【作者】 郝俊光；

【导师】 陈坚；

【作者基本信息】 江南大学 ， 发酵工程， 2007， 博士

【摘要】 蛋白酶A(EC3.4.23.6)是一种天冬氨酸族蛋白酶,由PEP4基因编码,可通过酵母的自溶或胁迫条件下的分泌而残存于成品啤酒中,从而分解纯生啤酒的泡沫蛋白而降低泡沫的稳定性。本文从检测啤酒泡沫蛋白的组成、探讨蛋白酶A对纯生啤酒泡沫的影响、跟踪生产中蛋白酶A的变化规律、构建蛋白酶A低水平或缺失的酵母突变菌株、筛选低蛋白酶A的酵母改良菌株及探讨其发酵性能等方面进行了系统的研究,旨在得到具有大生产应用潜力的纯生啤酒生产菌株,提高纯生啤酒的泡沫稳定性。啤酒中的蛋白质组成和含量是影响啤酒泡沫稳定性的主要因素之一,目前人们对泡沫蛋白组成的认识尚不统一。以往对啤酒泡沫蛋白的检测,大多局限于单向电泳、色谱、氨基酸分析等传统方法,但这些方法可能掩盖了性质相近蛋白的存在。本研究为探明啤酒泡沫蛋白的组成,除采用了传统的单向电泳和氨基酸分析技术外,还使用了双向电泳、MAIDI-TOF MS、LC/MS/MS等先进的检测技术对啤酒泡沫蛋白组成进行探讨。由组成氨基酸的分布规律和SDS-PAGE电泳检测可知啤酒泡沫不止含蛋白质Z和LTP1组成。双向电泳的检测,进一步了揭示啤酒泡沫蛋白组成的复杂性,发现纯生啤酒与其泡沫2蛋白分布相似,但含量和分布特点不同。利用MALDI-TOF MS对啤酒泡沫蛋白进行检测,虽然未得到期望的结果,但从中检测到糖基化LTP1的存在。利用LC/MS/MS,在啤酒泡沫中共检测到23种蛋白,除蛋白质Z和LTP1外,其它蛋白均未被报道。通过对比纯生啤酒与熟啤酒的泡沫变化规律,肯定了蛋白酶A是造成纯生啤酒泡沫不稳定的直接原因。青岛纯生啤酒生产菌株S.carlsbergensis 04-1和B在染色体水平上存在细微差异,二者使用代数对发酵液中蛋白酶A活力的影响趋势一致。第0代和1代的发酵液中蛋白酶A活力较低,2-4代酵母的蛋白酶A活力明显升高。将编码蛋白酶A的PEP4敲除是构建蛋白酶A低水平菌株或缺失菌株、解决纯生啤酒泡沫稳定性问题的有效途径。Lager啤酒酿造所用的S.carlsbergensis为四倍体,其编码蛋白酶A的基因序列和拷贝数不确定,需采用基因多重中断技术,才能得到蛋白酶A完全缺失菌株。本文构建了抗Zeocin的质粒pSH-Zeo,并以含抗G418基因的pUG6质粒为模板进行PCR介导的基因敲除,实现了工业菌株S.carlsbergensis转化子的显性筛选。进而将Cre-LoxP重组系统和PCR介导的基因中断技术结合,成功地敲除S.carlsbergensis的一个PEP4等位基因,然后将报告基因KanMX剔除,使突变菌株中不含任何外源基因,获得了基因安全性高的突变株pep4∷LoxP/PEP4。为避免相同PCR产物重复转化多倍体时引起的回复突变,提出采用缩进式敲除多个序列相同的等位基因、实现多重基因中断的观点。通过这种方式,成功地敲除了S.carlsbergensis的第二个PEP4等位基因,但由于S.carlsbergensis特殊的表达和调控机制,酵母在被敲掉第二个PEP4基因后不能生存。由此确定,青岛啤酒使用的S.carlsbergensis菌株中含有两个与S.cerevisiae的序列相同的编码蛋白酶A的PEP4等位基因、且对S.carlsbergensis的生存起关键作用。对得到的突变株pep4∷LoxP/PEP4进行筛选,主要参考发酵度和蛋白酶A活力指标,筛选出低蛋白酶A的改良菌株S.carlsbergensis 04-1-M和B-M。通过稳定性传代试验,证实pep4∷LoxP基因具有良好的遗传稳定性,从而保证了菌株的低蛋白酶A表达水平的遗传稳定性。改良菌株的特性与出发菌株相近,凝聚性较对应的原出发菌株略强。S.carlsbergensis 04-1-M的致死温度为51℃,而S.carlsbergensis 04-1、B、B-M的致死温度为52℃,蛋白酶A改良菌株和野生菌株有着相近的发酵性能和生理特性。在100 L中试应用试验中,改良菌株的低蛋白酶A特性被证实,具有工业化应用的潜力。发酵过程中,其在氨基酸和可发酵糖的利用、悬浮酵母数的变化、酵母形态方面与出发菌株表现相近,而其降糖速度略慢、发酵液中麦芽三糖的残留量略高、总氨基酸利用率略高。改良菌株终了发酵液的双乙酰、戊二酮、乙偶姻、乙醛含量均略高于对应的出发菌株,而醇酯比、发酵度、蛋白酶A活力均低于对应的出发菌株。改良菌株酿造出的啤酒柔和、协调、清爽、无异味。尽管如此,改良菌株在大生产应用之前尚还需做更多的实验验证。更多还原

【Abstract】 Protease A[EC3.4.23.6], an aspartic proteolytic enzyme, is encoded by PEP4 gene in S. cerevisiae. It can be excreted under stress conditions or leaked by autolysis into fermented wort during Lager beer brewing process using S. carlsbergensis, Lager beer yeast, which detriments foam stability of final draft beer. With an aim to improve foam stability of draft beer by managing to get a commercial strain with low-protease A level, following efforts were taken in this study: identify proteins profile of the draft beer foam, find out the difference of foam stability between draft beer and pasteurized beer, trace the change of protease A in larger scale production, construct low or non protease A strain by gene knock-out, select out low protease A upgraded strain within resultant transformants, and investigate fermentation performance of upgraded strains by 100L pilot trial.Composition and concentrations of foam proteins play a key role in foam stability of draft beer, while no unequivocal view on key foam proteins profile has been achieved by far. Traditional measurements such as SDS PAGE, chromatography, amino acids analysis, were employed to clarify foam proteins profile in previous investigations, which possibly hid the truth of it. To better understand foam proteins, advanced modern measurements were adopted including two dimensional electrophoresis、MALDI-TOF MS、LC/MS/MS in this study, besides the traditional SDS PAGE and amino acids analysis. Results of SDS PAGE and amino acids analysis suggested primarily that proteins were complex, not only confined to protein Z and LTP1. Results of two dimensional electrophoresis further clarified the complexity of foam proteins and uncovered the similar distributions of proteins between foam and beer with different content and characterizations. Glycosylatin forms of LTP1 were detected by MALDI -TOF MS in beer foam though no other valuable information was gained. Totally, there were 23 proteins detected in draft beer foam by LC/MS/MS, most of them were not reported by other before, which enlarges and updates the list of proteins related to foam.Foam stability of draft beer and pasteurized beer were compared which indicated that protease A is direct negative factor of foam stability of draft beer. Karyotypes of S. carlsbergensis 04-1 and B, used by different breweries in Tsingtao Brewery Group, were similar with a little difference. The relation between cropped generations and protease A activity of them was parallel. The protease A activity of drafted beer brewed with 1&2 cropped generation S. carlsbergensis 04-1 and B was low, while higher brewed with higher generation yeast. The mimic PU treatment was adopted to verify the effect of pasteurization units on protease A and invertase activity. There was no activity of protease A in beer treated with PU 7, while invertase activity still could be detected. The foam stability increased relatively as the treated PU value increased in the range of 0-7.It is effective and direct means to increase foam stability of draft beer by constructing low or no protease A activity brewing strain through encoding gene PEP4 deletion. S. carlsbergensis is a tetraploid whose detailed copy number and detailed sequence of PEP4 are uncertain. To achieve a PEP4 absent strain in tetraploid, multiple gene disruptions should be taken. In this study, dominant selective marker Sh ble and KanMX were applied by construction plasmid pSH-Zeo and PCR with pUG6 as template. Combined Cre-LoxP and PCR meditated gene disruption, mutants with pep4::LoxP/PEP4 were gained. As KanMX was deleted ultimately from transformant pep4::KanMX/PEP4 in this study, gene security of modified strain was improved without heterogenous gene remained.To avoid reverse mutation caused by repeated deleting allelic genes in allopliod with same PCR product, retractive primer design strategy was suggested and applied in this study. By this means, a pep4::LoxP/pep4::KanMX strain was gained. But it did not survive by its special expressive and regulative regime after second PEP4 deletion. It is indicated that there are only two PEP4 allelic genes with the same sequence to S. cerevisiae in S. carlsbergensis used by Tsingtao Brewery Group, which play a key role in S. carlsbergensis metabolism and survival.The low protease A upgraded strain, S. carlsbergensis 04-1-M and B-M, was gained by successive selection within pep4::LoxP/PEP4 transformants, mainly taking fermentation degree and protease A activity of fermented wort of laboratory brewing trials in account. PCR verified that the mutant gene pep4::LoxP remained stable in offspring strains which ensured the inheritance of corresponding low protease A characteristic of the upgraded strain. The fermentation degrees of upgraded strains were lower than their wild strains, while flocculation values are higher. Lethal temperature of S. carlsbergensis 04-1-M was 51℃, which was different from all the others.In 100 L pilot brewing trail, all the two upgraded strains perform similar to their counterparts, such as assimilation of amino acids & fermentable saccharides, propagation of yeast, ect. Relatively slow attenuation rate of upgraded strains resulted in a little more maltotriose left in theirs final fermented worts. The concentration of diacetyl, pentanedion, acetoin in final fermented worts brewed by upgraded strains were higher, while ratio of higher alcohol to ester, fermentation degree, protease A activity were lower, than those of their counterparts. The flavor and taste of final fermented worts brewed by upgraded ones were evaluated by experienced taste panel of Tsingtao Brewery Group, no defect were detected by them. More trails are suggested for industrial application though its promising prospective is implied by our 100 L pilot brew.更多还原