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.     

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.     

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。     

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。     

Factors influencing proteinase A activity during the production of unpasteurised beer

Compared with pasteurised beer, a decline in foam retention during storage is an issue for unpasteurised beer. The major reason for this is that proteinase A is able to slowly breakdown foam promoting proteins in beer. Therefore, controlling the activity of proteinase A is key to solving this problem. In this study, foam quality in unpasteurised beer was studied systematically on a commercial scale considering factors including yeast activity, strain, generation number and storage time. Accordingly, yeast handling procedures to manage proteinase A activity were established: (1) yeast strain P with reduced proteinase A should be used in production; (2) storage time of recovered yeast should be no more than two days; (3) proteinase A activity in recycled yeast slurry should be less than 10×10^‐5 U/mL and (4) the number of yeast generations should be less than three. With the application of these measures, proteinase A activity was significantly decreased, and the corresponding foam quality was improved. © 2020 The Institute of Brewing & Distilling     

啤酒酵母细胞活性检测的新方法——细胞分析仪法

提出了一种新的检测啤酒酵母细胞活性的方法———细胞分析仪检测法。通过这种方法 ,研究了啤酒酵母自溶对酵母细胞活性的影响 ,以及高低温贮存时酵母细胞活性的变化情况。结果表明 ,低温贮存较高温条件贮存细胞活性下降幅度小。另外 ,还对不同代数酵母在发酵过程中的细胞活性进行了跟踪测定与比较分析 ,指出了啤酒发酵过程中酵母细胞活性的变化趋势 ,同时发现酵母的细胞活性影响啤酒中双乙酰的还原。     

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.     

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 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.     

纯生啤酒泡沫稳定性的研究

纯生啤酒的泡持随着货架时间的延长会逐渐衰减 ,严重影响啤酒的外观质量。大量的文献资料证实 ,纯生啤酒泡持性的下降是由酒液中存在的蛋白酶A造成的。通过对成品酒泡持性的跟踪测定 ,重点讨论了发酵及啤酒过滤过程控制对泡持衰减趋势的影响。     

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.     

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.     

Impact of the Long‐Term Maintenance Method of Brewer's Yeast on Fermentation Course,Yeast Vitality and Beer Characteristics

The effect of the long‐term maintenance method used with a brewer's yeast on its technological properties was determined in laboratory fermentation trials with a 12°P all‐malt wort. The trials were performed at a constant temperature and under conditions of constant substrate concentration. Two cultures of a bottom fermenting yeast, Saccharomyces pastorianus RIBM 95, were tested — one culture was maintained by subculturing on wort agar slopes at 4°C and the other culture underwent a three year storage in liquid nitrogen at minus 196°C. Parameters under investigation included yeast vitality measured as acidification power (AP), fermentation time needed to reach an alcohol level of 4%, the yeast cell count, sedimentation of the yeast during the fermentation, and the production of beer flavour compounds in green beer. The yeast culture stored for three years in liquid nitrogen displayed a higher count of suspended cells, required a shorter time to attenuate the wort to produce 4% alcohol and produced a 1.5 to 2.5‐fold higher concentration of a number of flavour compounds. The long‐term storage method did not affect the sedimentation ability and vitality of the yeast strain tested.     

The effect of pitching rate on fermentation,maturation and flavour compounds of beer produced on an industrial scale

The aim of the study was to determine the effect of the initial number of yeast cells in the wort on the process of fermentation, maturation and the content of the volatile components of beer, as well as the viability and vitality of the yeast biomass. The experiments were performed on an industrial scale, with fermentation and maturation in cylindro‐conical fermentation tanks with a capacity of 3800 hL. Yeast for pitching was collected after secondary fermentation (third passage) and wort pitching levels were 5 × 10⁶, 7 × 10⁶ and 9 × 10⁶ cells/mL. During fermentation and maturation, the changes in the content of the extract, yeast growth, yeast vitality and selected volatile components were investigated. Experiments showed that the yeast inoculum had a significant impact on the course of the fermentation and metabolic changes. With increasing numbers of cells introduced into the wort, the content of the esters and fusel alcohols increased, while the acetaldehyde concentration decreased. These changes affected the final quality of the beer. Copyright © 2015 The Institute of Brewing & Distilling     

低浓度淡爽型啤酒的酿造

以改善低浓度淡爽型啤酒品质为目的 ,提出了一种新颖的低浓度淡爽型啤酒的酿造方法 .采用二次煮出二段式糖化法 ,用 70 %麦芽和 3 0 %大米的原料配比 ,提高麦芽汁中糖与非糖的比值 ,并在糖化过程中添加啤酒酵母提取物作啤酒发酵的补充氮源 .所酿造的啤酒口味纯正 ,泡沫洁白细腻 ,持久挂杯 .     

泡沫陶瓷固定化酵母细胞啤酒连续主发酵工艺

采用泡沫陶瓷固定化酵母细胞,并对其应用于啤酒连续主发酵进行了实验研究,获取了优化的工艺条件:发酵温度16℃,稀释率0.044h-1。在此工艺条件下,实际测得浓度降低为7.09%,嫩啤酒双乙酰含量为0.144mg/L。嫩啤酒主要理化指标的测定结果表明:采用固定化酵母细胞啤酒主发酵工艺不会对啤酒质量造成影响。     

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。     

THE ORIGIN OF THE MEDIUM CHAIN LENGTH FATTY ACIDS PRESENT IN BEER

The medium chain length fatty acids that are excreted during fermentation are produced by synthesis and not by degradation. The fermentation of a wort supplemented with propionic acid (C₃) or valeric acid (C₅) leads to the excretion of nonanoic acid (C₉) in addition to the usual even chain acids. C₉ acid was not detected in the beer when the inoculated yeasts contained a high proportion of pentadecanoic acid (C₁₆) and heptadecanoic acid (C₁₇) or when the C₁₇ acid was added to the wort, demonstrating that a degradative route is unimportant. The content of the medium chain length fatty acids in beer varies directly with their content in yeast; thus the fatty acid composition of the beer reflects changes in the content of these acids in yeast brought about by alteration in the supply of oxygen or by the addition of C3 acid to wort.     

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.     

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.