| Auricularia auricula (A. auricula) has been used as food and drug for a long time. A. auricula fruit-bodies are rich in melanin and melanin is considered to be one of the most important functional components. Melanin is a high molecular weight pigment (generally of red, black, or brown color) produced from oxidative polymerization of phenolic or indolic compounds. It has a number of healthful functions, such as antioxidation, anti-HIV activity, anti-aging, and immunomodulatory activity. These facts illuminated natural melanin has enormous development potentiality to be used as a healthful food colorant. However, A. auricula fruit-bodies growing on solid culture, the time to complete the fruit-bodies is too long and the extraction process of melanin is tedious and expensive. Thus, there is little information available in literature about utilization of A. auricula melanin. A large amount of melanin can also been produced by A. auricula through submerged culture, which has been proved to a desirable method with many advantages, such as short fermentation period, low producing cost, high product output and easy downstream processing.In this study, the strain of A. auricula produced high-yield melanin was screened. Through its fermentation test, preliminary mechanism of A. auricula’s melanin formation and effects of carbohydrate on tyrosinase and melanin were researched. The tyrosinase produced by A. auricula was isolated and purified, then the characteristics of the enzyme were investigated. Using tyrosinase, A. auricula fermentation broth melanin was preparationed by method of tyrosine fed in shake flash, and physicochemical properties and stability were compared with A. auricula dried fruit-body melanin. Various components of A. auricula fermentation broth melanin were isolated by gel filtration chromatography, and their color, molecular weight and structure composition were compared. Meanwhile, antioxidant activities and anti-ultraviolet of these components were investigated. The main results and findings were shown as follows:1. A. auricula RF201was screened and considered as excellent strain produced melanin from twenty two species of A. auricula. Through its fermentation test, it was showed that correlation between melanin yield and tyrosinase activity was positive effect significantly (r=0.622, P<0.01) and correlation between melanin yield and carbohydrate content was negative effect significantly (total sugar, r=-0.607, P<0.01; reducing sugar, r=-0.956, P<0.01). Meanwhile, tyrosinase was key enzyme during A. auricula’s melanin formation and melanin yield was influenced by concentration of substrate, tyrosine to some extent.2. The tyrosinase produced by A. auricula through submerged culture was isolated and purified by ammonium sulfate fraction, Sephadex G-100gel filtration chromatography and DEAE-Sephcrose-FF ion-exchange column chromatography and tyrosinase was purified21.43-fold from crude protein extracts with a yield of27.41%and specific activity of1283.82U/mg. The final preparation gave a single band on sodium dodecyl sulfate polyacrylamide gels.According to result of SDS-PAGE, the protein subunit molecular weight of purified tyrosinase produced by A. auricula was12.62kDa. The optimum pH of purified tyrosinase was7.0and it was stable at neutral and alkaline conditions. The optimum temperature of purified tyrosinase was40℃and it was rapidly inactivating at more than60℃.The results indicated the tyrosinase produced by A. auricula has high stereospecificity. The purified tyrosinase could catalyze tyrosine, DOPA, catechol, resorcinol, hydroquinone and gallic acid, and couldn’t react with phenylalanine, cysteine and ferulic acid.In metal salts, tyrosinase was actitaved by CUSO4and was inhibited by Al(NO3)3and MgSO4. The tyrosinase was also inhibited by Tween-80, Triton X-100and EDTA of surfactant. However, low concentration of SDS promoted tyrosinase activity, high concentration of SDS inhibited tyrosinase activity. Tyrosine, DOPA and catechol were used as substrate of tyrosinase, enzymatic reaction michaelis constant (Km) were5.88、4.06and1.94mmol/L respectively, and maximum reaction rate (Vmax) were64.10,42.74and21.51μmol/min respectively.3. The single-factor experiments results showed that various carbon sources (glucose, sucrose, maltose, fructose, lactose and soluble starch) had inhibitory effect on tyrosinase synthesis and natural substances (wheat bran extract), nitrogen source (casein) and inorganic salt (copper sulfate) had promotion effect significantly on tyrosinase synthesis. Therefore, wheat bran extract, casein and copper sulfate were chosen to further optimize using response surface methodology. The central composite experimental results showed the optimum medium components were as follows:wheat bran extract36.0%, casein1.1 g/L and copper sulfate0.13g/L. Under these conditions, the highest tyrosinase activity was17.22U/ml, which resulted in an overall2.1-fold increase compared with that using the original medium components.The Plackett-Burman experiments results showed that fermentation conditions including time, broth content, inoculation amount and initial pH had significant effect on tyrosinase production. These conditions were further optimized using central composite experimental design and the optimum fermentation conditions were as follows:time3.47d, broth content52.92mL, inoculation amount7.39%and initial pH7.9. Under these conditions, the highest tyrosinase activity was22.69U/mL, which wasn’t significantly different from the predicted value.A. auricula fermentation broth melanin was preparationed by method of tyrosine fed in shake flash, and the optimum adding time, addition amount, supplement time and supplement amount of tyrosine were2d,1g/L,4d and1.5g/L respectively. Under these conditions, the highest melanin yield was1035.71mg/L and conversion rate was41.42%.4. A. auricula fermentation broth melanin powder presented more dark colored than A. auricula dried fruit-body melanin in visual color. These two melanins were insoluble in both water and common organic solvents. They dissolved only in alkali aqueous solution and precipitated acidic aqueous solution (pH<3). A. auricula fermentation broth and dried fruit-body melanins were gradually oxidative bleached by oxidant and were stable to reducer. They exhibited strong optical absorbance in a wide UV-VIS spectral range.A. auricula fermentation broth and dried fruit-body melanins were easy soluble in alkaline solvents and were stable to higher pH conditions. They were sensitive to temperature and light and losses were increased with increasing strength and time prolonging. Some metal ions including Zn2+, Cu2+, Fe3+and Fe2+could decrease stability of A. auricula’s melanins. These two melanins were unstable in white granulated sugar and citric acid solutions, but were stable in other food additives.5. By Sephadex G-100gel filtration chromatography, two components of A. auricula fermentation broth melanin, F1and F2, the molecular weights were404.97and20.69kDa respectively, were obtained. They exhibited strong optical absorbance in a wide UV-VIS spectral range. Fl presented lower values (L*, b*, C*and h°), but exhibited higher value of ΔE than F2in visual color.Elemental composition analytical results showed that Fl and F2contained C, H, N, O and S elements. Thus, they belonged to phaeomelanins. IR spectra and1H NMR of F1and F2showed that structure of two melanins contained a lot of hydroxyl, carbonyl and aromatic groups. Through oxdative degradation, F1was composed of43.22%DHICA and9.78%DHI, and F2was composed of69.04%DHICA and17.47%DHI。6. Two components of A. auricula fermentation broth melanin, F1and F2, had significant stronger antioxidant abilities by determining reducing power, superoxide radical scavenging activity and hydroxyl radical scavenging activity than BHT and exhibited dose-dependent. They could protect lactic acid bacteria and decrease injury caused by ultraviolet, while protective effects were affected by some factors such as radiation distance and time and also exhibited dose-dependent. F1possessed a stronger anti-ultraviolet ability, which might be caused by different color. |
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