深红红螺菌1.5005菌株高产辅酶Q_（10）发酵工艺优化及诱变育种的研究
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摘要
辅酶Q10是脂溶性多烯醌类化合物,与维生素类有共同特征,以极低的含量广泛存在于许多亚细胞结构中。作为植物的光合作用和动物呼吸链中的递氢体,在生物体中具有重要的生理功能。辅酶Q10是细胞自身合成的天然抗氧化剂、细胞代谢激活剂,并能提高机体免疫力,增强抗体的产生,改善T细胞的功能,能抑制线粒体的过氧化,保护生物膜结构的完整性,在医药、保健品、食品添加剂、化妆品等领域都有广泛的用途。本文以深红红螺菌1.5005 (Rhodospirillum rubrum1.5005)为出发菌株,对高产辅酶Q10的发酵条件以及提取工艺,诱变育种进行了优化,大幅度提高了该菌辅酶Q10的发酵单位,主要研究结果有以下几方面：
1、通过对深红红螺菌1.5005种子培养基的优化,得出最佳种子培养基：麦芽糖10g/L、葡萄糖20g/L、尿素0.3g/L、酵母膏6g/L、NaHCO3 20g/L、MgSO4·7H2O1g/L、NaCl 5g/L。深红红螺菌1.5005的生长过程符合典型的微生物生长曲线,从32h到44h为对数期,菌体生长最旺盛,这时候可以作为生产菌种接入到发酵培养基中。
2、通过发酵工艺的优化,得出深红红螺菌1.5005最佳发酵条件为：接种量10%,培养温度32℃,初始pH7.0,250ml锥形瓶中的装液量100ml,发酵时间72h,光照静置发酵。优化后辅酶Q10发酵单位达到了12.969mg/L,比优化前提高了42.61%。
3、对深红红螺菌1.5005辅酶Q10的提取纯化工艺进行了各方面的优化,最佳破壁方法为碱辅助冻融破壁法,最适提取条件为：1M的氢氧化钠溶液常温处理20 min,沸水浴4 min,然后放入-36℃的冰箱中冷冻4h,冷冻结束后,迅速放到沸水浴中融化,皂化温度80℃,皂化时间60min,蒸馏温度80℃,优化后辅酶Q10的得率较优化前提高了52.71%。
4、研究了紫外线、亚硝基胍单因素对深红红螺菌1.5005的诱变效应,结果表明：在功率为15W的紫外灯下,距离30cm处进行照射130S时,致死率为95.9%；以浓度为0.2 mg/ml的NTP诱变处理40min时,致死率为96.94%。
5、深红红螺菌1.5005经过紫外—亚硝基胍复合诱变后,通过发酵实验,最终选育得到了一株遗传稳定性良好的辅酶Q10高产菌株UV6NTP4,其发酵产量达到了33.344mg/L,是出发菌株的1.67倍。经连续传代5次后,并进行发酵实验,结果证实菌株UV6NTP4产辅酶Q10的量具备良好的遗传稳定性。且辅酶Q10高产菌株UV6NTP4比出发菌株提前4h进入对数生长期,缩短了发酵周期,提高了辅酶Q10的生产效率。
Coenzyme Q10 is lipid soluble compound of quinone homolog,and have common characteristics with vitamins, generally exists in many subcellular structures with microamount. it plays important physiological functions as the delivery of hydrogen in the photosynthesis of plants and the respiratory chain of animals. Coenzyme Q10 have a general usage in medicine, healthcare products, food additives, cosmetics and other fields,it is the natural antioxidant that cell's own synthesis in vivo, activating agent of metabolism,it can improve the immunity of organism, improve the functions of T cells, inhibit the peroxidation of mitochondria, protect the integrity of the biofilm structure. As an original strain in this research,the mutagenic breeding for the high productivity Coenzyme Q10 strain with Rhodospirillum rubrum 1.5005 was studied in this paper, and optimized the fermentation conditions and the extract technology in every aspects,made a great progress in increasing the fermentation unit of Coenzyme Q10.The main research results to list in the follow:
1、Through the optimization of the seed culture medium,the suitable seed culture medium of Rhodospirillum rubrum 1.5005 were:maltose lOg/L, glucose 20g/L, urea 0.3g/L, yeast extract 6g/L, NaHCO3 20g/L, MgSO4·7H2O 1g/L, NaCl 5g/ L. Rhodospirillum rubrum 1.5005 has the typical growth process of the microbial growth curve, cell growth on the number of times from 32h to 44h in the most vigorous, as producing bacteria access to the culture medium in this time.
2、Through the optimization of the fermentation conditions,the suitable fermentationancan conditions of Rhodospirillum rubrum 1.5005 were::inoculum size 10%, temperature 32℃, initial pH7.0, the volume of medium was 100ml in 250ml flask volume, the best fermentation time was 72h, fermentation with lighting and stand. The Coenzyme Q10 production was higher than before,the Coenzyme Q10 fermentation unit reached 12.969mg/L,increased 42.61% than before.
3、Extraction methods were investigated in various aspects, the best method for cell wall disrupting method is the base broken assisted freezing and thawing method, the optimum extraction conditions was that:1M sodium hydroxide solution processing at room temperature 20min, boiling water bath 4 min, then placed in-36℃freezer refrigerator in 4h, the rapid melting into a boiling water bath after frozen, saponification temperature:80℃, saponification time:60min, distillation temperature: 80℃, the yield of Coenzyme Q10 more than before optimization increased 52.71%.
4、The mutagenic effect of Rhodospirillum rubrum 1.5005 was studied by using single factor UV-radiation and NTP. It is found that exposured with 15W UV lamp in the distance of 30cm for 130s,the lethality rate was 95.9%; dealed with 0.2mg/ml volume of concentration NTP for 40min, the lethality rate was 96.94%.
5、Through the UV-DES composite mutagenesis and fermentation with Rhodospirillum rubrum 1.5005, a high productivity coenzyme Q10 UV6NTP4 with good genetic stability was gained, and the coenzyme Q10 yield of the strain UV6NTP4 reached 33.344mg/L ultimatly, which was 1.67 times higher than the original strain. After continuous passage 5 times and fermentation experiments, the results confirm that the strain UV6NTP4 has good genetic stability. The high productivity coenzyme Q10 UV6NTP4 got into the logarithmic growth phase 4h earlier than the original strain, shortened the fermentation period, increased the production efficiency of Coenzyme Q10.
1、通过对深红红螺菌1.5005种子培养基的优化,得出最佳种子培养基：麦芽糖10g/L、葡萄糖20g/L、尿素0.3g/L、酵母膏6g/L、NaHCO3 20g/L、MgSO4·7H2O1g/L、NaCl 5g/L。深红红螺菌1.5005的生长过程符合典型的微生物生长曲线,从32h到44h为对数期,菌体生长最旺盛,这时候可以作为生产菌种接入到发酵培养基中。
2、通过发酵工艺的优化,得出深红红螺菌1.5005最佳发酵条件为：接种量10%,培养温度32℃,初始pH7.0,250ml锥形瓶中的装液量100ml,发酵时间72h,光照静置发酵。优化后辅酶Q10发酵单位达到了12.969mg/L,比优化前提高了42.61%。
3、对深红红螺菌1.5005辅酶Q10的提取纯化工艺进行了各方面的优化,最佳破壁方法为碱辅助冻融破壁法,最适提取条件为：1M的氢氧化钠溶液常温处理20 min,沸水浴4 min,然后放入-36℃的冰箱中冷冻4h,冷冻结束后,迅速放到沸水浴中融化,皂化温度80℃,皂化时间60min,蒸馏温度80℃,优化后辅酶Q10的得率较优化前提高了52.71%。
4、研究了紫外线、亚硝基胍单因素对深红红螺菌1.5005的诱变效应,结果表明：在功率为15W的紫外灯下,距离30cm处进行照射130S时,致死率为95.9%；以浓度为0.2 mg/ml的NTP诱变处理40min时,致死率为96.94%。
5、深红红螺菌1.5005经过紫外—亚硝基胍复合诱变后,通过发酵实验,最终选育得到了一株遗传稳定性良好的辅酶Q10高产菌株UV6NTP4,其发酵产量达到了33.344mg/L,是出发菌株的1.67倍。经连续传代5次后,并进行发酵实验,结果证实菌株UV6NTP4产辅酶Q10的量具备良好的遗传稳定性。且辅酶Q10高产菌株UV6NTP4比出发菌株提前4h进入对数生长期,缩短了发酵周期,提高了辅酶Q10的生产效率。
Coenzyme Q10 is lipid soluble compound of quinone homolog,and have common characteristics with vitamins, generally exists in many subcellular structures with microamount. it plays important physiological functions as the delivery of hydrogen in the photosynthesis of plants and the respiratory chain of animals. Coenzyme Q10 have a general usage in medicine, healthcare products, food additives, cosmetics and other fields,it is the natural antioxidant that cell's own synthesis in vivo, activating agent of metabolism,it can improve the immunity of organism, improve the functions of T cells, inhibit the peroxidation of mitochondria, protect the integrity of the biofilm structure. As an original strain in this research,the mutagenic breeding for the high productivity Coenzyme Q10 strain with Rhodospirillum rubrum 1.5005 was studied in this paper, and optimized the fermentation conditions and the extract technology in every aspects,made a great progress in increasing the fermentation unit of Coenzyme Q10.The main research results to list in the follow:
1、Through the optimization of the seed culture medium,the suitable seed culture medium of Rhodospirillum rubrum 1.5005 were:maltose lOg/L, glucose 20g/L, urea 0.3g/L, yeast extract 6g/L, NaHCO3 20g/L, MgSO4·7H2O 1g/L, NaCl 5g/ L. Rhodospirillum rubrum 1.5005 has the typical growth process of the microbial growth curve, cell growth on the number of times from 32h to 44h in the most vigorous, as producing bacteria access to the culture medium in this time.
2、Through the optimization of the fermentation conditions,the suitable fermentationancan conditions of Rhodospirillum rubrum 1.5005 were::inoculum size 10%, temperature 32℃, initial pH7.0, the volume of medium was 100ml in 250ml flask volume, the best fermentation time was 72h, fermentation with lighting and stand. The Coenzyme Q10 production was higher than before,the Coenzyme Q10 fermentation unit reached 12.969mg/L,increased 42.61% than before.
3、Extraction methods were investigated in various aspects, the best method for cell wall disrupting method is the base broken assisted freezing and thawing method, the optimum extraction conditions was that:1M sodium hydroxide solution processing at room temperature 20min, boiling water bath 4 min, then placed in-36℃freezer refrigerator in 4h, the rapid melting into a boiling water bath after frozen, saponification temperature:80℃, saponification time:60min, distillation temperature: 80℃, the yield of Coenzyme Q10 more than before optimization increased 52.71%.
4、The mutagenic effect of Rhodospirillum rubrum 1.5005 was studied by using single factor UV-radiation and NTP. It is found that exposured with 15W UV lamp in the distance of 30cm for 130s,the lethality rate was 95.9%; dealed with 0.2mg/ml volume of concentration NTP for 40min, the lethality rate was 96.94%.
5、Through the UV-DES composite mutagenesis and fermentation with Rhodospirillum rubrum 1.5005, a high productivity coenzyme Q10 UV6NTP4 with good genetic stability was gained, and the coenzyme Q10 yield of the strain UV6NTP4 reached 33.344mg/L ultimatly, which was 1.67 times higher than the original strain. After continuous passage 5 times and fermentation experiments, the results confirm that the strain UV6NTP4 has good genetic stability. The high productivity coenzyme Q10 UV6NTP4 got into the logarithmic growth phase 4h earlier than the original strain, shortened the fermentation period, increased the production efficiency of Coenzyme Q10.
引文
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