啤酒发酵过程中蛋白酶A的分泌及其与泡沫稳定性的关系

泡沫稳定性严重影响了纯生啤酒的质量。本文考察了蛋白酶A活性与泡沫稳定性的关系,结果显示二者呈明显负相关的关系。同时考察了影响蛋白酶A分泌的因素,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、发酵阶段末期、较低的酵母活力和较高的酵母死亡率都会导致发酵液中蛋白酶A活性偏高。因此在实际生产中,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以下麦汁发酵、尽早结束发酵、提高酵母活力、降低酵母死亡率都会对减少蛋白酶A的分泌量起到积极作用.     

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与啤酒泡沫稳定性(一)

鉴于对有关分泌蛋白酶 A(简写 PrA)的酿酒酵母基因组成、基因特点、蛋白酶 A 本身的生理生化特点、蛋白酶 A 的酶活检测方法、不同酵母菌株分泌蛋白酶 A(简写 PrA)的差异性、蛋白酶 A 影响啤酒泡沫的作用机理、啤酒生产过程中影响酵母分泌蛋白酶 A(简写 PrA)的各种工艺条件、如何降低蛋白酶 A(简写 PrA)对成品啤酒泡沫的影响等方面的知识,国内啤酒行业人员可能了解不多或了解不太全面,本文作者通过查阅大量的国外资料,对以上问     

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。     

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。      

蛋白酶A敲除对酿酒酵母细胞抗辐射特性的影响

蛋白酶A(PrA)是啤酒酵母体内一种重要的天冬氨酸蛋白酶,由PEP4基因编码。它能够催化细胞内蛋白质水解,参与液泡中多种酶的加工和成熟过程。本文考察了蛋白酶A对酿酒酵母的抗辐射性影响。以正常单倍体酿酒酵母菌株和敲除PEP4基因的突变菌株为研究对象,比较两者抗辐射的特性。试验结果表明:蛋PEP4基因的敲除对啤酒酵母的抗氧化酶,即超氧化物歧化酶(SOD)和过氧化氢酶(CAT)有显著影响。蛋白酶A的缺失和辐射导致突变株细胞内海藻糖的积累量大幅增加。     

蛋白酶A与啤酒泡沫稳定性(二)

有关蛋白酶A与啤酒泡沫稳定性的内容已经在<啤酒科技>2004年第1期中介绍有关分泌蛋白酶A的酿酒酵母基因组成、基因特点、蛋白酶A(PrA)本身的生理生化特点、检测蛋白酶A的合适底物、蛋白酶A的酶活检测方法等方面的内容.本篇文章中通过查阅大量的国外资料,陆续介绍不同酵母菌株分泌蛋白酶A(简写PrA)的差异性、蛋白酶A影响啤酒泡沫的作用机理、啤酒生产过程中影响酿酒酵母分泌蛋白酶A的各种工艺条件,就如何降低与消除蛋白酶A对成品啤酒泡沫的影响,提出了一些看法.     

蛋白酶A抑制剂GLPAI动力学性质的研究

对一种从灵芝发酵液中提取得到的蛋白酶A抑制剂GLPAI(Ganoderma Lucidum proteinase A inhibitor)的动力学性质进行了研究，分别以胃蛋白酶、胰蛋白酶和蛋白酶A为底物考察了GLPAI的动力学性质，实验结果：GLPAI对上述3种蛋白酶的抑制类型均属于混合型抑制模式，对胃蛋白酶的Ki-4．64(μmol／L)；对胰蛋白酶的Ki=33．5(μmol／L)；对蛋白酶A的Ki=2．7(μmol／L)．     

蛋白酶A敲除对酿酒酵母抗氧化性的影响

蛋白酶A(PrA)是酿酒酵母体内一种重要的非金属蛋白酶,参与液泡中多种酶的成熟加工和信号转导因子的调控等过程。本文考察蛋白酶A对单倍体酿酒酵母的抗氧化性影响。以正常单倍体酿酒酵母菌和蛋白酶A敲除的突变菌株为研究对象,探讨PEP4基因编码的蛋白酶A对酵母细胞氧化胁迫下的抗氧化调控作用。试验结果表明蛋白酶A敲除对酿酒酵母抗氧化能力有显著的负面影响。与对照菌相比,突变株在氧化应激下展现出抗氧化能力降低的特征。同时,逆境应激因子——海藻糖的积累也与酵母细胞中蛋白酶A的存在和氧化胁迫处理浓度有一定关联。     

高浓和低浓啤酒酿造中蛋白酶A对泡沫活性多肽的影响

啤酒产生丰富泡沫的能力受泡沫活性多肽水平的影响,含有疏水区域的特定多肽,如脂质转移蛋白(LTPl)是啤酒泡沫的重要组成成分。尽管高浓啤酒酿造在商业上是一项可行的技术,但同低浓酿造相比,其产品具有更低的泡沫稳定性。人们认为这主要是由于蛋白酶A对上述的疏水性多肽所起的降解作用造成的,本论文的研究目标是比较和确定在高浓(20°P)和低浓(12°P)麦汁发酵过程中疏水性多肽,尤其是泡沫-LTPl损失的数量,来评估蛋白酶A对这些多肽的影响,疏水性多肽和泡沫-LTPl,在高浓酿造中的损失更大,更进一步来说,已获得的结果表明蛋白酶A改变的是多肽的疏水性,而不是它们的分子量大小。大约有20％的疏水性多肽和57％的泡沫-LTPl表现出蛋白酶A抗性,这些疏水性多肽和泡沫-LTPl损失程度的差异直接影响到最终产品的泡沫稳定性。     

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。     

The structure and function of Saccharomyces cerevisiae proteinase A

Saccharomyces cerevisiae proteinase A (saccharopepsin; EC 3.4.23.25) is a member of the aspartic proteinase superfamily (InterPro IPR001969), which are proteolytic enzymes distributed among a variety of organisms. Targeted to the vacuole as a zymogen, its activation at acidic pH can occur by two different pathways, a one-step process to release mature proteinase A, involving the intervention of proteinase B, or a step-wise pathway via the autoactivation product known as pseudo-proteinase A. Once active, S. cerevisiae proteinase A is essential to the activities of other yeast vacuolar hydrolases, including proteinase B and carboxypeptidase Y. The mature enzyme is bilobal, with each lobe providing one of the two catalytically essential aspartic acid residues in the active site. The crystal structure of free proteinase A reveals that the flap loop assumes an atypical position, pointing directly into the S(1) pocket of the enzyme. With regard to hydrolysis, proteinase A has a preference for hydrophobic residues with Phe, Leu or Glu at the P1 position and Phe, Ile, Leu or Ala at P1', and is inhibited by IA(3), a natural and highly specific inhibitor produced by S. cerevisiae. This review is the first comprehensive review of S. cerevisiae PrA.     

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。     

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     

Purification of yeast proteinase A from fresh beer and its specificity on foam proteins

Yeast proteinase A from fresh beer was first purified with Sephadex G‐100 column chromatography and the active fractions reached to 5.3‐fold purification with 7% of yield. After purification with DEAE Sephadex A50, proteinase A activity increased to be 10.1 times of the initial with 1% of yield. When identifying the sample from chromatography by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), only one protein band with 42 kDa was observed, this indicated that the enzyme was purified. The pattern of electrophoresis of hydrolysed beer by crude proteinase A did not show lipid transfer protein (LTP) on the gel. The result of SDS‐PAGE of interaction mixture of purified proteinase A and beer also indicated that LTP was decomposed.     

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。     

Vacuolar and extracellular maturation of Saccharomyces cerevisiae proteinase A

The vacuolar aspartyl protease proteinase A (PrA) of Saccharomyces cerevisiae is encoded as a preproenzyme by the PEP4 gene and transported to the vacuole via the secretory route. Upon arrival of the proenzyme proPrA to the vacuole, active mature 42 kDa PrA is generated by specific proteolysis involving the vacuolar endoprotease proteinase B (PrB). Vacuolar activation of proPrA can also take place in mutants lacking PrB activity (prb1). Here an active 43 kDa species termed pseudoPrA is formed, probably by an autocatalytic process. When the PEP4 gene is overexpressed in wild-type cells, mature PrA can be found in the growth medium. We have found that prb1 strains overexpressing PEP4 can form pseudoPrA extracellularly. N-terminal amino acid sequence determination of extracellular, as well as vacuolar pseudoPrA showed that it contains nine amino acids of the propeptide, indicating a cleavage between Phe67 and Ser68 of the preproenzyme. This cleavage site is in accordance with the known substrate preference for PrA, supporting the notion that pseudoPrA is formed by autoactivation. When a multicopy PEP4 transformant of a prb1 mutant was grown in the presence of the aspartyl protease inhibitor pepstatin A, a significant level of proPrA was found in the growth medium. Our analyses show that overexpression of PEP4 leads to the secretion of proPrA to the growth medium where the zymogen is converted to pseudoPrA or mature PrA in a manner similar to the vacuolar processing reactions. Amino acid sequencing of secreted proPrA confirmed the predicted cleavage by signal peptidase between Ala22 and Lys23 of the preproenzyme.     

构建具有大麦脂转移蛋白1分泌能力的酿酒酵母

为了增强纯生啤酒的泡沫性能,从酿酒酵母表达质粒YEplac181出发,将大麦脂转移蛋白1(LTP1)成熟肽的编码序列置于酿酒酵母ADH1启动子(alcohol dehydrogenase promoter)和CYC1终止子(cytochrome C terminator)的调控下,构建大麦脂转移蛋白1的酿酒酵母表达质粒YEp181-KAMLC。通过酿酒酵母α-信息素信号肽的引导分泌,酿酒酵母表达的成熟大麦LTP1被分泌到发酵液中。对发酵液的检测表明,在发酵132h后LTP1的产量可达到29.45mg/L。     

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。