Genetic diversity of wine Saccharomyces cerevisiae strains in an experimental winery from Galicia (NW Spain)

Genetic diversity of wine Saccharomyces cerevisiae strains involved in spontaneous fermentations was studied by analysis of mitochondrial DNA restriction patterns. Yeasts were isolated at different stages of fermentations with must from three different white grapevine varieties, Albariño, Godello and Treixadura, which are autochthonous from Galicia. Nineteen different patterns out of a total of 446 strains analysed were identified, but only a few of them appeared at high frequency and therefore were able to lead the fermentation process. Some strains were common to all fermentations; however, most of them were a minority being only found at low frequency for one or two specific grape varieties. The dominant strain was different for each variety except in one case, suggesting that some strains are better adapted to certain must conditions.     

Effect of must characteristics on the diversity of Saccharomyces strains and their prevalence in spontaneous fermentations

Aims: The aim of this study was to investigate whether grapevine variety and must characteristics influence the diversity of Saccharomyces strains and their prevalence during spontaneous fermentations. Methods and results: Musts from different grapevine varieties, all of them autochthonous from Galicia, were used to perform spontaneous fermentations. Yeasts were isolated from the must and at the beginning, in the middle and at the end of fermentations. Those yeasts identified as Saccharomyces were characterized at the strain level by analysis of mtDNA‐RFLP. The results showed a low diversity of Saccharomyces strains, which was related to must sugar content and total acidity. Moreover, from a total of 44 different Saccharomyces strains, only eleven of them appeared at frequencies higher than 20% and were able to lead fermentations. A significant correlation between yeast strains and must acidity was observed, with the predominance of certain strains at high acidity values. Conclusions: Must characteristics, such as sugar content and acidity, influence the Saccharomyces strains diversity and the leader strains during fermentation. Significance and Impact of the Study: These results showed the adaptation of certain Saccharomyces strains to must with specific characteristics; this may be considered by winemakers for yeast inocula selection. Our findings have special relevance because this is the first study carried out in Galicia dealing with the influence of must properties on yeast strains that control fermentations.     

Genomic characterization and selection of wine yeast to conduct industrial fermentations of a white wine produced in a SW Spain winery

Aims: To analyse the diversity of wild yeast in spontaneous fermentations of a white wine and to select the most suitable autochthonous starter yeasts. The selected yeasts would be used for inoculation of industrial fermentations in several years. Methods and Results: Yeasts were characterized by applying electrophoretic karyotyping. This technique was chosen because it can reveal the large‐scale mutations in the yeast genome induced by gross chromosomal rearrangements. This type of mutation is considered one of the main forces behind the rapid evolution of industrial yeasts. A heterogeneous population of yeast strains was observed in the spontaneous fermentations during two consecutive years. Four of the most abundant strains were isolated and tested for microbiological features of industrial importance. The selected autochthonous strains were used as starter yeasts for the following 7 years. In the majority of these experiences, we obtained homogeneous yeast populations, in which the karyotype of one of the inoculated strains – karyotype V – emerged as clearly dominant. Conclusions: The inoculation of the selected strain with karyotype V and a proper handling of the inoculum scaling‐up process led to the substitution of the spontaneous fermentations by controlled fermentations producing a highly satisfactory final product. Significance and Impact of the Study: We monitored the wine yeast population of an industrial system for a total of 9 years. Our work is one of the first examples made at industrial scale showing how molecular techniques can be successfully applied to improve the efficiency of the winemaking process.     

Metabolic diversity of Saccharomyces cerevisiae strains from spontaneously fermented grape musts

One hundred and fifteen Saccharomyces cerevisiae strains from Aglianico del Vulture, a red wine produced in Southern Italy, were characterized for the production of some secondary compounds involved in the aroma and taste of alcoholic beverages. The strains exhibited a uniform behaviour in the production levels of n-propanol, active amyl alcohol and ethyl acetate, whereas isobutanol, isoamyl alcohol and acetaldehyde were formed with a wide variability. Only five strains produced wines close to the reference Aglianico del Vulture wine for the traits considered. Of these, two strains were selected, underwent to tetrad analysis and the single spore cultures were tested in grape must fermentation. The progeny of one strain showed a significant metabolic variability, confirming the necessity to test starter cultures for the segregation of traits of technological interest. Our findings suggest the selection of specific strains for specific fermentations as a function of the vine variety characteristics in order to take the major advantage from the combination grape must/S. cerevisiae strain.     

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.     

Biodiversity of wild strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> as tool to complement and optimize wine quality

The principal agent in winemaking is the yeast Saccharomyces cerevisiae, which is characterized by a significant strain biodiversity. Here we report the characterization of 80 wild S. cerevisiae strains, isolated from grapes of different varieties in southern Italy, for genetic and technological variability. By PCR amplification with M13 primer a significant polymorphism was recorded and 12 different biotypes were identified among the strains. The specific strain-pattern could be used to follow the dynamics of different biotypes during the fermentation process. The analysis of experimental wines obtained by inoculated fermentations with the 80 strains showed significant differences among the wines. The level of each compound was a function of the strain performing the fermentative process. The main variables for the strain differentiation were the production of acetaldehyde and acetic acid, which ranged from 53 to 282 mg/l and from 0.20 to 1.88 g/l, respectively. Selected strains were tested in fermentation with two different grape musts, yielding experimental wines differing in the levels of secondary compounds and polyphenol content, in function of the interaction “grape must composition/yeast strain”. This finding has an applicative value for the potentiality of utilizing the resource of strain variability as a tool to individuate suitable starter cultures, which are able to complement and optimize grape quality.     

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.     

Selection of an autochthonous Saccharomyces strain starter for alcoholic fermentation of Sherry base wines

Several indigenous Saccharomyces strains from musts were isolated in the Jerez de la Frontera region, at the end of spontaneous fermentation, in order to select the most suitable autochthonous yeast starter, during the 2007 vintage. Five strains were chosen for their oenological abilities and fermentative kinetics to elaborate a Sherry base wine. The selected autochthonous strains were characterized by molecular methods: electrophoretic karyotype and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and by physiological parameters: fermentative power, ethanol production, sugar consumption, acidity and volatile compound production, sensory quality, killer phenotype, desiccation, and sulphur dioxide tolerance. Laboratory- and pilot-scale fermentations were conducted with those autochthonous strains. One of them, named J4, was finally selected over all others for industrial fermentations. The J4 strain, which possesses exceptional fermentative properties and oenological qualities, prevails in industrial fermentations, and becomes the principal biological agent responsible for winemaking. Sherry base wine, industrially manufactured by means of the J4 strain, was analyzed, yielding, together with its sensory qualities, final average values of 0.9 g/l sugar content, 13.4 % (v/v) ethanol content and 0.26 g/l volatile acidity content; apart from a high acetaldehyde production, responsible for the distinctive aroma of “Fino”. This base wine was selected for “Fino” Sherry elaboration and so it was fortified; it is at present being subjected to biological aging by the so-called “flor” yeasts. The “flor” velum formed so far is very high quality. To the best of our knowledge, this is the first study covering from laboratory to industrial scale of characterization and selection of autochthonous starter intended for alcoholic fermentation in Sherry base wines. Since the 2010 vintage, the indigenous J4 strain is employed to industrially manufacture a homogeneous, exceptional Sherry base wine for “Fino” Sherry production.     

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...     

Wine fermentations using membrane processed hydrolyzed whey

A process for the utilization of modified whey syrups in wine alcohol fermentations is described. Palatable wines containing 10–12.5% alcohol were produced when various strains of S. cerevisiae var. ellipsoideus were fermented with hydrolyzed whey permeate syrups and grape juice concentrates. Experimental control of final alcohol and residual sugar levels in the wines by various chaptalization techniques is discussed. The possibility of utilizing this process in areas where both whey and grapes are abundant is mentioned.     

Selective effects of sulfur dioxide and yeast starter culture addition on indigenous yeast populations and sensory characteristics of wine

Riesling musts, with or without sulfur dioxide added, were fermented either with or without the addition of yeast. Uninoculated fermentations took much longer to finish than inoculated musts. There were no significant differences in growth of non- Saccharomyces yeasts in uninoculated musts with less than 50 mg l⁻¹ SO₂ added. The starter culture was completely dominant over indigenous Saccharomyces cerevisiae and strongly inhibitory to non- Saccharomyces . Alcohol and acetaldehyde were greater in the inoculated treatments ; titratable acidity and acetic acid were greater in the uninoculated fermentations. There were no statistically significant differences among any treatments in final pH, ammonia content, or colour (A₄₂₀). Uninoculated fermentations had higher sensory scores ( P > 0·95) for 'spicy', 'apple', 'melon', 'pear', and 'H₂S', while inoculated wines had higher scores ( P > 0·95) for 'paper', 'oxidized', and 'sweaty'. Sulfite treatment produced an assortment of significant sensory differences in the finished uninoculated wines, but in inoculated wines the additions of SO₂ to the must had no significant effect on indigenous yeast populations or on flavour.     

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.     

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.     

Yeast strain effect on the concentration of major volatile compounds and sensory profile of wines from Vitis vinifera var. Treixadura

The fermentative ability of five Saccharomyces cerevisiae strains and their influence on the aroma and sensory properties of wine from Treixadura were evaluated to determine the most suitable yeast that produces a high quality wine from this grapevine variety. The results indicated that all strains, except T2, were able to lead the vinification process and have good fermentative powers. The chemical composition of wines obtained with resident cellar yeasts consisted of a significant amount of glycerol, a compound that contributes to the structure and smoothness in taste of the wine. In addition, these strains were good producers of acetates, ethyl esters and fatty acids, compounds that positively influence wine aroma. Compounds with a direct contribution to the aroma of Treixadura wines (Odour Activity Value >1) included 2-methyl-1-butanol, 3-methyl-1-butanol, 2-phenylethanol, isoamyl and ethyl acetate and the ethyl esters, ethyl butyrate, ethyl hexanoate, ethyl octanoate and ethyl decanoate. Sensory analysis supported the fact that cellar strains produced more fruity and floral wines than commercial or spontaneous fermentations. We conclude that resident cellar yeasts enhanced sensory quality of wines from Treixadura.     

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.     

Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.     

Experiences with the use of a starter culture in the fermentation of maize for ‘kenkey’ production in Ghana

Controlled fermentation of maize was carried out using six strains of Lactobacillus fermentum and one strain of yeast, Saccharomyces cerevisiae, isolated from traditionally fermented maize dough as starter cultures for inoculum enrichement. The fermentations were monitored by pH, acidity, microbiological analysis and taste panel evaluation of two products, kenkey and koko, prepared from the fermented doughs. The strains of L. fermentum used as starter culture dominated the microflora during fermentation and in most inoculated doughs the required pH was attained by 24 h instead of 48 h of dough fermentation. Higher contents of lactic acid bacteria and yeasts were observed in inoculated doughs at the initial stages of fermentation but the spontaneously fermented doughs attained similar lactic acid bacteria and yeasts counts by 24 h of dough fermentation. The organoleptic quality of kenkey and koko prepared from doughs fermented with starter culture for 48 h was not significantly different from the traditional products. Kenkey prepared from doughs fermented for 24 h with starter culture were found to be unacceptable by the taste panel although similarly produced koko was acceptable.The authors are with the Food Research Institute, Council for Scientific and Industrial Research, P.O Box M 20. Accra, Ghana.     

Yeast species associated with spontaneous wine fermentation of Cabernet Sauvignon from Ningxia,China

The eastern base of the Helan Mountains in Ningxia is a fast developing wine production area in China. Of urgent necessity to the Ningxia wine industry is to be able to produce wines with typical regional characteristics. It is well known that autochthonous yeast species and strains play an important role in introducing local character or terroir into the winemaking practice. The aim of this study was to investigate indigenous yeast species diversity and preselect desirable S. cerevisiae strains in the Ningxia region. Four hundred wine-related yeast colonies were isolated from Cabernet Sauvignon musts in three vineyards at the beginning, middle and final stages of spontaneous fermentations. Yeast species were first classified according to colony morphologies on Wallerstein Laboratory Nutrient Agar (WL) and confirmed by sequencing of the D1/D2 domain of the 26S rRNA gene. Nine unique colony morphology types were profiled on WL agar and three new types were found. After sequence analysis of representative colonies from each WL group, nine yeast species were identified, namely H. uvarum, H. occidentalis, M. pulcherrima, C. zemplinina, H. vineae, I. orientalis, Z. bailii, P. kluyveri and S. cerevisiae. The non-Saccharomyces yeasts appeared mainly in the early stage of the fermentation while H. uvarum, I. orientalis and P. kluyveri were able to remain in the final stage. Fourty-six S. cerevisiae isolates were preselected according to physiological characteristics and twelve strains with valuable fermentation properties were obtained.     

Impact of different malolactic fermentation inoculation scenarios on Riesling wine aroma

During malolactic fermentation (MLF), lactic acid bacteria influence wine aroma and flavour by the production of volatile metabolites and the modification of aroma compounds derived from grapes and yeasts. The present study investigated the impact of different MLF inoculation strategies with two different Oenococcus oeni strains on cool climate Riesling wines and the volatile wine aroma profile. Four different timings were chosen for inoculation with bacteria to conduct MLF in a Riesling must/wine with a high acidity (pH 2.9–3.1). Treatments with simultaneous inoculation showed a reduced total fermentation time (alcoholic and malolactic) compared to the sequential inoculations. No negative impact of simultaneous alcoholic and malolactic fermentation on fermentation success and on the final wine volatile aroma composition was observed. Compared to sequential inoculation, wines with co-inoculation tended to have higher concentrations of ethyl and acetate esters, including acetic acid phenylethylester, acetic acid 3-methylbutylester, butyric acid ethylester, lactic acid ethylester and succinic acid diethylester. Results of this study provide some alternatives to diversify the number of wine styles by safely conducting MLF in low-pH, cool-climate white musts with potential high alcohol content.