【作者】 邓开野；

【导师】 任露泉；

【作者基本信息】 吉林大学 ， 农业机械化工程， 2004， 博士

【摘要】 透明质酸不仅可以从动物的眼玻璃体中提取出来，还可以从脐带、皮肤、关节滑液中提取得到。此外，尿液，血浆、皮肤胸淋巴中都含HA。HA是人体中不可缺少的一部分，随着年龄的增长，体内HA的含量逐渐减少，尤其是皮肤组织细胞和细胞间的水分含量减少，细胞间的以HA为主组成的胶状基质所充填的空间见小，导致细胞排列紧密，胶原蛋白失水硬化，使皮肤粗糙，失去弹性。因此水分对于皮肤健康是至关重要的。多年来，科学工作者一直将化妆品的保湿、保水作为研究的主要内容。而透明质酸的独特的生物学特性和流体力学特性，决定它在化妆品和医药领域中的地位。HA具有强烈的亲水性、保湿功能和良好的生物相容性，又是皮肤和其他组织中广泛存在的天然生物分子，从20世纪80年代开始用于化妆品中，被誉为理想的“天然保湿因子”。目前，添加了HA的化妆品被称为“仿生化妆品”和“第四代化妆品”，所以，在日用化工，特别是化妆品工业中有广阔的应用前景。本文突破以往的从生物组织中提取透明质酸的传统方法，从牛鼻黏膜中筛选得到产透明质酸的链球菌株，以玉米淀粉为原料，通过发酵手段制备透明质酸。通过物理和化学手段相结合，对该野生菌株进行诱变处理，得到了无溶血性，性能稳定的生产菌株。并探讨了利用该菌株发酵生产透明质酸的摇瓶工艺条件，分批发酵工艺条件，以及分批发酵的动力学的特点。同时对分批发酵的方式进行改进，采用单一补料分批发酵的方法，使HA的产量大有提高。最后考察了发酵液中提取透明质酸时，提取条件对提取得率的影响。本课题是2000年吉林省重点科技发展项目“利用玉米淀粉为原料制备透明质酸”的一部分。本论文主要针对以下的关键技术做了研究。确定了有效的菌种筛选和诱变方法。取刚宰杀牛的鼻黏膜，稀释后培养<WP=124>于血琼脂平板培养基进行培养，培养结束时，通过判断微生物细菌在血平板上的菌落及溶血性特征，来选择初筛菌株。复筛时，产物具有典型的HA红外吸收特性，即可将此菌株确定为产生透明质酸的出发菌株。利用紫外线和亚硝基胍进行诱变，得到性能稳定，无溶血环的变异株。确定该变异株的便利的染色方法，和保藏方法。对种子培养基的组成进行优化，确定了所用碳源、氮源、金属离子等的用量，可以采用2%淀粉的葡萄糖水解液作为碳源，利用2%蛋白胨和酵母粉的混合有机氮源及0.4%硝酸钠为无机氮源，MgSO4·7H2O为0.1%，KH2PO4 为0.2%，采用1：1的碳氮比的培养基作为种子培养基，有利于该菌株的生长。采用正交优化实验设计方法，得到了摇瓶发酵培养基的组成，为：4%的淀粉葡萄糖水解液，2%蛋白胨和酵母粉的混合有机氮源及0.5%硝酸钠为无机氮源，MgSO4·7H2O为0.1%，KH2PO4 为0.2%。采用淀粉的葡萄糖水解液为碳源，蛋白胨和酵母粉的复合氮源为主要营养成分的培养基，可以代替小牛血清培养基，大大地降低了发酵成本本文所筛选诱变得到的变异菌株在氧气供给充分和不充分的条件下均能大量生长，合成透明质酸。但是通风条件好时，HA产量要高。所以摇瓶培养时，500mL三角烧瓶装液量为20mL时，可获得的产量最大。采用5L小型发酵罐，对所筛选诱变后，获得的变异菌株的分批发酵生产HA的培养条件进行了研究。分别考察了初糖浓度、pH值、培养温度、氧的传递、加入HA的合成前体等对发酵的影响。结果表明，高的初糖浓度对菌体的生长和HA的合成产生抑制作用，发现5%的初糖浓度比较合适。认为比较理想的小罐分批发酵条件为：接种龄为14-16h，搅拌转速为200rpm、pH值7.0、培养温度37℃、通气量2L/L.min。HA合成前体UMP的添加可以促进菌体的繁殖和大幅度地提高HA的产量。本论文对所选育的菌株利用淀粉的葡萄糖水解液发酵生产透明质酸的发酵过程，以统计热力学为基础，结合一些经典的微生物动力学模型的建立方法，建立了描述整个发酵过程的生长、产物生成和基质消耗的动力学模型，并实验确定了模型参数。利用得到的数学模型和已有的实验数据比较，计算值与实验<WP=125>值拟和较好，表明，该模型可以较好地描述该菌株在正常条件下利用淀粉的葡萄糖水解液发酵生产透明质酸的生产过程。由于HA发酵有着底物和产物同时抑制的动力学特征。HA高水平的发酵可以通过补料方式来实现。本研究采用单一补料分批培养方式进行补料发酵时，通过对发酵过程中对菌体生长，产物合成，及副产物的影响来看，该方法可以通过改善发酵过程中环境条件，来提高HA的产量。和分批发酵相比，发酵液中HA的量提高大约3.5%。HA发酵可以采用单一补料分批发酵的方法来提高生产强度，缩短发酵周期，有效地降低生产成本，是一种可以应用有效提高HA发酵生产性能指标的现代发酵技术。提取出化妆品级透明质酸，其白色精提物HA中葡萄糖醛酸的含量为31.3%，比旋光度为[α]20D=-68°。但透明质酸产量较低，得到浅黄色初提物HA 2.77g/L,白色精提物HA 0.98g/L。本文考察了提取条件对提取率的影响，认为采用温度为30℃，而pH值为7的提取条件来提取发酵液中的透明质酸比较合适，确定了采用乙醇和CPC相结合的办法来提取HA的方法。采用红外分光光度计对透明质酸的标准品和本研究发酵液中的提取物进行了分析，由于更多还原

【Abstract】 Hyaluronic acid (HA) can not only be extracted from glass body of animals, but also from umbilical cord, skin, cartilage tissue and et al. HA is contained in urine, blood plasma, and lymph too. HA is the most important part in the humanbody. The content of HA will gradually reduce as the increasing of the age. Especially, the reduce of water content among the tissue cells and intercellular can induce the filling room of gel substrates which HA is main component get smaller, thus cells arranges closely, colloid protein hardens cause of losing water, so the skin gets roughness, and lost flexibility. The water is very important to the skin health. Scientists are thinking the moisturing and watering of cosmetics as the main researching contents. Because there is special biological feature and hydrodynamics feature, HA has important role in the fields of cosmetics and medicine. HA has strong hydrophilic property, moisturing feature and good biological compatibility. HA is nature biomolecule existed extensively in skin and other tissues. HA is honored by naming ‘natural moisturing factor, NMA’ and applied in cosmetics in 1980s. At present, the cosmetics containing HA goes by the name of ‘ Bionic Cometics’ and ‘the forth generation cosmetics’. HA has very wide applicable foreground in the industry of cosmetics. This article breaks through the traditional method to prepare HA from biological tissues. It prepares HA through fermentation method by using cornstarch as materials and Steptococcus, which is screening from viscous film of ox. The wild strain is induced by physical-chemical methods. A non-haemolysis and stable <WP=128>mutant was found. The flask shaking and batch fermentation techniques conditions were discussed. The characteristics of batch fermentation dynamics were discussed. Through the improving the mode of batch fermentation, the product of HA improved a lot by the method of single batch feeding.The influence of extraction condition to the extracted yield was discussed. This paper as part task of Science and Technology Department of Jilin Province did some useful studies on hyaluronic acid production by bacterial fermentation. Below works were chiefly completed and correlated conclusions were obtained. Effective screening and inducing method for bacterial were determined. Sampling the cattle tunica mucosanasi, culturing in the blood agar plate, and then choosing the strain through judging the colony and hemolytic features. The starting strain was determined by the typical HA infrared absorption spectrum. The compound mutation with UV and NTG was adopted. The mutant without haemolysis and stable mutant were found. The dyeing and preserved methods were determined. Optimized seed medium was determined by the single factor experiment. The best form is 2% starch hydrolysate as carbon source, and peptone and yeast extract as nitrogen source. The ratio of carbon and nitrogen glucose is 1:1. The other components are 1% peptone, 1% yeast, 0.4% NaNO3, 0.1% MgSO4·7H2O, 0.2% KH2PO4. By using compound nitrogen source instead the blood serum.Optimized fermentation medium in flask shaking was determined by orthogonal design. The components are 4% starch hydrolysate, 1% peptone, 1% yeast, 0.5 NaNO3, 0.1% MgSO4·7H2O, 0.2% KH2PO4.The culture medium is using the starch hydrolysate as carbon source, and peptone and yeast as compound nitrogen source. It can instead the ox blood serum medium and reduce the cost greatlyThe mutant can grow well under the sufficient and unsufficient of oxygen supply. But the HA yield is high under the better aeration conditions. So 20mL culture medium was cultured in 500mL flask and <WP=129>high yield of HA could be obtained during flask sharking culture. Batch fermentation condition was determined by using 5L fermenter. The affective factors to the fermentation were discussed, such as initial glucose concentration, pH value, temperature, oxygen impressing, adding synthesized precursor. The results showed that the higher initial glucose concentration could restrain the bacteria更多还原