Research On The Antibacterial Active Substances In Grifola Frondosa

Edible and medicinal fungi are the resources of natural active substances.Grifola frondasa,is a kind of edible and medicinal fungus that is high in nutrients and has a wide range of biological activities.Although,the substances extracted from G.frondasa have been reported to have antiviral,antitumor,antioxidant,immune regulating and hypoglycemic effects,little research has been conducted on the antibacterial activity.The structure of the antibacterial active substances as well as the mechanism of inhibition are not clear.In this work,G.frondasa was used as the research object to isolate the antibacterial active substances from the fermentation supernatant of G.frondasa.Eight strains of bacteria,such as Staphylococcus aureus,was employed as indicator to assess the antibacterial activity strength of each isolated component.The structure of the antibacterial active substances was thereby indentified by high performance liquid chromatography and liquid chromatography-mass spectrometry.The antibacterial mechanism was finally investigated for the antibacterial active substances.At the same time,selenium nanoparticles were synthesized using the fermentation supernatant concentrate as a medium to study their antibacterial activity.The main findings of the study are as follows.（1）The laboratory-preserved strain of G.frondasa,was identified by comparing the sequencing results with the samples in the NCBI database.A phylogenetic tree was further constructed to confirm the preserved strain as Grifola frondasa.The changes in flavonoids,polyphenols,polysaccharides and biomass were measured to obtain the fermentation time.The extracellular polysaccharide content peaked at 0.263 g·L^-1 on 8^th day,the polyphenol and flavonoid contents reached a maximum of 0.047 and 0.039 g·L^-1,respectively,on 9^th day,while the biomass likewise peaked at 7.98 g·L^-1 on 9^th day.（2）The fermentation supernatants from different fermentation days were lyophilized and concentrated to obtain crude extract of fermentation broth.The minimum inhibitory concentration（MIC）of these fermentation broth crude extrat were compared with each other to determine the appropriate fermentation time.The fermentation broth crude extract on 10^thday of fermentation revealed substantial inhibitory activity against the eight indicator bacteria.The most prominent inhibitory activity against S.aureus and Salmonella,with MICs of 8 and32 mg·m L^-1 respectively.As a result,the fermentation time for G.frondasa was determined to be 10 days.（3）To obtain a single component of the bacteriostatic active substance I-2,the fermentation broth crude extract was separated by using a series of methods including ethanol precipitation,ethyl acetate and n-butanol extraction,dextran gel column chromatography and semi-preparative liquid chromatography.The compound was identified as 5-（hydroxymethyl）-2-furancarboxylic acid by comparing the liquid chromatography peak time,maximum absorption wavelength and mass spectral fragmentation information of component I-2 with that of the 5-hydroxymethyl-2-furancarboxylic acid standard.（4）The growth curve and time-kill curve demonstrated that 5-hydroxymethyl-2-furancarboxylic acid is a time-and concentration-dependent bacteriostatic active substance,and the inhibition mechanism of 5-hydroxymethyl-2-furancarboxylic acid was determined to inhibit or kill bacteria by disrupting the cell membrane of S.aureus via pyridine iodide staining and field emission scanning electron microscope.（5）Using the crude extract of G.frondosa fermentation broth as a stabilizer,it reacted with selenium nanoparticles（Se NPs）to successfully synthesize G.frondosa nano-selenium complexes（GF-Se NPs）.GF-Se NPs exhibited high thermal stability and can be kept stably for at 4°C in the dark for 25 days.GF-Se NPs demonstrated significant antibacterial activity against S.aureus and B.cereus,with MICs of 20 and 80μg·m L^-1,respectively.