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.     

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.     

Influence of indigenous yeasts on the fermentation and volatile profile of plum brandies

The aim of this study was to determine the influence of different yeasts isolated from fresh blue plum fruits (Aureobasidium sp.) and spontaneously fermenting plum musts (Kloeckera apiculata and Saccharomyces cerevisiae), as well as commercial wine and distillery strains, on the fermentation and chemical composition of plum brandies. Gas chromatography methods were used to detect major volatile components. The most rapid fermentation occurred in musts inoculated with S. cerevisiae. However, the highest concentration of ethanol was detected in samples after spontaneous fermentation (8.40% v/v). Plum brandies obtained after distillation contained from 66.3 (K. apiculata) up to 74.3% v/v ethanol (spontaneous fermentation). The samples after spontaneous fermentation were distinguished by a high content of acetoin, ethyl acetate and total esters, accompanied by a low level of methanol and fusel alcohols. Non-Saccharomyces yeasts were responsible for higher concentrations of esters and methanol, while S. cerevisiae strains resulted in increased levels of higher alcohols. It was also found that isolated indigenous strains of S. cerevisiae synthesized relatively low amounts of higher alcohols compared to commercial cultures. Samples obtained using the distillery strain of S. cerevisiae received the highest score (18.2) during sensory analysis and were characterized by a well-harmonised taste and aroma.     

Diversity of Saccharomyces strains on grapes and winery surfaces: analysis of their contribution to fermentative flora of Malbec wine from Mendoza (Argentina) during two consecutive years

Spontaneous fermentations are still conducted by several wineries in different regions of Argentina as a common practice. Native Saccharomyces strains associated with winery equipment, grape and spontaneous fermentations of Malbec musts from "Zona Alta del Río Mendoza" region (Argentina) were investigated during 2001 and 2002 in the same cellar. Low occurrence of Saccharomyces on grapes and their limited participation during fermentation were confirmed. Strain sequential substitution during fermentation was observed. Between 30% and 60% of yeast population at the end of fermentation was coming from yeasts already present in the winery. A stable and resident Saccharomyces micro-flora in the winery was confirmed. It exhibited a dynamic behaviour during season and between years. Commercial strains were found during fermentation in different percentages, but their presence on winery equipment was low. The present work represents a first approach to winery yeast and spontaneous fermentation Saccharomyces population dynamics in an important viticultural region from Argentina that has never been characterized before. The results obtained have an important significance for the local industry, showing for the first time the real situation of the microbial ecology of alcoholic fermentation in an industrial winery from Mendoza, Argentina.     

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.     

Contribution by Saccharomyces cerevisiae Yeasts to Fermentation and Flavour Compounds in Wines from cv. Kalecik karasi Grape

The effect of indigenous and commercial S. cerevisiae yeasts on fermentation and flavour compounds of wines was examined in pasteurised grape juice. The flavour compounds were analysed and identified by GC‐FID and GC‐MS, respectively and in general, the amounts of these volatiles were increased by the use of both indigenous and commercial yeasts. The levels of isoamyl alcohol, isoamyl acetate, ethyl octanoate and ethyl deconoate exceeded flavour thresholds. All grape juices were fermented to dryness. Selected yeasts produced higher ethanol concentrations compared to spontaneous fermentations.     

Fermentation kinetics and chemical characterisation of vino tostado,a traditional sweet wine from Galicia (NW Spain)

BACKGROUND: Grapes after harvesting are air dried and pressed in order to concentrate sugars, acids and flavour compounds to produce vino tostado (toasted wine), a wine with intense aroma and flavour notes and high residual sugar concentration. In order to get a better knowledge of the difficulties involved, several fermentations were conducted at 12 and 28 °C using 0, 15 and 30 g hL^?1 ammonium sulfate and 0, 25 and 50 g hL^?1 exogenous commercial yeast (Saccharomyces cerevisiae var. bayanus) to study the kinetics of sugar consumption and ethanol, acetic acid and glycerol production. RESULTS: Fermentation kinetic parameters were calculated and metal concentrations and antioxidant activity were analysed. CONCLUSION: The spontaneous fermentation at 12 °C and all fermentations conducted with the commercial yeast gave vino tostado of adequate quality, while the spontaneous fermentation at 28 °C was sluggish. High‐temperature fermentations led to sweeter wines with higher volumetric productivities, although low‐temperature fermentations produced better wines in terms of higher glycerol and lower acetic acid levels. Fructose was the only sugar to be consumed during spontaneous fermentations, while both glucose and fructose were consumed during fermentations of the inoculated musts, with preference for each monosaccharide depending on temperature. Copyright © 2009 Society of Chemical Industry     

Contribution of wild yeasts to the formation of volatile compounds in inoculated wine fermentations

The aim of this work was to study the contribution of wild yeasts to the volatile composition of wine in inoculated fermentations. To do so, Parellada must, sterilized and inoculated with Saccharomyces cerevisiae strain Na33 (pure inoculated fermentation), inoculated Parellada must (mixed inoculated fermentation) and Parellada must that fermented with its wild yeasts (control fermentation) were used. From the results obtained in the pure inoculated fermentation it can be seen that S. cerevisiae produced appreciable quantities of isoamyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate. However, the wild yeasts also contributed to the synthesis of esters since the total concentration of these substances was higher in the mixed inoculated fermentation than in the pure inoculated fermentation. 2-Phenylethyl acetate was only synthesized by wild yeasts when they did not compete with S. cerevisiae. The concentration of total alcohols was similar in the three samples; the important production of isobutanol and 2-phenylethanol in the control fermentation is noteworthy. As regards the acids, the greatest concentration corresponded to the mixed inoculated fermentation. The wild yeasts contributed to the synthesis of these compounds to a significant extent and S. cerevisiae synthesized appreciable amounts of short-chain fatty acids.     

Indigenous Saccharomyces cerevisiae strains and their influence on the quality of Cataratto, Inzolia and Grillo white wines

The present paper deals with three new strains of Saccharomyces cerevisiae, isolated in old wineries of Sicily, which were microbiologically and molecularly characterized and tested for their ability to produce white wines. Examined in terms of their growth pattern, fermentation vigour, sulphite tolerance, fermentative power, spore formation, and production of acetic acid, hydrogen sulphide and phenolic off-flavours, the strains were utilized as starters in experimental fermentations of musts obtained from the cultivars Inzolia, Grillo and Catarratto. Further, the three musts were also fermented using two commercial S. cerevisiae strains. The quality of the wines produced was confirmed by their principal oenochemical parameters, by sensory analysis and qualitative and quantitative determination of the volatile aroma constituents. All the data were statistically elaborated. Interestingly, the new selected yeasts were able to increase the pear notes (Z)-ethyl-4-decenoate, (E)-ethyl-3-decenoate, and (Z)-ethyl-3-decenoate which are fundamental for the aroma of these Sicilian wines. From our results, the new yeast strains were found to produce white wines of a quality which was not inferior to those obtainable with the best commercial strains selected in other geographical areas, but also with a distinctive aromatic profile.     

The influence of pre‐fermentative practices on the dominance of inoculated yeast starter under industrial conditions

The influence of pre‐fermentative practices on the growth dynamics of a ‘natural’ starter culture with specific phenotype (H₂S^?) concurrently with wild yeast populations was evaluated under winery conditions. Different clarification procedures and added SO₂ strongly influenced species and cell numbers isolable at the pre‐fermentation stage. Independent treatments of must with sulphite addition or vacuum‐filtering clarification caused a 30‐fold reduction in viable cells. Clarification procedures, enhanced by the selective effect of SO₂ addition, induced the appearance of Saccharomyces cerevisiae ‘wild’ yeasts. Correct application of the inoculum generally guarantees the dominance of fermentation by starter cultures. However, inoculated fermentations using unclarified white and red musts exhibited a consistent presence and persistence of non‐Saccharomyces and/or Saccharomyces ‘wild’ yeasts during fermentation. The extent and composition of the initial wild microflora at the start of fermentation may affect the presence and persistence of wild Saccharomyces and non‐Saccharomyces yeasts during guided fermentations under commercial conditions. The above findings confirm the results of previous works carried out at laboratory‐ or pilot‐scale level. Furthermore, they suggest a clear correlation between the modality of pre‐fermentative practices and the presence and persistence of ‘wild’ yeasts during fermentation. © 2002 Society of Chemical Industry     

Saccharomyces cerevisiae associated with the spontaneous fermentation of tequila agave juice

Saccharomyces cerevisiae dominates the spontaneous fermentation of blue agave juice. Because of the batch heterogeneity, the aim of this work was to determine the strain diversity of S. cerevisiae among fermentations. During January and February 2015, agave juice was sampled in triplicate from four sampling points at a tequila distillery. The heterogeneity of yeast strains and the production of carbon dioxide were assessed during fermentation, whereas the amount of ethanol produced was measured at the end of the process. The fermentation cycle times varied widely (9 to 25 days), as did fermentation efficiency (2.5–45.5%). Yeast isolates were identified at the species level by ITS‐5.8S rRNA restriction fragment length polymorphism and differentiated at the strain level by random amplified polymorphic DNA. A total of 199 isolates were obtained and identified as S. cerevisiae, showing 69 different random amplified polymorphic DNA profiles. There was no clear dominance of any strain during fermentation. However, two strains (P1 and P2) were detected in all fermentation samples, suggesting their residency in the distillery, despite the deep‐cleaning applied to the tanks after each fermentation batch. According to the RAPD profiles, the number of strains isolated from fermentation samples increased from 17 in January to 25 in February. © 2018 The Institute of Brewing & Distilling     

Yeasts isolated from New Zealand vineyards and wineries

Background and Aims: The yeast flora from a range of New Zealand commercial wineries was surveyed to estimate the incidence of yeast species in grape juice. Methods and Results: Molecular analysis of the internal transcribed spacer region was performed for 1279 yeast colonies isolated from 17 different fresh grape juices sampled in eight New Zealand wineries between 2003 and 2009. The 17 juices contained at least 25 different species of yeast from nine genera. Microsatellite fingerprinting of Saccharomyces cerevisiae showed that some strains were identical to known commercial yeast varieties, but we also found evidence for local populations of S. cerevisiae common to individual wineries or regions. Five genotypes from Central Otago, New Zealand, were very closely related to a single sequenced strain derived from Chile, which in turn is related to European wine isolates. Conclusions: The yeast flora found in New Zealand grape juices is broadly similar to that found in wineries elsewhere around the world. Genotyping of S. cerevisiae suggests recent dispersal of both commercial and non-commercial yeast strains from Europe to New Zealand. Significance of the Study: These data are consistent with two human-mediated modes for the international dispersal of S. cerevisiae: one via the escape of strains traded commercially, and another via long distance dispersal of non-commercial strains.     

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.     

Electrospray Ionization Mass Spectrometry Characterization of Musts and Alembic Brazilian Cachaças Using Selected Yeast Strains

The choice of fermentation system during cachaça production can greatly influence the chemical composition of the beverage. In this work, Saccharomyces cerevisiae strains were selected based on fermentative properties and used as starters to produce alembic cachaça. In distillery scale production, the selected yeast strains exhibited greater adaptiblity to the fermentation environment and hence remained predominant throughout the process. Electrospray ionization mass spectrometry in the negative ion mode revealed that most of the compounds present in the must are different from those formed in the distillate for both cachaças obtained from spontaneous and selected strains. However, beverages produced using selected strains showed greater similarity in chemical profiles than those produced from spontaneous strain fermentation. Moreover, a smaller number of ions were detected in beverages produced by selected strain than from spontaneous strain fermentation. Our results indicate that the selected S. cerevisiae strains evaluated are able to produce cachaças less subject to variation in chemical composition. This could potentially improve brand consistency and thus commercial viability, particularly in the international market.     

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.     

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。     

Fermentation Characteristics and Aromatic Profile of Plum Wines Produced with Indigenous Microbiota and Pure Cultures of Selected Yeast

The aim of this study was to assess and compare fermentation characteristics and aromatic profile of plum wines produced with indigenous microbiota and pure cultures of different selected yeast. Experiments were carried out with plum (Prunus domestica L.) varieties of different fruit ripening times (?a?anska rana, ?a?anska lepotica, and Po?ega?a). Wine fermentations were conducted by the activity of indigenous microbiota, commercially available Saccharomyces cerevisiae and Saccharomyces bayanus yeast strains and joint activity of Schizosaccharomyces pombe and S. cerevisiae (sequential inoculation). Statistically significant differences in fermentative characteristics and the content of certain volatile compounds were observed as a result of metabolic activity of various indigenous and/or selected yeasts during fermentation of plum pomace. Minimal duration of fermentation (4 to 5 d) and fastest ethanol production rate (from 12.3 to 15.5 g/L/d) were the characteristics of the studied S. cerevisiae strains. Isobutanol, 3‐methyl‐1‐butanol, 1‐heptanol, and 1‐octanol were the most prevalent higher alcohols in the tested plum wine samples. The predominant ester in plum wines was ethyl acetate, ethyl lactate, amyl acetate, isoamyl acetate, and ethyl palmitate, esters responsible for the floral and fruity olfactory tones, were also present in large amounts. Also, the use of S. cerevisiae strains resulted in the production of plum wines with better sensory characteristics than ones produced with other investigated yeasts. Obtained results are significant since there is limited data on the compounds responsible for the unique flavor of plum wine, as well as on the impact of different yeast starter cultures application on the overall quality of fruit wines.     

Saccharomyces cerevisiae and Hanseniaspora uvarum mixed starter cultures: Influence of microbial/physical interactions on wine characteristics

The growing trend in the wine industry is the revaluation of the role of non-Saccharomyces yeasts, promoting the use of these yeasts in association with Saccharomyces cerevisiae. Non-Saccharomyces yeasts contribute to improve wine complexity and organoleptic composition. However, the use of mixed starters needs to better understand the effect of the interaction between these species during alcoholic fermentation. The aim of this study is to evaluate the influence of mixed starter cultures, composed by combination of different S. cerevisiae and Hanseniaspora uvarum strains, on wine characteristics and to investigate the role of cell-to-cell contact on the metabolites produced during alcoholic fermentation. In the first step, three H. uvarum and two S. cerevisiae strains, previously selected, were tested during mixed fermentations in natural red grape must in order to evaluate yeast population dynamics during inoculated fermentation and influence of mixed starter cultures on wine quality. One selected mixed starter was tested in a double-compartment fermentor in order to compare mixed inoculations of S. cerevisiae/H. uvarum with and without physical separation. Our results revealed that physical contact between S. cerevisiae and H. uvarum affected the viability of H. uvarum strain, influencing also the metabolic behaviour of the strains. Although different researches are available on the role of cell-to-cell contact-mediated interactions on cell viability of the strains included in the mixed starter, to our knowledge, very few studies have evaluated the influence of cell-to-cell contact on the chemical characteristics of wine.     

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     

Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance

Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus‐like strains. Lager yeasts are particularly adapted to low‐temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make‐up of lager yeast spore clones, we introduced molecular markers to analyse mating‐type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18°Plato at 18–25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. Copyright © 2012 John Wiley & Sons, Ltd.