Strategies To Promote The Production Of Total Triterpenoids In Submerged Fermentation Of Sanghuangporus Baumii And Its Mechanistic Study

Sanghuangporus species is a rare and precious medicinal and edible fungus.It contains many types of bioactive compounds such as polysaccharides,triterpenoids,phenols,etc.Triterpenoids,the major bioactive constituents of Sanghuangporus spp.are currently receiving a lot of attention from the scientific community due to their potent pharmacological activities.However,very few reports have been conducted about the possibility of adjusting the fermentation conditions to increase the triterpenoids yield from certain fungi such as Sanghuangporus baumii.In this study,we focused on the production of triterpenoids by submerged fermentation,proposed efficient fermentation protocols and carried out the corresponding mechanism explorations.Firstly,the fermentation conditions of various types and concentrations of carbon and nitrogen sources,initial pH,inoculum amount and time course were screened by single-factor experiments.Under the suitable culture condition(60 g·L^-1 glucose,10 g·L^-1 corn steep powder,initial pH 4,15%inoculum size on the fourth day),the maximum production of triterpenoids reached 236.8 mg·L^-1on day 8 of the fermentation,which was 3.0 times as high as that of control(78.1 mg·L^-1).In order to explore effective natural exogenous factors,high(0.2 g·L^-1)and low(0.04 g·L^-1)concentrations of four different mulberry shavings extracts（water,petroleum ether,ethyl acetate,methanol）were added to stimulate the production of triterpenoids by submerged fermentation of S.baumii.Results showed that 0.2 g·L^-1 ethyl acetate and methanol extract can significantly increase the yield of triterpenoids,which resulted in 24.76%and 16.8%higher yield respectively than that of the DMSO group.The following GC-MS analysis identified 74 and 42 substances from those two effective extracts,and also revealed the presence of oleamide as a key component responsible for promoting the production of triterpenoids.2 g·L^-1 oleamide could promote the production of triterpenoids to 324.7 mg·L^-1,which was 1.4-fold higher than that of the control.The role of fatty acids in enhancing the triterpenoids synthesis from S.baumii was further investigated because oleamide belongs to a typical fatty acid derivative.Results showed that unsaturated fatty acids can significantly promote the production of triterpenoids,also,a direct correlation was indicated between the degree of unsaturation of fatty acid and the triterpenoids yield.The best stimulatory additive was linolenic acid.It increased the triterpenoids yield to 573.3mg·L^-1,which was 2.5 times higher than the control.The detailed underlying mechanisms between linolenic acid and triterpenoids production were explored from the effects on nutrients supply,mycelial morphology,cell membrane and triterpenoids biosynthetic pathway.Firstly,the consumption of linolenic acid was monitored by HPLC,in which showed that linolenic acid was consumed rapidly at the early stage after addition and could be utilized as nutrient for mycelial growth and triterpenoids production.Secondly,SEM observation and MDA content determination were used to monitor the changes of mecylial morphology and its peroxidation degree.Results showed that the addition of linolenic acid could significantly enhance the degree of mycelial wrinkles and lipid peroxidation.In addition,GC-MS analysis and the release of cell constituents were conducted to compare the changes of cell membrane components and cell integrity.Results showed that the addition of linolenic acid could increase the unsaturated/saturated fatty acid ratio significantly and destroy the cell integrity partially,resulting in a better membrane permeability and fluidity.Finally,the measurement of key enzyme amounts and the expression levels of genes related to triterpenoids biosynthetic pathway were monitored at the molecular level.Results showed that the addition of linolenic acid promoted all these genes expression levels and enzyme amounts significantly at their particular period,which also indicated that gene expressions and enzyme amounts both showed temporal specificity.All of these mechanism exploratory experiments were conducted to provide more supports for the relationship between linolenic acid addition and triterpenoids productivity,in which laid a theoretical foundation for further investigating on the underlying mechanism of triterpenoids synthesis.