工业化酿造过程中酵母分泌蛋白酶A的规律及影响因素的研究

对啤酒工业化规模发酵过程中酵母分泌蛋白酶A的规律进行了探讨,对酵母代数及酵母贮存条件等因素对酵母分泌蛋白酶A的影响进行了研究,并对蛋白酶A活性不同的成品纯生啤酒的泡持值、泡沫活性蛋白含量及蛋白酶A活性进行了跟踪分析。结果表明:发酵过程中,蛋白酶A的活性呈上升趋势且接种酵母的蛋白酶A活性越高,与其对应的发酵液中蛋白酶A的活性越高,成品酒的泡沫稳定性越差。另外,随着酵母代数及贮存时间的增加,酵母分泌蛋白酶A的量增加。当酵母蛋白酶A活性控制在0.015U/m L以下且成品酒的初始蛋白酶A活性在15×10-5U/m L以下时,储存4个月的成品纯生啤酒的泡沫稳定性较好。      

啤酒发酵过程中影响蛋白酶A分泌的因素

纯生啤酒中残存的蛋白酶A严重影响泡沫稳定性,制约了纯生啤酒的质量提升。为了探索啤酒发酵过程中影响蛋白酶A分泌的因素,作者分别考察了菌种、酵母生理状态、酵母代数、麦汁浓度、发酵时间等对蛋白酶A分泌的影响。结果发现,蛋白酶A分泌量高的菌株,处于稳定期之后的酵母、较高的酵母代数、较高的原麦汁浓度和在发酵阶段末期都会导致发酵液中蛋白酶A活性偏高。建议在实际生产中,采用蛋白酶A分泌量少的菌种、调整酵母生理状态、使用小于3代的酵母、采用18°P以下的麦汁发酵和尽早结束发酵都会对降低蛋白酶A的分泌量起到积极作用。     

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.     

蛋白酶A对纯生啤酒泡持性的影响研究

该文研究蛋白酶A活力对纯生啤酒泡持性的影响,同时建立初始成品纯生啤酒及过滤前发酵液蛋白酶A活力限量值。结果表明,随着贮存时间的延长,纯生啤酒蛋白酶A活力和泡持值均呈下降趋势,最终残留蛋白酶A活力是影响其货架期泡持值的主要因素。纯生啤酒泡持值与蛋白酶A活力呈显著负相关（P＜0.01）。为了保证成品纯生啤酒在货架期内泡沫稳定性,发酵液出罐滤酒前的蛋白酶A活力应＜24×10-5 U/mL,相应成品纯生啤酒初始蛋白酶A活力应＜15×10-5 U/mL。该内控标准能为纯生啤酒生产企业控制泡持性提供指导。     

纯生啤酒存放过程中泡沫稳定性与酵母蛋白酶A以及蛋白含量与组成变化的研究

酵母蛋白酶A已经被证实对啤酒泡沫稳定性有负面作用。通过测定纯生啤酒存放过程中酵母蛋白酶A活性变化、泡持性衰减及蛋白含量的变化,进一步说明酵母蛋白酶A以及蛋白种类与含量对纯生啤酒泡沫稳定性的影响及其相互关系。对不同存放时期纯生啤酒样品中蛋白质进行电泳鉴定的结果显示,存放3月后的纯生啤酒中脂肪转运蛋白1(LTP1)完全消失,这一结果表明LTP1是影响啤酒泡沫稳定性的主要蛋白,该蛋白降解可能是酵母蛋白酶A作用的结果。     

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.     

Yeast Proteolytic Activity During High and Low Gravity Wort Fermentations and its Effect on Head Retention

The aim was to discover the effect of high gravity brewing on yeast protease activity during fermentation, on the loss of hydrophobic polypeptides from wort during fermentation, and on the foam stability of stored beer. The hydrophobic polypeptide content of low (10° Plato) gravity worts showed a steady decline throughout fermentation, but for the 20° Plato wort there was a rapid decline over the first 8 days of fermentation, followed by little change over the remaining period. The decrease in hydrophobic polypeptides was greater in the high gravity fermentation. Proteinase A increased during fermentations with the highest levels being present at the end of fermentations. High gravity fermentations exhibited levels of yeast protease that from the 3rd to 11th day of fermentation were at least twice the values of the low gravity fermentations. The high gravity brewed beer contained significantly higher levels of proteinase A activity than the low gravity brewed beer. The inclusion of FERMCAP™, an antifoam, in high gravity wort did not affect either the hydrophobic polypeptide levels or foam stability of the resultant beer. This suggests that proteinase A, rather than fermenter foaming, must be the major contributor to the lack of foam stability of high gravity brewed beer. Head retention measurements conducted on the high and low gravity brewed bottled beers, over a five month period, demonstrated a steady decline in foam stability for both beers. The declines in head retention did not occur in high and low gravity beers that had been pasteurised.     

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.     

A FLUOROMETRIC ASSAY FOR PROTEINASE A IN BEER AND ITS APPLICATION FOR THE INVESTIGATION OF ENZYMATIC EFFECTS ON FOAM STABILITY

A previously developed fluorometric assay using synthetic substrate, Succinyl-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide, for yeast proteinase A (PrA) was modified for the accurate and quick determination for the activity in unpasteurized beer. Employing simple HPLC for the determination of 7-amino-4-methylcoumarine (AMC), a final degradation product on this assay, the activity of PrA in beer was measured without the interference of the fluorogenic and photosensitive substance present in beer. The assay for common unpasteurized beers was completed within 5 hours without any concentration procedure. Its linearity and reproducibility were satisfactory for quantitative purposes. Using a purified PrA from brewer's yeast, the effect of the PrA activity on foam stability during storage was furthermore clarified. The exclusive effect of PrA on foam stability was also demonstrated by proteinase inhibitor test.     

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     

The Loss of Hydrophobic Polypeptides during Fermentation and Conditioning of High Gravity and Low Gravity Brewed Beer

The object of this study was to investigate the loss of hydrophobic polypeptides, which are important for foam quality and stability in finished beer. Loss of hydrophobic polypeptide due to fermenter foaming occurs during transfer of fermented wort since a gradient of hydrophobic polypeptides towards the surface is created during fermentation. Due to higher polyphenol levels in high gravity (20°Plato) wort, more hydrophobic polypeptides are lost due to cold break (cold trub) precipitation compared to low gravity (12°Plato) wort. Another important factor affecting the loss of hydrophobic polypeptides could be proteinase A activity during fermentation, especially in high gravity fermentation where the yeast is exposed the higher stress. During high gravity fermentation, where osmotic pressures are higher, ethanol levels become greater, and nitrogen‐carbohydrate ratios are lower, more proteinase A is released by the yeast. This release of proteinase A into fermenting wort could have implications for the foam stability of the finished product.     

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。     

纯生啤酒质量影响因素初探

针对纯生啤酒的质量稳定性进行了相关的研究。研究发现 ,纯生啤酒的货架保鲜期比相应熟啤酒延长了 10 %～ 2 0 %左右 ;纯生啤酒经常会遇到泡持性下降的问题 ,运用特殊合成的底物从泡持性较差的啤酒中检测出很高的蛋白酶A活性 ;啤酒灌装后的蔗糖转化酶活性在保存 3个月后 ,经检测能保存 80 %以上的酶活 ,5个月后检测仍能保存 6 0 %以上的酶活 ;外加葡萄糖氧化酶会造成啤酒中葡萄糖含量的异常增加 ,采用液相色谱分析法对加入葡萄糖氧化酶啤酒的蔗糖转化酶活性进行了分析 ;温瓶温度升高对蛋白酶A和蔗糖转化酶活性能产生相似的作用 ,蔗糖转化酶活性对温度升高的抵抗能力稍高于蛋白酶A ,在蛋白酶A失活的临界点 ,蔗糖转化酶仍能保持约30 %的活力     

The Effect of Proteinase A on Foam‐Active Polypeptides During High and Low Gravity Fermentation

The ability of beer to produce good foam is influenced by the level of foam‐active polypeptides. Specific polypeptides with hydrophobic domains, such as Lipid Transfer Protein (LTP1), are important components of beer foam. Although, high gravity brewing is a commercially viable technique, it has the disadvantage of producing beer with less foam stability compared to lower gravity brewed counterparts. It is thought that proteinase A plays a key role in the degradation of these hydrophobic polypeptides responsible the beer foam stability. The object of this study was to compare and quantify the loss of hydrophobic polypeptides and specifically foam‐LTP1 during high gravity (20°Plato) and low gravity (12°Plato) wort fermentations and to evaluate the effect of proteinase A on these polypeptides. The losses of hydrophobic polypeptides and foam‐LTP1 were generally greater in high gravity brews. Furthermore, the results obtained suggest that proteinase A alters the hydrophobicity of these polypeptides rather than their molecular size. Approximately 20% of hydrophobic polypeptides and approximately 57% of foam‐LTP1 appeared to be proteinase A resistant. These differential losses of hydrophobic polypeptide and foam‐LTP1 could have implications for the foam stability of the finished product.     

Detection of Proteinases in Saccharomyces cerevisiae by Flow Cytometry

The physiological state of a yeast population used for inoculation determines how rapidly the cells adapt to new environmental conditions, begin proliferating and utilising extract. The decision as to whether a yeast culture is suitable for re‐pitching should not be based only on viability determinations since this can be misleading. Increased proteolytic activity in a yeast population indicates the onset of senescence. A flow cytometric method has been developed for measuring a wide variety of proteinases in Saccharomyces cerevisiae employing a commercially available casein‐dye conjugate. The detection of intracellular proteinase activity gives an early indication of apoptotic events and allows improved assessment of the physiological state of a yeast population. This knowledge will assist the industry to optimize the selection of yeast and its subsequent fermentation performance. Yeast cell autolysis with all its negative consequences for beer quality and stability will thus be minimised.     

酵母自溶的成因及其对啤酒质量的影响

酵母自溶由酵母胞内蛋白分解酶外泄引起，影响酵母自溶的因素有：(1)酵母菌种；(2)麦汁营养成分组成不合理；(3)酵母使用代数过高；(4)酵母添加量过多；(5)温度、压力、pH值等发酵工艺条件控制不当；(6)酵母回收时间、方法、压力、酵母贮存条件；(7)微生物污染。酵母自溶会影响啤酒风味稳定性，使啤酒苦味、涩味加重；啤酒双乙酰含量增加；啤酒的泡持性下降；啤酒总酸偏高；啤酒pH值升高；增加啤酒过滤成本。防止酵母自溶的方法有：(1)选择优良强壮的出发菌株；(2)控制酵母添加量和使用代数；(3)制备营养丰富、组成合理的麦汁；(4)严格发酵工艺奈件；(5)加强酵母质量管理；(6)加强卫生管理，保证纯种发酵。     

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     

酿酒酵母蛋白酶A作用机理及影响因素研究综述

蛋白酶A是酿酒酵母体内一种重要的蛋白酶,参与液泡中多种酶的加工和成熟.它分泌到细胞外,会破坏纯生啤酒的泡沫蛋白,是影响纯生啤酒泡沫稳定性的主要因素之一.本文从蛋白酶A的基本特性、形成及作用机制、活性的检测以及改变或降低纯生啤酒中蛋白酶A活性的途径等方面叙述了蛋白酶A的研究进展,为纯生啤酒泡沫稳定性的改善提供理论支持,为解决目前啤酒行业普遍存在的纯生啤酒泡沫衰减问题提供参考.     

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.