Composition of Saccharomyces cerevisiae strains in spontaneous fermentations of Pinot Noir and Chardonnay

There is a lack of knowledge about the composition of Saccharomyces cerevisiae strains in spontaneous fermentations of Pinot Noir and Chardonnay cultivars. The objectives were to determine the relative abundance of indigenous and commercial S. cerevisiae strains in spontaneous fermentations at three wineries from the two cultivars and to compare the composition of the S. cerevisiae strains between cultivars and wineries. Three fermentation vessels were sampled at three stages of fermentation for each cultivar at each winery. Isolates were identified to the strain level using seven microsatellite loci. Commercial S. cerevisiae strains were isolated at a frequency higher than that of the indigenous strains at each winery for both cultivars. The composition of S. cerevisiae strains was different for each cultivar and at each winery. Our results illustrate the clear influence of inoculated commercial active dry yeast strains on the composition of S. cerevisiae strains in spontaneous fermentations at wineries conducting both inoculated and spontaneous fermentations.     

Application of fluorescence in situ hybridisation (FISH) to the analysis of yeast population dynamics in winery and laboratory grape must fermentations

To analyse the yeast population diversity during wine fermentations, specific fluorescein-labelled oligonucleotide probes targeted to the D1/D2 region of the 26S rRNA of different yeast species known to occur frequently in this environment were designed and tested with reference strains. The probes were then used to identify wine must isolates and to follow, in combination with plate counts, the evolution of yeast populations in two winery fermentations of white and red grape musts. In both cases, a high diversity of non-Saccharomyces yeast species was detected, including Candida stellata, Hanseniaspora uvarum, H. guilliermondii, Kluyveromyces marxianus, K. thermotolerans and Torulaspora delbrueckii. Some of these species (e.g., K. marxianus, K. thermotolerans and T. delbrueckii) were present in significant amounts during the tumultuous fermentation stage, despite the predominance of Saccharomyces cerevisiae cells following the inoculation of the wine musts with a starter strain. To further clarify the yeast population dynamics at the late phase of the fermentations, and because winery conditions do not allow a reliable control of experimental variables, strains isolated from the industrial musts were used to conduct two laboratory microvinifications in synthetic grape juice, using different ratios of S. cerevisiae/non-Saccharomyces in the inocula. Under these conditions, the results were similar to those obtained in the winery, showing a yeast profile with mixed species throughout the first fermentation stage, i.e. until about 40-50% of the total sugar was consumed. Non-Saccharomyces yeasts were outgrown by S. cerevisiae only after ethanol reached concentrations around 4-5% (v/v), which argues in favour of a potential important role of non-Saccharomyces in the final organoleptic characteristics of the wine.     

Indigenous yeast population from Georgian aged wines produced by traditional “Kakhetian” method

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

Quantitative and qualitative analysis of non-Saccharomyces yeasts in spontaneous alcoholic fermentations

In this study, we looked at the yeast population present in four spontaneous alcoholic fermentations in the Rioja appellation (D.O.Ca. Rioja, Spain). The study was conducted through the identification of the yeasts via the PCR–RFLP technique of the ITS region of rDNA. In a first harvest, the qualitative diversity of the species present in spontaneous alcoholic fermentation was studied, and in a second harvest, their quantitative significance. In spontaneous fermentations, 17 different yeast species were found, although only two of them, Candida stellata and Kloeckera apiculata, as well as Saccharomyces cerevisiae, appeared in major proportions during fermentation. The significance of the non-Saccharomyces yeasts during the spontaneous alcoholic fermentation was conditioned by the presence of Saccharomyces cerevisiae in the medium. Species not cited in literature in connection with winemaking and yeasts associated with wines spoilage have been detected in all the alcoholic fermentations carried out in the qualitative study.     

Selection of indigenous Saccharomyces cerevisiae strains for Nero d'Avola wine and evaluation of selected starter implantation in pilot fermentation

The present research studied Saccharomyces cerevisiae yeasts isolated from Nero d'Avola grapes, collected in different areas of the Sicily region. RAPD-PCR analysis with M13 primer was used for preliminary discrimination among 341 S. cerevisiae isolates. Inoculated fermentations with S. cerevisiae strains, exhibiting different RAPD-PCR fingerprinting, revealed the impact of selected strains on volatile compound concentration. Two selected strains were used in fermentation at cellar level and the restriction analysis of mtDNA on yeast colonies isolated during fermentation was used to control strain implantation. This study represents an important step to establish a collection of indigenous S. cerevisiae strains isolated from a unique environment, such as Nero d'Avola vineyards. Different starter implantation throughout inoculated fermentation represents an additional character, which might be considered during the selection program for wine starter cultures.     

Effect of grape indigenous Saccharomyces cerevisiae strains on Montepulciano d'Abruzzo red wine quality

The growth of selected, indigenous Saccharomyces cerevisiae added as starters (SRS1, MS72 and RT73) was monitored during Montepulciano d'Abruzzo wine production. In all the fermentations the addition of the starter, caused a decrease of the non-Saccharomyces yeasts. When strains MS72 and RT73 were used as starters they were detected in the first phases of fermentations, while strain SRS1 competed successfully with native yeasts during all the process. Wines obtained by fermentation with the indigenous starters showed some different characteristics, according to the chemical and sensory analyses. This study highlighted that among selected starters with high fermentative capacity, some are able to dominate better than other natural wine yeast biota, whereas some strains can interact and survive besides native yeast populations during the fermentation. As a consequence, the dominance character can have a positive or negative effect on wine quality and has to be considered in the frame of yeast selection in order to improve or characterize traditional wines. Winemakers could choose among different degrees of yeast dominance to modulate the interaction among starter and native wine yeast population.     

Alleviation of stuck wine fermentations using salt‐preconditioned yeast

The influence of salt (sodium chloride) on the cell physiology of wine yeast was investigated. Cellular viability and population growth of three wine‐making yeast strains of Saccharomyces cerevisiae, and two non‐Saccharomyces yeast strains associated with wine must microflora (Kluyveromyces thermotolerans and K. marxianus) were evaluated following salt pre‐treatments. Yeast cells growing in glucose defined media exposed to different sodium chloride concentrations (4, 6 and 10% w/v) exhibited enhanced viabilities compared with nontreated cultures in subsequent trial fermentations. Salt ‘preconditioning’ of wine yeast seed cultures was also shown to alleviate stuck and sluggish fermentations at the winery scale, indicating potential benefits for industrial fermentation processes. It is hypothesized that salt induces specific osmostress response genes to enable yeast cells to better tolerate the rigours of fermentation, particularly in high sugar and alcohol concentrations. Copyright © 2014 The Institute of Brewing & Distilling     

沙城产区酿酒酵母多样性研究

对沙城产区龙眼葡萄相关环境中分离筛选的酿酒酵母进行多样性研究。在连续3年(2008、2009、2010年)的实验中,共从葡萄园土壤、葡萄酒厂设备表面、接触过葡萄酒厂设备的葡萄汁和自然发酵过程中采集菌源样品227份,共分离得到1358株酵母菌。用5.8S-ITS区域RFLP方法进行分子水平的分类鉴定及赖氨酸培养基复筛,得到了270株酿酒酵母。再利用Interdelta PCR指纹图谱法将酿酒酵母区分为16类,其中土壤5类,自然发酵过程中第2、3、4期分别得到4类、10类和11类;酒厂设备表面3类;接触酒厂设备的葡萄汁3类。酿酒酵母的种类因样品采集时间、采集地点等不同有明显区别。自然发酵过程中得到的酿酒酵母被认为是本土酵母的可能性最大。     

接种不同嗜杀特性的酿酒酵母对赤霞珠发酵中酵母多样性的影响

以赤霞珠葡萄为原料,分别接种不同嗜杀特性的酿酒酵母(Saccharomyces cerevisiae)菌株NXU17-26(中性)、UCD522(敏感菌株)和UCD2610(嗜杀菌株),并以自然发酵为对照,研究各菌株对赤霞珠葡萄酒的发酵特征及发酵中酵母菌多样性的影响。结果表明,接种发酵在启酵和发酵速度上显著快于自然发酵。WLN培养基将分离到的480株酵母菌鉴定为7种类型,26S rDNA D1/D2序列分析进一步将其鉴定为4属5种:葡萄汁有孢汉逊酵母(Hanseniaspora uvarum)、克鲁维毕赤酵母(Pichia kluyveri)、伯顿丝孢毕赤酵母(Hyphopichia burtonii)、S.cerevisiae、库徳毕赤酵母(Pichia kudriavzevii)。这4属5种的酵母均存在于自然发酵中,而接种发酵中仅有H.uvarum和S.cerevisiae两种酵母,接种发酵中酵母菌多样性较低。Interdelta指纹图谱分析表明,所接种的酿酒酵母菌株是相应发酵中的优势菌株:接种中性酵母NXU17-26的发酵中,NXU17-26的基因型占比为63.46%;接种敏感菌株UCD522中,UCD522的基因型占比为44.68%,野生酿酒酵母NXU18-15表现出较强的竞争力,基因型占比为34.04%;接种嗜杀酵母UCD2610的发酵中,UCD2610的基因型占比为62.74%。非加权算术平均数法聚类分析表明,分离自同一发酵中的不同酿酒酵母菌株间的遗传差异性较小;分离自不同发酵中的酿酒酵母菌株间遗传差异性较大。     

Evolution of the population of Saccharomyces cerevisiae from grape to wine in a spontaneous fermentation

To determine the grape or winery origin of the Saccharomyces cerevisiae involved in spontaneous fermentation, musts were collected at different stages of wine-making process and fermented. First, grapes were collected in two different vineyards and crushed at the laboratory. Second, musts were collected after crushing and clarification in the cellar. Third, musts collected in the cellar were sterilized and inoculated with tartar deposit collected in the vats. The fourth fermentation was in the cellar. For the two vineyards, two hundred of S. cerevisiae clones were isolated for each of the four fermentations, driving to a library of 1600 clones. All the library was analysed by inter-delta PCR with a basic set of primers and about 20% of the library was further analysed by inter-delta PCR with an improved set of primers. Six, and more than 30 different PCR patterns were obtained from basic- and improved-PCR analysis, respectively. The amounts of each family were analysed at the different stages of wine making. Our study demonstrates that the two vineyards present different S. cerevisiae populations. Moreover the S. cerevisiae strains involved in spontaneous fermentation in the cellar originate partly from the vineyard and partly from the winery, in amounts varying with the must.     

Indigenous wine killer yeasts and their application as a starter culture in wine fermentation

Wine yeast strains were isolated from seven fermentations of the red wines ‘Refošk’ and ‘Teran’, produced in the southwestern part of Slovenia. Among 613 isolated yeast strains, 22 expressed killer activity against the supersensitive strain Saccharomyces cerevisiae. Killer strains were isolated at different stages of wine fermentation but did not dominate in any of them. Species identification was based on the combination of RFLP analysis of an amplified rDNA region and biochemical–physiological tests. Killer isolates were identified as S. cerevisiae, Pichia anomala, Pichia kluyveri, Pichia pijperi, Hanseniaspora uvarum and Candida rugosa. Electrophoretic karyotyping was used to differentiate strains of the same species. Fermentation properties of four S. cerevisiae strains that possessed stable killer activity were characterized in fermentations of Malvasia must by studying their population dynamics and chemical composition and by sensory analysis of the wines produced. In order to compare the results, spontaneous fermentations and fermentations induced by commercial yeast starters were performed concomitantly. The local killer strain S_S12/10 showed the best fermentation properties and produced wine with favourable characteristics.     

Indigenous lactic acid bacteria communities in alcoholic and malolactic fermentations of Tempranillo wines elaborated in ten wineries of La Rioja (Spain)

Indigenous lactic acid bacteria (LAB) communities have been analyzed for three years (2006, 2007 and 2008) during alcoholic (AF) and malolactic (MLF) fermentations of Tempranillo wines in ten wineries of La Rioja. The results showed that analytical composition of wines and physical–chemical conditions of elaboration influenced the LAB populations, the MLF duration and the percentage of each isolated species and strains. The highest diversity of LAB species was observed during AF in all the wineries. Oenococcus oeni was present in all studied stages of the fermentation process, being the predominant species at final AF stage. The study of 925 isolates of O. oeni by Pulsed Field Gel Electrophoresis (PFGE) allowed the detection of a total of 112 distinct genotypes. Most fermentation stages of both AF and MLF showed mixed O. oeni strain populations, so that there were different genotypes able to share their ecological niche or tank in spontaneous MLF. The frequency of participation of each genotype varied either from year to year or from winery to winery. Otherwise, seven genotypes were detected in the three studied years and in at least three out of the ten studied wineries, being four of them also present in the three studied subzones of this region. These results suggest the existence of an endemic microbiota in this region, the adaptation of indigenous O. oeni strains to the winery conditions every year and the interest of selecting predominant genotypes in order to preserve the biodiversity and peculiarity of these wines.     

Co-inoculation of different Saccharomyces cerevisiae strains and influence on volatile composition of wines

Wine is the result of the performance of different yeast strains throughout the fermentation in both spontaneous and inoculated processes. 22 Saccharomyces cerevisiae strains were characterized by microsatellite fingerprinting, selecting 6 of them to formulate S. cerevisiae mixed cultures. The aim of this study was to ascertain a potential benefit to use mixed cultures to improve wine quality. For this purpose yeasts behavior was studied during co-inoculated fermentations. Aromatic composition of the wines obtained was analyzed, and despite the fact that only one strain dominated at the end of the process, co-cultures released different concentrations of major volatile compounds than single strains, especially higher alcohols and acetaldehydes. Nevertheless, no significant differences were found in the type and quantity of the amino acids assimilated. This study demonstrates that the final wine composition may be modulated and enhanced by using suitable combinations of yeast strains.     

Permanence of yeast inocula in the winery ecosystem and presence in spontaneous fermentations

The oenological practice of systematic inoculation with active dried yeasts is commonly used by many wineries around the world. However, the use of these yeasts is not free from controversy, since this practice has occasionally been described as having a negative effect on the biodiversity of natural yeast present in the wineries. The purpose of this study is to analyse the presence of commercial yeasts used as inocula in the ecosystem of three D.O.Ca. (“Qualified” Designation of Origin) Rioja wineries. It studies the permanence of these yeasts in winery equipment and their participation in spontaneous fermentations where they have not been used for inoculation. The results indicate that the presence of the active dry yeasts used in the wineries was scarce or non-existent, both in the ecosystem of each winery and in the spontaneous fermentations where they had not been added. So, repeated inoculation with active dry yeasts allowed a high presence and development of autochthonous (Saccharomyces and non-Saccharomyces) yeasts, both in equipment and in the spontaneous fermentations carried out.     

Growth of non-Saccharomyces yeasts affects nutrient availability for Saccharomyces cerevisiae during wine fermentation

Yeast produces numerous secondary metabolites during fermentation that impact final wine quality. Although it is widely recognized that growth of diverse non-Saccharomyces (NS) yeast can positively affect flavor complexity during Saccharomyces cerevisiae wine fermentation, the inability to control spontaneous or co-fermentation processes by NS yeast has restricted their use in winemaking. We selected two NS yeasts from our Uruguayan native collection to study NS-S. cerevisiae interactions during wine fermentation. The selected strains of Hanseniaspora vineae and Metschnikowia pulcherrima had different yeast assimilable nitrogen consumption profiles and had different effects on S. cerevisiae fermentation and growth kinetics. Studies in which we varied inoculum size and using either simultaneous or sequential inoculation of NS yeast and S. cerevisiae suggested that competition for nutrients had a significant effect on fermentation kinetics. Sluggish fermentations were more pronounced when S. cerevisiae was inoculated 24h after the initial stage of fermentation with a NS strain compared to co-inoculation. Monitoring strain populations using differential WL nutrient agar medium and fermentation kinetics of mixed cultures allowed for a better understanding of strain interactions and nutrient addition effects. Limitation of nutrient availability for S. cerevisiae was shown to result in stuck fermentations as well as to reduce sensory desirability of the resulting wine. Addition of diammonium phosphate (DAP) and a vitamin mix to a defined medium allowed for a comparison of nutrient competition between strains. Addition of DAP and the vitamin mix was most effective in preventing stuck fermentations.     

Yeasts Isolated from the Alcoholic Fermentation of Agave duranguensis During Mezcal Production

Mezcal is a spirit produced in some regions of México. In the state of Durango, mezcal is produced via traditional fermentation of the Agave duranguensis plant. To better understand traditional fermentation processes, it is necessary to know which yeast species are present in fermentations in different producer regions. The aim of this research was to study yeasts involved in traditional mezcal fermentation in Durango, México, and investigate the phylogeny of the native Saccharomyces cerevisiae strains involved in this process. The 5.8S-ITS genomic region was analyzed to identify strains present in the fermentation process samples in this study. To differentiate strains belonging to the genus Saccharomyces, different molecular techniques were used, including analysis of mitochondrial DNA and δ elements and sequencing of the 5.8S-ITS genomic region. Although a high diversity of microorganisms was found at the beginning of fermentation, Saccharomyces cerevisiae was the only yeast present at the end of fermentation in region I, while Torulaspora delbrueckii was found in a higher number than S. cerevisiae at the end of fermentation in the region II. Molecular techniques demonstrated that Saccharomyces cerevisiae isolated in Durango are phylogenetically independent from the strains isolated in other regions of Latin America and Europe.     

Yeast population dynamics in five spontaneous fermentations of Malvasia must

The dynamics of the wine yeast strains presented in five spontaneous Malvasia wine fermentations have been studied. Samples were analysed for their microbiological characteristics and chemical substances. All 937 isolates were characterized using electrophoretic karyotyping and tested for their killer activity. The non- Saccharomyces population was identified using a combination of PCR-RFLP analysis of the rDNA spacer region and physiological testing. The total yeast population level in the must after sedimentation was 10⁵cfu ml⁻¹and included the following genera:Candida, Metschnikowia, Hanseniaspora, Rhodotorula, Issatchenkia and Debaryomyces. However, Saccharomyces sp. was not detected in fresh must samples plated on YEPD medium. Based on the chromosome length polymorphism among 649 isolates from the subsequent phases of fermentation, 46 different electrophoretic patterns of Saccharomyces cerevisiae were distinguished. The most abundant karyotypes were L₁, L₄, L₁₂, P₆. A sequential substitution of S. cerevisiae strains occurred during the different phases of fermentation. At the slow fermentation rate, karyotype L₄was most abundant in almost all fermenters. At the beginning of the tumultuous fermentation phase, the most frequent karyotype became L₁followed by karyotype L₄. Finally, during the fermentation process, pattern L₄was clearly replaced by karyotype L₁followed by pattern L₁₂. Despite the same fermentation source (grape must), differences among five spontaneous fermentations were observed. The population dynamics of S. cerevisiae yeasts, especially the dynamics of the major S. cerevisiae strains (L₁, L₄, and L₁₂) were quite similar in all five fermenters in opposite to the minor strains of S. cerevisiae.     

Dynamic study of yeast species and Saccharomyces cerevisiae strains during the spontaneous fermentations of Muscat blanc in Jingyang,China

The evolution of yeast species and Saccharomyces cerevisiae genotypes during spontaneous fermentations of Muscat blanc planted in 1957 in Jingyang region of China was followed in this study. Using a combination of colony morphology on Wallerstein Nutrient (WLN) medium, sequence analysis of the 26S rDNA D1/D2 domain and 5.8S-ITS-RFLP analysis, a total of 686 isolates were identified at the species level. The six species identified were S. cerevisiae, Hanseniaspora uvarum, Hanseniaspora opuntiae, Issatchenkia terricola, Pichia kudriavzevii (Issatchenkia orientalis) and Trichosporon coremiiforme. This is the first report of T. coremiiforme as an inhabitant of grape must. Three new colony morphologies on WLN medium and one new 5.8S-ITS-RFLP profile are described. Species of non-Saccharomyces, predominantly H. opuntiae, were found in early stages of fermentation. Subsequently, S. cerevisiae prevailed followed by large numbers of P. kudriavzevii that dominated at the end of fermentations. Six native genotypes of S. cerevisiae were determined by interdelta sequence analysis. Genotypes III and IV were predominant. As a first step in exploring untapped yeast resources of the region, this study is important for monitoring the yeast ecology in native fermentations and screening indigenous yeasts that will produce wines with regional characteristics.     

A new simplified AFLP method for wine yeast strain typing

A simplified method of AFLP (Amplified Fragment Length Polymorphisms) is presented for typing yeast present during wine fermentations. The changes introduced allowed analysis by gel electrophoresis and considerably reduced the need for equipment. Another remarkable improvement was the use of non-labelled primers which reduces the cost of the analysis. This method was applied to reference strains from culture collection to test the reliability of the technique. A total of 180 colonies isolated from a spontaneous fermentation were typed into eleven different strains of Hanseniaspora uvarum, six of Hanseniaspora vineae, four of Candida zemplinina, and eleven of Saccharomyces cerevisiae. This method is suitable for typing yeast strains for routine grape and wine ecology analysis.     

Allelic variants of hexose transporter Hxt3p and hexokinases Hxk1p/Hxk2p in strains of Saccharomyces cerevisiae and interspecies hybrids

The transport of sugars across the plasma membrane is a critical step in the utilization of glucose and fructose by Saccharomyces cerevisiae during must fermentations. Variations in the molecular structure of hexose transporters and kinases may affect the ability of wine yeast strains to finish sugar fermentation, even under stressful wine conditions. In this context, we sequenced and compared genes encoding the hexose transporter Hxt3p and the kinases Hxk1p/Hxk2p of Saccharomyces strains and interspecies hybrids with different industrial usages and regional backgrounds. The Hxt3p primary structure varied in a small set of amino acids, which characterized robust yeast strains used for the production of sparkling wine or to restart stuck fermentations. In addition, interspecies hybrid strains, previously isolated at the end of spontaneous fermentations, revealed a common amino acid signature. The location and potential influence of the amino acids exchanges is discussed by means of a first modelled Hxt3p structure. In comparison, hexokinase genes were more conserved in different Saccharomyces strains and hybrids. Thus, molecular variants of the hexose carrier Hxt3p, but not of kinases, correlate with different fermentation performances of yeast. Copyright © 2015 John Wiley & Sons, Ltd.