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     

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.     

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).     

Evaluation of ITS PCR and RFLP for Differentiation and Identification of Brewing Yeast and Brewery ‘Wild’ Yeast Contaminants

A reference library of ITS PCR/RFLP profiles was collated and augmented to evaluate its potential for routine identification of domestic brewing yeast and known ‘wild’ yeast contaminants associated with wort, beer and brewing processes. This library contains information on band sizes generated by restriction digestion of the ribosomal RNA‐encoding DNA (rDNA) internal transcribed spacer (ITS) region consisting of the 5.8 rRNA gene and two flanking regions (ITS1 and ITS2) with the endonucleases CfoI, HaeIII, HinfI and includes strains from 39 non‐Saccharomyces yeast species as well as for brewing and non‐brewing strains of Saccharomyces. The efficacy of the technique was assessed by isolation of 59 wild yeasts from industrial fermentation vessels and conditioning tanks and by matching their ITS amplicon sizes and RFLP profiles with those of the constructed library. Five separate, non‐introduced yeast taxa were putatively identified. These included Pichia species, which were associated with conditioning tanks and Saccharomyces species isolated from fermentation vessels. Strains of the lager yeast S. pastorianus could be reliably identified as belonging to either the Saaz or Frohberg hybrid group by restriction digestion of the ITS amplicon with the enzyme HaeIII. Frohberg group strains could be further sub‐grouped depending on restriction profiles generated with HinfI.     

THE INCIDENCE AND SIGNIFICANCE OF WILD SACCHAROMYCES CONTAMINANTS IN THE BREWERY

The persistence of low levels of contamination by non-brewing Saccharomyces through several batch fermentations establishes the immuno-fluorescent method as a very sensitive procedure for estimating the microbiological purity of pitching yeasts. Trade return figures for draught beers show that in this brewery the principal cause for high rejection rates has, on several occasions, been contamination of pitching yeasts with “wild” Saccharomyces. The recommendation is made that pitching yeasts should be discarded when the level of infection achieves 100 cells of wild Saccharomyces per million cells of brewing yeast.     

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     

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.     

Performance of wild Saccharomyces and Non-Saccharomyces yeasts as starter cultures in dough fermentation and bread making

The use of wild Saccharomyces and non-Saccharomyces yeasts might result in bread with different and attractive sensory characteristics. This study aimed to evaluate the performance of Saccharomyces and non-Saccharomyces yeasts as starter culture in dough fermentation to bread making and the physicochemical parameters and aromatic profile of bread. All 26 wild yeasts strains isolated from Brazilian Cerrado fruit and tree bark were osmotolerant, and 19.4% were able to ferment maltose. Candida tropicalis ART101.3 and Saccharomyces cerevisiae SC5952 had the best growth capacity under high concentrations of glucose and maltose. Also, they were resistant to lyophilisation. Kinetic parameters of bioreactor cultivations showed high cell growth and lower generation time with 10 g L⁻¹ maltose. Bread produced with C. tropicalis ART101.3 and the control bread had similar physicochemical properties and acceptance of consumers. Bread with S. cerevisiae SC5951 had a lower specific volume and a different colour than control bread; however, the consumers found no significant difference. More than 70% of the consumers demonstrated purchase intention of bread produced with both wild yeasts. The present study shows the potential of native Cerrado yeasts to be used and exploited in industrial processes and contributes to the diversification of bread starter cultures.     

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.     

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.     

Quantitative and qualitative analysis of non-Saccharomyces yeasts in spontaneous alcoholic fermentations

In this study, we looked at the yeast population present in four spontaneous alcoholic fermentations in the Rioja appellation (D.O.Ca. Rioja, Spain). The study was conducted through the identification of the yeasts via the PCR–RFLP technique of the ITS region of rDNA. In a first harvest, the qualitative diversity of the species present in spontaneous alcoholic fermentation was studied, and in a second harvest, their quantitative significance. In spontaneous fermentations, 17 different yeast species were found, although only two of them, Candida stellata and Kloeckera apiculata, as well as Saccharomyces cerevisiae, appeared in major proportions during fermentation. The significance of the non-Saccharomyces yeasts during the spontaneous alcoholic fermentation was conditioned by the presence of Saccharomyces cerevisiae in the medium. Species not cited in literature in connection with winemaking and yeasts associated with wines spoilage have been detected in all the alcoholic fermentations carried out in the qualitative study.     

Yeasts found in vineyards and wineries

Wine is a complex beverage, comprising thousands of metabolites that are produced through the action of a plethora of yeasts and bacteria during fermentation of grape must. These microbial communities originate in the vineyard and the winery and reflect the influence of several factors including grape variety, geographical location, climate, vineyard spraying, technological practices, processing stage and season (pre‐harvest, harvest, post‐harvest). Vineyard and winery microbial communities have the potential to participate during fermentation and influence wine flavour and aroma. Therefore, there is an enormous interest in isolating and characterising these communities, particularly non‐Saccharomyces yeast species to increase wine flavour diversity, while also exploting regional signature microbial populations to enhance regionality. In this review we describe the role and relevance of the main non‐Saccharomyces yeast species found in vineyards and wineries. This includes the latest reports covering the application of these species for winemaking; and the biotechnological characteristics and potential applications of non‐Saccharomyces species in other areas. In particular, we focus attention on the species for which molecular and genomic tools and resources are available for study. Copyright © 2016 John Wiley & Sons, Ltd.     

PERFORMANCE OF STRAINS OF SACCHAROMYCES CEREVISIAE IN BATCH FERMENTATION

The properties of 153 strains of Saccharomyces cerevisiae, isolated from British breweries, have been determined in batch fermentations. A wide range of expression of technically significant characteristics has been observed. Head formation is absent in over one-quarter of the yeasts. While physical aspects of yeast behaviour determine the degree of fermentation which occurs with some yeasts, the loss of fermentative ability is of major importance with others. Fermentation rate is determined during the first 24 hr. after inoculation. It is found impossible to predict fermentation characteristics from giant colony morphology.     

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.     

New applications for Schizosaccharomyces pombe in the alcoholic fermentation of red wines

The fermentation of grape must using non‐Saccharomyces yeasts with particular metabolic and biochemical properties is of growing interest. In the present work, red grape must was fermented using four strains of Schizosaccharomyces pombe (935, 936, 938 and 2139), Saccharomyces cerevisiae 7VA and Saccharomyces uvarum S6U, and comparisons were made over the fermentation period in terms of must sugar (glucose + fructose), malic acid, acetic acid, ammonia, primary amino nitrogen, lactic acid, urea (a possible fermentation activator or precursor of other metabolites) and pyruvic acid (a molecule affecting vitisin formation and therefore colour stability) concentration. The colour intensity of the fermenting musts was also recorded. The Schizosaccharomyces strains consumed less primary amino nitrogen and produced less urea and more pyruvic acid than other Saccharomyces species. Further, three of the four Schizosaccharomyces strains completed the breakdown of malic acid by day 4 of fermentation. The main negative effect of the use of Schizosaccharomyces was strong acetic acid production. The Schizosaccharomyces strains that produced most pyruvic acid (938 and 936) were associated with better ‘wine’ colour than the remaining yeasts. The studied Schizosaccharomyces could therefore be of oenological interest.     

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.     

Susceptibility and resistance to ethanol in Saccharomyces strains isolated from wild and fermentative environments

In this work, we apply statistical modelling techniques to study the influence of increasing concentrations of ethanol on the overall growth of 29 yeast strains belonging to different Saccharomyces and non‐Saccharomyces species. A modified Gompertz equation for decay was used to objectively estimate the noninhibitory concentration (NIC) and minimum inhibitory concentration (MIC) for the assayed strains to ethanol, which are related to the susceptibility and resistance of yeasts to this compound, respectively. A first ANOVA analysis, grouping strains as a function of their respective Saccharomyces species, revealed that S. cerevisiae was the yeast with the highest, and statistically significant, ethanol resistance value. Then, a second factorial ANOVA analysis, using the origin of strains (wild or fermentative) and their taxonomic classification (S. cerevisiae, S. paradoxus or S. bayanus var. uvarum) as categorical predictor variables, showed that no significant differences for the NIC and MIC parameters were found between both ecological niches within the same species, indicative that these physiological characteristics were presumably not modified throughout the adaptation to human‐manipulated fermentative environments. Finally, differences among selected strains with respect to ethanol tolerance were correlated to the initial contents of unsaturated fatty acids, mainly oleic acid. Copyright © 2010 John Wiley & Sons, Ltd.     

Permanence of yeast inocula in the winery ecosystem and presence in spontaneous fermentations

The oenological practice of systematic inoculation with active dried yeasts is commonly used by many wineries around the world. However, the use of these yeasts is not free from controversy, since this practice has occasionally been described as having a negative effect on the biodiversity of natural yeast present in the wineries. The purpose of this study is to analyse the presence of commercial yeasts used as inocula in the ecosystem of three D.O.Ca. (“Qualified” Designation of Origin) Rioja wineries. It studies the permanence of these yeasts in winery equipment and their participation in spontaneous fermentations where they have not been used for inoculation. The results indicate that the presence of the active dry yeasts used in the wineries was scarce or non-existent, both in the ecosystem of each winery and in the spontaneous fermentations where they had not been added. So, repeated inoculation with active dry yeasts allowed a high presence and development of autochthonous (Saccharomyces and non-Saccharomyces) yeasts, both in equipment and in the spontaneous fermentations carried out.     

Fermentation of grape must by sequential association of yeasts: Accumulation of pyruvic and α-ketoglutaric acid

Fermentations of grape (cv. Malvar) musts from two consecutive vintages were carried out using the autochthonous microflora, a sequential association of yeasts and conventional fermentations with addition of sulfur dioxide to the must. The pyruvic and α-ketoglutaric acid content over the course of fermentation was measured and showed that for both vintages tested the maximum accumulation of the ketoacid pyruvic acid took place several days earlier in fermentations using a sequential association of yeasts than in conventional fermentations. The accumulation of pyruvic acid was higher in the must made from grapes with a higher degree of ripening and the lowest level of added SO₂. In the fermentations using either a sequential association of yeasts or the autochthonous microflora with no added SO₂, accumulation of α-ketoglutaric acid was higher in the must with the higher nitrogen content when the species making the greatest percentage contributions at the start of fermentation presented high levels of proteolytic activity.     

Influence of yeast strain on binding of sulphur dioxide in wines,and on its formation during fermentation

The amounts of sulphur dioxide bound by acetaldehyde, pyruvic acid and α-ketoglutaric acid during fermentation of three grape juices by eight wine yeasts (Saccharomyces sp.) are reported. These constituents accounted for 49–83 % (mean 69) of the measured bound SO₂, depending on the yeast strain and juice. the maximum range of concentrations of the binding components for individual wines were 10–48 ppm for acetaldehyde, 9–77 ppm for pyruvic acid and 5–63 ppm for α-ketoglutaric acid, depending on yeast strain and grape juice. the validity of the calculations was verified by an experiment with SO₂ and the three binding compounds in a multicomponent model system. The acetaldehyde content was related to the total SO₂ present, which itself was determined by the strain of yeast. SOz bound in the wines after a further SO₂ addition was correlated significantly with pyruvic and α-ketoglutaric acids, but not with acetaldehyde. Certain yeasts produced SO₂ during fermentation in grape juice and in synthetic media with defined sulphur sources. More SO₂ was produced at pH 3.6 than 3.0 in the absence of added sulphate in grape juice. Sulphate was the best sulphur source for SO₂ production in synthetic media, although some yeasts were able to produce smaller amounts of SO₂ from l-cysteine and reduced glutathione.