Studies on acetate ester production by non-Saccharomyces wine yeasts

A double coupling bioreactor system was used to fast screen yeast strains for the production of acetate esters. Eleven yeast strains were used belonging to the genera Candida, Hanseniaspora, Metschnikowia, Pichia, Schizosaccharomyces and Zygosacharomyces, mainly isolated from grapes and wine, and two wine Saccharomyces cerevisiae strains. The acetate ester forming activities of yeast strains belonging to the genera Hanseniaspora (Hanseniaspora guilliermondii and H. uvarum) and Pichia (Pichia anomala) showed different substrate specificities and were able to produce ethyl acetate, geranyl acetate, isoamyl acetate and 2-phenylethyl acetate. The influence of aeration culture conditions on the formation of acetate esters by non-Saccharomyces wine yeast and S. cerevisiae was examined by growing the yeasts on synthetic microbiological medium. S. cerevisiae produced low levels of acetate esters when the cells were cultured under highly aeration conditions, while, under the same conditions, H. guilliermondii 11104 and P. anomala 10590 were found to be strong producers of 2-phenylethyl acetate and isoamyl acetate, respectively.     

Rational selection of non-Saccharomyces wine yeasts for mixed starters based on ester formation and enological traits

Thirty-eight yeast strains belonging to the genera Candida, Hanseniaspora, Pichia, Torulaspora and Zygosaccharomyces were screened for ester formation on synthetic microbiological medium. The genera Hanseniaspora and Pichia stood out as the best acetate ester producers. Based on the ester profile Hanseniaspora guilliermondii 11027 and 11102, Hanseniaspora osmophila 1471 and Pichia membranifaciens 10113 and 10550 were selected for further characterization of enological traits. When growing on must H. osmophila 1471, which displayed a glucophilic nature and was able to consume more than 90% of initial must sugars, produced levels of acetic acid, medium chain fatty acids and ethyl acetate, within the ranges described for wine. On the other hand, it was found to be a strong producer of 2-phenylethyl acetate. Our data suggest that H. osmophila 1471 is a good candidate for mixed starters, although the possible interactions with S. cerevisiae deserve further research.     

Monitoring a mixed starter of Hanseniaspora vineae-Saccharomyces cerevisiae in natural must: impact on 2-phenylethyl acetate production

The effect of simultaneous or sequential inoculation of Hanseniaspora vineae CECT 1471 and Saccharomyces cerevisiae T73 in non-sterile must on 2-phenylethyl acetate production has been examined. In both treatments tested, no significant differences in Saccharomyces yeast growth were found, whereas non-Saccharomyces yeast growth was significantly different during all days of fermentation. Independently of the type of inoculation, S. cerevisiae was the predominant species from day 3 till the end of the fermentation. The dynamics of indigenous and inoculated yeast populations showed H. vineae to be the predominant non-Saccharomyces species at the beginning of fermentation in sequentially inoculated wines, whereas the simultaneous inoculation of S. cerevisiae did not permit any non-Saccharomyces species to become predominant. Differences found in non-Saccharomyces yeast growth in both fermentations influenced the analytical profiles of final wines and specifically 2-phenylethyl acetate concentration which was two-fold increased in sequentially inoculated wines in comparison to those co-inoculated. In conclusion we have shown that H. vineae inoculated as part of a sequential mixed starter is able to compete with native yeasts present in non-sterile must and modify the wine aroma profile.     

Acetate ester formation in wine by mixed cultures in laboratory fermentations

Two non-Saccharomyces wine yeast strains, Hanseniaspora guilliermondii 11104 and Pichia anomala 10590, selected as good producers of acetate esters when grown on synthetic microbiological medium, have been tested in wine fermentations as mixed cultures together with Saccharomyces cerevisiae. Wines produced using mixed cultures showed levels of acetaldehyde, acetic acid, glycerol and total higher alcohols within the ranges described for wine, whereas an increase in acetate ester concentrations was found. Ethyl acetate was the main ester produced, and isoamyl acetate and 2-phenylethyl acetate made up the next largest group of ester compounds in the wines analysed. H. guilliermondii 11104 was found to be a strong producer of 2-phenylethyl acetate in both pure and mixed cultures whereas S. cerevisiae was the best producer of ethyl esters. Mixed cultures did not influence ethyl ester levels at all.     

Influence of sequential inoculum of Starmerella bacillaris and Saccharomyces cerevisiae on flavonoid composition of monovarietal Sangiovese wines

The selection of Starmerella bacillaris strains to be used with Saccharomyces cerevisiae as mixed cultures has been recently suggested in order to produce wines containing lower ethanol and higher glycerol concentrations and to promote fructose degradation due to their fructophilic character. However, studies about effects of such mixed starter cultures on phenolic compounds, which are responsible for the colour and health-enhancing properties in red wines, are currently lacking. Therefore, in this work, the influence of sequential inoculated fermentation (SIF) with Starm. bacillaris and S. cerevisiae on phenolic content of monovarietal Sangiovese wine was evaluated by fermentations at laboratory scale. Axenic fermentations (AXFs) with S. cerevisiae were performed as control. S. cerevisiae attained higher cell densities in AXF compared with SIF. The experimental wines obtained by SIF showed significant lower ethanol and higher glycerol concentrations, whereas no significant difference was detected in colour intensity. The total phenol index reached significantly lower values in SIF. Furthermore, the wines produced by SIF contained higher concentrations of vitisin A that has a greater colour stability than the anthocyanin monomer. Finally, a lower content of both free anthocyanins and flavan-3-ols, key compounds for wine quality possessing also health-enhancing properties, was found in wines obtained by SIF. On the contrary, no significant difference was detected on flavonol concentration between SIF and AXF. This study highlighted that the use of sequential inoculum of Starm. bacillaris and S. cerevisiae can contribute to increasing the colour stability of red wines, even if at the expense of compounds with health properties.     

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.     

Sweet Wine Production by Two Osmotolerant Saccharomyces cerevisiae Strains

The use of Saccharomyces cerevisiae to produce sweet wine is difficult because yeast is affected by a hyperosmotic stress due to the high sugar concentrations in the fermenting must. One possible alternative could be the coimmobilization of the osmotolerant yeast strains S. cerevisiae X4 and X5 on Penicillium chrysogenum strain H3 (GRAS) for the partial fermentation of raisin musts. This immobilized has been, namely, as yeast biocapsules. Traditional sweet wine (that is, without fermentation of the must) and must partially fermented by free yeast cells were also used for comparison. Partially fermented sweet wines showed higher concentration of the volatile compounds than traditionally produced wines. The wines obtained by immobilized yeast cells reached minor concentrations of major alcohols than wines by free cells. The consumption of specific nitrogen compounds was dependent on yeast strain and the cellular immobilization. A principal component analysis shows that the compounds related to the response to osmotic stress (glycerol, acetaldehyde, acetoin, and butanediol) clearly differentiate the wines obtained with free yeasts but not the wines obtained with immobilized yeasts.     

Higher alcohols concentration and its relation with the biological aging evolution

The production of 1-propanol, isobutanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol by two flor yeast strains during accelerated biological aging of sherry wines with different aging time has been evaluated. The S. cerevisiae G1 strain has shown a higher production of higher alcohols than the S. bayanus F12 strain highlighting the 1-propanol production in the youngest wine. Differences between the two studied yeasts decreased in relation with the aging time of the initial wine used. The multiple regression analysis carried out to relate the higher alcohols concentration and the values obtained from an aging equation showed R-squared values ranging from 81.1 to 96.5%. The higher alcohols that best described such relation were 1-propanol, isobutanol and 3-methyl-1-butanol.     

The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates

The fruity odours of wine are largely derived from the synthesis of esters and higher alcohols during yeast fermentation. The ATF1- and ATF2-encoded alcohol acetyltransferases of S. cerevisiae are responsible for the synthesis of ethyl acetate and isoamyl acetate esters, while the EHT1-encoded ethanol hexanoyl transferase is responsible for synthesizing ethyl caproate. However, esters such as these might be degraded by the IAH1-encoded esterase. The objectives of this study were: (a) to overexpress the genes encoding ester-synthesizing and ester-degrading enzymes in wine yeast; (b) to prepare Colombard table wines and base wines for distillation using these modified strains; and (c) to analyse and compare the ester concentrations and aroma profiles of these wines and distillates. The overexpression of ATF1 significantly increased the concentrations of ethyl acetate, isoamyl acetate, 2-phenylethyl acetate and ethyl caproate, while the overexpression of ATF2 affected the concentrations of ethyl acetate and isoamyl acetate to a lesser degree. The overexpression of IAH1 resulted in a significant decrease in ethyl acetate, isoamyl acetate, hexyl acetate and 2-phenylethyl acetate. The overexpression of EHT1 resulted in a marked increase in ethyl caproate, ethyl caprylate and ethyl caprate. The flavour profile of the wines and distillates prepared using the modified strains were also significantly altered as indicated by formal sensory analysis. This study offers prospects for the development of wine yeast starter strains with optimized ester-producing capability that could assist winemakers in their effort to consistently produce wine and distillates such as brandy to definable flavour specifications and styles.     

Outlining a future for non-Saccharomyces yeasts: selection of putative spoilage wine strains to be used in association with Saccharomyces cerevisiae for grape juice fermentation

The use of non-Saccharomyces yeasts that are generally considered as spoilage yeasts, in association with Saccharomyces cerevisiae for grape must fermentation was here evaluated. Analysis of the main oenological characteristics of pure cultures of 55 yeasts belonging to the genera Hanseniaspora, Pichia, Saccharomycodes and Zygosaccharomyces revealed wide biodiversity within each genus. Moreover, many of these non-Saccharomyces strains had interesting oenological properties in terms of fermentation purity, and ethanol and secondary metabolite production. The use of four non-Saccharomyces yeasts (one per genus) in mixed cultures with a commercial S. cerevisiae strain at different S. cerevisiae/non-Saccharomyces inoculum ratios was investigated. This revealed that most of the compounds normally produced at high concentrations by pure cultures of non-Saccharomyces, and which are considered detrimental to wine quality, do not reach threshold taste levels in these mixed fermentations. On the other hand, the analytical profiles of the wines produced by these mixed cultures indicated that depending on the yeast species and the S. cerevisiae/non-Saccharomyces inoculum ratio, these non-Saccharomyces yeasts can be used to increase production of polysaccharides and to modulate the final concentrations of acetic acid and volatile compounds, such as ethyl acetate, phenyl-ethyl acetate, 2-phenyl ethanol, and 2-methyl 1-butanol.     

Growth and fermentation kinetics of a mixed culture of Saccharomyces cerevisiae var. bayanus and Williopsis saturnus var. saturnus at different ratios in longan juice (Dimocarpus longan Lour.)

Cofermentation of longan juice by mixed cultures of Saccharomyces cerevisiae var. bayanus EC‐1118 and Williopsis saturnus var. saturnus CBS254 at two inoculation ratios (EC‐1118:CBS254 = 1:100 and 1:1000 cfu mL^?1) was performed to ascertain their impact on longan wine aroma compound formation. The results showed improved aroma compound profiles in the longan wine fermented with mixed yeasts in comparison with the longan wines fermented with single yeasts in terms of increased production of acetate esters, fatty acid ethyl esters, alcohols and carboxylic acids. The impact of cofermentation on longan wine aroma formation was affected by the ratio of S. cerevisiae EC‐1118 to W. saturnus CBS254 with 1:100 cfu mL^?1 being more effective. This research suggests that the inoculation ratio of mixed yeasts may be used as an effective means of manipulating longan wine aroma.     

Genetic and enological analysis of selected Saccharomyces cerevisiae strains for wine production

The non‐wine Saccharomyces cerevisiae strain of 96581 was found to be a promising candidate for the production of white wine. It produced wines with fusel alcohols that were 57% higher than those produced by the wine yeasts studied and was also more efficient in the production of 2‐phenethyl acetate and 3‐methyl‐1‐butanol acetate. This study also shows that there is a difference in the ester‐formation efficiency for acetates relative to C6, C8 and C10 fatty acid esters for all the studied yeast strains. Therefore, it supports the view that other unidentified enzymes besides those regulated by ATF1 and ATF2 genes are involved in the production of ethyl esters of C6–C10 fatty acids. DNA analysis of the 25S, 18S, 5.8S and 5S ribosomal DNA genes in these strains showed high conservation. Despite the closely related nature of these yeast strains, the chemical profiles of the wines produced were significantly different.     

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.     

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.     

Initial Saccharomyces cerevisiae concentration in single or composite cultures dictates bioprocess kinetics

The growth dynamics of three non-Saccharomyces strains in combination with Saccharomyces cerevisiae during fermentation of a sterile grape juice have been studied. The influence of the initial concentrations of S. cerevisiae on the whole yeast community was the main purpose of this research. The progression of S. cerevisiae within the first 5 days of fermentation was monitored by enumeration on selective and non-selective media. The population of each species was evaluated by morphological criteria. After 24 h, Hanseniaspora uvarum represented more than 50% of the whole yeast community, including ferments with the highest initial concentration of S. cerevisiae. As the population of S. cerevisiae increased, H. uvarum decreased. Metchnikowia pulcherrima was more inhibited by S. cerevisiae than H. uvarum, whereas the growth of Candida stellata was less inhibited. After thirty days, irrespective of the initial concentration of S. cerevisiae, only S. cerevisiae was detected in all ferments. Conversion of sugars to ethanol correlated with the initial population of S. cerevisiae. Glucose was almost completely exhausted in all cases, independent of the initial S. cerevisiae concentration used. Grape juices inoculated with composite inocula of non-S. cerevisiae and S. cerevisiae, with its initial concentration lower than 5 cfu/ml did not produce wines according to wine regulations. The concentration of ethanol in the wines did not reach the minimum amount of 9 vol%. Samples of wines fermented with a composite inoculum the concentration of S. cerevisiae represented 50 cfu/ml of grape juice, were judged to have the best sensorial properties.     

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.     

Heavy sulphur compounds, higher alcohols and esters production profile of Hanseniaspora uvarum and Hanseniaspora guilliermondii grown as pure and mixed cultures in grape must

Hanseniaspora guilliermondii and Hanseniaspora uvarum were tested in grape must fermentations as pure and mixed starter cultures with Saccharomyces cerevisiae. In pure cultures, the specific growth rates found were 0.29 h(-1) for H. uvarum, 0.23 h(-1) for H. guilliermondii and 0.18 h(-1) for S. cerevisiae. No significant differences were observed between these values and those obtained in mixed cultures. Results presented in this work show that growth of apiculate yeasts during the first days of fermentation enhances the production of desirable compounds, such as esters, and may not have a negative influence on the production of higher alcohols and undesirable heavy sulphur compounds. Growth of apiculate yeasts reduced the total content of higher alcohols in wines, when compared to those produced by a pure culture of S. cerevisiae. Furthermore, the highest levels of 2-phenylethyl acetate were obtained when H. guilliermondii was inoculated in grape musts, whereas H. uvarum increased the isoamyl acetate content of wines. Apiculate yeasts produced high amounts of ethyl acetate; however, the level of this compound decreased in mixed cultures of apiculate yeasts and S. cerevisiae. When S. cerevisiae was used as a starter culture, wines showed higher concentrations of glycerol, 2-phenylethanol and ethyl hexanoate. In mixed cultures of apiculate yeasts and S. cerevisiae, wines presented amounts of methionol, acetic acid-3-(methylthio)propyl ester, 4-(methylthio)-1-butanol, 2-mercaptoethanol and cis-2-methyltetrahydro-thiophen-3-ol similar to those produced by a pure culture of S. cerevisiae. An increase in the amounts of 3-(ethylthio)-1-propanol, trans-2-methyltetrahydro-thiophen-3-ol and 3-mercapto-1-propanol was obtained in wines produced from mixed cultures with H. guilliermondii.     

SPME-GC-MS分析不同商业葡萄酒酵母香气物质合成的差异性

采用顶空固相微萃取(HS-SPME)和气质联用法(GC-MS)分析了4株常用商业葡萄酒酵母的挥发性香气成分。对酵母代谢的19种香气成分进行定性定量分析,菌株CY3079具有较高的高级醇生成能力,其中异戊醇和2-苯乙醇的合成均高于其它酵母菌株。D254酵母具有较高的酯类和挥发酸类合成能力,其中乙酸乙酯和乙酸对酵母D254总酯和总挥发酸的含量贡献较大。EC1118高级醇生成量最低,但是具有较高的乙酸-2-苯乙酯生成能力。RC212酒样具有较低的酯类和挥发酸。感官品评试验表明,D254酒样的果香比较明显;CY3079酒样的刺激性大于其它菌株;EC1118酒样花香比较突出。     

Taqman real-time PCR for the detection and enumeration of Saccharomyces cerevisiae in wine

Saccharomyces cerevisiae is the main yeast species responsible for wine fermentation; however, its presence during maturing or barrel-ageing can sometimes result in a reduction in the quality of wine by refermentation. In this work, we developed a quantitative real-time PCR (QPCR) for the rapid detection and quantification of S. cerevisiae in wine. The primers and the hydrolysis probe (TaqMan^®) were designed from the sequence of a DNA fragment present only in S. cerevisiae and absent in other wine yeasts obtained from an RAPD-PCR analysis. The QPCR developed was highly reproducible, allowing the specific detection and quantification of this yeast in artificially contaminated wines, with a detection limit of 78 CFU/mL. Furthermore, the usefulness of the QPCR developed was evaluated through the quantification of the yeast in wine samples obtained from vineyards, confirming the quantitative capacity of the method. The methodology developed was specific, fast and a sensitive tool for the detection and enumeration of S. cerevisiae cells in wine.     

Production of Table Wine from Processed Tea Bags Using Different Strains of Saccharomyces cerevisiae

Wine samples were produced from locally available tea infusions (Lipton tea, Top tea and Highland tea) using baker’s yeast (Saccharomyces cerevisiae By1) and yeast cultures from pineapple (S. cerevisiae Py6) and cocoa (S. cerevisiae CY43). Physicochemical analyses and microbial evaluation were undertaken during fermentation. Lipton tea wine produced, using baker’s yeast, pineapple yeast and cocoa yeast had highest alcoholic contents of 7.88%, 6.25% and 7.20%, respectively. Top tea wine produced using the same set of yeasts had highest alcoholic contents of 9.78%, 5.43 and 8.15% respectively, while Highland tea wine produced highest alcoholic contents of 9.78%, 7.07% and 7.61% respectively. Physicochemical analyses for all the wines produced showed that the specific gravity, total solids and pH of the must decreased as fermentation progressed while the titratable acidity remained constant throughout the must fermentation. Colony counts showed a high biomass of yeast cells that decreased as it tended towards the end of fermentation. Sensory analysis of the wines showed that Lipton and Top tea are the most acceptable organoleptically when compared with the commercial wine used as control although the three tested teas were not significantly different statistically. Baker’s yeast was rated as the best yeast for wine fermentation irrespective of the type of tea used. All the wines produced were generally accepted as they were scored above average. This study highlights the potential of using different tea infusions as alternatives to grape and other fruit musts in wine making. It also confirms that commercial yeasts such as baker’s yeast can be used in homemade wine production.