Patagonian wines: the selection of an indigenous yeast starter

The use of selected yeasts for winemaking has clear advantages over the traditional spontaneous fermentation. The aim of this study was to select an indigenous Saccharomyces cerevisiae yeast isolate in order to develop a regional North Patagonian red wine starter culture. A two-step selection protocol developed according to physiological, technological and ecological criteria based on killer interactions was used. Following this methodology, S. cerevisiae isolate MMf9 was selected among 32 indigenous yeasts previously characterized as belonging to different strains according to molecular patterns and killer biotype. This isolate showed interesting technological and qualitative features including high fermentative power and low volatile acidity production, low foam and low sulphide production, as well as relevant ecological characteristics such as resistance to all indigenous and commercial S. cerevisiae killer strains assayed. Red wines with differential volatile profiles and interesting enological features were obtained at laboratory scale by using this selected indigenous strain.     

Analysis of yeast populations during alcoholic fermentation: a six year follow-up study

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.     

Yeast assessment during alcoholic fermentation inoculated with a natural “pied de cuve” or a commercial yeast strain

The present study has been carried out in an organic winery established in 2003 in the Denomination of Origin “Sierras de Málaga” (Southern Spain) region during the 2007 vintage. The aim of this work was to ascertain the yeast microflora present in the winery and during the vinifications and to obtain a collection of autochthonous S. cerevisiae strains from this area. Yeast populations from three vats containing fermenting musts from different grape varieties were analysed. Two of them were inoculated with a natural “pied de cuve” while the third one was sown with a rehydrated commercial yeast strain. A total of 382 yeasts were isolated and identified, initially by restriction analysis of ribosomal DNA and further by sequencing of this region. Non-Saccharomyces yeasts were found in all three musts but they practically disappeared as the fermentations progressed. Analysis of mitochondrial DNA RFLP revealed 13 different restriction patterns of Saccharomyces cerevisiae strains, five of them similar to those of commercial strains used in the winery. Commercial strains were found even in vats inoculated with a “pied de cuve” generated by spontaneous fermentation of a must sample. The analysis of samples recovered from different winery surfaces and equipments demonstrated that non-Saccharomyces and both commercial and autochthonous Saccharomyces strains were part of the resident microflora in the winery. Biodiversity of autochthonous S. cerevisiae in fermentation vats was low but two of them were able to compete with the commercial ones and they were isolated even at the end of the fermentation.     

Survival of commercial yeasts in the winery environment and their prevalence during spontaneous fermentations

Inoculation of active dry yeasts during the wine-making process has become a common practice in most wine-producing regions; this practice may affect the diversity of the indigenous population of Saccharomyces cerevisiae in the winery. The aim of this work was to study the incidence of commercial yeasts in the experimental winery of Estación de Viticultura e Enoloxía de Galicia (EVEGA) and their ability to lead spontaneous fermentations. To do this, 64 spontaneous fermentations were carried out in the experimental cellar of EVEGA over a period of 7 years. Samples were taken from must and at the beginning, vigorous and final stages of fermentation. A representative number of yeast colonies was isolated from each sample. S. cerevisiae strains were characterised by analysis of mitochondrial DNA restriction patterns. The results showed that although more than 40 different strains of S. cerevisiae were identified, only 10 were found as the dominant strain or in codominance with other strains in spontaneous fermentations. The genetic profiles (mtDNA-RFLPs) of eight of these strains were similar to those of different commercial yeasts that had been previously used in the EVEGA cellar. The remaining two strains were autochthonous ones that were able to reach implantation frequencies as high of those of commercial yeasts. These results clearly indicated that commercial wine yeasts were perfectly adapted to survive in EVEGA cellar conditions, and they successfully competed with the indigenous strains of S. cerevisiae, even during spontaneous fermentations. On the other hand, autochthonous dominant strains that presented desirable oenological traits could be of interest to preserve wine typicity.     

Influence of killer strains of Saccharomyces cerevisiae on wine fermentation

The effect of killer strains of Saccharomyces cerevisiae on the growth of sensitive strains during must fermentation was studied by using a new method to monitor yeast populations. The capability of killer yeast strains to eliminate sensitive strains depends on the initial proportion of killer yeasts, the susceptibility of sensitive strains, and the treatment of the must. In sterile filtered must, an initial proportion of 2-6% of killer yeasts was responsible for protracted fermentation and suppression of isogenic sensitive strains. A more variable initial proportion was needed to get the same effect with non-isogenic strains. The suspended solids that remain in the must after cold-settling decreased killer toxin effect. The addition of bentonite to the must avoided protracted fermentation and the suppression of sensitive strains; however, the addition of yeast dietary nutrients with yeast cell walls did not, although it decreased fermentation lag.     

Patagonian wines: implantation of an indigenous strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in fermentations conducted in traditional and modern cellars

In this work we evaluate the implantation capacity of the selected S. cerevisiae indigenous strain MMf9 and the quality of the produced wines in a traditional (T) and a modern (M) cellar with different ecological and technological characteristics in North Patagonia (Argentina). Red musts were fermented in 10,000 l vats using the indigenous strain MMf9 as well as the respective controls: a fermentation conducted with a foreign starter culture (BC strain) in M cellar and a natural fermentation in T cellar. Since commercial S. cerevisiae starters are always used for winemaking in M cellar and in order to compare the results, natural fermentations and fermentations conducted by the indigenous strain MMf9 were performed at pilot (200 l) scale in this cellar, concomitantly. Thirty indigenous yeasts were isolated at three stages of fermentation: initial, middle and end. The identification of the yeast biota associated to vinifications was carried out using ITS1-5.8S-ITS2 PCR-RFLP. The intra-specific variability of the S. cerevisiae populations was evaluated using mtDNA-RFLP analysis. Wines obtained from all fermentations were evaluated for their chemical and volatile composition and for their sensory characteristics. A higher capacity of implantation of the indigenous MMf9 strain was evidenced in the fermentation carried out in M cellar (80% at end stage) than the one carried out in T cellar (40%). This behaviour could indicate that each cellar differs in the diversity of S. cerevisiae strains associated to wine fermentations. Moreover a higher capacity of implantation of the native starter MMf9 with regard to the foreign (BC) one was also found in M cellar. The selected indigenous strain MMf9 was able to compete with the yeast biota naturally present in the must. Additionally, a higher rate of sugar consumption and a lower fermentation temperature were observed in vinifications conducted by MMf9 strain with regard to control fermentations, producing wines with favourable characteristics. Even when its implantation in T fermentation was lower than that observed in M one, we can conclude that the wine features from MMf9 fermentations were better than those from their respective controls. Therefore, MMf9 selected indigenous strain could be an interesting yeast starter culture in North Patagonian wines.     

Utilization of KHR killer as genetic marker for purity test of starter yeast during fermentation of grape musts

An excellent wine yeast, Saccharomyces cerevisiae W3, which had KHR killer, was added as a starter yeast into grape must and behavior of the starter strain and wild yeasts was investigated during fermentation by using KHR killer as a genetic marker. The KHR killer was detected only in the strain W3 and not in other wine and wild yeast strains. Accordingly, the frequency of starter yeast W3 was monitored throughout the fermentation of grape musts by using KHR killer, W3 was discriminated efficiently from wild yeasts during fermentation by KHR killer activity and proved to lead the fermentation as a dominant yeast until their termination.     

Biotechnology of natural and winery-associated strains of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

A new body of evidence challenges the original consolidated theory of Pasteur on the natural (vineyard) origin of wine strains of Saccharomyces cerevisiae and instead indicates a local, winery-restricted life cycle. The findings open novel biotechnological perspectives for obtaining autochthonous selected starters for the wine industry. A local, individual, and specific fermenting yeast flora, mass selected year after year through many generations of S. cerevisiae in grape must, is present on the surfaces of every winery. These yeast strains are endowed with exceptional enological properties and capable of producing an assortment of volatile compounds apparently contributing to the specific bouquet of locally produced wines.     

Occurrence of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> killer yeasts in wine-producing areas of Greece

The occurrence of the killer character was studied in 840 Saccharomyces cerevisiae isolates at the end of spontaneous fermentations in four wine-producing regions of Greece, Mantinia and Nemea in Peloponnese and the islands Limnos and Santorini. The incidence of killer strains varied from one region to another. Sensitive and neutral strains were also found among the S. cerevisiae strains. Using the plate bioassay at pH 4.5 two different killer phenotypes were detected among the killer isolates which differed in their degree of killer activity. They were designated as SK (strong killer) and WK (weak killer). The proportion of SK to WK phenotypes differed from one area to another. All killer isolates were assayed for expression of killer activity also at pH 3.5. The lower pH decreased the killer activity of all isolates and changed the proportion of SK to WK phenotypes. The percentage of WK phenotypes increased in all cases and some killer isolates lost their killer activity completely, but generally the killer activity remained significant, especially in the area of Mantinia where the SK phenotype remained dominant at the low pH found in musts and wine.     

Fermentation behaviour of controlled mixed and sequential cultures of Candida cantarellii and Saccharomyces cerevisiae wine yeasts

Behaviour of Candida cantarellii and Saccharomyces cerevisiae strains during the fermentation of Syrah grape must using pure, mixed and sequential yeast cultures was studied. Different kinds of inocula have been tested according to the type of culture. Inocula proportions used in mixed C. cantarellii and S. cerevisiae strains reflect the population levels in natural grape microbiota. Biomass evolution of both strains was analysed in relation to different byproduct levels. Saccharomyces cerevisiae overcame C. cantarellii in the different co-culture assays at 48 h of fermentation. The final concentration of ethanol was similar in mixed and both sequential tests and higher (from 7.8 to 10.6%) than in S. cerevisiae pure culture. In mixed and sequential cultures, the glycerol content of the final products was 44.3 to 52.8% higher than the one obtained with pure S. cerevisiae fermentation. Wine analytical profiles of experiments that involved S. cerevisiae and C. cantarellii strains differed from the pure ones mainly in acetoin, propanol and succinic acid contents. From an enological point of view, analysed byproducts are relevant. Considering this, mixed assay and the inoculation of S. cerevisiae after 3 days of pure C. cantarellii fermentation appear to be the more appropriate options to develop the particular characteristics of Syrah wines. This revised version was published online in July 2006 with corrections to the Cover Date.     

Saccharomyces cerevisiae wine yeast populations in a cold region in Argentinean Patagonia. A study at different fermentation scales

AIMS: To study the diversity and dynamics of indigenous Saccharomyces wine populations during Malbec spontaneous fermentation, a representative Patagonian red wine, at both industrial and laboratory scale. METHODS AND RESULTS: Two molecular techniques, including restriction fragment length polymorphism of mitochondrial (mt) DNA and polymorphism of amplified delta interspersed element sequences, were used for characterization of indigenous yeasts at strain level. The mtDNA restriction patterns showed the major discriminative power; however, by combining the two molecular approaches it was possible to distinguish a larger number of strains and, therefore, draw more representative conclusions about yeast diversity. Although a great diversity of wild Saccharomyces cerevisiae strains was observed, only nine represented more than half of the total Saccharomyces yeast biota analysed; five of these were common and took over the Malbec must fermentation in both vinifications. CONCLUSIONS: Many different indigenous S. cerevisiae strains were identified; nevertheless, the dominant strains in both industrial and laboratory vinification processes were just a few and the same. SIGNIFICANCE AND IMPACT OF THE STUDY: Small-scale fermentation appears to be a valuable tool in winemaking, one especially helpful in evaluating microbiological aspects of as well as possible interactions between inoculated selected strains and native strains.     

商业酵母在不同品种葡萄酒工业化生产中的定殖差异

[背景]酵母菌在葡萄酒酿造中起到重要的作用,接种商业活性干酵母(active dry yeast,ADY)进行葡萄酒酿造在国内较为普遍,然而商业酿酒酵母(Saccharomyces cerevisiae)对我国本土酵母菌资源的影响及二者竞争关系的相关报道不多.[目的]比较商业酿酒酵母在不同品种葡萄酒工业化生产中的定殖差...     

Cell-recycle batch fermentation using immobilized cells of flocculent Saccharomyces cerevisiae wine strains

Five, highly flocculeng strains of Saccharomyces cerevisiae, isolated from wine, were immobilized in calcium alginate beads to optimize primary must fermentation. Three cell-recycle batch fermentations (CRBF) of grape musts were performed with the biocatalyst and the results compared with those obtained with free cells. During the CRBF process, the entrapped strains showed some variability in the formation of secondary products of fermentation, particularly acetic acid and acetaldehyde. Recycling beads of immobilized flocculent cells is a good approach in the development and application of the CRBF system in the wine industry.     

Yeast population dynamics in spontaneous fermentations: Comparison between two different wine-producing areas over a period of three years

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.     

贺兰山东麓优良本土酿酒酵母筛选及发酵特性分析

【背景】商业酵母的使用造成葡萄酒同质化问题严重,发掘优良本土酿酒酵母具有十分重要的意义。【目的】从168株宁夏本土酿酒酵母菌株中筛选出性能优良、具有出色葡萄酒发酵能力的菌株。【方法】基于杜氏管发酵试验和乙醇、高糖等耐受性试验分析产H2S能力及生长曲线测定的方法,筛选出发酵力好、耐受性强、低产H2S的本土酿酒酵母进行赤霞珠葡萄酒发酵试验,测定葡萄酒样基础理化指标、酚类物质和挥发性成分,探究筛选出的酿酒酵母发酵特性。【结果】初步筛选出发酵快速,能适应13%乙醇、350 g/L葡萄糖、250 mg/L SO2、pH 1.0的生存环境且低产H2S的4株本土酿酒酵母YC-E8、QTX-D17、QTX-D7、YQY-E18。菌株YC-E8产甘油能力强,所发酵酒样香气与商业酵母XR、F33最为接近,适用于赤霞珠葡萄酒的发酵。菌株QTX-D17发酵酒样中酒精、单宁、总酚和花色苷含量最高,表现出本土酿酒酵母优良的发酵特性。菌株QTX-D7所发酵酒样香气中乙酸乙酯、辛酸乙酯、1-壬醇等物质含量较高,赋予了葡萄酒香蕉味、苹果味、菠萝味、椰子味等愉悦花果香。【结论】最终筛选出3株优良本土酿酒酵母QTX-D17...     

A quick screening method to identify β-glucosidase activity in native wine yeast strains: application of Esculin Glycerol Agar (EGA) medium

One hundred and fifty-four yeast strains were isolated from grapes and musts of Uruguayan vineyards and wineries. Only thirty strains showed β-glucosidase activity in Esculin Glycerol Agar (EGA) solid medium. Twenty-one were non-Saccharomyces and nine were Saccharomyces cerevisiae strains. The objective of this study was to evaluate the suitability of Esculin Glycerol Agar (EGA) solid medium for screening β-glucosidase activity in native yeasts strains. Halo sizes measured in the EGA solid medium were correlated to the Glycosyl-Glucose (GG) indexes measured after fermentation of grape musts with each strain. The two S. cerevisiae strains with the best performance were selected for further fermentations on a Muscat Miel grape must, rich in bound monoterpenes. The levels of free linalool, hodiol I and geraniol increased significantly as compared to fermentation with a commercial wine yeast strain. These results show the suitability of this simple and economic medium to identify S. cerevisiae glucosidase producers with a potential impact on real winemaking conditions. On the other hand, great variability was found for the non-Saccharomyces strains, and this would demand further studies for each species. In conclusion, the use of EGA solid medium shows that the screening method is suitable for exploring the glucosidase activity of native strains of S. cerevisiae and shows good correlation with its real impact on free aroma compounds in the final wine.     

Changes in the intracellular concentrations of the adenosine phosphates and nicotinamide adenine dinucleotides ofSaccharomyces cerevisiae during batch fermentation

Significant changes in the intracellular concentrations of adenosine phosphates and nicotinamide adenine dinucleotides were observed during fermentation of grape must by three different strains ofSaccharomyces cerevisiae: S. cerevisiae var.cerevisiae, a typical fermentative yeast strain and two flor-veil-forming strains,S. cerevisiae var.bayanus andS. cerevisiae var.capensis. The intracellular concentration of ATP was always higher inS. cerevisiae var.cerevisiae than in the flor-veil-forming strains. NAD⁺ and NADP⁺ concentrations decreased at faster rates in the flor-veil-forming yeasts than in the other yeast but NADH concentration was the same in all yeasts for the first 10 days of fermentation. NADPH concentration was always lower inS. cerevisiae var.cerevisiae than in the other yeasts and this yeast also showed higher rates of growth and fermentation during the early stages of the fermentation and the presence of non-viable cells at the end of fermentation. In contrast, the flor-veil-forming strains maintained growth and fermentation capabilities for a relatively long time and viable cells were present throughout the entire fermentation process (31 days).The authors are with the Department of Microbiology, Faculty of Sciences, University of Cordoba, Avda. San Alberto Magno s/n, 14004-Córdoba, Spain     

Combined use of killer biotype and mtDNA-RFLP patterns in a Patagonian wine Saccharomyces cerevisiae diversity study

The aim of this work was to characterize the indigenous wine Saccharomyces cerevisiae diversity within the Argentinean Patagonia. Two cellars with particular enological practices located in different winegrowing areas were selected and 112 indigenous S. cerevisiae isolates were obtained from spontaneous red wine fermentations carried out in them. Thirty-five and 19 patterns were distinguished among the total indigenous isolates using mtDNA-RFLP and killer biotype analysis, respectively. The combination of both typing techniques rendered a higher variability with 42 different patterns, i.e. 42 strains, evidencing a great diversity in S. cerevisiae populations associated with spontaneous red wine fermentations in Northwestern Patagonia. The analysis of the relatedness among strains using Principal Coordinates Analysis from combined data allowed the clustering of the strains into two populations significantly related to their origin fermentations. The combined use of the mtDNA-RFLP analysis together with the killer biotype method proved to be a powerful tool in the fingerprinting of the enological S. cerevisiae strains.     

接种发酵和自然发酵中酿酒酵母菌株多样性比较

[目的]探究自然发酵和接种发酵两种发酵方式,对霞多丽葡萄发酵中酵母菌种多样性和酿酒酵母菌株遗传多样性的影响.[方法]以霞多丽葡萄为原料,分别进行自然发酵和接种不同酿酒酵母菌株(NXU 17-26、UCD522和UCD2610)的发酵,利用26S rDNA D1/D2区序列分析和Interdelta指纹图谱技术分别进行酵...     

Killer toxin producing strains of the yeasts Hanseniaspora uvarum and Pichia kluyveri

By heat treatment killer strains of the type K1 of Saccharomyces cerevisiae that are known to harbour dsRNA plasmids were completely cured, whereas only a small fraction of the clones of the killer type K2 had lost the dsRNA dependent killer character. The K2 killers but not the strains of killer type K1 were easily cured by cycloheximide. Killer strains of Hanseniaspora uvarum were not curable by heat treatment. Curing was successfull with cycloheximide or 5-fluorouracil. Two double-stranded RNA plasmids were detected in the killer strains of H. uvarum. The smaller dsRNA plasmid was absent in the strains that were cured of their killer character by 5-fluorouracil. The killer character of H. uvarum was transferred to S. cerevisiae by spheroplast fusion. The fusion products showing the killer character contained both dsRNA plasmids, obviously the smaller plasmid (M-dsRNA) carries the genes for killer toxin formation. Killer strains of Pichia kluyveri were not curable of their killer character, in these strains no dsRNA plasmids were detected.This paper was kindly supported by a grant from the Deutsche Forschungsgemeinschaft