發(fā)酵法制備黑木耳色素及其功能特性研究
發(fā)布時間：2018-09-03 18:13
【摘要】：黑木耳屬于藥食兩用真菌,其子實體長期以來被人們普遍食用。黑木耳子實體含有對人體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,呈紅、黑或紅棕等顏色,具有抗氧化、抗病毒、抗衰老和增強人體免疫力等保健功能,是極具發(fā)展?jié)摿Φ奶烊还δ苄允称分珓�。但由于黑木耳子實體生長周期長、黑色素提取工藝復(fù)雜、成本高而未見利用黑木耳色素的報道。采用液體深層發(fā)酵法生產(chǎn)黑木耳色素具有周期短、成本低、產(chǎn)量高和便于分離純化等特點。 本研究篩選高產(chǎn)黑色素的黑木耳菌種,探討發(fā)酵法形成黑木耳色素的條件以及碳水化合物對酪氨酸酶和黑色素形成的影響；分離純化黑木耳酪氨酸酶,并對其酶學性質(zhì)進行研究；采用搖瓶補料添加酪氨酸的方法制備黑木耳色素,并與子實體黑色素進行理化性質(zhì)和穩(wěn)定性比較；采用凝膠柱層析分離黑木耳發(fā)酵液黑色素各組分,比較色澤、分子量和結(jié)構(gòu)組成等,并分析其抗氧化活性以及抗紫外線輻射能力。主要研究結(jié)果如下： 1、從22株黑木耳菌種中篩選得到RF201為黑木耳產(chǎn)黑色素的優(yōu)良菌株。通過發(fā)酵試驗,確定黑色素產(chǎn)量與酪氨酸酶活力呈極顯著正相關(guān)(r=0.622,P0.01),與碳水化合物含量呈極顯著負相關(guān)(總糖,r=-0.607,P0.01；還原糖,r=-0.956,P0.01)。酪氨酸酶是黑木耳色素形成過程中的關(guān)鍵酶,酪氨酸含量在一定程度上決定黑色素產(chǎn)量。 2、通過硫酸銨分級沉淀、Sephadex G-100葡聚糖凝膠和DEAE-Sephcrose-FF離子交換層析,分離并純化了黑木耳發(fā)酵液中的酪氨酸酶,SDS-PAGE電泳圖譜呈單一條帶,較粗酶液純化21.43倍,比活力達到1285.82U/mg,回收率為27.41%。 通過SDS-PAGE測定,黑木耳酪氨酸酶的蛋白亞基分子量為12.62kDa。該酶最適pH為7.0,在中性和堿性條件下穩(wěn)定；最適溫度為40℃,60℃以上高溫在短時間內(nèi)使其失活。 黑木耳酪氨酸酶具有很強的底物專一性,可催化酪氨酸、多巴、鄰苯二酚、間苯二酚、對苯二酚和沒食子酸等發(fā)生反應(yīng),但對苯丙氨酸、半胱氨酸和阿魏酸等沒有催化作用。金屬鹽中CuSO4對酪氨酸酶有激活作用,Al(NO3)3和MgSO4則起抑制作用。表面活性劑Tween-80、Triton X-100和EDTA對酪氨酸酶活力均有抑制作用,而SDS在低濃度時具有促進作用,高濃度時則起抑制作用。分別以酪氨酸、多巴和鄰苯二酚為底物,酶動力學反應(yīng)的Km分別為5.88、4.06和1.94mmol/L,Vmax分別為64.10、42.74和21.51μmol/mmin。 3、碳源中的葡萄糖、蔗糖、麥芽糖、果糖、乳糖和可溶性淀粉均對酪氨酸酶的合成有抑制作用,而天然復(fù)合物中的麥麩汁、氮源中的干酪素和無機鹽中的硫酸銅對酪氨酸酶產(chǎn)量具有顯著促進作用。采用中心組合試驗獲得最優(yōu)培養(yǎng)基組成為：麥麩汁36%、干酪素1.1g/L、硫酸銅0.13g/L。在此條件下,酪氨酸酶最大活力為17.22U/mL,比優(yōu)化前培養(yǎng)基中酪氨酸酶活力提高2.1倍。 Plackett-Burman試驗篩選結(jié)果表明,發(fā)酵條件中的時間、裝液量、接種量和起始pH是對酪氨酸酶產(chǎn)量有顯著影響的因素,經(jīng)中心組合試驗進一步優(yōu)化后,得到黑木耳產(chǎn)酪氨酸酶的最優(yōu)發(fā)酵條件為：時間3.47d、裝液量52.92mL、接種量7.39%和起始pH7.9。在此條件下,酪氨酸酶的最大產(chǎn)量為22.69U/mL,與預(yù)測值相符。 采用黑木耳搖瓶補料分批發(fā)酵技術(shù)制備黑色素時,酪氨酸最適添加時間為2d、添加量為1g/L、補加時間為4d、補加量為1.g/L,在此條件下,酪氨酸的轉(zhuǎn)化率為41.42%,黑色素產(chǎn)量達到1035.71mg/L。 4、黑木耳發(fā)酵液黑色素在表觀色澤上比子實體黑色素更趨近于黑色。兩種黑色素均不溶于水和常見的有機溶劑,僅溶于堿性溶液,且在pH小于3的酸性溶液中產(chǎn)生沉淀。黑木耳發(fā)酵液和子實體黑色素都能被氧化劑氧化漂白,而對還原劑穩(wěn)定。在紫外可見光譜范圍內(nèi)兩者均表現(xiàn)出強烈的吸收特性。 黑木耳發(fā)酵液黑色素和子實體黑色素均易溶于堿性溶液,在高pH環(huán)境中穩(wěn)定；兩者對溫度和光照敏感,隨強度的提高和時間的延長,損失變大；Zn2+、Cu2+、Fe3+和Fe2+等金屬離子可降低黑木耳色素的穩(wěn)定性。兩種黑木耳色素在白砂糖和檸檬酸溶液中不穩(wěn)定,但在其它食品添加劑溶液中較穩(wěn)定。 5、采用Sephadex G-100柱層析獲得黑木耳發(fā)酵液黑色素F1和F2兩組分,分子量分別為404.97和20.69kDa,兩者在紫外可見區(qū)域都有強烈的吸收。F1比F2具有更低的L*、b*、C*和h0,在外觀色澤上更趨近于黑色。 元素分析表明：F1和F2都含有C、H、N、O和S元素,屬于棕黑色素。紅外色譜和核磁氫譜表明F1和F2的結(jié)構(gòu)中均含有大量的羥基、羰基和芳環(huán)基團。經(jīng)氧化降解測得Fl中5,6-二羥基-2-吲哚羧酸占43.22%、5,6-二羥基吲哚占9.78%；F2中5,6-二羥基-2-吲哚羧酸占69.04%、5,6-二羥基吲哚占17.47%。 6、黑木耳色素組分F1和F2均具有較強的抗氧化能力,在還原力、超氧陰離子和羥自由基清除能力等方面均顯著高于BHT,并表現(xiàn)出劑量依賴性特征。黑木耳色素可有效保護乳酸菌免受紫外線照射導(dǎo)致的損傷,其效果受照射距離和照射時間等因素影響。
[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.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：Q936
本文編號：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.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：Q936
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