The effect of nitrogen source on yeast metabolism and H2S formation

The impacts of aspartic acid and glutamine used as nitrogen supplements for alcoholic fermentations conducted by Saccharomyces were studied. Synthetic grape juice media and commercially prepared grape juices were supplemented with diammonium phosphate, aspartic acid, or glutamine to increase yeast-assimilable nitrogen concentrations to 250 mg N/L prior to fermentation. Two yeast strains, UCD522 and EC1118, were inoculated at 10⁵ CFU/mL and fermentations were monitored for soluble solids, hydrogen sulfide (H₂S), and residual amino acids. In general, unsupplemented media/juices fermented slower than supplemented ones, produced more H₂S, and contained lower concentrations of amino acids after fermentation. Among the supplemented treatments, fermentation rates, H₂S production, and amino acid utilization varied depending on the nitrogen source and yeast strain. Those fermentations supplemented with aspartic acid were generally slower and sometimes did not achieve dryness. In contrast, glutamine additions yielded fermentation rates and H₂S production equivalent or better than other supplemented treatments. Based on these results, the use of glutamine appears promising as an alternative nitrogen supplement for wine production.     

Alleviation of stuck wine fermentations using salt‐preconditioned yeast

The influence of salt (sodium chloride) on the cell physiology of wine yeast was investigated. Cellular viability and population growth of three wine‐making yeast strains of Saccharomyces cerevisiae, and two non‐Saccharomyces yeast strains associated with wine must microflora (Kluyveromyces thermotolerans and K. marxianus) were evaluated following salt pre‐treatments. Yeast cells growing in glucose defined media exposed to different sodium chloride concentrations (4, 6 and 10% w/v) exhibited enhanced viabilities compared with nontreated cultures in subsequent trial fermentations. Salt ‘preconditioning’ of wine yeast seed cultures was also shown to alleviate stuck and sluggish fermentations at the winery scale, indicating potential benefits for industrial fermentation processes. It is hypothesized that salt induces specific osmostress response genes to enable yeast cells to better tolerate the rigours of fermentation, particularly in high sugar and alcohol concentrations. Copyright © 2014 The Institute of Brewing & Distilling     

Altered Patterns of Maltose and Glucose Fermentation by Brewing and Wine Yeasts Influenced by the Complexity of Nitrogen Source

Maltose and glucose fermentations by industrial brewing and wine yeasts strains were strongly affected by the structural complexity of the nitrogen source. In this study, four Saccharomyces cerevisiae strains, two brewing and two wine yeasts, were grown in a medium containing maltose or glucose supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low sugar concentration for brewing and wine strains, independent of nitrogen supplementation, and the type of sugar. At high sugar concentrations altered patterns of sugar fermentation were observed, and biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for brewing and wine strains. In maltose, high biomass production was observed under peptone and casamino acids for the brewing and wine strains, however efficient maltose utilization and high ethanol production was only observed in the presence of casamino acids for one brewing and one wine strain studied. Conversely, peptone and casamino acids induced higher biomass and ethanol production for the two other brewing and wine strains studied. With glucose, in general, peptone induced higher fermentation performance for all strains, and one brewing and wine strain produced the same amount of ethanol with peptone and casamino acids supplementation. Ammonium salts always induced poor yeast performance. The results described in this paper suggest that the complex nitrogen composition of the cultivation medium may create conditions resembling those responsible for inducing sluggish/stuck fermentation, and indicate that the kind and concentration of sugar, the complexity of nitrogen source and the yeast genetic background influence optimal industrial yeast fermentation performance.     

Selection and molecular characterization of wine yeasts isolated from the ‘El Penedès’ area (Spain)

A study of the microbiota present during the wine fermentation of five grape varieties from the ‘El Penedès’ area (Spain) was carried out to select autochthonous yeast strains for industrial wine production. In this study we identified members of the genera Candida, Dekkera, Hanseniaspora, Kluyveromyces, Torulaspora, Zygosaccharomyces and Saccharomyces in wine fermentation microbiota. Strains of Saccharomyces cerevisiae, as responsible agents of the alcoholic fermentation, were considered for a selection protocol. In this work we applied different enological criteria for selection, but previously we have characterized and differentiated Saccharomyces isolates by molecular methods to reduce the number of strains to analyse. Three strains were selected to conduct fermentations according to their characteristics. Finally, using mitochondrial DNA restriction analysis we demonstrated that the autochthonous selected strains are important contributors to the wine fermentation.     

Effect of grape indigenous Saccharomyces cerevisiae strains on Montepulciano d'Abruzzo red wine quality

The growth of selected, indigenous Saccharomyces cerevisiae added as starters (SRS1, MS72 and RT73) was monitored during Montepulciano d'Abruzzo wine production. In all the fermentations the addition of the starter, caused a decrease of the non-Saccharomyces yeasts. When strains MS72 and RT73 were used as starters they were detected in the first phases of fermentations, while strain SRS1 competed successfully with native yeasts during all the process. Wines obtained by fermentation with the indigenous starters showed some different characteristics, according to the chemical and sensory analyses. This study highlighted that among selected starters with high fermentative capacity, some are able to dominate better than other natural wine yeast biota, whereas some strains can interact and survive besides native yeast populations during the fermentation. As a consequence, the dominance character can have a positive or negative effect on wine quality and has to be considered in the frame of yeast selection in order to improve or characterize traditional wines. Winemakers could choose among different degrees of yeast dominance to modulate the interaction among starter and native wine yeast population.     

The hexose transporters of Saccharomyces cerevisiae play different roles during enological fermentation

We investigated the role of hexose transporters in a Saccharomyces cerevisiae strain derived from an industrial wine strain by carrying out a functional analysis of HXT genes 1-7 under enological conditions. A strain in which the sugar carrier genes HXT1-HXT7 were deleted was constructed and the HXT genes were expressed individually or in combination to evaluate their role under wine alcoholic fermentation conditions. No growth or fermentation was observed in winemaking conditions for the hxt1-7 delta strain. The low-affinity carriers Hxt1 and Hxt3 were the only carriers giving complete fermentation of sugars when expressed alone, indicating that these carriers play a predominant role in wine fermentation. However, these two carriers have different functions. The Hxt3 transporter is thought to play a major role, as it was the only carrier that gave an almost normal fermentation profile when produced alone. The hxt1 carrier was much less effective during the stationary phase and its role is thought to be restricted to the beginning of fermentation. The high-affinity carriers Hxt2, Hxt6 and/or Hxt7 were also required for normal fermentation. These high-affinity transporters have different functions: hxt2 is involved in growth initiation, whereas Hxt6 and/or Hxt7 are required at the end of alcoholic fermentation. This work shows that the successful alcoholic fermentation of wine involves at least four or five hexose carriers, playing different roles at various stages in the fermentation cycle.     

Screening for new brewing yeasts in the non‐Saccharomyces sector with Torulaspora delbrueckii as model

This study describes a screening system for future brewing yeasts focusing on non‐Saccharomyces yeasts. The aim was to find new yeast strains that can ferment beer wort into a respectable beer. Ten Torulaspora delbrueckii strains were put through the screening system, which included sugar utilization tests, hop resistance tests, ethanol resistance tests, polymerase chain reaction fingerprinting, propagation tests, amino acid catabolism and anabolism, phenolic off‐flavour tests and trial fermentations. Trial fermentations were analysed for extract reduction, pH drop, yeast concentration in bulk fluid and fermentation by‐products. All investigated strains were able to partly ferment wort sugars and showed high tolerance to hop compounds and ethanol. One of the investigated yeast strains fermented all the wort sugars and produced a respectable fruity flavour and a beer of average ethanol content with a high volatile flavour compound concentration. Two other strains could possibly be used for pre‐fermentation as a bio‐flavouring agent for beers that have been post‐fermented by Saccharomyces strains as a consequence of their low sugar utilization but good flavour‐forming properties. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

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.     

Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae

Non-Saccharomyces yeasts are metabolically active during spontaneous and inoculated must fermentations, and by producing a plethora of by-products, they can contribute to the definition of the wine aroma. Thus, use of Saccharomyces and non-Saccharomyces yeasts as mixed starter cultures for inoculation of wine fermentations is of increasing interest for quality enhancement and improved complexity of wines. We initially characterized 34 non-Saccharomyces yeasts of the genera Candida, Lachancea (Kluyveromyces), Metschnikowia and Torulaspora, and evaluated their enological potential. This confirmed that non-Saccharomyces yeasts from wine-related environments represent a rich sink of unexplored biodiversity for the winemaking industry. From these, we selected four non-Saccharomyces yeasts to combine with starter cultures of Saccharomyces cerevisiae in mixed fermentation trials. The kinetics of growth and fermentation, and the analytical profiles of the wines produced indicate that these non-Saccharomyces strains can be used with S. cerevisiae starter cultures to increase polysaccharide, glycerol and volatile compound production, to reduce volatile acidity, and to increase or reduce the total acidity of the final wines, depending on yeast species and inoculum ratio used. The overall effects of the non-Saccharomyces yeasts on fermentation and wine quality were strictly dependent on the Saccharomyces/non-Saccharomyces inoculum ratio that mimicked the differences of fermentation conditions (natural or simultaneous inoculated fermentation).     

Effect of growth temperature on yeast lipid composition and alcoholic fermentation at low temperature

The loss of viability of wine yeast strains due to low-temperature fermentations could be overcome by increasing their stress tolerance and adaptability. Changes in membrane lipid composition are one of the first responses to cold stress. The aim of this study was to analyze the various adaptation mechanisms to low temperatures by comparing the better adapted Saccharomyces species. The viability, vitality, fermentation capacity, and lipid composition of different Saccharomyces species (S. cerevisiae, S. bayanus, S. uvarum, and a hybrid S. cerevisiae/S. uvarum) with different fermentative origins (wine, beer, and baker’s strains together with a laboratory strain) were compared after culturing at low (13 °C) and optimal (25 °C) temperatures. In spite of specific responses of the different strains/species, the results showed that at low temperature, the medium-chain fatty acid and the triacylglyceride content increased, whereas the phosphatidic acid content and the phosphatidylcholine/phosphatidylethanolamine ratio decreased. Only the laboratory strain was not able to ferment the sugars, and after growing at both temperatures, its lipid composition was very different from that of the other strains. The hybrid strain showed the highest sugar consumption at 13 °C and the best vitality whatever the preculture temperature used. The rest of the species needed a preadaptation at low temperature involving a change in their lipid composition to improve their fermentation rate at 13 °C.     

Isolation and Characterisation of Sri Lankan Yeast Germplasm and Its Evaluation for Alcohol Production

Use of inferior yeast cultures represents one of the reasons for low fermentation efficiencies in Sri Lankan alcohol distilleries that use sugarcane molasses. The present study isolated and characterised yeast strains found in natural environments in Sri Lanka and evaluated their performance under laboratory conditions in an effort to select superior strains for industrial fermentations. Yeasts were characterised based on morphological and physiological features such as sugar fermentation and nitrate assimilation. Ethanol production, alcohol tolerance and growth rate of the most promising strains were monitored following laboratory fermentations of molasses. Over a thousand yeast cultures were collected and screened for fermentative activity and a total of 83 yeast isolates were characterised as higher ethanol producers. Most of these belonged to the genus Saccharomyces. Certain strains produced over 10% (v/v) alcohol in molasses media during 72 h laboratory fermentations. Only two strains, SL‐SRI‐C‐102 and 111, showed an appreciable fermentation efficiency of about 90%. The latter strain produced the highest level of ethanol, 11% (v/v) within a 48 h fermentation and exhibited improved alcohol tolerance when compared with the baker's yeast strains currently used in Sri Lankan alcohol distilleries. This study highlights the benefits of exploiting indigenous yeasts for industrial fermentation processes.     

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.     

Review: Pure non‐Saccharomyces starter cultures for beer fermentation with a focus on secondary metabolites and practical applications

Recently there has been increased interest in using non‐Saccharomyces yeasts to ferment beer. The worldwide growth of craft beer and microbreweries has revitalised the use of different yeast strains with a pronounced impact on aroma and flavour. Using non‐conventional yeast gives brewers a unique selling point to differentiate themselves. Belgian brewers have been very successful in using wild yeasts and mixed fermentations that often contain non‐Saccharomyces yeasts. Historically, ancient beers and beers produced before the domestication of commonly used Saccharomyces strains most likely included non‐Saccharomyces species. Given the renewed interest in using non‐Saccharomyces yeasts to brew traditional beers and their potential application to produce low‐alcohol or alcohol‐free beer, the fermentation and flavour characteristics of different species of non‐Saccharomyces pure culture yeast were screened for brewing potential (Brettanomyces anomalus and bruxellensis, Candida tropicalis and shehatae, Saccharomycodes ludwigii, Torulaspora delbrueckii, Pichia kluyveri, Zygosaccharomyces rouxii). Alcohol‐free beer is already industrially produced using S. ludwigii, a maltose‐negative species, which is a good example of the introduction of non‐Saccharomyces yeast to breweries. Overall, non‐Saccharomyces yeasts represent a large resource of biodiversity for the production of new beers and have the potential for wider application to other beverage and industrial applications. Almost all of the trials reviewed were conducted with varying fermentation parameters, which plays an important role in the outcome of the studies. To understand these impacts all trials were described with their major fermentation parameters. Copyright © 2016 The Institute of Brewing & Distilling     

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.     

Glucose and Fructose Fermentation by Wine Yeasts in Media Containing Structurally Complex Nitrogen Sources

Glucose and fructose fermentations by industrial yeasts strains are strongly affected by both the structural complexity of the nitrogen source and the availability of oxygen. In this study two Saccharomyces cerevisiae industrial wine strains were grown, under shaken and static conditions, in a media containing either a) 20% (w/v) glucose, or b) 10% (w/v) fructose and 10% (w/v) glucose or c) 20% (w/v) fructose, all supplemented with nitrogen sources varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Data suggest that a complex structured nitrogen source is not submitted to the same control mechanisms as those involved in the utilization of simpler structured nitrogen sources, and mutual interaction between carbon and nitrogen sources, including the mechanisms involved in the regulation of aerobic/anaerobic metabolism, may play an important role in defining yeast fermentation performance and the differing response to the structural complexity of the nitrogen source, with a strong impact on fermentation performance.     

Biotechnological potential of non-Saccharomyces yeasts isolated during spontaneous fermentations of Malvar (Vitis vinifera cv. L.)

Non-Saccharomyces yeast species assume an important role in wine flavor. Notwithstanding, the chemical basis for the flavor characteristics of wines from some grape varieties is not yet defined. The value of this work lies in the use of Malvar white grape, an autochthonous variety from Madrid (Spain) winegrowing region to conduct spontaneous fermentations. This is the first time that a comparative characterization of a wide range of non-Saccharomyces species and a comprehensive analysis of these yeast-derived volatiles has been carried out in this grape variety. β-glucosidase and pectinase (polygalacturonase) extracellular activities were tested on agar plates as primary selection criteria among the 504 non-Saccharomyces isolated from Malvar spontaneous fermentations during four consecutive harvests. Analysis of the wines obtained after fermentation using the selected yeast strains indicates that non-Saccharomyces yeasts isolated along the fermentative process seem that could have a positive impact, showing a high variability in the volatile compounds contributing to the organoleptic characteristics of Malvar wines. Torulaspora delbrueckii CLI 918 was defined as the yeast strain with potential interest for its contribution to the aromatic wine profile with flowery and fruity aromas and could be used in mixed starter cultures with Saccharomyces cerevisiae. However, Hanseniaspora guilliermondii increased the volatile acidity and ethyl acetate, but this species along with the genus Pichia and Candida seem to provide a high quantity of extracellular enzymes which may be beneficial in wine making.     

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.     

Lipid composition of wine strains of Saccharomyces kudriavzevii and Saccharomyces cerevisiae grown at low temperature

Some species of the Saccharomyces genus have shown better adaptation at low temperature than the wine yeast Saccharomyces cerevisiae. That is the case of the cryophilic yeast Saccharomyces kudriavzevii. Several studies have revealed the importance of the lipid composition in the yeast adaptive response at different environmental temperatures. Thus we analysed the lipid composition of three S. kudriavzevii strains during growth at optimum (28°C) and low temperature (12°C), and compared them with different commercial strains; one S. cerevisiae strain and two hybrids between S. cerevisiae and S. kudriavzevii. Our results show a general increase in the medium-chain fatty acid, triacylglyceride, sterol esters and squalene and a decrease in the chain length of the fatty acids, in phosphatidic acid and in the ratio phosphatidylcholine/phosphatidylethanolamine at low temperatures. The S. kudriavzevii strains had higher percentages of medium-chain fatty acids and squalene and shorter chain lengths regardless of the growth temperature. This differential lipid composition may partially explain the better adaptation of S. kudriavzevii at low temperatures. We have also confirmed the better fermentation performance of the strains of this species at low temperature, being an appealing alternative to S. cerevisiae for cold fermentations.