细菌发酵法生产透明质酸的研究

【作者】 于雷；

【导师】 张凤宽；

【作者基本信息】 吉林农业大学 ， 食品科学， 2003， 硕士

【摘要】 透明质酸是由Myer和Palmer于1934年从牛眼的玻璃体中最先分离出此物质并加以命名的。它是生物体内普遍存在的酸性粘多糖类物质，化学本质为(1-β-4)D-葡糖醛酸和(1-β-3)N-乙酰基-D-氨基葡糖组成的双糖单位重复连接构成的大分子糖胺聚糖。透明质酸主要分布于动物的结缔组织中，但不同来源的透明质酸化学结构完全相同，仅存在相对分子量的差异，而没有种属特异性。 由于特殊的生理作用，独特的流变学特性和极强的持水保湿能力，透明质酸在化妆品工业，医学研究，临床治疗等领域有着广泛的应用。 目前，透明质酸的生产方法逐渐由动物组织提取法转向微生物发酵法。细菌发酵法生产透明质酸具有产量不受原料资源限制，成本低，产量高，有较高的相对分子量，分离纯化工艺简便，易于大规模生产等特点成为透明质酸生产的发展方向，应当进一步深入研究。 本文作为吉林省科技厅“深层发酵法生产透明质酸”课题的部分内容，对细菌发酵法生产透明质酸进行了有益的探索，主要完成了以下工作，并得到了相关结论。 1、确定了有效的菌种筛选方法，从140份牛鼻粘膜中成功地筛选到了三株产透明质酸的菌株，为生产透明质酸奠定了基础。在初筛过程中，该菌株经血琼脂平板培养后的形态观察及荚膜染色镜检，证明为带有荚膜的乙型溶血性链球菌。在复筛过程中，利用红外吸收光谱法以目的代谢产物即透明质酸为目标，以Sigma公司生产的透明质酸为标样进行筛选。Sigma公司HA标准样品的红外谱图与发酵液精提物的红外谱图各自吸收峰的位置和形状基本相同，峰的相对强度相近。上述结果提示筛选菌株的产物呈较典型的HA红外吸收图谱。 2、确定了菌种诱变方法。紫外线诱变和亚硝基胍诱变，即物理诱变和化学试剂诱变相结合的复合处理，使血琼脂平板上的菌落形态明显变大，呈较大透明黏液状，并得到一株不具有溶血性的突变菌。 用紫外分光光谱法对发酵液精提物和标样透明质酸的水溶液进行分析，发酵液精提物的紫外光谱在196nm处有特征吸收，与sigma标样的相同。并且随着HA含量的增加吸光度值也变大，表明利用其最大波长处紫外光谱分析可以作为衡量发酵液中HA含量多少的依据。但发酵液中提取物在257nm和280nm处存在紫外吸收，这表明发酵液中提取物较sigma标样存在核酸和蛋白质等杂质。 将诱变处理前后的菌株发酵液精提物分别在波长196nm处进行紫外分光测吉林农业大学硕士学位论文细菌发酵法生产透明质酸的研究量，其吸收分别为1.813和2.071。表明诱变后透明质酸的产量提高幅度不大。 3、确定了菌株种子培养基的最佳组成。基于菌体生长和透明质酸合成相偶联，选择透明质酸的产量为试验指标，进行了碳源，氮源，碳氮比，有机氮和无机氮协同作用对应指标的单因素试验。试验结果表明在其它控制条件不变的情况下，以2%葡萄糖作为碳源，蛋白栋和酵母膏作为氮源，碳氮比为1:1，有机氮与无机氮的比为5:1时，试验指标最高。 4、确定了摇瓶发酵培养基的最佳组成。同样选择透明质酸产量作为试验指标，以葡萄糖，蛋白陈和酵母膏，NaNo3为试验因素设计正交试验玩必，通过对获得数据进行分析和综合实际，摇瓶发酵培养基最佳组成确定为葡萄糖1%，蛋白陈1%，酵母膏1%，NaNO3 0.5%。通过对种子培养基和发酵培养基优化得到以蛋白陈和酵母膏复合氮源为主要成分的培养基，它们可以代替血清培养基，菌体的生长良好。 5、确定了菌株的最佳培养条件。绘制出种子培养基中菌体的生长曲线，取14一16h为接种的最佳种龄，发酵培养基中的最佳装液量确定为20nil/250d。培养基和培养条件优化后，菌株发酵液精提物的紫外吸收由2.071提高至2.562，表明透明质酸的产量有所提高。 6、确定了透明质酸的分离提纯工艺。通过乙醇沉淀和CPC络合等手段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36.2%，比旋光度为间ZOD二一680。但透明质酸产量较低，每100onu新鲜发酵液中得到浅黄色初提物I诬A 2.779白色精提物H人0.9490更多还原

【Abstract】 Hyaluronic acid was first discovered through the vitreous body of the cattle eyes in 1934 by Myer and Palmer. Hyaluronic acid is comprised of linear,unbranching,polyanionic disaccharide units consisting of D-glucuronic acid and N-acetyl glucosamine joined alternately by beta 1-3 and 1-4 glycosidic bonds. Hyaluronic acid is mainly isolated from the intercellular matrix of aninal connective tissues. The chemical composition of hyaluronic acid from different sources is completely same oppositing to the molecular weigh and specific characteristics between genuses are not existed.Because of its special physiological action,extraordinary rheoldgy character and moister-holding function, hyaluronic acid is extensively applied to areas including medical research,clinical therapy and cosmetic industry.Today,traditional hyaluronic acid producing method by extration of animal tissues has began to turn to microbial route.Because of sufficient sources,low cost,high output,relatively high molecular weigh and easy purification, hyaluronic acid by bacterial fermentation should be paid more attention and studied furtherly.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 completeled and correlated conclusions were obtained.1. Efficient screening method for bacteria was determined.Three strains of hyaluronic acid-producing bacteria were isolated from 140 cattle tunica mucosanasi successfully.This laid a foundation for hyaluronic acid production. (3 -hemolytic streptococcus with capsules was discovered through observation of bacterial conformation on blood agar flat plate and examination to dyeing capsule with microscope.Infrared spectrophotometry was used in rescreening.lt demonstrated that hyaluronic acid from Sigma corporation and extract from fermentation broth had the same absorption spectrum .The above-mentioned result promted that the extract from fermentation broth of screened bacteria had the typical infrared absorption spectrum of hyaluronic acid.2. Mutation method for bacteria was determined.The compound mutaion with UV and NTG made the colony form obviously large and presented transparent mucus shape and a mutant without haemolysis was found.UV spectrophtometry was applied to analyzing both extract from fermentation broth and hyaluronic acid from Sigma corportation.They had the same maximum absorption at 196nm wavelength.The more hyaluronic acid existed the more absorbency increased.So UV spectrophtometry can be used as warranty for measuring the content of hyaluronic acid.But extract from fermentation broth had the absorption at 257nm and 280nm wavelength.This indicated that there were impurity such as nucleic acid and protein and so on.The absorbance of extract from fermentation broth of original bacteria was 1.813,while that of mutant was 2.071.It showed that the yield of hyaluronic acid after mutation did not improve too much.3. Optimizd seed midium was determined. Hyaluronic acid fermentation was a kind of associated fermentation in which hyaluronic acid output and growth of bacteria were of the same importance.The single factor tests on carbon source ,nitrogen source,ratio of carbon source and nitrogen source.synergy of organic and inorganic nitrogen influencing hyaluronic acid output was done.The result indicated that hyaluronic acid output was highest when carbon source was 2% glucose,nitrogen source was peptone and yeast,ratio of carbon source and nitrogen source was 1:1,ratio of organic and inorganic nitrogen was 5:1 and other conditions were controlled invariably.4. Optimized fermentation medium in culture flask was determined.Chosing hyaluronic acid output as indexes,glucose,peptone and yeast, NaNO3 as factors,orthogonal design L9(34) was made.By analyzing the data and considering the practices,optimizd fermentation medium was composed of 1 % glucose, 1 % peptone, 1% yeast and 0.5% NaNO3.It found that the medium更多还原