Comparison between Yeasts from Grape and Agave Musts for Traits of Technological interest

Summary In Mexico there are different alcoholic beverages produced from agave juices from different agave plants, which are cooked, fermented and distilled. For tequila production only Agave tequilana is allowed. In this study we compared yeast strains of different species from different origin (agave and grape juice) for parameters of technological interest, such as SO₂ and copper resistance, ethanol tolerance and enzymatic activities. All agave strains were found to be more resistant to SO₂ and agave non-Saccharomyces yeasts were more tolerant to ethanol, whereas grape strains exhibited positive results for β-glucosidase and β-xylosidase activities. As regards fermentations of Agave tequilana juice with ethanol added at different concentrations, only agave Saccharomyces strains were more tolerant to ethanol than grape strains.     

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.     

Kinetics of growth and sugar consumption in yeasts

An overview is presented of the steady- and transient state kinetics of growth and formation of metabolic byproducts in yeasts.Saccharomyces cerevisiae is strongly inclined to perform alcoholic fermentation. Even under fully aerobic conditions, ethanol is produced by this yeast when sugars are present in excess. This so-called Crabtree effect probably results from a multiplicity of factors, including the mode of sugar transport and the regulation of enzyme activities involved in respiration and alcoholic fermentation. The Crabtree effect inS. cerevisiae is not caused by an intrinsic inability to adjust its respiratory activity to high glycolytic fluxes. Under certain cultivation conditions, for example during growth in the presence of weak organic acids, very high respiration rates can be achieved by this yeast.S. cerevisiae is an exceptional yeast since, in contrast to most other species that are able to perform alcoholic fermentation, it can grow under strictly anaerobic conditions.Non-Saccharomyces yeasts require a growth-limiting supply of oxygen (i.e. oxygen-limited growth conditions) to trigger alcoholic fermentation. However, complete absence of oxygen results in cessation of growth and therefore, ultimately, of alcoholic fermentation. Since it is very difficult to reproducibly achieve the right oxygen dosage in large-scale fermentations, non-Saccharomyces yeasts are therefore not suitable for large-scale alcoholic fermentation of sugar-containing waste streams. In these yeasts, alcoholic fermentation is also dependent on the type of sugar. For example, the facultatively fermentative yeastCandida utilis does not ferment maltose, not even under oxygen-limited growth conditions, although this disaccharide supports rapid oxidative growth.     

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.     

Experimental approach for target selection of wild wine yeasts from spontaneous fermentation of “Inzolia” grapes

The aim of this research was the study of indigenous yeasts isolated from spontaneous fermentation of Inzolia grapes, one of the most widespread native white grapes in Sicily (Italy). The use of selective medium for the isolation and the screening for sulphur dioxide tolerance were useful for the first selection among 640 isolates. The yeasts characterized by high SO₂ tolerance were identified at species level by restriction analysis of ITS region; although the majority of isolates were identified as S. cerevisiae, some non-Saccharomyces yeasts were found. Forty-seven selected yeasts, both S. cerevisiae and non-Saccharomyces yeasts, were characterized for genetic and technological diversity. The genetic polymorphism was evaluated by RAPD-PCR analysis, whereas the technological diversity was analyzed by determining the main secondary compounds in the experimental wines obtained by inoculating these yeasts. Both the molecular and metabolic profiles of selected yeasts were able to clearly discriminate S. cerevisiae from non-Saccharomyces yeasts. This research was useful for the constitution of a collection of selected indigenous yeast strains, including S. cerevisiae and non-Saccharomyces species possessing interesting enological traits. This collection represents a source of wild yeasts, among of which it is possible to select indigenous starters able to maintain the specific organoleptic characteristics of Inzolia wine.     

Interactions between Kluyveromyces marxianus and Saccharomyces cerevisiae in tequila must type medium fermentation

Traditional tequila fermentation is a complex microbial process performed by different indigenous yeast species. Usually, they are classified in two families: Saccharomyces and Non-Saccharomyces species. Using mixed starter cultures of several yeasts genera and species is nowadays considered to be beneficial to enhance the sensorial characteristics of the final products (taste, odor). However, microbial interactions occurring in such fermentations need to be better understood to improve the process. In this work, we focussed on a Saccharomyces cerevisiae/Kluyveromyces marxianus yeast couple. Indirect interactions due to excreted metabolites, thanks to the use of a specific membrane bioreactor, and direct interaction due to cell-to-cell contact have been explored. Comparison of pure and mixed cultures was done in each case. Mixed cultures in direct contact showed that both yeast were affected but Saccharomyces rapidly dominated the cultures whereas Kluyveromyces almost disappeared. In mixed cultures with indirect contact the growth of Kluyveromyces was decreased compared to its pure culture but its concentration could be maintained whereas the growth of Saccharomyces was enhanced. The loss of viability of Kluyveromyces could not be attributed only to ethanol. The sugar consumption and ethanol production in both cases were similar. Thus the interaction phenomena between the two yeasts are different in direct and indirect contact, Kluyveromyces being always much more affected than Saccharomyces.     

A comparison of the killer character in different yeasts and its classification

The interactions between 20 killer yeasts of various genera and species were examined. Ten distinct groups were recognised with respect to killer activity and 10 distinct groups with respect to resistance to killer action. Using both killing and resistance phenotypes, 13 classes of killer yeast were found. With the exception of Torulopsis glabrata NCYC 388, non-Saccharomyces strains of yeast were not killed by a member of the genus Saccharomyces.The killer character of the 3 killing groups of Saccharomyces identified could be cured by treatment with cycloheximide or incubation at elevated temperature and the effectiveness of these procedures was indicative of the category of killer yeast examined. Killer yeasts not belonging to the genus Saccharomyces could not be cured of their activity. Double-stranded ribonucleic acids were extracted only from Saccharomyces spp. and the molecular weights of the species present were a function of the killer class to which a strain belonged.By an analysis of the effects of proteolytic enzymes, temperature and pH on killer activity and by gel chromatography of crude preparations of killer factors, the toxins of different killer classes were shown to be biochemically distinct. However all toxins had certain properties in common consistent with there being a protein component essential to killer action.     

Utilization of organic nitrogen compounds by yeasts of the genusSaccharomyces

Yeasts belonging to 27 species ofSaccharomyces were tested for their ability to used-amino acids,l-lysine and various amines and amides as nitrogen source. Most yeasts capable of growing onl-lysine or amines could utilized-amino acids.Saccharomyces (sensu strictu) have a very limited ability to grow on the organic nitrogen compounds tested. However, there is no obvious relationship between the utilization of these compounds and the proposed divisions of the genusSaccharomyces.Issued as N.R.C. No. 9845.     

Identification of yeasts within Saccharomyces sensu stricto complex by PCR-fingerprinting

Biological relatedness makes species characterization of the industrially important Saccharomyces sensu stricto complex difficult. In this paper we present a set of PCR-fingerprinting markers based in Single Primer Amplification Reactions (SPAR) that, together with PCR-ribotyping and single gene RFLP analysis, can effectively identify individual species and fully characterize the hybrid nature of industrial isolates. With those markers, all six yeast species of the sensu stricto complex could be discriminated and we also identified errors in the previous taxonomic characterization of certain wine yeasts. The unique patterns generated by the SPAR markers could be useful in monitoring yeast populations during industrial fermentation processes and can be used to detect the appearance of yeast hybrids in these environments.     

The growth kinetics and fermentation behaviour of some non-Saccharomyces yeasts associated with wine-making

Summary The growth kinetics and fermentation behaviour of five non-Saccharomyces yeast species associated with wine-making were evaluated.The results showed that the Candida stellata and Torulspora delbrueckii species are interesting for biotechnological applications in wine-making, whereas small-size apiculate yeasts could be profitably used in the production of wine for vinegar manufacture.     

Evolution of ecological dominance of yeast species in high‐sugar environments

In budding yeasts, fermentation in the presence of oxygen evolved around the time of a whole genome duplication (WGD) and is thought to confer dominance in high‐sugar environments because ethanol is toxic to many species. Although there are many fermentative yeast species, only Saccharomyces cerevisiae consistently dominates wine fermentations. In this study, we use coculture experiments and intrinsic growth rate assays to examine the relative fitness of non‐WGD and WGD yeast species across environments to assess when S. cerevisiae’s ability to dominate high‐sugar environments arose. We show that S. cerevisiae dominates nearly all other non‐WGD and WGD species except for its sibling species S. paradoxus in both grape juice and a high‐sugar rich medium. Of the species we tested, S. cerevisiae and S. paradoxus have evolved the highest ethanol tolerance and intrinsic growth rate in grape juice. However, the ability of S. cerevisiae and S. paradoxus to dominate certain species depends on the temperature and the type of high‐sugar environment. Our results indicate that dominance of high‐sugar environments evolved much more recently than the WGD, most likely just prior to or during the differentiation of Saccharomyces species, and that evolution of multiple traits contributes to S. cerevisiae's ability to dominate wine fermentations.     

Yeast Diversity and Persistence in Botrytis-Affected Wine Fermentations

Culture-dependent and -independent methods were used to examine the yeast diversity present in botrytis-affected (“botrytized”) wine fermentations carried out at high (~30°C) and ambient (~20°C) temperatures. Fermentations at both temperatures possessed similar populations of Saccharomyces, Hanseniaspora, Pichia, Metschnikowia, Kluyveromyces, and Candida species. However, higher populations of non-Saccharomyces yeasts persisted in ambient-temperature fermentations, with Candida and, to a lesser extent, Kluyveromyces species remaining long after the fermentation was dominated by Saccharomyces. In general, denaturing gradient gel electrophoresis profiles of yeast ribosomal DNA or rRNA amplified from the fermentation samples correlated well with the plating data. The direct molecular methods also revealed a Hanseniaspora osmophila population not identified in the plating analysis. rRNA analysis also indicated a large population (>10⁶ cells per ml) of a nonculturable Candida strain in the high-temperature fermentation. Monoculture analysis of the Candida isolate indicated an extreme fructophilic phenotype and correlated with an increased glucose/fructose ratio in fermentations containing higher populations of Candida. Analysis of wine fermentation microbial ecology by using both culture-dependent and -independent methods reveals the complexity of yeast interactions enriched during spontaneous fermentations.     

刺梨自然发酵过程中非酿酒酵母多样性分析

【目的】分析刺梨果实自然发酵过程中非酿酒酵母菌群特征,为筛选优质刺梨非酿酒酵母提供参考。【方法】基于Illumina MiSeq高通量测序技术和WL营养琼脂鉴定培养基纯种分离技术,分析刺梨果实自然发酵1 d (F1)、3 d (F3)、5 d (F5)和15 d (F15) 4个阶段及YPD培养基富集培养样本中非酿酒酵母种群组成和多样性。【结果】高通量测序分析结果共获得182个OTUs (operational taxonomic units,OTUs),归属于81个属107个种;物种多样性分析结果表明,刺梨果实自然发酵前期,优势非酿酒酵母为汉逊酵母(Hanseniasporasp.)和伯顿丝孢毕赤酵母(Hyphopichiaburtonii),二者在样本F1中分别占42.59%和26.85%;随着自然发酵的不断进行,二者的比例逐渐降低,在第15天(F15),Hanseniaspora sp.和H. burtonii比例降低至7.73%和0.52%。相反,Pichia sporocuriosa和未培养的酵母,随着自然发酵不断进行所占比例逐渐增大,分别由F1中的0.23%和0.33%增至F15中的37.26%和32.62%。此外,采用WL营养琼脂鉴定培养基纯种分离和鉴定技术,从刺梨上分离到Hanseniasporasp.、H.burtonii、克鲁维毕赤酵母(Pichia kluyveri)、P. sporocuriosa和异常威克汉姆酵母(Wickerhamomyces anomalus) 5种类型的可培养非酿酒酵母。【结论】刺梨果实上存在着丰富的非酿酒酵母菌资源,研究刺梨自然发酵过程中非酿酒酵母多样性,为酵母资源开发和利用奠定基础。     

The Finding of the Yeast Species Saccharomyces bayanusin Far East Asia

The genetic analyses of nine Far East Asian Saccharomyces isolates allowed us to identify three species (S. cerevisiae, S. paradoxus, and S. bayanus). The occurrence of the last species in Far East Asia was shown for the first time. A new methodology for the molecular genetic differentiation of Saccharomyces sensu stricto species is described. The ecogeographical distribution of Saccharomyces yeasts is discussed.     

Fermentation of Agave tequilana juice by Kloeckera africana: influence of amino-acid supplementations

This study aimed to improve the fermentation efficiency of Kloeckera africana K1, in tequila fermentations. We investigated organic and inorganic nitrogen source requirements in continuous K. africana fermentations fed with Agave tequilana juice. The addition of a mixture of 20 amino-acids greatly improved the fermentation efficiency of this yeast, increasing the consumption of reducing sugars and production of ethanol, compared with fermentations supplemented with ammonium sulfate. The preference of K. africana for each of the 20 amino-acids was further determined in batch fermentations and we found that asparagine supplementation increased K. africana biomass production, reducing sugar consumption and ethanol production (by 30, 36.7 and 45%, respectively) over fermentations supplemented with ammonium sulfate. Therefore, asparagine appears to overcome K. africana nutritional limitation in Agave juice. Surprisingly, K. africana produced a high concentration of ethanol. This contrasts to poor ethanol productivities reported for other non-Saccharomyces yeasts indicating a relatively high ethanol tolerance for the K. africana K1 strain. Kloeckera spp. strains are known to synthesize a wide variety of volatile compounds and we have shown that amino-acid supplements influenced the synthesis by K. africana of important metabolites involved in the bouquet of tequila. The findings of this study have revealed important nutritional limitations of non-Saccharomyces yeasts fermenting Agave tequilana juice, and have highlighted the potential of K. africana in tequila production processes.     

The importance of aeration strategy in fuel alcohol fermentations contaminated with Dekkera/Brettanomyces yeasts

Whole corn mash fermentations infected with industrially-isolated Brettanomyces yeasts were not affected even when viable Brettanomyces yeasts out-numbered Saccharomyces yeasts tenfold at the onset of fermentation. Therefore, aeration, a parameter that is pivotal to the physiology of Dekkera/Brettanomyces yeasts, was investigated in mixed culture fermentations. Results suggest that aeration strategy plays a significant role in Dekkera/Brettanomyces-mediated inhibition of fuel alcohol fermentations. Although growth of Saccharomyces cerevisiae was not impeded, mixed culture fermentations aerated at rates of ≥20 ml air l⁻¹ mash min⁻¹ showed decreased ethanol yields and an accumulation of acetic acid. The importance of aeration was examined further in combination with organic acid(s). Growth of Saccharomyces occurred more rapidly than growth of Brettanomyces yeasts in all conditions. The combination of 0.075% (w/v) acetic acid and contamination with Brettanomyces TK 1404W did not negatively impact the final ethanol yield under fermentative conditions. Aeration, however, did prove to be detrimental to final ethanol yields. With the inclusion of aeration in the control condition (no organic acid stress) and in each fermentation containing organic acid(s), the final ethanol yields were decreased. It was therefore concluded that aeration strategy is the key parameter in regards to the negative effects observed in fuel alcohol fermentations infected with Dekkera/Brettanomyces yeasts.     

Identification and characterization of yeasts in brem, a traditional Balinese rice wine

Fifty-one yeast strains isolated from fermented mash of Balinese rice wine, brem, fermented using five different types of starters, ragi tape, were identified on the basis of their internal transcribed spacer (ITS) regions and their 18S rDNA sequences. The results revealed that Saccharomyces cerevisiae(35 strains), Candida glabrata(six strains), Pichia anomala(three strains) and Issatchenkia orientalis(seven strains) were the main yeasts in the fermentation of the rice wine. These yeasts undergo succession during the fermentation in which S. cerevisiae was mostly found as the principal yeast at the end of fermentation. Phylogenetic analysis based on the 18S rDNA sequences of selected strains placed the isolated S. cerevisiae strains in the Saccharomyces sensu stricto group. Karyotype analysis of the S. cerevisiae strains resolved using pulsed field gel electrophoresis (PFGE) showed that the strains are typically associated with different types of starters.     

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.     

Analysis of non‐Saccharomyces yeast populations isolated from grape musts from Sicily (Italy)

Aims: The aim of this study was to identify the non‐Saccharomyces yeast populations present in the grape must microflora from wineries from different areas around the island of Sicily. Methods and Results: Yeasts identification was conducted on 2575 colonies isolated from six musts, characterized using Wallerstein Laboratory (WL) nutrient agar, restriction analysis of the amplified 5·8S‐internal transcribed spacer region and restriction profiles of amplified 26S rDNA. In those colonies, we identified 11 different yeast species originating from wine musts from two different geographical areas of the island of Sicily. Conclusions: We isolated non‐Saccharomyces yeasts and described the microflora in grape musts from different areas of Sicily. Moreover, we discovered two new colony morphologies for yeasts on WL agar never previously described. Significance and Impact of the Study: This investigation is a first step in understanding the distribution of non‐Saccharomyces yeasts in grape musts from Sicily. The contribution is important as a tool for monitoring the microflora in grape musts and for establishing a new non‐Saccharomyces yeast collection; in the future, this collection will be used for understanding the significance of these yeasts in oenology.