Fermentation Production,biological Activity And Biosynthetic Pathway Of Melanin From Streptomyces Sp. ZL-24

Melanin is a heterogeneous polyphenol polymer that is widely found in animals,plants and microorganisms.Microbial melanin is a natural pigment,which has the advantages of safety,non-toxicity,and abundant resources,and has broad application prospects in the fields of food,cosmetics,and pharmaceuticals.This thesis focuses on the research of fermentation conditions,melanin characterization,biological activity detection and melanin biosynthesis pathway of Streptomyces sp.ZL-24 to produce melanin.The main results and conclusions are as follows:The results of the single-factor experiment of the medium composition showed that soybean peptone,NiCl₂ and FeSO₄ were the significant factors affecting black yield.When soybean peptone was used as the nitrogen source,the melanin production increased from 5.9mg/L to 45.9 mg/L;when NiCl₂ was added to the medium,the maximum melanin production was 56.2 mg/L;after adding FeSO₄,the melanin production Reached 64.8 mg/L.Through the central composite design experiment,the optimal medium composition for melanin fermentation was determined to be soybean peptone?20.31 g/L?,FeSO₄?1.33 g/L?and NiCl₂?3.05 mM?.Using the best medium above,according to the inoculation volume 3%?v/v?,medium pH 7.0,30?,liquid fermentation for 5 days,the maximum melanin production reached 189.9 mg/L.However,this data is far lower than the actual production of melanin,because the above melanin production is mainly determined by the extraction method of hydrochloric acid precipitation,and there is still a large amount of melanin in the fermentation broth that has not been precipitated.Therefore,the above data only represents the production of water-insoluble melanin.Further using the above optimal medium,the water-soluble melanin with higher purity was prepared by the solid plate method,and its maximum yield reached 4.24g/L,which was higher than the data reported in the literature.The water-insoluble melanin and water-soluble black produced by strain ZL-24 were purified by acid precipitation,enzymatic hydrolysis and other steps.UV-vis,FT-IR,and NMR pop analysis results showed that the above melanin had typical melanin spectral characteristics.The melanin biological activity test results showed that water-soluble melanin showed strong antibacterial effect on Gram-positive bacteria Staphylococcus aureus ATCC6538 and Mycobacterium smegmatis ATCC10231 and Gram-negative bacteria Pseudomonas aeruginosa ATCC9027,while water-insoluble melanin only inhibited Positive bacteria.Both water-insoluble and water-soluble melanin can inhibit the biofilm formation of Gram-positive bacteria and Gram-negative bacteria.Among them,Staphylococcus aureus ATCC6538 and Pseudomonas aeruginosa ATCC9027 have higher anti-biofilm activity,and the inhibition rates are 79.2%and 71.7%,67.5%and 74.6%.Both melanins showed strong hydroxyl radical scavenging activity,DPPH scavenging activity,and superoxide radical scavenging activity.The scavenging activities of the three free radicals above 50 mg/mL water-soluble and water-insoluble melanin were 96%and 88.7%,65.3%and 55.7%,59.9%and 60.7%,respectively.Water-soluble melanin has the protective effects of low cytotoxicity and H₂O₂-induced oxidative damage of nerve cells SYHY-5Y.The biosynthetic pathways of strain ZL-24 melanin composition analysis,metabolic intermediates HPLC,LC-MS analysis,key enzyme activity determination,key enzyme gene deletion mutation and complementation were analyzed.The results showed that strain ZL-24melanin mainly It consists of dopa melanin and pus melanin,mainly pus melanin.Tyrosinase is a key enzyme in the former synthesis and is responsible for the synthesis of melanin through the dopa pathway,and 4-hydroxyphenylpyruvate oxygenase is a key enzyme in the latter synthesis,and is responsible for the synthesis of melanin through the uric acid pathway.