Use Of non-Saccharomyces Yeasts Research Articles

Torulaspora delbrueckii for secondary fermentation in sparkling wine production

In the search for the desired oenological features and flavour complexity of wines, there is growing interest in the potential use of non-Saccharomyces yeast that are naturally present in the winemaking environment. Torulaspora delbrueckii is one such yeast that has seen profitable use in mixed fermentations with Saccharomyces cerevisiae and with different grape varieties. T. delbrueckii can have positive and distinctive impacts on the overall aroma of wines, and has also been used at an industrial level. Here, T. delbrueckii was successfully used in pure and mixed secondary fermentations for sparkling wine. The two selected T. delbrueckii strains used completed the secondary fermentation ‘prise de mousse’ in these pure and mixed fermentations. The sparkling wines obtained with T. delbrueckii showed different aromatic compositions and sensory profiles to those of S. cerevisiae. T. delbrueckii strain DiSVA 130 showed high esters production and significantly high scores for some of the aromatic descriptors that positively influence the sensory profile of sparkling wine. Thus, the use of T. delbrueckii in pure and mixed fermentations is a suitable strategy to further develop the flavour complexity during secondary fermentation of sparkling wines.

Effect of Saccharomyces, Non-Saccharomyces Yeasts and Malolactic Fermentation Strategies on Fermentation Kinetics and Flavor of Shiraz Wines

The use of non-Saccharomyces yeasts to improve complexity and diversify wine style is increasing; however, the interactions between non-Saccharomyces yeasts and lactic acid bacteria (LAB) have not received much attention. This study investigated the interactions of seven non-Saccharomyces yeast strains of the genera Candida, Hanseniaspora, Lachancea, Metschnikowia and Torulaspora in combination with S. cerevisiae and three malolactic fermentation (MLF) strategies in a Shiraz winemaking trial. Standard oenological parameters, volatile composition and sensory profiles of wines were investigated. Wines produced with non-Saccharomyces yeasts had lower alcohol and glycerol levels than wines produced with S. cerevisiae only. Malolactic fermentation also completed faster in these wines. Wines produced with non-Saccharomyces yeasts differed chemically and sensorially from wines produced with S. cerevisiae only. The Candida zemplinina and the one L. thermotolerans isolate slightly inhibited LAB growth in wines that underwent simultaneous MLF. Malolactic fermentation strategy had a greater impact on sensory profiles than yeast treatment. Both yeast selection and MLF strategy had a significant effect on berry aroma, but MLF strategy also had a significant effect on acid balance and astringency of wines. Winemakers should apply the optimal yeast combination and MLF strategy to ensure fast completion of MLF and improve wine complexity.

Non-Saccharomyces Yeasts Nitrogen Source Preferences: Impact on Sequential Fermentation and Wine Volatile Compounds Profile.

Nitrogen sources in the must are important for yeast metabolism, growth, and performance, and wine volatile compounds profile. Yeast assimilable nitrogen (YAN) deficiencies in grape must are one of the main causes of stuck and sluggish fermentation. The nitrogen requirement of Saccharomyces cerevisiae metabolism has been described in detail. However, the YAN preferences of non-Saccharomyces yeasts remain unknown despite their increasingly widespread use in winemaking. Furthermore, the impact of nitrogen consumption by non-Saccharomyces yeasts on YAN availability, alcoholic performance and volatile compounds production by S. cerevisiae in sequential fermentation has been little studied. With a view to improving the use of non-Saccharomyces yeasts in winemaking, we studied the use of amino acids and ammonium by three strains of non-Saccharomyces yeasts (Starmerella bacillaris, Metschnikowia pulcherrima, and Pichia membranifaciens) in grape juice. We first determined which nitrogen sources were preferentially used by these yeasts in pure cultures at 28 and 20°C (because few data are available). We then carried out sequential fermentations at 20°C with S. cerevisiae, to assess the impact of the non-Saccharomyces yeasts on the availability of assimilable nitrogen for S. cerevisiae. Finally, 22 volatile compounds were quantified in sequential fermentation and their levels compared with those in pure cultures of S. cerevisiae. We report here, for the first time, that non-Saccharomyces yeasts have specific amino-acid consumption profiles. Histidine, methionine, threonine, and tyrosine were not consumed by S. bacillaris, aspartic acid was assimilated very slowly by M. pulcherrima, and glutamine was not assimilated by P. membranifaciens. By contrast, cysteine appeared to be a preferred nitrogen source for all non-Saccharomyces yeasts. In sequential fermentation, these specific profiles of amino-acid consumption by non-Saccharomyces yeasts may account for some of the interactions observed here, such as poorer performances of S. cerevisiae and volatile profile changes.

Use of non-Saccharomyces yeasts and oenological tannin in red winemaking: Influence on colour, aroma and sensorial properties of young wines

Today, many non-Saccharomyces strains have been verified can be positive for the development of wine anthocyanin and aroma in different fermentation scenarios. Moreover, oenological tannins are widely used in wine industry to improve the colour profile and aroma complexity. The aim of this work is to analyze the fermentation characters of non-Saccharomyces strains and investigate the effects of pre-fermentative addition of oenological tannins on the wine components as well as sensory properties. For this purpose, five selected non-Saccharomyces strains and grape seed tannin were used to carry out the different fermentation trials. As a result, the grape seed tannin were less likely to influence growth kinetics of non-Saccharomyces strains. Schizosaccharomyces pombe has been proved can be effective to reduce the malic acid content while increase the level of vinylphenolic pyranoanthocyanin, which is positive for wine colour stability. Pre-fermentative use of oenological tannin was verified could be beneficial for the wines fermented with non-Saccharomyces regarding the improvement of wine colour, anthocyanin composition and the complexity of volatile compounds. Nevertheless, sensory analysis showed that oenological tannin could be less effective to modify the aroma impression of non-Saccharomyces wines.

Sequential Inoculation of Native Non-Saccharomyces and Saccharomyces cerevisiae Strains for Wine Making.

The use of non-Saccharomyces yeast for wine making is becoming a common trend in many innovative wineries. The application is normally aimed at increasing aromas, glycerol, reducing acidity, and other improvements. This manuscript focuses on the reproduction of the native microbiota from the vineyard in the inoculum. Thus, native selected yeasts (Hanseniaspora uvarum, Metschnikowia pulcherrima, Torulaspora delbrueckii, Starmerella bacillaris species and three different strains of Saccharomyces cerevisiae) were inoculated sequentially, or only S. cerevisiae (three native strains together or one commercial) was used. Inoculations were performed both in laboratory conditions with synthetic must (400 mL) as well as in industrial conditions (2000 kg of grapes) in red winemaking in two different varieties, Grenache and Carignan. The results showed that all the inoculated S. cerevisiae strains were found at the end of the vinifications, and when non-Saccharomyces yeasts were inoculated, they were found in appreciable populations at mid-fermentation. The final wines produced could be clearly differentiated by sensory analysis and were of similar quality, in terms of sensory analysis panelists’ appreciation.

Use of non-Saccharomyces in single-culture, mixed and sequential fermentation to improve red wine quality

The use of non-Saccharomyces yeasts can provide advantages in winemaking such as: a reduction of ethanol content, the synthesis of certain metabolites that are important for color stability or a good yield in the production of certain volatile compounds (ethyl lactate, 2,3-butanediol, 2-phenylethyl acetate). The main objective was general evaluation of wines made by non-Saccharomyces yeast under different fermentation trials compared to wines made by Saccharomyces cerevisiae. Single pure fermentations were performed by two strains of S. cerevisiae (used as controls) and two strains of Schizosaccharomyces pombe. The yeasts Lachancea thermotolerans and Torulaspora delbrueckii were tested in sequential fermentations with S. cerevisiae strains. The effect on acidity due to the interaction between L. thermotolerans and S. pombe in a mixed fermentation was studied. Sequential fermentations with L. thermotolerans and S. cerevisiae can reduce the ethanol content and increase the content of glycerol and pyruvic acid; in addition, the use of L. thermotolerans can increase the lactic acid content, while the mixed fermentations of S. pombe and L. thermotolerans may increase the acetaldehyde content and may also reduce the ethanol content. However, these samples require more time to complete the fermentation. Single pure fermentations by S. pombe can increase the amounts of vitisins, acetaldehyde and glycerol and the amounts of acetic acid. The use of L. thermotolerans in a sequential fermentation allowed an increase in fruitiness and body character of red wine.

The Role and Use of Non-Saccharomyces Yeasts in Wine Production

The contribution by the numerous grape-must-associated non-Saccharomyces yeasts to wine fermentation has been debated extensively. These yeasts, naturally present in all wine fermentations, are metabolically active and their metabolites can impact on wine quality. Although often seen as a source of microbial spoilage, there is substantial contrary evidence pointing to a positive contribution by these yeasts. The role of non-Saccharomyces yeasts in wine fermentation is therefore receiving increasing attention by wine microbiologists in Old and New World wine producing countries. Species that have been investigated for wine production thus far include those from the Candida, Kloeckera, Hanseniaspora, Zygosaccharomyces, Schizosaccharomyces, Torulaspora, Brettanomyces, Saccharomycodes, Pichia and Williopsis genera. In this review the use and role of non-Saccharomyces yeast in wine production is presented and research trends are discussed.

Monitoring volatile compounds production throughout fermentation by Saccharomyces and non-Saccharomyces strains using headspace sorptive extraction.

Currently, there is a growing interest in the use of non-Saccharomyces yeast to enhance the aromatic quality of wine, with pure or mixed cultures, as well as sequential inoculation. Volatile components of wines were closely related to their sensory quality. Hence, to study the evolution of volatile compounds during fermentation was of great interest. For this, sampling methods that did not alter the volume of fermentation media were the most suitable. This work reports the usefulness of headspace sorptive extraction as non-invasive method to monitor the changes in volatile compounds during fermentation. This method allowed monitoring of 141 compounds throughout the process of fermentation by Saccharomyces cerevisiae and Lachancea thermotolerans strains. Both strains showed a similar ability to ferment a must with high sugar content. The S. cerevisiae strain produced higher amount of volatile compounds especially esters that constitutes fruity aroma than L. thermotorelans.

Characterization of polymeric pigments and pyranoanthocyanins formed in microfermentations of non-Saccharomyces yeasts.

To assess the influence of non-Saccharomyces yeasts on the pyranoanthocyanins and polymeric pigments formation after the addition of (+)-catechin and procyanidin B2 to fresh red grape must. The fermentation of red grape musts was done with non-Saccharomyces yeasts either alone or in sequential fermentations with the Saccharomyces cerevisiae species. The characterization of both pyranoanthocyanin and polymeric pigments has been carried out with liquid chromatography coupled to mass spectroscopy (HPLC-DAD-ESI/MS). Red wines were also characterized by infrared spectroscopy (FTIR), gas chromatography (GC-FID) and spectrophotometry (UV-Vis). It has been observed that fermentation with the species Schizosaccharomyces pombe led to higher concentrations of pigments of all types: anthocyanins, polymeric pigments and pyranoanthocyanins, particularly vitisin A. The use of non-Saccharomyces yeasts improve the formation of stable pigments in red wines thanks to the differences in the microbial metabolism from among the yeasts studied. Colour stability as one of the main organoleptic properties in red wines, may be improved by the controlled use of selected non-Saccharomyces yeasts during red must fermentation.

Impact of sequential co-culture fermentations on flavour characters of Solaris wines

Nowadays, the use of non-Saccharomyces yeasts in combination with Saccharomyces cerevisiae is being recognised to enhance the analytical composition of the wines. The aim of this work was to evaluate the influence of indigenous non-Saccharomyces yeasts on the flavour character of wines from the cool-climate grape cultivar Solaris in Denmark. The volatile and non-volatile compounds as well as the sensory properties of wines were evaluated. Solaris wines with Hanseniaspora uvarum sequentially inoculated with S. cerevisiae produced a larger amount of glycerol as well as heptyl acetate and 2-phenylethyl acetate. This co-culture fermentation also produced higher amounts of ethyl acetate and acetic acid, reducing the possibility of its use in winemaking. Three Metschnikowia strains, a M. chrysoperlae strain and two M. fructicola strains, gave a comparable production of volatile compounds. These wines were characterised by several floral and fruity attributes. The Metschnikowia strains turned out to be promising in winemaking from Solaris grapes.

Comparison of Fermentation and Wines Produced by Inoculation of Hanseniaspora vineae and Saccharomyces cerevisiae.

Interest in the use of non-Saccharomyces yeasts in winemaking has been increasing due to their positive contributions to wine quality. The non-Saccharomyces yeast Hanseniaspora vineae is an apiculate yeast that has been associated with the production of wine with good aromatic properties. However, little is known about the fermentation dynamics of H. vineae in natural must and its interaction with autochthonous yeasts. In the present study, we performed semi industrial fermentations of Macabeo and Merlot musts inoculated with either H. vineae or S. cerevisiae. The yeast population dynamics were monitored by plate culturing, PCR-DGGE and massive sequencing techniques. The results obtained with these techniques show that H. vineae was able dominate the autochthonous microbiota in Macabeo must but not in Merlot must, which exhibited a larger, more diverse yeast population. The presence of H. vineae throughout most of the Macabeo fermentation resulted in more fruity and flowery wine, as indicated by the chemical analysis of the final wines, which demonstrated a strong presence of phenyl ethyl acetate at concentrations higher than the threshold of perception and approximately 50 times more than that produced in wines fermented with S. cerevisiae. This compound is associated with fruity, floral and honey aromas.

Use of Schizosaccharomyces pombe and Torulaspora delbrueckii strains in mixed and sequential fermentations to improve red wine sensory quality

One of the main opportunities in the use of non-Saccharomyces yeasts is its great intraspecific variability in relation to the synthesis of secondary products of fermentation. Thus, mixed or sequential fermentation with non-Saccharomyces can increase the synthesis of certain metabolites that are important for colour stability, such as acetaldehyde and pyruvic acid (vitisin precursors) or vinylphenols (vinylphenolic pyranoanthocyanin precursors). Furthermore, the selection and use of non-Saccharomyces yeast strains with good yields in the production of certain volatile compounds (ethyl lactate, 2,3-butanediol, 2-phenylethyl acetate), with limited formation of higher alcohols, is a way to improve the aromatic profile of red wine. The main aim of this work was to evaluate the influence of sequential and mixed fermentations with Schizosaccharomyces pombe and Torulaspora delbrueckii strains on red wine's sensory quality. Anthocyanins and aromatic profiles, as well as glycerol and organic acid content, were analysed in the red wines obtained. Results show that, in general, mixed fermentations can promote an increment in polyols synthesis, while sequential fermentations can enhance the herbaceous aroma. Moreover, the use of T. delbrueckii in mixed fermentations allowed an increase to the fruity character of red wine. The use of S. pombe in sequential fermentations increased the stability of the colouring matter by favouring vitisins and vinylphenolic pyranoanthocyanin formation.

Sequential culture with Torulaspora delbrueckii and Saccharomyces cerevisiae and management of fermentation temperature to improve cherry wine quality.

There has been limited research on the use of non-Saccharomyces yeasts for the production of cherry wines. This work used an autochthonous Torulaspora delbrueckii strain 49 (TD49) in association with a commercial S. cerevisiae RC212 yeast, to investigate the effect of multi-starter culture (sequential inoculation and simultaneous inoculation) and fermentation temperature on the quality of cherry wines. Both TD49 and RC212 proliferated during alcoholic fermentation (AF) under sequential inoculation conditions, whereas in the case of simultaneous inoculation, TD49 increased slowly at first and then declined sharply near the fermentation end. The analytical profile showed that both mixed fermentations produced lower levels of volatile acidy and higher levels of aromatic compounds than those from RC212 mono-culture. During sensory analysis, wines from sequential fermentation obtained the highest score, mainly due to the higher intensity in 'fruity' and 'floral' characters. As for the influence of temperature, a low temperature (20 °C) enhanced TD49 persistence during AF, but the sensory quality decreased anyway; 30 °C resulted in decreases in most measured descriptors. Therefore, 25 °C was selected as the best culture temperature. TD49/RC212 sequential inoculation and fermentation at 25 °C significantly enhanced the cherry wine quality.

Exploitation of the non-Saccharomyces yeast Starmerella bacillaris (synonym Candida zemplinina) in wine fermentation: Physiological and molecular characterizations

Nowadays, the use of non-Saccharomyces yeasts in combination with Saccharomyces cerevisiae is a state-of-the-art strategy to improve complexity and enhance the analytical composition of the wines. This application has stimulated the interest of understanding how the non-Saccharomyces yeasts can contribute to the quality of the wines. The study presented here explores the potential use of Starmerella bacillaris (synonym Candida zemplinina) under winemaking conditions. Physiological and genetic characterizations of sixty-three isolates of Starm. bacillaris, previously isolated from four different varieties of grapes, were carried out. Both analyses revealed a low level of diversity between the isolates of Starm. bacillaris, while the fermentation trials in laboratory scale demonstrated the good enological performance of this species. The strong fructophilic character of this species and its ability to produce low quantities of ethanol and acetic acid and high amounts of glycerol were confirmed. The results, presented here, demonstrated a potential application of this non-Saccharomyces species in mixed wine fermentations with S. cerevisiae.

Oenological consequences of sequential inoculation with non-Saccharomyces yeasts (Torulaspora delbrueckii or Metschnikowia pulcherrima) and Saccharomyces cerevisiae in base wine for sparkling wine production

The use of non-Saccharomyces yeasts for the production of quality wine has become increasingly frequent in recent years. Several studies of the influence of Torulaspora delbrueckii and Metschnikowia pulcherrima on chemical composition have been reported, especially in aspects concerning aroma. The aim of this article was to study the influence of sequential inoculation of these non-Saccharomyces yeasts and Saccharomyces cerevisiae on the composition and quality of base wine for sparkling wine production. The results indicate that sequential inoculation with non-Saccharomyces yeasts may be an interesting tool for obtaining base wines with different characteristics. On the one hand, T. delbrueckii Biodiva™ strain increased glycerol concentration, reduced volatile acidity and exerted a positive effect on foaming properties improving foamability (Hm) and foam persistence (Hs). On the other hand, M. pulcherrima Flavia® strain also increased foam persistence (Hs) and changed the aromatic profile, increasing smoky and flowery notes.

Fermentative aptitude of non-Saccharomyces wine yeast for reduction in the ethanol content in wine

Over the last few decades, there has been a progressive increase in the ethanol content in wines due to global climate change and to the new wine styles that are associated with increased grape maturity. This increased ethanol content can have negative consequences on the sensory properties of the wines, human health, and economic aspects. Several microbiological approaches for decreasing the ethanol content have been suggested, such as strategies based on genetically modified yeasts, the adaptive evolution of yeasts, and the use of non-Saccharomyces yeast. In the present study, we investigated the interspecies and intraspecies variability of some non-Saccharomyces wine yeast species under anaerobic fermentation conditions. Across different grape juices and fermentation trials, Hanseniaspora uvarum, Zygosaccharomyces sapae, Zygosaccharomyces bailii, and Zygosaccharomyces bisporus promoted significant reductions in ethanol yield and fermentation efficiency in comparison with Saccharomyces cerevisiae. The diversion of alcoholic fermentation and the abundant formation of secondary compounds might explain the marked reduction in ethanol yield, as determined through the segregation of the majority of the strains according to their species attributes observed using principal component analysis. These data suggest that careful evaluation of interspecies and intraspecies biodiversity can be carried out to select yeast that produces low-ethanol yields.

Non-Saccharomyces wine yeasts have a promising role in biotechnological approaches to winemaking

Biotechnology as applied to winemaking includes several aspects of the fermentation industry, such as monitoring of microbial populations, use of selected starter cultures, and control of undesired yeasts. Over the last few decades, the control of microorganisms using biotechnological approaches has become of increasing importance in the winemaking field. The profusion of selected starter strains has allowed more extensive use of inoculated fermentations, with a consequent improvement in the control of fermentation combined with the use of new biotechnological processes in winemaking. As a consequence of this re-evaluation of the role of non-Saccharomyces yeasts in winemaking over the last few years, several studies have evaluated the use of controlled mixed fermentations using Saccharomyces and different, non-Saccharomyces, yeast species that are a part of the winemaking environment. In this context, mixed fermentations using controlled inoculations of Saccharomyces cerevisiae starter cultures and non-Saccharomyces yeasts represent a practical way towards improving wine complexity and enhancing specific characteristics of a wine. Another trait in the use of non-Saccharomyces yeasts in winemaking relates to the control of spoilage microorganisms. Indeed, more strict control of undesirable yeasts is required during the various phases of wine fermentation. Moreover, there is now increasing interest in the use of natural antimicrobial agents in foods and, in this context, killer yeasts might have important roles in the control of spontaneous and/or spoilage microflora. Thus, killer toxins appear to represent an attractive solution for use as antimicrobial agents, to partially, or even completely, substitution for chemical agent use even if application costs could limit their use in winemaking.

Chapter 4: Safety evaluation of foods and food ingredients derived from microorganisms

The application of non-Saccharomyces yeasts in beer as a natural tool for innovation, to create different aroma profiles and flavoured non-alcoholic beers, has attracted great interest from both researchers and commercial brewers. As a result, a higher diversity of non-Saccharomyces yeasts for beer production is expected on the market in the coming years. However, the safe use of non-Saccharomyces yeasts has not been broadly investigated and no guidance for the safety assessment of yeasts is published. The fundamentals of a safety assessment include an accurate taxonomic species identification using up-to date methods, along with a literature study regarding the yeast species in question. The strain-specific safety concerns that should be assessed involve pathogenic potential, antifungal resistance, production of biogenic amines and possible allergic reactions. However, yeast safety assessment is in its infancy compared to bacterial safety assessment and research is needed to set cut-off values for antifungal resistance, identify potential virulence genes and validate screening tools to assess yeast strains. Finally, the individual breweries are responsible for the safety related to the process in which yeasts are applied and throughout the shelf life of the beer. The application of non-Saccharomyces yeasts for industrial beer production is promising in terms of defining new prototypes and developing healthier and safer beers, but only if good food safety measures, i.e., both for the strain and the production process, are in place throughout the food value chain. In this way, the ancient role of yeasts in making beverages safer and thereby improving food safety is emphasized.