【作者】 李娟；

【导师】 李华；

【作者基本信息】 西北农林科技大学 ， 葡萄与葡萄酒学， 2009， 硕士

【摘要】 酿酒酵母(Saccharomyces cerevisiae)和酒酒球菌(Oenococcus oeni)是重要的两类葡萄酒工业微生物,其分别能够进行酒精发酵和苹果酸-乳酸发酵,为了获得酒精发酵与苹果酸-乳酸发酵同时进行的新型工程菌株,本研究基于前人对酿酒酵母1450和酒酒球菌SD-2a原生质体跨界融合的研究,对两亲株原生质体制备与再生、融合方法及条件进行研究,并尝试采用灭活单、双亲原生质体技术并结合电诱导融合法进行不同灭活原生质体的融合探索,以进一步对其融合的可行性和重复性作验证性探讨,为获得具有高效降酸且遗传性更加稳定的融合子提供理论与试验依据。主要研究内容和结论如下:1.对实验室保存的酿酒酵母1450与酒酒球菌SD-2a两亲本进行抗药性的筛选,确定完全抑制酵母1450和酒酒球菌SD-2a生长的抗生素浓度分别为100μg/mL的放线菌酮和20μg/mL的氨苄青霉素。2.通过单因素和正交试验,分别确定亲本酵母1450和酒酒球菌SD-2a原生质体制备与再生的最佳条件。酿酒酵母1450原生质体制备的最佳条件为:菌龄6 h,预处理剂3%β-巯基乙醇,蜗牛酶浓度1.5%,酶解温度42℃,酶解渗透压稳定剂0.5 mol/L蔗糖。在此条件下,酵母1450原生质体形成率与再生率可达14.14%。酒酒球菌SD-2a原生质体制备的最佳条件为:菌龄34 h,预处理剂青霉素G钠0.3μg/mL,溶菌酶浓度1 mg/mL,酶解温度37℃、酶解渗透压稳定剂0.8 mol/L KCl。在此条件下,酒酒球菌SD-2a原生质体形成率与再生率达10.65%。3.本试验初次对两亲本原生质体进行紫外与热灭活条件的研究,确定了两亲本原生质体的灭活条件:在20W紫外灯10 cm处的照射条件下,酵母1450和酒酒球菌SD-2a原生质体的灭活时间分别以180 s和100 s为最佳;在水浴55℃的条件下,1450和SD-2a原生质体热灭活时间分别为15 min和9 min。4.对酵母1450和酒酒球菌SD-2a原生质体进行化学PEG融合,采用不同融合温度和不同PEG融合浓度条件对两亲本原生质体融合的研究,最终确定在浓度30%PEG-4000,融合温度32℃的条件下,两亲本融合率效果最好。5.初次对两亲本原生质体采用热灭活方法进行不同灭活原生质体电诱导融合,确定以两亲本活体和SD-2a单灭活方法处理后,在电压30V,脉冲时间30us,脉冲数5次,脉冲间隙600 ms的融合条件下,融合效果较为理想。更多还原

【Abstract】 Saccharomyces cerevisiae and Oenococcus oeni are the two of the most important microorganisms in wine industry, the before carries out and latter performs malolactic fermentation. To select the new yeast strain with maloalcoholic fermentation, single and biparental inactivated protoplast were initially combined to electrofusion to proceed fusion condition of the two strain protoplst on the basis of previous studies and to further investigate feasibility and repeatability of protoplast fusion and to provide theoretical and experimental basis for the obtaintion of high-efficient and inheritance stable fusant.The main results are as follows:1. We tested the susceptibility to antibiotics in 1450 and SD-2a and results showed that the inhibitory concentration of 1450 and SD-2a were respectively 100μg/mL Actidione and 20μg/mL Ampicillin.2. Discussing different factors and orthogonal test which affected the protoplast formation rate and regeneration rate of 1450 and SD-2a, the optimum preparation conditions of 1450 protoplast were: 1450 cells cultured 6h, pretreatment solution of 3%β-mercaploethanol, 1.5% Snailase, treatment at 42℃, 0.5 mol/L sucrose as osmotic stabilizer, 1mol/L sorbitol for the regeneration , the protoplast formation was 99.85% and regeneration ratio reached 14.31%, the protoplast formation and regeneration ratio reached 14.14%. And the optimum preparation conditions of SD-2a protoplast were: SD-2a cells cultured 34h, pretreatment solution of 0.3μg/mL penicillin G sodium salt, 1mg/mL lysozyme, treatment at 37℃, 0.8 mol/L KCl as osmotic stabilizer, the protoplast formation and regeneration ratio reached 10.65%3. The regeneration modes of 1450 and SD-2a protoplast showed: monolayer regeneration medium could enhance the regeneration of 1450 protoplast and SD-2a protoplast were perfectly cultivated on the double-layer medium。4. Inactivating conditions of strains 1450 and SD-2a were determined by lethal rate: In 55℃, strains 1450 and SD-2a protoplast were treated for 15 mins and 9 mins respectively; In the distance of 10cm, 180 seconds and 100 seconds radiation treatments to strains 1450 and SD-2a protoplast with 20W UV-light were the best.5. After different temperatures and PEG concentations for chemical fusion of 1450 and SD-2a protoplasts, the results indicated the optimal fusion conditions of 30%PEG4000, fusion temperature 32℃at 30min.6. The single-(double)parents inactivated protoplast methods for electrofusion of 1450 and SD-2a protoplast heated-inactivated were compared in order to establish the proper electrofusion way for deacidfication Wine Yeast breeding. We found the two ways of biparental activated and SD-2a inactivated protoplast electrofusion showed perfect fusion effect as the condition of voltage30V, plength 30 us, pulse time 5 and inverval of 600 ms.更多还原