| Higher fungi are one of the important sources of antibiotics and pesticide leading compounds.The compounds contained in it have novel structure and remarkable activity.Pholiota nameko belongs to basidiomycetes,laminaria,umbrellae,bulimidae,cycadus,also known as Pholiota nameko.Mushroom has many advantages,such as wide material source and strain can be isolated and cultured.In this paper,the study object of this paper is Pholiota nameko.The literature reports that the extract,polysaccharide and protein of Pleurotus ostreatus have many pharmacological effects on enhancing immunity,anti-oxidation,anti-inflammatory and anti-tumor.Through the previous studies of other members of this group,it was found that the mushroom belongs to sensitive fungi,that is,changing its fermentation conditions can induce it to produce different secondary metabolites,and the fermentation liquid of Pleurotus ostreatus contains different chemical structure types and the active chemical components are the same.In order to fully explore the potential of secondary metabolites and explore the mechanism of secondary metabolites,we have made deep exploration of secondary metabolites from chemical composition,metabology and transcriptomics,in order to obtain a large number of secondary metabolites with new structure and significant activity.(1)The chemical components of ethyl acetate extract of Pleurotus ostreatus were purified and identified by various means of separation and purification:(1)Fermentation conditions: the sterilization water per liter contains 200 g of peeled potatoes,20 g glucose,1.5g of Mg SO4,KH2PO4 3G,vb110 mg,peptone 1.0g,and p H adjusted to 6.0-6.5 with citric acid.The culture conditions: 24 ℃ constant temperature,rotating speed 150 r/min,dark culture fermentation 25 days.A new compound was identified from the ethyl acetate layer extract 27 g.A new compound was identified from the extract: 1-hydroxy-6-bromo-2,2,5,7-tetramethyllindan.(2)Fermentation conditions: rice and water(1:1.4),culture conditions: room temperature,45 days.The ethyl acetate layer extract of the fermentation liquid of Pleurotus ostreatus was 62.1g.Five compounds were isolated and identified.They were:(22e,24R)-ergosta-7,22-dien-3 β-ol(1)、(22E,24R)-5 α,eight α-Epidioxyergosta-6,22-dien-3 β-ol(2)、 β-Sitosterin(3)、(22E,24R)-ergosta-4,6,8(14),22-tetra en-3-one(4)、22E,24R)-Ergosta-4,6,8(14),22-tetraen-3-one(5).(3)Fermentation conditions: glucose 5.00%,yeast powder 0.50%,peptone 0.15%,Mg SO4 and kh2po40.05% were added in each litre of ultra pure water.The culture conditions were 24 ℃,150 R / min,and the incubation conditions were 25-30 days in the dark bed.Four known compounds were identified from 36 g ethyl acetate extract,which were donacinol B(6),donacinol C(7),trichapargins a(8),trichapargins B(9).(4)The sensitivity and hypoglycemic activity of insulin were studied in the fermentation liquid of Pleurotus ostreatus.The results showed that compound 8 had weak activity after 24 hours of single action at 10 um concentration;Compound 7,compound 6 and compound 9 showed no activity after 24 hours of single action at10 um concentration.(2)Because of the limitations of the laboratory culture conditions and the purification methods,it is difficult for us to get the compounds with little content but novel chemical structure from the fermentation liquid of Pleurotus ostreatus.LC combined with ms/ms with high selectivity and high sensitivity can analyze complex samples in real time.The ultra high performance liquid chromatography tandem mass spectrometry(UPLC MS / MS)can be used to determine the quality and quantity of complex samples.Metabolomics analysis is mainly aimed at detecting and screening metabolites with significant biological significance and statistical differences from biological samples,and then expounding the metabolic process and mechanism of organism.The secondary metabolites produced by fermentation broth of Pleurotus ostreatus under different culture conditions were analyzed qualitatively and quantitatively by lc-ms/ms.The results showed that seven types of secondary metabolites were detected from the mixed samples,342 metabolites in total.Among them,the types of interest are terpenoids(9),coumarins(15),and other classes(64).The samples of each group were compared with each other,and the different metabolites were screened out.The number of different metabolites selected among them was 231,211,230,199,133 and211 respectively.The results showed that the content of metabolites in potato medium 3 was the most abundant and the content of GP medium was the worst.The metabolic pathway of the two metabolites was analyzed.Through KEGG metabolic pathway analysis,it is found that the enrichment of differential metabolites is used in 63 pathways,of which there are two pathways related to our research: metabolism pathway and secondary metabolite biosynthesis,and the number of differential metabolites enriched is the most,respectively 80 and 35.(3)The second metabolites of mycelium of mushroom fermentation broth were studied by transcriptional sequencing,and the mechanism of different secondary metabolites was explored under different fermentation conditions,which provided a theoretical basis for further development and utilization of Pleurotus.The results showed that 15480 genes were compared with the public database by blast software,and 10900 Uni Gene were annotated.The difference genes among the groups were screened.The results showed that the number of differential genes screened out by GP medium vs rice medium was 2450,and the minimum number of differential genes screened by GP medium vs potato medium was 1020.The results of the enrichment analysis of the different gene go can be used to identify the differential expression genes in the cell components,biological processes and molecular functions of mycelium of mushroom fermentation broth under different culture conditions.Finally,KEGG enrichment analysis was carried out for differential expression genes,and the significant enrichment pathway of differential expression genes was determined.Meanwhile,the pathway related to secondary metabolites of Pleurotus ostreatus was found,and the results were displayed in the form of bubble chart,The gene number and gene name of the pathway related to secondary metabolites were found in KEGG database. |
PDF Full Text Request