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高密度培养裂殖壶菌生产DHA
时间:2011-02-10 浏览次数:1562次 无忧论文网
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    二十二碳六烯酸(DHA)是一种极其重要的ω-3系列高度不饱和脂肪酸,具有促进婴幼儿脑部发育、保护视力、提高机体免疫能力等重要生理功能。商业鱼油因DHA含量较低、有鱼腥味等诸多不利因素,难以满足市场需求,因此寻找微生物来源的DHA就显得日益迫切。本研究从浙江温州地区红树林中筛选得到一株产DHA真菌WZ-3,并通过18S rDNA鉴定为Schizochytrium sp.,经测定,Schizochytrium sp.油脂中几乎不含有EPA,是婴幼儿和孕期妇女比较理想的DHA来源。通过单因素实验对影响Schizochytrium sp.生长、油脂以及DHA积累的主要因素进行了选择,结果表明:葡萄糖、酵母粉分别是Schizochytrium sp.产DHA的最适碳源和氮源,最适摇瓶生长条件为:温度25℃、装液量100 ml/500 ml、初始pH6.0、种龄48h、接种量4%-6%、培养基盐度为自然海水的一半。 采用Box–Behnken实验设计和响应面分析方法对Schizochytrium sp.发酵产DHA复合氮源培养基进行了优化,得到最佳培养基配方为:葡萄糖126 g·L-1、酵母粉10 g·L-1、玉米浆2 g·L-1、大豆蛋白胨5 g·L-1以及0.5倍自然海水。采用优化后的培养基及培养条件,发酵120 h菌体生物量干重最大可达42.9 g·L-1,油脂34.1 g·L-1,DHA13.8 g·L-1。以合成培养基为基础,对B族维生素对Schizochytrium sp.油脂积累的影响进行了考察,发现:除维生素B2对菌体油脂有一定的抑制作用以外,其余考察维生素对Schizochytrium sp.油脂积累都有不同程度的促进作用,其中生物素、硫辛酸、叶酸对油脂有比较好的促进作用。正交实验优化得到最佳维生素组合为:维生素B1 5 mg·L-1、维生素B1214 mg·L-1、生物素6 mg·L-1、硫辛酸30 mg·L-1、叶酸40 mg·L-1。通过单一缺失实验设计对合成培养基中氨基酸氮源进行了考察,从20种基本氨基酸中挑选出10种Schizochytrium sp.发酵生产DHA必需的氨基酸种类,通过测量发酵过程中必须氨基酸的消耗量以及均匀实验设计对这10种必需氨基酸的用量进行了合理的优化,由多元线性逐步回归所得方程获得发酵DHA的最佳氨基酸组合为:丙氨酸2.0 g·L-1、蛋氨酸1.2 g·L-1、半胱氨酸0.2 g·L-1、赖氨酸0.3 g·L-1、组氨酸1.3 g·L-1、谷氨酸1.4 g·L-1、谷氨酰胺1.4 g·L-1、异亮氨酸1.3 g·L-1、苏氨酸1.0 g·L-1、色氨酸1.6 g·L-1。 [英文摘要]:     Docosahexaenoic Acid (DHA) is one of ω-3-polyunsaturated fatty acids, plays an important role in the development of brain and eyes for infants, and it is beneficial for cardiovascular disease, and it also can improve body immunity. Conventional fish oils may not be suitable to meet the increasing demand for DHA owing to their limited supply, lower content of DHA and peculiar taste and odor. Thus the search for oleagiuous microorganisms rich in DHA as commercial sources is being more urgent. In this study, Strain WZ-3 was isolated from a mangrove, Wenzhou, China, and identified as Schizochytrium sp. by 18S rDNA analysis. DHA is accumulated in the cell lipid of Schizochytrium sp., and EPA is almost not existed in it, so it is a good source for infant and pregnant women. The variables with the biggest effect on the growth, lipid and DHA accumulation are medium compositions and culture conditions. Through one-factor-at-a-time experiments, glucose and yeast extract were found to be most suitable carbon and nitrogen source for DHA production by Schizochytrium sp., respectively. The optimized operational culture conditions by shaken flasks were temperature 25℃, culture volume 100 ml/500 ml, initial pH 6.0, inoculating age 48h, inoculating quantity 4%-6% and in seawater at a half salt concentration. Statistical analysis of the effect of carbon and mix-nitrogen source on DHA production by employing statistical optimization methods such as Box–Behnken design and surface response methodology indicated that the optimized medium should contain (g·L-1) glucose 126, yeast extract 10, corn steep liquor 2, soy peptone 5 and the seawater at a half salt concentration. By using the optimized medium, dry cell mass of 42.9 g·L-1, total fatty acid of 34.1 g·L-1 and DHA accumulation of 13.8 g·L-1 could be achieved after 120 h of incubation, respectively. The effect of Vitamin B Complex on the oil accumulation of Schizochytrium sp. were considered in synthetical medium, when vitamin B2 was added at the beginning of fermentation, lipid accumulation was decreased, incading vitamin B2 adverse to lipid accumulation. Vitamin B1, Vitamin B12, biotin, lipoic acid and folic acid were favorable for growth, and these helpful vitamin were optimized consist of Vitamin B15 mg·L-1, Vitamin B1214 mg·L-1, biotin 6 mg·L-1, lipoic acid 30 mg·L-1 and folic acid 40 mg·L-1 by orthogonal experimental design. Amino acids act as nitrogen source in synthetical medium. Using single factor absence design, 10 kinds of amino acids were selected out of 20 elementary amino acids as indispensable amino source for Schizochytrium sp. produce DHA. Then each necessary amino acid was analyzed by HPLC and calculated wastage during fermentation course. Uniform Design and Multiple stepwise linear regression analysis were employed to optimize these amino acids. The optimal necessary amino acids were contained: alanine 2.0 g·L-1, methionine 1.2 g·L-1, cysteine 0.2 g·L-1, lysine 0.3 g·L-1, histidine 1.3 g·L-1, glutamic acid 1.4 g·L-1, glutamine 1.4 g·L-1, isoleucine 1.3 g·L-1, threonine 1.0 g·L-1 and tryptophan 1.6 g·L-1, respectively.     
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