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.     

A novel approach for the production of a non‐alcohol beer (≤0.5% abv) by a combination of limited fermentation and vacuum distillation

Despite the increasing demand, the production of non‐alcohol beers is still limited by unsatisfactory or artificial flavour and taste. In this study, a novel approach to producing non‐alcohol beer is presented, in which the alcohol‐reducing techniques, limited fermentation and vacuum distillation were combined. Starting from barley and wheat malts, wort with a low level of fermentable sugars was prepared by infusion mashing and lautering. Limited fermentation was carried out by Saccharomycodes ludwigii at 18°C. When the level of fermentable sugar was reduced by 25%, the fermented wort was quickly cooled from 18 to 0°C and held at that temperature for two days. The young beer was obtained after degassing and removal of yeast and was then subjected to vacuum distillation at 0.06 MPa to remove the alcohol. The concentrated extract is suitable for storage and transportation. The final product of non‐alcohol beer was obtained by dilution with deoxygenated water and carbonation with 6.0 g/L CO₂, followed by addition of 8–12% of regular beer and equilibration for 2–3 days to develop normal beer aroma. The results showed that the non‐alcohol beer had several favourable properties, including the alcohol level of <0.5% (v /v), colour 7.0 (EBC), thiobarbituric acid value of 1.05 and ratio of alcohols to esters of 1.08. Compared with other methods for the production of non‐alcohol beer, this novel approach produced a favourable alternative to regular beers with similar flavour characteristics and satisfactory stability. Copyright © 2017 The Institute of Brewing & Distilling     

Screening of new strains of Saccharomycodes ludwigii and Zygosaccharomyces rouxii to produce low‐alcohol beer

Low‐alcohol beer (0.5–1.2% v/v ethanol) is a less common brewing industry output than standard beer but there is an increasing interest in this product, as evidenced by increased attention to health and safety and government policies on alcohol and diet. The main challenge in the production of low‐alcohol beer is the achievement of a product as similar as possible to regular beer, particularly concerning the content of the volatile compounds. These compounds can be lost during the physical removal of alcohol by dialysis, reverse osmosis and vacuum rectification. Consequently, an alternative technique is the use of biological methods, which involve the employment of non‐conventional yeasts. In this paper, 11 non‐conventional yeast strains were tested for low‐alcohol beer production. The strains used belonged to two different species: Saccharomycodes ludwigii and Zygosaccharomyces rouxii. The beer samples produced by these strains were analysed for their ethanol content and main volatile compounds. The S. ludwigii strains were more suitable for brewing low‐alcohol beer, especially strain DBVPG 3010, which also showed a higher content of esters and a lower amount of diacetyl compared with previous reports. The Z. rouxii strains produced an ethanol and diacetyl content above the taste threshold. This screening project can be considered as a first step towards the production of low‐alcohol beer by means of new selected non‐conventional yeasts. Copyright © 2015 The Institute of Brewing & Distilling     

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.     

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.     

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.     

Could non-Saccharomyces yeasts contribute on innovative brewing fermentations?

With the advances in the production of beer worldwide, more challenges arise each year in the search for new approaches to the development of distinctive beverages. Attempts to obtain products with more complex sensory characteristics have led experts and brewers to prospect for non-conventional yeasts, i.e., non-Saccharomyces yeasts that may provide a new range of perspectives in terms of techniques and approaches. Besides the widespread use of Dekkera/Brettanomyces for the production of sour beers, other species are emerging for presenting unusual metabolic features that include the production of fruity esters, and a distinctive enzymatic apparatus. Wickerhamomyces anomalus and Torulaspora delbrueckii stand out as the most promising yeasts in brewing processes. Such new tendencies in the use of non-Saccharomyces yeasts comprise the production of low-alcohol beers, functional beers, and bioflavoring approaches. This is a little explored field in brewing practice, still requiring extensive research with practical application. In this sense, this review aims to present the main points for the application of non-conventional yeasts in beer production, and thus contribute to future advances in the topic.     

CAPRYLIC FLAVOUR AS A FEATURE OF BEER FLAVOUR

Caprylic flavour is part of the distinctive overall flavour of a large proportion of beers in trade. The flavour occurs in the majority of lager beers and in about 20% of ales. It is correlated with the levels of octanoic acid and decanoic acid present in beer. Lager yeasts (S. uvarum) tend to liberate larger amounts of these two fatty acids during fermentation than do ale yeasts (S. cerevisiae). The flavour significance of these acids has been determined by correlating the results of sensory and instrumental analyses and also by adding the purified acids to beer. Implications of these two methods for assessing the flavour significance of chemical constituents of a complex product are discussed.     

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 Influence of Inoculum Level on Fermentation and Flavour Compounds of White Wines Made from cv. Emir

In this study, the influence of the addition of a commercial wine yeast (Saccharomyces cerevisiae) at inocula of 1 × 10⁴ to 1 × 10⁷ cells/ml in Emir must was investigated with a focus on yeast growth, fermentation rate, ethyl alcohol and flavour compound formation. Spontaneous fermentation without inoculation was also performed. Higher peak counts were observed with higher amounts of S. cerevisiae yeast. Addition of various amounts of yeast led to the earlier disappearance of non‐Saccharomyces yeasts. The fermentation rate was improved with higher amounts of yeast, but ethanol production was not affected. Concentrations of higher alcohols increased with increasing inoculum levels, especially inoculum sizes of 1 × 10⁶ cells/ml and 1 × 10⁷ cells/ml. The amount of ethyl acetate was reduced with increased inoculum levels.     

Evaluation of the potential of commercial non-Saccharomyces yeast strains of Torulaspora delbrueckii and Lachancea thermotolerans in beer fermentation

This work evaluated the potential of three commercial non-Saccharomyces yeast strains Torulaspora delbrueckii (Biodiva and Prelude) and Lachancea thermotolerans (Concerto) for beer fermentation. The fermentation performance, volatile and non-volatile profiles were compared. Growth behaviours of all three yeast strains exhibited similar trends during the initial fermentation phase although a marked population decline was detected in strains Prelude and Concerto, which also showed a rapid utilisation of maltose, while strain Biodiva was unable to consume maltose and consumed lesser amounts of amino acids. Additionally, terpenoids inherently absent in the wort such as β-caryophyllene and geranyl acetone were produced in all beers, significantly higher in beers fermented with strain Prelude. For volatile profiles, Prelude and Concerto produced more ethanol and significantly higher amounts of acetate esters and long-chained ethyl esters. Strain Biodiva, on the other hand, produced higher amounts of isoamyl alcohol and ethyl butanoate.     

Microbial succession and metabolite changes during the fermentation of Chinese light aroma‐style liquor

A combination of culture‐dependent and culture‐independent methods and SPME–GC–MS were used to monitor changes of bacterial and yeast communities, and flavour compounds during the fermentation process of Chinese light aroma‐style liquor. Bacillus and Lactobacillus were the main bacterial genera. Pichia anomala, Saccharomyces cerevisiae and Issatchenkia orientalis were the dominant yeast species. There was a close relationship between fermentation time and the shift of microbial community. Compared with the microbiota in the fermentation of other style liquors, higher bacterial diversity and different non‐Saccharomyces composition led to a variety of metabolites. Metabolite analysis showed that esters, acids, alcohols, aromatic compounds and phenols were the main flavour components and most of them were synthesised in the latter phase of fermentation. Principal component analysis further demonstrated that Bacillus and yeast were the most influential microorganisms in the first 10 days of fermentation, and lactic acid bacteria predominated in the later phase. Lactic acid bacteria regulated the composition of other bacteria and yeast, and synthesised flavour compounds to affect the organoleptic properties of liquor. S. cerevisiae and P. anomala were two important yeast species responsible for the characteristic aroma of liquor. These results present a comprehensive understanding of microbial interaction and potential starter cultures to produce desirable liquor quality. © 2018 The Institute of Brewing & Distilling     

Optimization of alcohol‐free beer production by lager and cachaça yeast strains using response surface methodology

Alcohol‐free beers (AFBs) are an attractive segment of the beer market both for the brewing industry and for consumers. While AFBs produced by arrested/limited fermentation often suffer from a lack of volatile compounds, beer flavour can be improved by yeast selection and optimization of fermentation conditions. The yeast selection strategy was demonstrated by comparing traditional lager yeast with selected cachaça yeast strains. Correspondingly, response surface methodology was used to enhance the formation of the flavour‐active volatile compounds by optimization of the fermentation conditions (original wort extract, fermentation temperature, pitching rate). Statistical analysis of the experimental data revealed the relative significance of process variables and their interactions. The developed quadratic model describing the responses of total esters and higher alcohols to changes in process variables was used to predict the ideal fermentation conditions in terms of flavour formation. The predicted conditions were experimentally verified and alternative strategies of AFB production are suggested. Copyright © 2016 The Institute of Brewing & Distilling     

The Occurrence of Beer Spoilage Lactic Acid Bacteria in Craft Beer Production

Beer is one of the world's most ancient and widely consumed fermented alcoholic beverages produced with water, malted cereal grains (generally barley and wheat), hops, and yeast. Beer is considered an unfavorable substrate of growth for many microorganisms, however, there are a limited number of bacteria and yeasts, which are capable of growth and may spoil beer especially if it is not pasteurized or sterile‐filtered as craft beer. The aim of this research study was to track beer spoilage lactic acid bacteria (LAB) inside a brewery and during the craft beer production process. To that end, indoor air and work surface samples, collected in the brewery under study, together with commercial active dry yeasts, exhausted yeasts, yeast pellet (obtained after mature beer centrifugation), and spoiled beers were analyzed through culture‐dependent methods and PCR‐DGGE in order to identify the contaminant LAB species and the source of contamination. Lactobacillus brevis was detected in a spoiled beer and in a commercial active dry yeast. Other LAB species and bacteria ascribed to Staphylococcus sp., Enterobaceriaceae, and Acetobacter sp. were found in the brewery. In conclusion, the PCR‐DGGE technique coupled with the culture‐dependent method was found to be a useful tool for identifying the beer spoilage bacteria and the source of contamination. The analyses carried out on raw materials, by‐products, final products, and the brewery were useful for implementing a sanitization plan to be adopted in the production plant.     

The Production of Isoamyl Acetate from Amyl Alcohol by Saccharomyces cerevisiae

Isoamyl acetate is a natural flavour ester, widely used as a source of banana flavour by the food industry. Fusel alcohols such as amyl alcohol are produced in significant quantities as a waste product, sometimes referred to as “lees oil” or “fusel oil”, of the alcohol distilling industry. By manipulation of brewing yeast fermentation conditions, a significant portion of added amyl alcohol was shown to be converted to isoamyl acetate. This was achieved by the addition of L‐leucine and amyl alcohol in fermentations carried out by a high ester‐producing brewing yeast strain of Saccharomyces cerevisiae and by the use of alkaline fermentation conditions coupled with high gravity media. Mutant strains selected on 5,5,5 trifluoro‐DL‐leucine produced substantially high levels of isoamyl acetate. The adjustment of fermentation conditions outlined in this paper may act as a stepping stone for the potential use of Saccharomyces cerevisiae and other yeasts to produce high levels of natural flavour esters.     

THE ADDITION OF PROTEASES TO THE FERMENTER TO CONTROL CHILL-HAZE FORMATION

The addition of papain and proteases isolated from various yeast strains to fermentation to reduce chill-haze formation is discussed. Particular attention is paid to the behaviour of the yeast throughout fermentation and to the character of the final beers. The results suggest that fermentation in the presence of papain and a protease preparation prepared from Candida olea 148 progress normally and in the case of C. olea 148 protease-treated fermentation no change in beer flavour was detected.     

Investigation of beer‐spoilage ability of Dekkera/Brettanomyces yeasts and development of multiplex PCR method for beer‐spoilage yeasts

There have been many beer‐spoilage incidents caused by wild yeasts. Saccharomyces cerevisiae, Dekkera anomala and D. bruxellensis have been recognized as beer‐spoilage yeasts in the brewing industry. In contrast, the beer spoilage ability of Brettanomyces custersianus has not been well characterized, although this species was isolated from beer. In this study, the beer‐spoilage ability of currently described Dekkera/Brettanomyces yeast species was investigated. As a consequence, D. anomala, D. bruxellensis and B. custersianus were shown to grow in commercial beers. On the other hand, the remaining two Brettanomyces species, B. naardenensis and B. nanus, did not grow in beer. These results indicate that B. custersianus should be recognized as a beer‐spoilage species, in addition to S. cerevisiae, D. anomala, and D. bruxellensis. Therefore we developed multiplex polymerase chain reaction (PCR) for the simultaneous detection and identification of B. custersianus and the other beer‐spoilage yeast species. For this purpose, PCR primers were designed in the internal transcribed spacer region or 26S rDNA, and each PCR product was made in different sizes to easily discriminate the species from electrophoretic results. Specificity, reactivity and sensitivity of the designed primers were evaluated. As a result, the developed multiplex PCR method was shown to have high specificity and reactivity, and therefore was considered as an effective tool to identify beer‐spoilage yeast species. This tool can contribute to microbiological quality assurance in breweries. Copyright © 2015 The Institute of Brewing & Distilling     

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).     

Construction of self‐cloning bottom‐fermenting yeast with low vicinal diketone production by the homo‐integration of ILV5

The vicinal diketones (VDK), such as diacetyl and 2,3‐pentandione, impart an unpleasant butter‐like flavour to beer. Typically, these are required to be reduced below the flavour thresholds during the maturation (lagering) stages of the brewing process. To shorten beer maturation time, we constructed a self‐cloning, bottom‐fermenting yeast with low VDK production by integrating ILV5, a gene encoding a protein that metabolizes α‐acetolactate and α‐aceto‐α‐hydroxybutyrate (precursors of VDK). A DNA fragment containing Saccharomyces cerevisiae‐type ILV5 was inserted upstream of S. cerevisiae‐type ILV2 in bottom‐fermenting yeast to construct self‐cloning strains with an increased copy number of ILV5. Via transformation, ILV2 was replaced with the sulfometuron methyl (SM) resistance gene SMR1B, which differs by a single nucleotide, to create SM‐resistant transformants. The wort fermentation test, using the SC‐ILV5‐homo inserted transformant, confirmed a consecutive reduction in VDK and a shortening period during which VDK was reduced to within the threshold. The concentrations of ethyl acetate, isoamyl acetate, isoamyl alcohol, 1‐propanol, isobutyl alcohol and active isoamyl alcohol (flavour components) were not changed when compared with the parent strain. We successfully constructed self‐cloning brewer's yeast in which SC‐ILV5 was homo‐inserted. Using the transformed yeast, the concentration of VDK in fermenting wort was reduced, whereas the concentrations of flavour components were not affected. This genetically stable, low VDK‐producing, self‐cloning bottom‐fermenting yeast would contribute to the shortening of beer maturation time without affecting important flavour components produced by brewer's yeast. Copyright © 2012 John Wiley & Sons, Ltd.