绿豆抗氧化活性肽的制备及其抗氧化活性研究
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摘要
绿豆在我国的种植范围广、产量高,其富含淀粉及蛋白质。目前对于绿豆的加工应用主要限于淀粉的生产,而忽略蛋白的开发,造成浪费和环境污染。绿豆蛋白功效比高,具有较高的营养价值及可开发性。本文以绿豆为原料,研究了绿豆抗氧化活性肽的酶解制备工艺,采用SephadexG-25凝胶柱色谱对酶解产物进行分离纯化,采用高效液相色谱和氨基酸分析仪对绿豆抗氧化活性肽的分子量分布及氨基酸组成进行了分析,对绿豆抗氧化活性肽的体外抗氧化能力进行了评价,结果如下：
(1)采用传统碱提酸沉法制备绿豆蛋白,一次提取率可达73.49％,所制备的绿豆蛋白粉中蛋白质的含量为75.97％。
(2)中性蛋白酶为绿豆抗氧化活性肽制备的理想蛋白酶,其最佳酶解工艺参数为：底物浓度2%,酶解pH 6.5,酶解温度50℃,加酶量5000u/g,酶解时间120min。在此工艺下得到的酶解产物抗氧化能力最高,其对羟自由基的清除率为58.02％。
(3)绿豆酶解产物经超滤分离,被分离成分子量大小不同的五种组分,其分子量范围分别为：<1KDa、1KDa-5KDa、5KDa-10KDa、10KDa-30KDa、>30KDa。其中1KDa-5KDa的组分所占的质量比重最大,且其抗氧化能力最强,清除羟自由基能力和清除DPPH自由基的能力分别达到了65.91％和40.89％。
(4)分子量范围为1KDa-5KDa的绿豆酶解产物经SephadexG-25凝胶柱色谱进一步分离纯化后,得到了两种分子量分别为3426Da和1272Da的绿豆抗氧化活性肽T,和T2。T1和T2均有较强的抗氧化能力,T1对羟自由基和DPPH自由基的清除率分别为69.14％和58.62％；T2对羟自由基和DPPH自由基的清除率分别为91.70％和74.68％。
(5)采用HPLC法对绿豆抗氧化活性肽T1和T2的纯度进行了检测,T1的纯度为85.92%,T2的纯度为94.99％。
(6)用全自动氨基酸分析仪对制备的绿豆抗氧化活性肽T1和Tz的氨基酸组成进行了检测分析,检测结果显示绿豆抗氧化活性肽T1和T2的氨基酸组成合理,且富含各种与抗氧化活性相关性较强的氨基酸残基。
(7)以Vc和BHT作为对照,对所制备的绿豆抗氧化活性肽的总还原能力进行测定。结果表明绿豆抗氧化活性肽具有较强的总还原能力,其还原能力和BHT的还原能力接近。
(8)绿豆抗氧化活性肽对超氧阴离子具有较强的清除能力,当浓度为1.Omg/mL时,绿豆抗氧化活性肽对超氧阴离子的的清除率可达60.07％,高于同浓度的BHT对超氧阴离子的清除能力。
(9)采用邻二氮菲-Fe2-法测定了绿豆抗氧化活性肽对羟自由基的清除能力。测定结果表明,绿豆抗氧化活性肽对羟自由基具有较强的清除能力。当浓度为25mg/mL时,绿豆抗氧化活性肽对羟自由基的清除率为70.51％, Vc对羟自由基的清除率79.83％, BHT对羟自由基的清除率53.27％,在该浓度下,绿豆抗氧化活性肽对羟自由基的清除能力和Vc的接近,而显著大于BHT的。
(10)以Vc和BHT为对照,研究了绿豆抗氧化活性肽对DPPH自由基的清除能力。结果表明,绿豆抗氧化活性肽对DPPH自由基具有较强的清除能力。当浓度1.Omg/mL时,绿豆抗氧化活性肽对DPPH自由基的清除率为32.54％,BHT对DPPH自由基的清除率25.67％,在该浓度下,绿豆抗氧化活性肽对DPPH自由基的清除能力强于BHT的。
(11)体外抗氧化活性研究结果表明,本实验所制备的绿豆抗氧化活性肽具有较强的体外抗氧化活性,抗氧化能力高于BHT,而低于Vc。
Mung bean has high output, rich starch and protein, it is cultivation that is wide range in our country. Currently, processing applications for the mung bean mainly is confined to the starch's production, while ignoring the development of protein, resulting in wasting and environmental pollution. Mung bean's protein ratio are efficient, is had a high nutritional value and can be developed resistance. This paper mainly is studied antioxidant activity of mung bean peptide preparation, purification, composition and in vitro antioxidant capacity of preliminary, the results as follows:
(1)Preparation of the traditional protein (alkaline extraction and acid precipitation) prepared mung bean protein, the first extraction, the extraction rate can be reached 73.49%, the preparation of the mung bean protein purity of 75.97%.
(2)Neutral protease for mung bean autioxidant activity of the preparation of the ideal protease, peptide its optimum parameters is enzymatic hydrolysis:substrate concentration of 2%, hydrolysis pH 6.5, reaction temperature 50℃, enzyme concentration 5000u/g, reaction time 120min. In the process hydrolysates is obtained under the highest antioxidant capacity, its rate of hydroxyl radical scavenging 58.02%.
(3)Mung bean hydrolysates by ultrafiltration, is separated into five different molecular weight fractions, the molecular weight range:<1KDa, 1KDa-5KDa,5KDa-10KDa,10KDa-30KDa,> 30KDa.1KDa-5KDa which share the quality of the components of the largest share, and the strongest antioxidant, scavenging DPPH radical capacity and ability to remove 65.91%, respectively, and reached 40.89%.
(4)Molecular weight range of the mung bean enzyme 1KDa-5KDa SephadexG-25 gel products were further purified by column chromatography, the two molecular weights were obtained 3426Da, 1272Da antioxidant activity of mung bean peptide T1 and T2. T1 and T2 have a strong antioxidant capacity, T1 of the hydroxyl radical and DPPH radical scavenging rate was 69.14% and 58.62%; T2 on hydroxyl radicals and DPPH radical scavenging rate was 91.70% and 74.68%.
(5)Using HPLC method antioxidant activity of mung bean T1 and T2 of the purity of peptide were detected, chromatography results show that:T1 purity of 85.92%, T2 purity of 94.99%.
(6)With automatic amino acid analyzer on the preparation of antioxidant activity of mung bean peptide amino acid composition of T1 and T2 were examined analysis results showed that antioxidant activity of mung bean and T2 peptide T1 reasonable amino acid composition, and rich in variety and strong antioxidant activity relationships of amino acid residues.
(7)To Vc and BHT contrast, prepared for the antioxidant activity of mung bean total reducing capacity of peptide were determined. The results show that the antioxidant activity of mung bean total peptide is had strong reducing power, reducing power and its ability to close the reduction of BHT.
(8)Mung bean antioxidant superoxide anion activated carbon is had a strong scavenging, when the concentration of 1.0mg/mL, mung bean antioxidant peptide on superoxide anion scavenging rate of 60.07%, higher than the same concentration BHT scavenging of superoxide anion.
(9)Using phenanthroline-Fe2+method for the determination of the antioxidant activity of mung bean peptide to hydroxyl radical scavenging. Test showed that antioxidant activity of mung bean peptide is had a strong hydroxyl radical scavenging. When the concentration of 25mg/mL, the antioxidant activity of mung bean peptide clearance rate of hydroxyl radical 70.51%, Vc rate of hydroxyl radical scavenging 79.83%, BHT on hydroxyl radical scavenging rate 53.27%, the concentration, mung bean peptide antioxidant activity on hydroxyl radical scavenging capacity and Vc close to, but significantly greater than BHT's.
(10)To Vc and BHT as a control to study the antioxidant activity of mung bean peptide on DPPH radical scavenging. The results showed that the antioxidant activity of mung bean peptide on strong DPPH radical scavenging ability. When the concentration of 1.0mg/mL, samples of DPPH radical scavenging rate was 32.54%, BHT on DPPH radical scavenging rate of 25.67%, the concentration of mung bean peptide antioxidant free radical scavenging DPPH stronger than BHT's.
(11)ln vitro results show that the antioxidant activity, Mung bean is prepared in this experiment antioxidant peptide is had strong antioxidant activity, antioxidant activity than BHT, but lower than Vc.
(1)采用传统碱提酸沉法制备绿豆蛋白,一次提取率可达73.49％,所制备的绿豆蛋白粉中蛋白质的含量为75.97％。
(2)中性蛋白酶为绿豆抗氧化活性肽制备的理想蛋白酶,其最佳酶解工艺参数为：底物浓度2%,酶解pH 6.5,酶解温度50℃,加酶量5000u/g,酶解时间120min。在此工艺下得到的酶解产物抗氧化能力最高,其对羟自由基的清除率为58.02％。
(3)绿豆酶解产物经超滤分离,被分离成分子量大小不同的五种组分,其分子量范围分别为：<1KDa、1KDa-5KDa、5KDa-10KDa、10KDa-30KDa、>30KDa。其中1KDa-5KDa的组分所占的质量比重最大,且其抗氧化能力最强,清除羟自由基能力和清除DPPH自由基的能力分别达到了65.91％和40.89％。
(4)分子量范围为1KDa-5KDa的绿豆酶解产物经SephadexG-25凝胶柱色谱进一步分离纯化后,得到了两种分子量分别为3426Da和1272Da的绿豆抗氧化活性肽T,和T2。T1和T2均有较强的抗氧化能力,T1对羟自由基和DPPH自由基的清除率分别为69.14％和58.62％；T2对羟自由基和DPPH自由基的清除率分别为91.70％和74.68％。
(5)采用HPLC法对绿豆抗氧化活性肽T1和T2的纯度进行了检测,T1的纯度为85.92%,T2的纯度为94.99％。
(6)用全自动氨基酸分析仪对制备的绿豆抗氧化活性肽T1和Tz的氨基酸组成进行了检测分析,检测结果显示绿豆抗氧化活性肽T1和T2的氨基酸组成合理,且富含各种与抗氧化活性相关性较强的氨基酸残基。
(7)以Vc和BHT作为对照,对所制备的绿豆抗氧化活性肽的总还原能力进行测定。结果表明绿豆抗氧化活性肽具有较强的总还原能力,其还原能力和BHT的还原能力接近。
(8)绿豆抗氧化活性肽对超氧阴离子具有较强的清除能力,当浓度为1.Omg/mL时,绿豆抗氧化活性肽对超氧阴离子的的清除率可达60.07％,高于同浓度的BHT对超氧阴离子的清除能力。
(9)采用邻二氮菲-Fe2-法测定了绿豆抗氧化活性肽对羟自由基的清除能力。测定结果表明,绿豆抗氧化活性肽对羟自由基具有较强的清除能力。当浓度为25mg/mL时,绿豆抗氧化活性肽对羟自由基的清除率为70.51％, Vc对羟自由基的清除率79.83％, BHT对羟自由基的清除率53.27％,在该浓度下,绿豆抗氧化活性肽对羟自由基的清除能力和Vc的接近,而显著大于BHT的。
(10)以Vc和BHT为对照,研究了绿豆抗氧化活性肽对DPPH自由基的清除能力。结果表明,绿豆抗氧化活性肽对DPPH自由基具有较强的清除能力。当浓度1.Omg/mL时,绿豆抗氧化活性肽对DPPH自由基的清除率为32.54％,BHT对DPPH自由基的清除率25.67％,在该浓度下,绿豆抗氧化活性肽对DPPH自由基的清除能力强于BHT的。
(11)体外抗氧化活性研究结果表明,本实验所制备的绿豆抗氧化活性肽具有较强的体外抗氧化活性,抗氧化能力高于BHT,而低于Vc。
Mung bean has high output, rich starch and protein, it is cultivation that is wide range in our country. Currently, processing applications for the mung bean mainly is confined to the starch's production, while ignoring the development of protein, resulting in wasting and environmental pollution. Mung bean's protein ratio are efficient, is had a high nutritional value and can be developed resistance. This paper mainly is studied antioxidant activity of mung bean peptide preparation, purification, composition and in vitro antioxidant capacity of preliminary, the results as follows:
(1)Preparation of the traditional protein (alkaline extraction and acid precipitation) prepared mung bean protein, the first extraction, the extraction rate can be reached 73.49%, the preparation of the mung bean protein purity of 75.97%.
(2)Neutral protease for mung bean autioxidant activity of the preparation of the ideal protease, peptide its optimum parameters is enzymatic hydrolysis:substrate concentration of 2%, hydrolysis pH 6.5, reaction temperature 50℃, enzyme concentration 5000u/g, reaction time 120min. In the process hydrolysates is obtained under the highest antioxidant capacity, its rate of hydroxyl radical scavenging 58.02%.
(3)Mung bean hydrolysates by ultrafiltration, is separated into five different molecular weight fractions, the molecular weight range:<1KDa, 1KDa-5KDa,5KDa-10KDa,10KDa-30KDa,> 30KDa.1KDa-5KDa which share the quality of the components of the largest share, and the strongest antioxidant, scavenging DPPH radical capacity and ability to remove 65.91%, respectively, and reached 40.89%.
(4)Molecular weight range of the mung bean enzyme 1KDa-5KDa SephadexG-25 gel products were further purified by column chromatography, the two molecular weights were obtained 3426Da, 1272Da antioxidant activity of mung bean peptide T1 and T2. T1 and T2 have a strong antioxidant capacity, T1 of the hydroxyl radical and DPPH radical scavenging rate was 69.14% and 58.62%; T2 on hydroxyl radicals and DPPH radical scavenging rate was 91.70% and 74.68%.
(5)Using HPLC method antioxidant activity of mung bean T1 and T2 of the purity of peptide were detected, chromatography results show that:T1 purity of 85.92%, T2 purity of 94.99%.
(6)With automatic amino acid analyzer on the preparation of antioxidant activity of mung bean peptide amino acid composition of T1 and T2 were examined analysis results showed that antioxidant activity of mung bean and T2 peptide T1 reasonable amino acid composition, and rich in variety and strong antioxidant activity relationships of amino acid residues.
(7)To Vc and BHT contrast, prepared for the antioxidant activity of mung bean total reducing capacity of peptide were determined. The results show that the antioxidant activity of mung bean total peptide is had strong reducing power, reducing power and its ability to close the reduction of BHT.
(8)Mung bean antioxidant superoxide anion activated carbon is had a strong scavenging, when the concentration of 1.0mg/mL, mung bean antioxidant peptide on superoxide anion scavenging rate of 60.07%, higher than the same concentration BHT scavenging of superoxide anion.
(9)Using phenanthroline-Fe2+method for the determination of the antioxidant activity of mung bean peptide to hydroxyl radical scavenging. Test showed that antioxidant activity of mung bean peptide is had a strong hydroxyl radical scavenging. When the concentration of 25mg/mL, the antioxidant activity of mung bean peptide clearance rate of hydroxyl radical 70.51%, Vc rate of hydroxyl radical scavenging 79.83%, BHT on hydroxyl radical scavenging rate 53.27%, the concentration, mung bean peptide antioxidant activity on hydroxyl radical scavenging capacity and Vc close to, but significantly greater than BHT's.
(10)To Vc and BHT as a control to study the antioxidant activity of mung bean peptide on DPPH radical scavenging. The results showed that the antioxidant activity of mung bean peptide on strong DPPH radical scavenging ability. When the concentration of 1.0mg/mL, samples of DPPH radical scavenging rate was 32.54%, BHT on DPPH radical scavenging rate of 25.67%, the concentration of mung bean peptide antioxidant free radical scavenging DPPH stronger than BHT's.
(11)ln vitro results show that the antioxidant activity, Mung bean is prepared in this experiment antioxidant peptide is had strong antioxidant activity, antioxidant activity than BHT, but lower than Vc.
引文
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