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.     

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.     

THE RAPID DETECTION OF BREWERY CONTAMINANTS BELONGING TO THE GENUS SACCHAROMYCES—EXAMINATION OF LAGER YEASTS

For the detection of wild Saccharomyces contaminants in lager yeasts, two different sera combinations, absorbed by two different strains of Sacch. carlsbergensis, are required. The most satisfactory test system applies to lager yeasts belonging to sub-group I, where 60% of Sacch. cerevisiae contaminants are detected, all Sacch. carlsbergensis strains belonging to sub-group II, and all other brewery contaminants belonging to the genus Saccharomyces. The system used for lager yeasts belonging to sub-group II is less satisfactory: 50% of Sacch. cerevisiae strains and all Sacch. carlsbergensis strains belonging to sub-group I were detected, but only 60% of strains belonging to other Saccharomyces spp. The limitations of the antigenic structures ascribed to the various species of this genus are demonstrated, and it is suggested that an immuno-fluorescent test procedure should be used in any further studies relating to antigenic inter-relationships.     

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.     

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     

THE IMMUNOFLUORESCENT STAINING TECHNIQUE FOR THE DETECTION OF WILD YEASTS - PRACTICAL PROBLEMS

An apparent heavy wild yeast infection in pitching yeast has been detected using the immunofluorescent detection method. This infection could not be detected by conventional liquid forcing or plating techniques. The yeasts responsible were isolated and identified as Saccharomyces cerevisiae. The yeasts were very similar to the pitching yeast but varied in a number of respects associated with the cell wall such as flocculation character and giant colony morphology. The results suggest that the immunofluorescent-positive (IP) yeasts are variants of the culture yeast. As a result of this work it is felt that although immunofluorescence is of value for the rapid detection of infection, it must always be used in association with more conventional microbiological techniques.     

SELECTIVE SACCHAROMYCES MEDIA CONTAINING ERGOSTEROL AND TWEEN 80

A mixture (ET80) of ergosterol and Tween 80 stimulated the anaerobic growth on solid media of almost all strains of Saccharomyces tested. Yeasts from other genera have only produced micro-colonies. It is possible to detect Saccharomyces yeasts after 40 h anaerobic incubation and subsequently to estimate total infection after a further 24 h aerobic incubation. Saccharomyces contaminants can be differentiated from other yeasts and from bacteria within 24 h by a simple staining procedure using malachite green. Anaerobic incubation for 4 days on ET80-supplemented crystal violet agar is selective for wild Saccharomyces only, thus allowing a clear differentiation between lysine-positive and lysine-negative yeasts. This medium is suitable for use with membrane filter-discs.     

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     

A CONVENIENT DOMINANT SELECTION MARKER FOR GENE TRANSFER IN INDUSTRIAL STRAINS OF SACCHAROMYCES YEAST: SMRI ENCODED RESISTANCE TO THE HERBICIDE SULFOMETURON METHYL

The SMR1-410 gene of S. cerevisiae, encoding resistance to the herbicide sulfometuron methyl (SM), was used as a dominant selection marker in yeast replicating and yeast integrating vectors for the transformation of wild type strains of baking, brewing (ale and lager), distilling, wine and sake Saccharomyces yeasts. Transformation of lithium treated cells by a YEp vector resulted in transformation frequencies ranging from 200 to 8,000 transformants per 10 ug of DNA. Utilizing a yeast integrating vector with SMR1–410 as the only yeast DNA sequences, it was demonstrated that a single copy of SMR1–410 is sufficient to confer stably inherited SM resistance. Thus the SMR1–410 sequence has the unique ability to act as a selectable marker and to also provide a site for chromosomal integration. Since transformants were resistant to levels at least seven fold higher than wild type strains the resistance phenotype was clearly expressed and easily scored in all industrial strains tested. Unlike other selection markers derived from mammalian or bacterial cells, SMR1–410 is derived from S. cerevisiae. Thus industrial utilization of this marker as a means of genetically improving food and beverage strains of Saccharomyces yeasts by recombinant DNA technology is enhanced, as government regulatory agencies are likely to view it in a more favourable light.     

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.     

APPLICATION OF PULSED FIELD CHROMOSOME ELECTROPHORESIS IN THE STUDY OF CHROMOSOME XIII AND THE ELECTROPHORETIC KARYOTYPE OF INDUSTRIAL STRAINS OF SACCHAROMYCES YEASTS

Pulsed field chromosome electrophoresis is a powerful new technique in yeast genetics which permits the resolution of intact yeast chromosomes in an agarose gel matrix. We utilized contour-clamped homogeneous electric field electrophoresis (CHEF) to survey representative strains of Saccharomyces yeasts from the brewing, baking, distilling, sake and wine industries for their electrophoretic karyotypes. All of the strains tested were found to have a unique chromosomal profile, indicating the potential of this technology for “fingerprinting” prototrophic strains of Saccharomyces yeasts. By employing an ILV2 gene probe specific for chromosome XIII, we determined that all of the industrial strains of Saccharomyces yeasts possessed a chromosome XIII which migrated in an identical fashion to chromosome XIII from a reference haploid strain of Saccharomyces cerevisiae. While one lager yeast strain, Saccharomyces carlsbergensis M244, was found to contain two alleles of ILV2 when digested genomic DNA was probed with ILV2, the presence of a novel independently migrating chromosome XIII could not be detected. A homeologous chromosome XIII in this yeast will therefore have to be determined by genetic analysis. Pulsed field chromosome electrophoresis is concluded to be a technology with immediate application to Quality Control and Research and Development programs in industries using Saccharomyces yeasts.     

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.     

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.     

Coding repeat instability in the FLO11 gene of Saccharomyces yeasts

In Saccharomyces yeasts, the FLO11 gene encodes an adhesin involved in filamentation, invasive growth, flocculation and adherence to solid surfaces. In wild Saccharomyces flor yeasts, a particularly expanded FLO11 allele also confers to these yeasts the ability to float under stressing liquid environments. We report here that, under optimal laboratory conditions, the repeats domain of the FLO11 gene in these wild yeasts is extremely unstable. Changes in length in the FLO11 coding repeats domain affected Flo11p‐associated functions but, interestingly, some of these functions were affected more than others. Therefore, length variations in this single gene provide a combinatorial diversity, which may contribute to a very rapid adaptation to fluctuating environments. Functional analysis of contracted alleles indicated that buoyancy was not associated to FLO11 length. In contrast, this property depended on the different types of repetitive units found in this gene. Thus, not only variations in the number of intragenic repeats but also the abundance and/or distribution of the different repetitive units may have phenotypic and evolutionary implications. Copyright © 2009 John Wiley & Sons, Ltd.     

THE EFFECT OF YEAST TREHALOSE CONTENT AT PITCHING ON FERMENTATION PERFORMANCE DURING BREWING FERMENTATIONS

The effect of yeast trehalose content at pitching on the fermentation performance during brewing fermentations was studied using a commercial strain of lager yeast, Saccharomyces cerevisiae (AJL 2155). Pitching yeasts with different trehalose contents were obtained by collecting cells in suspension after 96 h and 144 h of fermentation in EBC tubes in 10.8°P brewers wort at 14°C. The trehalose content of the pitching yeast had no effect on growth, specific gravity and ethanol production during the subsequent fermentation. A high trehalose content of the pitching yeast, however, sustained cell viability during the initial stage of fermentation, increased the carbohydrate utilisation rate and increased the production of isoamyl alcohol and isobutanol. For these aspects of fermentation performance, the trehalose content of the pitching yeast may prove useful in evaluating the vitality of pitching yeasts within the brewery .     

INFLUENCE OF NITRATE AND BACTERIAL CONTAMINATION ON THE FORMATION OF APPARENT TOTAL N-NITROSO COMPOUNDS (ATNC) DURING FERMENTATION

Formation of Apparent Total N-Nitroso Compounds (ATNC) was monitored throughout fermentations of all-malt ale worts supplemented with nitrate (0–100 mg Litre^?1 (0–100 ppm)). The pitching yeasts were obtained from commercial breweries and contained different levels of the contaminant bacterium Obesumbacterium proteus (0–2.1% by number). Levels of ATNC present at the end of fermentation were dependent on both initial wort nitrate levels and the initial level of bacterial contamination of the pitching yeast. Only relatively low nitrate levels were required to produce ATNC levels greater than the Brewers' Society recommended limit of 20 μg Litre^?1 (ppb), provided that the bacteria were present. Indeed, the use of whole hops alone would contribute sufficient nitrate to the wort to produce excessive amounts of ATNC, provided O.proteus was present. The only feasible solution to ATNC production during fermentation is to remove the contaminating bacteria from both the pitching yeast and brewing plant. Effective removal of O.proteus can be achieved by acid washing the pitching yeast under carefully controlled conditions, prior to fermentation.     

Influence of different yeasts on the growth of lactic acid bacteria in wine

The influence of various yeasts on the growth of lactic acid bacteria in wine was tested by inoculating Lactobacillus hilgardii, L. brevis and two strains of Leuconostoc mesenteroides into experimental wines made with twelve different yeasts of the genus Saccharomyces. Wines made from juice which had been infected with several spoilage yeasts and then fermented with a wine yeast were also tested in this way. It was found that the yeasts differed considerably in their effects on bacterial growth. In some of the experimental wines bacterial growth was delayed or failed altogether. Generally, the unfavourable influence of any yeast on bacterial growth was much reduced if the wines were left in contact with the yeast cells for some weeks after the fermentation. The significance of these results in relation to the occurrence of malo-lactic fermentation in commercial wineries is discussed.     

FATTY ACIDS AND ESTERS PRODUCED DURING THE SPONTANEOUS FERMENTATION OF LAMBIC AND GUEUZE

Lambic and gueuze are Belgian beers obtained by spontaneous fermentation of wort. During previous studies it was found that they result from the successive development of enterobacteria, Kloeckera and Saccharomyces yeasts, bacteria of the genus Pediococcus, and Brettanomyces yeasts. The beers are characterized by high concentrations of acetic and lactic acid, ethyl acetate and ethyl lactate. This study of the content of the higher fatty acids during a 20 month fermentation period confirms the succession of the different micro-organisms. Pure cultures of isolated yeasts and bacteria produced fatty acids which were also found in the fermenting wort at periods when these organisms were active. Lambic and Gueuze are especially rich in caprylic (C₈) and capric (C₁₀) acids. These are probably produced by Saccharomyces and Brettanomyces. Important amounts of ethyl caprylate and ethyl caprate were also found. As ethyl caprate is almost absent in other beers, it might be considered as another typical aroma component of lambic and gueuze.     

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.     

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.