Domesticating brewing yeast for decreasing acetaldehyde production and improving beer flavor stability

High level of acetaldehyde produced by yeast in beer industry, especially in China, is still an important issue. To obtain a brewing strain with low acetaldehyde, a novel approach, based on domestication by disulfiram–ethanol plates, was developed. A mutant D-A-14 with lower acetaldehyde was obtained with UV mutagenesis. Through longtime domestication, the mutant D-A-14 produced 60.57 % less (2.20 mg/L) than the Wt strain MA12. Besides, the ratio of higher alcohols to esters in beer fermented by the mutant D-A-14 (3.7) was lower than that of MA12 (5.8), which exhibited harmonious flavor. For further study, DNA microarray technique was exploited to analyze the changes in genes involved in metabolism between the Wt and mutant. The data of DNA microarray were consistent with the decrease in acetaldehyde, organic acids, and the ratio of higher alcohols to esters of mutant D-A-14. Therefore, the newly screened strain has the potential to be applied in the brewing industry.     

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     

CHILL PROOFING BY A STRAIN OF SACCHAROMYCES CEREVISIAE SECRETING PROTEOLYTIC ENZYMES

The attempted exploitation of a strain of yeast with extracellular protease activity in the fermentation of brewers' wort is described. S. cerevisiae strain YS01 was previously shown to secrete proteases with activity against a variety of substrates. It is shown here that these included some of the tannin precipitable proteins involved in the formation of chill haze in beer and consequently, more stable beers resulted from incubation with added YS01 cell-free culture supernatants. The proteases were also produced in an active form at low levels during fermentation of brewers' wort using established commercial conditions. Concomitantly, a marginally more stable beer resulted. Respiratory deficient, auxotrophic rare-mating was used to transfer the extracellular proteolytic characteristic (epr1.1) to a commercial ale yeast. Segregants of the recombinants obtained showed great instability in protease secretion and were unsuitable for brewing purposes. The potential advantages and drawbacks to this approach are discussed.     

啤酒酵母乙醛代谢关键酶相关性研究

乙醛是啤酒中的主要风味物质,其代谢主要来自酵母细胞。酵母中乙醇脱氢酶及乙醛脱氢酶是乙醛代谢的关键酶,对乙醛变化起着重要作用。跟踪啤酒酵母发酵过程中相对酶活力及乙醛变化,发现两种乙醇脱氢酶和乙醛脱氢酶的相对酶活力与发酵过程乙醛含量变化具有一定相关性。同时对低产乙醛啤酒酿酒酵母kb2-4与出发菌株啤酒酵母kb进行发酵试验,跟踪检测相对酶活力及乙醛含量,其乙醇脱氢酶Ⅰ和乙醇脱氢酶Ⅱ及乙醛脱氢酶相对酶活力均高于出发菌株,平均增幅分别为15.5%,11.6%和5%。3种酶活性的变化协同作用可以使乙醛含量降幅最大为33.8%。     

改良Genomeshuffling技术选育埃博霉素B高产菌株

目的：用改genomeshuffling技术选育埃博霉素高产菌株。方法：本研究对genomeshu御ing技术进行了改良,具体是在递归原生质体融合过程中对原生质体进行了紫外诱变;通过UV和DES复合诱变方法获得T4株出发菌株,并用改良genomeshuffling技术选育埃博霉素B高产菌株。结果：通过三轮基因组重组成功选育到了2株遗传稳定的高产埃博霉素B重组菌株,其中一株重组菌株S0073-45的埃博霉素B产量达到T42．5mg／L。结论：该研究证明改良后的基因组重组技术不仅能有效提高埃博霉素B的产量,而且比传统的基因组重组技术更为高效。     

Influence of yeast strain on binding of sulphur dioxide in wines,and on its formation during fermentation

The amounts of sulphur dioxide bound by acetaldehyde, pyruvic acid and α-ketoglutaric acid during fermentation of three grape juices by eight wine yeasts (Saccharomyces sp.) are reported. These constituents accounted for 49–83 % (mean 69) of the measured bound SO₂, depending on the yeast strain and juice. the maximum range of concentrations of the binding components for individual wines were 10–48 ppm for acetaldehyde, 9–77 ppm for pyruvic acid and 5–63 ppm for α-ketoglutaric acid, depending on yeast strain and grape juice. the validity of the calculations was verified by an experiment with SO₂ and the three binding compounds in a multicomponent model system. The acetaldehyde content was related to the total SO₂ present, which itself was determined by the strain of yeast. SOz bound in the wines after a further SO₂ addition was correlated significantly with pyruvic and α-ketoglutaric acids, but not with acetaldehyde. Certain yeasts produced SO₂ during fermentation in grape juice and in synthetic media with defined sulphur sources. More SO₂ was produced at pH 3.6 than 3.0 in the absence of added sulphate in grape juice. Sulphate was the best sulphur source for SO₂ production in synthetic media, although some yeasts were able to produce smaller amounts of SO₂ from l-cysteine and reduced glutathione.     

Application of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry as a monitoring tool for in‐house brewer's yeast contamination: a proof of concept

Contamination of brewer's pitching yeast cultures with wild‐type yeasts or bacteria is unwanted as it can corrupt the fermentation outcome and causes huge economic losses for the brewing industry. The applicability of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) as a fast tool to monitor the purity of brewer's yeast cultures was investigated. This proof of concept was examined for a brewer's yeast strain contaminated with wild‐type yeast and for bottled beer produced by fermentation with that particular contaminated brewer's yeast strain. The data demonstrated that MALDI‐TOF MS is very suitable to discriminate between brewing and non‐brewing yeast strains. Copyright © 2014 The Institute of Brewing & Distilling     

耐受性黄酒酵母YS6.2.5的选育及初步应用

黄酒酵母的耐受性对黄酒酿造至关重要。对原始菌株YS 分别采用乙醇- 热冲击法、细胞紫外诱变法和原生质体紫外诱变法,结合高温驯育进行选育,筛选到一株在38℃能正常生长发酵的黄酒酵母突变株YS6.2.5。YS6.2.5体积小,近似圆形,产香好,产酸少,30℃时产酒精能力、酒精耐性、高糖耐性分别为9.0%、19%、30°Bx,38℃时相应指标分别为6.5%、20%、28°Bx。酿酒实验表明,其生理耐性强,酿酒性能好,遗传性状稳定,具有较好的工业应用前景。     

Breeding of high malate‐producing diploid sake yeast with a homozygous mutation in the VID24 gene

Malate is an important taste component of sake (a Japanese alcoholic beverage) that is produced by the yeast Saccharomyces cerevisiae during alcoholic fermentation. A variety of methods for generating high malate‐producing yeast strains have been developed to date. We recently reported that a high malate‐producing strain was isolated as a mutant sensitive to dimethyl succinate (DMS), and that a mutation in the vacuolar import and degradation protein (VID) 24 gene was responsible for high malate productivity and DMS sensitivity. In this work, the relationships between heterozygous and homozygous mutants of VID24 and malate productivity in diploid sake yeast were examined and a method was developed for breeding a higher malate‐producing strain. First a diploid yeast was generated with a homozygous VID24 mutation by genetic engineering. The homozygous integrants produced more malate during sake brewing and grew more slowly in DMS medium than wild‐type and heterozygous integrants. Thus, the genotype of the VID24 mutation influenced the level of malate production and sensitivity to DMS in diploid yeast. Then a homozygous mutant from a heterozygous mutant was obtained without genetic engineering by ultraviolet irradiation and culturing in DMS with nystatin enrichment. The non‐genetically modified sake yeast with a homozygous VID24 mutation exhibited a higher level of malate productivity than the parent heterozygous mutant strain. These findings provide a basis for controlling malate production in yeast, and thereby regulating malate levels in sake. Copyright © 2016 The Institute of Brewing & Distilling     

植物乳杆菌细菌素高产菌株的诱变选育及其对肉丸的防腐保鲜作用

为了提高细菌素产量并研究细菌素对肉制品的保鲜效果,本研究以植物乳杆菌JL-A65为出发菌株,对其进行常温等离子(ARTP)诱变、甲基硝基亚硝基胍(MNNG)诱变与基因组改组,并将细菌素与双乙酸钠复配后添加到肉丸中。结果表明,ARTP诱变最佳处理时间为40 s,经筛选得到两株突变株A7-10和A8-110,细菌素产量提高率分别为45.1%和48.9%。MNNG诱变的最佳处理浓度为1.5 mg/mL,经筛选得到两株突变株M2-58和M7-111,细菌素产量提高率分别为46.6%和31.3%。对植物乳杆菌进行基因组改组最终得到一株融合菌株F4-23,细菌素产量为413 mg/L,较原始菌株提高了103.48%。细菌素与双乙酸钠之间存在协同作用,可以使肉制品保质期较对照延长5 d。结论:利用理化诱变结合基因组改组可以获得细菌素高产菌株,细菌素与双乙酸钠复配后可以使肉丸保质期较对照延长5 d。细菌素对肉丸具有较好的防腐保鲜效果。     

Genome shuffling to improve fermentation properties of acetic acid bacterium by the improvement of ethanol tolerance

Whole‐cell biocatalysts are commonly limited by low tolerance of extreme process conditions such as alcoholic strength, temperature, pH and concentration of solute. Here, we describe the use of genome shuffling to improve the ethanol tolerance of acetic acid bacterium, which is a poorly characterised industrial strain. The best strain A3‐11 was constructed and selected after three rounds of shuffling from the wild‐type strain of acetic acid bacterium. A3‐11 could grow in liquid medium with 13% ethanol. During the vinegar fermentation, the organic acid components produced were distinctly improved by A3‐11. It was found that the content of acetic acid was twice as much as the control. Furthermore, the concentration of tartaric acid was 4.04‐fold more than the control. Notably, A3‐11 increased in butanedioic acid production by up to 71.73% compared with the control.     

脲基酰胺酶基因在黄酒酵母中的整合型表达

氨基甲酸乙酯是一种人类的潜在致癌物,在许多发酵酒中均有存在,主要来源于发酵过程中酵母代谢产生的尿素。以pYX212为载体,将脲基酰胺酶基因DUR1,2克隆到TPI强启动子和终止子之间的位点,再通过同源重组的方式将受强启动子调控的目的基因整合到黄酒酵母的基因组中,最终获得一株低产尿素的胞内脲基酰胺酶基因组成型高表达的黄酒酵母85#DUR1,2。在实验室规模的黄酒酿造实验中,85#DUR1,2产尿素量为8.34mg/L,比出发菌株降低了69.9%,贮存一段时间后的酒液中氨基甲酸乙酯含量比出发菌株降低了40.5%,而发酵性能、酒精度、总酸及氨基态氮与出发菌株无显著差异。     

Genome shuffling of Zygosaccharomyces rouxii to accelerate and enhance the flavour formation of soy sauce

BACKGROUND: The purpose of this study was to achieve rapid improvement of the flavour of soy sauce by increasing the salt stress resistance of Zygosaccharomyces rouxii. Here, we describe genome shuffling to improve the salt tolerance of Z. rouxii while simultaneously enhancing and accelerating flavour formation of soy sauce. RESULTS: A mutant, S3‐2, with a stronger resistance to salt, was selected after three rounds of genome shuffling. S3‐2 not only grew well in peptone/yeast extract/dextrose medium containing a high salt content with wide range of pH, but also exhibited stronger stress resistance to potassium chloride and lithium chloride. In high‐salt liquid fermentation, S3‐2 obviously accelerated flavour formation of soy sauce, thus decreasing the total time required for development of the aroma. In addition, S3‐2 gave high amino acid nitrogen and good flavour. In particular, the ethyl acetate content was 2.38 times that in the control. S3‐2 distinctly improved the formation of 4‐hydroxy‐2 (or 5) ‐ethyl‐5 (or 2) ‐methyl‐3 (2H) ‐furanone by up to 75%. Another important flavour component, 4‐ethylguaiacol, was also detected. CONCLUSIONS: Genome shuffling was successfully used to achieve significant improvements in flavour formation. The selected strain improved the main flavour components and amino acid nitrogen, thereby enhancing the quality of soy sauce. Copyright © 2009 Society of Chemical Industry     

Fed‐batch fermentation with glucose syrup as an adjunct for high‐ethanol beer brewing

To produce a beer with a high ethanol content, preliminary research on fed‐batch fermentation profiles with glucose syrup as an adjunct during the primary fermentation period was conducted. The ethanol concentration of the beer was elevated by feeding a glucose syrup into the fermentors at a later stage of primary fermentation. Fermentation trials were carried out using a typical lager strain, SC‐9, with a pitching rate at 7.0 × 10⁶ cells/mL. An all‐malt wort (12.5°P) was employed and the primary fermentation temperature was 14 °C. Glucose syrup was supplemented when the concentration of residual reducing sugars was decreased to ~10 g/L. Results showed that the supplemented glucose was consumed rapidly and that the ethanol concentration in the final beer was raised to 67.9 g/L. Additional growth of yeast was observed after feeding accompanied by a low yield of ethanol (~0.46 g/g). Formation of diacetyl was enhanced by yeast growth and two additional peaks were obtained after feeding. The peak value of the diacetyl concentration was 1.90 mg/L. The fed‐batch fermentation resulted in a beer with an overproduction of higher alcohols and esters, indicating that brewing under these experimental conditions led to an unbalanced flavour profile. Results of optimization demonstrated that the optimal conditions were found to be 15°P for initial wort extract, 10 °C for fermentation temperature and 20 × 10⁶ cells/mL for yeast pitching rate, leading to total higher alcohols of 173.8 mg/L, total esters of 22.8 mg/L and an acetaldehyde concentration of 40.5 mg/L. A 12 day maturation and fermentation temperature of 8 °C was needed to reduce the acetaldehyde to 14.3 mg/L. Copyright © 2014 The Institute of Brewing & Distilling     

Influence of yeast strains on the physicochemical characteristics,methanol and acetaldehyde profiles and volatile compounds for Korean rice distilled spirit

The objective of this study was to investigate the influence of yeast strains on the physicochemical characteristics, methanol and acetaldehyde profiles, and volatile compounds of Korean rice distilled spirits. Ten yeast strains were employed for the brewing of distilled spirits and the resulting products were filtered and distilled twice. The amounts of methanol and acetaldehyde for the ten yeasts showed different profiles. Higher amounts of methanol were detected for strains CL, CY, DV, BD, ED and LP, while EC, D2, D4 and RH had <2 mg L^?1 methanol content. Strains D2, BD and ED produced the lowest amounts of acetaldehyde. The head portions of the spirits, which started in the fraction that contained <5 mg L^?1 of acetaldehyde, were between 7.7% (BD) and 18.2% (LP) of the total fractions. Strains D2, CL and CY produced more alcohol in the body fraction than the other yeasts. The major volatile compounds were esters in the form of fatty acid ethyl esters, such as ethyl palmitate, ethyl myristate and ethyl oleate. Isoamyl alcohol, which is an important volatile compound for rice wine, occupied 0.91–2.24% of the relative peak areas. Strain D2, of the strains tested, appeared to be the most appropriate yeast for Korean distilled spirit based on alcohol production and the high relative peak area of volatile compounds, except for ethanol. Strains CL and CY could also be considered for producing high‐quality Korean rice distilled spirits with efficiency and flavour. Copyright © 2015 The Institute of Brewing & Distilling     

Improvement of <Emphasis Type="Italic">Candida parapsilosis</Emphasis> by genome shuffling for the efficient production of arabitol from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

Arabitol is used in the food industry as a low-calorie sweetener. It is produced by yeasts during the biotransformation process of l-arabinose. Genome shuffling was performed in Candida parapsilosis DSM 70125, an efficient producer of arabitol, to obtain fusants with improved arabitol production ability. Four mutants from the parental library were used for the first round of genome shuffling. The best fusants, GSI-1 and GSI-10A, were subjected to a second round of genome shuffling. Finally, two fusants, GSII-3 and GSII-16, produced concentrations of arabitol that were 50% higher than that of the wild-type strain during selection culture. Under the optimal conditions established for C. parapsilosis, the two fusants produced 11.83 and 11.75 g/L of arabitol and were approximately 15–16% more efficient than the wild-type strain. Flow cytometry analysis showed that the ploidy of the new strains did not change.     

Genome shuffling to improve fermentation properties of top-fermenting yeast by the improvement of stress tolerance

Fermentation properties of top-fermenting yeast are under the control of multiple genes difficult to manipulate directly by classical breeding, metabolic engineering, or other genetic methods with specific genes or pathways as target. Here, genome shuffling is introduced to improve fermentation performance (such as the viability of the yeast, flavor of beer, and the fermentation time) by improving wort and ethanol tolerance of top-fermenting yeast. The strategy was performed not based on polyethylene glycol (PEG)-mediated protoplast fusion but using yeast sexual and asexual reproduction by itself. The best performing strain W3-8 was selected on the selective plates after 3 rounds of genome shuffling. The fermentation time of W3-8 was not only markedly shortened, but also, most flavor compounds were distinctly improved. In particular, ethanol yield was increased by up to 67% after the 3^rd pitching compared with the control. Furthermore, W3-8 promoted desired amounts of esters and higher alcohols, in accordance with specific consumer preferences. Significant improvement in the fermentation traits of the top-fermenting yeast was achieved using genome shuffling.     

QTL mapping of sake brewing characteristics of yeast

A haploid sake yeast strain derived from the commercial diploid sake yeast strain Kyokai no. 7 showed better characteristics for sake brewing compared to the haploid laboratory yeast strain X2180-1B, including higher production of ethanol and aromatic components. A hybrid of these two strains showed intermediate characteristics in most cases. After sporulation of the hybrid strain, we obtained 100 haploid segregants of the hybrid. Small-scale sake brewing tests of these segregants showed a smooth continuous distribution of the sake brewing characteristics, suggesting that these traits are determined by multiple quantitative trait loci (QTLs). To examine these sake brewing characteristics at the genomic level, we performed QTL analysis of sake brewing characteristics using 142 DNA markers that showed heterogeneity between the two parental strains. As a result, we identified 25 significant QTLs involved in the specification of sake brewing characteristics such as ethanol fermentation and the production of aromatic components.     

THE GENETIC MANIPULATION OF KILLER CHARACTER INTO BREWING YEAST

A procedure is described whereby the cytoplasmically-inherited killer character of a laboratory strain of Saccharomyces cerevisiae is transferred to a brewing yeast strain. Neither preparation of protoplasts of the brewing yeast nor mutation of its nuclear genes are required for this process. The brewing yeast killer strains produced have the advantages over their parent brewing cell that they kill sensitive yeasts and are immune to the killing action of certain killer yeasts. The method described offers significant advantages over the process of transformation as a means of genetically manipulating commercial yeasts.