CN108250291B - 一种抗氧化骨胶原多肽及其制备方法 - Google Patents
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Abstract
本发明涉及一种抗氧化骨胶原多肽及其制备方法，其解决了现有产品来源单一、存在免疫原性的技术问题，其氨基酸序列如序列表1。本发明同时提供一种抗氧化骨胶原多肽的制备方法，其以金枪鱼骨为原料来提取胶原蛋白，然后采用胰‑糜复合酶对其进行酶解，分离纯化、冷冻干燥得到抗氧化骨胶原多肽。本发明可用于抗氧化多肽的制备领域。
Description
技术领域
本发明涉及一种多肽及其制备方法，具体地说是一种抗氧化骨胶原多肽及其制备方法。
背景技术
生物分子的氧化是一个自由基介导的过程，它会对食品和生物系统造成许多不利的影响。动脉硬化、癌症等多种疾病相关的有力自由基会不可避免的随着养代谢的过程而产生。在食品中，食品营养成分的氧化会产生过氧化物，其不仅会影响是食用价值，引起食品品质降低，严重的甚至会导致摄入者的身体发生疾病。因此，寻找安全的抗氧化剂一致时生化营养学的研究热点。虽然人工合成的抗氧化剂相对于天然抗氧化剂相比要便宜很多，但是会对人体产生伤害，因此，需要寻找一种安全的天然抗氧化剂。
胶原蛋白是动物体中普遍存在的一种大分子蛋白质，其主要存在于动物的结缔组织中。胶原蛋白因其具有良好的生物相容性、生物活性及生物可降解性被广泛地应用在食品、医药、组织工程、化妆品等领域中。胶原水解物是胶原蛋白在一定条件下适度水解而得到的水解物。与胶原蛋白相比，胶原水解物的分子量低、水溶解性较好，易被人体吸收。与此同时，胶原蛋白的水解物也具有了更多的生物活性，如：抗氧化性，延缓衰老，补钙促进骨骼发育提高免疫力，促进皮肤细胞再生等。
目前的胶原水解物主要来源于猪、牛等动物。但是，这些胶原水解物可能存在免疫原性、宗教信仰等问题。随着国内水产加工业的发展，水产加工企业产生的下脚料如鱼头、鱼皮、鱼鳞、鱼骨、鱼鳍等逐年增长。但是，水产加工下脚料的利用并不充分，造成资源浪费。
发明内容
本发明就是为了解决现有产品来源单一、存在免疫原性的技术问题，提供一种抗氧化性能良好、不存在免疫原性的抗氧化骨胶原多肽及的制备方法。
为此，本发明提供一种抗氧化骨胶原多肽，其氨基酸序列如序列表1所示，所述抗氧化骨胶原多肽肽链含有两个蛋氨酸残基。
本发明提供一种抗氧化骨胶原多肽的制备方法，其以金枪鱼骨为原料来提取胶原蛋白，然后采用胰-糜复合酶对其进行酶解，分离纯化、冷冻干燥得到抗氧化骨胶原多肽。
优选的，酶解条件为：pH为8，温度37℃，酶解时间为2h，胰蛋白酶的质量浓度为0.5％，糜蛋白酶质量浓度为0.1％。
优选的，分离纯化手段包括DEAE-52纤维素离子交换色谱，RP-HPLC反相高效液相色谱。
优选的，纯化步骤为：(1)酶解产物用DEAE-52纤维素阴离子交换色谱进行分离，上样后洗去未吸附组分，再以pH 7.2磷酸缓冲液进行以0.1，0.5，1.0mol/L的NaCl溶液进行洗脱，流速为1.0mL/min，在280nm下进行测量，测定各吸收峰对应的洗脱组分的抗氧化活性；(2)收集具有最佳抗氧化活性的峰，再利用RP-HPLC反相高效液相色谱进一步分离，所用的色谱柱为Zorbax SB-C18，上样量为20μL，流速为1.0mL/min，检测波长为280nm，洗脱液自含体积比2％乙腈和98％水的混合液开始，至体积比为98％乙腈和2％水，进行梯度洗脱；收集在27.6min处的洗脱峰，即得到抗氧化骨胶原多肽。然后利用MS/MS鉴定多肽的氨基酸序列。
本发明同时提供一种抗氧化骨胶原多肽在清除DPPH自由基中的应用。
优选的，本发明提供的抗氧化骨胶原多肽在清除DPPH自由基中的应用，抗氧化骨胶原多肽半抑制浓度IC50为3.149mM。
本发明所选用的金枪鱼胶原中含有大量的具有抗氧化活性的氨基酸，比如芳香族氨基酸，赖氨酸，精氨酸等等。这些氨基酸暴露在多肽一端可以增强多肽的抗氧化性能。胰蛋白酶可以剪切蛋白酶的赖氨酸、精氨酸位点。糜蛋白酶可以剪切芳香族氨基酸位点。因此我们选用胰-糜复合蛋白酶来水解金枪鱼骨胶原，得到高抗氧化性能的水解物，并进一步分离提纯出高抗氧化性能的多肽。
DPPH自由基是一种合成的、具有单电子、稳定的、以氮为中心的顺磁化合物。当自由基清除剂存在时，DPPH自由基接受一个电子或氢原子，形成稳定的DPPH-H化合物，使其甲醇(或乙醇)溶液从深紫色变为黄色，变色程度与其接受的电子数量(自由基清除活性)成定量关系，因而可用分光光度计进行快速的定量分析DPPH自由基清除率测定方法，并将其作为评价纯化合物的抗氧化性能的标准之一。
本发明立足于寻找一种天然高校的抗氧化剂，以来自于金枪鱼骨胶原为出发点，通过胰-糜蛋白酶的切割控制，切割为具有特定结构组成的活性多肽，而使抗氧化性能得以高效的实现；并且本发明还改善了我国对金枪鱼骨利用率偏低的情况，既可解决大量金枪鱼骨资源的高效利用问题，又能接触消费者对抗氧化剂在食品安全方面的顾虑，对科技、经济和食品业的发展将具有深远的意义。
附图说明：
图1为本发明抗氧化骨胶原多肽的RP-HPLC图谱；
图2为本发明纯化的抗氧化骨胶原多肽清除DPPH自由基的“量-效”关系曲线。
具体实施方式
下面结合实施例，对本发明做进一步说明，但本发明的保护范围并不限于此。
实例例1
本发明提供的制备方法如下：
(1)金枪鱼骨胶原水解物的提取
取金枪鱼骨10g清洗干净，用1.25mol/L盐酸浸泡12小时，每1.5小时换一次酸除去无机盐。然后将金枪鱼骨清洗至pH为7.0，然后在酶解条件为pH为8，温度37℃，酶解时间为2h，按照胰蛋白酶占体积浓度为0.5％，糜蛋白酶占体积浓度为0.1％条件加入胰蛋白酶和糜蛋白酶，收集上清液，经过滤、浓缩和冷冻干燥后得到金枪鱼骨胶原水解物。
(2)酶解产物的分离、纯化
将酶解产物用DEAE-52纤维素阴离子胶原色谱进行分离，上样后洗去未吸附组分，再以pH 7.2磷酸缓冲液进行以0.1，0.5，1.0mol/L的NaCl溶液进行洗脱，流速为1.0mL/min，在280nm下进行测量，测定各吸收峰对应的洗脱组分的抗氧化活性；收集具有最佳抗氧化活性的峰，再利用RP-HPLC反相高效液相色谱进一步分离，所用的色谱柱为Zorbax SB-C18，上样量为20μL，流速为1.0mL/min，检测波长为280nm，洗脱液自含体积比2％乙腈和98％水的混合液开始，至体积比为98％乙腈和2％水，进行梯度洗脱。收集在27.6min处的洗脱峰，即得到本发明多肽。纯化后的多肽具有很强的抗氧化性能力，如图2所示。
(3)抗氧化骨胶原多肽的氨基酸序列测定
利用LC-MS/MS并搜索MASCOT谱库鉴定抗氧化骨胶原多肽的氨基酸序列为SSGPPVPGPMGPMGPR。
(4)DPPH自由基清除能力的测定
利用DPPH自由基清除率测定法研究。配置浓度为0.2mM的DPPH乙醇溶液，避光保存。将100μL样品，25μL DPPH溶液，75μL污水乙醇混合语96孔板中。在室温下放置60min，与517nm处测定吸光度，吸光值越小，表明自由基清除能力越强。
从图2中可以看到，当抗氧化骨胶原多肽浓度为3mM时，其对DPPH自由基的清除率为47.6％，当抗氧化骨胶原多肽浓度为5mM时，其对DPPH自由基的清除率为67.4％。当浓度大于3mM时，清除DPPH自由基的清除率大于50％。计算可得其对DPPH自由基半抑制浓度IC50值为3.149mM。相对于其他多肽具有较低的半抑制浓度,如GPP(IC50 7.155mM)[1]。说明抗氧化骨胶原具有较强的抗氧化能力。
[1]Chi C F,Wang B,Wang Y M,et al.Isolation and characterization ofthree antioxidant peptides from protein hydrolysate of bluefin leatherjacket(Navodon septentrionalis)heads[J].Journal of Functional Foods,2015,12:1-10.
序列表
<110> 北京化工大学
<120> 一种抗氧化骨胶原多肽及其制备方法
<130> WPFC1180020
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> PRT
<213> 金枪鱼骨(tuna fish bone)
<400> 1
Ser Ser Gly Pro Pro Val Pro Gly Pro Met Gly Pro Met Gly Pro Arg
1 5 10 15
Claims (5)
1.一种抗氧化骨胶原多肽，其特征是：所述抗氧化骨胶原多肽的氨基酸序列如序列表1所示，所述抗氧化骨胶原多肽肽链含有两个蛋氨酸残基。
2.如权利要求1所述的抗氧化骨胶原多肽的制备方法，其特征是：以金枪鱼骨为原料来提取胶原蛋白，然后采用胰-糜复合酶对其进行酶解，分离纯化、冷冻干燥得到抗氧化骨胶原多肽。
3.根据权利要求2所述的抗氧化骨胶原多肽的制备方法，其特征在于：所述酶解条件为：pH为8，温度37℃，酶解时间为2h，胰蛋白酶的质量浓度为0.5％，糜蛋白酶质量浓度为0.1％。
4.根据权利要求2所述的抗氧化骨胶原多肽的制备方法，其特征在于所述分离纯化手段包括DEAE-52纤维素离子交换色谱，RP-HPLC反相高效液相色谱。
5.根据权利要求2所述的抗氧化骨胶原多肽的制备方法，其特征在于：所述纯化步骤为：
(1)酶解产物用DEAE-52纤维素阴离子交换色谱进行分离，上样后洗去未吸附组分，再以pH 7.2磷酸缓冲液进行以0.1，0.5，1.0mol/L的NaCl溶液进行洗脱，流速为1.0mL/min，在280nm下进行测量，测定各吸收峰对应的洗脱组分的抗氧化活性；
(2)收集具有最佳抗氧化活性的峰，再利用RP-HPLC反相高效液相色谱进一步分离，所用的色谱柱为Zorbax SB-C18，上样量为20μL，流速为1.0mL/min，检测波长为280nm，洗脱液自含体积比2％乙腈和98％水的混合液开始，至体积比为98％乙腈和2％水，进行梯度洗脱；收集在27.6min处的洗脱峰，即得到抗氧化骨胶原多肽。
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Non-Patent Citations (3)
| Title |
|---|
| 《Isolation and identification of an antioxidantcollagen peptide from skipjack tuna (Katsuwonuspelamis) bone》;Ding Ding等;《RSC Advances》;20190828;第9卷;第27032-27041页 * |
| 《金枪鱼骨抗氧化酶解物的工艺优化》;胡静等;《食品研究与开发》;20150831;第36卷(第15期);第64-69页 * |
| 《金枪鱼鱼骨胶原肽的制备及抗氧化活性研究》;谭洪亮等;《水产学报》;20140131;第38卷(第1期);第143-148页 * |
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