Study On Natural Products And Biological Activity Of Marine Fungus(Penicillium Sclerotiorum F93-1)based On OSMAC Strategy

As a special ecological environment in the transitional zone between land and sea,mangroves are characterized by high biodiversity and rich species resources.Many literature reports reported that microbial metabolites derived from mangrove have rich biological activity which are one of the important sources of novel compounds with novel structure and unique skeleton.Meanwhile,marine fungi have emerged from the research on marine natural products as a resource treasure house for new drug discovery and research due to their short growth cycle,convenient industrial-scale culture,green and sustainable advantages.One strain many compounds strategy（OSMAC）,namely"single strain multiple secondary metabolites strategy",is a method to activate the silent biosynthesis gene cluster and stimulate the metabolic potential of microorganisms as much as possible by changing the culture conditions and medium composition of the same strain,which is currently an effective means to develop active compounds with novel structures.In this study,marine fungi were selected as the research object,which isolated from mangrove sediments in Beilun estuary national nature reserve,Fangchenggang,Guangxi Province.The optimal fermentation medium was screened out by using OSMAC strategy of different medium and different salt concentrations with the growth state of the bacteria,the abundance of secondary metabolites and the antioxidant activity of the crude extract as the screening indicators.The natural products were separated and purified by modern chromatography technology,and the biological activity of the compounds was tested after their structures were confirmed by spectral analysis.The specific research results are as follows:（1）A total of 85 culturable fungi were isolated from mangrove sediment samples collected from Beilun Hekou national nature reserve,Fangchenggang,Guangxi Province.They were identified in 26 genera,23families,19 orders,10 classes,3 phyla.The Fusarium oxysporum was used as the pathogen to conduct the preliminary antibacterial screening,F93-1and F93-2 with good antibacterial activity and easy to culture were screened out,then the antibacterial activity of 6 plant pathogenic fungi as the indicating strains was re-screened by using the five-point confrontation method.The results showed strain F93-1 had a high inhibitory effect on three pathogenic bacteria of Fusarium oxysporum,Bipolaris sorokiniana and Rhizoctonia solani,while strain F93-2 had high inhibitory effect on Fusarium oxysporum and Colletotrichum musae.Based on morphological characteristics and ITS-r DNA sequence,F93-1 and F93-2 were identified as Penicillium sclerotiorum and Talaromyces allahabadensis,respectively.（2）The optimal medium for the strains F93-1 and F93-2 was screened using the OSMAC strategy of selecting five different media and adding 3different sea salt concentrations.By observing the growth state of the bacteria during the fermentation,and comparing the differences of high performance liquid chromatography（HPLC）fingerprints of secondary metabolites under different culture conditions and the free radical scavenging activity of each crude extract,the fermentation medium with rich metabolites and strong antioxidant activity was screened out.The optimal medium for F93-1 was MEM medium with sea salt concentration of 30‰,under which the DPPH radical scavenging activity EC₅₀ of the secondary metabolite was 126.1μg/m L.The optimal medium for F93-2was MYA medium with sea salt concentration of 30‰,and under these conditions,the DPPH radical scavenging activity EC₅₀ of the secondary metabolite was 82.7μg/m L.（3）The screened optimal medium was used for batch fermentation of strain F93-1,and the fermentation product was extracted with ethyl acetate to obtain a crude extract.22 compounds were separated by modern chromatographic separation techniques such as normal phase silica gel column,reverse phase silica gel column,dextran gel column,and preparative liquid chromatography,and their structures were confirmed by spectral analysis.The 22 compounds included 5 new compounds（3 new amide skeleton compounds and 2 new sterol saponins compounds）.1benzoic acid derivative（phthalate）;1 terpene compound（phylalnembioid F）;3 polyketides（penicilazaphilone D,penicillazaphilone G）;1 alkaloid（methylprednisolone A）;5 heterocyclic compounds（geumsanol F,（+）-（2R,3S）-2-chloro-3-hydroxy-3-yl-γ-buty-rolacton,hypocrellone A,Rimboxo,cyclo（L-Pro-L-phe））;2 pentanols（methylpentanol,erythritol）.（4）The antibacterial and antioxidant activities of the isolated compounds were measured.The results showed that total 11 compounds had significant antibacterial effects on food-borne pathogens（Staphylococcus aureus,Escherichia coli,and Bacillus cereus）.The MIC of compound 3 against Escherichia coli was 7.8μg/m L,and the MIC of compound 18 against Staphylococcus aureus and Bacillus cereus were 62.5μg/m L and 15.6μg/m L,respectively.Compounds 10,13,14 and 4exhibited good activities of DPPH radical scavenging.The EC₅₀ of compound 4 was 749.89μg/m L.The results showed that mangrove rhizosphere sediments contained a large number of culturable fungal resources,and some of its strains also had rich antibacterial activity against plant pathogens and high DPPH free radical antioxidant activity.There were 3 new amide skeleton compounds,2 new compounds and 3 compounds first isolated from Penicillium sclerotiorum.Some of the compounds had high broad-spectrum food-borne pathogen inhibition and antioxidant activity.It further indicated that the natural products of fungi from mangrove special habitats had the characteristics of novel structure and significant activity,which enriched the natural product database and provided a number of potential active small molecular compounds for the development of antibacterial and antioxidant marine drugs.