富含DHA的裂殖壶菌的工业化生产试验、脂肪酸提取及应用研究
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摘要
论文主要对海洋真菌裂殖壶菌(Schizochytrium limacinum OUC88)的发酵培养,脂肪酸提取及其饵料效果进行了研究。主要探讨了发酵条件对细胞生长和产物形成的影响,并对菌株在发酵中的动力学特性进行了分析,分别在10L、100L微生物发酵罐中进行了扩大实验。利用有机溶剂进行了裂殖壶菌胞内脂肪酸的提取研究。随后,进行了裂殖壶菌对轮虫卤虫营养强化及大菱鲆育苗的饵料实验。
结果表明:
1利用10L生物反应器优化裂殖壶菌的发酵条件。首先进行单因子优化试验,试验因子有温度、pH、通气量、搅拌转速、接种量、发酵时间、接种菌龄,分别对试验因子设立一系列水平梯度,结果得到一条包含拐点的曲线,得出单个试验因子的最佳水平;
依据单因子优化试验,对发酵温度,通气量,pH,转速,接种量,发酵体积,发酵压力,接种菌龄,发酵时间等条件进行Plackett-Burman试验设计,得到对裂殖壶菌发酵产量起决定作用的因子是发酵时间,通气量,转速和接种菌龄;接下来对这四个因子进行旋转中心组合设计并结合响应面曲线得到最佳的发酵条件;
得到最佳发酵条件后,对裂殖壶菌的发酵动力学进行了初步研究,利用数学公式对试验结果进行非线性拟合获得了裂殖壶菌发酵过程中的菌株生长的动力学模型,产物生成的动力学模型和底物消耗的动力学模型。
利用100L生物反应器进行裂殖壶菌发酵的中型试验,基本证实了小试得到的结论,中试取得了较好的结果。
最终产率为每升发酵液获得24.1g裂殖壶菌干粉并含有二十二碳六烯酸(DHA)4.7g。建立的菌株生长动力学模型为:产物生成动力学模型为:
2用裂殖壶菌干粉强化轮虫卤虫,提高饵料体内不饱和脂肪酸特别是DHA的含量,增加饵料的营养价值。高压气相色谱检测显示:经过12小时的强化,饵料体内的DHA含量都显著提高,分别达到9.36mg/g和15.8mg/g。用强化后的饵料进行大菱鲆育苗时,仔鱼的成活率达到12%和正常体色率85%,较之原来都有显著提高。
3用有机溶剂和CO2超临界两种方法萃取细胞内的脂肪酸。利用正交试验设计分别获得两种提取方法的最佳条件组合。两种方法各有优点,有机溶剂法能提取细胞内全部的脂肪酸且萃取率较高,而CO2超临界法可以得到单一的DHA组分,没有有机溶剂残留,安全可靠。
This thesis is about a heterotrophic marine fungal Schizochytrium limacinum OUC88. It mainly studied the effect envirmental factors on both the bacterial cell growth and products synthesis in suspension culture. In order to industrialize the whole process, the middle-scale culture in 10L、100L fermentor and the extraction methods were studied. The result are as follow:
1. Fermentation parameters for biomass and DHA production of Schizochytrium limacinum OUC88 in a fermenter (working volume 7 L) were optimized using Plackett-Burman and central composite rotatable design. Out of 10 factors studied by Plackett-Burman design, four influenced the biomass production significantly. Central composite rotatable design was used to optimize the significant factors and response surface plots were generated. Using these response surface plots and point prediction, optimized values of the factors were determined as follows temperature 23℃, aeration rate 1.48 L min-1 L-1, agitation 250 rpm and inoculum cells in mid-exponential phase, the maximum yield of DCW and DHA were 24.1 g L-1 and 4.7 g L-1, respectively. These predicted values were also verified by validation experiments. The kinetic equation of bacterium cultured was: X=9.19*e0.08t/(24.46-0.37*e0.08t); The kinetics of DHA production was P = -0.25 + 0.2X
2. The rotifer and Artemia were harvested at 3, 6, 9, 12, 18 and 24 h intervals, analyzed for their fatty acid composition, and compared with the control which fed on yeast only. The highest DHA content resulted from the enrichment period of 12 h for both fed organisms, reached to 13.4% and 10.9% of the total fatty acids (TFAs) in rotifers and Artemia nauplii, respectively, and the DHA level has reduced sharply if enrichment time longer than 12 h. The pseudoalbinism rate of turbot Scophthatmus maximus juveniles which fed on enriched rotifers and Artemia nauplii has reduced significantly (40% lower than in control group), suggesting that enriched rotifers and Artemia nauplii by DHA-rich Schizochytrium limanium OUC88 may provide a practical strategy for fish juveniles in aquaculture.
3. In DHA extraction process, the optimum values of parameters of the extraction were determined by Orthogonal design experiment.
结果表明:
1利用10L生物反应器优化裂殖壶菌的发酵条件。首先进行单因子优化试验,试验因子有温度、pH、通气量、搅拌转速、接种量、发酵时间、接种菌龄,分别对试验因子设立一系列水平梯度,结果得到一条包含拐点的曲线,得出单个试验因子的最佳水平;
依据单因子优化试验,对发酵温度,通气量,pH,转速,接种量,发酵体积,发酵压力,接种菌龄,发酵时间等条件进行Plackett-Burman试验设计,得到对裂殖壶菌发酵产量起决定作用的因子是发酵时间,通气量,转速和接种菌龄;接下来对这四个因子进行旋转中心组合设计并结合响应面曲线得到最佳的发酵条件;
得到最佳发酵条件后,对裂殖壶菌的发酵动力学进行了初步研究,利用数学公式对试验结果进行非线性拟合获得了裂殖壶菌发酵过程中的菌株生长的动力学模型,产物生成的动力学模型和底物消耗的动力学模型。
利用100L生物反应器进行裂殖壶菌发酵的中型试验,基本证实了小试得到的结论,中试取得了较好的结果。
最终产率为每升发酵液获得24.1g裂殖壶菌干粉并含有二十二碳六烯酸(DHA)4.7g。建立的菌株生长动力学模型为:产物生成动力学模型为:
2用裂殖壶菌干粉强化轮虫卤虫,提高饵料体内不饱和脂肪酸特别是DHA的含量,增加饵料的营养价值。高压气相色谱检测显示:经过12小时的强化,饵料体内的DHA含量都显著提高,分别达到9.36mg/g和15.8mg/g。用强化后的饵料进行大菱鲆育苗时,仔鱼的成活率达到12%和正常体色率85%,较之原来都有显著提高。
3用有机溶剂和CO2超临界两种方法萃取细胞内的脂肪酸。利用正交试验设计分别获得两种提取方法的最佳条件组合。两种方法各有优点,有机溶剂法能提取细胞内全部的脂肪酸且萃取率较高,而CO2超临界法可以得到单一的DHA组分,没有有机溶剂残留,安全可靠。
This thesis is about a heterotrophic marine fungal Schizochytrium limacinum OUC88. It mainly studied the effect envirmental factors on both the bacterial cell growth and products synthesis in suspension culture. In order to industrialize the whole process, the middle-scale culture in 10L、100L fermentor and the extraction methods were studied. The result are as follow:
1. Fermentation parameters for biomass and DHA production of Schizochytrium limacinum OUC88 in a fermenter (working volume 7 L) were optimized using Plackett-Burman and central composite rotatable design. Out of 10 factors studied by Plackett-Burman design, four influenced the biomass production significantly. Central composite rotatable design was used to optimize the significant factors and response surface plots were generated. Using these response surface plots and point prediction, optimized values of the factors were determined as follows temperature 23℃, aeration rate 1.48 L min-1 L-1, agitation 250 rpm and inoculum cells in mid-exponential phase, the maximum yield of DCW and DHA were 24.1 g L-1 and 4.7 g L-1, respectively. These predicted values were also verified by validation experiments. The kinetic equation of bacterium cultured was: X=9.19*e0.08t/(24.46-0.37*e0.08t); The kinetics of DHA production was P = -0.25 + 0.2X
2. The rotifer and Artemia were harvested at 3, 6, 9, 12, 18 and 24 h intervals, analyzed for their fatty acid composition, and compared with the control which fed on yeast only. The highest DHA content resulted from the enrichment period of 12 h for both fed organisms, reached to 13.4% and 10.9% of the total fatty acids (TFAs) in rotifers and Artemia nauplii, respectively, and the DHA level has reduced sharply if enrichment time longer than 12 h. The pseudoalbinism rate of turbot Scophthatmus maximus juveniles which fed on enriched rotifers and Artemia nauplii has reduced significantly (40% lower than in control group), suggesting that enriched rotifers and Artemia nauplii by DHA-rich Schizochytrium limanium OUC88 may provide a practical strategy for fish juveniles in aquaculture.
3. In DHA extraction process, the optimum values of parameters of the extraction were determined by Orthogonal design experiment.
引文
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