【作者】 陈诚；

【导师】 梅乐和；

【作者基本信息】 浙江大学 ， 生物化工， 2007， 硕士

【摘要】 全顺式-4，7，10，13，16，19-二十二碳六烯酸（docosahexaenoic acid，简称DHA）是一种具有重要生理意义和经济价值的多不饱和脂肪酸。它不仅是婴幼儿大脑、视觉正常发育的必需物质，而且还具有延缓老年人大脑和视觉功能衰退和降低血脂、抑制血栓生成、抗自身免疫反应、抑制肿瘤等功效。因此，DHA已广泛用于生产婴幼儿食品、保健食品和辅助治疗剂等产品，其制备和加工方法也日受关注。目前DHA的主要制备方法是从深海鱼油中提取。由于鱼油资源有限、成份复杂，这种制备方法存在供应不稳定、产量有限、成本较高等问题，限制了DHA的进一步应用。更不利的是，由于海洋环境污染的恶化，这种方法的安全性受到了越来越多的质疑。因此，开发安全、可靠的DHA新来源已成为当前研究的热点。裂殖壶菌Schizochytrium limacinum SR21是一种海洋真菌，该菌DHA含量高、生长快、易于培养、对人畜无毒害，是一种极具生产前景的DHA微生物资源。本文系统研究了利用Schizochytrium limacinum SR21制备DHA的相关过程及工艺。论文首先对菌体中的DHA提取和检测过程进行了研究、确定了较佳的DHA提取路线和检测方法；其后，在摇瓶中对Schizochytrium limacinum SR21的液体发酵条件进行了考察和优化，显著提高了DHA的产量；最后，为了改进菌体生长最适温度（25℃）低于常温这一不利于工业化生产的缺点、同时也为了进一步提高DHA产量，根据设定的选育路线和筛选模型对原始菌株进行了初步诱变育种并取得了一定进展。首先对DHA的提取过程和检测方法进行了研究。比较了六种破胞方法，确定了120℃、干燥3h的高温干燥方法，该法在尽量破碎菌体细胞的同时避免DHA的破坏；改进了酸性氯仿甲醇法，采用盐酸水解干菌体后用体积比为2：1的氯仿—甲醇溶液萃取脂质的方法，实现了脂质的充分、高效提取；优化了甲酯化DHA的三氟化硼乙醚催化法，将对甲酯化效果有重要影响的0.8mol·L^-1的KOH-MeOH用量优化为0.0707ml·mg^-1脂质；最后确定了DHA的气相色谱检测参数。其次，在摇瓶中对Schizochytrium limacinum SR21制备DHA的液体发酵条件进行了考察和优化。首先，考察了碳源、氮源以及金属离子等培养基组成对菌体生长和DHA产量的影响。发现葡萄糖、酵母粉和蛋白胨浓度对单位菌体的DHA含量无显著影响、但对细胞干重具有正效应；而K⁺能促进菌体生长，但会降低单位菌体中的DHA含量。其后，采用单因素实验考察了种龄、接种量、装液量、发酵液初始pH值和发酵温度等操作条件的效应，初步确定了各操作条件的最适参数，并发现变温培养方式有利于提高DHA在脂质中的含量。最后，采用均匀设计方法对等关键因素进行了进一步的优化，通过对实验结果回归建模的方法确定最适培养基组成为：葡萄糖145g·L^-1，酵母粉2.6g·L^-1，蛋白胨5.2g·L^-1，K⁺浓度10mM·L^-1，海水晶20g·L^-1，磷酸盐缓冲体系，pH6.8；最适操作条件为：装液量为250ml的三角烧瓶中装20ml的发酵液，种龄24h，接种量4％，培养时间5天，前三天25℃培养，后两天20℃培养。在此优化条件下，细胞干重达到55.64g·L^-1，DHA产量达到12.61g·L^-1。与优化前的DHA产量4.15g·L^-1相比，优化使DHA产量提高了2.04倍。最后，对SR21菌株进行了诱变育种以改变其最适温度低于常温这一不利于工业化生产的缺点、同时也试图进一步提高该菌株的DHA产量。采用自行设计的低温驯化方法和筛选模型，对紫外诱变菌株进行了初步选育，得到了一株DHA产量比原始菌株提高了26.73％的突变株。更多还原

【Abstract】 DHA （docosahexaenoic acid） is a kind of polyunsaturated fatty acids （PUFAs） which has important physiological functions and high economic value. It is essential in promoting the development of brain and eyes of infants and it can also decrease blood lipid, choke back thrombosis, reject self-immune reactions and inhibit tumour for the old. Therefore DHA has been widely used in producing infants’ food, health food and assistant therapeutic dose, etc. Thus more and more attention has been payed on the preparation process of DHA. Nowadays, DHA is mainly extracted from deep sea fish oil. Due to the lack of fish oil, the low producion of DHA from fish oil and the high cost, DHA can not be widely applied in the food industry and the pharmaceutical industry. What is worse, the polution of the sea is deteriorating day after day, DHA extracted from fish oil is being doubted of safety problems. As a result, more and more people are focusing their eyes on how to find a new source for DHA, which should not only be stable and secure but also with low cost.DHA production by microorganism fermentation has lots of advantages compared to fish oil. Schizochytrium limacinum SR21 is a promising microorganism in producing DHA as it has high content of DHA and the composement of PUFAs in SR21 is simple. In this dissertation, the extraction process of lipid, the transmethylation method of DHA and the culture conditions of Schizochytrium limacinum SR21 in shake flask were studied and optimized and then the original strain was mutagenized under UV and a DHA high-yield strain was found.Firstly, the extraction process of lipid and transmethylation method of DHA were studied. After 5 days-cultivation, cells were dried under the temperature of 120 ℃ for 3 hours after they were centrifuged. Chloroform :methaol=2:1 were used to extract lipids from the dry cells, after they were disposed by hydrochloric acid for 40min at the temperature of 65℃. This method is not only fast but also efficient. Before DHA was detected by gas chromatography （GC）, it needed to be transmethylated. The dosage of KOH-MeOH （0.8M） is quite important in the transmethylation process. The most suitable amount would be 0.0707ml per mg lipid. The parameters of GCwere determined finally.Secondly, the cultural conditions of producing DHA with Schizochytrium limacinum SR21 in shake flask were investigated. First of all, the medium components such as carbon source, nitrogen source and metallic ions were studied. The concentrations of glucose, yeast extract and peptone have positive effect on dry cell weight.Dry cell weight increased with K⁺ in the medium, but lipid content decreased. After that, other factors including inoculum age, inoculum size, liquid volume, initial pH value and temperature were ulteriorly optimized and determined. Enventually, Uniform Design was used to optimize DHA yield. According to the results of the designed experiments, the recursive equation gave us an opitimized medium:Glucose 145g·L^-1,Yeast extract 2.6g·L^-1,Peptone 5.2g·L^-1, K⁺ 10mM·L^-1 in half the concentration of sea wa-ter,phosphate buffer.The optimum culture conditions would be: initial pH 6.8,inoculum age 24h, inoculum size 4%, culture time 5days, incubated at the temperature of 25℃ in the first three days, 20℃ in the remaining two days. Under this condition, dry cell weight achieved 55.64g/L, DHA yield 12.61g/L. DHA yield was increased by more than two times compared to pre-optimization conditions.Finally, the original strain was mutagenized under UV in order to get a strain which could grow at a normal temperature and also with higher DHA content. The mutagenized strains were screened with the designed screening model. A high-yield strain was screened out, whose DHA yield increased by 26.73%.更多还原