蛹虫草腺苷深层发酵优化及其中药活性研究
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摘要
本论文是在对蛹虫草大量实验研究的基础上,确定以蛹虫草中活性成分——腺苷作为主要研究对象。本实验首先通过单因素和响应面法确定了腺苷最优的提取条件。然后,对蛹虫草培养基的碳氮源种类进行了筛选,并对筛选出的碳氮源浓度进行了单因素和响应面的优化,在培养基优化的基础上,进一步优化蛹虫草的培养条件,分别对装液量、接种量、温度、转速、初始pH进行了单因素实验,并从中选出影响较显著的三个因素进行中心组合实验优化。在摇瓶培养的基础上,进行10-L发酵罐放大实验,分别进行了分批发酵、间歇补料分批(补充碳源)发酵、间歇补料分批(补充氮源)发酵、控制pH发酵和间歇补料分批(同时补充碳、氮源和不控制pH)发酵。最后,对蛹虫草提取物抑制肿瘤细胞的中药活性进行了初步的研究。
Cordyceps militaris is a rarity wild Chinese traditional medicine which con-tain lots of medicinal value. Adenosine is one of the major bioactive compounds in Cordyceps militaris. There are a great deal of physiological and pharmacol-ogical activities. Because the cost of adenosine production is very expensive, most of it depended on importment which used large amounts of foreign ex-change. On account of lacking of wild resources and hard to cultivation, the submerged cultivation will have obvious technological superiority in produce bioactive compounds from Cordyceps militaris. Now the most of the reports are mainly focused on, the optimization of cordycepin and biomass, however, the research of the optimization of adenosine submerged fermentation is much rare. The content of this paper is that improve the content of adenosine throμgh opti-mizing the extraction, medium composition and culture conditions. On the basis of the front results, we processed fermenter experiment and pharmacological activity of Chinese medicine.
The extraction of adenosine from Cordyceps militaris was optimized by the single factor tests and central composite design. The optimum conditions for ex-traction process were as follows: extracting temperature was 44℃, extraction time was 2.3h, the ratio of sample to water was 1:38; the optimized extraction conditions produced theoretically extraction rate of 1.42mg/g and in practice 1.44mg/g. The good agreement between the predicted and experimental results verified the validity of the model and existence of an optimal point.
The sort of carbon and nitrogen sources was optimized. Considering the im-pact of pH, dry weight, residual sμgar, adenosine, the results showed that the best fermentation media is maltose and yeast powder. At the same time, the best fermentation time four-day cultivation. The best concentration of carbon and nitrogen sources is maltose 20g/L yeast powder 45g/L.On the basis of the front results ,the result of central composite design is : maltose 20.3g/L yeast powder 51g/L, the result of the optimized conditions produced theoretically content of adenosine 53.27mg/L and in practice 53.92mg/L. the content of adenosine in optimized medium is four times higher than the original PDA integrated media.
The fermentation conditions of adenosine from Cordyceps militaris were op-timized by the single factor tests and central composite design. The optimum conditions for fermentation process were as follows: medium capacity 80mL/250mL, inoculum density 4%, initial pH = 6, temperature, agitation speed 120r/min. Then we optimized the three more significant factors which were me-dium capacity, temperature, initial pH by central composite design. The result of central composite design is temperature 23.9℃, pH5.45, medium capacity 82.4mL. The result of the optimized conditions produced theoretically content of adenosine 62.01mg/L and in practice 62.74 mg/L. According to the results of previous optimization, the kinetics curve of optimized medium and original me-dium was detected for 10 days. The content of adenosine and dry weight in op-timized medium is higher than original medium. The maximum were 63.21mg/L and 12.73g/L respectively.
According to the results of shake flasks,the scale-up experiment w ere done by the10-L fermentor. First, batch fermentation was done to detect the parame-ters as follow: pH, viscosity, dry weight, residual sμgar concentration, residual protein concentration, content of adenosine. The tests results of batch fermenta-tion, intermission fed-batch (carbon) fermentation, intermission fed-batch (ni-trogen) fermentation and control pH fermentation were compare: 1. Feeding carbon is more conducive to the dry weight. When the culture time is 4 days, the maximum dry weight is 9.82g/L. At the same time, the decreasing rate of adeno-sine was significantly reduced. 2. Feeding nitrogen is more conducive to adeno-sine. When the culture time is 4 days, the maximum content of adenosine and dry weight were 64.36mg/L and 9.41g/L, respectively. At the same time, the de-creasing rate of adenosine was significantly reduced.3. Controlling the pH was not conducive to content of adenosine and dry weight. On the basis of the front results, intermission fed-batch (carbon, nitrogen and non- control pH) fermenta-tion was done. When the culture time is 4 days, the maximum content of adeno-sine and dry weight were 78.59mg/L and 12.42g/L respectively which were bet-ter than the batch fermenter.
The pharmacological activity of Cordyceps militaris was preliminarily tested. MTT assay was used to test the exo-extracts and mycelium extracts against two cancer cell lines, MCF-7 breast cancer and B16 mouse melanoma in different concentrations and different time. The solvent exo-extracts showed much higher inhibition on the proliferation of B16 mouse melanoma than mycelium extracts. The inhibition rate against B16 mouse melanoma could be up to 90% in the concentration of 10mg/mL and in 72h of treatment. Mycelium extracts had a more significant inhibition effect on MCF-7 breast cancer than exo-extracts. The inhibition rate against MCF-7 breast cancer could reach the highest value of about 90% in the concentration of 8mg/mL and in 24h of treatment. As can be seen from the comparison that exo-extracts and mycelium extracts had different effects on different cancer cell lines .
Cordyceps militaris is a rarity wild Chinese traditional medicine which con-tain lots of medicinal value. Adenosine is one of the major bioactive compounds in Cordyceps militaris. There are a great deal of physiological and pharmacol-ogical activities. Because the cost of adenosine production is very expensive, most of it depended on importment which used large amounts of foreign ex-change. On account of lacking of wild resources and hard to cultivation, the submerged cultivation will have obvious technological superiority in produce bioactive compounds from Cordyceps militaris. Now the most of the reports are mainly focused on, the optimization of cordycepin and biomass, however, the research of the optimization of adenosine submerged fermentation is much rare. The content of this paper is that improve the content of adenosine throμgh opti-mizing the extraction, medium composition and culture conditions. On the basis of the front results, we processed fermenter experiment and pharmacological activity of Chinese medicine.
The extraction of adenosine from Cordyceps militaris was optimized by the single factor tests and central composite design. The optimum conditions for ex-traction process were as follows: extracting temperature was 44℃, extraction time was 2.3h, the ratio of sample to water was 1:38; the optimized extraction conditions produced theoretically extraction rate of 1.42mg/g and in practice 1.44mg/g. The good agreement between the predicted and experimental results verified the validity of the model and existence of an optimal point.
The sort of carbon and nitrogen sources was optimized. Considering the im-pact of pH, dry weight, residual sμgar, adenosine, the results showed that the best fermentation media is maltose and yeast powder. At the same time, the best fermentation time four-day cultivation. The best concentration of carbon and nitrogen sources is maltose 20g/L yeast powder 45g/L.On the basis of the front results ,the result of central composite design is : maltose 20.3g/L yeast powder 51g/L, the result of the optimized conditions produced theoretically content of adenosine 53.27mg/L and in practice 53.92mg/L. the content of adenosine in optimized medium is four times higher than the original PDA integrated media.
The fermentation conditions of adenosine from Cordyceps militaris were op-timized by the single factor tests and central composite design. The optimum conditions for fermentation process were as follows: medium capacity 80mL/250mL, inoculum density 4%, initial pH = 6, temperature, agitation speed 120r/min. Then we optimized the three more significant factors which were me-dium capacity, temperature, initial pH by central composite design. The result of central composite design is temperature 23.9℃, pH5.45, medium capacity 82.4mL. The result of the optimized conditions produced theoretically content of adenosine 62.01mg/L and in practice 62.74 mg/L. According to the results of previous optimization, the kinetics curve of optimized medium and original me-dium was detected for 10 days. The content of adenosine and dry weight in op-timized medium is higher than original medium. The maximum were 63.21mg/L and 12.73g/L respectively.
According to the results of shake flasks,the scale-up experiment w ere done by the10-L fermentor. First, batch fermentation was done to detect the parame-ters as follow: pH, viscosity, dry weight, residual sμgar concentration, residual protein concentration, content of adenosine. The tests results of batch fermenta-tion, intermission fed-batch (carbon) fermentation, intermission fed-batch (ni-trogen) fermentation and control pH fermentation were compare: 1. Feeding carbon is more conducive to the dry weight. When the culture time is 4 days, the maximum dry weight is 9.82g/L. At the same time, the decreasing rate of adeno-sine was significantly reduced. 2. Feeding nitrogen is more conducive to adeno-sine. When the culture time is 4 days, the maximum content of adenosine and dry weight were 64.36mg/L and 9.41g/L, respectively. At the same time, the de-creasing rate of adenosine was significantly reduced.3. Controlling the pH was not conducive to content of adenosine and dry weight. On the basis of the front results, intermission fed-batch (carbon, nitrogen and non- control pH) fermenta-tion was done. When the culture time is 4 days, the maximum content of adeno-sine and dry weight were 78.59mg/L and 12.42g/L respectively which were bet-ter than the batch fermenter.
The pharmacological activity of Cordyceps militaris was preliminarily tested. MTT assay was used to test the exo-extracts and mycelium extracts against two cancer cell lines, MCF-7 breast cancer and B16 mouse melanoma in different concentrations and different time. The solvent exo-extracts showed much higher inhibition on the proliferation of B16 mouse melanoma than mycelium extracts. The inhibition rate against B16 mouse melanoma could be up to 90% in the concentration of 10mg/mL and in 72h of treatment. Mycelium extracts had a more significant inhibition effect on MCF-7 breast cancer than exo-extracts. The inhibition rate against MCF-7 breast cancer could reach the highest value of about 90% in the concentration of 8mg/mL and in 24h of treatment. As can be seen from the comparison that exo-extracts and mycelium extracts had different effects on different cancer cell lines .
引文
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