发酵法制备黑木耳色素及其功能特性研究
发布时间：2018-09-03 18:13
【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,P0.01),与碳水化合物含量呈极显著负相关(总糖,r=-0.607,P0.01；还原糖,r=-0.956,P0.01)。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。
[Abstract]:Auricularia auricula is a kind of edible and medicinal fungi. Its fruiting body has been widely eaten for a long time. Auricularia auricula fruiting body contains melanin which has physiological effects on human body. It is a natural functional food colorant with great potential for development. However, due to the long growth cycle of Auricularia auricula fruiting body, the complex extraction process of melanin and the high cost, it has not been reported to use Auricularia auricula pigment.
In this study, high-melanin-producing strains of Auricularia auricula were screened, and the conditions for the formation of Auricularia auricula pigment by fermentation and the effects of carbohydrates on the formation of tyrosinase and melanin were investigated. The physical and chemical properties and stability of black fungus melanin were compared with that of fruiting body melanin. The components of black fungus melanin were separated by gel column chromatography, and their color, molecular weight and structure were compared.
1. RF201 was screened from 22 strains of Auricularia auricula to produce melanin. Through fermentation test, it was found that melanin production was positively correlated with tyrosinase activity (r = 0.622, P 0.01) and negatively correlated with carbohydrate content (total sugar, r = - 0.607, P 0.01; reducing sugar, r = - 0.956, P 0.01). Tyrosine content is a key enzyme in the formation process of Auricularia auricula pigment, to a certain extent, determines melanin production.
2. Tyrosinase from Auricularia auricula fermentation broth was separated and purified by ammonium sulfate fractional precipitation, Sephadex G-100 glucan gel and DEAE-Sephcrose-FF ion exchange chromatography. SDS-PAGE electrophoresis showed a single band, the crude enzyme was purified 21.43 times, the specific activity was 1285.82 U/mg, and the recovery was 27.41%.
The protein subunit molecular weight of Auricularia auricula tyrosinase was 12.62 kDa determined by SDS-PAGE. The optimum pH of the enzyme was 7.0, which was stable in neutral and alkaline conditions. The optimum temperature was 40 C, and the temperature above 60 C made it inactivated in a short time.
Auricularia auricula tyrosinase has strong substrate specificity. It can catalyze the reaction of tyrosine, dopa, catechol, resorcinol, hydroquinone and gallic acid, but has no catalytic effect on phenylalanine, cysteine and ferulic acid. CuSO4 in metal salts activates tyrosinase, while Al (NO3) 3 and MgSO4 inhibit it. Tween-80, Triton X-100 and EDTA all inhibited the activity of tyrosinase, while SDS promoted the activity of tyrosinase at low concentration and inhibited it at high concentration. The Km of enzyme kinetics reaction was 5.88, 4.06 and 1.94 mmol/L with tyrosine, DOPA and catechol as substrates, and Vmax was 64.10, 42.74 and 21.51 micromol/mmin, respectively.
3. Glucose, sucrose, maltose, fructose, lactose and soluble starch in carbon source inhibited the synthesis of tyrosinase, while wheat bran in natural compound, casein in nitrogen source and copper sulfate in inorganic salt significantly promoted the production of tyrosinase. The maximum activity of tyrosinase was 17.22 U/mL in wheat bran juice, 1.1 g/L casein and 0.13 g/L cupric sulfate, which was 2.1 times higher than that in the optimal medium.
The results of Plackett-Burman test showed that time, liquid loading, inoculation amount and initial pH had significant effects on tyrosinase production. After further optimization by central combination test, the optimal fermentation conditions for tyrosinase production from Auricularia auricula were obtained as follows: time 3.47 d, liquid loading 52.92 mL, inoculation amount 7.39% and initial pH 7.9. Under these conditions, the maximum yield of tyrosinase was 22.69U/mL, which was consistent with the predicted value.
The optimum addition time of tyrosine was 2 days, 1 g/L, 4 days and 1.g/L. Under these conditions, the conversion of tyrosine was 41.42% and the yield of melanin was 1035.71 mg/L.
4. Black fungus fermentation broth melanin is more black than fruiting body melanin in apparent color. Both melanins are insoluble in water and common organic solvents, only in alkaline solution, and precipitate in acidic solution with pH less than 3. Black fungus fermentation broth and fruiting body melanin can be oxidized and bleached by oxidant, but stable to reducing agent. In both ultraviolet and visible spectra, both of them exhibit strong absorption characteristics.
Both melanin and fruiting body melanin of Auricularia auricula fermentation broth are soluble in alkaline solution and stable in high pH environment; they are sensitive to temperature and light, and the loss increases with the increase of strength and time; Zn2+, Cu2+, Fe3+ and Fe2+ ions can reduce the stability of Auricularia auricula pigment. Two kinds of Auricularia auricula pigment are stable in white granulated sugar and lemon. The acid solution is unstable but stable in other food additive solutions.
5. Sephadex G-100 column chromatography was used to obtain melanin F1 and F2 from Auricularia auricula fermentation broth with molecular weights of 404.97 and 20.69kDa, respectively. Both of them have strong absorption in ultraviolet-visible region. F1 has lower L*, b*, C* and H0 than F2, and tends to black in appearance and luster.
Elemental analysis showed that both F1 and F2 contained C, H, N, O and S elements, belonging to brown melanin. Infrared spectrum and nuclear magnetic resonance hydrogen spectrum showed that both F1 and F2 contained abundant hydroxyl, carbonyl and aromatic ring groups. Acid accounted for 69.04%, 5,6- two hydroxyindole accounted for 17.47%.
6. Auricularia auricula pigment components F1 and F2 have strong antioxidant capacity. They are significantly higher than BHT in reducing power, superoxide anion and hydroxyl radical scavenging capacity, and show dose-dependent characteristics. Auricularia auricula pigment can effectively protect lactic acid bacteria from ultraviolet radiation damage, and its effect is affected by irradiation distance and irradiation time. Factor effect.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：Q936
本文编号：2220772
[Abstract]:Auricularia auricula is a kind of edible and medicinal fungi. Its fruiting body has been widely eaten for a long time. Auricularia auricula fruiting body contains melanin which has physiological effects on human body. It is a natural functional food colorant with great potential for development. However, due to the long growth cycle of Auricularia auricula fruiting body, the complex extraction process of melanin and the high cost, it has not been reported to use Auricularia auricula pigment.
In this study, high-melanin-producing strains of Auricularia auricula were screened, and the conditions for the formation of Auricularia auricula pigment by fermentation and the effects of carbohydrates on the formation of tyrosinase and melanin were investigated. The physical and chemical properties and stability of black fungus melanin were compared with that of fruiting body melanin. The components of black fungus melanin were separated by gel column chromatography, and their color, molecular weight and structure were compared.
1. RF201 was screened from 22 strains of Auricularia auricula to produce melanin. Through fermentation test, it was found that melanin production was positively correlated with tyrosinase activity (r = 0.622, P 0.01) and negatively correlated with carbohydrate content (total sugar, r = - 0.607, P 0.01; reducing sugar, r = - 0.956, P 0.01). Tyrosine content is a key enzyme in the formation process of Auricularia auricula pigment, to a certain extent, determines melanin production.
2. Tyrosinase from Auricularia auricula fermentation broth was separated and purified by ammonium sulfate fractional precipitation, Sephadex G-100 glucan gel and DEAE-Sephcrose-FF ion exchange chromatography. SDS-PAGE electrophoresis showed a single band, the crude enzyme was purified 21.43 times, the specific activity was 1285.82 U/mg, and the recovery was 27.41%.
The protein subunit molecular weight of Auricularia auricula tyrosinase was 12.62 kDa determined by SDS-PAGE. The optimum pH of the enzyme was 7.0, which was stable in neutral and alkaline conditions. The optimum temperature was 40 C, and the temperature above 60 C made it inactivated in a short time.
Auricularia auricula tyrosinase has strong substrate specificity. It can catalyze the reaction of tyrosine, dopa, catechol, resorcinol, hydroquinone and gallic acid, but has no catalytic effect on phenylalanine, cysteine and ferulic acid. CuSO4 in metal salts activates tyrosinase, while Al (NO3) 3 and MgSO4 inhibit it. Tween-80, Triton X-100 and EDTA all inhibited the activity of tyrosinase, while SDS promoted the activity of tyrosinase at low concentration and inhibited it at high concentration. The Km of enzyme kinetics reaction was 5.88, 4.06 and 1.94 mmol/L with tyrosine, DOPA and catechol as substrates, and Vmax was 64.10, 42.74 and 21.51 micromol/mmin, respectively.
3. Glucose, sucrose, maltose, fructose, lactose and soluble starch in carbon source inhibited the synthesis of tyrosinase, while wheat bran in natural compound, casein in nitrogen source and copper sulfate in inorganic salt significantly promoted the production of tyrosinase. The maximum activity of tyrosinase was 17.22 U/mL in wheat bran juice, 1.1 g/L casein and 0.13 g/L cupric sulfate, which was 2.1 times higher than that in the optimal medium.
The results of Plackett-Burman test showed that time, liquid loading, inoculation amount and initial pH had significant effects on tyrosinase production. After further optimization by central combination test, the optimal fermentation conditions for tyrosinase production from Auricularia auricula were obtained as follows: time 3.47 d, liquid loading 52.92 mL, inoculation amount 7.39% and initial pH 7.9. Under these conditions, the maximum yield of tyrosinase was 22.69U/mL, which was consistent with the predicted value.
The optimum addition time of tyrosine was 2 days, 1 g/L, 4 days and 1.g/L. Under these conditions, the conversion of tyrosine was 41.42% and the yield of melanin was 1035.71 mg/L.
4. Black fungus fermentation broth melanin is more black than fruiting body melanin in apparent color. Both melanins are insoluble in water and common organic solvents, only in alkaline solution, and precipitate in acidic solution with pH less than 3. Black fungus fermentation broth and fruiting body melanin can be oxidized and bleached by oxidant, but stable to reducing agent. In both ultraviolet and visible spectra, both of them exhibit strong absorption characteristics.
Both melanin and fruiting body melanin of Auricularia auricula fermentation broth are soluble in alkaline solution and stable in high pH environment; they are sensitive to temperature and light, and the loss increases with the increase of strength and time; Zn2+, Cu2+, Fe3+ and Fe2+ ions can reduce the stability of Auricularia auricula pigment. Two kinds of Auricularia auricula pigment are stable in white granulated sugar and lemon. The acid solution is unstable but stable in other food additive solutions.
5. Sephadex G-100 column chromatography was used to obtain melanin F1 and F2 from Auricularia auricula fermentation broth with molecular weights of 404.97 and 20.69kDa, respectively. Both of them have strong absorption in ultraviolet-visible region. F1 has lower L*, b*, C* and H0 than F2, and tends to black in appearance and luster.
Elemental analysis showed that both F1 and F2 contained C, H, N, O and S elements, belonging to brown melanin. Infrared spectrum and nuclear magnetic resonance hydrogen spectrum showed that both F1 and F2 contained abundant hydroxyl, carbonyl and aromatic ring groups. Acid accounted for 69.04%, 5,6- two hydroxyindole accounted for 17.47%.
6. Auricularia auricula pigment components F1 and F2 have strong antioxidant capacity. They are significantly higher than BHT in reducing power, superoxide anion and hydroxyl radical scavenging capacity, and show dose-dependent characteristics. Auricularia auricula pigment can effectively protect lactic acid bacteria from ultraviolet radiation damage, and its effect is affected by irradiation distance and irradiation time. Factor effect.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：Q936
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