Study On Biological Characteristics Of Extracellular Melanin Produced By Eurotium Cristatum

Eurotium cristatum is the dominant species in the fermentation process of brick tea,and can be used to produce a variety of extracellular enzymes(polyphenol oxidase,pectinase,cellulase,protease,etc.),extracellular polysaccharides and pigments Strains.Melanin is a multifunctional biopolymer,which is made by oxidative polymerization of phenols or indole compounds in animal and plant microorganisms.Melanin has a variety of functions such as anti-oxidation,anti-ultraviolet radiation,anti-proliferation,elimination of excessive free radicals in the human body,and regulation of immunity.Its re-industry,agriculture,medicine and other aspects have broad application prospects.When the growth of Eurotium cristatum,a large number of pigments are metabolized.The main pigment is yellow pigment,which will be accompanied by the production of melanin as the culture time increases.In this paper,Eurotium cristatum was used as raw material to study the extracellular melanin produced by fermentation.The extraction process and culture conditions of this melanin were optimized,and its stability,antioxidant activity and anti-proliferative effect were studied.The findings are as follows:1.Alkaline extraction process optimization of Eurotium cristatum extracellular melaninIn this experiment,the extraction conditions of the extracellular melanin method of Eurotium cristatum were optimized by response surface methodology.The results showed that the optimal extraction process of extracellular melanin from Eurotium cristatum was:Na OH concentration was 1 mol/L,temperature was 74?,p H was 2.5,and material-liquid ratio was 1:1.4.Under the optimal extraction conditions,the extracellular melanin extraction of Eurotium cristatum reached 3.982 mg/m L.2.Optimization of Fermentation Conditions and Stability of Eurotium cristatum extracellular melaninThe potato glucose liquid culture medium was used as the basic medium,and the response conditions were used to optimize the extracellular melanin fermentation conditions of Eurotium cristatum.The results showed that the optimal fermentation conditions for Eurotium cristatum extracellular melanin were:shaking rate184 r/min,culture temperature29,medium p H 6.8.Under these fermentation conditions,extracellular melanin was obtained at 4.27 mg/m L.The obtained melanin has a maximum absorption peak at 298 nm.The melanin has strong heat resistance below 70?and excessive temperatures will cause its decomposition.Mg²⁺,Zn²⁺,Fe³⁺metal ions can enhance their stability,the melanin should be protected from light and should not be contacted with strong oxidants,reducing agents and compounds containing Ca²⁺and Cu²⁺during use.3.Study on the antioxidative activity and anti-proliferative ability of melanin from Eurotium cristatumIn this experiment,DPPH radical scavenging method,ABTS radical scavenging method,superoxide anion scavenging method,hydroxyl radical scavenging method,iron ion reduction ability,oxygen radical absorption ability method(ORAC)and peroxy radical scavenging ability method(PSC)were used to analyze the antioxidant activities of two kinds of melanin from Eurotium cristatum.At the same time,its anti-proliferative effect on Hep G2cells was also analyzed.The results showed that the two melanin of Eurotium cristatum had good scavenging effects on DPPH free radicals,ABTS free radicals,superoxide anion free radicals,and hydroxyl free radicals,and had certain reducing ability.The melanin antioxidant activity of Eurotium cristatum is extracellular melanin>intracellular melanin.When the melanin concentration reached 100?g/m L,the scavenging rates of extracellular melanin of Eurotium cristatum against DPPH free radicals,ABTS free radicals,superoxide anion free radicals and hydroxyl free radicals were 75.55%,70.01%,87.99%,86.53%,Intracellular melanin is 69.09%,63.26%,79.74%,78.44%,the FRAP value of the extracellular melanin of Eurotium cristatum is 1.57,and the intracellular melanin is 0.84.The results of oxygen free radical absorption capacity(ORAC)experiments showed that the extracellular melanin ORAC value of Eurotium cristatum was 66.59?mol TE/g,and the intracellular melanin ORAC value was 49.29?mol TE/g.Peroxide free radical scavenging capacity(PSC)experiments showed that the IC₅₀ values of the extracellular and intracellular melanin of Eurotium cristatum on free radical scavenging were 5.79?g/m L and 11.19?g/m L respectively,and the PSC values were 22.46?mol VCE/g DW,11.66?mol VCE/g DW.The anti-proliferative ability of human hepatoma cells showed that the extracellular melanin of Eurotium cristatum had the strongest anti-proliferative ability of hepatoma cells,with an IC₅₀ value of 30.54?g/m L and an intracellular melanin IC₅₀ of Eurotium cristatum 31.11?g/m L.The antioxidant capacity and anti-proliferative effect of extracellular melanin of Eurotium cristatum is superior to that of intracellular melanin of Eurotium cristatum.