Study On The Melanin Preparation, Structure Analysis And Prebiotic Activity Of Boletus Griseus

In this paper,melanin（BgM）was prepared from Boletus griseus.The structure of BgM was analyzed to obtain its possible structural formula.The oxidative degradation products of melanin were analyzed.The effects of BgM on probiotics were also studied.The results are as follows:1.Melanin form Boletus griseus was prepared through alkali-soluble acid precipitation method,and the yield was 10.38%.Purified melanin BgM was obtained after acid hydrolysis and organic solvent treatment.BgM was insoluble in acidic solutions,aqueous solutions and common organic reagents,and was dissolved in alkaline solutions.The maximum absorption peak of the UV-visible absorption spectrum of BgM was 214 nm,which was consistent with the maximum UV absorption of natural melanin.The color values of melanin before and after purification were 213 and 954.8,respectively,which further indicated that the purity of BgM had been greatly improved.2.FESEM,elemental analysis,FTIR,NMR ¹H and ¹³C spectra,Py-GC/MS and UPLC-MS/MS were used to analyze the structure of BgM.The FESEM of BgM was found to be irregular flaky particles with an average particle size of about 1.25μm.Elemental analysis showed that the percentages of C,H,N,S and O elements of the BgM were 56.38%,5.86%,6.17%,2.44%and 28.04%,respectively,and the S/N and C/N ratios were 0.17 and 10.66,respectively.Characteristic absorption peaks were observed at 3426 cm^-1,1600 cm^-1,and 1105 cm^-1in the FTIR spectrum,and it was found BgM contained phenolic hydroxyl groups,amino groups,carbonyl groups,methylene groups,and methyl groups.BgM was an eumelanin whose main skeleton has both benzene ring and indole,and the branched chain mainly consisted of alkanes,alcohols,and fatty acids.The BgM ammonia-hydrogen peroxide degradation product BgM1 was analyzed by UPLC-MS/MS and four primary mass spectral peaks were obtained.Combined with the above analysis results,the chemical structure of BgM was estimated,and the condensation formula was[C₂₈（OR₁）₄（OR₂）₃H₁₁O₆N₄]_n.3.Degradation products BgM1 and BgM2 were prepared by ultrasonic-assisted ammonia-hydrogen peroxide and hydrogen peroxide to degrade BgM.The results showed that BgM1 and BgM2 had broad solubility compared to BgM,and BgM1retained the original color of melanin to a greater extent.The molecular weight and molecular weight range of BgM1 was higher than those of BgM2.By Py-GC/MS analysis,it was found that there were 52 pyrolysis products of BgM1,among which indole and its derivatives are more abundant.There were 61 pyrolysis products of BgM2,mainly amides,carboxylic acids,ketones and benzene,without indole and its derivatives.The above results show that appropriate degradation of melanin can improve its solubility,and ammonia-hydrogen peroxide degradation products retain the original color and skeleton structure of melanin to a certain extent.4.The non-prescription human intestinal flora probiotic bacteria tablets were used as fermentation strains,and artificial gastric juice and artificial intestinal juice were used as simulated digestive juices to study BgM in vitro digestion and intestinal fermentation.The proliferation of probiotics and the change of short-chain fatty acid content of fermentation products were studied by simulating BgM in vitro.The study found that DM had a certain promoting effect on the proliferation of probiotics,and the highest content of acetic acid in the fermentation products,followed by butyric acid and isovaleric acid.The contents of acetic acid,butyric acid,isobutyric acid,valeric acid and isovaleric acid in DM group were higher than those in FOS group for most of the fermentation time.The results indicated that BgM may be a potential probiotic and has great potential for development.