Construction of recombinant industrial brewer’s yeast with lower diacetyl production and proteinase A activity

The characteristic buttery taste of diacetyl has long been a major problem in the brewing industry, and the foam stability of unpasteurized beer is often influenced by proteinase A (PrA), which is encoded by PEP4 and released from yeast cells into beer during brewing. A recombinant industrial brewer’s yeast strain that reduces the diacetyl content of beer and improves foam stability was constructed. We constructed a PGK1p-ILV5-PGK1t expression cassette, which was introduced into one of the PEP4 alleles via PCR-mediated homologous recombination. Then, the second PEP4 allele was disrupted using the Cre-loxP recombination system, and the recombinant strain was designated as S-CSIK12. The results show that the diacetyl production of S-CSIK12 is always lower than that of the host strain at all stages of beer fermentation. In addition, brewing with S-CSIK12 reduced the PrA activity of the final beer by 44 % compared with that using the wild-type strain. The head retention of the beer brewed with S-CSIK12 (260 ± 2 s) was better than that of the host strain S-6 (212 ± 3 s). Considering that more PrA is released from yeast cells during the final stage of main fermentation and that the timing of yeast cropping is determined by diacetyl reduction, brewing with strains that have low diacetyl production also reduced the PrA activity of the beer and improved its head retention. The present study provides reference for the brewing industry as well as research on the diacetyl reduction and foam stability of beer.     

Construction of recombinant industrial <Emphasis Type="Italic">S. cerevisiae</Emphasis> strain with barley lipid-transfer protein 1 secretion capability and lower PrA activity

Beer barley LTP1 in beer is an important component of beer foam, and it participates in the formation of beer foam. The digestion of beer barley LTP1 by proteinase A from brewing yeast leads to the decline of beer foam stability, especially for the unpasteurized beer. The objective of this study was to construct an industrial brewing yeast strain to secrete recombinant barley LTP1 into fermenting wort during beer fermentation for the foam stability improvement. We constructed barley LTP1 expression cassette and transformed into the host industrial yeast cells to replace partial PEP4 alleles using homologous recombination method. The expression of b-LTP1 was under control of the constitutive yeast ADH1 promoter, and the concentration of recombinant barley LTP1 secreted by recombinants reached 26.23 mg/L after incubation in YEPD medium for 120 h. The PrA activity of the recombinant strain declined compared with the host strain. The head retention of beer brewed with the recombinant industrial strain (326 ± 12 s) was improved when the host strain WZ65 (238 ± 7 s) and the constructed strain S.c-P-1 (273 ± 10 s) with partial PEP4 gene deficiency were used as control. The present study may provide reference for brewing industries and researches on beer foam stability.     

High‐Throughput Isolation of Bacteriocin‐Producing Lactic Acid Bacteria,with Potential Application in the Brewing Industry

Lactic acid bacteria (LAB) were isolated from malted cereals by means of a high‐throughput screening approach and investigated for antimicrobial activity against a range of beer‐spoiling bacteria. Putative bacteriocin‐producing strains were identified by 16S rRNA analysis and the inhibitory compounds were partially characterized. Following determination of the inhibitory spectra of the strains, an unspeciated Lactobacillus sp. UCC128, with inhibitory activity against a range of beer‐spoiling strains was subjected to further characterization. A bacteriocin was purified from this strain and analyzed by mass spectrometry to determine the weight of the protein. The result indicated that the bacteriocin was highly similar to pediocin AcH/PA‐1 from Pediococcus acidilactici. The bacteriocin‐producers identified in this study have the potential to be used in the brewing industry to enhance the microbiological stability of beer in conjunction with hurdles already in place in the brewing process.     

EXPRESSION OF THE STA2 GLUCOAMYLASE GENE OF SACCHAROMYCES CEREVISIAE IN BREWERS' YEAST

Two fragments of DNA containing the Saccharomyces cerevisiae STA2 glucoamylase gene, with differing lengths of 5î non-coding DNA, were separately subcloned into a yeast centromeric plasmid. Of these two subclones, only the shorter one (containing 127 base-pairs of 5î non-coding DNA) was able to confer glucoamylase production on a standard laboratory strain of S. cerevisiae. The longer subclone (containing 465 bp of 5î non-coding DNA) did, however, confer glucoamylase production on a strain of S. cerevisiae lacking a functional STA10 gene (which encodes a repressor of STA2 gene expression). All-yeast plasmids lacking bacterial DNA were constructed from the two STA2 subclones for the transformation of a lager brewing yeast. Only the shorter STA2 subclone conferred glucoamylase activity on this yeast. The level of enzyme activity was comparable to that produced by the same yeast strain containing STA2 expressed from the PGK1 (that is, PGK1) promoter.     

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     

The development of bactericidal yeast strains by expressing the Pediococcus acidilactici pediocin gene (pedA) in Saccharomyces cerevisiae

The excessive use of sulphur dioxide and other chemical preservatives in wine, beer and other fermented food and beverage products to prevent the growth of unwanted microbes holds various disadvantages for the quality of the end‐products and is confronted by mounting consumer resistance. The objective of this study was to investigate the feasibility of controlling spoilage bacteria during yeast‐based fermentations by engineering bactericidal strains of Saccharomyces cerevisiae. To test this novel concept, we have successfully expressed a bacteriocin gene in yeast. The pediocin operon of Pediococcus acidilactici PAC1·0 consists of four clustered genes, namely pedA (encoding a 62 amino acid precursor of the PA‐1 pediocin), pedB (encoding an immunity factor), pedC (encoding a PA‐1 transport protein) and pedD (encoding a protein involved in the transport and processing of PA‐1). The pedA gene was inserted into a yeast expression/secretion cassette and introduced as a multicopy episomal plasmid into a laboratory strain (Y294) of S. cerevisiae. Northern blot analysis confirmed that the pedA structural gene in this construct (ADH1_P‐MFα1_S‐pedA‐ADH1_T, designated PED1), was efficiently expressed under the control of the yeast alcohol dehydrogenase I gene promoter (ADH1_P) and terminator (ADH1_T). Secretion of the PED1‐encoded pediocin PA‐1 was directed by the yeast mating pheromone α‐factor's secretion signal (MFα1_S). The presence of biologically active antimicrobial peptides produced by the yeast transformants was indicated by agar diffusion assays against sensitive indicator bacteria (e.g. Listeria monocytogenes B73). Protein analysis indicated the secreted heterologous peptide to be approximately 4·6 kDa, which conforms to the expected size. The heterologous peptide was present at relatively low levels in the yeast supernatant but pediocin activity was readily detected when intact yeast colonies were used in sensitive strain overlays. This study could lead to the development of bactericidal yeast strains where S. cerevisiae starter cultures not only conduct the fermentations in the wine, brewing and baking industries but also act as biological control agents to inhibit the growth of spoilage bacteria. Copyright © 1999 John Wiley & Sons, Ltd.     

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     

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.     

125th Anniversary Review: Diacetyl and its control during brewery fermentation

Diacetyl is a butter‐tasting vicinal diketone produced as a by‐product of yeast valine metabolism during fermentation. Concentration is dependent on a number of factors including rate of formation of the precursor α‐acetolactate by yeast, spontaneous decarboxylation of this acetohydroxy acid to diacetyl and removal of diacetyl by yeast via the action of various reductase enzymes. Lowering concentrations of diacetyl in green beer represents an expensive and time‐consuming part of the brewing process and strategies to minimize diacetyl formation or hasten its reduction have potential for improving overall efficiency of the lager brewing system. Here we review the processes that determine diacetyl levels in green beer as well as the various ways in which diacetyl levels can be controlled. The amount of diacetyl produced during fermentation can be affected by modifying process conditions, wort composition or fermentation technique, or by yeast strain development through genetic engineering or adaptive evolution. The process of diacetyl reduction by yeast is not as well understood as the process of formation, but is dependent on factors such as physiological condition, cell membrane composition, temperature and pH. The process of diacetyl removal is typically rate‐limited by the reaction rate for the spontaneous decarboxylation of α‐acetolactate to diacetyl. Copyright © 2013 The Institute of Brewing & Distilling     

Construction of a brewing yeast expressing the glucoamylase gene STA1 by mating

Standard brewing yeast cannot utilize larger oligomers or dextrins, which represent about 25% of wort sugars. A brewing yeast strain that could ferment these additional sugars to ethanol would be useful for producing low‐carbohydrate diabetic or low‐calorie beers. In this study, a brewing yeast strain that secretes glucoamylase was constructed by mating. The resulting Saccharomyces cerevisiae 278/113371 yeast was MAT a/α diploid, but expressed the glucoamylase gene STA1 . At the early phase of the fermentation test in malt extract medium, the fermentation rate of the diploid STA1 strain was slower than those of both the parent strain S. cerevisiae MAFF113371 and the reference strain bottom‐fermenting yeast Weihenstephan 34/70. At the later phase of the fermentation test, however, the fermentation rate of the STA1 yeast strain was faster than those of the other strains. The concentration of ethanol in the culture supernatant of the STA1 yeast strain after the fermentation test was higher than those of the others. The concentration of all maltooligosaccharides in the culture supernatant of the STA1 yeast strain after the fermentation test was lower than those of the parent and reference strains, whereas the concentrations of flavour compounds in the culture supernatant were higher. These effects are due to the glucoamylase secreted by the constructed STA1 yeast strain. In summary, a glucoamylase‐secreting diploid yeast has been constructed by mating that will be useful for producing novel types of beer owing to its different fermentation pattern and concentrations of ethanol and flavour compounds. Copyright © 2017 The Institute of Brewing & Distilling     

Effect of bacteriocin‐producing Pediococcus acidilactici K10 on beer fermentation

Beer is generally considered to be a beverage that has high microbiological stability. However, some undesirable lactic acid bacteria (LAB) can grow in beer and consequently spoil this beverage. In this study, bacteriocin‐producing Pediococcus acidilactici K10 was used as a means of bio‐acidifying the mash and reducing the spoilage LAB content of the beer. The K10 strain had antimicrobial activity against two beer spoilage LAB strains in wort and did not grow in a beer environment. The K10 strain was inoculated before the mashing step. The effect of K10 as a starter culture was investigated and compared with a control. As a result, filtration time was shortened by 17 min, alcohol content was increased by 137%, foam stability was increased by 156%, bitterness was increased by two bitterness units and there was a significant difference (p < 0.05) in aromatic and sour odour. The feasibility of using bacteriocin‐producing LAB strain in beer brewing is envisaged. Copyright © 2016 The Institute of Brewing & Distilling     

Saccharomyces cerevisiae Biodiversity During the Brewing Process of an Artisanal Beer: A Preliminary Study

In this study we investigated the biodiversity of Saccharomyces cerevisiae during the brewing of an artisanal beer, as well as during its storage in the bottle for 107 days at 20°C. After inoculation with an active dried yeast (ADY), the yeast counts were followed during fermentation and after bottling. Yeast loads remained stable at 10⁶–10⁷ colony forming units (cfu)/mL, and only after day 21, were they were reduced to 10⁴ cfu/mL. After three months in the bottle they spanned 10²–10⁵ cfu/mL. Almost all isolated yeasts were identified as S. cerevisiae and after molecular characterization, unexpected results were obtained. The ADY did not take over the fermentation process and only after 21 days did isolates from the beer share similarities with the inoculated strain. During storage, a high diversity was found, underlining that each bottle developed its own micro‐ecosystem. This study highlighted the necessity for better investigations of S. cerevisiae population dynamics during artisanal brewing. Even when the chemical parameters measured confirmed a correct fermentation process, the inoculated strain was not the main yeast involved in the fermentation and consequently, the final product may have different sensory characteristics from the ones expected by the producers.     

Humulus lupulus – a story that begs to be told. A review

The hop cones of the female plant of the common hop species Humulus lupulus L. are grown almost exclusively for the brewing industry. Only the cones of the female plants are able to secrete the fine yellow resinous powder (i.e. lupulin glands). It is in these lupulin glands that the main brewing principles of hops, the resins and essential oils, are synthesized and accumulated. Hops are of interest to the brewer since they impart the typical bitter taste and aroma to beer and are responsible for the perceived hop character. In addition to the comfortable bitterness and the refreshing hoppy aroma delivered by hops, the hop acids also contribute to the overall microbial stability of beer. Another benefit of the hop resins is that they help enhance and stabilize beer foam and promote foam lacing. In an attempt to understand these contributions, the very complex nature of the chemical composition of hops is reviewed. First, a general overview of the hop chemistry and nomenclature is presented. Then, the different hop resins found in the lupulin glands of the hop cones are discussed in detail. The major hop bitter acids (α‐ and β‐acids) and the latest findings on the absolute configuration of the cis and trans iso‐α‐acids are discussed. Special attention is given to the hard resins; the known δ‐resin is reviewed and the ε‐resin is introduced. Recent data on the bittering potential and the antimicrobial properties of both hard resin fractions are disclosed. Attention is also given to the numerous essential oil constituents as well as their contributions to beer aroma. In addition to the aroma contribution of the well‐known essential oil compounds, a number of recently identified sulfur compounds and their impact on beer aroma are reviewed. The hop polyphenols and their potential health benefits are also addressed. Subsequently, the importance of hops in brewing is examined and the contributions of hops to beer quality are explained. Finally, the beer and hop market of the last century, as well as the new trends in brewing, are discussed in detail. Hop research is an ever growing field of central importance to the brewing industry, even in areas that are not traditionally associated with hops and brewing. This article attempts to give a general overview of the different areas of hop research while assessing the latest advances in hop science and their impact on brewing. Copyright © 2014 The Institute of Brewing & Distilling     

The effect of hopping regime,cultivar and β‐glucosidase activity on monoterpene alcohol concentrations in wort and beer

Previous studies show that the complexity of hop aroma in beer can be partly attributed to the hydrolysis of glycosidically bound monoterpene alcohols extracted from hops during the brewing process to release volatile aglycones. However, fundamental studies that examine the extraction of glycosides during brewing and their subsequent hydrolysis by yeast have not been performed. Furthermore, extraction of other hop‐derived compounds into beer shows a strong dependency on the hop cultivar being used and the point at which it is added. This study focused on the extent of glycoside extraction owing to hopping regime and cultivar, and their hydrolysis by yeast β‐glucosidase activity. Glycoside concentrations of wort made with three different hopping regimes and three cultivars were measured by the difference in volatile aglycone concentrations between samples treated with purified β‐glucosidase and untreated samples. Aglycone concentrations were measured by solid‐phase microextraction gas chromatography–mass spectrometry. Additionally, β‐glucosidase activities for 80 different yeast strains and their effect on aglycone concentration in wort were determined. Results showed that yeast have a wide range of abilities to hydrolyse glycosides with a maximum hydrolysis occurring after 3 days of fermentation regardless of yeast activity. Although it was shown that yeast are capable of glycoside hydrolysis, glycoside concentrations in wort are low and make small contributions to hop aroma. These results help explain the extent to which different brewing yeasts and hopping regimes contribute to hoppy beer aroma through the hydrolysis of non‐volatile hop‐derived compounds. Copyright © 2017 The Institute of Brewing & Distilling     

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.     

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.     

Investigating the Antimicrobial Efficacy of a Lactococcal Bacteriocin for the Development of Microbiologically Stable Beer

Barley isolate Lactococcus lactis M30 produces an antimicrobial proteinaceous activity, which at least under laboratory conditions was shown to target beer spoiling lactic acid bacteria, including Lactobacillus brevis BSH9. The aim of this study was to investigate the application of this antibacterial activity at various stages of the brewing process and in packaged beer. Lactococcus lactis M30 was shown to produce the antimicrobial activity during growth under specific conditions in fortified unhopped wort. However, this activity was lost during wort boiling and yeast fermentation. When the bacteriocin was added directly to beer it retained in vitro activity following pasteurisation, while it was also shown to inhibit growth in situ when pasteurised beer was challenged with low levels of the beer spoiling Lactobacillus brevis BSH9 culture. The capacity of the bacteriocin to prevent microbial spoilage of beer was tested at various temperatures over a period of seven weeks. Storage of bacteriocin‐containing beer at 30°C or room temperature resulted in a decrease in antimicrobial activity over time, but when refrigerated or frozen, this beer retained sufficient activity to be effective against Lactobacillus brevis BSH9.     

The use of atmospheric and room temperature plasma mutagenesis to create a brewing yeast with reduced acetaldehyde production

High acetaldehyde levels in beer from yeast metabolism is a major concern for brewers in China. To obtain a strain with lower acetaldehyde production, this work reports a novel approach based on atmospheric and room temperature plasma mutagenesis and high‐throughput screening using 4‐methylpyrazole + disulphiram plating. A mutant LAL‐8a with lower acetaldehyde‐producing capability was obtained. The alcohol dehydrogenase activity decreased by 54% compared with the wild‐type M14 and the aldehyde dehydrogenase activity increased by 64% of the wild‐type strain. Through domestication and fermentation in EBC tubes, the mutant LAL‐8a was shown to produce 2.2 mg/L acetaldehyde, 88.2% less than the wild‐type strain M14. In addition, the ratio of higher esters to alcohols in beer fermented by the mutant LAL‐8a (0.28) was higher than M14 (0.16). The fermentation performance of LAL‐8a was similar to that of the wild‐type M14. This work suggests strain LAL‐8a a promising option for the brewing industry. © 2018 The Institute of Brewing & Distilling