γ-亚麻酸油脂发酵和改性的研究
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摘要
γ-亚麻酸是多价不饱和脂肪酸中的一种，它在人体可转化成前列腺素和白三烯等一系列生理活性物质。γ-亚麻酸在心脑血管系统、呼吸系统、肝脏、糖尿病性神经病变和生殖系统具有非常重要的调节作用。
发酵法是生产γ-亚麻酸的一种重要方法，本文研究了被孢霉332发酵的条件，表明pH值为5.5适合发酵，高于10~6孢子／mL的接种量有利于提高GLA产量，溶氧水平过低是造成γ-亚麻酸产量低的重要原因。本研究进行了500升发酵实验，发酵条件为：搅拌速度300r／min，通气量为0.75v／v，pH5.5左右，发酵溶氧高于10％，在此条件下菌体干重达到了63克／升，油脂产量达到24.6克／升，γ-亚麻酸产量达2.45克／升，各项指标均比摇瓶有大幅度提高，在此基础上又进行了5000升罐的发酵，单位体积产量接近500升罐水平。
在培养体系中添加吐温和橄榄油可诱导被孢霉脂肪酶的产生，二甲基硅油没有诱导脂肪酶产生的作用，脂肪酶的存在可导致菌体油脂降解和油脂酸值增加，外部碳源饥饿也可诱导脂肪酶的产生，脂肪酶可通过80℃以上的高温处理而灭活，高温灭活处理对油脂中γ-亚麻酸无明显影响，但可有效地保存菌体中的油脂，避免油脂酸值增加。
用薄板层析分离了被孢霉油脂的主要成分，被孢霉油脂主要含三甘酯、脂肪酸、双甘酯、单甘酯和磷脂，各成分中γ-亚麻酸含量依次为8.1％、6.2％、10.3％、14.7和23.5％。采用5种溶剂体系进行了油脂提取实验，分别为为氯仿-甲醇(2∶1)、正己烷、异丙醇、正己烷-异丙醇(2∶1)和乙醇、正己烷两步抽提法，其中正己烷法方法简单、成熟，提取所得油脂中三甘酯含量高，是适合被孢霉油脂的提取方法。冬化处理有利于提高油脂中γ-亚麻酸含量。
研究考察了菌体含水量、颗粒度和压坯处理对超临界法萃取油脂的影响，表明，低于5％的含水量有利于油脂的萃取，含水量较低时萃取油脂中的三甘酯含量相对较高；菌体颗粒度对萃取影响不大，说明菌体油脂萃取的主要障碍是菌体细胞壁；压坯处理可有效地破坏细胞结构，非常有利于油脂萃取，压坯后的菌体经3小时萃取可获得80％的萃取率，而未压坯的菌体只能获得50％的萃取率。
和三甘酯相比，双甘酯具有特殊的代谢方式，多价不饱和脂肪酸双甘酯的应用领域正在开发中，本文进行了甘油解法和酯化法合成双甘酯的研究。Novozyme
Gamma linolenic acid (GLA) is a member of polyunsaturated fatty acids (PUFAs), it can be transformed to PGs and LTs in human bodies, which exert important regulation function in the cardiac and brain blood vessel system, breath system, liver, diabetic neuropathy and genital system.
Fermentation conditions were studied in this thesis, pH5.5 was the optimized pH value, inoculum higher than 10~6spore/mL could enhance the GLA yield, and too low level of dissolved oxygen was an important reason of low GLA yield. 500 L culture research was carried out and the fermentation conditions were: agitation speed 300 r/min, aeration 0.75v/v, pH5.5 and dissolved oxygen was higher than 10%. The dried biomass could reach 63g/L, oil yield 24.6g/L and the GLA yield reached 2.45g/L, all the indexes were a lot higher than the vibration culture. 5000L culture was carried out based on the above research, and the yield level was close to the level of 500L culture.
Tween-80 and olive oil can promote Mortierella to produce lipases, which could lead to oil decomposition and rising of acid value, while dimethyl silicone oil could not induce the lipases. The lipases could also be induced by the hunger of carbon source and could be deactivated by treatment of temperature higher than 80℃. Deactivation treatment brought no obvious effect on the GLA content, but could preserve the oil in the mycelium effectively and avoid rising of acid value.
Mortierella oil was separated by TLC. The main compositions were triacylglycerols, fatty acids, diacylglycerols, monoacylglycerols and phospholipids, the gamma linolenic acid contents in them were 8.1%. 6.2%, 10.3% 14.7 and 23.5% respectively. The oil extraction was performed with 5 solvent systems, which were chlorlform-methanol(2:1), hexane, isopropanol, hexane-isopropanol(2:1) and ethanol-hexane two steps extractions. Hexane extraction method was a simple and mature one, which could give high triacylglycerol content, so it was the suitable extraction method of Mortierella oil. Winterization could enhance the gamma linolenic acid content in the oil.
Effects of water content, granularity and compact on the super critical CO_2
发酵法是生产γ-亚麻酸的一种重要方法，本文研究了被孢霉332发酵的条件，表明pH值为5.5适合发酵，高于10~6孢子／mL的接种量有利于提高GLA产量，溶氧水平过低是造成γ-亚麻酸产量低的重要原因。本研究进行了500升发酵实验，发酵条件为：搅拌速度300r／min，通气量为0.75v／v，pH5.5左右，发酵溶氧高于10％，在此条件下菌体干重达到了63克／升，油脂产量达到24.6克／升，γ-亚麻酸产量达2.45克／升，各项指标均比摇瓶有大幅度提高，在此基础上又进行了5000升罐的发酵，单位体积产量接近500升罐水平。
在培养体系中添加吐温和橄榄油可诱导被孢霉脂肪酶的产生，二甲基硅油没有诱导脂肪酶产生的作用，脂肪酶的存在可导致菌体油脂降解和油脂酸值增加，外部碳源饥饿也可诱导脂肪酶的产生，脂肪酶可通过80℃以上的高温处理而灭活，高温灭活处理对油脂中γ-亚麻酸无明显影响，但可有效地保存菌体中的油脂，避免油脂酸值增加。
用薄板层析分离了被孢霉油脂的主要成分，被孢霉油脂主要含三甘酯、脂肪酸、双甘酯、单甘酯和磷脂，各成分中γ-亚麻酸含量依次为8.1％、6.2％、10.3％、14.7和23.5％。采用5种溶剂体系进行了油脂提取实验，分别为为氯仿-甲醇(2∶1)、正己烷、异丙醇、正己烷-异丙醇(2∶1)和乙醇、正己烷两步抽提法，其中正己烷法方法简单、成熟，提取所得油脂中三甘酯含量高，是适合被孢霉油脂的提取方法。冬化处理有利于提高油脂中γ-亚麻酸含量。
研究考察了菌体含水量、颗粒度和压坯处理对超临界法萃取油脂的影响，表明，低于5％的含水量有利于油脂的萃取，含水量较低时萃取油脂中的三甘酯含量相对较高；菌体颗粒度对萃取影响不大，说明菌体油脂萃取的主要障碍是菌体细胞壁；压坯处理可有效地破坏细胞结构，非常有利于油脂萃取，压坯后的菌体经3小时萃取可获得80％的萃取率，而未压坯的菌体只能获得50％的萃取率。
和三甘酯相比，双甘酯具有特殊的代谢方式，多价不饱和脂肪酸双甘酯的应用领域正在开发中，本文进行了甘油解法和酯化法合成双甘酯的研究。Novozyme
Gamma linolenic acid (GLA) is a member of polyunsaturated fatty acids (PUFAs), it can be transformed to PGs and LTs in human bodies, which exert important regulation function in the cardiac and brain blood vessel system, breath system, liver, diabetic neuropathy and genital system.
Fermentation conditions were studied in this thesis, pH5.5 was the optimized pH value, inoculum higher than 10~6spore/mL could enhance the GLA yield, and too low level of dissolved oxygen was an important reason of low GLA yield. 500 L culture research was carried out and the fermentation conditions were: agitation speed 300 r/min, aeration 0.75v/v, pH5.5 and dissolved oxygen was higher than 10%. The dried biomass could reach 63g/L, oil yield 24.6g/L and the GLA yield reached 2.45g/L, all the indexes were a lot higher than the vibration culture. 5000L culture was carried out based on the above research, and the yield level was close to the level of 500L culture.
Tween-80 and olive oil can promote Mortierella to produce lipases, which could lead to oil decomposition and rising of acid value, while dimethyl silicone oil could not induce the lipases. The lipases could also be induced by the hunger of carbon source and could be deactivated by treatment of temperature higher than 80℃. Deactivation treatment brought no obvious effect on the GLA content, but could preserve the oil in the mycelium effectively and avoid rising of acid value.
Mortierella oil was separated by TLC. The main compositions were triacylglycerols, fatty acids, diacylglycerols, monoacylglycerols and phospholipids, the gamma linolenic acid contents in them were 8.1%. 6.2%, 10.3% 14.7 and 23.5% respectively. The oil extraction was performed with 5 solvent systems, which were chlorlform-methanol(2:1), hexane, isopropanol, hexane-isopropanol(2:1) and ethanol-hexane two steps extractions. Hexane extraction method was a simple and mature one, which could give high triacylglycerol content, so it was the suitable extraction method of Mortierella oil. Winterization could enhance the gamma linolenic acid content in the oil.
Effects of water content, granularity and compact on the super critical CO_2
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