Metabolome And Radical Scavenging Activity Comparison Between Hirsutella Huangshanensis At Different Culturing Time

At present,apart from a few species of entomogenous fungi such as Cordyceps sinensis,Cordyceps militaris,Beauveria,Metarhizium and Paecilomyces etc,have been reported on metabolites,but metabolites of the other entomogenous fungi are still unknown.Most of the literatures about entomogenous fungi still stay in sample collection,classification,distribution of survey or flora research stages.Insufficient imformation on metabolites of entomogenous fungi make us lacking comprehensive basic information during physiology and ecology studies on the fungi.It results in that many problems about fungi preservation,degradation,varation and application can not be resolved or clarified.It makes also the precious fungal resources not been reasonablly used.Hirsutella huangshanensis is the asexual form of the Cordyceps formosana.Reports show that the fungal metabolites contain anti-cancer and anti-oxidation and other bioactive components.But the metabolic discipline of the bioactive compounds in the fungus during fermentation process is still unknown.In this study,metabolic disciplines of extracellular metabolites and intracellular metabolites were studied in the fermentation process of Hirsutella Huangshanensis.It will provide theoretical basis for further development and utilization of the fermentation products,and technological improvement of liquid fermentation process of the fungus.It is the first time for studying on metabolomic variation in mycelia and fermentation broth of H.huangshanensis using metabolomic method.Results will lay a solid foundation for the rational utilization of the fungus.By comparing free radical scavenging activity of methanol extracts from mycelia of different culture time,we found that there are significant scavenging activity differences between the extracts against DPPH radicals.The mycelial extract reached the highst scavenging rate 93.45% when cultured for 40 days.From 30 days to 45 days the mycelial methanol extracts remain relative higher radical scavenging activity.Before 30 days or after 45 days the mycelial extracts have relatively lower scavenging activities.Before 30 days during the culture process,the nutrient-rich culture medium makes mycelia proliferate fastly and metabolite,and the bioactive metabolites were gradually accumulated,therefore,the DPPH radical scavenging activities were gradually increased.After 45 days culture the mycelia were gradually getting aging,and metabolism slowing down,and bioactive metabolites gradually reduced and the radical scavenging activities slowed down.The methanol extracts from the fermentation broth at different culture times showed also different scavenging activity against DPPH radicals.The methanol extract reached the highest scavenging rate 86.46% against DPPH radicals when cultured for 45 days.As a result of gradual accumulation of metabolites in the fermentation broth,the DPPH scavenging activity of the fermentation broth had a rising trend.At the last stage of fermentation the radical scavenging activity trended to stable.This trend is consistent with the basic rule of metabolites accumulation in a fermentation broth.There was significant difference of radical scavenging activity between the mycelia and the fermentation broth.On the whole,the radical scavenging activity of the mycelial extract was higher than that of the fermentation broth.At the later stage,with the mycelialaging and metabolites accumulation in the fermentation broth,the scavenging activities of the mycelia and the broth tend to be consistent.PCA study showed significant different metabolome between the mycelia and the fermentation broth at different culture time.But metabolome of the mycelia or the fermentation broth at different culture time were sometimes close.Metabolome of the mycelia cultured for 20,25,30 or 35 days can be classified as one group,while 40,45,50 or 55 days be classified as another class.Metabolome of the fermentation broth with 20,25,30 or 35 days cultivation can be classified as one group,while broth with 40,45,50 or 55 days cultivation be classified as another class.For the purpose of finding out the different metabolotes precisely we selected 20 and40 days cultured fermentation broth and mycelia as representatives of the two fermentation stages.The differential metabolites between the two stages were analysed by using OPLS-DA to remove the system differences and identify the metabolitesfrom.The identified differential metabolites were clustered by HCA method.Results of metabolomic analysis showed many metabolism up-regulated at the last stage such as metabolism of glycolysis,citrate cycle(TCA),pentose phosphate pathway,pyrimidine metabolism,cysteine and methionine metabolism,pantothenate and CoA biosynthesis,valine,leucine and isoleucine biosynthesis and arginine biosynthesis etc.Through identification of the differencial metabolites,as well as the original data analysis we found that hyphae contains more active substances such as ergosterol,threonine,stearic acid,oleic acid,PC(16: 0/0: 0),PC(16: 0/0: 0),PC(18: 1(9E)/ 0: 0)),PE(18: 1(9Z)/ 0: 0),ricinelaidic acid,botrydial,testosterone glucuronide and polyporusterone D,some of which have antibacterial,antioxidant,anti-cancer and other pharmacological activity,indicating that hyphae and these active substances has great potential in the development of related drugs.The study found also that contents of metabolites such as L-methionine,lysine,Lys Gly Gly,Gln Arg Val Arg,tryptophyl-arginine,L-histidinal,Asp Trp Gly,N-formylmethionine,L-histidinal and 2-aminophenol etc.which mainly related to amino acid metabolism were higher in the fermentation broth than in the hyphae.And,contents of these substances was slightly higher in the 40 days cultured fermentation broth than in the20 days cultured fermentation broth.The reason may also be associated with the accumulation of substances in the fermentation broth.The study found also that the fermentation broth contained slightly higher content of glutamic acid,tryptophan,tyrosine,aspartic acid,proline,isoleucine,phenylalanine,lysine,histidine and other free amino acids.And there was no common fungal toxins detected from the fermentation broth.Because containing flavorous amino acids and no toxins the fermentation broth has a greater beverage development potential.