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%。     

Reduced acetaldehyde production by genome shuffling of an industrial brewing yeast strain

High levels of acetaldehyde produced by yeast during fermentation can be of concern to product quality. A novel approach, based on genome shuffling, was applied to reduce the production of acetaldehyde by industrial brewing strain YS86. Four isolates with different impacts of acetaldehyde concentration were obtained from populations generated by ultraviolet irradiation and nitrosoguanidine mutagenesis. These yeast strains were then subjected to recursive pool‐wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a recombinant YSF2–9 strain produced less acetaldehyde than wild‐type strain YS86, by 64.5 and 66.2% in laboratory and pilot plant fermentations, respectively. The shuffled yeast strain YSF2–9 was genetically stable and may have a potential application in brewing industry for managing acetaldehyde in beer. Copyright © 2017 The Institute of Brewing & Distilling     

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.     

用分子生物学手段控制啤酒中乙醛含量的研究

以F718,R719为引物,以质粒pFA6a—kanMX4为模板进行PCR扩增,采用基因转化法获得1株乙醇脱氢酶Ⅱ基因突变型工业酿酒酵母。驯养后的突变株啤酒生产小试表明,突变株乙醛含量为5．386mg／L,比原菌株乙醛含量7．932mg／L有所降低;发酵液发酵结束时,双乙酰含量为0．058mg／L,比原菌株双乙酰含量0．034mg／L有所升高;突变株发酵度为63％,比原菌株66％略有降低。     

高产SO2啤酒酵母菌株发酵性能的研究

主要对高产二氧化硫啤酒酵母突变株M8的发酵性能和遗传稳定性进行了研究.啤酒发酵实验结果表明,突变株M8发酵性能较好.突变株M8第20代菌株发酵的啤酒的二氧化硫生成量比原株S-5提高了20.72%,硫化氢生成量降低了49.3%,遗传稳定性良好.     

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     

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     

高产SO_2啤酒酵母菌株抗氧化性能的研究

对高产SO_2啤酒酵母突变株M8在发酵不同时期的SO_2和H_2S生成量,以及抗氧化性能进行了研究。啤酒发酵实验结果表明,突变株M8各代菌株的SO_2生成量在发酵第5d达到最高峰(平均22.17 mg/L);突变株M-20的累计H_2S生成量比原株下降了62.5%。0℃贮酒7d,突变株M-20发酵的啤酒的SO_2生成量比原株S -5提高了13.1%,DPPH自由基清除率提高了12.4%,TBA值降低了4.6%,啤酒的风味稳定性有所改善。     

改进酿酒酵母谷物发酵性能的诱变研究

酿酒酵母经诱变获得有效利用麦汁糖的优良性能。突变菌株经5d摇床培养使YNB基础培养基的麦芽三糖发酵度从32.4%提高到81.8%。薄层层析证明突变株麦芽三糖转运能力得到改善。模拟工业发酵实验证明突变菌株的麦汁糖发酵性能大大改善,而原有发酵优良性状未受到影响。说明该突变株益于工业啤酒生产和低热啤酒的生产。     

扣囊复膜酵母菌高产突变株转化可溶性淀粉生产海藻糖的研究

以前的研究曾发现扣囊复膜酵母菌 (Saccharomycopsisfibuligerasdu)能利用可溶性淀粉发酵累积质量浓度为 180 g/L的海藻糖。然而 ,该菌株含有高活性的酸性和中性海藻糖酶 ,使发酵过程中海藻糖的累积下降。为了提高海藻糖的产量 ,去除海藻糖酶的活性是必要的。野生型菌株通过EMS的诱变作用 ,筛选出了 1株不能同化海藻糖 ,但生长速率不变的突变株。该突变株与野生型菌株相比能够利用淀粉发酵累积更高含量的海藻糖 ,与野生型菌株相比 ,突变株细胞的酸性海藻糖酶的活性低 3倍 ,中性海藻糖酶的活性低 3 7倍。这意味着酸性和中性海藻糖酶活性的降低对该突变株的海藻糖产量的提高起关键作用     

Part III: the influence of serial repitching of Saccharomyces pastorianus on the production dynamics of some important aroma compounds during the fermentation of barley and gluten‐free buckwheat and quinoa wort

The present paper is the last report of a comprehensive study regarding the influence of the serial repitching of Saccharomyces pastorianus TUM 34/70 on the composition of a barley, buckwheat or quinoa fermentation medium. In particular, it focuses on the production dynamics of important volatile compounds typically associated with the aroma of beer. Samples were taken every 24 h after 11 serial repitchings of a single starter culture, analysed for the particular aroma compound content by distillation followed by gas chromatography with flame ionization detection. The term ‘serial repitching factor’ is used for the first time to support the visual evaluation of the influence of serial repitching. Results showed that the levels of methanol in the quinoa wort fermentation were only slightly higher than in barley and in practical terms independent of successive fermentation. The behaviour of acetaldehyde in quinoa was similar to that in barley. However, there was a final 2‐fold lower production of some important aroma compounds compared with barley and buckwheat and for this reason quinoa cannot be recommended as a gluten‐free substitute to produce a bottom‐fermented beer. Regarding the buckwheat wort fermentation, a 2‐ to 3‐times lower final acetaldehyde content than in barley is desirable, whereas a relatively high methanol content is not desirable. Barley and buckwheat showed comparable sum concentrations and similar overall profiles of some important aroma compounds. From this perspective, buckwheat appears to be a promising substitute for barley as a brewing raw material. The overall conclusions of our comprehensive study (Parts I–III) are that buckwheat shows adequate brewing properties to substitute for barley in the commercial preparation of a bottom‐fermented gluten‐free beer‐like beverage, and yeast can be repitched at least 11 times. In contrast, quinoa in practical terms shows no substitutional potential for barley in beer; however, it has many nutritious advantages, thus the commercial preparation of a unique, bottom‐fermented gluten‐free ‘non‐beer‐like’ beverage – where the yeast could be repitched six times at most – appears feasible. Copyright © 2015 The Institute of Brewing & Distilling     

低产乙酸啤酒酵母菌株A12的选育及中试

以啤酒酿造生产菌株啤酒酵母JM-36为出发菌株,用甲基磺酸乙酯(EMS)诱变,用含浅蓝菌素的麦芽汁琼脂平板分离抗浅蓝菌素的突变株,在低温下发酵,以发酵液的乙酸、双乙酰、乙醛、高级醇、发酵度和凝聚性为筛选指标,得到1株发酵特性优良的菌株A12。以13°BX麦芽汁为培养基,用100L发酵罐在10℃下发酵14d,菌株A12发酵液的发酵度为68.2%,乙酸、双乙酰、乙醛和高级醇的含量分别为62.3mg/L、0.081mg/L、5.321mg/L和76.43mg/L。菌株A12的主要发酵特性优良且稳定,啤酒口感良好。     

Determination of several flavours in beer with headspace sampling–gas chromatography

In order to determine flavours (diacetyl, pentanedione, acetoin and acetaldehyde) in beer, gas chromatography coupled with headspace sampling technique was developed in our laboratory. The calibrations were accomplished by six level standard addition methods, and relative coefficients were ?0.999. Repeatability experiments were accomplished by detecting 20 successive same standard samples, which showed that detection precision was <2% relative standard deviation (RSD) for three flavours (diacetyl, pentanedione and acetaldehyde), and detection precision of acetoin was <2.5% (RSD). Four flavours gradually increased and then decreased during the process of fermentation, and they leveled off when the fermentation was over. The ratio (diacetyl/pentanedione) reflected the degree of contamination: normal beer had a ratio of approximate 1; the ratio (diacetyl/pentanedione) was higher than 1, displaying that beer was contaminated by microbes. Our data suggested that gas chromatography coupled with headspace sampling technique could be used successfully to determine flavours (diacetyl, pentanedione, acetoin and acetaldehyde) in beer.     

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.     

Construction of self‐cloning industrial brewer's yeast with SOD1 gene insertion into PEP4 prosequence locus by homologous recombination

Superoxide dismutase (SOD, encoded by SOD1), which can scavenge active oxygen free radicals, is an ideal endogenous antioxidase in beer. In this study, the SOD1 expression cassette was constructed, and this cassette contained the PGK1 promoter, the PGK1 terminator and the SOD1 gene fused to the signal sequence of the yeast mating pheromone α‐factor (MFα1_s). One of the prosequences of the PEP4 gene (encoding proteinase A, PrA) in Saccharomyces cerevisiae strain S‐6 was replaced by the SOD1 expression cassette via homologous recombination and the self‐cloning strain S54PS, which could improve the antioxidant capability and foam stability of beer, was successfully obtained. Fermentation results showed that the SOD activity of the final beer brewed with S54PS was increased by 21.06%. Accordingly, the DPPH‐radical scavenging activity of S54PS increased by 30.6% compared with that yielded by the parental strain S‐6. Furthermore, the PrA activity of S54PS was always lower than that of the parental strain at all stages of beer fermentation. The head retention of the beer (255 ± 4 s) was better than that of the parental strain (224 ± 1 s). Hence, this research implies that S54PS exhibits good brewing performance and can be applied to improve the industrial brewing process. Copyright © 2016 The Institute of Brewing & Distilling     

Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae

A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β‐Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9‐fold higher than that of the wild‐type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild‐type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild‐type strain. The relative value (mnn2 deletion mutant/wild‐type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β‐glucosidase activity using p‐nitrophenyl‐β‐glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high‐molecular‐weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.     

Reduced pyruvate decarboxylase and increased glycerol-3-phosphate dehydrogenase [NAD+] levels enhance glycerol production in Saccharomyces cerevisiae

This investigation deals with factors affecting the production of glycerol in Saccharomyces cerevisiae. In particular, the impact of reduced pyruvate-decarboxylase (PDC) and increased NAD-dependent glycerol-3-phosphate dehydrogenase (GPD) levels was studied. The glycerol yield was 4·7 times (a pdc mutant exhibiting 19% of normal PDC activity) and 6·5 times (a strain exhibiting 20-fold increased GPD activity resulting from overexpression of GPD1 gene) that of the wild type. In the strain carrying both enzyme activity alterations, the glycerol yield was 8·1 times higher than that of the wild type. In all cases, the substantial increase in glycerol yield was associated with a reduction in ethanol yield and a higher by-product formation. The rate of glycerol formation in the pdc mutant was, due to a slower rate of glucose catabolism, only twice that of the wild type, and was increased by GPD1 overexpression to three times that of the wild-type level. Overexpression of GPD1 in the wild-type background, however, led to a six- to seven-fold increase in the rate of glycerol formation. The experimental work clearly demonstrates the rate-limiting role of GPD in glycerol formation in S. cerevisiae.     

Production of alcohol‐free beer with elevated amounts of flavouring compounds using lager yeast mutants

Arrested or limited fermentation process is a widespread method used for production of alcohol‐free beer (AFB). However, it usually leads to worty off‐flavour and a lack of pleasant fruity flavour/aroma. The aim of this study was to isolate spontaneous mutants of Saccharomyces pastorianus strain 2 resistant to 5,5,5‐trifluoro‐dl ‐leucine, since the resistance is related to overproduction of flavour active isoamyl alcohol (IAAL) and isoamyl acetate (IAAC). The mutants were the subject of selection during series of screening tests aimed at selecting the best producer of target compounds (IAAL and IAAC). Subsequently, the overproduction stability of the selected mutant strain was verified in four consecutive fermentations of AFBs. A higher pitching rate and increased fermentation temperature did not result in an overall improvement in the production of the target compounds. Sensory analysis proved the flavouring effect of the spontaneous lager yeast mutant on the AFB produced by arrested fermentation. Copyright © 2013 The Institute of Brewing & Distilling     

Construction of a Single PEP4 Allele Deletion in Saccharomyces carlsbergensis and a Preliminary Evaluation of Its Brewing Performance

The secretion of proteinase A (encoded by PEP4) from brewer's yeast is detrimental to the foam stability of unpasteurized beer. The aim of this study was to construct mutants of the allopolyploid Saccharomyces carlsbergensis strain TT, which were partially or completely deficient in proteinase A activity. Allelic PEP4 genes were consecutively disrupted by using the Cre‐loxP recombination system combined with PCR‐mediated gene disruption. A single PEP4 deletion mutant TT‐M was successfully constructed. However, no viable mutant could be obtained when the second allelic PEP4 gene was deleted. The brewing performances of the parent strain and the modified strain were compared on a 100 L pilot fermenter scale. Proteinase A activity in fermented wort brewed with mutant strain TT‐M was significantly lower (p<0.05) than that of the parent strain TT, whereas no significant difference on either maltose or maltotriose assimilation (p>0.05) was found. The mutant TT‐M remained genetically stable, as shown by diagnostic PCR, after re‐streaking for 20 generations. The flavor and taste of the final fermented wort, brewed with the mutant strain TT‐M, was evaluated by the Tsingtao expert sensory panel, and found to be comparable to that of the parent strain and exhibited no distinct defects. The flavor component profiles of these two finished products were also comparable. The study demonstrated allelic genes in polyploid industrial yeasts could be efficiently and consecutively deleted by the retractive primer disruption strategy, and the mutant of Saccharomyces carlsbergensis partially deficient in proteinase A contributed to an improvement in foam stability.