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.     

Evaluation of the fermentative potential of Candida zemplinina yeasts for craft beer fermentation

The fermentative potential of Candida zemplinina Y.01667 and Y.01670 was evaluated to explore the potential use of these yeasts for craft beer fermentations. Fermentation experiments were carried out at different temperatures and soluble solid concentrations, using synthetic media with glucose syrup as a sugar source and with a laboratory malt wort plus different adjuncts. Results showed that both strains fermented well at 14 °C and had improved fermentative activity at 20 °C. The fermentative kinetics of C. zemplinina Y.01667 and Y.01670 were not affected when experiments at higher concentrations of soluble solids were conducted. Furthermore, C. zemplinina strains had better growth, higher viable cells counts, less free amino nitrogen consumption, lower sedimentation rates and slighter changes in pH values, when compared with results of the lager beer yeast Saccharomyces cerevisiae S‐23 in the synthetic medium tested. Fermentations in a malt wort with different adjuncts indicated that C. zemplinina Y.01670 could possibly be used as a yeast in craft beer production. Copyright © 2016 The Institute of Brewing & Distilling     

Maltose Transport by Brewer's Yeasts in Brewer's Wort

The kinetics of maltose transport by two industrial yeasts were studied. The ale and lager strain each showed both high and low affinity transport. For the lager strain, maltose transport was only weakly inhibited by maltotriose, sucrose and trehalose, suggesting that its dominant maltose transporter is the maltose‐specific type coded by MALx1 genes. For the ale strain, maltose transport was strongly inhibited by maltotriose, sucrose and trehalose, suggesting that its dominant maltose transporter may be the AGT1‐encoded type that also carries these sugars. Also glucose inhibited transport by the ale strain more than that by the lager strain. Instantaneous inhibition by ethanol at concentrations met in brewery fermentations was moderate (about 25% at 50 g ethanol · L^?1). The apparent Vmax for high affinity transport increased about 100‐fold between 0 and 30°C, whereas the Km (3 ± 1 mM) was constant. Standard activities of maltose transport and maltase were followed through pilot fermentations of 11–24°P worts. Rapid (20 s) measurements of the zero‐trans‐rate of maltose uptake were also made with each day's yeast (rapidly harvested and washed) in reaction mixtures containing the same day's wort labelled with tracer ¹⁴C‐maltose. Results suggested that maltose uptake is the dominant factor controlling the rate of maltose utilization in these wort fermentations.     

Fermentation kinetics and chemical characterisation of vino tostado,a traditional sweet wine from Galicia (NW Spain)

BACKGROUND: Grapes after harvesting are air dried and pressed in order to concentrate sugars, acids and flavour compounds to produce vino tostado (toasted wine), a wine with intense aroma and flavour notes and high residual sugar concentration. In order to get a better knowledge of the difficulties involved, several fermentations were conducted at 12 and 28 °C using 0, 15 and 30 g hL^?1 ammonium sulfate and 0, 25 and 50 g hL^?1 exogenous commercial yeast (Saccharomyces cerevisiae var. bayanus) to study the kinetics of sugar consumption and ethanol, acetic acid and glycerol production. RESULTS: Fermentation kinetic parameters were calculated and metal concentrations and antioxidant activity were analysed. CONCLUSION: The spontaneous fermentation at 12 °C and all fermentations conducted with the commercial yeast gave vino tostado of adequate quality, while the spontaneous fermentation at 28 °C was sluggish. High‐temperature fermentations led to sweeter wines with higher volumetric productivities, although low‐temperature fermentations produced better wines in terms of higher glycerol and lower acetic acid levels. Fructose was the only sugar to be consumed during spontaneous fermentations, while both glucose and fructose were consumed during fermentations of the inoculated musts, with preference for each monosaccharide depending on temperature. Copyright © 2009 Society of Chemical Industry     

Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance

Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus‐like strains. Lager yeasts are particularly adapted to low‐temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make‐up of lager yeast spore clones, we introduced molecular markers to analyse mating‐type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18°Plato at 18–25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. Copyright © 2012 John Wiley & Sons, Ltd.     

Comparing the Impact of Environmental Factors During Very High Gravity Brewing Fermentations

The impact of the initial dissolved oxygen, fermentation temperature, wort concentration and yeast pitching rate on the major fermentation process responses were evaluated by full factorial design and statistical analysis by JMP 5.01 (SAS software) software. Fermentation trials were carried out in 2L‐EBC tall tubes using an industrial lager brewing yeast strain. The yeast viability, ethanol production, apparent extract and real degree of fermentation were monitored. The results obtained demonstrate that very high gravity worts at 22°P can be fermented in the same period of time as a 15°P wort, by raising the temperature to 18°C, the oxygen level to about 22 ppm, and increasing the pitching rate to 22 × 10⁶ cell/mL. When diluting to obtain an 11.5°P beer extract, the volumetric brewing capacity increased 91% for the 22°P wort fermentation and 30% using the 15°P wort. After dilution, the fermentation of the 22°P wort resulted in a beer with higher esters levels, primarily the compound ethyl acetate.     

Effect of the respiro-fermentative balance during yeast propagation on fermentation and wort attenuation

Breweries use different yeast strains to create beers with different flavours and aromas. Yeast propagation must produce yeast that performs consistently from the first fermentation to harvesting and re-pitching in subsequent fermentations. Breweries propagate yeast in wort leading to low efficiency fermentative growth in Crabtree-positive yeast. There is limited knowledge on the impact on beer production when fermenting with yeast propagated in sugar limited and nutrient supplemented wort. It was hypothesised that propagating yeast in this way would have a positive impact on subsequent fermentation performance. Saccharomyces cerevisiae was propagated at the laboratory scale in standard wort with a high carbon to nitrogen (C:N) ratio (850) or in modified wort supplemented with yeast extract to achieve a low C:N ratio (100) and at varying sugar concentrations. Propagation in low C:N wort with 2°P sugar yielded a 27% decrease in fermentation efficiency and a 46% increase in cell production compared to 2°P high C:N wort. This suggests nitrogen is critical to the respiro-fermentative balance during growth. Yeast propagated in standard wort resulted in slower fermentations and significant under-attenuation compared to yeast grown in the modified wort with low sugar and high nitrogen. The results of this study suggest the nitrogen and sugar content drive the respiro-fermentative balance during yeast propagation. The metabolism of yeast during propagation induces significant downstream impacts on the subsequent fermentation performance and wort attenuation. © 2020 The Institute of Brewing & Distilling     

Investigation of mashing regimes for low‐alcohol beer production

Low‐alcohol beer can be obtained by physical and biological methods. The group of biological methods includes modification of the mashing regimes and changes in the fermentation process. The aim of the present work was to study two mashing regimes for low‐alcohol beer production. The increase in the mashing duration at 50 °C led to a linear increase in the extract and the concentration of reducing and fermentable sugars in the wort. It was found that the rate of formation of reducing sugars was higher than that of the formation of fermentable sugars, which can be used for the optimization of the mashing process. The introduction of a pause at 77 °C did not lead to a substantial increase in the concentration of fermentable extract, but did lead to an increase in the total and non‐fermentable extract. The available nitrogen content in the laboratory wort was in the range of 120–150 mg/dm³. As a result of conducting fermentation processes with the top‐fermenting yeast strain Saccharomyces cerevisiae S‐33, it was found that the combination of a small amount of fermentable sugars and a low fermentation temperature led to a beer being obtained that met the requirements for a low‐alcohol beverage. 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.     

Production of Beer with a Genetically Engineered Strain of S. cerevisiae with Modified Beta Glucanase Expression

This study used a recombinant Saccharomyces cerevisiae strain, which expressed both β‐glucanase enzyme and reduced Pro‐teinase A expression during wort fermentations. The genetic stability and fermentation features of the strain were examined. The recombinant strain's proteinase A activity was reduced compared to the parent strain; β‐glucanase was produced throughout the fermentation. The fermentation with the recombinant S. cerevisiae strain exhibited a larger reduction in β‐glucan content than what was observed with the control strain, with β‐glucan degradation above 80%. The foam stability period was reduced when the beer produced by the recombinant S. cerevisiae was stored for 3 months. SDS‐PAGE analysis of the beer proteins indicated that lipid transfer protein 1 had disappeared. Fermentation studies indicated that based on the parameters examined, this recombinant strain was suitable for industrial beer production.     

Impact of the Long‐Term Maintenance Method of Brewer's Yeast on Fermentation Course,Yeast Vitality and Beer Characteristics

The effect of the long‐term maintenance method used with a brewer's yeast on its technological properties was determined in laboratory fermentation trials with a 12°P all‐malt wort. The trials were performed at a constant temperature and under conditions of constant substrate concentration. Two cultures of a bottom fermenting yeast, Saccharomyces pastorianus RIBM 95, were tested — one culture was maintained by subculturing on wort agar slopes at 4°C and the other culture underwent a three year storage in liquid nitrogen at minus 196°C. Parameters under investigation included yeast vitality measured as acidification power (AP), fermentation time needed to reach an alcohol level of 4%, the yeast cell count, sedimentation of the yeast during the fermentation, and the production of beer flavour compounds in green beer. The yeast culture stored for three years in liquid nitrogen displayed a higher count of suspended cells, required a shorter time to attenuate the wort to produce 4% alcohol and produced a 1.5 to 2.5‐fold higher concentration of a number of flavour compounds. The long‐term storage method did not affect the sedimentation ability and vitality of the yeast strain tested.     

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     

Proteolytic and angiotensin‐converting enzyme‐inhibitory activities of selected probiotic bacteria

This study was carried out to examine the proteolytic and angiotensin‐converting enzyme (ACE‐I) activities of probiotic lactic acid bacteria (LAB) as influenced by the type of media, fermentation time, strain type and media supplementation with a proteolytic enzyme (Flavourzyme^®). Lactobacillus casei (Lc210), Bifidobacterium animalis ssp12 (Bb12), Lactobacillus delbrueckii subsp. bulgaricus (Lb11842) and Lactobacillus acidophilus (La2410) were grown in 12% of reconstituted skim milk (RSM) or 4% of whey protein concentrates (WPC‐35) with or without combination (0.14%) of Flavourzyme^® for 12 h at 37 °C. All the strains were able to grow in both media depending on type of strains used and fermentation time. All the strains showed higher proteolytic activity and produced more antihypersensitive peptides when grown in RSM medium at 12 h, when compared to WPC. Combination with Flavourzyme^® also increased LAB growth and proteolytic and ACE‐I activities. Of the four strains used, Bb12 and La2410 outperformed Lc210 and Lb11842. The highest ACE‐I activity and proteolytic activity were found in B. animalis ssp12 combined with Flavourzyme^®. Flavourzyme^® led to an increase in the production of bioactive peptides with ACE‐I activity during 12 h at 37 °C.     

Direct Evidence That Maltose Transport Activity Is Affected by the Lipid Composition of Brewer's Yeast

A brewer's yeast strain was grown with maltose as sole carbon source under strictly anaerobic conditions with and without ergosterol and/or unsaturated fatty acid (Tween 80) supplements. Under all these conditions the MALx1 genes for maltose transporters were strongly expressed during growth. The fatty acid unsaturation indices of growing and stationary phase yeast were increased from about 20% to 56–69% by supplementation with Tween 80. Ergosterol contents were increased up to at least 4‐fold by supplementation with ergosterol and Tween 80. Maltose transport activity measured at 20°C was not increased by supplementation with Tween 80 alone, but was increased 2‐fold and 3‐fold, respectively, in growing and stationary phase yeast by supplementation with ergosterol together with Tween 80. The stimulation of maltose transport by ergosterol was greater when the transport was measured at temperatures (10°C and 0°C) lower than 20°C. The results show that proper function of maltose transporters requires adequate amounts of ergosterol in the yeast. This effect may partly explain the low maltose (and maltotriose) uptake rates both in the second half of brewery fermentations, when the sterol content of yeast has fallen, and when fresh wort is pitched with sterol‐deficient cropped yeast.     

WORT ENTEROBACTERIA—A REVIEW

Of the family Enterobacteriaceae, Citrobacter freundii, Enterobacter aerogenes, Ent. cloacae, Hafnia alvei, Klebsiella aerogenes and Serratia species have been detected in fermenting wort. Escherichia coli and animal parasites have not been isolated. As shown by G.C. ratio, DNA base sequence comparison, numerical taxonomy and phage typing, Obesumbacterium proteus shares the same family and is placed in the genus Hafnia as H. protea, with two subspecies. H. protea survives brewery fermentations better than other members of the family and is therefore common in pitching yeast. However, all wort enterobacteria are sensitive to pH values below 4.4 and to a lesser degree to ethanol concentrations over 2% (w/v). Rates of brewery fermentations may be retarded by enterobacteria and the beer pH elevated. Other wort bacteria isolated, species of Achromobacter, Acinetobacter and Pseudomonas, are sensitive to pH and ethanol, and present in smaller numbers in wort than the enterobacteria. Beer flavour with respect to fusel alcohols and esters, volatile sulphur compounds, carbonyl compounds and volatile phenols is seriously influenced by wort enterobacteria. Isolation and enumeration of the enterobacteria in fermenting wort are conveniently carried out on MacConkey agar medium; H. protea colonies grow in 48–72 h at 30°C but the other enterobacteria produce colonies in 20–30 h.     

CLONING OF A YEAST GENE WHICH CAUSES PHENOLIC OFF-FLAVOURS IN BEER

A gene (POF1) has been cloned, which confers upon yeast (Saccharomyces cerevisiae) the ability to decarboxylate phenolic acids such as ferulic and trans-cinnamic acid. This property was previously shown to be a cause of phenolic off-flavour production in wort fermentations. The identity of the cloned gene was confirmed as POF1 by gene disruption techniques. Southern blotting of total genomic DNA revealed that sequences homologous to POF1 are conserved in Pof^? brewing strains of Sacch. cerevisiae. The transformation of a Pof^? lager strain with the cloned POF1 gene led to the production of an aroma characteristic of a phenolic off-flavour, when the transformed strain was used in wort fermentations. This latter observation suggests that the Pof^? phenotype of brewers' yeast is specifically due to the absence of a functional POF1 gene.