CN105646700B - 赤魟软骨抗氧化胶原肽及其用途 - Google Patents
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Abstract
本发明公开了一种赤魟软骨抗氧化胶原肽及其用途。本发明以赤魟软骨为原料，通过胶原蛋白提取、胰蛋白酶和中性蛋白酶双酶酶解得酶解液，酶解液采用超滤、离子交换树脂层析、凝胶柱层析和反相高效液相色谱分离纯化得到抗氧化胶原肽Gly‑Ile‑Glu‑Gly‑Glu‑Glu‑Gly‑Trp，ESI‑MS测定分子量为875.85 Da；制备的高活性抗氧化胶原肽对DPPH自由基、羟自由基、ABTS自由基和超氧阴离子自由基具有良好的清除作用；同时亦显示出良好的脂质过氧化抑制作用，可以作为药品、保健食品或食品添加剂进行开发。
Description
技术领域
本发明涉及一种动物软骨胶原肽，具体涉及一种赤魟软骨抗氧化胶原肽及其用途。
背景技术
胶原肽是胶原蛋白被蛋白酶降解后的产物，分子量大约在180-3000 Da，被认为比较普通胶原蛋白更利人体消化吸收。同时，现有研究证明胶原肽具有多种生物活性，例如抑制血管紧张素转化酶活性、抗氧化活性，减少膝关节或髋关节等骨关节炎患者的疼痛，增强骨密度，维持骨代谢平衡等。
赤魟（Dasyatis akajei），属软骨鱼纲，下孔总目，鲼形目，魟科，魟属。赤魟为常见软骨鱼类，分布于我国沿海。研究表明赤魟软骨含有大量胶原蛋白，可以作为胶原肽的原料进行开发利用。
发明内容
本发明所要解决的技术问题是提供一种能够清除自由基和抑制脂质过氧化作用的赤魟软骨抗氧化胶原肽。
本发明为解决上述技术问题所采取的技术方案为：一种赤魟软骨抗氧化胶原肽：该赤魟软骨抗氧化胶原肽为八肽化合物，氨基酸序列为Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW)，ESI-MS测定分子量为875.85 Da。
该赤魟软骨抗氧化胶原肽的制备方法为：
1）赤魟软骨胶原蛋白的制备：将赤魟软骨晒干、粉碎，按照料液比1 g:15～20 mL加入到NaOH溶液（0.1 mol/L）于1～4℃下浸泡2～4 h，脱除非胶原蛋白；处理后赤魟软骨用蒸馏水反复洗涤至中性、沥干，按照料液比1 g: 5～8 mL加入pH 7.4的EDTA溶液（0.5 mol/L），于1～4℃下浸泡2～3天（每天更换1次EDTA溶液），蒸馏水洗涤2～3次，干燥、粉碎，得脱钙赤魟软骨粉。将脱钙赤魟软骨粉按照料液比1 g:5～8 mL加入到0.5 mol/L乙酸溶液并在1～4℃下动态浸泡3 d，10 000g离心20～25 min，取上清液，并加入NaCl至溶液终浓度为1.0 mol/L，静置60～90 min，12 000g离心25～30 min得沉淀物，冷冻干燥即为赤魟软骨胶原蛋白。
2）赤魟软骨胶原蛋白的酶解：将赤魟软骨胶原蛋白按照料液比1 g:15～20 mL加入到磷酸氢二钠-磷酸二氢钠缓冲液（pH 7.5，0.2 mol/L），按照胶原蛋白质量的1.0～1.5%向溶液中加入胰蛋白酶（1.9×104 U/g），于温度45～50℃酶解3～4 h后，将溶液升温至90～95℃，并于此温度保持8～10 min后，冷却至45～50℃；按照胶原蛋白质量的1.5～1.8%向溶液中加入中性蛋白酶（1.0×105 U/g），于温度45～50℃下酶解6～8 h，将溶液升温至90～95℃，并于此温度保持15～20 min后，10 000g离心20～25 min，取上清液，即为酶解产物；
3）赤魟软骨抗氧化胶原肽的制备：将上述酶解产物采用3.5 kDa超滤膜进行超滤处理，收集分子量小于3.5 kDa部分，冻干，得超滤酶解物；将超滤酶解物次经离子交换树脂层析、凝胶柱层析和反相高效液相色谱（RP-HPLC）纯化，得抗氧化胶原肽。
作为优选，所述的步骤1）中的赤魟为赤魟（Dasyatis akajei）。
作为优选，所述步骤4）的离子交换树脂层析、凝胶柱层析和RP-HPLC纯化的具体过程为：
离子交换树脂层析：将上述超滤酶解物溶于双蒸水配成浓度为25～30 mg/mL的溶液，经过DEAE-52离子交换树脂层析柱，用水、0.5 mol/L和1.0 mol/L NaCl溶液进行洗脱，流速为0.9～1.2 mL/min，洗出溶液每3 min收集一管并于225 nm检测，按峰合并试管内溶液，比较各峰的羟自由基的清除活性，选择活性最强组分冻干，即为离子交换层析酶解物；
凝胶柱层析：将上述离子交换层析酶解物溶于双蒸水配成浓度为25～30 mg/mL的溶液，经过葡聚糖凝胶Sephadex G-25柱层析分离，用双蒸水进行洗脱，流速为0.8～1.0mL/min，洗出溶液每3 min收集一管并于225 nm检测，按峰合并试管内溶液，比较各峰的羟自由基的清除活性，选择活性最强组分冻干，即为凝胶层析酶解物；
RP-HPLC纯化：将上述凝胶层析酶解物用双蒸水配成40～50 μg/mL的溶液，利用RP-HPLC进行纯化，根据对羟自由基的清除活性得1个高活性抗氧化胶原肽Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW)，ESI-MS测定分子量为875.85 Da。
再优选，所述RP-HPLC条件为：进样量15～20 μL；色谱柱Zorbax SB- C18（250 mm×4.6 mm，5 μm）；流动相：40%乙腈；洗脱速度0.8~1.0 mL/min；紫外检测波长225 nm。
本发明还提供赤魟软骨抗氧化胶原肽的用途：由于其具有安全无毒副作用、抗氧化活性强和易于消化吸收等优点，因而可以作为药品、保健食品和食品的添加剂。
与现有技术相比，本发明所提供的赤魟软骨抗氧化胶原肽对DPPH自由基、羟自由基和超氧阴离子自由基具有良好的清除作用；同时，Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp(GIEGEEGW)亦显示出良好的脂质过氧化抑制作用；Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp(GIEGEEGW)具有安全无毒副作用、抗氧化活性强和易于消化吸收等优点，可以作为药品、保健食品和食品添加剂。
附图说明
图1是本发明的DEAE-52离子交换树脂层析图。
图2是本发明的葡聚糖凝胶Sephadex G-25层析图。
图3葡聚糖凝胶Sephadex G-25制备酶解物的RP-HPLC分析。
图4 Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW)的质谱图。
图5 Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW)的抗脂质氧化能力。
具体实施方式
以下结合附图实施例对本发明作进一步详细描述。
实施例：
一种赤魟软骨抗氧化胶原肽的制备方法，制备工艺流程如下：赤魟软骨→软骨胶原蛋白→胰蛋白酶和中性蛋白酶双酶酶解→酶解物→超滤→离子交换层析→凝胶过滤层析→高效液相色谱制备→抗氧化胶原肽。
1）赤魟软骨胶原蛋白的制备：将赤魟（Dasyatis akajei）软骨晒干、粉碎，按照料液比1 g:18 mL加入到NaOH溶液（0.1 mol/L）于3℃下浸泡3 h，脱除非胶原蛋白；处理后赤魟软骨用蒸馏水反复洗涤至中性、沥干，按照料液比1 g: 6 mL加入pH 7.4的EDTA溶液（0.5mol/L），于4℃下浸泡3天（每天更换1次EDTA溶液），蒸馏水洗涤3次，干燥、粉碎，得脱钙赤魟软骨粉。将脱钙赤魟软骨粉按照料液比1 g:6 mL加入到0.5 mol/L乙酸溶液并在3℃下动态浸泡3 d，10 000g离心20 min，取上清液，并加入NaCl至溶液终浓度为1.0 mol/L，静置90min，12 000g离心25 min得沉淀物，冷冻干燥即为赤魟软骨胶原蛋白。
2）赤魟软骨胶原蛋白的酶解：将赤魟软骨胶原蛋白按照料液比1 g:15 mL加入到磷酸氢二钠-磷酸二氢钠缓冲液（pH 7.5，0.2 mol/L），按照胶原蛋白质量的1.3%向溶液中加入胰蛋白酶（1.9×104 U/g），于温度48℃酶解3.5 h后，将溶液升温至90℃，并于此温度保持10 min后，冷却至45℃；按照胶原蛋白质量的1.6%向溶液中加入中性蛋白酶（1.0×105U/g），于温度45℃下酶解6 h，将溶液升温至90℃，并于此温度保持15 min后，10 000g离心20 min，取上清液，即为酶解产物；
3）赤魟软骨抗氧化胶原肽的制备：将上述酶解产物采用3.5 kDa超滤膜进行超滤处理，收集分子量小于3.5 kDa部分，冻干，得超滤酶解物；将超滤酶解物次经离子交换树脂层析、凝胶柱层析和反相高效液相色谱（RP-HPLC）纯化，得抗氧化胶原肽。
①离子交换树脂层析：将上述超滤酶解物溶于双蒸水配成浓度为28 mg/mL的溶液，经过DEAE-52离子交换树脂层析柱，用水、0.5 mol/L和1.0 mol/L NaCl溶液进行洗脱，流速为1.0～1.2 mL/min，洗出溶液每3 min收集一管并于225 nm检测，按峰合并试管内溶液，比较各峰的羟自由基的清除活性，选择活性最强组分冻干，即为离子交换层析酶解物DCP-III（图1）；
②凝胶柱层析：将上述离子交换层析酶解物溶于双蒸水配成浓度为25 mg/mL的溶液，经过葡聚糖凝胶Sephadex G-25柱层析分离，用双蒸水进行洗脱，流速为1.0 mL/min，洗出溶液每3 min收集一管并于225 nm检测，按峰合并试管内溶液，比较各峰的羟自由基的清除活性，选择活性最强组分冻干，即为凝胶层析酶解物DCP-III-3（图2）；
③RP-HPLC纯化：将上述凝胶层析酶解物用双蒸水配成40 μg/mL的溶液，利用RP-HPLC进行纯化（RP-HPLC条件为：进样量18 μL；色谱柱Zorbax SB- C18（250 mm×4.6 mm，5 μm）；流动相：40%乙腈；洗脱速度0.8 mL/min；紫外检测波长225 nm)，根据对羟自由基的清除活性得1个高活性抗氧化胶原肽Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW)（图3）。
④结构检测：收集DPPH自由基和羟自由基清除活性最高的抗氧化胶原肽，经RP-HPLC检测为单一峰，利用蛋白/多肽序列分析仪测定氨基酸序列为Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW)，ESI-MS测定分子量为875.85 Da（[M+H]+ 876.81 Da）（图4）。
将制得的赤魟软骨抗氧化胶原肽Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp(GIEGEEGW)进行自由基清除实验和脂质过氧化抑制实验，实验结果表明：Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW)对DPPH 自由基（EC50 1.93 mg/mL）、羟自由基（EC500.35 mg/mL）、ABTS自由基（EC50 0.16 mg/mL）和超氧阴离子自由基（EC50 0.24 mg/mL）具有良好清除作用；同时，Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW)亦显示出良好的脂质过氧化抑制作用（图5）。
最后，还需要注意的是，以上列举的仅是本发明的一个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。
SEQUENCE LISTING
<110> 浙江海洋学院
<120> 赤魟软骨抗氧化胶原肽及其用途
<130> zjou-wb-201511-301
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 8
<212> PRT
<213> 人工合成
<400> 1
Gly Ile Glu Gly Glu Glu Gly Trp
1 5
Claims (1)
1.赤魟软骨抗氧化胶原肽，其特征在于：该抗氧化胶原肽为八肽化合物，氨基酸序列为Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp，ESI-MS测定分子量为875.85Da。
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