贻贝蛋白酶解制备活性肽的工艺研究及活性评价
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摘要
本文以紫贻贝和厚壳贻贝为实验材料，采用酶解、超滤、离子交换层析、凝胶渗透层析和高效液相色谱法等分离纯化手段，得到了贻贝活性肽，并对贻贝蛋白的酶解工艺、活性肽的制备工艺及其抗氧化和血管生成抑制活性进行了研究。
首先采用正交实验法，以羟自由基清除率为评价指标，分别研究了厚壳贻贝中性蛋白酶酶解工艺和紫贻贝碱性蛋白酶酶解工艺。结果如下：厚壳贻贝中性蛋白酶最佳工艺为酶解时间3h，酶解温度60℃，料液比1:2，加酶量3%；紫贻贝碱性蛋白酶最佳工艺为酶解温度45℃，酶解时间3h，pH8.0，加酶量3%，料液比1:1。在最佳条件下，10mg/mL厚壳贻贝和紫贻贝水解物溶液的羟自由基清除率分别为66.82%和80.97%。
分别将厚壳贻贝中性蛋白酶酶解物(MCNH)、紫贻贝碱性蛋白酶酶解物(MEAH)、紫贻贝木瓜蛋白酶酶解物(MEPH)和紫贻贝中性蛋白酶酶解物(MENH)通过截留分子量为10kDa和3kDa的超滤膜进行超滤分段，并分别测定其浓度10mg/mL时羟自由基清除率，选取清除率高的部分，即MCNH-1、MEAH-1、MEPH-1、MENH-2通过DEAE-52阴离子交换色谱进一步分离，选取羟自由基清除率高的部分即MCNH-1-1、MEAH-1-4、MEPH-1-6、MENH-2-1经Sephadex G-15进一步分离，对所得组分的羟自由基清除率进行评价；将紫贻贝中性蛋白酶酶解产物经超滤后得到的MW<3kDa的部分(MENH-3)直接经Sephadex G-15分离，得率最高的组分（MENH-3-3）经反相高效液相色谱Zorbax C18纯化后得到了纯度较高的活性肽（MNH），其分子量为574.68Da。随着浓度递增，MNH对羟自由基清除率也增加，浓度在5mg/mL时对羟自由基清除率为23.49%。
将以上各步骤中得到的抗氧化活性较高的部分，包括粗蛋白（MCNH-1、MEAH-1、MEPH-1、MENH-2、MENH-3）、离子峰（MCNH-1-1、MEAH-1-4、MEPH-1-6、MENH-2-1）、凝胶峰（MCNH-1-1-2、MEAH-1-4-1、MEPH-1-6-1、MENH-2-1-1、MENH-3-3）、不同浓度MNH进行鸡胚绒毛尿囊膜（CAM）血管生成抑制实验，结果表明：各样品对鸡胚绒毛尿囊膜血管的生成均有显著抑制作用，其中碱性蛋白酶酶解紫贻贝得到的粗蛋白、离子峰和凝胶峰，对鸡胚绒毛尿囊膜血管的抑制略优于其他蛋白酶酶解产物。
In the present experiment, the antioxidative hydrolysates from Mytilus edulis and Mytiluscoruscus muscle proteins were prepared by proteinase using single-factor and orthogonal tests. Thehydrolysates subsequently separated and purified by ultrafiltration (UF) memebrane, DEAE-52celluloseanion-exchange chromatography, Sephadex G-25gel filtration column and Reversed Phase HighPerformance Liquid Chromatography (RP-HPLC), and one new antioxidative peptide (MNH) wasisolated from the hydrolysate and and its molecular mass and N-terminal amino-acid sequencewas determined by using ESI-MS and Procise Protein/Peptide Sequencer. The antioxidantand angiogenesis inhibitory activities of hydrolysates, their fractions and purified peptidewere evaluated.
Inspired by OH scavenging assay, the efficient methods had been developed toacquire protein hydrolysates (MCNH and MEAH) from M. coruscus and M. edulis bysingle-factor and orthogonal tests, respectively. Under optimal hydrolysis parameters ofneutral protease (hydrolysis time3h, the hydrolysis temperature60℃, the solid-liquidratio1:2, enzyme dose3%), the OH scavenging rate of MCNH reached66.82%at theconcentration of10mg/mL. The OH scavenging rate of MEAH reached80.97%at theconcentration of10mg/mL under the optimum conditions of temperature45℃, hydrolysistime3h, pH8.0, the enzyme dose3%, solid-liquid ratio1:1for alcalase.
Based on the molecular weight (MW), MCNH, MEAH, MEPH (the hydrolysate fromM. edulis by papain) and MENH (the hydrolysate from M. edulis by neutral protease) werefractionated by ultrafiltration membrane system. The·OH scavenging rate of fractions atconcentration of10mg/mL were measured, and the selected fractions (MCNH-1,MEAH-1,MEPH-1, MENH-2) with higher antioxidant activities were separated through DEAE-52 cellulose anion-exchange chromatography and gel filtration chromatography, and the antioxidantactivities of resulted subfractions were evaluated. MENH-3was purified through aSephadex G-15gel filtration and RP-HPLC on Zorbax C18, and a new peptide (MNH) withmolecular weight of574.68Da was obtained. The hydroxyl radical scavenging ratesshowed dose dependency and reached23.49%at the concentration of5mg/mL.
The fractions with higher antioxidative activities, including crude protein (MCNH-1,MEAH-1, MEPH-1, MENH-2, MENH-3), fractions through ion chromatography(MCNH-1-1, MEAH-1-4, MEPH-1-6-2-1), fractions through gel chromatography(MCNH-1-1-2the MEAH-1-4-1, MEPH-1-6-1, MENH-2-1-1MENH-3-3) and MNH, werecarried on inhibiting formation of the blood vessels of the chick embryo chorioallantoicmembrane (CAM). The results indicated that all of the samples showed strength activitieson angiogenesis inhibitory, especially the fractions obtained from MEAH, which wasslightly better than the other protease hydrolysates.
首先采用正交实验法，以羟自由基清除率为评价指标，分别研究了厚壳贻贝中性蛋白酶酶解工艺和紫贻贝碱性蛋白酶酶解工艺。结果如下：厚壳贻贝中性蛋白酶最佳工艺为酶解时间3h，酶解温度60℃，料液比1:2，加酶量3%；紫贻贝碱性蛋白酶最佳工艺为酶解温度45℃，酶解时间3h，pH8.0，加酶量3%，料液比1:1。在最佳条件下，10mg/mL厚壳贻贝和紫贻贝水解物溶液的羟自由基清除率分别为66.82%和80.97%。
分别将厚壳贻贝中性蛋白酶酶解物(MCNH)、紫贻贝碱性蛋白酶酶解物(MEAH)、紫贻贝木瓜蛋白酶酶解物(MEPH)和紫贻贝中性蛋白酶酶解物(MENH)通过截留分子量为10kDa和3kDa的超滤膜进行超滤分段，并分别测定其浓度10mg/mL时羟自由基清除率，选取清除率高的部分，即MCNH-1、MEAH-1、MEPH-1、MENH-2通过DEAE-52阴离子交换色谱进一步分离，选取羟自由基清除率高的部分即MCNH-1-1、MEAH-1-4、MEPH-1-6、MENH-2-1经Sephadex G-15进一步分离，对所得组分的羟自由基清除率进行评价；将紫贻贝中性蛋白酶酶解产物经超滤后得到的MW<3kDa的部分(MENH-3)直接经Sephadex G-15分离，得率最高的组分（MENH-3-3）经反相高效液相色谱Zorbax C18纯化后得到了纯度较高的活性肽（MNH），其分子量为574.68Da。随着浓度递增，MNH对羟自由基清除率也增加，浓度在5mg/mL时对羟自由基清除率为23.49%。
将以上各步骤中得到的抗氧化活性较高的部分，包括粗蛋白（MCNH-1、MEAH-1、MEPH-1、MENH-2、MENH-3）、离子峰（MCNH-1-1、MEAH-1-4、MEPH-1-6、MENH-2-1）、凝胶峰（MCNH-1-1-2、MEAH-1-4-1、MEPH-1-6-1、MENH-2-1-1、MENH-3-3）、不同浓度MNH进行鸡胚绒毛尿囊膜（CAM）血管生成抑制实验，结果表明：各样品对鸡胚绒毛尿囊膜血管的生成均有显著抑制作用，其中碱性蛋白酶酶解紫贻贝得到的粗蛋白、离子峰和凝胶峰，对鸡胚绒毛尿囊膜血管的抑制略优于其他蛋白酶酶解产物。
In the present experiment, the antioxidative hydrolysates from Mytilus edulis and Mytiluscoruscus muscle proteins were prepared by proteinase using single-factor and orthogonal tests. Thehydrolysates subsequently separated and purified by ultrafiltration (UF) memebrane, DEAE-52celluloseanion-exchange chromatography, Sephadex G-25gel filtration column and Reversed Phase HighPerformance Liquid Chromatography (RP-HPLC), and one new antioxidative peptide (MNH) wasisolated from the hydrolysate and and its molecular mass and N-terminal amino-acid sequencewas determined by using ESI-MS and Procise Protein/Peptide Sequencer. The antioxidantand angiogenesis inhibitory activities of hydrolysates, their fractions and purified peptidewere evaluated.
Inspired by OH scavenging assay, the efficient methods had been developed toacquire protein hydrolysates (MCNH and MEAH) from M. coruscus and M. edulis bysingle-factor and orthogonal tests, respectively. Under optimal hydrolysis parameters ofneutral protease (hydrolysis time3h, the hydrolysis temperature60℃, the solid-liquidratio1:2, enzyme dose3%), the OH scavenging rate of MCNH reached66.82%at theconcentration of10mg/mL. The OH scavenging rate of MEAH reached80.97%at theconcentration of10mg/mL under the optimum conditions of temperature45℃, hydrolysistime3h, pH8.0, the enzyme dose3%, solid-liquid ratio1:1for alcalase.
Based on the molecular weight (MW), MCNH, MEAH, MEPH (the hydrolysate fromM. edulis by papain) and MENH (the hydrolysate from M. edulis by neutral protease) werefractionated by ultrafiltration membrane system. The·OH scavenging rate of fractions atconcentration of10mg/mL were measured, and the selected fractions (MCNH-1,MEAH-1,MEPH-1, MENH-2) with higher antioxidant activities were separated through DEAE-52 cellulose anion-exchange chromatography and gel filtration chromatography, and the antioxidantactivities of resulted subfractions were evaluated. MENH-3was purified through aSephadex G-15gel filtration and RP-HPLC on Zorbax C18, and a new peptide (MNH) withmolecular weight of574.68Da was obtained. The hydroxyl radical scavenging ratesshowed dose dependency and reached23.49%at the concentration of5mg/mL.
The fractions with higher antioxidative activities, including crude protein (MCNH-1,MEAH-1, MEPH-1, MENH-2, MENH-3), fractions through ion chromatography(MCNH-1-1, MEAH-1-4, MEPH-1-6-2-1), fractions through gel chromatography(MCNH-1-1-2the MEAH-1-4-1, MEPH-1-6-1, MENH-2-1-1MENH-3-3) and MNH, werecarried on inhibiting formation of the blood vessels of the chick embryo chorioallantoicmembrane (CAM). The results indicated that all of the samples showed strength activitieson angiogenesis inhibitory, especially the fractions obtained from MEAH, which wasslightly better than the other protease hydrolysates.
引文
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