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中国被毛孢超声强化液体发酵技术研究
Alternative TitleThe submerged fermentation of Hirsutella sinensis under ultrasonic stimulation
孙秀萍
Subtype工程硕士
Thesis Advisor王晓东
2014-05
Degree Grantor中国科学院研究生院
Degree Discipline生物工程
Keyword中国被毛孢   液体发酵   超声刺激   腺苷含量
Abstract本研究以中国被毛孢为试验菌株,优化了摇瓶发酵培养基配方和发酵条件,研究了超声对中国被毛孢发酵过程的影响,并对发酵液的抑菌活性做了分析,对菌丝体粉、和腺苷标准品对Caco-2肠癌细胞和HepG-2肝癌细胞的抑制作用进行了比较。 在单因素实验的基础上,进行了正交试验,结果表明最佳发酵培养基配方为:葡萄糖3%,蚕蛹粉2%,土豆20%,KH2PO4 0.2%,MgSO4 0.1%,水解酪蛋白0.1%,复合维生素0.05%。最佳摇瓶发酵条件:初始pH 6.5,装液量100mL/500mL,接种量为5%,温度为16 oC,摇床转速150r/min,在此条件下,得到菌丝体干重为16.53g/L,腺苷含量为1.853mg/g,与未优化前的菌丝体干重12.14g/L和腺苷含量1.261mg/g相比,菌丝体干重提高了36.2%,腺苷含量提高了46.8%。 采用优化后的培养基配方在5L发酵罐上进行深层发酵,优化之后确定最终发酵条件为:罐压为0.05MPa,接种量5%,通气量0.75vvm,装料量为4L,温度16oC,搅拌速度200r/min,与未优化前相比,菌丝体干重增长了14.10%,腺苷含量增加了16.61%。 采用Box-Behnken中心组合实验和响应面分析法,对超声刺激中国被毛孢的摇瓶发酵进行了研究,确定超声刺激中国被毛孢发酵的最佳工艺条件:超声时间44min,开始进行超声刺激的时间即超声起始作用时间为发酵进行到33h时,超声功率100w。在此条件下,腺苷含量为2.573mg/g,与未进行超声刺激时(腺苷含量为1.944mg/g)相比,腺苷含量增加32.4%。而菌丝体的干重由原来的16.57g/L增至19.83g/L,增加了19.7%。 抑菌试验表明中国被毛孢的菌丝体和发酵液中均含有抑菌物质,但是未经浓缩的发酵液中的抑菌物质的浓度比较低,观察不到抑菌性。在高度浓缩时才会表现出抑菌性。 抗癌试验结果:菌丝体粉和腺苷标准品均对Caco-2肠癌细胞和HepG-2肝癌细胞的生长具有抑制作用。对Caco-2细胞的抑制作用:菌丝体粉<腺苷标准品。对HepG-2细胞的抑制作用:菌丝体粉<腺苷标准品。两种物质对Caco-2细胞的抑制作用要强于对HepG-2细胞的抑制作用。 关键词:中国被毛孢;液体发酵;超声刺激;腺苷含量
Other AbstractIn this study, Hirsutella sinensis was used as the experimental strain. The medium components and the fermentation conditions in flask were optimized, and the effect of ultrasonic stimulation on the fermentation of Hirsutella sinensis was studied. The study also investigated the antibacterial effects of mycelia and fermentation liquid, and the antitumor effects of mycelia powder and standard sample of adenosine on Caco-2 and HepG-2. The optimal medium determined by the results of orthogonal experiment which were based on the results of signal factor experiments was as follows: glucose 3%, silkworm chrysalis powder 2%, potato 20%, KH2PO4 0.2%, MgSO4 0.1%, hydrolysis casein 0.1%, and multivitamin 0.05%. The optimized fermentation conditions in flask were as follows: initial pH 6.5, medium volume 100mL/500mL, inoculum concentration 5%, the temperature 16 oC, and shaker's rotating speed 150r/min. Under above conditions, the dry weight of mycelia was up to 16.53g/L and the adenosine content reached 1.853mg/g. Compared with the conditions without optimization, the dry weight of mycelia and adenosine content increased by 36.2% and 46.8%, respectively. The fermentation experiments in 5L fermenter were carried out. The results showed that suitable pressure of the fermenter was 0.05MPa, inoculum concentration was 5%, air flow rate was 0.75vvm, temperature was 16 oC and stirred velocity was 200r/min. The dry weight of mycelia and adenosine content increased 14.10% and 16.61% compared with the result without optimization respectively. Box-Behnken center united experiment design and response surface methodology were introduced to study the ultrasonic stimulation of the shake flask fermentation of H.sinensis. The effects of ultrasonic time, ultrasonic initial time, ultrasonic power and ultrasonic temperature on dry weight of mycelia and adenosine content were investigated. The optimal ultrasonic conditions were determined as follows: ultrasonic time 44min, ultrasonic initial time 33h, ultrasonic power 100w and ultrasonic temperature 20 oC. Under these conditions, the dry weight of mycelia was up to 19.83g/L and the adenosine content reached 2.573mg/g. Compared with the results under the condition without ultrasonic stimulation (dry weight of mycelia was 16.57g/L and the adenosine content was 1.944mg/g), the dry weight of mycelia and adenosine content increased by 19.7% and 32.4%, respectively. Antibacterial experiment showed that both of the mycelia and fermentation liquid had antibacterial effects. But the antibacterial effect of fermentation liquid with low concentration of antibacterial ingredients was not remarkable. Fermentation liquid should be highly condensed to show this property. The results of antitumor experiment showed that mycelia powder and standard sample of adenosine had antitumor effects on Caco-2 and HepG-2. For both Caco-2 and HepG-2, the antitumor effect of standard sample of adenosine was greater than mycelia powder. The antitumor effects of mycelia powder and standard sample of adenosine on Caco-2 were greater than on HepG-2. Key words:Hirsutella sinensis;Liquid Fermentation;Ultrasonic Stimulation; Adenosine Content
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/15569
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
孙秀萍. 中国被毛孢超声强化液体发酵技术研究[D]. 中国科学院研究生院,2014.
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