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.     

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.     

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.     

青岛啤酒酵母与高浓酵母筛选高浓酿造酵母融合亲株

以青岛啤酒酵母和高浓精酵母为供试菌株,筛选出生长良好的酵母,为选育具有青岛啤酒风味的高浓酵母做准备.比较了7株酵母不同糖类发酵、离子抗性、二氧化碳减重、发酵液风味品评等指标.结果表明:T1、T2和T3是传统的青岛啤酒发酵菌株,其发酵液口味符合青岛啤酒口味要求,且对Cu2+均不耐受;而G4和G6发酵减重试验和风味物质分析中的乙醛含量指标的评价均优于G5和G7菌株,且它们的发酵液的风味也接近青啤口味.因此,选择T1、T2、T3和G4、G6作100L酿造试验,进一步确定融合亲株.     

CHARACTERISATION OF AMYLOLYTIC BREWING YEAST

Amylolytic brewing yeast can be used for the production of low carbohydrate beer and for maximizing fermentation efficiency. In this paper we describe the characterisation of amylolytic brewing yeast in which the STA2 (DEXI) gene, which codes for an extracellular glucoamylase, was cloned under two different promoters; PGK (phosphoglycerate kinase) and GPD1 (sn-glycerol-3-phosphate dehydrogenase) present on episomal plasmids. Both amylolytic strains were shown to ferment and degrade wort as efficiently as the control strain supplemented with an exogenous commercial glucoamylase, despite reduced intracellular glycogen levels (30% of wild-type). However, the nature of the promoter on the expression plasmid was shown to influence both the growth rate of the amylolytic strains and the stability of the plasmids during non-selective growth. One of the strains containing plasmid pDVX4 (GPD promoter) was found to show high levels of stability when tested in ten successive pilot scale (8Hlitre) fermentations.     

SO2与啤酒的风味稳定性

啤酒中含有一定量的SO2,SO2可由酵母产生,也可由外源添加的亚硫酸盐类物质产生。SO2对于保护啤酒的风味生物稳定性具有重要的作用,阐述了啤酒中SO2的来源、含量及在保护啤酒的风味生物稳定性等方面的作用。     

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.     

提高啤酒风味稳定性的几点措施

本文从菌种、生产工艺、添加剂等方面总结了提高啤酒风味稳定性的几点措施：改造啤酒生产菌种、发酵结束之前向发酵液中添加酵母、在生产过程中添加抗氧化剂，同时精选原料，并结合无氧化酿造，利于提高啤酒风味稳定性。     

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     

采用复合诱变选育低分泌蛋白酶A啤酒酵母菌株的研究

啤酒酵母在发酵过程中分泌胞外的蛋白酶A是影响纯生啤酒泡沫稳定性的关键因素，本研究通过采用亚硝基胍（TNG）和甲基黄酸乙酯（EMS）复合诱变选育满足食品安全性的低蛋白酶A、发酵性能正常的优良啤酒酵母菌株，为解决纯生啤酒泡沫稳定性奠定基础。     

Self-cloning brewing yeast: a new dimension in beverage production

Since the mid-1990s, biotechnology has advanced, and there has been an increased focus on using genetically modified yeast in the production of fermented beverages and the manufacturing of bioethanol. Yeast is the primary microorganism for fermented beverages such as beer, wine and sake. However, existing individual strains will not completely fulfill future demands for an efficient and high-quality fermentation. In this case, several research groups have been working on genetic modifications of yeast to create an up-to-date application. Genetically modified organisms (GMO) such as yeast, crops and plants in the food and beverage production are not desired by the consumer. A possible solution to overcome the consumer distaste of products labeled as containing GMO could be the application of self-cloning yeasts. Thus, connotated, the modification of the genome occurs without heterologous DNA. This review is an overview of current research regarding the use of self-cloning yeast in brewing, wine making, baked goods and sake production. The main focus of this paper concerns the possibilities of promoter usage and the construction of self-cloning yeast and the monitoring of self-cloning yeast.     

The Horace Brown Medal Lecture: Forty Years of Brewing Research

Horace Brown spent fifty years conducting brewing research in Burton‐on‐Trent, Dublin and London. His contributions were remarkable and his focus was to solve practical brewing problems by employing and developing fundamental scientific principles. He studied all aspects of the brewing process including raw materials, wort preparation, fermentation, yeast and beer stability. As a number of previous presenters of the Horace Brown Lecture have discussed Brown's achievements in detail, the focus of this paper is a review of the brewing research that has been conducted by the author and his colleagues during the past forty years. Similar to Horace Brown, fundamental research has been employed to solve brewing problems. Research studies that are discussed in this review paper include reasons for premature flocculation of ale strains resulting in wort underattenuation including mechanisms of co‐flocculation and pure strain flocculation, storage procedures for yeast cultures prior to propagation, studies on the genetic manipulation of brewer's yeast strains with an emphasis on the FLO1 gene, spheroplast fusion and the respiratory deficient (petite) mutation, the uptake and metabolism of wort sugars and amino acids, the influence of wort density on fermentation characteristics and beer flavour and stability, and finally, the contribution that high gravity brewing has on brewing capacity, fermentation efficiency and beer quality and stability.     

Advanced Method for Measuring Proteinase A in Beer and Application to Brewing*

Proteinase A, excreted from yeast cells into beer during fermentation in the brewing process, has been shown to degrade foam-active proteins and to decrease foam stability. In order to improve the measurement of this enzyme in beer, a new fluorescent peptide, MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys (Dnp)-NH₂, was synthesised and applied to the accurate and rapid estimation of proteinase A in commercial beer and fermenting wort. This novel substrate is several hundred times more sensitive to proteinase A than other previously reported synthetic substrates or native protein substrates. The concentration of proteinase A in beer is closely related to foam stability and proteinase A activity was found to increase gradually during fermentation. The concentration of proteinase A excreted from yeast cells is also closely related to the vitality of pitching yeast cells. This new method was successfully applied to the evaluation of yeast vitality and the development of optimum yeast handling procedures.     

不同类型酵母对精酿啤酒化学和感官特性的影响

为研究不同类型的酵母对精酿啤酒的化学和感官特性的影响,选取5株酿酒酵母（4株上面发酵酵母、1株下面发酵酵母）进行酿造发酵实验,采用顶空固相微萃取（HS-SPME）法以及离子色谱（IC）法提取并鉴定分析啤酒中的香气成分和有机酸成分,采用偏最小二乘法-判别分析（PLS-DA）进行挥发性化合物的定量与鉴别,并用电子舌对成品酒进行滋味评价。结果表明,4株上面啤酒酵母（A1-A4）与下面啤酒酵母L5相比,上面啤酒酵母的起发速度更快,发酵度更高,发酵液风味物质含量较高,酯香醇厚。综合发酵性能指标可以看出,菌株A2的发酵速度较快,最早达到发酵峰值,发酵液感官风味协调,是一株发酵性能优良的精酿啤酒酵母;菌株A3具有较高的发酵度为80.87%,乙酸乙酯的产量高达45.64 mg/L,是一株具有高发酵度的啤酒酵母。     

Cloning and characterization of the Hansenula polymorpha PEP4 gene encoding proteinase A

The Hansenula polymorpha PEP4 gene encoding proteinase A was cloned by Southern blot hybridization using the Saccharomyces cerevisiae PEP4 gene as probe and characterized by gene disruption and overexpression. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1239 nucleotides corresponding to a polypeptide of 413 amino acids, sharing about 67.2% sequence similarity with that of S. cerevisiae proteinase A. That the cloned H. polymorpha PEP4 gene encodes proteinase A was supported by a gene disruption experiment, which showed that the H. polymorpha pep4 mutant strain showed significantly reduced level of carboxypeptidase Y activity when assayed with an artificial substrate. When the PEP4 gene is overproduced in pep4 mutant strain, mature proteinase A could be found in the growth medium. N-terminal amino acid sequencing of extracellular proteinase A revealed the presence of a putative propeptide of 55 amino acids ending with a dibasic peptide (Lys-Arg), probably processed by Kex2p-like endopeptidase of H. polymorpha. The nucleotide sequence of the H. polymorpha PEP4 gene has been submitted to GenBank under Accession No. U67173.     

Construction of self-cloning industrial brewing yeast with high-glutathione and low-diacetyl production

Self‐cloning strains of industrial brewing yeast were constructed, in which one allele of α‐acetohydroxyacid synthase (AHAS) gene (ILV2) was disrupted by integrating Saccharomyces cerevisiae genes, γ‐glutamylcysteine synthetase gene (GSH1) and copper resistant gene (CUP1) into the locus of ILV2. The self‐cloning strains were selected for their resistance to CuSO₄ and identified by PCR amplification. The results of AHAS and glutathione (GSH) assay from fermentation with the self‐cloning strains in 500‐mL conical flask showed that AHAS activity decreased and GSH content increased compared with that of host yeasts. The results of pilot scale brewing in 5‐L fermentation tank also indicated that GSH content in beer fermented with self‐cloning strains T5‐3 and T31‐2 was 1.3 fold and 1.5 fold of that of host QY5 and QY31, respectively; and diacetyl content decreased to 64% and 58% of their hosts, respectively. The self‐cloning strains do not contain any heterologous DNA, they may be more acceptable to the public.     

抗老化啤酒酵母的选育

以无锡轻工大学生物工程学院保藏的一株啤酒酵母为出发菌株，经紫外线诱变及蛋氨酸连续驯养后，选育得到一株抗老化性能较为优良的菌株Ｍ４．在１ｍ３发酵罐中的中试结果表明，与出发菌株相比，其羰基化合物（ＴＢＡ）含量降低１９．１％，谷胱甘肽（ＧＳＨ）含量增加２９．６％．在不改变其它生产条件的情况下，啤酒风味稳定性提高９２％，工业应用前景良好。     

果酿啤酒的酿造工艺和品质研究进展

果酿啤酒的品质受众多因素影响,特别是水果特性差异和产品质量标准缺失使得果酿啤酒的酿造工艺存在较大差别,产品质量难以稳定。为进一步提升果酿啤酒品质,该文对近年来果酿啤酒的研究现状进行综述,探讨水果榨汁处理、灭菌方式,发酵过程中原麦汁浓度、主发酵温度、果汁添加量及添加阶段、酵母菌种选择等因素对果酿啤酒品质的影响,分析果酿啤酒的风味成分及风味劣变、活性成分及抗氧化活性,并对果酿啤酒的发展趋势及品质提升进行展望,以期实现不同果酿啤酒的精准调控,为实际生产中果酿啤酒的酿造提供借鉴。     

基于顶空气相-离子迁移谱技术对不同酵母发酵的百香果果啤香气比较

酵母菌种对啤酒的风味与品质有着重大的影响.本文以皮尔森麦汁、啤酒花、啤酒酵母、百香果原浆和水等为主要原料酿造百香果果啤,采用气相-离子迁移谱(Gas chromatography-ion mobility spectrometry,GC-IMS)技术测定不同啤酒酵母(S-04、US-05、S-33、CS31和CN36)...     

Degradation of a Foam‐Promoting Barley Protein by a Proteinase from Brewing Yeast

The degradation of a major protein component in beer, the lipid transfer protein (LTP1), by the yeast proteinase A was determined. Another major protein fraction in beer, the protein Z‐fraction, was not degraded by this enzyme. Protein preparations from beer and barley containing LTP1 were examined for degradation by proteinase A using SDS‐PAGE, immunoblotting and RP‐HPLC. LTP1 from barley was completely resistant to proteinase A, whereas LTP1 concentrated from beer was cleaved. We conclude that LTP1 was modified during the brewing process, thus rendering it more susceptible to proteinase A degradation.