| ObjectiveThe solid state fermentation system was established by using Monascus purpureus as fermentation strain,and Salvia miltiorrhiza as the fermentation medium.We studied on the adaptability of the fermentation system based on the growth status of Monascus purpureus and the rate of drying and consumption of fermentation medium.The content of and Tanshinone II A is used as reference index to optimize the fermentation condition.We used modern analytical technology to conform components of fermentation system transformed in the process of fermentation,separate and purify the target compounds and determine the structures of these copounds.The changes of antioxidant and antibacterial activities of fermentation medium were investigated.The antitumor activity of the isolated products was also studied.Our study provided experimental data for the research of bi-directional solid state fermentation of Monascus purpureus-Salvia Miltiorrhiza.It also provided certain theoretical basis for secondary development of Salvia miltiorrhiza,promoted the development of medical products industry about Monascus and Salvia miltiorrhiza.MethodEvaluation of adaptability and medium optimization,extraction process optimization We builded system of bi-directional solid state fermentation of Monascus purpureus-Salvia Miltiorrhiza,according to the growth state of Monascus purpureus in Salvia miltiorrhiza fermentation substrate,drying rate,consumption rate of the medium,the content changes of Monacolin K to study the adaptability of fermentation system.The influencing factors of Monacolin K and Tanshinone ?A content in fermentation products were investigated by single factor test,including the initial water content,inoculation amount,pH,culture temperature,substrate ratio,loading rate and fermentation cycle.On the basis of this,four factors,the initial water content,inoculation amount,substrate ratio and fermentation cycle,were designed to do orthogonal test.We used the content of Monacolin K and Tanshinone?A as the technological parameters to obtain the optimum fermentation conditions.We selected material liquid ratio,extraction temperature,extraction time,extraction times to investigate single factor test.The four factors and the corresponding level were used to make an orthogonal design to determine the optimum extraction of Tanshinone ?A in fermentation productExtracting target compunds and researching biological activity on fermentation product:We used TLC and HPLC to qualitatively analyze the composition of the fermentation,repeatly use of silica and gel column chromatography to separate the target product.We used potassium ferricyanide method to determine the reduction power of fermentation medium,DPPH method to determine its antioxidant activity,used the filter bacteriostatic method to evaluate antibacterial activities of the fermentation extractions on staphylococcus aureus,escherichia coli,and yeast.We used MTT method to evaluate the inhibitory effect of the isolated target compound on the proliferation of lung cancer A549 cells in vitro.ResultsWe established the Monascus purpureus-salvia miltiorrhiza bi-directional solid state fermentation system.Through the growth of Monascus purpureus and the rate of drying and consumption of fermentation medium showed that the adaptability of fermentation system was good,and it had produced content of Monacolin K in the fermentation process,proved that the fermentation system exists the possibility of material transformation.The optimum fermentation conditions of Monascus purpureus-Salvia miltiorrhiza bi-directional solid fermentation system is as fol lows:20g frmentation medium in 250mL triangle flask,the ratio of rice and Salvia miltiorrhiza in fermentation medium is 1:1,initial water content was adjusted to 120%,inoculum size 40%,pH 5,20 days culture incubator under 30?Under the best fermentation conditions,the tanshinone ?A content is 1.32 mg/g,Monacolin K content was 1.69 mg/g.The best extraction method of tanshinone ?A inMonascus purpureus-salvia miltiorrhiza bi-directional solid fermentation is as follow extraction time is 90 min,extraction temperature is 60?,and solid-liquid ratio is 1:5,extract for two times.The concentration of tanshinone ?A under the best extract conditions is 1.78 mg/gThe HPLC atlas results showed that there were significant changes of composition and content in the fermentation medium,and these changes indicated that there had material transformation between the fermentation systems.The target product was locked through analysing the extract of fermentation product by TLC.After separation,The target products were identified as 5a,8a-erythroergeno-6,22-diene-3?-ol and the other was a long chain fatty acid containing one double bond.After the transformation of Monascus purpureus,the antioxidant capacity of fermentation medium was increased.The IC50 value of the antioxidant capacity of fermentation sample was 4.39mg/mL,and he IC50 value of the negative control was 13.76mg/mL.The antibacterial activities of the ferment-ation product for StapHylococcus aureus and Escherichia coli was significant,the minimum inhibitory concentration was 125mg/mL.The anti-tumor experiments of MTT show that compound 5a,8a-epidioxyergosta-6,22-dien-3?-ol has obvious inhibitory effect to the A549 lung cancer cell.2 mg/mL 5a,8a-epidioxyergosta-6,22-dien-3?-ol,dosage of 2 ?l,4?l,6?l,the inhibition rate were 49.13%,67.88%,88.90%ConclusionThe Monascus purpureus grew well on the culture medium of rice and Salvia miltiorrhiza and can produce amount of Monacolin K during the fermentation process There has a certain degree of material transformation in the growth and metabolism of Monascus purpureusThe antioxidant activity of the fermentation medium was enhanced through the transformation of Monascus purpureus,and it had a certain antibacterial effect.The fatty acids isolated from the fermentation products had some inhibitory effects on StapHylococcus aureus and Escherichia coli,and 5a,8a-epidioxyergosta-6,22-dien-3?-ol had a significant inhibitory effect on the proliferation of lung cancer A549 cells. |
PDF Full Text Request