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.     

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。     

α-氨基氮对纯生啤酒发酵过程中酸性蛋白酶分泌的影响

初始α-氨基氮含量是影响纯生啤酒发酵过程中酸性蛋白酶分泌的重要因素.通过调节麦汁中初始α-氨基氮含量,考察其对酸性蛋白酶分泌和高级醇生成的影响.结果表明,α-氨基氮在190mg/L时,酸性蛋白酶分泌量较低,啤酒风味较好.     

发酵条件对纯生啤酒中酸性蛋白酶分泌的影响

发酵条件是影响纯生啤酒发酵过程中酸性蛋白酶分泌的重要因素。研究了发酵温度、培养基pH、酵母接种量和CO2含量等发酵条件对酸性蛋白酶分泌的影响。结果表明,在8℃、pH6.4、接种量5%和不带压发酵的条件下,酸性蛋白酶分泌量少,酶活低,啤酒泡沫稳定性好。     

蛋白酶A检测技术在啤酒酵母选育中的应用

应用人工合成的荧光肽底物考察了五株啤酒酵母的蛋白酶A水平,对其中一株蛋白酶A含量较高的菌种进行了紫外诱变分离,通过发酵试验筛得低蛋白酶A菌种,酵母回收后的发酵液中的蛋白酶A水平较出发菌株下降了70%以上,为纯生啤酒泡沫衰减问题提供了菌种解决方案。     

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。     

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。     

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。     

酿造工艺对啤酒中有机酸的影响

有机酸是啤酒中重要的风味物质,其组成与含量同酿造工艺有着密切的关系.采用离子色谱法探索了不同酵母菌种、酵母接种景、发酵温度、麦汁pH值以及原麦汁浓度5个因素对啤酒中有机酸的影响.结果表明,酵母菌种、酵母接种量以及原麦汁浓度是有机酸组成和含量的主要影响因素;啤酒最终的pH值和总酸受原麦汁浓度的影响显著,且呈正相关性.     

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。     

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.     

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.     

高浓高温对啤酒酵母发酵性能的影响

以啤酒酵母S-6为实验菌株,研究了主发酵温度和原麦汁浓度对啤酒发酵的残糖、酒精度、风味物质和絮凝性等性能指标的影响。结果表明,原麦汁浓度一定时,主发酵温度对高级醇和乙酸酯的含量影响较大,主发酵温度由10 ℃提高至16 ℃时,高级醇含量提高了10%～20%,乙酸酯含量提高了8%～16%,但CO2累积质量损失、残糖、酒精度和絮凝性基本不受温度的影响;主发酵温度一定时,原麦汁浓度对酵母絮凝性影响较大,原麦汁浓度越高,酵母絮凝性越低,将高浓（18 °Bx）发酵液稀释50%至常浓（12 °Bx）,残糖、酒精度和高级醇的含量与常浓发酵液基本相同。该研究为选育高温高浓发酵低产高级醇同时强絮凝性酵母菌株提供了重要依据。     

Optimisation of High Gravity and Diet Beer Production in a German Brewery by Flow Cytometry

Increasing the quantity of beer production without diminishing the quality of the product is a key concern of the beer producing industry. Modifications to the brewery's equipment and settings are the most commonly used methods to improve the brewing process, while the supreme importance of the physiological state of the beer producing organisms, the yeast cells, for the productivity of the brewing process is often poorly recognised. The work described here was designed to optimise two processes: the inoculation regime used to produce high gravity bottom-fermenting beer, and the production of high quality diet beer. To achieve these aims, flow cytometry was used to follow changes in the distribution of DNA, neutral lipid and 3β-hydroxsterol contents in Saccharomyces carlsbergensis strains during inoculation, fermentation and storage. This allowed potential time-saving alterations in the process to be identified. Double staining techniques proved that vigorous fermentative activity and long-term survival capacity during main and secondary fermentation requires intense multiplication of the yeast cells during inoculation. The production of high gravity beer was then enhanced by altering the schedule of the wort additions, and thus increasing the yeast's activities related to multiplication. To produce diet beer, oligosaccharides that remain after the standard brewing process are degraded by adding small amounts of wort, usually during secondary fermentation. However, during this period of fermentation the physiological activity of the yeast cells is hampered by low carbon and high ethanol concentrations. Adding small batches of wort at carefully defined time points and in optimised amounts, even during the main fermentation, improves the physiological state of the yeast cells and rapidly decreases the carbon concentration within the fermentation tank. Both of these factors help to promote quick fermentation to a high quality diet beer. Thus, the flow cytometric investigations provided a reliable basis for identifying effective means of improving the process regime for brewing both of these products.     

不同麦汁浓度及酵母代数的电导率在线监测

采用电导率测量的方法对啤酒发酵过程进行了在线监测，并且对不同麦汁浓度、不同酵母代数进行了研究。结果表明：对于不同浓度的麦汁，其发酵过程中电导率的变化呈现明显的规律性，酵母代数对电导率的影响较大，1代酵母要比0代酵母提前约50min出现第1个拐点，即结束迟滞期；提前3h出现第2个拐点。这为研究啤酒发酵提供了一种有效的方法。     

酵母形态指标百分数离散度与啤酒质量的关联性

通过跟踪不同发酵罐相同发酵时间的啤酒发酵液中的酵母形态指标百分数离散度,检测发酵结束后各发酵罐的啤酒的质量指标,得出啤酒酵母指标百分数离散度与啤酒质量的关联性,即酵母形态指标百分数离散度与酒精度、发酵度、总酸这三个啤酒质量指标成负相关,与原麦芽汁浓度、外观糖度、乙醛浓度、pH值、双乙酰这五个啤酒质量指标成正相关.     

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