Effect of pure and mixed cultures of the main wine yeast species on grape must fermentations

Mixed inoculation of non-Saccharomyces yeasts and S. cerevisiae is of interest for the wine industry for technological and sensory reasons. We have analysed how mixed inocula of the main non-Saccharomyces yeasts and S. cerevisiae affect fermentation performance, nitrogen consumption and volatile compound production in a natural Macabeo grape must. Sterile must was fermented in triplicates and under the following six conditions: three pure cultures of S. cerevisiae, Hanseniaspora uvarum and Candida zemplinina and the mixtures of H. uvarum:S. cerevisiae (90:10), C. zemplinina:S. cerevisiae (90:10) and H. uvarum:C. zemplinina:S. cerevisiae (45:45:10). The presence of non-Saccharomyces yeasts slowed down the fermentations and produced higher levels of glycerol and acetic acid. Only the pure H. uvarum fermentations were unable to finish. Mixed fermentations consumed more of the available amino acids and were more complex and thus better able to synthesise volatile compounds. However, the amount of acetic acid was well above the admissible levels and compromises the immediate application of mixed 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.     

Effects of pulsed electric fields on the extraction of phenolic compounds during the fermentation of must of Tempranillo grapes

The effects of a pre-treatment of the grape skin with electric field treatments (PEF) at 5 and 10 kV/cm on the evolution of colour, anthocyanin and index of total polyphenols during the Tempranillo grape vinification and on the wine characteristics after fermentation have been investigated.Results showed that the permeabilization of the grape skin by application of a PEF treatment at room temperature caused an increment of the colour intensity, anthocyanin content and of total polyphenolic index with respect to the control during all the vinification process. Generally along the maceration process the values of these three parameters increased when the electric field strength raised from 5 to 10 kV/cm.Colour intensity and anthocyanin content of the wine significantly increased when the PEF treatment was applied at 10 kV/cm. However, the total phenolic index increased considerably with the application of a PEF treatment at 5 kV/cm. A further increment on the electric field strength did not appreciably augment this attribute. PEF did not affect the ratio between the components of the red wine colour (tint and yellow, red and blue components) and other wine characteristics such as alcohol content, total acidity, pH, reducing sugar concentration and volatile acidity.These results indicate that the PEF treatment previous to the maceration step in the winemaking process of the red wine can contribute to reduce the duration of the maceration during vinification or to increase the quantity of these attributes in the final wine.

Industrial relevance

This study investigates the effect of the application of a PEF pre-treatment of the grape skin on the evolution of colour, anthocyanin and index of total polyphenols during the Tempranillo grape vinification. Results obtained indicate that the application of a PEF treatment preceding the maceration step in the winemaking process of red wine could contribute to reduce the duration of the maceration during vinification or to increase the quantity of these parameters in the final wine. Latter effect could be of interest in grapes poor in polyphenols.     

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.     

A Focus on Quality and Safety Traits of Saccharomyces cerevisiae Isolated from Uva di Troia Grape Variety

The aim of this work was to study Saccharomyces cerevisiae strains isolated from vineyards of the autochthonous grape variety “Uva di Troia” located in different geographical areas of Apulian region (Southern Italy). Four hundred isolates were studied in relation to H₂S production, β‐glucosidase activity, and pigments adsorption from grape skin. Thus, 81 isolates were selected, identified through the amplification of the interdelta region, and grouped in 19 biotypes (from I to XIX). The enological performances were assessed to determine the content of residual sugars, ethanol, glycerol, and volatile acidity, after a microfermentation in Uva di Troia must for each isolate. The ability to remove ochratoxin A (OTA) was studied as an additional tool to select promising strains. A geographical‐dependent technological variability was found for glycerol and volatile acidity, suggesting that the different indigenous yeasts can have a peculiar impact on the final characteristics of the corresponding wine (“Nero di Troia”). Only 2 biotypes (VI and XVII) were able to remove OTA throughout fermentation, with the highest reduction achieved by the biotype XVII (ca. 30%).     

Selenium biotransformation by Saccharomyces cerevisiae and Saccharomyces bayanus during white wine manufacture: Laboratory-scale experiments

The main purpose of this laboratory-scale study was to evaluate the transformation of inorganic selenium, as sodium selenite, when added to white grape juice as part of the fermentation process of white wine. The participation of yeast, added in the fermentation of the must, is necessary to convert inorganic selenium into organoseleno species. Two different yeasts, Saccharomyces cerevisiae and Saccharomyces bayanus were used for fermentation. The effects of different Se concentrations on cells and their viability during fermentation were evaluated. The alcoholic fermentation that produced wine was not affected by the presence of selenium, regardless of the type of Saccharomyces used. After 21 days of fermentation, the white wine and residual yeast were separated and analysed by ICP-MS and LC–ICP-MS for determination of total selenium and speciation. Selenomethionine was found to be the main Se-species in the selenised white wine. The results obtained are preliminary but they could be considered for future studies using both pilot and full-scale vinification processes.     

The application of malolactic fermentation process to create good-quality grape wine produced in cool-climate countries: a review

The work presents the essence of the secondary fermentation occurring in the course of grape wines production—malolactic fermentation (MLF). The selection of wine yeast and bacteria preparations, scenario of malolactic bacteria inoculation as well as control system of the process conditions are proposed to create the specific character and style of grape wines produced in cool-climate countries, in which the acidity of grape must is often significantly enhanced and the concentration of aroma compounds is considerably low. The role played by appropriately run MLF is presented, along with the advantages and disadvantages of the process, benefits, and technological drawbacks resulting from the introduction of this procedure to the vinification process. Moreover, methods to initiate and run MLF as well as interactions occurring between microorganisms used in wine production, i.e., wine yeasts from the genus Saccharomyces and wine bacteria from the genus Oenococcus, are also presented and discussed.     

Role of non-Saccharomyces yeasts in Korean wines produced from Campbell Early grapes: Potential use of Hanseniaspora uvarum as a starter culture

Several yeasts were isolated from Campbell Early grapes (Vitis labrusca cultivar Campbell Early), the major grape cultivar in Korea, grown in two different regions. PCR-RFLP analysis of the ITS I-5.8S-ITS II region showed that 34 isolates out of a total of 40 were in the same group. Phylogenetic analysis revealed that the major strain belonged to one species, Hanseniaspora uvarum, although they displayed some nucleotide mismatches between them. During spontaneous alcohol fermentation at 20 °C, the two grape musts containing 24 °Brix sugar exhibited similar fermentation patterns with differences in final alcohol production and yeast viable counts. PCR analysis of the yeasts randomly isolated during the fermentation using an intron splice site primer showed changes in yeast flora between 8 and 10 days of fermentation. We found that the dominant yeasts displaying various PCR patterns using the primer remained the same throughout the early stages of fermentation, as determined by molecular typing of their ITS regions using PCR-RFLP, and these yeasts were identical to those isolated from grape berries. Among the isolates, the strain designated SS6 was selected based on its potassium metabisulfite resistance, alcohol production (distillation method), and flavor (by sniffing test) of grape juice. When Campbell Early grape must was inoculated with H. uvarum SS6 cells, no differences in fermentation pattern were observed compared with that inoculated with cells of Saccharomyces cerevisiae W-3, an industrial wine yeast strain. However, SS6 wine showed higher levels of organic acid (especially lactic acid), aldehydes, and minor alcohols (except n-propyl alcohol), as well as a higher score in sensory evaluation, compared to those of W-3 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.     

Fast identification of wine related lactic acid bacteria by multiplex PCR

The microflora of must and wine consists of yeasts, acetic acid bacteria and lactic acid bacteria (LAB). The latter group plays an important role for wine quality. The malolactic fermentation carried out by LAB leads to deacidification and stabilisation of wines. Nevertheless, LAB are often associated with wine spoilage. They are mainly responsible for the formation of biogenic amines. Furthermore, some strains produce exopolysaccharide slimes, acetic acid, diacetyl and other off-flavours. In this context a better monitoring of the vinification process is crucial to improve wine quality. Moreover, a lot of biodiversity studies would also profit from a fast and reliable identification method.     

Production of white wine by Saccharomyces cerevisiae immobilized on grape pomace

White wine was produced with Saccharomyces cerevisiae cells immobilized on grape pomace, by natural adsorption. The support, the main solid waste from the wine industry, consisted of the skins, seeds and stems. Immobilization was tested using different media, namely complex culture medium, raw grape must and diluted grape must. Grape pomace was revealed to be an appropriate support for yeast cell immobilization. Moreover, grape must was shown to be the most suitable medium as immobilized cells became adapted to the conditions in the subsequent alcoholic fermentation in the wine‐making process. The wines produced, either with immobilized cells or with free cells, were subjected to chemical analysis by HPLC (ethanol, glycerol, sugars and organic acids) and by gas chromatography (major and minor volatile compounds); additionally, colour (CIELab) and sensory analysis were performed. The use of immobilized systems to conduct alcoholic fermentation in white wine production proved to be a more rapid and a more efficient process, especially when large amounts of SO₂ were present in the must. Furthermore, the final wines obtained with immobilized cells demonstrated improved sensory properties related to the larger amounts of ethanol and volatile compounds produced. The more intense colour of these wines could be a drawback, which could be hindered by the reutilization of the biocatalyst in successive fermentations. Copyright © 2012 The Institute of Brewing & Distilling     

Effects of mediums on fermentation behaviour and aroma composition in pure and mixed culture of Saccharomyces cerevisiae with Torulaspora delbrueckii

The nutrient status and composition in mediums have a significant effect on yeast metabolism and phenotypic characteristics in wine fermentation. In this study, the effects of three frequently used mediums, including synthetic grape juice (SGJ), grape juice without grape pericarp and seeds (GJ) and grape must with grape pericarp and seeds (GMPS), on yeast fermentation behaviour and aroma compounds produced by pure and mixed culture of Saccharomyces cerevisiae T73 with Torulaspora delbrueckii TD20 were investigated after alcoholic fermentation. The results showed that high fermentation activities and cell population were always found in GJ medium irrespective of inoculated approach. More esters and higher alcohols were produced in GMPS medium fermented by pure S. cerevisiae, while SGJ medium had increased levels of fatty acids. Consistent with previous literatures, the mixed fermentation of T. delbrueckii and S. cerevisiae produced more acetate esters and fatty acids than the pure culture of S. cerevisiae, while this enological trait was only found in SGJ and GJ, not in GMPS. Our results highlighted that more attention should be paid to the fermentation medium when evaluating the enological and aromatic properties of selected yeasts used in industrial winemaking. In this regard, the combined use of GJ and GMPS medium might be a suitable choice.     

SCREENING FOR EXTRACELLULAR ACID PROTEASE(S) PRODUCTION BY WINE YEASTS*

Ninety-four Saccharomyces wine yeasts were screened for their ability to produce extracellular acid protease(s) under simulated vinification conditions. Seventeen strains were selected on the basis of their higher proteolytic activity on haemoglobin and casein. These strains showed maximum activity after 20 days of fermentation on haemoglobin and 25 days on casein. Wines obtained from grape juice fermented by the above 17 strains showed significative differences in the residual protein content.     

Molecular characterization and oenological properties of wine yeasts isolated during spontaneous fermentation of six varieties of grape must

Fermentation by naturally occurring yeasts may produce wines of complex oenological properties that are unique to a specific region. The present work analyses the population dynamics of the yeasts during spontaneous fermentation of six varieties of grape must from the “Valle del Andarax” area (Spain). In this study we identified members of the genera Candida, Hanseniaspora, Issatchenkia, Metschnikowia, Pichia and Saccharomyces by PCR-RFLP of the ITS region. The capability of these yeasts to ferment grape must of the Macabeo variety was studied, and the volatile profile of the wine from each microvinification has been determined. Of all the yeasts isolated Candida stellata and Saccharomyces cerevisiae were able to consume virtually all the initial glucose, producing ethanol contents typical of table wines. The best profile of higher alcohols was given by Saccharomyces cerevisiae followed by Hanseniaspora uvarum, Issatchenkia orientalis and Candida stellata. Metschnikowia pulcherrima and Pichia fermentans showed the highest production of ethyl caprilate and 2-phenyl ethanol, compounds associated with pleasant aromas.     

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.     

酵母菌的生存因子及其特性

麦角甾醇是酵母的“生存因子”之一,它能够延长处于衰减阶段的酵母生存时间,增强酵母菌株对酒精的抵抗能力,使酵母进一步发酵,降低葡萄酒中的残糖,但酵母细胞内麦角甾醇的富积与其生存条件、发酵环境、选择与保存条件之间有密切的关系,因而,酵母的“生存因子”对葡萄酒的酿造具有重要意义。     

Flow cytometric analysis of Saccharomyces cerevisiae populations in high‐sugar Chardonnay fermentations

Fermentation of high‐sugar grape juice by five commercial strains of Saccharomyces cerevisiae was analyzed in real time both microbiologically and chemically. Viable, dead and ‘injured’ yeast were enumerated by flow cytometry. Chemical changes in the fermentations were measured by Fourier transform infrared spectroscopy. Sensory testing of wines was conducted at 3 and 6 months post bottling. Fermentation kinetics varied among the five strains of yeast, with two of the yeasts unable to completely ferment the juice. Wines differed in glycerol and volatile acidity production. Sensorial differences were detected in all wine pairs tested at 3 months, but only in one of the four wine pairs tested at 6 months. Copyright © 2006 Society of Chemical Industry     

Biodiversity and safety aspects of yeast strains characterized from vineyards and spontaneous fermentations in the Apulia Region,Italy

This work is the first large-scale study on vineyard-associated yeast strains from Apulia (Southern Italy). Yeasts were identified by Internal Transcribed Spacer (ITS) ribotyping and bioinformatic analysis. The polymorphism of interdelta elements was used to differentiate Saccharomyces cerevisiae strains. Twenty different species belonging to 9 genera were identified. Predominant on the grape surface were Metschnikowia pulcherrima, Hanseniaspora uvarum and Aureobasidium pullulans, whereas M. pulcherrima and H. uvarum were dominant in the early fermentation stage. A total of 692 S. cerevisiae isolates were identified and a number of S. cerevisiae strains, ranging from 26 to 55, was detected in each of the eight fermentations. The strains were tested for biogenic amines (BAs) production, either in synthetic media or grape must. Two Pichia manshurica, an Issatchenkia terricola and a M. pulcherrima strains were able to produce histamine and cadaverine, during must fermentation. The production of BAs in wine must was different than that observed in the synthetic medium. This feature indicate the importance of an “in grape must” assessment of BAs producing yeast. Overall, our results suggest the importance of microbiological control during wine-making to reduce the potential health risk for consumer represented by these spoilage yeasts.     

Differentiation between fermenting and spoilage yeasts in wine by total free fatty acid analysis

Differentiation between fermenting and spoilage yeasts in wine was estimated by cellular fatty acid profiles. Forty-two strains of yeasts representing 17 genera were grown on a defined liquid medium for 48 h at 25°C in a rotary shaker. After saponification of yeast cells, free fatty acid extracts were analysed by gas chromatography. Multivariate analysis was performed by Principal Components Analysis (PCA) to define clusters of fatty acids and yeasts. Strains were characterised especially by long-chain fatty acids, palmitic (C16) to linolenic (C18:3) acid under aerobic culture. Nevertheless, most of Saccharomyces cerevisiae and also Dekkera bruxellensis (former names D intermedia and Brettanomyces lumbicus) synthesized medium-chain fatty acids, octanoic (C8) acid to dodecanoic (C12) acid. With this method it was possible to differentiate fermenting grape yeasts such as S cerevisiae from spoilage yeasts on the basis of the absence of linoleic (C18:2) and linolenic (C18:3) acids. However, the method seemed unreliable for the identification of strains, more particularly those of species of S cerevisiae.     

Influence of SO<Subscript>2</Subscript> on the evolution of volatile compounds through alcoholic fermentation of must stabilized by pulsed electric fields

The aim of this work was to study the influence of sulphur dioxide (SO₂) on the formation of volatile compounds by yeast through wine alcoholic fermentation. Thus Parellada must was microbiologically stabilized using a pulsed electric field (PEF) treatment and inoculated with Saccharomyces cerevisiae Na33 strain. Fermentation was carried out with or without SO₂ and the results showed that the evolution of the volatile compounds profile throughout the process was similar. The content of volatile acids in wine obtained by using sulphur dioxide was not significantly different from that fermented without adding the compound. However, the final content of total alcohols and esters was significantly different even thought the differences were small. Consequently, when grape must is treated by PEF the sulphur dioxide concentration usually used in winemaking could be reduced to safer levels or even eliminated without an important effect on the volatile compounds content of the final product. Therefore, the absence of sulphur dioxide should not have a negative impact on the sensory characteristics of wine.