用细胞融合的方法选育耐高酒精度的酿酒酵母

Selecting High Alcohol Concentration Restrains of Saccharomyces Cerevisiae

选择耐高渗透压、耐高酒精度和发酵终了产酒精量较高的黄酒酵母H—1和目前酒精工业生产用菌K号酒精酵母K—1,运用细胞融合技术选育发酵速度快、产酒精量高的工业用酒精酵母.双倍体黄酒酵母H—1和双倍酒精酵母K—1经过产孢前预培养和产孢培养后,蜗牛酶水解子囊孢子壁,离心收集单倍体子囊孢子,培养后得到单倍体黄酒酵母H—2和单倍体酒精酵母K—2.用硫酸二乙醇诱变处理单倍体细胞,得到单倍体黄酒酵母的维生素缺陷型H—3和单倍体酒精酵母的氨塞骏营养缺陷型K—3通过正交试验找出了H—3和K—3原生质体形成及再生的较优条件是:对数生长后期的细胞33℃、0.2%的β—巯基乙醇预处理15分钟,然后4%蜗牛酶作用2小时.用35%聚乙二醇和10mMCaC1_2诱导融合40分钟,于再生基本夹层培养基上培养获得营养互补融合子,并且考查了融合子的遗传稳定性.通过耐酒精度、一发酵速度和最终产酒精量的测定,筛选出融合子HK—6.HK—6与生产用K号酒精酵母相比,发酵速度相接近,而最终产酒精量提高了11%.可耐受16%的酒精度.

The protoplast fusions of Saccharomyces cereviae H-1 which resist high osmotic pressure and high alcohol concentration and K -1 which is alcohol production strains in the current industry obtained Saccharo myces cerevision HK - 6 which had not only the faster far mentaion speed dut also had the higher alcohol concentration. The haploid cells of sacckaromyces cerevisiae H-2 and K -2 was isolated after the diploid cells of saccharomyces cerevisiae H -2 and K -1 had been cultured in the pre-sporogenous and sporogenous medium, and the ascospores had deen under reaction snail enzyme. Then we obtained the haploid cell of saccharo myces cerevisiae aux-otroph H -3 lack pf vitamin and K - 3 lack of amino acid. By the cut cross tests we discovered the suitable conditions of fusion parents' pnoto-plast formation and regeneration. It was by using past-exponential phase cellsunder the reaction of 0.2% β-mencaptoethand for 5 minutes and 4 % snail enzyme in the water of 33 C for 2 hours. The fusions of parental protoplast were induced by using 35% polyt-hyleneglycol and 10 mm Gacl for 40 minutes. The fusion strains of nutritional complamentation were obtained and their genetic stability was tested. The fusion strain HK - 6 was identified after checking the resistant alcohol concentration, tne fermentation. Speed and the production of alcohol. Compared with K -1, HK-6 has close fermentation speed but its alcohol production increased 11% and it could resist 16% alcohol concentration.