Genetically modified industrial yeast ready for application

Tremendous progress in the genetic engineering of yeast had been achieved at the end of 20th century, including the complete genome sequence, genome-wide gene expression profiling, and whole gene disruption strains. Nevertheless, genetically modified (GM) baking, brewing, wine, and sake yeasts have not, as yet, been used commercially, although numerous industrial recombinant yeasts have been constructed. The recent progress of genetic engineering for the construction of GM yeast is reviewed and possible requirements for their application are discussed. 'Self-cloning' yeast will be the most likely candidate for the first commercial application of GM microorganisms in food and beverage industries.     

Constructing an amylolytic brewing yeast Saccharomyces pastorianus suitable for accelerated brewing

An amylolytic brewing yeast Saccharomyces pastorianus, free of vector sequences and drug-resistance genes, was constructed by disrupting the alpha-acetolactate synthase gene and introducing the alpha-amylase gene as a selective marker. The resulting recombinant strain was able to utilize starch as the sole carbon source and its alpha-acetolactate synthase activity was lowered by 30%. Fermentation tests confirmed that the diacetyl concentration and the residual oligosaccharide were reduced by 70% and 25%, respectively, in fermented wort by the recombinant strain, while the brewing performance of the recombinant strain was retained.     

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     

超高浓酿造下抗葡萄糖阻遏啤酒酵母的筛选

根据高浓发酵下(16°P)发酵度的高低,挑选下面啤酒酵母C12作为出发菌株。经过2-去氧-D-葡萄糖的定向驯养、抗性平板分离初筛以及复筛验证等步骤,筛选出一株抗葡萄糖阻遏效应的菌株CM23。将该菌株在18°P麦汁15℃条件下进行3L的EBC小型啤酒发酵实验并测定发酵指标。结果表明:与出发菌株相比,CM23的降糖速度提高了37%,达到1.8°P/d,真正发酵度达到66%,且双乙酰还原能力以及啤酒中主要风味物质含量基本不变。CM23是一株具有工业应用前景的啤酒超高浓酿造酵母菌株。     

低产乙醛啤酒酵母的定向驯化筛选

为降低啤酒中乙醛含量,采用紫外线对1株啤酒工业生产菌株MI4进行诱变,经双硫仑平板初筛、乙醛培养基驯化复筛,获得了1株低产乙醛的啤酒酵母D-A-14。与出发菌株MI4相比,采用该突变株酿制的啤酒中乙醛含量为2.86 mg/L,降低了76%;且高级醇总量降低而酯含量升高,风味更加协调。这表明筛选得到的低乙醛突变株适于啤酒工业生产。     

Optimised quantification of the antiyeast activity of different barley malts towards a lager brewing yeast strain

The brewing of beer involves two major biological systems, namely malted barley (malt) and yeast. Both malt and yeast show natural variation and assessing the impact of differing malts on yeast performance is important in the optimisation of the brewing process. Currently, the brewing industry uses well-established tests to assess malt quality, but these frequently fail to predict malt-associated problem fermentations, such as incomplete fermentations, premature yeast flocculation (PYF) and gushing of the final beer product. Antimicrobial compounds, and in particular antiyeast compounds in malt, may be one of the unknown and unmeasured malt factors leading to problem fermentations. In this study, the adaptation of antimicrobial assays for the determination of antiyeast activity in malt is described. Our adapted assay was able to detect differing antiyeast activities in nine malt samples. For this sample set, malts associated with PYF during fermentation and gushing activity in beer showed high antiyeast activity. Both PYF and gushing are malt quality issues associated with fungal infection of barley in the field which may result in elevated antimicrobial activity in the barley grain. Also, two more malts that passed the normal quality control tests were also observed to have high antiyeast activity and such malts must be considered as suspect. Based on our results, this assay is a useful measure of malt quality as it quantifies the antiyeast activity in malt which may adversely impact on brewery fermentation.     

Construction of proteinase A deficient transformant of industrial brewing yeast

Yeast proteinase A is detrimental to beer foam. The proteinase A deficient transformant of industrial brewing yeast, WZ65/a, was constructed using PCR-mediated gene disruption, and the transformant was verified to be genetically stable. The PCR analysis showed that PEP4 gene coding for proteinase A in the WZ65/a was disrupted. No matter in the yeast cells or in the fermenting liquor of WZ65/a, proteinase A activity could not be detected. Analysis of the main charicteristics indexes of beer also showed that proteinase A activity and foam performance in the beer brewed with WZ65/a were better than that of the host strain, WZ65.     

Synthesis of superoxide dismutase,catalase and other enzymes and oxygen and superoxide toxicity during changes in oxygen concentration in cultures of brewing yeast

The physiological effects on brewing yeast, growing in semi-defined wort medium, of a sudden transition from aerobiosis to anaerobiosis were studied. Two yeast strains were examined, used for ale and lager fermentations respectively. The reverse transition (from anaerobiosis to aerobiosis) was also examined. Transitions were applied by changing the sparging gas during growth or in stationary phase, and the effects on the specific activities of certain key enzymes and on the viability of the cultures were examined. Neither type of transition led to significant changes in growth rate, the rate of ethanol production or the specific activities of alcohol dehydrogenase and pyruvate decarboxylase. The most significant change was in the specific activity of CuZn-superoxide dismutase, which showed a rapid increase in activity on transition from anaerobiosis to aerobiosis, and a decrease in activity on the reverse transition. Catalase activity in the ale yeast generally followed that of CuZn-superoxide dismutase, whereas in the lager yeast it remained unchanged by the transitions. The transition from anaerobiosis to aerobiosis caused increases in citrate synthase and Mn-superoxide dismutase, though only after a significant lag period. Aerobic to anaerobic transitions caused a decrease in Mn-superoxide dismutase activity, while citrate synthase remained unchanged. Anaerobically grown cells showed a rapid loss in viability on exposure to oxygen (5–7% in the first hour), while aerobically grown cells were unaffected. When anaerobically grown cells were exposed to 0·25 mM -potassium superoxide, there was an 8% loss of viability within 10 min, whereas aerobic cells were not affected. It is concluded that the toxic effect of oxygen is due to superoxide (or a species derived from it) and that the CuZn-superoxide dismutase (but not the Mn-isoenzyme) plays a role in protecting the cells. The de novo synthesis of the CuZn-enzyme is not always rapid enough to confer full protection.     

Overview of craft brewing specificities and potentially associated microbiota

The brewing process differs slightly in craft breweries as compared to industrial breweries, as there are fewer control points. This affects the microbiota of the final product. Beer contains several antimicrobial properties that protect it from pathogens, such as low pH, low oxygen and high carbon dioxide content, and the addition of hops. However, these hurdles have limited power controlling spoilage organisms. Contamination by these organisms can originate in the raw materials, persist in the environment, and be introduced by using flavoring ingredients later in the process. Spoilage is a prominent issue in brewing, and can cause quality degradation resulting in consumer rejection and product waste. For example, lactic acid bacteria are predominately associated with producing a ropy texture and haze, along with producing diacetyl which gives the beer butter flavor notes. Other microorganisms may not affect flavor or aroma, but can retard fermentation by consuming nutrients needed by fermentation yeast. Quality control in craft breweries today relies on culturing methods to detect specific spoilage organisms. Using media can be beneficial for detecting the most common beer spoilers, such as Lactobacillus and Pediococci. However, these methods are time consuming with long incubation periods. Molecular methods such as community profiling or high throughput sequencing are better used for identifying entire populations of beer. These methods allow for detection, differentiation, and identification of taxa.     

Disruption of ubiquitin-related genes in laboratory yeast strains enhances ethanol production during sake brewing

Sake yeast can produce high levels of ethanol in concentrated rice mash. While both sake and laboratory yeast strains belong to the species Saccharomyces cerevisiae, the laboratory strains produce much less ethanol. This disparity in fermentation activity may be due to the strains' different responses to environmental stresses, including ethanol accumulation. To obtain more insight into the stress response of yeast cells under sake brewing conditions, we carried out small-scale sake brewing tests using laboratory yeast strains disrupted in specific stress-related genes. Surprisingly, yeast strains with disrupted ubiquitin-related genes produced more ethanol than the parental strain during sake brewing. The elevated fermentation ability conferred by disruption of the ubiquitin-coding gene UBI4 was confined to laboratory strains, and the ubi4 disruptant of a sake yeast strain did not demonstrate a comparable increase in ethanol production. These findings suggest different roles for ubiquitin in sake and laboratory yeast strains.     

浓香型白酒酿造微生物研究进展

在传统固态白酒酿造过程中,微生物对白酒的产量、品质、风味等都起着重要作用。该文以浓香型白酒生产的关键工艺为依据,对酒曲、窖泥、酒醅微生物的研究现状进行阐述,从参与酿造过程微生物的角度了解白酒发酵、生香、产酯等过程的实质,为浓香型白酒生产中酿造微生物的进一步研究提供思路。提出酿造微生物与白酒品评中"色、香、味、格"之间的联系,尤其是酿造微生物与白酒中呈香呈味物质的相关性研究,将是今后白酒风味化学研究的重要方向与趋势。     

Inhibition of mitochondrial fragmentation during sake brewing causes high malate production in sake yeast

We previously demonstrated the presence and fragmentation of mitochondria during alcohol fermentation. Here, we show that Fis1p induces mitochondrial fragmentation, and inhibition of mitochondrial fragmentation causes higher malate production during sake brewing. These findings indicate that mitochondrial morphology affects the metabolism of constituents, providing a breeding strategy for high-malate-producing yeasts.     

微型啤酒的酿造与酵母扩培技术

在微型啤酒设备中,一般不设计酵母扩培罐,所用酵母以外购为主,接种酵母的优劣就成了啤酒质量的关键。介绍了一种简捷方便、自主进行酵母逐级扩大培养的实用技术,争取做到酵母自备自用。同时,选用优质原料,采用科学合理的糖化、发酵工艺,确保酿制出优质的鲜啤酒,以满足广大消费者的需要。     

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.     

浅谈现代酿造酱油调配生产的体会

以生产实践中所观察到的基本现象浅述了发酵酱汁存在的缺陷和原因，介绍了合理使用食品添加剂的体会及酱汁配兑简易计算公式、操作应注意的问题。同时提出了现代酿造酱油调配生产的工艺流程。     

“SP-3”与“SP-2”在全小麦啤酒酿造中应用研究

“SP-3”是新选育的啤酒酵母菌株,而“SP-2”为通常生产大麦芽啤酒使用的啤酒酵母菌株,“SP-3”与“SP-2”啤酒酵母菌株在全小麦啤酒生产中应用对比试验结果表明,“SP-3”啤酒酵母菌株在全小麦芽啤酒的酿造中适用性较强,各项指标均优于“SP-2”啤酒酵母菌株,用其酿制的啤酒口感纯正、清爽、柔和,能够较好地适应当前消费者的口感需求.     

活性干酵母在酱油中的应用

酵母菌在酱油及酱类生产中能产生特殊的香味,是酱香的重要来源;在酱油生产中添加酱油活性干酵母,能使酱油中的乙醇和总酯的含量等指标明显提高,酱油品质显著改善;酱油活性干酵母作为一种活性干菌体使用、存储也非常方便.     

Factors influencing ethanol production rates at high-gravity brewing

The aim of this work was to investigate the influences of different fermentation factors on ethanol production rates by Saccharomyces cerevisiae (lager strain), at high-gravity brewing using response surface methodology. An empirical linear polynomial model was developed to describe the behaviour of the dependent variable as a function of significant factors. The resultant functional relationship in terms of coded values for predicting ethanol production rates was: Y=0.421+0.155X₂+0.0575X₂X_3, where Y represents the ethanol production rate (g/lh), and X₂ and X₃ are coded levels for fermentation temperature and nutrient supplementation, respectively. Patterns of yeast growth, decrease in wort gravity and ethanol production were also evaluated at the maximum ethanol production rate (0.694 g/lh). It was concluded that higher ethanol production rates could be achieved by increasing fermentation temperature and supplementing high-gravity worts with yeast extract, ergosterol and Tween 80.