【作者】 李中超；

【导师】 李华；

【作者基本信息】 西北农林科技大学 ， 发酵工程， 2009， 硕士

【摘要】 苹果酸-乳酸发酵(Malolactic Fermentation, MLF)是葡萄酒生产中非常重要的二次发酵过程,是葡萄酒生产中主要的生物降酸方法,也是酿造优质干红葡萄酒必须进行的工艺环节。自然启动MLF时,启动和发酵时间难以预测,并且增加了葡萄酒发生病害的几率。所以现在人们大都用商业化活性乳酸菌进行MLF。由于葡萄酒环境恶劣,而实验室培养的菌体由于环境良好,导致其天然抗性降低;同时因为冷冻干燥时的超极端环境对菌体的损害,冻干后活菌数减少,所以造成人工接种诱导MLF时,不能成功启动。因此研究用胁迫培养条件恢复和提高菌体的抗性,以提高菌体的接种存活率和冻干存活率以及研究其中的胁迫适应机制对直投式酒酒球菌的开发和生产有重要的理论和实践意义。本试验选用西北农林科技大学葡萄酒学院优选酒酒球菌SD-2a,研究并优化了酒酒球菌质膜H+-ATPase的制备和活性测定方法,并在此基础上研究了不同胁迫处理下质膜H+-ATPase活性的大小,同时研究了培养基组分和温度胁迫处理对酒酒球菌SD-2a菌体的生长、接种存活率、冻干存活率和质膜H+-ATPase活性的影响,以观察胁迫处理对菌体存活率提高的有效性,并探讨酒酒球菌SD-2a质膜H+-ATPase在菌体胁迫适应性方面的作用机制。主要研究结果如下:1.试验采用0.05%TritonX-100,-20℃,24h低温通透细胞膜获得酒酒球菌SD-2a的质膜H+-ATPase,用分光光度法研究了酶活的测定条件。通过单因素试验和正交试验结果分析得知,酒酒球菌细胞膜H+-ATPase的最佳酶活测定条件为:Tris-HCl反应缓冲体系,pH7.0,37℃水浴中反应30min。2.酒酒球菌SD-2a质膜H+-ATPase在酸胁迫抗性中起关键的作用。无DL-苹果酸的ATB培养基,在培养菌体过程中,pH缓冲能力弱,细菌生长量大,所获菌体接种模拟酒后的接种和冻干存活率以及质膜H+-ATPase的活性也最高;酸胁迫处理明显抑制了酒酒球菌SD-2a的生长速率,但酸胁迫处理pH3.6和pH3.2处理与对照相比,质膜H+-ATPase的活性显著增加,分别是对照的1.19和1.41倍,同时最大菌体密度也达到了对照的94.4%和90.6%。所以酸胁迫处理是酒酒球菌SD-2a直投式发酵剂生产的有效胁迫处理方式,胁迫处理平衡点在pH3.5左右。3.酒精胁迫降低了酒酒球菌SD-2a质膜H+-ATPase的活性,5%和10%酒精胁迫处理的菌体的质膜H+-ATPase活性分别是对照处理的80.1%和76.3%。并且酒精胁迫处理所得菌体产量低,10%酒精处理最大菌密度只有对照的38%。质膜H+-ATPase不在抗酒精胁迫的直接主要的作用机制,但其活性大小可以作为菌体生理状态的指示物。4.温度胁迫显著影响酒酒球菌SD-2a生长,在35℃胁迫处理下,菌体生长很快到达稳定期,但最大OD600值只有对照的70%;在10℃胁迫处理下,细菌的生长时间明显延长,延滞期变长,但最终菌体最大OD600值达到了对照的83.3%。高低温胁迫处理都增加了质膜H+-ATPase的活性,说明质膜H+-ATPase在抗温度胁迫中起一定作用。通过冷冻干燥试验表明,低温处理可以显著增强菌体的抗冻干能力,但其接种发酵活性很低,所以温度胁迫也不是制备直投式乳酸菌发酵剂的有效的处理方式。更多还原

【Abstract】 Malolactic Fermentation (MLF), an essential part for making wine with good quality, performed by Malolactic bacteria (MLB), can improve the quality of wine by acid degradation. And the resulted wine obtains a favorable balance of acidity, organoleptic properties and microbiological stability.Due to the harsh conditions of wine for bacterial survival and growth, but strains lose its natural resistance in the lab; at the same time, lyophilization offers stress conditions such as freezing and other extremeness environment diminish the cell viability. So it creates the artificial infection induced MLF can’t succeed. Therefore research on the stress culture conditions to restore and enhance the resistance of strains witch can enhance the inoculation and freeze-dry livability as well as study the stress adaptation mechanism have important theory and practice significance to freeze-dried ready-to-use MLF starter exploitation and production.In this paper, activity determination conditions of plasma membrane H+-ATPase were studied and optimized using O. oeni SD-2a. Based on this, the effect of stress treatment on plasma membrane H+-ATPase activity were studied, simultaneously effect of medium composition and temperature stress treatment on bacterium growth, MLF activity after directly inoculated into wine-like medium and freeze-drying viability were studied in order to evaluate the validity of strains livability elevated after stress treatment and discuss the stress adaptive mechanism of plasma membrane H+-ATPase of O.oeni SD-2a.The main results were as follows:1. Plasma membrane H+-ATPase of O.oeni SD-2a was obtained using frozen permeate plasma membrane, and its activity was determined with spectrophotometry. The experiment results showed that the optimal activity determination conditions of plasma membrane H+-ATPase of O.oeni SD-2a is Tris-HCl buffer system, pH7.0 and 30 minutes’water bath reaction at 37℃.2. Plasma membrane H+-ATPase of O.oeni SD-2a plays an important role in acid stress fastness. ATB medium without DL-malic acid has weak pH buffer capability, so it can induce self acid stress response, ATB medium is in favor of the growth of O.oeni SD-2a. Strains cultured in this medium have the highest inoculation and freeze-dry livability and H+-ATPase activity. Acid stress treatment obviously restrains the growth speed of O.oeni SD-2a, but the activity of H+-ATPase under pH3.6 and pH3.2 treatments were 1.19 and 1.41 times compared with the control treatment. At the same time the tiptop OD600 can achieved 94.4% and 90.6% of control treatment. So we believe that acid stress is an efficiency treatment to prepare the freeze-dried ready-to-use starter, balance point of acid stress is about pH3.5.3. The alcohol stress condition reduced the Plasma membrane H+-ATPase activity of O.oeni SD-2a, under 5% and 10% alcohol stress treatment is only 80.1% and 76.3% compared with control treatment, respectively. Although alcohol stress can decrease the yield of O.oeni SD-2a, we don’t believe H+-ATPase is the directness function mechanism in resist alcohol stress.4. Under 35℃stress treatment condition, the bacteria grow into the stabilization period very quickly, but the biggest OD600 value is very low, only 70% of the control treatment; Under 10℃condition, the bacterium growth time is prolonged obviously, the lag phase prolonged too, but the final biggest OD600 value has achieved 83.3% of control treatment. High and low temperature treatment all increased the activity of plasma membrane H+-ATPase, which show H+-ATPase may plays a role in resist temperature stress.Low temperature can enhance the freeze-drying viability notability but has a low MLF activity,so we also don’t believe temperature stress is an efficiency treatment to prepare the freeze-dried ready-to-use starter.更多还原