| Cyclocarya paliurus contains multiple biological activities, is a good health food resource. However, its development and exploitation were limited since lack of resource and breeding difficulties. In our research, with old and tender leaves from different years of Cyclocarya paliurus as explants for callus induction,cell lines with high yield triterpenic acid were got through visual method and dichotomy screening, and to establish a stable cells suspension culture system of Cyclocarya paliurus, then expanded with an agitator bioreactor. The optimization of inoculum density, agitation speed of blade and flow rate of air were based on the research of dissolved oxygen of the agitator bioreactor, the rules of nutrients consumption were explored under the optimization of cultivation conditions,which are intended to provide theoretical support for Large-scale industrial production triterpenic acid with cells suspension of Cyclocarya paliurus. The results indicated as follows:Callus were induced from the leaves of Cyclocarya paliurus and subcultured merit,rules of cell biomass and triterpenoid accumulation were investigatived.(1) There are significant differences in growth status and triterpenoid content of callus induced from different Leaf explants and high-yielding callus line was available by selection.(2)Subculture have an effect on cells biomass and triterpenoid accumulation, when subcultured around to the10 th generation, the growth of different cell lines and triterpenoid accumulation were keeping to be stable.(3) Cell line 6 with high yield triterpenic acid was found from nine callus line, and the highest content of triterpenoid acid can reach to 2.55mg/g.The dissolved oxygen performance of 5L stirred bioreactor was researched. The influence of mass transfer coefficient KLa with flow rate of air, agitation speed of blade,temperature, blade combination and installation position was investigated. The results indicated that the above five parameters have a greater impact on Kla, and there was a very significant affect with different levels of flow rate of air and agitation speed, different level of temperature and blade installation position also exist significant affect. When flow rate of air with 1000 ml/min, agitation speed of blade with 200 rpm, temperature of 35℃,installation height H =8 cm, blade in combination with 2FDI, the oxygen mass transfer coefficient KLa achieves the maximum value of 7.32, 7.20, 6.48, 6.36, 5.82 H–1,respectively.Optimization parameters to expand culture of suspension cells of cyclocarya paliurus with a stirred bioreactor. The optimum culture situations were obtained as follows: 28℃,pH 5.8±0.2, inoculum density 5%, agitation speed of blade 80 rpm, flow rate of air 800ml/min. With the condition, the fresh weight, dry weight, total triterpenoid content and total triterpenic yield achieved the hightest value were 275.16 g /L、12.99 g /L、4.64 mg /Land 60.21 mg /L, respectively.The rules of nutrients consumption was investigated, when cells was cultured in stirred bioreactor.(1) Total sugar content was declined steadily in the whole culture, 4.6g/L was detected at the end of cultivation;(2) At the early stage of, sucrose concentration was decreased rapidly in 0-4 day, only 2.72 g/L was detected on day of 8;(3)The content of reducing sugar increased clearly in 0-4 day, reached the highest value of 37.64 g/L, then decreased and almost used up at the end of cultivation;(4) Nitrate concentration was sharply decreased on days of 8-16,but 15.84 mmol/L(which was 40.41% of the intitial concentration) of nitrate was detected at the end of culture cycle;(5) The concentrations of ammonium and phosphate was stably decreased in the whole time and almost used up after16 days of cultivation. |
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