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Influence of grape treatment on the wine yeast populations isolated from spontaneous fermentations
Abstract
To study the influence of different methods of grape treatment in wineries on the diversity of the yeast species in spontaneous fermentations.
Grapes were crushed and pressed in three different ways followed by spontaneous fermentation. The same grape material picked and crushed aseptically directly in the vineyard served as control. Yeasts isolated at different stages of the fermentation were characterized by 5.8S-ITS-RFLP. Yeasts of the Saccharomyces sensu stricto complex were additionally analysed by microsatellite polymerase chain reaction fingerprinting. The diversity of yeast species isolated from winery fermentations was much greater than from the vineyard fermentation in respect to yeasts of the genus Saccharomyces as well as non-Saccharomyces.
Oenonogical methods alter significantly the yeast diversity in spontaneous fermentations of grape juice.
Managing spontaneous fermentations successfully depends not only on choosing the suitable grapes but also on the crushing and pressing techniques leading to different yeast populations.
... Данные тематики работ характерны для традицион ных европейских регионов производства вин, но появляются подобные исследования и для новых развивающихся регионов виноделия, таких как ази атские и южно американские. Так, с начала 2000 х годов теме биоразнообразия дрожжевых грибов, выделяемых из виноградников, а также при сбра живании виноградного сока было посвящено около полусотни работ, выполненных в европейских стра нах (Австрия [2][3][4], Венгрия [5][6][7], Германия [8], 1 Автор для корреспонденции (e mail: kachalkin_a@mail.ru). ...
... Таким об Виды AUT HUN DEU GRC ESP ITA PRT RUS SVN FRA CHE ISR IND CHN ZAF AUS NZL ARG Примечание. AUT -Австрия [2][3][4], HUN -Венгрия [5][6][7], DEU -Германия [8], GRC -Греция [9,10], ESP -Испания [11][12][13][14][15][16], ITA -Италия [17][18][19], PRT -Португалия [20][21][22][23], RUS -Россия (Дагестан) [51, 52, данное исследование], SVN -Словения [24][25][26], FRA -Франция [27][28][29][30][31], CHE -Швейцария [32], ISR -Израиль [33], IND -Индия [34][35][36], CHN -Китай [37][38][39], ZAF -ЮАР [40,41], AUS -Австралия [42], NZL -Новая Зеландия [43], ARG -Аргентина [44][45][46][47][48], BRA -Бразилия [49][50][51]. Безусловно на представленное "эталонное" сообщество накладываются и некоторые поправ ки, влияющие на присутствие тех или иных видов на винограде или на их разобщенность в верти кально ярусном ряду. ...
... Since the early 2000s, about fifty works on yeast biodiversity in vineyards and in fermented grape juice have been published. They were carried out in European countries (Austria [2-4], Hungary [5][6][7], Germany [8], Greece [9,10], Spain [11][12][13][14][15][16], Italy [17][18][19], Portugal [20][21][22][23], Slovenia [24][25][26], France [27][28][29][30][31], Switzerland [32]), in Asia (Israel [33], India [34][35][36], China [37][38][39]), in South Africa [40,41], Australia [42], and New Zealand [43], as well as in South America (Argentina [44][45][46][47][48], Brazil [49][50][51]). ...
... Other previously reported often isolated genera are usually represented by one or two species. Thus, a shift from the genus to the species character ization of the vineyard yeast community becomes pos [8]; GRC, Greece [9,10]; ESP, Spain [11][12][13][14][15][16]; ITA, Italy [17][18][19]; PRT, Portugal [20][21][22][23]; RUS, Russia (Dagestan) [51, 52, present study]; SVN, Slovenia [24][25][26]; FRA, France [27][28][29][30][31]; CHE, Switzerland [32]; ISR, Israel [33]; IND, India [34][35][36]; CHN, China [37][38][39]; ZAF, South Africa [40,41]; AUS, Australia [42]; NZL, New Zealand [43]; ARG, Argentina [44][45][46][47][48]; BRA, Brazil [49][50][51]. ...
Long-term studies of yeast species diversity in the vineyards of the Republic of Dagestan using various isolation techniques and various substrates in the vertical tier dynamics revealed 38 species. The most diverse species complex including ~80% of the isolated species was formed on the berries. A list of 160 yeast species isolated from grapes, spontaneously fermented fresh juice, and other vineyard substrates was compiled using the results of the present work and the literature data on yeast occurrence. Analysis of generalized data revealed considerable similarity in the taxonomic composition of yeasts from different countries and continents and made it possible to shift from the genus to the species characterization of the grape-associated yeast community.
... The D. bruxellensis presence on berries can be limited by using anti-Botrytis treatment that contains procymidone. Procymidone, as well as benomyl, dichofluanide, vinclozoline, and iprodione are active compounds present in vine antifungal treatments with inhibiting properties to yeast (Sturm et al., 2006). This could support the fact that VP production by D. bruxellensis is less common during the early phases of winemaking, when treatments for D. bruxellensis inhibition are done. ...
The aim of this research has been to examine the microbiological and technological parameters influencing volatile phenols (VP) production in wine of the autochthonous Montenegrin grape varieties Kratošija and Vranac and the international grape variety Cabernet Sauvignon. Within two consecutive vintages (2012 and 2013) grape must and wines have been analysed on the presence of Dekkera bruxellensis yeast and on hydroxycinnamic acids (HCA) as precursors of VP. Among technological parameters, the influence of commercial yeast and lactic acid bacteria (LAB) and the addition of oak alternatives on wine chemical composition and sensory profile has been examined. Caffeic acid represented the prevalent HCA compound for all varietal wines, with the exception of Kratošija in the 2012 vintage in which ferulic acid (93.2%) was the main HCA. Higher total content of all three examined HCA was observed in all varietal wines inoculated with commercial yeast in 2012, as compared to 2013, due to the high content of caffeic acid in the 2012 vintage. Statistically significant differences in VP content were found between commercial yeast, LAB and oak alternatives. The presence of Dekkera bruxellensis spp. has been noticed only in the 2013 vintage for Vranac control wine after alcoholic fermentation (AF), confirming that these yeasts are present in extremely low numbers at the beginning of AF only. Vranac wines, inoculated with commercial yeast, contained the highest content of p-coumaric acid in 2012 (1.54 mg/L), followed by Kratošija wines in the 2013 vintage (1.03 mg/L). The highest number of samples with detected VP from the vintage 2012 were found for Vranac wines (12 out of 28), while in 2013 vintage, Kratošija had the highest number of samples (18 out of 28). VP have appeared only in wines of autochthonous grape varieties, after long bottle aging and the number of infected samples were vintage dependent. Comparing autochthonous Montenegrin wines to Cabernet Sauvignon, it can be concluded that this international variety grown in agro-ecological conditions of Ćemovsko field does not show increased VP even after three or four years of aging in bottles. It has been concluded that VP are formed predominantly in wines with high alcohol content, low pH, low reducing sugars content, and even in wines with free SO2 above 30 mg/L
... In various investigations in countries like China (Sun et al. 2014), Czech-Republic, Slovakia, Hungary (Minarik 1964), Slovenia (Jemec et al. 2001), Austria (Lopandic et al. 2008), Germany (Sturm et al. 2006), Switzerland (Díaz et al. 2013;Schütz and Gafner 1993), Italy (Tofalo et al. 2009), France (Fleet et al. 1984;Zott et al. 2008), Spain (Beltran et al. 2002;Clemente-Jimenez et al. 2004;Hierro et al. 2006), Portugal (Barata et al. 2008;Couto et al. 2005), USA (Egli et al. 1998;van Keulen et al. 2003), Brazil (Bezerra-Bussoli et al. 2013) and Australia (Heard and Fleet 1985), S. pombe was not detected. Most likely this is due to the fact that S. pombe is a week competitor against other indigenous yeast in spontaneous grape must fermentations or against inoculated starter cultures (Brugirard and Roques 1972;Delteil 1988;Ribereau-Gayon and Peynaud 1962). ...
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.
... Although it is possible that the S. uvarum population in this study was brought in from the vineyards with the grapes, we consider it more likely that the majority of these yeasts have established themselves as winery residents as opposed to vineyard residents. As mentioned above, Saccharomyces yeasts are typically rare on healthy grapes in the vineyard [40], and when Saccharomyces strains are identified in the vineyard, their presence is inconsistent between vintages [38,98], and they are not necessarily the strains that conduct alcoholic fermentation in the winery [99,100]. The presence of wineryresident yeast populations and communities is well-established in the literature [72,91,[101][102][103], and these yeasts are capable of over-wintering in the winery and entering fermentations to which they have not been inoculated [4,6,7,104]. ...
Saccharomyces cerevisiae is the primary yeast species responsible for most fermentations in winemaking. However, other yeasts, including Saccharomyces uvarum , have occasionally been found conducting commercial fermentations around the world. S . uvarum is typically associated with white wine fermentations in cool-climate wine regions, and has been identified as the dominant yeast in fermentations from France, Hungary, northern Italy, and, recently, Canada. However, little is known about how the origin and genetic diversity of the Canadian S . uvarum population relates to strains from other parts of the world. In this study, a highly diverse S . uvarum population was found dominating uninoculated commercial fermentations of Chardonnay grapes sourced from two different vineyards. Most of the strains identified were found to be genetically distinct from S . uvarum strains isolated globally. Of the 106 strains of S . uvarum identified in this study, four played a dominant role in the fermentations, with some strains predominating in the fermentations from one vineyard over the other. Furthermore, two of these dominant strains were previously identified as dominant strains in uninoculated Chardonnay fermentations at the same winery two years earlier, suggesting the presence of a winery-resident population of indigenous S . uvarum . This research provides valuable insight into the diversity and persistence of non-commercial S . uvarum strains in North America, and a stepping stone for future work into the enological potential of an alternative Saccharomyces yeast species.
... 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.
... Another method involves intentionally inoculating vineyardderived non-Saccharomyces yeasts prior to the onset of AF. Pied de cuve is a French term that refers to two different methods of indirect inoculation: (i) an inoculum made from must that is already fermenting in the winery (Ubeda Iranzo, González Magaña and González Viñas 2000;Clavijo, Calderón and Paneque 2011;Li, Liu and Liu 2012) or (ii) an inoculum made from spontaneously fermenting must in the vineyard, away from the influence of winery-resident yeast strains (Sturm, Grossmann and Schnell 2006). In this study, we use the second definition of pied de cuve inoculation, which is intended to increase the contribution of non-Saccharomyces yeasts from the vineyard by intentionally adding them in higher numbers to increase their competitive edge at the onset of AF. ...
The microbial consortium of wine fermentations is highly dependent upon winemaking decisions made at crush, including the decision to inoculate and the decision to add sulfur dioxide (SO2) to the must. To investigate this, Chardonnay grape juice was subjected to two inoculation treatments (uninoculated and pied de cuve inoculation) as well as two SO2 addition concentrations (0 and 40 mg/L). The bacterial communities, fungal communities, and Saccharomyces populations were monitored throughout fermentation using culture-dependent and culture-independent techniques. After fermentation, the wines were evaluated by a panel of experts. When no SO2 was added, the wines underwent alcoholic fermentation and malolactic fermentation simultaneously. Tatumella bacteria were present in significant numbers, but only in the fermentations to which no SO2 was added, and were likely responsible for the malolactic fermentation observed in these treatments. All fermentations were dominated by a genetically diverse indigenous population of Saccharomyces uvarum, the highest diversity of S. uvarum strains to be identified to-date; 150 unique strains were identified, with differences in strain composition as a result of SO2 addition. This is the first report of indigenous S. uvarum strains dominating and completing fermentations at a commercial winery in North America.
... Saccharomyces cerevisiae Querol et al., 1992Munoz et al., 2009Brettanomyces bruxellensis Ibeas et al., 1996Martorell et al., 2006Pichia guilliermondii Martorell et al., 2006 Saccharomyces cerevisiae Legras et al., 2007Richards et al., 2009Almeida et al., 2015 Liti et al., 2009Schacherer et al., 2009Almeida et al., 2015Gallone et al., 2016Saccharomyces uvarum Almeida et al., 2014 a CGH (array-CGH or "microarray karyotyping); MLST (multilocus sequence typing); REA-PGFE (restriction enzyme analysis with pulsed field gel electrophoresis); RAPD (rapid amplification of polymorphic DNA); RFLP (restriction fragment length polymorphism); AFLP (amplified fragment length polymorphism); mtDNA (mitochondrial DNA); mtRFLP (mitochondrial DNA restriction length polymorphism) ;TRtRNA (tandem repeat tRNA);FT-IR (fourier transform infrared spectroscopy); SNP (single nucleotide polymorphism) b (Albertin et al., 2014a, 2014b, Almeida et al., 2014, 2015, 2015Ayoub et al., 2006;Barquet et al., 2012;Börlin et al., 2016;Cadez et al., 2002;Cubillos et al., 2009;C. Curtin et al., 2012b;Dubourdieu et al., 1987;Dunn et al., 2005;Erny et al., 2012;Fay and Benavides, 2005;Gallone et al., 2016;Grangeteau et al., 2015;Guillamón et al., 1998;Hranilovic et al., 2017;Legras et al., 2007;Legras and Karst, 2003;Liti et al., 2009;Lopandic et al., 2007;Lopes et al., 2009;Martorell et al., 2006;Masneuf-Pomarede et al., 2015;Miot-Sertier and Lonvaud-Funel, 2007;Muñoz et al., 2009;Querol et al., 1992;Richards et al., 2009;Salinas et al., 2010;Schacherer et al., 2009;Schuller et al., 2004Schuller et al., , 2004Sturm et al., 2006;Tofalo et al., 2012;Urso et al., 2008) Among unicellular eukaryotes, S. cerevisiae is the most broadly studied model species and this mirrors its importance as industrial yeast with high contribution to fundamental knowledge of micro-and macro-organisms. Population genetics of the species highlighted great genetic diversity and grouping according to type of industrial fermentation environment (Aa et al., 2006;Fay and Benavides, 2005;Gallone et al., 2016;Legras et al., 2007;Liti et al., 2009;Schacherer et al., 2009). ...
Brettanomyces bruxellensis is a microorganism described as the first cause of microbial spoilage of wine. Its industrial relevance is highlighted by the fact that this yeast is isolated from different substrates such as beer, kombucha, bioethanol fermentation molasses and others. This project aims to explore the genetic diversity of the species by studying a large population of isolates from various geographical and ecological niches. For this purpose, a robust genotyping method (microsatellite analysis) was optimized and applied on the population, thus highlighting the coexistence of diploid and triploid populations worldwide. Further, the relation between genotypic clustering and physiological traits was studied. Namely, sulphite tolerance assay was performed on a subset of strains representative of the total population. The results reveal a link between genetic group and growth profile in the presence of sulphur dioxide. Competition experiments in presence of sulphites highlight a selective advantage of sulphite tolerant strains compared to sulphite sensitive ones, thus suggesting a specific adaptation to the main antimicrobial used in winemaking. This work contributes to a deeper understanding of this wine spoilage microorganism in means of genetic and phenotypic diversity and sheds light on putative evolutionary strategies for adaptation to human related environment of this non-conventional model yeast species.
... For musts obtained from white grapes (Chenin Blanc and Prensal White), cold settling reduces the overall yeast population and particularly affects the growth of certain species such as Hansenula anomala, Issatchenkia terricola and S. cerevisiae, instead of other species such as Candida zemplinina and Hanseniaspora uvarum, not very sensitive to this process and which become or remain the major species after racking (Mora and Mulet, 1991). Sturm et al. (2006) observed for Riesling grape must that non-Saccharomyces yeasts persist longer during fermentation if pressing is preceded by crushing or maceration. The temperature during pre-fermentation maceration of red grape varieties (Cabernet sauvignon and Malbec) also seems to play a role in the evolution of yeast populations (Maturano et al., 2015). ...
The effects of different anthropic activities (vineyard: phytosanitary protection; winery: pressing and sulfit-ing) on the fungal populations of grape berries were studied. The global diversity of fungal populations (moulds and yeasts) was performed by pyrosequenc-ing. The anthropic activities studied modified fungal diversity. Thus, a decrease in biodiversity was measured for three successive vintages for the grapes of the plot cultivated with Organic protection compared to plots treated with Conventional and Ecophyto pro-tections. The fungal populations were then considerably modified by the pressing-clarification step. The addition of sulfur dioxide also modified population dynamics and favoured the domination of the species Saccharomyces cerevisiae during fermentation. The non-targeted chemical analysis of musts and wines by FT-ICR-MS showed that the wines could be discriminated at the end of alcoholic fermentation as a function of adding SO 2 or not, but also and above all as a function of phytosanitary protection, regardless of whether these fermentations took place in the presence of SO 2 or not. Thus, the existence of signatures in wines of chemical diversity and microbiology linked to vineyard protection has been highlighted.
... Moreover the use of an anti-Botrytis treatment containing procymidone limits the presence of B. bruxellensis on berries. Procymidone, as well as dichofluanide, benomyl, iprodione and vinclozoline are the active substances found in vine antifungal treatments and have yeast inhibitory properties (NAVARRO et al., 1999;STURM et al., 2006). This could explain why in years where Botrytis development is favoured and treatments done, volatile phenol production due to B. bruxellensis occurs less during the early stages of winemaking. ...
Aims: This work reviews the latest knowledge concerning the role of Brettanomyces bruxellensis in red wine alteration.
Results and conclusion: The origin of this yeast species and its place in the wine microbial consortium are discussed as well as microbial equilibriums with the other species, notably Saccharomyces cerevisiae and lactic acid bacteria. As a consequence, fermentations are described as key steps in Brettanomyces development management. Furthermore, the influence of ageing through the use of traditional winemaking practices is explained.
Significance and impact of study: Finally, this paper emphases the need for a better understanding of chemical and microbial analysis together in order to better control this undesirable yeast and prevent the production of volatile phenols.
... In den letzten Jahren hat sich der Einsatz von verschiedenen molekularbiologischen Methoden zu Artbestimmung von Hefen als geeignet gezeigt. Für die Hefe-Spezies-Bestimmung ist vor allem die RFLP (Restriktions-Fragment-Längen-Polymorphismus)-Analyse der 5,8S rRNA-Gene mit den sich anschließenden "internaltranscribed-spacer"-Regionen (ITS) gut einsetzbar (Abb. 1) (Guillamón et al., 1998;Esteve-Zarzoso et al., 1999;Sturm et al., 2006). Quantitative und qualitative Daten über tatsächlich vorhandene mikrobielle Kontaminationen sind in der Literatur rar. ...
Zusammenfassung Beim Wein handelt es sich um ein traditionell durch das konservierende Gärungsprodukt Ethanol als keimarm eingestuftes Getränk. Durch Fehler im Fermentationsmanagement oder der folgenden " down stream "-Pro-zesse besteht aber ein potenzielles Risiko biologischer Verunreinigungen, verbunden mit ökonomischen Verlusten. Die Weinbranche, insbesondere abfüllende Betriebe, haben ein großes Interesse an schnellen und ein-fachen biologischen Kontrollen im Rahmen der Qualitätssicherung, um das Gefährdungspotenzial von Wein zu beurteilen. Quantitative und qualitative Daten über tatsächlich vorliegende mikrobielle Kontaminationen in gehandeltem Wein sind rar. Im Rahmen dieser Studie wird anhand der Überprüfung von 86 zufäl-lig ausgesuchten, unterschiedlichen Weiß-, Rotund Roséweinen, die in Deutschland gehandelt werden, gezeigt wie eine zweistufige Quali-tätskontrolle funktioniert. Unter Verwendung von Membranfiltration und RFLP-Analyse der ITS-Regionen im Bereich der 5,8S rRNA-Gene konn-ten mikrobielle Kontaminationen detektiert und auftretende Hefen der Spezies Candida albicans, Cryptococcus magnus, Dekkera bruxellensis, Pichia anomala, Pichia guillermondii, Pichia membranaefaciens, Rho-dotorula mucilaginosa, Saccharomyces cerevisiae und Torulaspora del-brueckii identifiziert werden. Summary Wine is due to the fermentation product ethanol traditionally a relatively biological stable beverage. But there is potential risk of biological contaminations by mismanagement during fermentations and following down stream processes associated with economic losses. The wine industry, especially bottling companies, are interested in fast and easy methods to monitor biological contaminants during quality control to assess the potential risks of the bottled product wine. At time quantitative and qualitative data about microbiological risks in traded wines are rare. The aim of this study was to show how a two step quality control could work on the example of 86 different wines traded in Germany. Membrane filtration and RFLP analysis of the ITS region of the 5.8S rRNA genes were the tools for detection of microbiological contaminations and yeast belonging to the species Candida albicans, Cryptococcus magnus, Dek-kera bruxellensis, Pichia anomala, Pichia guillermondii, Pichia mem-branaefaciens, Rhodotorula mucilaginosa, Saccharomyces cerevisiae and Torulaspora delbrueckii could be identified.
... This was likely because spontaneous fermentations contain diverse populations of yeasts including S. cerevisiae (Molina et al., 2009). Common species in spontaneous fermentations include: S. cerevisiae, S. bayanus, Candida spp., Hanseniaspora uvarum, Issatchenkia spp., Metschnikowia pulcherrima, Pichia anomal, and T. delbrueckii, among many others (Sturm, Grossmann, & Schnell, 2006). Yeasts have varying capabilities to produce diverse concentrations of aroma compounds due to genetic differences between S. cerevisiae strains, and S. cerevisiae vs. non-S. ...
... Population dynamics of indigenous yeasts have been reported to be affected by a multitude of viticultural as well as winery factors, including vineyard environment and management practices, harvest conditions, juice/ must processing conditions and fermentation conditions (inoculation, SO2, macro-and micronutrients availability, temperature and microbial interactions) (Fleet and Heard 1993, Charoenchai et al. 1997, Torija et al. 2001, Jemec and Raspor 2005, Sturm et al. 2006. ...
Background and Aims: Winemakers are constantly searching for new techniques to modulate wine style. Exploiting indigenous yeasts present in grape must is re-emerging as a commercial option in New World wine regions. Wines made with indigenous or ‘wild’ yeasts are perceived to be more complex by showing a greater diversity of flavours; however, the chemical basis for the flavour characteristics is not yet defined. In order to evaluate techniques for making wine with the ‘wild yeast fermentation’ character more reliably, it is necessary to define the salient chemical characteristics of such wines.Methods and Results: Pairs of Chardonnay wines were prepared from the same must and subjected to similar fermentation conditions in the wineries of origin, except for the mode of inoculation. Reference wines were made by inoculation with a Saccharomyces cerevisiae starter culture, whereas companion wines were allowed to undergo fermentation with the indigenous microflora. Of all wine chemicals analysed, only yeast-derived volatile fermentation products showed significant differences between the yeast treatments.Conclusions: Inoculated wines were associated with the esters ethyl hexanoate and 3-methylbutyl acetate and formed a clear cluster by principal component analysis. By comparison with inoculated wines, ‘wild’ yeast fermented wines showed high variability in volatile compounds that contribute to wine aroma, with higher concentrations of 2-methylpropanol, 2-methylbutanoic acid, ethyl 2-methylpropanoate, ethyl decanoate and ethyl dodecanoate potentially being sensorially important.Significance of the Study: This study shows that yeast-derived volatile fermentation products are a key difference between inoculated and uninoculated ferments and provides a chemical basis for the ‘wild yeast fermentation’ character.
... The isolation from grape juices obtained in the winery (industrial or experimental) may only be regarded as an approximation of the natural grape microbiota. In fact, when grape juice is only sampled in the winery, even at the beginning of fermentation, a different picture of yeasts species may be obtained, due to bulk transport, crushing or pressing in the winery (Barata et al., in press;Sturm et al., 2006). Therefore, it is mandatory to isolate yeasts from grapes aseptically collected in the vineyard and they must be processed as quickly as possible because grapes damaged during transport to laboratory may accumulate higher numbers of yeasts (Yanagida et al., 1992). ...
Grapes have a complex microbial ecology including filamentous fungi, yeasts and bacteria with different physiological characteristics and effects upon wine production. Some species are only found in grapes, such as parasitic fungi and environmental bacteria, while others have the ability to survive and grow in wines, constituting the wine microbial consortium. This consortium covers yeast species, lactic acid bacteria and acetic acid bacteria. The proportion of these microorganisms depends on the grape ripening stage and on the availability of nutrients. Grape berries are susceptible to fungal parasites until véraison after which the microbiota of truly intact berries is similar to that of plant leaves, which is dominated by basidiomycetous yeasts (e.g. Cryptococcus spp., Rhodotorula spp. Sporobolomyces spp.) and the yeast-like fungus Aureobasidium pullulans. The cuticle of visually intact berries may bear microfissures and softens with ripening, increasing nutrient availability and explaining the possible dominance by the oxidative or weakly fermentative ascomycetous populations (e.g. Candida spp., Hanseniaspora spp., Metschnikowia spp., Pichia spp.) approaching harvest time. When grape skin is clearly damaged, the availability of high sugar concentrations on the berry surface favours the increase of ascomycetes with higher fermentative activity like Pichia spp. and Zygoascus hellenicus, including dangerous wine spoilage yeasts (e.g. Zygosaccharomyces spp., Torulaspora spp.), and of acetic acid bacteria (e.g. Gluconobacter spp., Acetobacter spp.). The sugar fermenting species Saccharomyces cerevisiae is rarely found on unblemished berries, being favoured by grape damage. Lactic acid bacteria are minor partners of grape microbiota and while being the typical agent of malolactic fermentation, Oenococcus oeni has been seldom isolated from grapes in the vineyard. Environmental ubiquitous bacteria of the genus Enterobacter spp., Enterococcus spp., Bacillus spp., Burkholderia spp., Serratia spp., Staphylococcus spp., among others, have been isolated from grapes but do not have the ability to grow in wines. Saprophytic moulds, like Botrytis cinerea, causing grey rot, or Aspergillus spp., possibly producing ochratoxin, are only active in the vineyard, although their metabolites may affect wine quality during grape processing. The impact of damaged grapes in yeast ecology has been underestimated mostly because of inaccurate grape sampling. Injured berries hidden in apparently sound bunches explain the recovery of a higher number of species when whole bunches are picked. Grape health status is the main factor affecting the microbial ecology of grapes, increasing both microbial numbers and species diversity. Therefore, the influence of abiotic (e.g. climate, rain, hail), biotic (e.g. insects, birds, phytopathogenic and saprophytic moulds) and viticultural (e.g. fungicides) factors is dependent on their primary damaging effect.
... The analysis of wine samples requires the same attention in selecting the different yeast isolates responsible for the fermentation and maturation of the final product. Frequently, the first selecting step of yeast isolates includes the macro-and micromorphological observation, biochemical characterization or cultivation on specific agar media able to recognize, for example, the Saccharomyces isolates from non-Saccharomyces counterparts ( Caridi et al. 2002;Comitini and Ciani 2006;Sturm et al. 2006). ...
The investigation of yeast microflora during the must fermentation of two wine varieties (Frankovka modra - Blaufränkisch and Veltlinske zelene - Grüner Veltliner) from two consecutive vintages was performed using a three-step approach.
The investigation strategy consisted of the combination of yeast cultivation, selection of the isolated yeasts based on the amplification of internal transcribed spacer 2 using a fluorescence-labelled primer (f-ITS-PCR) and a final identification step based on amplification and sequencing of the ITS1-5.8S rDNA-ITS2 region of the selected yeasts. By this three-step approach, it was possible to screen 433 yeasts isolates that belonged to 13 different species.
The f-ITS-PCR allowed the unambiguous differentiation of all isolated yeast species that produced their typical f-ITS-PCR profile.
This is one of few reports that treat the yeast diversity in Slovakian wines and in two varieties largely cultivated in Central Europe. The three-step approach permitted the rapid and reliable identification of isolated yeasts. The f-ITS-PCR with its good discrimination power can represent a suitable molecular tool for the selection of yeast members recovered from food or other environments.
... Although data are still very little, it seems that S. cerevisiae strains on the grape may have only minor contributions to the overall fermentation (Mercado et al., 2007). However, this could depend on their initial populations and how these are influenced by grape-crushing procedures, cold-maceration processes and grape maturity (Hierro et al., 2006;Sturm et al., 2006). Presumably, the Saccharomyces flora in the winery originally came from grapes and evolved with time. ...
International competition within the wine market, consumer demands for newer styles of wines and increasing concerns about the environmental sustainability of wine production are providing new challenges for innovation in wine fermentation. Within the total production chain, the alcoholic fermentation of grape juice by yeasts is a key process where winemakers can creatively engineer wine character and value through better yeast management and, thereby, strategically tailor wines to a changing market. This review considers the importance of yeast ecology and yeast metabolic reactions in determining wine quality, and then discusses new directions for exploiting yeasts in wine fermentation. It covers criteria for selecting and developing new commercial strains, the possibilities of using yeasts other than those in the genus of Saccharomyces, the prospects for mixed culture fermentations and explores the possibilities for high cell density, continuous fermentations.
Having more information on yeast ecology during fermentation process is important for producing wine with typical characteristics. Till now, it is unclear how agricultural practices of grape influence yeast succession and wine quality. In this study, high-throughput sequencing was implemented to study yeast succession during spontaneous fermentation of grapes from both organic and conventional agricultural systems. Basic physicochemical parameters and sensory quality of wine samples were also examined. Results showed a total of 18 yeast genera (including 36 species) were identified, and these two kinds of samples differed a lot in yeast community compositions and succession. Hanseniaspora and Saccharomyces were the top two dominant genera, between which Hanseniaspora was superior at the beginning while the relative abundance of Saccharomyces boomed during fermentation and reached 99.96 ± 0.005% (organic) and 79.94 ± 7.76% (conventional) ultimately. Some species which were rare in vine and wine also emerged in this work. Yeast alpha diversity of organic sample fell more sharply than that of conventional sample during fermentation. Besides, organic sample had advantages in fermentation speed and wine sensory quality. This study enhanced our knowledge about yeast ecology during spontaneous fermentation of wine and demonstrated the influence of viticultural practices on yeast succession and wine quality.
The effects of different anthropogenic activities (vineyard, winery) on fungal populations from grape to wine were studied. To characterize these effects, it was necessary to access to the overall diversity of populations (pyrosequencing and spectroscopy FT-IR) but also to intra-specific diversity (FT-IR). Spectroscopy FT-IR has been validated for their ability to characterize the global population and to discriminate the strains for three species of non-Saccharomyces yeasts (NS). For the first time, it is shown that the grape berry is a limited source for NS yeasts while the winery seems to be a significant source; the air is an important vector for dissemination of these yeasts. In addition, persistence of NS yeast strains from year to year in the winery has been demonstrated. The studied anthropogenic activities modify the fungal diversity. Thus, lower biodiversity of grapes from organic modality was measured for the three vintages considered. The pressing / clarification step revises strongly fungal populations and the influence of the winery flora is confirmed. The addition of SO2 changes the population dynamics and favors the dominance of the species S. cerevisiae. The non-targeted chemical analysis shows, for the first time, that these wines can be distinguished at the end of the alcoholic fermentation (with or without SO2) depending on plant protection. Thus, the existence in wines of chemical and microbiological signatures associated with vineyard protection mode is highlighted.
Nowadays to know the spontaneous yeasts microbiota represents a very important desideratum not only for strictly research activity so as practical operation with direct implication in wine industry. Based on this aspect it was necessary to begin a series of studies concerning the composition of the yeasts spontaneous microbiota. This study developed between 2006 - 2007 in the Cotnari vineyard followed yeasts isolation from grapes and its persistence on musts. Also, was made a study regarding yeasts incidence and its dynamics on grapes and musts in the early or late stage of fermentation. In accordance with results the conclusions was that Ascomycota species has prevailed compared with Deuteromycota; the prevalent Ascomycota species was Saccharomyces ellipsoideus; the prevalent non - Saccharomyces species was Hansenula anomala. It also was registered many differences between species isolated from grapes and that one was isolated from musts regarding occurrence of its or number. The explanation consist in poor yeasts number on grapes and also possible contamination from vinery equipments. Candida mycoderma was noted as ubiquitous species based on its presence on grapes after that in musts and above all in wine.
Wine is due to the fermentation product ethanol traditionally a relatively biological stable beverage. But there is potential risk of biological contaminations by mismanagement during fermentations and following down stream processes associated with economic losses. The wine industry, especially bottling companies, are interested in fast and easy methods to monitor biological contaminants during quality control to assess the potential risks of the bottled product wine. At time quantitative and qualitative data about microbiological risks in traded wines are rare. The aim of this study was to show how a two step quality control could work on the example of 86 different wines traded in Germany. Membrane filtration and RFLP analysis of the ITS region of the 5.8S rRNA genes were the tools for detection of microbiological contaminations and yeast belonging to the species Candida albicans, Cryptococcus magnus, Dekkera bruxellensis, Pichia anomala, Pichia guillermondii, Pichia membranaefaciens, Rhodotorula mucilaginosa, Saccharomyces cerevisiae and Torulaspora delbrueckii could be identified.
The current study evaluated the diversity of yeast species in Cabernet Sauvignon grape must derived from three neighbouring vineyards from a similar terroir but on which significantly different management practices are employed. The fermentation kinetics and yeast population dynamics were monitored from the beginning to the end of spontaneous fermentation. The grape musts were characterised by distinct yeast populations comprising oxidative, weakly fermentative and strongly fermentative yeasts. Different combinations of dominant non-Saccharomyces yeasts were observed in each must, with significantly different assortments of dominant species, including Starmerella bacillaris (synonym Candida zemplinina), Lachancea thermotolerans, Hanseniaspora uvarum, Candida parapsilosis and Wickerhamomyces anomalus. None of these yeast consortia appeared to affect the growth of Saccharomyces cerevisiae or inhibit the overall progress of fermentation. However, the percentage of fermentative yeasts was positively correlated with the fermentation rate. Glucose and fructose consumption rates suggested active participation of both glucophilic and fructophilic yeasts from the onset of fermentation. The data highlight two parameters, viz. initial cell concentration and yeast community composition, as important fermentation drivers and open the possibility to predict fermentation behaviour based on the initial composition of the yeast community.
Spontaneous fermentation has been used for several thousands of years as an affordable source to preserve and enhance the quality of foods and beverages. Many fermented products are prepared in different parts of South America using raw materials such as milk, cassava, maize, cacao, coffee, grape, sugar cane, banana, and others. These traditional products can be used to make cachaça, wine, pisco, sour cassava starch, cheese, coffee, chocolate, vinegar and chichas which are important beverages and fermented foods consumed in South America. These fermented products have peculiar nutritional and sensory properties that derive from the biochemical transformations of specific raw materials. The diversity of the products varies according to the geographical area, cultural preference, fermentation techniques, local customs, and religious beliefs. Most traditional fermented foods and beverages of South America are produced on a minor scale using traditional recipes. In general, fermentations are conducted by complex microbial communities with the occurrence of yeasts, lactic and acetic acid bacteria and molds. Different microorganism species developed during fermentation, and their dynamics and frequencies of appearance determine a big fraction of the taste and flavour characteristics of these traditional foods and beverages. However, the microbial ecology of the fermentation processes is poorly studied in South America, and only in the last years several studies dealing with this subject are being published. The knowledge about the microbial ecology of food and beverage ecosystems is essential to understand the production process. In this chapter, we will discuss the production technologies and the microorganisms involved in the fermentation processes of several traditional foods and beverages produced in South America.
The use of gene technology to modify the genome of wine yeasts belonging to the species Saccharomyces cerevisiae began in the early 1990s. From a purely scientific point of view, many yeast constructs [genetically modified organisms (GMO)]
have been made so far, covering more or less all stages of the wine making process in which microorganisms or commercial enzymes
play a key role. The range of theoretical applications is summarised in this report. So far, only two wine-producing countries
worldwide allow the use of engineered wine yeasts; the changing situation in Germany regarding consumers’ attitudes towards
gene technology, and foodstuffs thus produced, will be outlined here. Experiments at the Geisenheim Research Center have highlighted
the essential stages of the wine making process where yeasts are involved by using engineered wine yeasts in comparison with
non-engineered yeast strains. Greenhouse simulations revealed the persistence of genetically modified (gm) yeasts when these
were used as fertilizers, as vintners do with yeast lees after the fermentation process. Furthermore, the persistence of engineered
yeast was also monitored in fermentations, after bottling, and after biological treatment of winery waste water. It turned
out that engineered wine yeast strains behave like non-engineered wine yeasts. They also persist in the winery interior and
installations as well as becoming part of the yeast flora on grape vines in a vineyard with annual fluctuations in the composition
of the yeast populations.
Keywords
Saccharomyces cerevisiae
–Alcoholic fermentation–Wine making–Genetic engineering–Risk assessment
We developed a method which allows electrophoretic fractionation of DNA in an agarose matrix according to an increasing current gradient, using a previously designed [R. Barbieri, V. Izzo, M.A. Costa, G. Giudice, G. Duro, Anal. Biochem. 212 (1993) 168; M.R. Asaro, V. Izzo, R. Barbieri, J. Chromatogr. A 855 (1999) 723] voltage gradient apparatus. This method allows the separation of different DNA fragments by increasing the distances of the components fractionated in the gel, revealing small differences in the length of different DNA components.
A method is presented to directly characterize the yeast diversity in wine fermentations using denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified ribosomal RNA genes. PCR-DGGE analysis of a commercial sweet wine fermentation clearly profiled the shifts in microbial diversity that occurred throughout the fermentation. Botrytis populations identified in press pan samples were absent from the settling tank and ensuing fermentation samples. Indigenous yeasts including Candida, Metschnikowia, and Pichia species were distinguished in the early stages of the fermentation prior to emergence of a Saccharomyces population. Surprisingly, the PCR-DGGE signature of Candida species persisted well into the fermentation long after the development of a dominant Saccharomyces population. By direct identification of yeast populations, PCR-DGGE can provide a rapid and comprehensive view of the microbial diversity present in wine fermentations without the necessity for enrichment plating.
The selection of wine yeasts is usually carried out within the species Saccha- romyces cerevisiae. It aims at identifying the yeast strains that, besides fermenting grape juice vigorously and producing high ethanol yield, can also positively influence the com- position and the sensorial characteristics of wine. The natural availability of yeast strains possessing an ideal combination of oenological characteristics is highly improbable. Moreover, selected S. cerevisiae wine strains usually produce wines with a plain aromatic profile. The extension of the selection of wine yeasts to S. cerevisiae not growing in oeno- logical environments or to non-Saccharomyces yeasts has provided strains possessing novel and interesting oenological characteristics. Nevertheless, these strains cannot be directly used as starter cultures in wine fermentations, mainly because they are not vigo- rous or competitive in oenological conditions. Wine strains possessing innovative oenolo- gical traits that can influence the sensorial characteristics of wine can be constructed using genetic or molecular methods. Intraspecific S. cerevisiae hybridisation has provided useful oenological strains. Nevertheless, the traits of oenological interest that can be exchanged or introduced using this technique are only those commonly found in the species S. cerevisiae. Innovative oenological traits can be introduced or exchanged by hybridising strains belon- ging to different species but with a sufficient genetic affinity for them to mate. Interspeci- fic Saccharomyces hybrids were found to be stable, vigorous and possessing the parental oenological traits in novel and interesting combinations. Nevertheless, they are sterile; the genetic improvement cannot therefore be taken further than the first generation. Moreover, the combination of the parental traits cannot be specifically programmed and a combina- tion of positive traits is often the result of chance. The recent development of recombinant DNA technology has overcome the limitations of traditional genetic techniques as well as broadening the potential of wine yeast improvement.
The PCR amplification and subsequent restriction analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene was applied to the identification of yeasts belonging to the genus Saccharomyces. This methodology has previously been used for the identification of some species of this genus, but in the present work, this application was extended to the identification of new accepted Saccharomyces species (S. kunashirensis, S. martiniae, S. rosinii, S. spencerorum, and S. transvaalensis), as well as to the differentiation of an interesting group of Saccharomyces cerevisiae strains, known as flor yeasts, which are responsible for ageing sherry wine. Among the species of the Saccharomyces sensu lato complex, the high diversity observed, either in the length of the amplified region (ranged between 700 and 875 bp) or in their restriction patterns allows the unequivocal identification of these species. With respect to the four sibling species of the Saccharomyces sensu stricto complex, only two of them, S. bayanus and S. pastorianus, cannot be differentiated according to their restriction patterns, which is in accordance with the hybrid origin (S. bayanus S. cerevisiae) of S. pastorianus. The flor S. cerevisiae strains exhibited restriction patterns different from those typical of the species S. cerevisiae. These differences can easily be used to differentiate this interesting group of strains. We demonstrate that the specific patterns exhibited by flor yeasts are due to the presence of a 24-bp deletion located in the ITS1 region and that this could have originated as a consequence of a slipped-strand mispairing during replication or be due to an unequal crossing-over. A subsequent restriction analysis of this region from more than 150 flor strains indicated that this deletion is fixed in flor yeast populations.
Discrimination of strains within the species Saccharomyces cerevisiae was demonstrated by the use of four different techniques to type 15 strains isolated from spoiled wine and beer. Random amplified polymorphic DNA with specific oligonucleotides and PCR fingerprinting with the microsatellite oligonucleotide primers (GAC)5 and (GTG)5 enabled discrimination between the strains tested. Additionally, restriction enzyme analysis, with TaqI and MseI, of PCR-amplified fragments from the complete internal transcribed spacer and nontranscribed spacer, both present in the rRNA-encoding gene cluster, proved to be suitable for generating intraspecies-specific patterns. Random amplified polymorphic DNA with primers 24 and OPA-11 and PCR fingerprinting with primer (GTG)5 appeared to generate the highest degree of diversity. However, the results indicated that there was no single PCR-mediated typing technique enabling discrimination on the strain level. Discrimination of each individual strain was nevertheless possible by combining the results obtained with all typing techniques.
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.
Genetic relationships of 24 phenotypically different strains isolated from sorghum beer in West Africa and the type cultures of the Saccharomyces sensu stricto species were investigated by universally primed polymerase chain reaction (PCR) analysis, microsatellite fingerprinting and PCR-restriction fragment length polymorphism (RFLP) of the ribosomal internal transcribed spacers. The results demonstrate that internal transcribed spacer (ITS) PCR-RFLP analysis with the endonucleases HaeIII, HpaII, ScrFI and TaqI is useful for discriminating S. cerevisiae, S. kudriavzevii, S. mikatae from one another and from the S. bayanus/S. pastorianus and S. cariocanus/S. paradoxus pairs. The sorghum beer strains exhibited the same restriction patterns as the type culture of S. cerevisiae CBS 1171. PCR profiles generated with the microsatellite primer (GTG)(5) and the universal primer N21 were almost identical for all isolates and strain CBS 1171. Despite phenotypic peculiarities, the strains involved in sorghum beer production in Ghana and Burkina Faso belong to S. cerevisiae. However, based on sequencing of the rDNA ITS1 region and Southern hybridisation analysis, these strains represent a divergent population of S. cerevisiae.
The distinctive flavors of beer, wine, and distilled or fortified alcoholic beverages are affected by many variables: raw material, flavor additives, and processing steps, which include fermentation, distillation, and subsequent aging. In this chapter on flavor chemistry of alcoholic beverages, the most important contributions of raw material, processing and aging will be surveyed for beer, wine and distilled spirits.
Recent ecological surveys indicate that the wine yeast Saccharomyces cerevisiae may be isolated with extreme difficulty from natural substrates, such as vineyard soil or the surface of mature grapes, conventionally believed to be its elected habitat. Conversely, it is amply demonstrated that its preferential location as the only fermenting colonizer is the surfaces of the wineries. Non-conventional methods for the separation, isolation, and enumeration of yeast cells from mature grapes of different varieties produced additional and unequivocal evidence on the numerical inconsistency of S. cerevisiae cells in nature. The taking over of musts by the natural yeast flora was also followed in microfermentations.
The aim of this work is to establish the influence of some enological treatments: grape juice clarification (by settling and by vacuum filtering), fermentation temperature (14°C and 18°C) and nitrogen addition (DAP 30 g/hL and commercial activator 15.5 g/hL), on the natural selection of wild Saccharomyces cerevisiae strains during the spontaneous fermentation of white grape juice, of the Viura variety from La Rioja (Spain). Mitochondrial DNA analysis is applied to the study of 240 isolates from two consecutive harvests. The results reveal one new effect of the clarification treatment: the selection effect on the wild yeast strain of Saccharomyces cerevisiae in spontaneous fermentation. However, the strain distribution is independent of fermentation temperature and nitrogen addition.
Will Australian winemakers follow the trends in winemaking from overseas? Are indigenous yeast species potentially useful in Australian winema king practices? This paper discusses the history behind the new interest in indigenous wine species and the current areas of research in wine microbiology, induding the role of innovation and technology in the future of winemaking.
Restriction fragment length polymorphisms in the rDNA internal transcribed spacer region (ITS) of 18 yeast strains currently assigned toSaccharomyces cerevisiae, S. pastorianus, andS. bayanus were examined. Primers complementary to the ITS region were used to amplify the ITS rDNA by the polymerase chain reaction (PCR). The products were digested with 10 endonucleases and cluster analysis was used to generate a phenogram from the restriction fragment data. Three strains ofS. cerevisiae (ATCC 10609, 26250, and 66162) exhibited restriction patterns that were different from the type strain but identical to those of theS. bayanus-S. pastorianus cluster. In contrast,S. pastorianus (ATCC 76671) showed restriction profiles that were different from its type strain but were identical to the type strain ofS. cerevisiae (ATCC 18824). These results suggest that the three strains ofS. cerevisiae should be reassigned to eitherS. pastorianus orS. bayanus, and the strain ofS. pastorianus (ATCC 76671) should be reclassified asS. cerevisiae.
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.
Recent ecological evidence points to a circulation model for Saccharomyces cerevisiae in nature which is different from that proposed at the end of the last century. The wine yeast ‘par excellence’ is isolated with extreme difficulty from conventional habitats, such as vineyard soil or the surface of ripe grapes, while it is almost the only species colonising the surfaces of the winery equipment.
The diversity of yeast species and strains was monitored by physiological tests and a simplified method of karyotyping of yeast chromosomes. During the first phase of investigated alcoholic fermentations, the yeast species Metschnikowia pulcherrima and Hanseniaspora uvarum were predominant, irrespective of the origin of the grape must. At the beginning of fermentation H. uvarum was even present in the case of induced fermentations with dried yeast. Middle and end phase of the alcoholic fermentation were clearly dominated by the yeast species Saccharomyces cerevisiae. In the case of spontaneous fermentations, several different strains of S. cerevisiae were present and competed with each other, whereas in induced fermentations only the inoculated strain of S. cerevisiae was observed. A competition of strains of S. cerevisiae also occurred during the fermentation with dried yeast product consisting of two different strains. An effect of H. uvarum on taste and flavour of wines can be postulated according to the frequency of its appearance during the first phase of fermentation. With the method of rapid karyotyping and supplementary physiological tests it was possible to make reliable assertions about the yeast diversity during alcoholic fermentation.
1999.The ecology of spontaneous fermentation in a new winery in La Rioja (Spain) was studied during 5 consecutive years using mitochondrial DNA restriction analysis. The number of different strains detected for each vintage and their appearance frequency varied from one year to another. A small number of strains were present in consecutive years, but the presence of each one varied in function of the specific year. Only one strain was present in all the 5 years studied. For the 1997 vintage, an unusual dominance of non-Sacharomyces yeasts in the vigorous fermentation was detected; this may explain the abnormal analytical data for the wines of that year.
The aim of the present study was to design species-specific primers capable of distinguishing between Saccharomyces cerevisiae, Saccharomyces bayanus/Saccharomyces pastorianus. The 5′-specific primers were designed from the ITS-1 region (between positions 150 and 182 from the 3′-SSU end) and the 3′-specific primers were located in the LSU gene (positions 560–590 from the 5′-end of this gene). These primers were tested with different collections and wild strains of these species and the results showed that the primers were capable of distinguishing between S. cerevisiae strains and S. bayanus/S. pastorianus. Not enough sequence differences were found between S. bayanus and S. pastorianus to design specific primers for these species using this region. This method offers an effective tool for a quick differentiation of the Saccharomyces strains of the most common species involved in industrial processes.
Numerous studies have described the yeast biota of grapes, and grape must in order to understand better the succession of yeasts during fermentation of wine. The origin of the wine yeasts has been rather controversial. By using more elaborate isolation methods, classical genetic analysis and electrophoretic karyotyping of monosporic clones, with this study, credible proof now exists that the vineyard is the primary source for the wine yeasts and that strains found on the grapes can be followed through the fermentation process.
A few days before vintage, yeasts were isolated quantitatively from three varieties of wine grape from three chteaux of the Graves region of the Gironde. There were about 105 viable yeasts/g of grape, from which only Kloeckera apiculata, Metschnikowia pulcherrima and one or more species of Rhodotorula were isolated.
A study of the microbiota present during the wine fermentation of five grape varieties from the ‘El Penedès’ area (Spain) was carried out to select autochthonous yeast strains for industrial wine production. In this study we identified members of the genera Candida, Dekkera, Hanseniaspora, Kluyveromyces, Torulaspora, Zygosaccharomyces and Saccharomyces in wine fermentation microbiota. Strains of Saccharomyces cerevisiae, as responsible agents of the alcoholic fermentation, were considered for a selection protocol. In this work we applied different enological criteria for selection, but previously we have characterized and differentiated Saccharomyces isolates by molecular methods to reduce the number of strains to analyse. Three strains were selected to conduct fermentations according to their characteristics. Finally, using mitochondrial DNA restriction analysis we demonstrated that the autochthonous selected strains are important contributors to the wine fermentation.
Most real-time video recognition systems have historically relied upon region of interest processing based on low level properties such as pixel color to quickly reduce the computational load required for real-time performance. Because these low levelproperties are quite sensitive to lighting variations, they are limited in their application scope. This paper presents an approach which segments objects of interest via higher level relational properties overcoming such limitations. These relations are autoassociatively and incrementally constructed through a processing structure called raster scan video processing in a hierarchical manner. Relational properties of interest are then obtained with local object specific processes. The compactness of this processing structure has allowed it to be implemented on one TMS320C40 digital signal processing chip at a processing rate of three frames per second. The approach is applied to the problem of road sign recognition in realistic outdoor scenes. Video frame recognition results for stop and pedestrian signs are presented along with details of the DSP implementation.
There is still a lack of agreement concerning the relative contribution of wine yeast that may originate in the vineyard compared to that which may originate in the cellar. Part of this controversy is due to the extreme difficulty of finding Saccharomyces cerevisiae on the grapes. We estimate that only about one in one-thousand grape berries carries wine yeast. However, we have found that grape berries that are damaged (i.e. the skin is broken) are very rich depositories of microorganisms including S. cerevisiae, and that one in four such berries is S. cerevisiae-positive. These positive berries have between 100,000 and 1,000,000 wine yeast cells on them, and there is evidence that these yeasts are clonal. We believe that the yeasts are brought to the berries by insects such as bees, wasps, and Drosophila and that they multiply in the rich medium of the grape interior. Even though there are many cells of S. cerevisiae on the damaged berries, they are in a definite minority. All the other organisms that are found in wine fermentations are also present on these berries, and their total numbers are in the range of 10 million to 100 million cells per berry.
PCR-RFLP analysis of the rDNA-ITS (internal transcribed spacer) region was applied to 174 yeast strains belonging to 30 species of oenological significance and including 27 type strains in order to define a rapid identification protocol for yeast colonies. DraI-or HaeIII-PCR-RFLP patterns were species-specific with the exception of teleomorphic and anamorphic forms. An improved protocol taking about 30 h was used for the detection and quantification of yeast species occurring in the course of a spontaneous wine fermentation at industrial level. Wine samples were taken and plated daily on an agar medium and the developed colonies were analysed by PCR-RFLP after 24 h of incubation. A representative sample of these colonies was also identified by traditional methods. Both procedures gave identical results. However, PCR-RFLP analysis allowed a more precise enumeration of the yeast populations, proving to be a reliable and simple method for monitoring the development of the yeast community throughout wine fermentation.
Yeasts are predominant in the ancient and complex process of winemaking. In spontaneous fermentations, there is a progressive growth pattern of indigenous yeasts, with the final stages invariably being dominated by the alcohol-tolerant strains of Saccharomyces cerevisiae. This species is universally known as the 'wine yeast' and is widely preferred for initiating wine fermentations. The primary role of wine yeast is to catalyze the rapid, complete and efficient conversion of grape sugars to ethanol, carbon dioxide and other minor, but important, metabolites without the development of off-flavours. However, due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing quest for specialized wine yeast strains possessing a wide range of optimized, improved or novel oenological properties. This review highlights the wealth of untapped indigenous yeasts with oenological potential, the complexity of wine yeasts' genetic features and the genetic techniques often used in strain development. The current status of genetically improved wine yeasts and potential targets for further strain development are outlined. In light of the limited knowledge of industrial wine yeasts' complex genomes and the daunting challenges to comply with strict statutory regulations and consumer demands regarding the future use of genetically modified strains, this review cautions against unrealistic expectations over the short term. However, the staggering potential advantages of improved wine yeasts to both the winemaker and consumer in the third millennium are pointed out.
The aim of the present study was to design species-specific primers capable of distinguishing between Saccharomyces cerevisiae, Saccharomyces bayanus/Saccharomyces pastorianus. The 5'-specific primers were designed from the ITS-1 region (between positions 150 and 182 from the 3'-SSU end) and the 3'-specific primers were located in the LSU gene (positions 560-590 from the 5'-end of this gene). These primers were tested with different collections and wild strains of these species and the results showed that the primers were capable of distinguishing between S. cerevisiae strains and S. bayanus/S. pastorianus. Not enough sequence differences were found between S. bayanus and S. pastorianus to design specific primers for these species using this region. This method offers an effective tool for a quick differentiation of the Saccharomyces strains of the most common species involved in industrial processes.
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.
Wine yeasts were isolated from fermenting Garnatxa and Xarel.lo musts fermented in a newly built and operated winery between 1995 and 2000. The species of non-Saccharomyces yeasts and the Saccharomyces cerevisiae strains were identified by ribosomal DNA and mitochondrial DNA RFLP analysis respectively. Non-Saccharomyces yeasts, particularly Hanseniaspora uvarum and Candida stellata, dominated the first stages of fermentation. However Saccharomyces cerevisiae was present at the beginning of the fermentation and was the main yeast in the musts in one vintage (1999). In all the cases, S. cerevisiae took over the process in the middle and final stages of fermentation. The analysis of the S. cerevisiae strains showed that indigenous strains competed with commercial strains inoculated in other fermentation tanks of the cellar. The continuous use of commercial yeasts reduced the diversity and importance of the indigenous S. cerevisiae strains.
The identification, differentiation and characterization of indigenous Saccharomyces sensu stricto strains isolated from Croatian vineyards and the evaluation of their oenological potential.
A total of 47 Saccharomyces sensu stricto strains were isolated from Chardonnay grapes and identified by physiological and molecular genetic methods. By using the standard physiological and biochemical tests, six isolates were identified as Saccharomyces cerevisiae and 41 as Saccharomyces paradoxus. However, PCR-RFLP analyses of the internal transcribed spacer (ITS1) region of the 18S ribosomal DNA identified 12 of the isolates as S.cerevisiae and 35 as S. paradoxus. Fermentation trials in a grape juice medium showed that these isolates ferment vigorously at 18 degrees C and display tolerance to high levels of ethanol. None of these isolates appeared to produce either hydrogen sulphide or killer toxins.
Saccharomyces paradoxus, possessing potentially important oenological characteristics, occurs in much higher numbers than S. cerevisiae in the indigenous population of Saccharomyces sensu stricto strains in Croatian vineyards.
This study forms an essential step towards the preservation and exploitation of the hidden oenological potential of the untapped wealth of yeast biodiversity in the Croatian grape-growing regions. The results obtained demonstrate the value of using molecular genetic methods, such as PCR-RFLP analyses, in conjunction with the traditional taxonomic methods based on phenotypic characteristics in such ecotaxonomic surveys. The results also shed some light on the ecology and oenological potential of S.paradoxus, which is considered to be the natural parent species of the domesticated species of the Saccharomyces sensu stricto group.
To evaluate whether intraspecific diversity of Saccharomyces cerevisiae in wine fermentations is affected by initial assimilable-nitrogen content.
Saccharomyces cerevisiae isolates from two spontaneous commercial wine fermentations started with adequate and inadequate nitrogen amounts were characterized by mitochondrial DNA restriction analysis. Several strains occurred in each fermentation, two strains, but not the same ones, being predominant at frequencies of about 30%. No significant differences were detected by comparing the biodiversity indices of the two fermentations. Cluster analysis demonstrated that the strain distribution was independent of nitrogen content, the two pairs of closely related dominant strains grouping into clusters at low similarity.
The genetic variability of S. cerevisiae in wine fermentations seemed not to depend on the nitrogen availability in must.
Nitrogen content did not affect the genetic diversity but may have induced a 'selection effect' on S. cerevisiae strains dominating wine fermentations, with possible consequences on wine properties.
Yeast isolates from orange fruit and juice in a spontaneous fermentation were identified and classified by two molecular techniques. The first was analysis of the restriction pattern generated from the polymerase chain reaction (PCR)-amplified 5.8S rRNA gene and the two internal transcribed spacers (ITS) using specific primers. The second technique was sequence analysis of the ITS regions using the same two primers. Nine different restriction profiles were obtained from the size of the PCR products and the restriction analyses with three endonucleases (CfoI, HaeIII and HinfI). These groups were identified as Candida tropicalis, Clavispora lusitaniae, Hanseniaspora uvarum, Pichia anomala, Pichia fermentans, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Saccharomyces unisporus, and Trichosporon asahii. Checking against identification according to morphological, physiological and biochemical traits corroborated this molecular identification. A total concordance was found in the identification with PCR-restriction fragment length polymorphism of the ITS region after analysing certified yeast strains from two different culture collections. Consequently, a rapid and reliable identification of the yeast populations was achieved by using molecular techniques.
The diversity and the composition of the yeast micropopulation significantly contribute to the sensory characteristics of wine. The growth of each wine yeast species is characterized by a specific metabolic activity, which determines concentrations of flavour compounds in the final wine. However, it must be underlined that, within each species, significant strain variability has been recorded. The wide use of starter cultures, mainly applied to reduce the risk of spoilage and unpredictable changes of wine flavour, can ensure a balanced wine flavour, but it may also cause a loss of characteristic aroma and flavour determinants. Thus, the beneficial contribution from the yeast increases when starter cultures for winemaking are selected on the basis of scientifically verified characteristics and are able to complement and optimise grape quality and individual characteristics. Here we report the characterization of a large number of strains of different wine yeast species, isolated from spontaneous wine fermentations and included in the culture collection of the Basilicata University.
The origin of the Saccharomyces cerevisiae strains that are responsible for spontaneous grape must fermentation was investigated in a long-established industrial winery by means of two different approaches. First, seven selected components of the analytical profiles of the wines produced by 58 strains of S. cerevisiae isolated from different sites and phases of the production cycle of a Grechetto wine were subjected to Principal Components Analysis. Secondly, the same S. cerevisiae isolates underwent PCR fingerprinting by means of delta primers. The results obtained by both methods demonstrate unequivocally that under real vinification conditions, the S. cerevisiae strains colonising the winery surfaces are the ones that carry out the natural must fermentation.
The performance of denaturing gradient gel electrophoresis (DGGE) for analysing yeasts associated with wine grapes was compared with cultural isolation on malt extract agar (MEA). After optimisation of PCR and electrophoretic conditions, the lower limit of yeast detection by PCR-DGGE was 10(2) cfuml(-1), although this value was affected by culture age and the relative populations of the species in mixed culture. In mixed yeast populations, PCR-DGGE detected species present at 10-100-fold less than other species but not when the ratio exceeded 100-fold. Aureobasidium pullulans was the main species isolated from immature, mature, and both damaged and undamaged grapes. It was not detected by PCR-DGGE when present at populations less than 10(3) cfug(-1). When approaching maturity, damaged grapes gave a predominance of Metschnikowia and Hanseniaspora species (10(5)-10(7) cfug(-1)), all detectable using PCR-DGGE. However, various species of Rhodotorula, Rhodosporidium and Cryptococcus were not detected by this method, even when populations were as high as 10(4) cfug(-1). PCR -DGGE was less sensitive than culture on MEA for determining the yeast ecology of grapes and could not reliably detect species present at populations less than 10(4) cfug(-1). However, this method detected a greater diversity of species than agar plating.
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