【作者】 李晗；

【导师】 康建雄；

【作者基本信息】 华中科技大学 ， 环境工程， 2008， 硕士

【摘要】 普鲁兰(Pullulan)又称为短梗霉多糖,是由α-(1,6)-葡萄糖苷键连接麦芽三糖构成的线性分子,在环境污染治理、食品工业等领域具有广泛的应用前景。本课题的研究主要集中于茁芽短梗霉的复合诱变、筛选高产变异株、探索变异株的最佳发酵条件,并测定变异株的生长曲线。以短梗霉N3.837为原始株,液体摇床预培养16—20h,收获菌体,用无菌生理盐水洗涤制成菌悬液。对菌悬液进行微波辐射,稀释涂布培养,计菌落数,并与未经辐射的菌落数进行比较,计算致死率并绘制微波诱变致死率曲线,得到致死率在95%以上仍存活的菌株16株。对16株诱变获得菌株进行发酵实验,用硫酸蒽酮法测定发酵培养基原液总糖量,用无水乙醇沉淀多糖,并观察发酵液颜色。其中,KL15多糖产率最高,达到17.45%,是原始株(8.87%)的2倍左右。以KL15为出发菌株,制备菌悬液,分别添加DES和EMS进行化学诱变,稀释涂布培养,绘制致死率曲线,得到经DES诱变后致死率在90%以上仍存活的菌株17株,以及经EMS后致死率在90%以上仍存活的菌株15株,对这32株诱变获得菌株进行发酵筛选。其中,KL22的黑色素产量最少,多糖产率高,达到23.88%,且产多糖能力稳定。利用单因素实验方法对变异株KL22的发酵条件进行优化研究,研究因素为发酵培养基的初始pH值,得到最佳初始pH值为6.6,且KL22的产普鲁兰能力对初始pH值的变化相当敏感,初始pH值的微小改变就会导致多糖产率的明显变化。对KL22进行生长曲线测定实验,培养时间为75h,每5h测定一次,观测指标包括发酵液颜色、多糖产率、发酵液pH值、细胞形态和数量变化。通过测定,分析了短梗霉发酵培养过程中细胞增殖、普鲁兰产量和发酵液pH值之间的变化关系,对今后的普鲁兰发酵生产提出了优化建议。另外,变异菌株KL22的黑色素产量在整个发酵过程中一直维持在较低水平,性状优良稳定,且多糖最高产率在约培养65h时获得,达到26.63%,经诱变后发酵产糖周期较出发菌株有所缩短,有利于普鲁兰的大规模生产。更多还原

【Abstract】 Pullulan is an unbranched homopolysaccharide which consists of maltotriose units withα-(1,6)-glucosidic bonds. There is extensive applying prospect for Pullulan in the fields, such as environment polluted disposal and food industry. We discussed combined mutagenizing to Aureobasidium Pullulans and screening for mutant strain with low pigment content and high Pullulan yield, and also investigated the conditons of the fermentation and the growth curve of the excellent mutant strain.By using Aureobasidium Pullulans N3.837 as the original strain, the strain was cultured in liquid basal medium for 16-20 hours, then washed by physiological saline twice and suspended. Suspensions, radiated by microwave, were diluted and spread on solid basal medium plates and cultured. After 48-72h, we counted the colonies, compared with no microwave mutagenize colonies, calculated the lethal rate and drew the lethal rate curve. Then, 16 strains which were alive while the lethal rate reached or surpassed 95% were selected to ferment for screening. After measureing the concentration of total sugar and extropolysaccharide, mutant KL15, of which the extropolysaccharide yield was 17.45%, was obtained. The Pullulan yield of KL15 is nearly one times more than that of Aureobasidium Pullulans N3.837(8.87%).By adding DES and EMS severally to the suspensions of mutant KL15 as chemistry mutation, 32 strains were taken out from culture dishes where the lethal rate reached or surpassed 90% and deepfreezed at 4℃. After screening by fermenting these strains, we knowed that mutant KL22, of which the extropolysaccharide yield reaches 23.88%, has the lowest pigment content. Repeated fermentative experiments proved the character of KL22 is stabile.Then, single factor experiment was used to study the optimum fermentation condition by fermenting mutant KL22. Initial pH of fermentative medium was selected to be the single factor. The result of experiment showed that 6.6, uder which the extropolysaccharide yield of KL22 reaches 25.41%, is the most suitable initial pH. What’s more, we know that a small deviation of initial pH will affect the Pullulan productivy of KL22 observably. At last, we fermented mutant KL22 for 75 hours to determine the growth curve. The extropolysaccharide yield, the color and pH of fermentative culture, the variation of the cells’morphological forms and density were surveyed and recorded every 5 hours. By analyzing the relationships between these variable factors, we submitted optimizational suggestions for the fermentative production of Pullulan in future. Meanwhile, it has been proved that when KL22 is cultivated nearly 65 hours, the highest extropolysaccharide yield appears(26.63%) . This fact shows that the Pullulan producting cycle of mutant KL22 is shower than that of Aureobasidium Pullulans N3.837, and will bring many benefits for the industrial production of Pullulan by shortening the fermentative cycle of Aureobasidium Pullulans.更多还原