大米蛋白酶法制备抗氧化肽的研究
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摘要
以大米蛋白为原料研究抗氧化肽的制备技术及其性能。
采用酶水解大米蛋白的方法生产高活性的抗氧化肽。以酶解液对DPPH自由基清除率为考察指标,从七种商品蛋白酶中筛选出三种蛋白酶:风味酶、中性酶、木瓜蛋白酶;分别进行正交试验,得出三种酶在各自最佳作用条件下对DPPH自由基清除率分别为75.2%、89.5%和58.8%;大米抗氧化肽的EC_(50)值分别为2.92 mg/mL、2.17 mg/mL和3.86 mg/mL。在各自最佳反应条件下得到的三种酶的水解产物,进行DPPH自由基清除率和EC_(50)值的横向比较,最终确定中性蛋白酶为本试验用酶,其最佳制备工艺参数为,底物浓度10g/100ml,加酶量8%,酶解pH 6.0,酶解温度60℃,时间30min。
在大米蛋白单酶水解实验基础上,将选出的三种酶两两组合,进行双酶分步水解试验,发现加酶顺序对水解产物的抗氧化活性有较大的影响;同时第一步酶解后灭酶明显优于不灭酶。最终比较得到最优组合:大米蛋白先经中性酶水解30min,灭酶后再由木瓜酶水解15min,得到的酶水解产物EC_(50)为1.23mg/mL,与单酶相比下降了43.3%。
在超声功率1500W、超声初始温度40℃条件下超声处理大米蛋白溶液20min,再用中性酶水解制备抗氧化肽,其DPPH自由基清除率可达到74.8%,EC_(50)为0.932 mg/mL。超声处理原料使水解产物的EC_(50)值比没做处理的显著下降了57.1%。
研究了制备得到多肽的抗氧化性能试验,发现大米抗氧化肽具有较强的清除O~(2。-)、DPPH自由基、羟自由基,其EC_(50)分别为0.0678 mg/mL、0.932 mg/mL和0.117mg/mL。
以732阳离子交换树脂、SepHadex-G25和HPLC制备柱纯化大米抗氧化肽,最终研究得到两种抗氧化肽。使用美国ABI的串联飞行质谱仪4700 TOF/TOF对分离到的抗氧化肽进行结构分析,得到两个多肽序列分别为Gly-Val-Pro-Ser-Ala-Val-Ser-Arg-Asn-Met-Thr-Arg-Ser-Arg和Leu-Ala-Ser-Gln-His-Leu-Val-Ala-Ile-Leu-Asp-Pro-Pro-Arg,其EC_(50)值分别为:4.91μM和5.39μM。
The preparation technology of anti-oxidant peptides,which derived from hydrolysis of rice protein by enzyme,and the peptides's characteristics were investigated in this study.
Three commercial proteases,flavor,neutral,and papain,were selected from seven ones according to the elimination rates of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical of hydrolytes.Orthogonal tests were performed to determin the respective optimum hydrolysis condition of each protease.And the elimination rate of DPPH free radical of each optimum hydrolyzed solution were 75.2%,89.5%and 58.8%,respectively.The values of EC_(50) of the anti-oxidant peptide products were 2.92 mg/mL,2.17 mg/mL,and 3.86 mg/mL,respectively.The neutral protease was preferred because the hydrolate by the neutral protease showed the highest anti-oxidant activity in the three ones.And the optimal enzymatic hydrolysis technologic parameters were as followings:the concentration of substrate 10g/100ml, the quantity of protease 8%,pH 6.0 at 60℃for 30 min.
The combinations of two enzymatic hydrolysis were tested based on single enzymatic hydrolysis for rice protein.It was found that the adding order of enzymes affected the anti-oxidant activity of hydrolyte obviously.It is advantageous to de-active the enzyme after the first enzymatic hydrolysis.For enzymatic hydrolysis of rice protein,the optimal combination was:firstly neutral protease hydrolysis for 30 min,then papain hydrolysis for 15min after de-active neutral protease.The EC_(50) of hydrolysate solution was 1.23mg/mL,decreased 43.3%compared with single enzymatic hydrolysis.
The raw material of rice protein was pretreated by ultrasound(power 1500W,at initial temperature 40℃for 20min),then hydrolyzed with neutral protease.The results showed that the elimination rate of DPPH and EC_(50) were 74.8%and 0.932 mg/mL,respectively.The EC_(50) dropped 57.1%compared with non-pretreated sample. The peptides were applied for the eliminations of O~(2。),DPPH and OH°free radicals and the EC_(50) were 0.0678 mg/mL,0.932 mg/mL and 0.117 mg/mL,respectively.
732 cation exchange resin chromatography,Sephadex-G25 chromatography,and HPLC were employed for the purification of enzymatic hydrolyte.Two anti-oxidant peptides were obtained after the purification.According to the structural analysis by ABI 4700 TOF/TOF,the amino acid sequences of the two peptides were Gly-Val-Pro-Ser-Ala-Val-Ser-Arg-Asn-Met-Thr-Arg-Ser-Arg and Leu-Ala-Ser-Gln-His-Leu-Val-Ala-Ile-Leu-Asp-Pro-Pro-Arg. The EC_(50) were 4.91μM and 5.39μM, respectively.
采用酶水解大米蛋白的方法生产高活性的抗氧化肽。以酶解液对DPPH自由基清除率为考察指标,从七种商品蛋白酶中筛选出三种蛋白酶:风味酶、中性酶、木瓜蛋白酶;分别进行正交试验,得出三种酶在各自最佳作用条件下对DPPH自由基清除率分别为75.2%、89.5%和58.8%;大米抗氧化肽的EC_(50)值分别为2.92 mg/mL、2.17 mg/mL和3.86 mg/mL。在各自最佳反应条件下得到的三种酶的水解产物,进行DPPH自由基清除率和EC_(50)值的横向比较,最终确定中性蛋白酶为本试验用酶,其最佳制备工艺参数为,底物浓度10g/100ml,加酶量8%,酶解pH 6.0,酶解温度60℃,时间30min。
在大米蛋白单酶水解实验基础上,将选出的三种酶两两组合,进行双酶分步水解试验,发现加酶顺序对水解产物的抗氧化活性有较大的影响;同时第一步酶解后灭酶明显优于不灭酶。最终比较得到最优组合:大米蛋白先经中性酶水解30min,灭酶后再由木瓜酶水解15min,得到的酶水解产物EC_(50)为1.23mg/mL,与单酶相比下降了43.3%。
在超声功率1500W、超声初始温度40℃条件下超声处理大米蛋白溶液20min,再用中性酶水解制备抗氧化肽,其DPPH自由基清除率可达到74.8%,EC_(50)为0.932 mg/mL。超声处理原料使水解产物的EC_(50)值比没做处理的显著下降了57.1%。
研究了制备得到多肽的抗氧化性能试验,发现大米抗氧化肽具有较强的清除O~(2。-)、DPPH自由基、羟自由基,其EC_(50)分别为0.0678 mg/mL、0.932 mg/mL和0.117mg/mL。
以732阳离子交换树脂、SepHadex-G25和HPLC制备柱纯化大米抗氧化肽,最终研究得到两种抗氧化肽。使用美国ABI的串联飞行质谱仪4700 TOF/TOF对分离到的抗氧化肽进行结构分析,得到两个多肽序列分别为Gly-Val-Pro-Ser-Ala-Val-Ser-Arg-Asn-Met-Thr-Arg-Ser-Arg和Leu-Ala-Ser-Gln-His-Leu-Val-Ala-Ile-Leu-Asp-Pro-Pro-Arg,其EC_(50)值分别为:4.91μM和5.39μM。
The preparation technology of anti-oxidant peptides,which derived from hydrolysis of rice protein by enzyme,and the peptides's characteristics were investigated in this study.
Three commercial proteases,flavor,neutral,and papain,were selected from seven ones according to the elimination rates of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical of hydrolytes.Orthogonal tests were performed to determin the respective optimum hydrolysis condition of each protease.And the elimination rate of DPPH free radical of each optimum hydrolyzed solution were 75.2%,89.5%and 58.8%,respectively.The values of EC_(50) of the anti-oxidant peptide products were 2.92 mg/mL,2.17 mg/mL,and 3.86 mg/mL,respectively.The neutral protease was preferred because the hydrolate by the neutral protease showed the highest anti-oxidant activity in the three ones.And the optimal enzymatic hydrolysis technologic parameters were as followings:the concentration of substrate 10g/100ml, the quantity of protease 8%,pH 6.0 at 60℃for 30 min.
The combinations of two enzymatic hydrolysis were tested based on single enzymatic hydrolysis for rice protein.It was found that the adding order of enzymes affected the anti-oxidant activity of hydrolyte obviously.It is advantageous to de-active the enzyme after the first enzymatic hydrolysis.For enzymatic hydrolysis of rice protein,the optimal combination was:firstly neutral protease hydrolysis for 30 min,then papain hydrolysis for 15min after de-active neutral protease.The EC_(50) of hydrolysate solution was 1.23mg/mL,decreased 43.3%compared with single enzymatic hydrolysis.
The raw material of rice protein was pretreated by ultrasound(power 1500W,at initial temperature 40℃for 20min),then hydrolyzed with neutral protease.The results showed that the elimination rate of DPPH and EC_(50) were 74.8%and 0.932 mg/mL,respectively.The EC_(50) dropped 57.1%compared with non-pretreated sample. The peptides were applied for the eliminations of O~(2。),DPPH and OH°free radicals and the EC_(50) were 0.0678 mg/mL,0.932 mg/mL and 0.117 mg/mL,respectively.
732 cation exchange resin chromatography,Sephadex-G25 chromatography,and HPLC were employed for the purification of enzymatic hydrolyte.Two anti-oxidant peptides were obtained after the purification.According to the structural analysis by ABI 4700 TOF/TOF,the amino acid sequences of the two peptides were Gly-Val-Pro-Ser-Ala-Val-Ser-Arg-Asn-Met-Thr-Arg-Ser-Arg and Leu-Ala-Ser-Gln-His-Leu-Val-Ala-Ile-Leu-Asp-Pro-Pro-Arg. The EC_(50) were 4.91μM and 5.39μM, respectively.
引文
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