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).     

FORMULATION AND TESTING OF CUPRIC SULPHATE MEDIUM FOR WILD YEAST DETECTION

A new differential medium, cupric sulphate medium, used for the detection of wild yeasts has been formulated and tested. This medium suppressed the growth of culture yeasts and supported that of most non-Saccharomyces wild yeasts. It is not suitable for the detection of Saccharomyces wild yeasts. The contaminating wild yeasts in yeast samples and swab samples were easily detected by this medium. Since Lin's medium or modified Lin's medium is suitable for the detection of Saccharomyces wild yeasts, it is suggested that they be used in conjunction with cupric sulphate medium for detecting a more complete spectrum of wild yeasts.     

Wine phenolic profile altered by yeast: Mechanisms and influences

Grape phenolic compounds undergo various types of transformations during winemaking under the influences of yeasts, which further impacts the sensory attributes, thus the quality of wine. Understanding the roles of yeasts in phenolics transformation is important for controlling wine quality through fermentation culture selection. This literature review discusses the mechanisms of how yeasts alter the phenolic compounds during winemaking, summarizes the effects of Saccharomyces cerevisiae and non-Saccharomyces yeasts on the content and composition of phenolics in wine, and highlights the influences of mixed cultural fermentation on the phenolic profile of wine. Collectively, this paper aims to provide a deeper understanding on yeast–phenolics interactions and to identify the current literature gaps for future research.     

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.     

The influence of pre‐fermentative practices on the dominance of inoculated yeast starter under industrial conditions

The influence of pre‐fermentative practices on the growth dynamics of a ‘natural’ starter culture with specific phenotype (H₂S^?) concurrently with wild yeast populations was evaluated under winery conditions. Different clarification procedures and added SO₂ strongly influenced species and cell numbers isolable at the pre‐fermentation stage. Independent treatments of must with sulphite addition or vacuum‐filtering clarification caused a 30‐fold reduction in viable cells. Clarification procedures, enhanced by the selective effect of SO₂ addition, induced the appearance of Saccharomyces cerevisiae ‘wild’ yeasts. Correct application of the inoculum generally guarantees the dominance of fermentation by starter cultures. However, inoculated fermentations using unclarified white and red musts exhibited a consistent presence and persistence of non‐Saccharomyces and/or Saccharomyces ‘wild’ yeasts during fermentation. The extent and composition of the initial wild microflora at the start of fermentation may affect the presence and persistence of wild Saccharomyces and non‐Saccharomyces yeasts during guided fermentations under commercial conditions. The above findings confirm the results of previous works carried out at laboratory‐ or pilot‐scale level. Furthermore, they suggest a clear correlation between the modality of pre‐fermentative practices and the presence and persistence of ‘wild’ yeasts during fermentation. © 2002 Society of Chemical Industry     

Presence of non-Saccharomyces yeasts in cellar equipment and grape juice during harvest time

The purpose of this study was to analyze the presence of different yeasts in the facilities of four wineries from the D.O.Ca. Rioja region in Spain. The study was conducted through the identification of the yeasts via the PCR-RFLP technique of the ITS region of rDNA. The diversity of non-Saccharomyces yeasts found in wineries has previously only been studied to a limited extent, despite the fact that these yeasts take part both in the start of spontaneous fermentation and in the changes which occur in the wines during their subsequent conservation. Most earlier studies carried out on cellar ecosystems have focussed on the clonal diversity of Saccharomyces cerevisiae. The results obtained in this study indicated that the presence of non-Saccharomyces yeasts in facilities is higher than that of the S. cerevisiae, with percentages of over 60% in all the wineries analyzed. Yeasts belonging to 10 genera and 18 species were isolated, but the only genera present in all four wineries were Cryptococcus, Pichia, and Saccharomyces. The Zygosaccharomyces bailii yeast responsible for taint was detected in one cleaned winery, in both the winemaking equipment and the fermenting must. It was also noted that the quantity and type of yeasts present in the facilities are related to the product used for cleaning them. It is also necessary to point out that the cleaning of the cellars prior to the reception of the grapes does not completely eliminate the yeasts present, so that these can subsequently become part of the vinification process.     

DETECTION OF WILD YEASTS IN THE BREWERY EFFICIENCY OF DIFFERENTIAL MEDIA

The efficiency of five differential media in the detection of wild yeasts was compared. On the basis of the types of wild yeast they are able to detect, these differential media were classified into two groups. Group I consists of actidione medium and lysine medium, and is suitable for the detection of non-Saccharomyces wild yeasts. In this group, the lysine medium detected more species and a higher percentage of wild yeasts than the actidione medium. Group II consists of crystal violet medium, SDM, and Lin's medium, and is suitable for the detection of Saccharomyces wild yeasts. In this group, Lin's medium is superior to crystal violet medium and SDM for wild yeast detection. It is suggested that lysine medium and Lin's medium be employed together for the detection of wild yeasts in the brewery.     

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.     

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.     

LONG-CHAIN FATTY ACID COMPOSITION AS A CRITERION FOR YEAST DISTINCTION IN THE BREWING INDUSTRY

The diversity of yeasts isolated from brewing plants and its role on beer quality makes yeast distinction a major concern in industrial microbiological control. Several approaches have been tried to develop rapid and simple methods to perform such tasks. Among these, stands the utilization of long-chain fatty acid composition of total yeast biomass. In this paper results are reported showing the potential of this technique to characterize yeast flora isolated from industrial plants. Fatty acid profiles of brewing species are clearly differentiated from those of non-Saccharomyces strains using statistical data treatment by principal component analysis (PCA). Distinction between brewing and wild strains of Saccharomyces spp. was not apparent. In comparison, fatty acid profiling showed higher discriminating ability than growth on lysine medium for non-Saccharomyces strains. For distinction of S. cerevisiae var. diastaticus from other Saccharomyces strains, growth on starch medium showed to be necessary.     

The ecology and evolution of non‐domesticated Saccharomyces species

Yeast researchers need model systems for ecology and evolution, but the model yeast Saccharomyces cerevisiae is not ideal because its evolution has been affected by domestication. Instead, ecologists and evolutionary biologists are focusing on close relatives of S. cerevisiae, the seven species in the genus Saccharomyces. The best‐studied Saccharomyces yeast, after S. cerevisiae, is S. paradoxus, an oak tree resident throughout the northern hemisphere. In addition, several more members of the genus Saccharomyces have recently been discovered. Some Saccharomyces species are only found in nature, while others include both wild and domesticated strains. Comparisons between domesticated and wild yeasts have pinpointed hybridization, introgression and high phenotypic diversity as signatures of domestication. But studies of wild Saccharomyces natural history, biogeography and ecology are only beginning. Much remains to be understood about wild yeasts' ecological interactions and life cycles in nature. We encourage researchers to continue to investigate Saccharomyces yeasts in nature, both to place S. cerevisiae biology into its ecological context and to develop the genus Saccharomyces as a model clade for ecology and evolution. © 2014 The Authors. Yeast published by John Wiley & Sons Ltd.     

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.     

Grape Processing by High Hydrostatic Pressure: Effect on Use of Non-<Emphasis Type="Italic">Saccharomyces</Emphasis> in Must Fermentation

Vitis vinifera (variety Tempranillo) grapes were pressurized at 400 MPa by high hydrostatic pressure for 10 min and the effect on wild microbial populations, phenol extraction and wine composition was monitored. After treatment, the grapes were inoculated and fermented with Saccharomyces cerevisiae and several non-Saccharomyces yeasts: Schizosaccharomyces pombe, Torulaspora delbrueckii, Metschnikowia pulcherrima and Lachancea thermotolerans. S. pombe was used as sole fermentative yeast, but T. delbrueckii, M. pulcherrima and L. thermotolerans were used in sequential cultures with S. cerevisiae to completely ferment the sugars. The HHP treatment reduces strongly or eliminates wild microorganisms, especially yeasts, facilitating the growth and development of non-Saccharomyces yeasts. Thus, it helps to get either a better expression of enzymatic activities or metabolites production of non-Saccharomyces affecting wine quality.     

GROWTH OF AEROBIC WILD YEASTS

Wild yeasts of the genera Debaryomyces, Hansenula and Pichia are commonly considered to be associated with spoilage only under aerobic conditions. However, in pure cultures in either wort or a synthetic medium of yeast nitrogen base + 10% glucose, yeasts of these genera grew as well as a brewing strain of Saccharomyces cerevisiae under anaerobic conditions. Growth of S. cerevisiae was increased by the addition of unsaturated fatty acid (Tween 80) or ergosterol to the medium for anaerobic culture. No equivalent requirement was observed for the wild yeasts examined. Indeed, growth of the wild yeasts was often reduced by the addition of Tween 80, which as a surfactant prevented formation of the surface film of growth. Even under anaerobic conditions, these yeasts grew best with a surface pellicle. Although capable of good anaerobic growth in pure culture, growth of the wild yeasts was suppressed under anaerobic conditions in mixed culture with S. cerevisiae, simulating a contaminated brewery fermentation. However, the contaminants competed successfully with S. cerevisiae under aerobic conditions. There was no evidence of a “killer” effect, but prevention of pellicle formation, or production of inhibitory levels of pH or ethanol under anaerobic conditions could explain the suppression of wild yeasts under anaerobic fermentation conditions.     

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.     

Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: A strategy to enhance acidity and improve the overall quality of wine

In the last few years there is an increasing interest on the use of mixed fermentation of Saccharomyces and non-Saccharomyces wine yeasts for inoculation of wine fermentations to enhance the quality and improve complexity of wines. In the present work Lachancea (Kluyveromyces) thermotolerans and Saccharomyces cerevisiae were evaluated in simultaneous and sequential fermentation with the aim to enhance acidity and improve the quality of wine.     

Bioprospecting for brewers: Exploiting natural diversity for naturally diverse beers

The burgeoning interest in archaic, traditional, and novel beer styles has coincided with a growing appreciation of the role of yeasts in determining beer character as well as a better understanding of the ecology and biogeography of yeasts. Multiple studies in recent years have highlighted the potential of wild Saccharomyces and non-Saccharomyces yeasts for production of beers with novel flavour profiles and other desirable properties. Yeasts isolated from spontaneously fermented beers as well as from other food systems (wine, bread, and kombucha) have shown promise for brewing application, and there is evidence that such cross-system transfers have occurred naturally in the past. We review here the available literature pertaining to the use of nonconventional yeasts in brewing, with a focus on the origins of these yeasts, including methods of isolation. Practical aspects of utilizing nondomesticated yeasts are discussed, and modern methods to facilitate discovery of yeasts with brewing potential are highlighted.     

Studies on acetate ester production by non-Saccharomyces wine yeasts

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

Yeasts Isolated from the Alcoholic Fermentation of Agave duranguensis During Mezcal Production

Mezcal is a spirit produced in some regions of México. In the state of Durango, mezcal is produced via traditional fermentation of the Agave duranguensis plant. To better understand traditional fermentation processes, it is necessary to know which yeast species are present in fermentations in different producer regions. The aim of this research was to study yeasts involved in traditional mezcal fermentation in Durango, México, and investigate the phylogeny of the native Saccharomyces cerevisiae strains involved in this process. The 5.8S-ITS genomic region was analyzed to identify strains present in the fermentation process samples in this study. To differentiate strains belonging to the genus Saccharomyces, different molecular techniques were used, including analysis of mitochondrial DNA and δ elements and sequencing of the 5.8S-ITS genomic region. Although a high diversity of microorganisms was found at the beginning of fermentation, Saccharomyces cerevisiae was the only yeast present at the end of fermentation in region I, while Torulaspora delbrueckii was found in a higher number than S. cerevisiae at the end of fermentation in the region II. Molecular techniques demonstrated that Saccharomyces cerevisiae isolated in Durango are phylogenetically independent from the strains isolated in other regions of Latin America and Europe.     

Flavour of sourdough wheat bread crumb

This study investigates the effect of adding sourdough to wheat bread dough on the production of flavour compounds in wheat bread crumb. The sourdoughs were fermented with starter cultures of lactic acid bacteria alone and in combination with sourdough yeasts. The volatile compounds in the bread crumb were isolated by a dynamic headspace technique and extraction analysis, analysed by gas chromatography (GC), and identified on the basis of GC retention times for reference compounds and mass spectrometry (MS). The chemical analyses were combined with sensory evaluation. The volume of the loaves increased significantly when the doughs had 5–20% sourdough added compared with the control bread (bread without sourdough). In the sourdough bread, the content of acetic acid, 2-methylpropanoic acid, and 3-methylbutanoic acid was generally higher, and loaves made with the addition of sourdoughs fermented withLactobacillus plantarum, L. delbrueckii, orL. sanfrancisco had a higher content of 2- or 3-methyl-1-butanol than control bread. Interactions were seen between the starter cultures and the sourdough yeasts, and the production of the following compounds was increased depending on the starter culture used and on the sourdough yeast: ethanol, 2-methylpropanol, 2/3-methyl-1-butanol, 2-phenylethanol, benzyl alcohol, acetic acid, 2-methylpropanoic acid, and 3-methylpropanoic acid. Bread made with an addition of 5% to 15% sourdough fermented withL. sanfrancisco had a pleasant, mild and sour odour and taste.L. plantarum bread had a strong, sour and unpleasant odour and a metallic sour taste with a sour aftertaste, but when the sourdough was also supplemented with the sourdough yeastSaccharomyces cerevisiae, the bread attained a more aromatic wheat bread flavour, which may be caused, in part, by a higher content of 2/3-methyl-1-butanol, 2-methylpropanoic acid, 3-methylbutanoic acid and 2-phenylethanol.