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。     

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。     

温瓶处理对纯生啤酒泡沫稳定性的影响研究

在温度不断提高的情况下，纯生啤酒中蛋白酶A及蔗糖转化酶的活力都呈下降趋势，但蔗糖转化酶对温度升高的抵抗力比蛋白酶A强，在蛋白酶A失活的临界点，蔗糖转化酶仍能保持约35％的活力。实验发现，在灌装过程中，对成品纯生啤酒采用60℃、2．5min的“温瓶”处理，可在部分保留蔗糖转化酶活力的同时，将蛋白酶A的活力降低到最低限度，有效地防止纯生啤酒泡沫稳定性的衰减。温瓶操作既可保持纯生啤酒的新鲜度，又可防止其泡持性的衰减。     

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。     

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.     

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     

国产纯生啤酒质量稳定性的研究

对国产纯生啤酒的质量稳定性进行了相关的研究。研究发现,国产纯生啤酒的货架保鲜期比相应熟啤酒延长了10%20%,但与国外品牌啤酒的保鲜期相比仍然存在较大的差距;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;纯生啤酒灌装后的蔗糖转化酶活性在保存三个月后仍保存80%以上的酶活,五个月后检测仍能保存60%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,采用液相色谱分析方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类似的作用,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。     

纯生啤酒泡沫稳定性的研究

活酵母细胞的蛋白酶胞内渗出，进一步降解泡沫蛋白，并导致最终产品的泡持性贫乏，这种观点已经被普遍报道和接受。本文通过对酿造过程酵母蛋白酶A活力的跟踪和最终产品的泡持性差异追溯，重点讨论了控制酵母胞外分泌蛋白酶从而提高纯生啤酒泡沫稳定性的工艺措施。     

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。     

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.     

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。     

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。     

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.     

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.     

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。     

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.