Aneuploidy,copy number variation and unique chromosomal structures in bottom‐fermenting yeast revealed by array‐CGH

DNA microarray for comparative genome hybridization (CGH) of bottom‐fermenting yeast was performed based on our in‐house DNA sequence data. Aneuploidy, copy number variation and unique chromosomal structures were observed among bottom‐fermenting yeast strains. Our array experiments revealed a correlation between copy number variation and mRNA expression levels. Chromosomal structures in a Saccharomyces carlsbergensis‐type strain and in a S. monacensis‐type strain that both belong to S. pastorianus phylogenetically differed greatly from those in contemporary industrial bottom‐fermenting yeast strains. The knowledge gained in this study contributes to a more precise genomic characterization of bottom‐fermenting yeast strains. Copyright © 2014 The Institute of Brewing & Distilling     

Overexpression of vacuolar H+‐ATPase‐related genes in bottom‐fermenting yeast enhances ethanol tolerance and fermentation rates during high‐gravity fermentation

Vacuolar H⁺‐ATPase (V‐ATPase) is thought to play a role in stress tolerance. In this study it was found that bottom‐fermenting yeast strains, in which the V‐ATPase‐related genes DBF2, VMA41/CYS4/NHS5 and RAV2 were overexpressed, exhibited stronger ethanol tolerance than the parent strain and showed increased fermentation rates in a high‐sugar medium simulating high‐gravity fermentation. Among the strains examined, the DBF2‐overexpressing bottom‐fermenting yeast strain exhibited the highest ethanol tolerance and fermentation rate in YPM20 medium. Using this strain, high‐gravity fermentation was performed by adding sugar to the wort, which led to increased fermentation rates and yeast viability compared with the parent strain. These findings indicate that V‐ATPase is a stress target in high‐gravity fermentation and suggests that enhancing the V‐ATPase activity increases the ethanol tolerance of bottom‐fermenting yeast, thereby improving the fermentation rate and cell viability under high‐gravity conditions. Copyright © 2012 The Institute of Brewing & Distilling     

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.     

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 Method to Detect Anti‐metabolic Factors in Fermentations

Premature yeast flocculation (PYF) has been described as the rapid settling of yeast cells during fermentation despite the presence of sufficient nutrients. PYF can cause negative impacts on beer quality and thus can be quite costly to brewers and maltsters. To investigate the causative agent of PYF, small‐scale fermentations were undertaken in both test tubes and cuvettes (15 and 3.5 mL respectively) using worts prepared from PYF‐positive and PYF‐negative malt samples. Fermentations were carried out using six malts, for up to seven days. Turbidity and extract values were monitored for all samples. The small scale (test tube) assay exhibited clear yeast cell flocculation differences between malts. In the cuvette assay the wort fermented, but the yeast cells settled out of suspension rapidly. While this property made the cuvette assay unsuitable for detecting PYF malt, it did allow for measurement of impaired sugar uptake by the yeast independent of yeast in suspension effects. All wort samples fermented in the cuvette assay showed a similar decline in apparent extract (p > 0.05), indicating that (at least in the samples studied) premature yeast flocculation was not caused by a decline in yeast activity. We believe the simple cuvette assay reported here could have application in the measurement of anti‐metabolic factors in fermenting media.     

Simultaneous utilization of ammonia,free amino acids and peptides during fermentative growth of Saccharomyces cerevisiae

The efficiency of nitrogen use by yeast is one of the key determinants of the successful completion of alcoholic fermentations. In this work the growth of Saccharomyces cerevisiae S288c in a synthetic medium containing ammonia and free amino acids, supplemented with yeast hydrolysate, was studied. Experiments with ¹⁵NH₄Cl and ¹⁵N‐labelled yeast hydrolysate were carried out to gain insight into which of these three classes of assimilable nitrogen sources yeast cells prefer. Co‐consumption of all three sources was observed; approximately 40% of the total nitrogen in the yeast protein fraction originated from yeast hydrolysate, while free amino acids and ammonia contributed 40 and 20%, respectively. The results indicate that several amino acids are more readily obtained from peptides, most likely when the uptake of their free forms is competitively inhibited and/or repressed. During the second half of each fermentation, a decrease in the incorporation of yeast hydrolysate‐derived nitrogen was observed. These results highlight the nutritional role of peptides in various yeast fermentations. Copyright © 2016 The Institute of Brewing & Distilling     

Pilot‐scale brewing using self‐cloning bottom‐fermenting yeast with high SSU1 expression

A pilot‐scale fermentation was performed using SSU1‐overexpressing bottom‐fermenting yeast strains constructed by ‘self‐cloning’. In these strains, the gene SSU1, encoding a plasma membrane protein that excretes sulphite, was highly expressed. The rate of fermentation of the two SSU1‐overexpressing strains tested showed some reduction during the mid‐fermentation phase as compared with the parental strain. These differences, however, did not affect overall fermentation and the final apparent extracts had decreased to a level normally obtained during brewing. The concentration of hydrogen sulphide in the wort remained low during fermentation in the case of the two self‐cloning strains compared with the parent. The concentration of 2‐mercapto‐3‐methyl‐1‐butanol, a sulphur compound that causes an ‘onion‐like’ off‐flavour, was also reduced in the case of the self‐cloning strains, a result confirmed by sensory evaluation of the beer immediately after bottling. Furthermore, with these strains the anti‐oxidation potential of bottled beer, as measured by electron spin resonance, was improved and the concentration of trans‐2‐nonenal in bottled beer after 7 days of accelerated aging at 37°C was decreased. These observations, together with the lower stale flavour score determined by sensory evaluation of bottled beer after a month of aging at 25°C, indicated that the flavour stability of the beer had been successfully improved. Copyright © 2013 The Institute of Brewing & Distilling     

FLOCCULATION MECHANISMS OF TOP AND BOTTOM FERMENTING BREWING YEAST

The flocculation behaviour of a large set of top and bottom fermenting brewing yeasts was investigated. Bottom and top fermenting strains flocculated according to different mechanisms. Bottom strains flocculated in the stationary growth phase in the presence of sufficiently high Ca²⁺ and sufficiently low sugar concentrations; these strains possessed a lectin-mediated flocculation mechanism. Top strains flocculated in the stationary growth phase without addition of Ca²⁺, only in the presence of sufficiently high concentrations of ethanol. Some of the top strains were inhibited with mannose, but not with sucrose or galactose, while others were not inhibited by any of these sugars. The different sensitivity of flocculation of top and bottom strains with respect to ethanol may be related to the hydrophobicity of the cell surface. Flocculation models for bottom and top fermenting yeasts were proposed. It is suggested that, besides the sugar inhibition pattern, the sensitivity of flocculation with respect to ethanol should be included as an additional parameter for classification of brewing yeasts .     

Oxylipin Associated Co‐Flocculation in Yeasts

According to the lectin‐theory, the yeast Schizosaccharomyces pombe lacks the specific receptors (α‐mannans) necessary to facilitate co‐flocculation with Saccharomyces cerevisiae species. In this study we demonstrate oxylipin associated co‐flocculation between Sacch. cerevisiae and S. pombe strains using differential cell staining, immunofluoresence and ultrastructural studies. Using a 3‐hydroxy (OH) oxylipin specific antibody coupled to a fluorescing compound, 3‐OH oxylipins were found to be present on the cell surfaces of Sacch. cerevisiae and S. pombe. The presence of 3‐OH oxylipins was confirmed using gas chromatography‐mass spectrometry. Strikingly, when acetylsalicylic acid (aspirin), a 3‐OH oxylipins inhibitor, was added to Sacch. cerevisiae which was then mixed with S. pombe strains grown in complex media, co‐flocculation was significantly inhibited. We conclude that aspirin‐sensitive 3‐OH 8:0 is probably involved in co‐flocculation.     

Flocculation in industrial strains of Saccharomyces cerevisiae: role of cell wall polysaccharides and lectin‐like receptors

Yeast flocculation is the reversible aggregation of yeast cells promoted by the interaction between lectin‐like protein receptors with mannose side chains on adjacent cell walls. Flocculation is governed by several physiological factors, including the type of nutrient sugar available to yeast. We grew four industrial strains of Saccharomyces cerevisiae , representing applications in the brewing, winemaking and bioethanol sectors, to late stationary phase and quantified the cellular content of mannans, glucans and lectin‐like proteins on yeast cell surfaces. Results indicated that brewing and champagne strains showed moderate to high flocculation ability when grown with glucose, fructose, maltose or galactose, whereas winemaking and fuel alcohol strains only showed moderate flocculation when grown on maltose and galactose. All yeast strains studied were weakly flocculent when grown on mannose. With regard to lectin‐like receptors, their number played a more important role in governing yeast flocculation than the mannan and glucan contents in yeast cell walls. We conclude that all the industrial strains of S. cerevisiae belonged to New‐Flo type on the basis of their flocculation behaviour observed when cultured on different sugars. Quantification of yeast cell wall polysaccharides and receptor sites indicates that mannan and glucan levels remain almost constant, irrespective of the strain under investigation. The main difference in flocculation characteristics in industrial yeast strains appears to be due to variations in concentrations of lectin‐like cell surface receptors. Our findings may benefit brewers, winemakers and other yeast‐based technologies in design of media to prevent premature flocculation during fermentation. Copyright © 2017 The Institute of Brewing & Distilling     

Structural Complexity of the Nitrogen Source and Influence on Yeast Growth and Fermentation

The structural complexity of the nitrogen source strongly affects both biomass and ethanol production by industrial strains of Saccharomyces cerevisiae, during fermentation in media containing glucose or maltose, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low glucose and maltose concentrations independent of nitrogen supplementation. At high sugar concentrations diauxie was not easily observed, and growth and ethanol production depended on the nature of the nitrogen source. This was different for baking and brewing ale and lager yeast strains. Sugar concentration had a strong effect on the shift from oxido‐fermentative to oxidative metabolism. At low sugar concentrations, biomass production was similar under both peptone and casamino acid supplementation. Under casamino acid supplementation, the time for metabolic shift increased with the glucose concentration, together with a decrease in the biomass production. This drastic effect on glucose fermentation resulted in the extinction of the second growth phase, probably due to the loss of cell viability. Ammonium salts always induced poor yeast performance. In general, supplementation with a nitrogen source in the peptide form (peptone) was more positive for yeast metabolism, inducing higher biomass and ethanol production, and preserving yeast viability, in both glucose and maltose media, for baking and brewing ale and lager yeast strains. Determination of amino acid utilization showed that most free and peptide amino acids present, in peptone and casamino acids, were utilized by the yeast, suggesting that the results described in this work were not due to a nutritional status induced by nitrogen limitation.     

Carrier‐free,continuous primary beer fermentation

Developing a sustainable continuous fermentation reactor is one of the most ambitious tasks in brewing science, but it could bring great benefits regarding volumetric productivity to modern breweries. Immobilized cell technology is often applied to reach the large densities of yeast needed in a continuous fermentation process. However, the financial cost associated with the use of carriers for yeast immobilization is one of the major drawbacks in the technology. This work suggests that yeast flocculation could address biomass immobilization in a gas‐lift reactor for the continuous primary fermentation of beer. Nearly 25 g dry wt L^?1 of yeast was flocculated in the reactor before interruption of the fermentation. Stable sugar consumption and ethanol production (4.5% alcohol by volume) from an 11°P wort was evidenced. The key esters and higher alcohols measured in the young beer met the standards of a finished primary beer fermentation. Copyright © 2014 The Institute of Brewing & Distilling     

Construction of a URA3 deletion strain from the allotetraploid bottom-fermenting yeast Saccharomyces pastorianus

The bottom‐fermenting lager yeast Saccharomyces pastorianus has been proposed to be allotetraploid, containing two S. cerevisiae (Sc)‐type and two S. bayanus (Sb)‐type chromosomes. This chromosomal constitution likely explains why recessive mutants of S. pastorianus have not previously been reported. Here we describe the construction of a ura3 deletion strain derived from the lager strain Weihenstephan34/70 by targeted transformation and subsequent loss of heterozygosity (LOH). Initially, deletion constructs of the Sc and Sb types of URA3 were constructed in laboratory yeast strains in which a TDH3p‐hygro allele conferring hygromycin B resistance replaced ScURA3 and a KanMX cassette conferring G‐418 resistance replaced SbURA3. The lager strain was then transformed with these constructs to yield a heterozygous URA3 disruptant (ScURA3⁺/Scura3Δ::TDH3p‐hygro, SbURA3⁺/Sbura3Δ::KanMX), which was plated on 5‐fluoroorotic acid (5‐FOA) plates to generate the desired Ura^– homozygous disruptant (Scura3Δ::TDH3p‐hygro/Scura3Δ::TDH3p‐hygro Sbura3Δ::KanMX/Sbura3Δ::KanMX) through LOH. This ura3 deletion strain was then used to construct a bottom‐fermenting yeast transformant overexpressing ATF1 that encodes an enzyme that produces acetate esters. The ATF1‐overexpressing transformant produced significantly more acetate esters than the parent strain. The constructed ura3? lager strain will be a useful host for constructing strains of relevance to brewing. Copyright © 2012 John Wiley & Sons, Ltd.     

Effects of Fermentation Parameters and Cell Wall Properties on Yeast Flocculation1

Industrial wort was fermented with a NewFlo phenotype ale yeast in lab‐scale cylindrical fermenters. The effects of various fermentation parameters and yeast cell wall properties on yeast flocculation were studied during 120 h fermentation. The evaluation of the cell volume during the fermentation revealed a non‐normal distribution (p < 0.05) at most fermentation times. Overall yeast cell size initially decreased during the first 24 h of fermentation then increased during the 24–60 h fermentation period. Cell size subsequently declined until the end of fermentation presumably due to floc settling. While yeast flocculation began after 24 h fermentation, most flocs remained in suspension until 60 h when the average turbulent shear rate caused by CO₂ evolution declined to below 8 s^?1. Both the Helm's flocculence and cell surface hydrophobicity values rapidly increased to high numbers from 24 h onward. Changes in the orthokinetic capture coefficient (α₀) value with fermentation time, measured in fermenting worts, indicated a significant increase (p < 0.001) after 24 h of fermentation. Presumably, this change was due to increases in ethanol and the decline in sugar concentration with time. Although a significant positive correlation (p < 0.05) was observed between zymolectin densities and cell surface areas, the total zymolectin level on yeast cell walls did not change significantly with fermentation time (p > 0.05). Interestingly, no significant difference existed in Helm's flocculation values of suspended and settled yeast cells (p > 0.05). The flocculation rate of LCC125 was readily inhibited by addition of glucose or maltose. Results suggest that fermentable sugar levels and shear force exert major influences on yeast flocculation during beer fermentations.     

Nutrient limitation leads to penetrative growth into agar and affects aroma formation in Pichia fabianii,P. kudriavzevii and Saccharomyces cerevisiae

Among fermentative yeast species, Saccharomyces cerevisiae is most frequently used as a model organism, although other yeast species may have special features that make them interesting candidates to apply in food‐fermentation processes. In this study, we used three yeast species isolated from fermented masau (Ziziphus mauritiana) fruit, S. cerevisiae 131, Pichia fabianii 65 and Pichia kudriavzevii 129, and determined the impact of nitrogen and/or glucose limitation on surface growth mode and the production of volatile organic compounds (VOCs). All three species displayed significant changes in growth mode in all nutrient‐limited conditions, signified by the formation of metafilaments or pseudohyphae. The timing of the transition was found to be species‐specific. Transition in growth mode is suggested to be linked to the production of certain fusel alcohols, such as phenylethyl alcohol, which serve as quorum‐sensing molecules. Interestingly, we did not observe concomitant increased production of phenylethyl alcohol and filamentous growth. Notably, a broader range of esters was found only for the Pichia spp. grown on nitrogen‐limited agar for 21 days compared to nutrient‐rich agar, and when grown on glucose‐ and glucose‐ plus nitrogen‐limited agar. Our data suggest that for the Pichia spp., the formation of esters may play an important role in the switch in growth mode upon nitrogen limitation. Further biological or ecological implications of ester formation are discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Use of spent brewer's yeast in L‐(+) lactic acid fermentation

The application of by‐products from the brewing industry in lactic acid (LA) production was investigated in order to replace expensive nitrogen sources (such as yeast extract) with cheaper and renewable nitrogenous materials such as brewer's yeast (BY). In this study, brewer's spent grain (BSG) hydrolysate was used for L‐(+)‐LA fermentation by Lactobacillus rhamnosus ATCC 7469. The effect of pH control during the fermentation and the addition of various BY contents (5–50 g/L) in BSG hydrolysate on fermentation parameters was evaluated. BY addition significantly increased free amino nitrogen (FAN) concentration (by 25.2% at 5 g/L to 616% at 50 g/L). A strong positive correlation between FAN concentration in the hydrolysate and concentration of L‐(+)‐LA produced was observed (correlation coefficient of 0.913). A high cell viability of L. rhamnosus ATCC 7469 (1.95–3.32 × 10⁹ CFU/mL at the end of fermentation) was achieved in all fermentations with the addition of brewer's yeast. The addition of BY increased L‐(+)‐lactic acid yield and volumetric productivity up to 8.4% (5 g/L) and 48.3% (50 g/L). The highest L‐(+)‐LA yield (89%) and volumetric productivity (0.89 g/L h^?1) were achieved in fermentation of BSG hydrolysate with 50 g/L of BY. © 2019 The Institute of Brewing & Distilling     

Flocculation,cell surface hydrophobicity and 3‐OH oxylipins in the SMA strain of Saccharomyces pastorianus

Three‐hydroxy‐oxylipins (3‐OH oxylipins) have been previously detected in brewing yeast production strains at flocculation onset. In this work, the SMA strain of Saccharomyces pastorianus was characterized during growth in a miniature fermentation assay by measuring flocculation and cell surface hydrophobicity (CSH). Proportions of 3‐OH oxylipin were also measured concurrently during growth in the miniature fermentation assay and a defined 3‐OH oxylipin extraction protocol using ethyl acetate is presented along with a novel derivatization and gas chromatography–mass spectrometry (GC‐MS) detection approach. When the SMA strain was grown in the assay, near maximal CSH and flocculation levels were achieved by a 36 h fermentation time. Under the same culture conditions, the oxylipin 3‐OH decanoic acid (3‐OH 10:0) was identified. This oxylipin could not be detected early in the fermentation, but elevated relative levels of 3‐OH 10:0 were reached by 36 h, coinciding with increased CSH levels. It was previously presumed that the formation of 3‐OH oxylipins at flocculation onset might increase the CSH. However, results from this study suggest that 3‐OH 10:0 may not contribute to cell wall hydrophobicity. The flocculation behaviour of the SMA strain was also monitored in the presence of 3‐OH 10:0, but exposure to this oxylipin did not impact the sedimentation of this yeast, suggesting that 3‐OH oxylipins may not act as mediators of quorum sensing in this strain. Copyright © 2015 The Institute of Brewing & Distilling