture Collection, according to mtDNA restriction patterns and chromosomal profiles. By using this kind of information we analyze the correlation between genetic distances and ecological or geographical factors by means of a cluster analysis, assessed by an analysis of the molecular variance (AMOVA). From these analyses, red wine strains are significantly grouped according to their geographic origin, independently of the wine type and the grapevine cultivar, and white wine strians according to ecological factors (wine type of grapevine cultivars). This study also confirms the usefulness of the analysis of interpopulation genetic polymorphism, such as that resulting from mtDNA restriction fragment analysis of wine yeast strains, to determine the distribution of variation in natural populations of Saccharomyces cerevisiae.
Must and wine from grapes harvested in two vintages (1986 and 1987) were anlyzed during vinification for physicochemical and microbiological characteristics. The 1986 vintage would be considered abnormal or poor vintage because of higher rainfall at harvest, and the 1987 one a normal vintage. Low reducing sugars and high volatile acidity at the beginning of the poor fermentation was observed as compared to normal vinification. The yeast population showed atypical evolution through the process since oxidative yeasts were isolated in the first stages of the poor vinification.
The effects of sulphur dioxide and a commercial starter inoculum on yeast population dynamics have been analysed by a molecular approach. Yeast identification from fermenting Carinyena grape musts was performed by RFLP's of mtDNA and rRNA-coding DNA. As expected, the use of a commercial inoculum speeded up the start of fermentation, while SO2 addition limited the development of non-Saccharomyces species. However, this effect was also observed with yeast inoculation. Further analysis of population dynamics could lead to a recommendation for the reduction of the dosage of SO2 by the addition of appropriate inoculum of yeasts in the must. Furthermore, the timing of inoculum addition could be modified to allow a proper contribution of non-Saccharomyces species. Molecular biology analysis of population dynamics could provide a tool to efficiently reduce the dosage of SO2 and adjust the timing of inoculum addition.
Wine yeasts were isolated from fermenting of Garnatxa and Xarel.lo musts prepared in a newly established winery during the 1994 and 1995 vintages. Individual strains were identified by mitochondrial DNA or rRNA coding DNA restriction analysis. A commercial starter, Saccharomyces cerevisiae, which was used during the first year of operation of the plant, took over the fermentations (100% of the analyzed colonies belonged to this strain) in both grape musts. This strain remained in the winery and appeared in non-inoculated fermentations during the following year. Two other main indigenous S. cerevisiae strains, designated as S. cerevisiae MF02 and S. cerevisiae MF03, competed with the commercial starter. Analysis of non-Saccharomyces strains from the spontaneous fermentation of Garnatxa must showed the presence of Hanseniaspora uvarum and Candida stellata at the beginning of the process, although strains of S. cerevisiae began to predominate after a few days and completed the fermentation.
Using mitochondrial DNA restriction endonuclease analysis, the dynamics of the natural Saccharomyces cerevisiae strains present in spontaneous wine fermentations have been studied. We observed a sequential substitution of Sacch. cerevisiae strains along fermentation agreeing with different fermentation phases. When the restriction pattern's similarity (measured as the fraction of shared restriction fragments) was high, a clear sequential substitution of the strains was seen. However, when the similarity was low, although a sequential substitution could be also observed between secondary strains, a clearly predominant strain was present along the whole fermentation process.
Volatile substances of wines obtained by fermentation of musts from 'Monastrell' grapes (Alicante, Spain) was studied for yeast isolated from such musts. The results of the statistical treatment performed show the importance of yeasts of low fermentative power, particularly Kloeckera apiculata, in the production of volatile substances. Saccharomyces cerevisiae var. chevalieri was found to be the most important yeast of high fermentative power.
Archaic speculations and firmly established legends regarding the origin of the yeast Saccharomyces cerevisiae and related species are revisited in light of past and recent ecological evidence pointing to a strict association with artificial, man-made environments such as wineries and fermentation plants. The nomenclature within this industrially important group is also discussed in view of the modifications imposed from application of molecular techniques to classification.
An ecological study of Saccharomyces cerevisiae strains in spontaneous alcoholic fermentation has been conducted in the same winery for two consecutive years (1994 and 1995). Yeast cells were identified and characterized using mitochondrial DNA restriction analysis. Although a great diversity of wild strains was observed, a sequential substitution of S. cerevisiae strains during the different phases of fermentation was detected. Furthermore, the most frequent strains were encountered in both years, and the dynamic populations were not influenced by climatic conditions. Finally, the RsaI restriction enzyme produced a species-specific pattern which allowed the identification of all the isolates as S. cerevisiae.
The levels of yeasts and lactic acid bacteria that naturally developed during the vinification of two red and two white Bordeaux wines were quantitatively examined. Yeasts of the genera Rhodotorula, Pichia, Candida, and Metschnikowia occurred at low levels in freshly extracted grape musts but died off as soon as fermentation commenced. Kloeckera apiculata (Hanseniaspora uvarum), Torulopsis stellata, and Saccharomyces cerevisiae, the dominant yeasts in musts, proliferated to conduct alcoholic fermentation. K. apiculata and eventually T. stellata died off as fermentation progressed, leaving S. cerevisiae as the dominant yeast until the termination of fermentation by the addition of sulfur dioxide. At least two different strains of S. cerevisiae were involved in the fermentation of one of the red wines. Low levels of lactic acid bacteria (Pediococcus cerevisiae, Leuconostoc mesenteroides, and Lactobacillus spp.) were present in grape musts but died off during alcoholic fermentation. The malolactic fermentation developed in both red wines soon after alcoholic fermentation and correlated with the vigorous growth of at least three different strains of Leuconostoc oenos.
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