【作者】 彭帮柱；

【导师】 岳田利；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2004， 硕士

【摘要】 :本研究立足于国内和国际研究现状，利用原生质体融合技术构建增香型苹果酒酿造酵母。虽然原生质体融合技术已经是一个成熟、系统的实验体系，利用该技术进行微生物遗传育种的研究报道国内外都很多，但用于构建增香型酿造酵母的研究并不多见。本试验以5株优良的酵母菌株为试验材料，通过对其各自发酵苹果酒的理化指标和感官指标的综合分析，得出5#酵母菌株较适合酿造优良的苹果酒；将5#酵母菌株作为诱变出发菌株，利用甲基磺酸乙酯（EMS）对其进行诱变，从突变株中筛选出1株遗传标记稳定的突变株E2，然后利用原生质体融合技术将其和发酵能力较强的3#酵母的灭活原生质体进行了融合，通过GC-MS对优良融合子及亲本发酵的苹果酒香气成分进行了鉴定分析，并结合感官品评，筛选得到3株优良的增香型苹果酒酿造菌株。主要研究结果如下：(1) 通过对5株试验菌株发酵苹果酒理化指标和感官指标的比较分析，筛选出1株发酵苹果酒口感、风味较好，具有苹果酒典型风格的优良酵母菌株（5#菌株），然后利用EMS对其进行诱变，筛选出1株赖氨酸缺陷型突变株E2，将其确定为原生质体融合时产香能力好的亲本菌株Y。(2) 通过对5株试验菌株的发酵速率的测定和发酵醪液理化指标的测定，发现3#菌株的发酵能力较强，将其确定为原生质体融合时发酵能力强的亲本菌株X。(3) 根据两亲本的生长曲线图，得到两亲本的对数生长期均约为第2～10h，其对数生长早期均约为第2～4h。因此确定两亲本在制备原生质体时的前培养时间为4h。(4) 在酶解温度35℃，蜗牛酶终浓度为1%的条件下，酶解80min时，亲本菌株Y的原生质体形成率和再生率分别可达93.5%和30.7%,亲本菌株X的原生质体形成率和再生率分别可达92.4%和29.5%,适合进行原生质体融合。(5) 在60℃下，对亲本菌株X的原生质体进行水浴灭活，发现灭活15min，即可100%的抑制或钝化亲本菌株X原生质体活性。(6) 将筛选的融合子及亲本酿造的苹果酒香气成分进行GC-MS鉴定分析，并结合感官品评，得到3株产香能力、发酵能力均较强的优良增香型苹果酒酵母菌株。更多还原

【Abstract】 This study was based upon the internal and external research situation, constructed cider yeast strain having aroma-improving ability and intense fermentability by protoplast fusion. Though protoplast fusion was already a ripe systematic technique, and many researchers in home and abroad wildly used it to breed new microorganism strains, there were a few research reports of constructing aroma-improving yeast strain.Five yeast strains were selected as experimental material in this study. According to the synthetic analysis of physic-chemical characteristics and organoleptic evaluation on ciders, strain 5# was chosen as optimal cider yeast. Then strain 5# was treated by ethyl methanesulfonate(EMS), strain E2, which has a stable hereditary marker, was isolated from mutants.Protoplasts of strain E2 were fused with inactivated protoplasts of strain 3# having intense fermentability, many stable fusants were obtained. A brewing trail with the excellent fusants and the parental strains was carried out, and the aroma components of different ciders were analyzed and identified by gas chromatography-mass spectrometry. Three fusants having beneficial characters of parental strains and aroma-improving ability were selected out. The main results in this study showed:(1) strain 5# was chosen as optimal cider yeast according to the synthetic analysis of different ciders brewed by the five yeast strains, and treated by EMS, strain E2, which is a lysine auxotrophic strain, was selected out from mutants for one parental stain in protoplast fusion.(2) strain 3#, which has intense fermentability, was chosen as another parental stain in protoplast fusion according to fermentative velocities of different strains and characteristics of different fermented apple juices.(3) The logarithmic growth phase of parental stains was from 2nd hour to 10th hour and the forepart phase was from 2nd hour to 4th hour according to the growth curve, therefore we used the parental cells incubated 4 hour to make protoplast.(4) The protoplast formation rate was 93.5% for parental stain of E2 and 92.4% for parental stain of 3# in the optimal reaction condition of at 35℃, 80min of reaction time, 1% final concentration of snailenzyme. and the protoplast regeneration <WP=7>rate was 30.7% and 29.5%,respectively.(5) The protoplast of strain 3# could be completely inactivated by 60℃ of water bath treatment for 15min.(6) The results obtained from analysis of different ciders by gas chromatography -mass spectrometry and sensory taste suggest that three fusants have better aroma-improving ability.更多还原