发酵法制备黑木耳色素及其功能特性研究
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摘要
黑木耳属于药食两用真菌,其子实体长期以来被人们普遍食用。黑木耳子实体含有对人体具有生理作用的黑色素。黑色素是由多酚和吲哚等化合物氧化聚合而成,呈红、黑或红棕等颜色,具有抗氧化、抗病毒、抗衰老和增强人体免疫力等保健功能,是极具发展潜力的天然功能性食品着色剂。但由于黑木耳子实体生长周期长、黑色素提取工艺复杂、成本高而未见利用黑木耳色素的报道。采用液体深层发酵法生产黑木耳色素具有周期短、成本低、产量高和便于分离纯化等特点。
本研究筛选高产黑色素的黑木耳菌种,探讨发酵法形成黑木耳色素的条件以及碳水化合物对酪氨酸酶和黑色素形成的影响；分离纯化黑木耳酪氨酸酶,并对其酶学性质进行研究；采用摇瓶补料添加酪氨酸的方法制备黑木耳色素,并与子实体黑色素进行理化性质和稳定性比较；采用凝胶柱层析分离黑木耳发酵液黑色素各组分,比较色泽、分子量和结构组成等,并分析其抗氧化活性以及抗紫外线辐射能力。主要研究结果如下：
1、从22株黑木耳菌种中筛选得到RF201为黑木耳产黑色素的优良菌株。通过发酵试验,确定黑色素产量与酪氨酸酶活力呈极显著正相关(r=0.622,P<0.01),与碳水化合物含量呈极显著负相关(总糖,r=-0.607,P<0.01；还原糖,r=-0.956,P<0.01)。酪氨酸酶是黑木耳色素形成过程中的关键酶,酪氨酸含量在一定程度上决定黑色素产量。
2、通过硫酸铵分级沉淀、Sephadex G-100葡聚糖凝胶和DEAE-Sephcrose-FF离子交换层析,分离并纯化了黑木耳发酵液中的酪氨酸酶,SDS-PAGE电泳图谱呈单一条带,较粗酶液纯化21.43倍,比活力达到1285.82U/mg,回收率为27.41%。
通过SDS-PAGE测定,黑木耳酪氨酸酶的蛋白亚基分子量为12.62kDa。该酶最适pH为7.0,在中性和碱性条件下稳定；最适温度为40℃,60℃以上高温在短时间内使其失活。
黑木耳酪氨酸酶具有很强的底物专一性,可催化酪氨酸、多巴、邻苯二酚、间苯二酚、对苯二酚和没食子酸等发生反应,但对苯丙氨酸、半胱氨酸和阿魏酸等没有催化作用。金属盐中CuSO4对酪氨酸酶有激活作用,Al(NO3)3和MgSO4则起抑制作用。表面活性剂Tween-80、Triton X-100和EDTA对酪氨酸酶活力均有抑制作用,而SDS在低浓度时具有促进作用,高浓度时则起抑制作用。分别以酪氨酸、多巴和邻苯二酚为底物,酶动力学反应的Km分别为5.88、4.06和1.94mmol/L,Vmax分别为64.10、42.74和21.51μmol/mmin。
3、碳源中的葡萄糖、蔗糖、麦芽糖、果糖、乳糖和可溶性淀粉均对酪氨酸酶的合成有抑制作用,而天然复合物中的麦麸汁、氮源中的干酪素和无机盐中的硫酸铜对酪氨酸酶产量具有显著促进作用。采用中心组合试验获得最优培养基组成为：麦麸汁36%、干酪素1.1g/L、硫酸铜0.13g/L。在此条件下,酪氨酸酶最大活力为17.22U/mL,比优化前培养基中酪氨酸酶活力提高2.1倍。
Plackett-Burman试验筛选结果表明,发酵条件中的时间、装液量、接种量和起始pH是对酪氨酸酶产量有显著影响的因素,经中心组合试验进一步优化后,得到黑木耳产酪氨酸酶的最优发酵条件为：时间3.47d、装液量52.92mL、接种量7.39%和起始pH7.9。在此条件下,酪氨酸酶的最大产量为22.69U/mL,与预测值相符。
采用黑木耳摇瓶补料分批发酵技术制备黑色素时,酪氨酸最适添加时间为2d、添加量为1g/L、补加时间为4d、补加量为1.g/L,在此条件下,酪氨酸的转化率为41.42%,黑色素产量达到1035.71mg/L。
4、黑木耳发酵液黑色素在表观色泽上比子实体黑色素更趋近于黑色。两种黑色素均不溶于水和常见的有机溶剂,仅溶于碱性溶液,且在pH小于3的酸性溶液中产生沉淀。黑木耳发酵液和子实体黑色素都能被氧化剂氧化漂白,而对还原剂稳定。在紫外可见光谱范围内两者均表现出强烈的吸收特性。
黑木耳发酵液黑色素和子实体黑色素均易溶于碱性溶液,在高pH环境中稳定；两者对温度和光照敏感,随强度的提高和时间的延长,损失变大；Zn2+、Cu2+、Fe3+和Fe2+等金属离子可降低黑木耳色素的稳定性。两种黑木耳色素在白砂糖和柠檬酸溶液中不稳定,但在其它食品添加剂溶液中较稳定。
5、采用Sephadex G-100柱层析获得黑木耳发酵液黑色素F1和F2两组分,分子量分别为404.97和20.69kDa,两者在紫外可见区域都有强烈的吸收。F1比F2具有更低的L*、b*、C*和h0,在外观色泽上更趋近于黑色。
元素分析表明：F1和F2都含有C、H、N、O和S元素,属于棕黑色素。红外色谱和核磁氢谱表明F1和F2的结构中均含有大量的羟基、羰基和芳环基团。经氧化降解测得Fl中5,6-二羟基-2-吲哚羧酸占43.22%、5,6-二羟基吲哚占9.78%；F2中5,6-二羟基-2-吲哚羧酸占69.04%、5,6-二羟基吲哚占17.47%。
6、黑木耳色素组分F1和F2均具有较强的抗氧化能力,在还原力、超氧阴离子和羟自由基清除能力等方面均显著高于BHT,并表现出剂量依赖性特征。黑木耳色素可有效保护乳酸菌免受紫外线照射导致的损伤,其效果受照射距离和照射时间等因素影响。
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.
本研究筛选高产黑色素的黑木耳菌种,探讨发酵法形成黑木耳色素的条件以及碳水化合物对酪氨酸酶和黑色素形成的影响；分离纯化黑木耳酪氨酸酶,并对其酶学性质进行研究；采用摇瓶补料添加酪氨酸的方法制备黑木耳色素,并与子实体黑色素进行理化性质和稳定性比较；采用凝胶柱层析分离黑木耳发酵液黑色素各组分,比较色泽、分子量和结构组成等,并分析其抗氧化活性以及抗紫外线辐射能力。主要研究结果如下：
1、从22株黑木耳菌种中筛选得到RF201为黑木耳产黑色素的优良菌株。通过发酵试验,确定黑色素产量与酪氨酸酶活力呈极显著正相关(r=0.622,P<0.01),与碳水化合物含量呈极显著负相关(总糖,r=-0.607,P<0.01；还原糖,r=-0.956,P<0.01)。酪氨酸酶是黑木耳色素形成过程中的关键酶,酪氨酸含量在一定程度上决定黑色素产量。
2、通过硫酸铵分级沉淀、Sephadex G-100葡聚糖凝胶和DEAE-Sephcrose-FF离子交换层析,分离并纯化了黑木耳发酵液中的酪氨酸酶,SDS-PAGE电泳图谱呈单一条带,较粗酶液纯化21.43倍,比活力达到1285.82U/mg,回收率为27.41%。
通过SDS-PAGE测定,黑木耳酪氨酸酶的蛋白亚基分子量为12.62kDa。该酶最适pH为7.0,在中性和碱性条件下稳定；最适温度为40℃,60℃以上高温在短时间内使其失活。
黑木耳酪氨酸酶具有很强的底物专一性,可催化酪氨酸、多巴、邻苯二酚、间苯二酚、对苯二酚和没食子酸等发生反应,但对苯丙氨酸、半胱氨酸和阿魏酸等没有催化作用。金属盐中CuSO4对酪氨酸酶有激活作用,Al(NO3)3和MgSO4则起抑制作用。表面活性剂Tween-80、Triton X-100和EDTA对酪氨酸酶活力均有抑制作用,而SDS在低浓度时具有促进作用,高浓度时则起抑制作用。分别以酪氨酸、多巴和邻苯二酚为底物,酶动力学反应的Km分别为5.88、4.06和1.94mmol/L,Vmax分别为64.10、42.74和21.51μmol/mmin。
3、碳源中的葡萄糖、蔗糖、麦芽糖、果糖、乳糖和可溶性淀粉均对酪氨酸酶的合成有抑制作用,而天然复合物中的麦麸汁、氮源中的干酪素和无机盐中的硫酸铜对酪氨酸酶产量具有显著促进作用。采用中心组合试验获得最优培养基组成为：麦麸汁36%、干酪素1.1g/L、硫酸铜0.13g/L。在此条件下,酪氨酸酶最大活力为17.22U/mL,比优化前培养基中酪氨酸酶活力提高2.1倍。
Plackett-Burman试验筛选结果表明,发酵条件中的时间、装液量、接种量和起始pH是对酪氨酸酶产量有显著影响的因素,经中心组合试验进一步优化后,得到黑木耳产酪氨酸酶的最优发酵条件为：时间3.47d、装液量52.92mL、接种量7.39%和起始pH7.9。在此条件下,酪氨酸酶的最大产量为22.69U/mL,与预测值相符。
采用黑木耳摇瓶补料分批发酵技术制备黑色素时,酪氨酸最适添加时间为2d、添加量为1g/L、补加时间为4d、补加量为1.g/L,在此条件下,酪氨酸的转化率为41.42%,黑色素产量达到1035.71mg/L。
4、黑木耳发酵液黑色素在表观色泽上比子实体黑色素更趋近于黑色。两种黑色素均不溶于水和常见的有机溶剂,仅溶于碱性溶液,且在pH小于3的酸性溶液中产生沉淀。黑木耳发酵液和子实体黑色素都能被氧化剂氧化漂白,而对还原剂稳定。在紫外可见光谱范围内两者均表现出强烈的吸收特性。
黑木耳发酵液黑色素和子实体黑色素均易溶于碱性溶液,在高pH环境中稳定；两者对温度和光照敏感,随强度的提高和时间的延长,损失变大；Zn2+、Cu2+、Fe3+和Fe2+等金属离子可降低黑木耳色素的稳定性。两种黑木耳色素在白砂糖和柠檬酸溶液中不稳定,但在其它食品添加剂溶液中较稳定。
5、采用Sephadex G-100柱层析获得黑木耳发酵液黑色素F1和F2两组分,分子量分别为404.97和20.69kDa,两者在紫外可见区域都有强烈的吸收。F1比F2具有更低的L*、b*、C*和h0,在外观色泽上更趋近于黑色。
元素分析表明：F1和F2都含有C、H、N、O和S元素,属于棕黑色素。红外色谱和核磁氢谱表明F1和F2的结构中均含有大量的羟基、羰基和芳环基团。经氧化降解测得Fl中5,6-二羟基-2-吲哚羧酸占43.22%、5,6-二羟基吲哚占9.78%；F2中5,6-二羟基-2-吲哚羧酸占69.04%、5,6-二羟基吲哚占17.47%。
6、黑木耳色素组分F1和F2均具有较强的抗氧化能力,在还原力、超氧阴离子和羟自由基清除能力等方面均显著高于BHT,并表现出剂量依赖性特征。黑木耳色素可有效保护乳酸菌免受紫外线照射导致的损伤,其效果受照射距离和照射时间等因素影响。
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.
引文
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