刺孢小克银汉霉γ-亚麻酸发酵新工艺研究
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摘要
多不饱和脂肪酸(PUFAs)对人体一系列生理活动的调节具有至关重要的作用,γ-亚麻酸(GLA)是PUFAs的一种,是人体必需脂肪酸之一。利用真菌在处理淀粉废水的同时获取GLA是废水资源化途径之一。本文利用刺孢小克银汉霉(Cunninghamella echinulata FR)对模拟土豆淀粉废水进行GLA发酵工艺研究,可为探索以废水作为廉价培养基,低成本获取γ亚麻酸提供研究基础及工艺。
通过对液体种培养条件的优化,获得种子液培养的最佳条件:菌种以三角瓶固体培养基活化6d,利用孢子悬液方式接入装液量为100mL(250mL摇瓶)PDY中,使孢子终浓度为108个/mL,加入6颗4-5mm的玻璃珠,在28℃、转速150r/min的条件下培养48h,最终获得了最佳菌球直径为0.5-2mm的致密菌丝球种子液。
通过摇瓶发酵条件的优化研究,明确了摇瓶发酵过程中最佳的溶氧条件为500mL摇瓶中装液量为150mL以150r/min的转速进行培养;同时利用响应面优化分别对培养基组分中C/N和补料方法进行了优化,最佳条件是:将(NH4)2SO4与黄豆粉以氮源质量比3:7混合为氮源,总氮元素添加量为0.238%,培养初始葡萄糖浓度为7.128%,在第4.2 d时补加浓度为4.67%的葡萄糖溶液进行发酵。最终发酵的生物量、油产量及GLA产量分别达到了35.6g/L、11.81g/L、10.36%,并建立了摇瓶发酵动力学模型。
在生物反应器培养过程中,对发酵过程的调控参数进行了优化。最优发酵温度及pH控制工艺为:发酵过程中的前4d保持菌株的最适生长温度28℃和最适生长pH 6.0,发酵过程中的后3d控制为最适积累油脂、合成GLA的温度18℃及最适pH 5.0;最优发酵罐转速控制工艺为:第1d和3d停止搅拌,第2d及第4d至第7d进行搅拌,搅拌转速为200 r/min,发酵期间通气量保持400L/h;最优补料工艺为:发酵第2d起每天补加葡萄糖1%,补加总浓度为5%。在最优发酵控制工艺下,生物量、产油量和GLA产量分别达到了38 g/L、13.46 g/L、10.12%。
Theγ-linolenic acid (GLA) is a kind of polyunsaturated fatty acids (PUFAs) which play an important role in the metabolism control of human physiology. In order to explore the low-cost PUFA-producing processes by Cunninghamella echinulata (labled FR), potato starch-industrial wastes (PSW) were used as a cheap nutrient source in this thesis.
It was found that the pellet diameter of FR mycelia was important to the control of liquid culture. The better condition for controlling liquid culture was shown as below. Firstly, PDA medium was used for culturing the FR for 6 days. Secondly, the cultured spores were used to inoculate 100 mL of PDY medium in 250 mL round flask with the final concentration of 10 8/mL. The contents of the flask were incubated at 28℃for 48 h on a reciprocal shaker (150 r/min) with six glass beads ( diameter 4-5 mm). Finally, we got the pellet mycelium with the diameter from 0.5mm to 2mm.
By improving the culture condition, we found that the best condition for dissolving oxygen in the medium is culturing the microorganisms in the 150 mL PSW medium of 500 mL round flask on a reciprocal shaker (150 r/min). By developing the C/N ratio and feeding methods, it was suggested that the optimal nitrogen resource were the mixture of (NH4) 2SO4 and soybean with the ratio 3:7. The total nitrogen atoms concentration and the glucose concentration were 0.238 % and 7.128 %, respectively. After 4.2 days culturing, the 4.67 % glucose was added to medium for feeding fermentation. The final biomass, oil yield and GLA content reached 35.6 g/L,11.81g/L and 10.36 %, respectively. Besides, the kinetics model had been established.
In the process of bioreactor fermentation, the parameter of different controlling methods were studied and optimized. During the first 4 days of the fermentation, the best culture temperature is 28℃and the optimized pH value is 6. During the last 3 days, the best condition for lipid accumulation and GLA synthesis is fermenting at 18℃and pH 5. The best fermenting condition for dissolving oxygen was by culturing organisms at the rotate speed of 200 r/min in the second day and the last 4 days, without shaking in the first day and the third day. During the whole process, the air feed speed kept at 400 L/h. The best feeding methods was adding 1 % glucose each day from the second day to the sixth day, and making the final feeding glucose concentration to 5 %. These methods greatly increased the biomass, oil content and the proportion of GLA in the oil, which reached 38 g/L, 13.46 g/L and 10.12 % .respectively.
通过对液体种培养条件的优化,获得种子液培养的最佳条件:菌种以三角瓶固体培养基活化6d,利用孢子悬液方式接入装液量为100mL(250mL摇瓶)PDY中,使孢子终浓度为108个/mL,加入6颗4-5mm的玻璃珠,在28℃、转速150r/min的条件下培养48h,最终获得了最佳菌球直径为0.5-2mm的致密菌丝球种子液。
通过摇瓶发酵条件的优化研究,明确了摇瓶发酵过程中最佳的溶氧条件为500mL摇瓶中装液量为150mL以150r/min的转速进行培养;同时利用响应面优化分别对培养基组分中C/N和补料方法进行了优化,最佳条件是:将(NH4)2SO4与黄豆粉以氮源质量比3:7混合为氮源,总氮元素添加量为0.238%,培养初始葡萄糖浓度为7.128%,在第4.2 d时补加浓度为4.67%的葡萄糖溶液进行发酵。最终发酵的生物量、油产量及GLA产量分别达到了35.6g/L、11.81g/L、10.36%,并建立了摇瓶发酵动力学模型。
在生物反应器培养过程中,对发酵过程的调控参数进行了优化。最优发酵温度及pH控制工艺为:发酵过程中的前4d保持菌株的最适生长温度28℃和最适生长pH 6.0,发酵过程中的后3d控制为最适积累油脂、合成GLA的温度18℃及最适pH 5.0;最优发酵罐转速控制工艺为:第1d和3d停止搅拌,第2d及第4d至第7d进行搅拌,搅拌转速为200 r/min,发酵期间通气量保持400L/h;最优补料工艺为:发酵第2d起每天补加葡萄糖1%,补加总浓度为5%。在最优发酵控制工艺下,生物量、产油量和GLA产量分别达到了38 g/L、13.46 g/L、10.12%。
Theγ-linolenic acid (GLA) is a kind of polyunsaturated fatty acids (PUFAs) which play an important role in the metabolism control of human physiology. In order to explore the low-cost PUFA-producing processes by Cunninghamella echinulata (labled FR), potato starch-industrial wastes (PSW) were used as a cheap nutrient source in this thesis.
It was found that the pellet diameter of FR mycelia was important to the control of liquid culture. The better condition for controlling liquid culture was shown as below. Firstly, PDA medium was used for culturing the FR for 6 days. Secondly, the cultured spores were used to inoculate 100 mL of PDY medium in 250 mL round flask with the final concentration of 10 8/mL. The contents of the flask were incubated at 28℃for 48 h on a reciprocal shaker (150 r/min) with six glass beads ( diameter 4-5 mm). Finally, we got the pellet mycelium with the diameter from 0.5mm to 2mm.
By improving the culture condition, we found that the best condition for dissolving oxygen in the medium is culturing the microorganisms in the 150 mL PSW medium of 500 mL round flask on a reciprocal shaker (150 r/min). By developing the C/N ratio and feeding methods, it was suggested that the optimal nitrogen resource were the mixture of (NH4) 2SO4 and soybean with the ratio 3:7. The total nitrogen atoms concentration and the glucose concentration were 0.238 % and 7.128 %, respectively. After 4.2 days culturing, the 4.67 % glucose was added to medium for feeding fermentation. The final biomass, oil yield and GLA content reached 35.6 g/L,11.81g/L and 10.36 %, respectively. Besides, the kinetics model had been established.
In the process of bioreactor fermentation, the parameter of different controlling methods were studied and optimized. During the first 4 days of the fermentation, the best culture temperature is 28℃and the optimized pH value is 6. During the last 3 days, the best condition for lipid accumulation and GLA synthesis is fermenting at 18℃and pH 5. The best fermenting condition for dissolving oxygen was by culturing organisms at the rotate speed of 200 r/min in the second day and the last 4 days, without shaking in the first day and the third day. During the whole process, the air feed speed kept at 400 L/h. The best feeding methods was adding 1 % glucose each day from the second day to the sixth day, and making the final feeding glucose concentration to 5 %. These methods greatly increased the biomass, oil content and the proportion of GLA in the oil, which reached 38 g/L, 13.46 g/L and 10.12 % .respectively.
引文
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[3]薛照辉,吴谋成.微生物油脂进展[J].山西食品工业, 2002, (2):10-11.
[4]颜治,陈晶.微生物油脂及其开发利用研究进展[J].粮食与油脂, 2003(7):13-15.
[5] Ratledge C . Microbial oils and fats: an assessment of their commercial potential [J]. Prog Ind Microbiol. 1982, (16):119-206.
[6] Sandra D. Dyal, Laziz Bouzidi. Maximizing the production ofγ-linolenic acid in ortierella ramanniana var. ramanniana as a function of pH, temperature and carbon source, nitrogen source, metal ions and oil supplementation[J]. Food Research International 2005, 38(10):815-829.
[7] Mona K. Gouda, Sanaa H. Omar. Single cell oil production by Gordon-ia sp.DG using agro-industrial wastes [J]. World J Microbiol Biotechnol, 2008, 13(24):1703-1711.
[8]薛照辉,吴谋成.微生物油脂进展[J].山西食品工艺, 2002, 12(2):10-11.
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