纯生啤酒泡沫稳定性的影响因素及改善策略

简要介绍了纯生啤酒泡沫稳定性的主要影响因素———泡沫阳性蛋白和酵母蛋白酶A对啤酒泡沫的影响及影响机理,并简要介绍了改善纯生啤酒泡沫的主要策略。     

酿酒酵母蛋白酶A的生化特性及生物学功能

酿酒酵母蛋白酶A是影响纯生啤酒泡沫稳定性的关键因素。由于纯生啤酒的广阔市场前景,对酵母蛋白酶A的研究越来越受到国内外研究者的关注。本文从蛋白酶A的结构、分泌、成熟、激活机制、催化机制、对啤酒泡沫的破坏作用以及酿造过程中蛋白酶A活性的变化等方面综述了蛋白酶A的研究进展,希望能为解决纯生啤酒泡沫稳定性问题提供理论参考。     

蛋白酶A与啤酒蛋白的相互作用

蛋白酶A是影响纯生啤酒泡沫稳定性的重要原因。本文利用相关蛋白质分析技术,SDS—PAGE电泳、HPLC技术和疏水色谱技术,研究蛋白酶A与啤酒蛋白质的相互作用。结果表明,蛋白酶A破坏的并不是某个分子量的啤酒泡沫蛋白,而是通过分解啤酒中疏水蛋白,从而降低了啤酒的泡沫稳定性。     

蛋白酶A与啤洒蛋白的相互作用

蛋白酶A是影响纯生啤酒泡沫稳定性的重要原因。本文利用相关蛋白质分析技术,SDS—PAGE电泳、HPLC技术和疏水色谱技术,研究蛋白酶A与啤酒蛋白质的相互作用。结果表明,蛋白酶A破坏的并不是某个分子量的啤酒泡沫蛋白,而是通过分解啤酒中疏水蛋白,从而降低了啤酒的泡沫稳定性。     

纯生啤酒泡沫稳定性的衰减规律研究

目前,市售的纯生啤酒在泡沫稳定性方面普遍存在明显的缺陷,即泡沫稳定性差,并随着货架时间的延长而逐渐下降。有的纯生啤酒存放一个月以后,其泡持性几乎衰竭。纯生啤酒泡持性的衰减主要是由成品啤酒中残留的活性蛋白酶A造成的。真正的纯生啤酒常常会因未经巴氏灭菌而残留一些酶类,其中蛋白酶A会破坏啤酒泡沫蛋白,从而使纯生啤酒的泡沫稳定性降低。本研究旨在寻找货架期间纯生啤酒泡沫稳定性衰减的一般规律,探究引起纯生啤酒泡沫稳定性衰减的根本原因,并提出相应的控制措施。     

蛋白酶抑制剂对纯生啤酒泡沫稳定性影响

纯生啤酒因未经巴氏灭菌而残留一些酶类,其中具有活性的蛋白酶A及非活性的蛋白酶A前驱物的存在会直接或间接地破坏纯生啤酒的泡沫蛋白,从而使纯生啤酒的泡沫稳定性降低。通常情况下,纯生啤酒中PrA的含量为10^-3TU～10^-5TU。纯生啤酒中蛋白酶A的含量与泡沫稳定性之间有着直接的关系,蛋白酶A及其前驱物的总量决定着降解成品啤酒中泡沫蛋白的综合能力。当蛋白酶A的含量大于10^-4TU时,纯生啤酒的泡沫稳定性就会受到较大的影响。本实验采用热水抽提、乙醇分级沉淀等步骤从灵芝真菌中分离提取一种特异性的蛋白酶抑制剂,用于抑制纯生啤酒中PTA的活性。实验表明,一定条件下,抑制率达75％。在成品纯生啤酒中添加适量的蛋白酶A抑制剂可使纯生啤酒的泡沫稳定性明显提高。     

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.     

高分子蛋白和蛋白酶A对纯生啤酒泡沫稳定性的影响

利用考马斯亮蓝法、荧光底物法分别跟踪检测啤酒酿造和贮存过程中高分子蛋白含量及蛋白酶A活力变化,研究影响纯生啤酒泡沫稳定性的关键因素。结果表明,各发酵罐因发酵阶段工艺参数的不同,导致高分子蛋白含量及蛋白酶A活力变化趋势存在明显差异。发酵阶段高分子蛋白含量缓慢降低,由入罐麦汁时的350~407.6 mg/L降到成品酒时的180.1~243.1 mg/L;蛋白酶A活力在回收酵母前增加,后达到最高值,其范围是18.27~30.13 U/m L,回收酵母后蛋白酶A活力下降,最终在成品酒中的蛋白酶A活力检测值为发酵过程中最高值的19.06%~36.4%。通过对成品纯生啤酒中高分子蛋白含量、蛋白酶A活力的跟踪,分析各自对泡持性的作用发现,高分子蛋白含量与泡持性(r=0.794,P0.01)显著正相关;蛋白酶A活力与泡持性及高分子蛋白含量之间没有显著相关性。     

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。     

纯生啤酒的泡沫稳定性及原因分析

利用中国国标测定方法,分析比较市场上销售的普通熟啤酒和纯生啤酒的泡沫稳定性,跟踪其在3个月货架期的泡沫稳定性的变化。结果表明,在3个月的货架期内,熟啤酒泡沫稳定性变化不大,纯生啤酒的泡沫稳定性随时间逐渐下降;贮藏条件对泡沫稳定性有影响,贮藏温度越高,纯生啤酒泡沫稳定性下降越快;对全国啤酒市场上的不同品牌的纯生啤酒进行了泡沫稳定性的跟踪检测,表明泡沫稳定性差是纯生啤酒的共同问题;蛋白酶A是导致纯生啤酒泡沫稳定性问题的关键因素。     

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.     

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。     

小麦面筋蛋白质酶解产物用作啤酒发泡蛋白的研究

为改善啤酒的泡沫性能,作者分别采用木瓜蛋白酶、胃蛋白酶以及碱性蛋白酶对小麦面筋蛋白进行适度酶解改性,并对其产物用作啤酒发泡蛋白的可行性进行了研究.结果表明,经适度酶解作用后,小麦面筋蛋白在pH 4.5条件下溶解性和泡沫性能得到显著改善(P<0.05),且小麦面筋蛋白酶解产物在啤酒环境中热稳定性较好,经30 min的热处理,含100 mg/L小麦面筋蛋白酶解产物的啤酒浊度与加热前相比增加不显著(p>0.05).小麦面筋蛋白胃蛋白酶酶解产物和碱性蛋白酶酶解产物对啤酒初始泡持性的改善效果都较好,但胃蛋白酶酶解产物对酵母蛋白酶A作用较敏感,对纯生啤酒货架期内泡持性的改善效果不太理想,而碱性蛋白酶酶解产物可明显改善纯生啤酒货架期内的泡沫稳定性.     

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     

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.     

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。     

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.     

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.