【作者】 王丹；

【导师】 陈光；

【作者基本信息】 吉林农业大学 ， 生物物理学， 2011， 硕士

【摘要】 苯基乳酸(Phenyllactic acid, PLA)是一种天然抑菌化合物,具有食品防腐保鲜的功效；同时作为丹参素的衍生物,苯基乳酸的体外抗血小板聚集活性与丹参素有着同等的药理作用,在食品和医药领域市场应用前景十分广阔。本研究使用基因组改组技术选育苯基乳酸高产菌株,旨在提高苯基乳酸的产率,推动苯基乳酸微生物发酵产业的发展,增加社会效益和经济效益。基因改组技术成功地将传统诱变育种技术与微生物细胞融合技术有机地结合,使重组对象从单个基因扩大到整个基因组,同时将多个亲本的优良表型随机的重组于同一株菌株,可以高效、快速地筛选出发酵性状优良的正向突变菌株,在很大程度上弥补了经过长期的传统诱变菌株对诱变剂产生钝化,生产性能很难再提高的缺陷。本研究首先从酸奶渣中分离筛选出一株发酵性能良好的苯基乳酸生产菌株Lactobacillus paracasei W2,该菌株在MRS培养基上生长良好,抑制食品中腐败菌的能力较强。在发酵培养基中添加苯基丙酮酸可使苯基乳酸的产量提高,摇瓶发酵实验结果表明苯基丙酮酸最适添加浓度为4 g/L,苯乳酸最高产量可达0.81g/L。经生理生化和16SrDNA序列测定鉴定此菌株为副干酪乳杆菌,GenBank登录号为HM101340。以副干酪乳杆菌Lactobacillus paracasei W2作为出发菌株,通过传统的物理诱变和化学诱变方法对苯基乳酸生产菌株进行菌种改良。结合牛津杯抑菌实验和碳酸钙平板筛选从141株存活菌株中初筛得到7株正突变菌株,经摇瓶测定苯基乳酸含量复筛得到5株生产性能较出发菌株显著提高的突变株,分别为U4、U7、U12和N3、N6。将这些正向突变株连续培养10代,检测每代菌株发酵液中的苯基乳酸含量,保存苯基乳酸产量变化不大,遗传性稳定的菌株。通过对Lactobacillus paracasei W2菌株生长曲线的测定,确定最佳诱变菌龄为18-20h。将紫外诱变获得的正向突变株U4、U7、U12和亚硝基胍诱变筛选的菌株N3、N6作为亲本菌株,进行多轮递推式原生质体融合。确定了最佳破壁条件为：溶菌酶浓度20g/L,变溶菌素浓度为15μg/m L；最佳融合条件为：常温下,聚乙二醇的浓度为50%,作用时间为10min。以双亲灭活原生质体得到损伤互补的方法筛选融合子,确定了紫外灭活最佳时间为20s,热灭活最佳条件为60℃水浴加热30min。最终筛选出四株正向突变株F5、F7、F8和F13,其中融合菌株F8苯基乳酸产量提高幅度最大,发酵液中苯基乳酸含量为1.92g/L,比原始菌株提高了1.37倍。更多还原

【Abstract】 Phenyllactic acid, PLA is a natural antimicrobial compounds, which has the efficacy to preserve fresh food from rotting;also as a derivative of Danshensu Salvianic acid A, PLA has the same pharmacological effects in vitro anti-platelet aggregation activity, in the food and pharmaceutical fields, its market prospect is very broad. In this study, genome shuffling was used to breed the producing strain to improve the PLA production rate of fermentation to promote the development of the industry, increase the social and economic benefits. The traditional mutation breeding techniques and microbial protoplast fusion were successfully reorganized by Gene technology for combination to the reorganization of objects from a single gene to the entire genome, while many parent strains were excellent phenotype with a reorganization of the strains,which could be efficiently and quickly filter out the fermentation of mutant strains for positive good character, instead of the mutants that is difficult to improve the performance of the defects after a long tradition of mutagen glazing.Firstly, a strain Lactobacillus paracasei W2 producing phenyllactic acid was isolated from the yogur brought, which grew well in MRS medium, inhibited the ability of food spoilage. The production of lactic acid could be increased by adding the ppa in the culture medium, the fermentation results show that the optimal concentration of ppa was 4 g/L, while the highest yield of PLA is 0.81g/L.The strain was identified by the physiological and biochemical method and 16SrDNA,sequenced as Lactobacillus paracasei, the GenBank accession number is HM101340.Lactobacillus paracasei W2,as parental strain, was mutagenized UV-ray and NTG.10 mutants were selected by the isolation on the calcium carbonate plates and Oxford Cup inhibition experiments from141 survival mutants. As a result of fermentation in Erlenmeyer flask, U4, U7, U12, and N3, N6,were more increased of Pla producing compared with the original mutant.These mutants were cultured for forward 10 generations, each generation of strains detected in the fermentation broth of Pla, little change saved with genetic stability. The optimum age for the mutation strain was 18-20h,by determining the growth curve of Lactobacillus paracasei W2.The genome shuffing was used to improve the production of phenyllactic acid by Lactobacillus paracasei W2. The starting population (U4、U7、U12、N3、N6) mutated by ultraviolet irradiation and nitrosoguanidine were used as the parental strains of protoplast fusion. After several rounds of genome shuffing, a forward mutant F8 was screened, its phenyllactic acid production was 1.92mg/ml,which increased 1.37 times more than the original strain.The optimum concentration of mutanolysin is 15μg/ml and the lysozyme is 20mg/mL. The optimum conditions of protoplast fusion were found, room temperature,PEG50%,the time of protoplast fusion is 10min.更多还原