【作者】 方华；

【导师】 陆健；

【作者基本信息】 江南大学 ， 发酵工程， 2006， 硕士

【摘要】 本文采用3种培养基分离麦曲中的微生物,共得到16株丝状真菌。真菌总菌落数达2.7×106个/g曲,伞枝犁头霉、米根霉、微小毛霉、米曲霉、烟曲霉占了所有分离真菌的89%。比较了超声波法,Yatalase酶法和氯化苄法提取麦曲中微生物总DNA的效果后,采用氯化苄法作为提取麦曲样品中微生物总DNA的方法。分析了绍兴地区3个黄酒厂麦曲样品中真菌的RISA图谱,建立3个麦曲RISA图谱的克隆文库,通过条带的测序得到3个麦曲样品的主要微生物均有伞枝犁头霉、酿酒酵母、微小毛霉、米曲霉、构巢裸孢壳。同时,发现地域环境影响着麦曲中的优势微生物。采用RISA指纹图谱技术分析了在制曲过程中麦曲中数量占优势的真菌变化的过程。根据图谱条带的变化,发现在制曲过程中,麦曲中优势真菌先增加后下降。初步研究了麦曲7株优势真菌的产酶情况,其中Aspergillus oryzae AO-01产α-淀粉酶、糖化酶、蛋白酶,活力分别为112.5 u/g干曲、1359.4 u/g干曲、817.6 u/g干曲,能够用作为黄酒纯种酿造的菌株。而其它的6株真菌由于液化酶和糖化酶不产或产量很低,不能有效地将淀粉原料分解为酵母能利用的葡萄糖。在真菌混合培养对产酶的影响的研究中,结果显示除Aspergillus oryzae AO-01和Aspergillus niger AN-13混合培养时,蛋白酶活力提高了3.7倍。其它真菌的混合培养物,对产酶的影响不大。通过响应面分析法和典型性分析得出Aspergillus oryzae AO-01产糖化酶的最优发酵条件:培养时间32.8 h,培养温度31.2℃,固水比1:1.24,预测糖化酶的最大酶活为1625.4 u/g干曲。验证性实验证明在优化条件下,Aspergillus oryzae AO-01产糖化酶最高可达1645.1 u/g干曲。更多还原

【Abstract】 This paper studied on the microorganisms in the Wheat Qu of shaoxin and the character of the major filamentous fungus. Three mediums, PDA, MEA, CDA, were used to seperated mould from Wheat Qu. Sixteen kinds of mould were obtained. Chromosome DNA of every kind of the pure culture was extracted by benyl chloride lysis, then the ITS rDNA fragment was amplified and sequenced. The results of identification were obtained after blastn with the GenBank database. According to the quantity of the single clones, Absidia corymbifera, Rhizopus oryzae, Rhizomucor Pusillus, Aspergillus orgyze and Aspergillus fumigatus were the major fungi in Wheat Qu.To obtain the total chromosome DNA of microbial mixture in Wheat Qu, three DNA extraction methods were applied. According to the quality, purity and integrality of DNA fragements, the benzyl chloride lysis was considered as the most effective method to extract the total chromosome DNA of microbial mixture in Wheat Qu. Funal diversity of Wheat Qu was analysed with an unclutred method RISA ( Ribosomal intergenic spacer analysis ), which based on the ITS fragement length polymorphism. ITS rDNA of microbial mixture in Wheat Qu were seperated by polyacrylmide gel electrophoresis after PCR amplification.RISA were carried out to analyze the fungal community in wheat Qu during the process of making Qu. Results showed that with increasing Qu-making the diversity of fungi in wheat Qu increased first and decreased subsequently. Gene clone libraries (ITS rDNA) containing 12 clonal sequences were successfully constructed. The fungal diversity shift observed by RISA was also shown by the clone library analysis. Consequently, the predominant fungus of wheat Qu were Absidia corymbifera, Saccharomyces cerevisiae, Rhizopus oryzae, Rhizomucor pusillus, Aspergillus oryzae, Emericella nidulans.The enzyme system of seven mold, which had been isolated from wheat Qu, was studied. Three enzymes includingα-amylase (112.5 u/g?Qu), glucoamylase (1359.4 u/g?Qu), proteinase (817.6 u/g?Qu) were produced by the Aspergillus oryzae AO-01. It could be used for starch saccharification in rice wine brewing. In addition, the effects of mixed mold culture on enzyme production were studied. The results showed that when Aspergillus oryzae AO-01 and Aspergillus niger AN-13 were incubated together, the activity of proteinase was 4.7-folds of initial activity.Box-Behnken experimental design was used to optimize the condition levels of incubation time, incubation temperature and moisture content. By response surface methodology and canonical analysis, the optimal conditions for higher production of glucoamylase were: incubation time, 32.8 h; incubation temperature, 31.2℃; moisture更多还原