啤酒酵母突变株发酵性能比较及随机扩增多态DNA分析

对9株啤酒酵母菌种及经过诱变获得的突变株进行了发酵试验，比较了不同菌株的发酵能力、产高级醇能力、双乙酰还原能力以及菌株稳定性。同时利用随机引物对不同啤酒酵母株的基因组DNA进行了随机扩增多态DNA（RAPD）分析，比较不同突变株之间基因组的分子差异。结果表明：不同酵母发酵14d后外观发酵度在72．3％-76．8％，其中酵母YZB具有较高的发酵能力，最终发酵产物的高级醇含量最低，双乙酰峰值最低为0．36mg／L，而酵母Y1110最终发酵产物的高级醇含量最低为67．4mg／L，但双乙酰峰值达0．41mg／L，后酵结束后这2株酵母的双乙酰均可降至0．1mg／L以下。菌株稳定性实验结果表明，在传代7次以后和第1代的主要性能没有明显变化。利用随机引物OPG-5对不同酵母的基因组进行RAPD分析，酵母Y1110、YZB和YZD可以通过特异的扩增谱带区别于其它菌株，该结果为啤酒酵母特异的分子标记奠定了基础。     

低产硫化氢啤酒酵母菌株的选育

啤酒酵母出发菌株S-5经紫外诱变后,以醋酸铅平板初筛,后经在不同硫源平板上培养筛选,再经啤酒发酵复筛,最终获得一株低产硫化氢的啤酒酵母突变株M8.结果表明,突变株M8的二氧化硫生成量为30.4 mg/L,硫化氢生成量为4.6μg/L.与出发菌株相比,M8硫化氢生成量下降了78.2%.     

粗毛革孔菌及其高产漆酶突变株的比较分析

漆酶是一种广泛分布的多酚氧化酶,在食品、能源和环保等领域具有重要的应用价值。本实验室所筛的高产漆酶粗毛革孔菌经诱变后,生长形态变化大,突变株漆酶活力可达303 U/mL,是野生菌的3倍多。本文比较分析了原始菌株和突变菌株的生理生化差异,并通过RAPD比较分析了它们的差异基因。用140条随机引物扩增获得20条稳定的特异条带,并对其中的3条进行回收、克隆,测序结果与BLASTX、NCBI数据库的比对表明引物S381扩增出的809 bp的特异条带对应于云芝的假想蛋白,引物S225扩增出的654 bp的特异条带对应于30 S核糖体蛋白S4,引物S193扩增的1027 bp的特异条带对应于糖苷水解酶。该试验为探讨粗毛革孔菌突变株高产漆酶的分子机制,以及通过定向突变构建高产漆酶工程菌提供理论依据。     

超声波诱变选育低双乙酰啤酒酵母的研究

降低啤酒酵母的双乙酰的合成水平,可缩短啤酒发酵周期,提高生产效率。为获得低双乙酰生成量的菌株,本研究对出发菌株S.Cerevisiae SH进行超声波诱变处理,通过苯磺隆抗性初筛和发酵复筛,筛选到4株发酵6 d的双乙酰生成量低于阈值(0.15 mg/L)的突变株,其中突变株SH-8的双乙酰生成量为0.092 mg/L,相比于出发菌株降低了61.8%。研究表明,突变株保持了出发菌株的优良性状并具有良好的遗传稳定性。     

超高压诱变选育富锌啤酒酵母菌株的研究

以啤酒酵母为出发菌株,采用超高压诱变方法诱变选育高富集锌的酵母菌株。优化了超高压处理的工艺条件,并基于菌株对锌离子的耐受性和富集性,对菌株进行筛选。结果表明,培养10 h的啤酒酵母菌处于对数生长期,适宜于诱变;超高压处理压力250 MPa、保压时间20 min为诱变的最佳工艺条件;成功选育出一株高富集锌的突变菌株,其细胞锌含量达2.75 mg/g,是出发菌的6.55倍。     

脉冲强光对啤酒酵母的诱变效应

采用脉冲强光对啤酒酵母菌种进行诱变处理,脉冲处理电压分别为1 000、1 500、2 000、2 500、3 000 V,闪照次数分别为4、8、16、32、48。测定出发菌株和筛选出的变异菌株的凝聚性、双乙酰产量、发酵速率、发酵结束理化性质等指标,比较变异菌株与出发菌株综合指标的差异。结果表明：经过初筛和复筛,筛选出10#和12#两株发酵性能较好的菌株。脉冲强光诱变处理并未对啤酒酵母的发酵度产生负面效应,而是有所提高或保持原酵母菌株的优良发酵性能。     

琥珀酸产生菌原生质体的复合诱变

利用酶解法制备琥珀酸产生菌的孢子原生质体,对其进行紫外线与He-Ne激光的复合诱变,筛选到了一株突变株MS-23,其琥珀酸产量为1.27g/L,是出发菌株产酸量的20.16倍。将此菌株连续传代5次,具有较好的遗传稳定性。     

复合诱变选育细菌纤维素耐酸性高产菌株的研究

首先用紫外线对出发菌株的菌体进行诱变,然后再用硫酸二乙酯对突变株进行诱变,最后经筛选获得耐酸突变株。该突变株在pH值为3.1以下生长时,产纤维素水平为9.2 g/L,比突变株U31提高了12.2%,比出发菌株提高了31.43%。对突变株UE26进行传代和稳定性试验,结果表明该菌株具有遗传稳定性,适宜作为研究细菌纤维素的高产菌株。     

60 Co-γ辐照对琥珀酸产生菌原生质体的诱变选育

采用不同剂量的^60Co-γ辐照对琥珀酸产生菌MS-23的原生质体进行诱变处理,结果表明,辐照剂量为30kGy时致死率超过90％,具有很好的诱变效果。经多次筛选得到1株高产琥珀酸的突变株MSCo-24,高效液相色谱法测定其琥珀酸产量为2．08g／L,比出发菌株产酸量提高了63．78％,遗传性状稳定。     

漆酶产生菌的原生质体诱变选育

利用紫外线对杂色云芝原生质体进行诱变,经原生质体再生培养和筛选再生菌株,获得一株高产漆酶突变株,突变株比出发菌株的漆酶酶活提高了54.32%.而且,突变株继代遗传稳定,说明利用原生质体紫外诱变育种是获得漆酶高产菌的一种有效方法.     

Detection of a point mutation in FAS2 gene of sake yeast strains by allele-specific PCR amplification

To identify yeast mutants with a point mutation, detection of the specific mutant alleles is necessary. For this purpose, we applied allele-specific polymerase chain reaction (PCR) to detect the FAS2-1250S dominant mutant allele that encodes an altered fatty acid synthase in Japanese brewer's yeast strains. These strains are known to produce a higher amount of ethyl caproate in Japanese sake. The mutant strains were supposed to be diploid and to contain heterozygous alleles, including wild-type FAS2 and a dominant FAS2-1250S. A set of oligonucleotide primers was designed to contain different nucleotides at their 3' termini: one type was identical to the wild type and the other to the mutant FAS2. Another set of primers was designed to have an additional mismatch at the second nucleotide from their 3' termini. By testing with control strains, we established PCR conditions for specific amplification. Using these conditions and a simple template preparation procedure with SDS, the presence of the allele was detected in commercially used sake yeast strains. The method presented here will be useful for the identification of specific yeast strains.     

复合诱变选育壳聚糖酶高产菌种及产酶条件优化

以具有产壳聚糖酶能力的枯草芽孢杆菌（Bacillus subtilis）G10为出发菌,利用紫外和微波对菌株G10进行复合诱变,选育壳聚糖酶高产菌株,并采用正交试验设计对突变株产酶条件进行优化。结果选育出一株产酶活相对较高的突变株W1-32,优化后的产酶条件为果糖1.3%,胶体壳聚糖0.5%,酵母粉2.0%,MgSO4·7H2O 0.3%,初始pH 7.2,温度28 ℃,转速200 r/min。在此优化条件下,菌株W1-32产壳聚糖酶活为11.82 U/mL,是出发菌株G10的6.9倍。该研究为菌种的选育和进一步研究应用提供理论依据。     

自选菠萝果酒酵母菌的诱变育种

以1株自行分离的菠萝果酒酵母菌H10为出发菌株,制备原生质体后进行紫外诱变选育优良突变菌株。优化了酶法制备菠萝果酒酵母原生质体的工艺条件和紫外照射时间,并对诱变菌株进行初筛和复筛。结果表明:菌龄10 h、在酶解温度32℃、酶解液pH6.2、酶解时间90 min为原生质体制备的最佳工艺条件;最优紫外灯照射时间为150 s;诱变菌株经过初筛和复筛后,得到1株对菠萝汁发酵能力强,产香能力好,耐乙醇和SO2,遗传稳定性好,适合热带地区菠萝酒生产的的优良酵母菌株H10-15。     

APPLICATION OF THE POLYMERASE CHAIN REACTION TO THE RAPID ANALYSIS OF BREWERY YEAST STRAINS

The rapid discrimination of closely-related Saccharomyces cerevisiae strains can pose a significant problem to breweries, in particular where closely related strains are being used simultaneously to manufacture different products. In this study, two PCR approaches have been examined to assess their usefulness for the discrimination of brewery ale and lager yeast strains. PCR using arbitrary primers (RAPD PCR) was found unsuitable for such an application since the DNA profiles generated from brewery strains were generally found to be identical, due presumably to the close genetic relatedness of these yeasts. In contrast, PCR using δ sequence primers could rapidly differentiate between many ale and lager strains and characteristic profiles for these were generated. This method could also be applied directly to yeasts isolated from brewery worts or from active dried yeast preparations. Results of such analyses were available within the working day.     

Isolation of a novel mutant strain of Saccharomyces cerevisiae by an ethyl methane sulfonate-induced mutagenesis approach as a high producer of bioethanol

In order to obtain mutant strains showing higher bioethanol production than wild-type strains, a commercial Saccharomyces cerevisiae type was subjected to mutagenesis using ethyl methane sulfonate (EMS). After adding EMS to a shaken yeast suspension, the viability of yeast cells was assessed by diluted sample inoculation to solid yeast-extract peptone glucose (YEPG) medium at 15-min intervals. At 45 min, the viability of yeast cells was estimated to be about 40%. Mutagenized cells were recovered from YEPG broth after incubation at 30 degrees C for 18 h. After this period, EMS-treated yeast cells were grown on solid aerobic low-peptone (ALP) medium containing 2-12% (v/v) ethanol. All plates were incubated at 30 degrees C for 2-6 d in order to form colonies. The mutant strains that tolerated high concentrations of ethanol were selected for bioethanol production in microfuge tubes containing fermentation medium. Formation of bioethanol in small tubes was detected by the distillation-colorimetric method. In addition, trehalose content and invertase activity were determined in each mutant strain. Among many isolated mutant strains, there were six isolated colonies that grew on ALP medium supplemented with 10% (v/v) ethanol and one of them produced bioethanol 17.3% more than the wild type.     

具高乙醇耐受力酵母菌的选育及其在猕猴桃果酒中的应用

以呼市周边各旗县果园的果实和土壤中筛选出的具有乙醇耐受力酵母菌株HJ-1为实验菌株,猕猴桃为原料,探究了猕猴桃果酒的发酵工艺。通过紫外诱变处理后经TTC法筛选、杜氏小管筛选等四级筛选,挑选正向突变酵母菌株,并应用于猕猴桃果酒发酵。后经单因素实验与正交实验,研究了发酵温度、接种量、初始pH等因素对猕猴桃果酒品质的影响。经紫外诱变最终筛得一株高乙醇耐受力正向突变菌株HJ-1E,其乙醇耐受力可达18% vol,产酒精能力为11.9% vol。猕猴桃果酒生产的最佳工艺为菌株接种量3.5%,初始pH5.7,SO₂添加量70 mg/L,发酵温度为21℃。诱变所得正向突变菌株HJ-1E在此发酵条件下生产的猕猴桃果酒果香浓郁,酒体醇厚,酒精度为13.3% vol,为猕猴桃果酒的酿造提供了参考。     

奶牛源鼠李糖乳杆菌的12C6+重离子束诱变选育

本研究利用中科院重离子加速器释放的12C6+重离子束作为辐射诱变源,以酸斑值（HC）和抑菌圈值为指标,对鼠李糖乳杆菌JF12-1进行功能性诱变。通过致死率和正负突变率确定诱变的最佳辐照剂量;对最佳辐照剂量下的突变菌株利用酸斑法进行初筛,抑菌圈法复筛,而后通过连续传代之后检测乳酸含量变化来测定遗传稳定性,并对遗传稳定菌株进行16S rDNA测序,定位其突变位点。结果显示辐照剂量为300 Gy时,致死率为79.86%,正突变率为30.33%,负突变率为5.38%,确定为最佳诱变剂量;酸斑法初筛最佳诱变剂量下的突变菌株,得到20株HC值较原始野生菌株提高25%以上的突变株;抑菌法复筛得到8株体外抑菌性较原始野生菌株提高15%以上的菌株;遗传稳定性测定发现这8株突变乳酸菌株产乳酸稳定性良好;16S rDNA测序发现原始野生型菌株JF12-1的可能突变位点不在16S rRNA基因上,促使其产酸和抑菌性能增强的突变位点可能发生在其他基因区段。利用12C6+重离子束成功诱变选育出了高产乳酸及体外抑菌性优良的功能性鼠李糖乳杆菌稳定株,为下一步深入开发该菌株提供了较好的理论基础和应用依据。     

硫酸二乙酯化学诱变选育高核糖核酸酿酒酵母及培养基组成优化

该研究以酿酒酵母（Saccharomyces cerevisiae）BY23为出发菌株,采用硫酸二乙酯（DES）对其进行化学诱变,筛选出一株生长性能好、胞内核糖核酸（RNA）含量高的突变株BY23-195,并以胞内RNA含量为评价指标,通过单因素及正交试验对其糖蜜培养基成分进行优化。结果表明：突变株BY23-195生长性能较好,在酵母浸出粉胨葡萄糖（YEPD）培养基中,RNA含量较出发菌株BY23提高了18.85%。最优糖蜜培养基组分为糖蜜（糖度调至12 °Bx）、酵母浸粉5%、硫酸铵0.05%、磷酸二氢钾0.05%、硫酸亚铁0.05%、硫酸锌0.10%。在此最优培养基组成下,突变株BY23-195胞内其RNA含量达到16.01%,较优化前（13.66%）提高了17.20%。     

普鲁兰糖无色素发酵工艺研究

目的获取普鲁兰糖产量高且色素分泌少的突变菌株,优化普鲁兰糖的发酵工艺。方法紫外法诱变出芽短梗霉;通过测定普鲁兰糖的产量优化培养基组成及发酵条件。结果获得了高产普鲁兰糖且色素分泌量少的突变菌株;优化后培养基组成为:蔗糖10%,酵母膏0.2%,(NH4)2SO4 0.06%。初始pH 6.5。工艺优化后发酵过程无色素分泌,普鲁兰糖产量达到67.2 g/L。结论得到了普鲁兰糖产量高且色素分泌少的突变菌株,工艺优化后发酵过程无色素分泌。