| The fungal infection caused by Candida albicans in clinical practice gradually worsens,and it is also the main source of infection in Candida infection,which poses a certain threat to human health.Wild edible and medicinal mushrooms are a valuable library of natural active substances,with rich nutrients,in addition to anti-tumor,antioxidant,hypoglycemic,antiviral,bacteriostatic and other functional active substances,but because most wild edible and medicinal mushrooms are difficult to achieve artificial cultivation and fruiting bodies are not easy to obtain,most resources have not been exploited.In the early research of large-scale preliminary screening of antibacterial activity of wild edible and medicinal bacteria,our laboratory found that Microporus vernicipes has a significant bacteriostatic effect on C.albicans,However,the active ingredient and mechanism of action are not clarified..In this study,the fermentation products of wild edible and medicinal mushroom M.vernicipes were obtained by two different fermentation methods: solid fermentation and liquid fermentation,and the extracts of the two fermentation products were evaluated in vitro to inhibit the activity of C.albicans.After that,the combination of chemistry and biology was used to track and separate the activity of the solid fermentation extract by liquid-liquid extraction and column chromatography,and the active components of the solid fermentation extract of M.vernicipes were preliminarily determined,and the chemical components of the active components were quickly identified by UHPLC-HR-MS technology to clearly inhibit the main active substances of C.albicans,and the effects of Mv5 components on the growth and reproduction of C.albicans were observed in combination with biology.The formation of early biofilms,the destruction of mature biofilms,the integrity of cell membranes,and transcriptomics techniques were used to explore the mechanism of action of active components to inhibit C.albicans.Results: 1.Solid fermentation and liquid fermentation of M.vernicipes were extracted by ethyl acetate.The results of antibacterial test showed that the diameter of the inhibition circle of solid fermentation crude extract at a concentration of 1mg/m L was 19.34±0.37 mm,and the lowest inhibitory concentration was 62.5μg/m L,and the liquid fermentation extract had no obvious antibacterial effect,which determined that solid fermentation was the optimal fermentation method.2.It was found that Mv5 in the column chromatography separation component had significantly inhibited the activity of C.albicans,and 23 compounds such as fatty acids and their derivatives,alkaloids,phenols and others were analyzed by UHPLC-HRMS,among which the main components were fatty acids.3.The mechanism of action study showed that the MIC of Mv5 components against C.albicans was 15.63 μg/m L,and the MFC of Mv5 components against C.albicans was 31.25 μg/m L;MV5 components can inhibit the formation of early biofilms,destroy the structure of mature biofilms,and have a destructive effect on C.albicans cell membranes.It can inhibit the bud tube growth of C.albicans,there by inhibiting the transformation of yeast morphology to hyphae.4.Transcriptomic analysis of C.albicans after the action of Mv5 components showed that a total of 193 differentially expressed genes were detected in the MIC concentration group,156 differentially up-regulated expression genes and 37 differentially down-regulated expression genes.A total of 391 differentially expressed genes were detected in the MFC concentration group,334 differentially up-regulated expression genes and 57 differentially down-regulated expression genes.Among these differentially expressed genes,hyphae-forming genes and biofilm formrelated genes were significantly downregulated.GO enrichment analysis showed that monosomal processes,metabolic processes,and cellular processes were the biological processes with the most gene enrichment.KEGG enrichment analysis showed that the differential genes of C.albicans after the action of Mv5 components were mainly enriched in metabolic pathways such as meiosis-yeast and amino acid metabolism.q RT-PCR results showed that the expression of TEC1,HWP1,ALS3 and SRR1 genes was significantly downregulated,CAALFM_C206890CA,HSP30,LDG3 and MRV8 gene expression were significantly upregulated compared with the blank control group in C.albicans after the action of Mv5 fraction.The research in this topic revealed for M.vernicipes has the effect of inhibiting C.albicans,solid fermentation is the optimal fermentation method for obtaining the active substance,the active ingredients and the mechanism of action are preliminarily analyzed,and the antibacterial mechanism of Mv5 components against C.albicans at multiple targets is revealed through transcriptomics,which lays a foundation for further research on the mechanism of bacteriostatic activity. |
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