共轭不饱和脂肪酸生物半合成法的初步研究
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摘要
共轭不饱和脂肪酸是一类含有两个以上共轭双键的脂肪酸,具有抗癌、抗动脉粥样硬化、减脂、增强免疫、促进骨骼形成、抗糖尿病等丰富的药理活性。其中,尤其以共轭亚油酸广受关注。此外,产油微生物能产生并贮存质量超过其细胞干重20%的微生物油脂,其脂肪酸组成与植物油脂相似,业已成为工业微生物领域的研究热点之一。本文对共轭亚油酸的生物半合成法开展了初步的研究。
42株云南红豆杉内生真菌用于筛选产油菌株。首先,采用苏丹Ⅳ染色法,光学显微镜观察到18株含油菌株菌丝内的被染成棕红色的油脂滴。以Folch法提取测定初步筛选得到的菌株油脂产率。油脂产率达到15%以上的丝状真菌有6株,其中菌株E1.3的油脂产率最高,达到25.2%。
为了增加E1.3油脂产量,考察了碳源、氮源、碳氮比、温度、培养周期等因素的影响。优化后的发酵培养基如下:60 g葡萄糖,2 g酵母浸粉,2 g硝酸钾,2 g磷酸二氢钾,0.2 g氯化钠,0.4 g硫酸镁,10 mL微量元素溶液及1 L水。优化后的培养条件为:100 mL液体培养基在250 mL摇瓶中,180 r/min,28℃培养6 d。在此条件下,生物量为10.4 g干菌丝/L,油脂含量为32.2%。
此外,恰当的油脂提取步骤对生产真菌油脂也有重要的影响。为了确定合适的真菌油脂提取工艺,选择了6种破碎菌丝的方法和5种经典的油脂提取方法进行试验。结果表明:机械匀浆破碎菌丝的效果最佳,而Folch法优于其它油脂提取方法。
气质联用分析了E1.3菌油脂肪酸构成,其中亚油酸含量最高,达到31.6%。曲霉E1.3菌油作为原料,乙二醇单丁醚作为溶剂,氢氧化钾作催化剂,碱催化异构化法制备共轭亚油酸。紫外光谱监控反应进程,考查多种因素对亚油酸转化率的影响,优化的反应条件:温度140℃,反应周期3 h,强碱催化剂与原料质量比为0.3:1,原料与溶剂质量比为1:5。在此条件下,共轭亚油酸的收率达到86%。结果表明:菌油可替代植物油作为碱催化异构化法制备共轭亚油酸的原料。
Conjugated unsaturated fatty acids containing two or more conjugated double bonds have been found numerous biological actions, including: anticarcinogenic effect, antiatherogenic effect, stimulating the immune response, reducing body fat, enhancing bone formation, and anti-diabetic effect. Particularly Conjugated linoleic acid (CLA) has attracted wide interest. Meanwhile, Oleaginous microorganisms have been one of the hot issues in the field of industrial industrial microbiology owing to the ability of accumulating microbial oil with similar fatty acid composition to that of plant-based oil over 20% of their biomass. The semi-biological synthesis method of CLA was studied initially in this dissertation.
42 endophytic fungi of Taxus yunnanensis were selected to screening for oleaginous strains. according to the Sudan IV staining method, red brown intracellula lipid bodies of 18 strains were observed through optical microscope firstly. And secondly the Folch method was adopted to determine the total lipid content of these strains. The number of strains whose lipid yield exceeds 15% is 6, and the lipid yield of strain No. E1.3 is highest, reaching 25.2%.
In order to increase the lipid output, the effects of different influence factors including carbon sources、nitrogen sources、carbon-nitrogen ratio、temperature、inoculation period and oxygen quantity dissolved in broth were examined. The better medium for the fermentation was found as follow: 60 g dextroglucose, 2 g yeast extract, 2 g Potassium Nitrate, 2 g Potassium Dihydrogen Phosphate, 0.2 g Sodium Chloride, 0.4 g Magnesium Sulfate, and 5 mL solution containing trace component in 1 L water. The optimal culture condition was 100 mL liquid medium being inoculated in 250-mL Erlenmeyer-flask, rotation speed as 180 r/min, 28℃for 6 day. Under the optimized conditions, the biomass was 10.4 g dry cell /L and the lipid yield is 0.322 g oil/g dry cell.
In the meanwhile, a suitable lipid extraction procedure is also very important on fungi lipid production. 6 different mycelium destruction methods and 5 different lipid extraction methods were tested to determine a suitable process. The results show that mechanical homogenizing is the best mycelium destruction method and the Folch method is superior to the other lipid extraction method.
Gas chromatography /mass Spectrometry was used to study the fatty acid profiles of the fungal oil of E1.3. And the relative content of LA up to 31.6% is highest according to the normalization method of area. Then conjugated linoleic acids were prepared from fungal oil by alkali isomerization using KOH as catalyst and 2-Butoxy Ethanol as solvent for the first time. UV spectra analyzed the effects of 3 factors on the rate of conversion of LA, and the suitable conditions for the preparation process were found: reaction temperature of 140℃, reaction time of 3 h, m(KOH):m(oil) = 0.3:1. Under the optimum conditions, the average rate of conversion of LA could reaches 86%. The results show that fungal oil maybe a potential alternative source of vegetable oil as the raw materials of CLA.
42株云南红豆杉内生真菌用于筛选产油菌株。首先,采用苏丹Ⅳ染色法,光学显微镜观察到18株含油菌株菌丝内的被染成棕红色的油脂滴。以Folch法提取测定初步筛选得到的菌株油脂产率。油脂产率达到15%以上的丝状真菌有6株,其中菌株E1.3的油脂产率最高,达到25.2%。
为了增加E1.3油脂产量,考察了碳源、氮源、碳氮比、温度、培养周期等因素的影响。优化后的发酵培养基如下:60 g葡萄糖,2 g酵母浸粉,2 g硝酸钾,2 g磷酸二氢钾,0.2 g氯化钠,0.4 g硫酸镁,10 mL微量元素溶液及1 L水。优化后的培养条件为:100 mL液体培养基在250 mL摇瓶中,180 r/min,28℃培养6 d。在此条件下,生物量为10.4 g干菌丝/L,油脂含量为32.2%。
此外,恰当的油脂提取步骤对生产真菌油脂也有重要的影响。为了确定合适的真菌油脂提取工艺,选择了6种破碎菌丝的方法和5种经典的油脂提取方法进行试验。结果表明:机械匀浆破碎菌丝的效果最佳,而Folch法优于其它油脂提取方法。
气质联用分析了E1.3菌油脂肪酸构成,其中亚油酸含量最高,达到31.6%。曲霉E1.3菌油作为原料,乙二醇单丁醚作为溶剂,氢氧化钾作催化剂,碱催化异构化法制备共轭亚油酸。紫外光谱监控反应进程,考查多种因素对亚油酸转化率的影响,优化的反应条件:温度140℃,反应周期3 h,强碱催化剂与原料质量比为0.3:1,原料与溶剂质量比为1:5。在此条件下,共轭亚油酸的收率达到86%。结果表明:菌油可替代植物油作为碱催化异构化法制备共轭亚油酸的原料。
Conjugated unsaturated fatty acids containing two or more conjugated double bonds have been found numerous biological actions, including: anticarcinogenic effect, antiatherogenic effect, stimulating the immune response, reducing body fat, enhancing bone formation, and anti-diabetic effect. Particularly Conjugated linoleic acid (CLA) has attracted wide interest. Meanwhile, Oleaginous microorganisms have been one of the hot issues in the field of industrial industrial microbiology owing to the ability of accumulating microbial oil with similar fatty acid composition to that of plant-based oil over 20% of their biomass. The semi-biological synthesis method of CLA was studied initially in this dissertation.
42 endophytic fungi of Taxus yunnanensis were selected to screening for oleaginous strains. according to the Sudan IV staining method, red brown intracellula lipid bodies of 18 strains were observed through optical microscope firstly. And secondly the Folch method was adopted to determine the total lipid content of these strains. The number of strains whose lipid yield exceeds 15% is 6, and the lipid yield of strain No. E1.3 is highest, reaching 25.2%.
In order to increase the lipid output, the effects of different influence factors including carbon sources、nitrogen sources、carbon-nitrogen ratio、temperature、inoculation period and oxygen quantity dissolved in broth were examined. The better medium for the fermentation was found as follow: 60 g dextroglucose, 2 g yeast extract, 2 g Potassium Nitrate, 2 g Potassium Dihydrogen Phosphate, 0.2 g Sodium Chloride, 0.4 g Magnesium Sulfate, and 5 mL solution containing trace component in 1 L water. The optimal culture condition was 100 mL liquid medium being inoculated in 250-mL Erlenmeyer-flask, rotation speed as 180 r/min, 28℃for 6 day. Under the optimized conditions, the biomass was 10.4 g dry cell /L and the lipid yield is 0.322 g oil/g dry cell.
In the meanwhile, a suitable lipid extraction procedure is also very important on fungi lipid production. 6 different mycelium destruction methods and 5 different lipid extraction methods were tested to determine a suitable process. The results show that mechanical homogenizing is the best mycelium destruction method and the Folch method is superior to the other lipid extraction method.
Gas chromatography /mass Spectrometry was used to study the fatty acid profiles of the fungal oil of E1.3. And the relative content of LA up to 31.6% is highest according to the normalization method of area. Then conjugated linoleic acids were prepared from fungal oil by alkali isomerization using KOH as catalyst and 2-Butoxy Ethanol as solvent for the first time. UV spectra analyzed the effects of 3 factors on the rate of conversion of LA, and the suitable conditions for the preparation process were found: reaction temperature of 140℃, reaction time of 3 h, m(KOH):m(oil) = 0.3:1. Under the optimum conditions, the average rate of conversion of LA could reaches 86%. The results show that fungal oil maybe a potential alternative source of vegetable oil as the raw materials of CLA.
引文
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