| Inonotus obliquus is a common medicinal fungus with a wide distribution.It can be found in the Russian Far East,Nordic Europe,Korea,Heilongjiang Province of China and other regions.Inonotus obliquus has significant effects on inhibiting the growth of tumor cells,enhancing immune function,antioxidant and antiviral.It can be used for the prevention and treatment of major diseases such as malignant tumors,diabetes mellitus and cardiovascular diseases.It is a kind of medicinal and edible fungus with great development value.Wild resources of Inonotus obliquus are very limited,and its price has gradually increased in recent years.Therefore,it is necessary to cultivate Inonotus obliquus artificially.Liquid fermentation is an effective way to produce Inonotus obliquus mycelium,which can provide sufficient raw materials for the research and development of Inonotus obliquus.How to improve the yield of active ingredients and optimize the culture technology is still a hot research topic in the process of artificial culture.At present,most of the studies focus on optimizing culture conditions and detecting active ingredients,but few reports have been reported on how several kinds of active ingredients accumulate and change during fermentation.In this study,Inonotus obliquus was studied for liquid fermentation.The active ingredients in Inonotus obliquus were analyzed by phenol sulfuric acid method and thin layer chromatography.The content of polysaccharides,triterpenoids and sterols in Inonotus obliquus during liquid fermentation was determined,and the optimum time of fermentation was determined.The germination of Inonotus obliquus was understood by comparing with fruiting bodies.The difference of active ingredients between fermentation culture and fruiting body showed that:(1)Identification of triterpenoids and steroids by TLC can quickly and accurately distinguish fruiting bodies from mycelia of Confucius obliquus and further optimize TLC development conditions.The results showed that the fruiting bodies and mycelia of Confucius obliquus could be separated well under the chromatographic conditions of dichloromethane: ethyl acetate(5:1).At the same time,the TLC results of sample extracts with 3,5 and 7 microliters of dot size were compared.When the dot size was 7 microliters,the band was clearer,so 7 microliters was taken as the best dot size.Through this experiment,the identification and comparison method of fruiting body and mycelium TLC of Inonotus obliquus was basically established.The better strain was screened as cfcc6584,which laid a solid foundation for the next experiment.(2)The effects of fermentation time on mycelia and fermentation broth,biomass and crude polysaccharide content were studied by liquid shaking flask culture.The results showed that more mycelia could be obtained by Inonotus obliquus during shaking flask fermentation for 8 days,and then the mycelia could autolysis.The content of polysaccharide in the mycelium of Inonotus obliquus after fermentation was higher than that in the fruiting body.The content of polysaccharide in the mycelium did not increase significantly on the 3rd to 5th day of fermentation,but increased significantly on the 6th day.According to the curve of biomass change,it was concluded that the polysaccharide of Inonotus obliquus was mainly produced in the logarithmic growth stage of mycelium fermentation.The highest polysaccharide yield was obtained from fermentation to the eighth day.After that,the polysaccharide content of Inonotus obliquus mycelium began to decrease,which indicated that the fermentation time needed to be controlled in order to obtain more polysaccharide production.The contents of triterpenoids and sterols in methanol extracts from mycelia of Inonotus obliquus during fermentation were compared by HPTLC,and the contents of fruiting bodies were also compared.The results showed that the fermented mycelium of Inonotus obliquus could produce the same components as the fruiting body.The main active components increased with the prolongation of fermentation time.The total amount of triterpenoids and sterols reached the highest at 9 days of fermentation,and the relative content of triterpenoids and sterols reached 77% of the fruiting body.(3)By comparing the composition of mycelium and fruiting body of Inonotus obliquus with three different color rendering methods on TLC,it can be seen that the mycelium produces antioxidant components which are not contained in fruiting body during fermentation,and this component accounts for a higher proportion in the total composition,but there is no obvious band in the color rendering of Indigo.The results of correlation analysis showed that the content of polysaccharides in Inonotus obliquus was closely related to DPPH free radical scavenging rate and total reducing power,which indicated that the reason for the increase of antioxidant activity in liquid fermentation of Inonotus obliquus was mainly due to the change of polysaccharide content in mycelium caused by fermentation.Therefore,combined with the experimental results,it is concluded that the 6th day should be chosen as the end point of fermentation when large-scale liquid culture of Inonotus obliquus is selected. |
