耐高糖酿酒酵母原生质体制备与再生过程研究

Research on the Preparation and Regeneration of Protoplast from High-concentration Mash Resistant Saccharomyces cerevisia

对1株耐高糖酿酒酵母的原生质体制备和再生条件进行了研究。结果表明,发酵7h后为对数生长中期,适宜原生质体化;L1(65)确定制备原生质体的最佳条件为蜗牛酶浓度(1.0%)、KCl高渗缓冲液(0.7mol/L)、酶解时间(1.5h)、预处理剂(0.1%β-巯基乙醇)和酶解温度(26℃),在此条件下原生质体形成率和再生率分别为80.84%和36.03%;原生质体形成后在7%蔗糖高渗培养基上夹层培养再生率较高(39.78%)。

The preparation and regeneration conditions of protoplast from a high-concentration mash resistant S. cerevisia strain was studied. The results showed that the optimum time for protoplast preparation was the exponential growth of 7 h after the inoculum, and the optimum enzyme for cell wall hydrolysis was snailase. Through L16 （5）^4 orthogonal experiment, the optimum preparation conditions ofS. cerevisia protoplast were identified as follows： 1.0 % of snailase, 0.7mol/L of KCl high osmotic buffer, 1.5 h hydrolysis, 0.1% of β-mercaptoethanol protectant and hy- drolysis temperature at 26 ℃. Under the above Conditions, the formation rate and the regeneration rate ofprotoplast were 80.84 % and 36.03 % respectively. A further study showed that higher regeneration rate （39.78 %） of S. cerevisia protoplast could be obtained when the prepared protoplast was regenerated on the sandwich culture of 7 % sucrose media.