CN116284341A - 一种低免疫原性、降血压、抗氧化的深海鱼皮胶原蛋白肽制备及应用 - Google Patents
一种低免疫原性、降血压、抗氧化的深海鱼皮胶原蛋白肽制备及应用 Download PDFInfo
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Abstract
本发明提供了一种降血压、抗氧化的低免疫原性深海鱼皮小分子胶原蛋白肽的制备方法和应用。所述胶原蛋白肽的制备方法包括:鱼皮的物理预处理、去脂、去杂蛋白;去除端肽;复合蛋白酶解‑超滤膜耦合工艺获取小分子胶原蛋白肽;色谱纯化获得降血压、抗氧化小分子胶原蛋白肽;辐射灭活细菌和内毒素,最终获得一种降血压、抗氧化的低免疫原性小分子胶原蛋白肽。本发明通过多种手段,去除端肽、杂蛋白、内毒素等物质,极大降低了胶原蛋白肽的免疫原性,减少对生物体的刺激和副反应。所述低免疫原性的胶原蛋白肽具有分子量小、纯度高、降血压、抗氧化活性强、免疫原性低、安全性好的特点,可用于食品、保健品和医学相关领域。
Description
技术领域
本发明涉及海洋生物技术产品开发及应用领域,具体涉及一种具有低免疫原性、降血压和抗氧化活性的深海鱼皮小分子胶原蛋白肽的制备及其应用。
背景技术
胶原蛋白肽是胶原蛋白的水解产物,具有诸多胶原蛋白所不具有的生物活性,其分子量小,比胶原蛋白更易为人体吸收,已报导的功效包括抗氧化、抗肿瘤、降血压、保肝护肝、促进钙吸收、调节激素水平和美容护肤等,作为一种新兴的生物活性物质在国内外发展迅速,被广泛应用于功能性食品、保健品、医疗和化妆品等领域。
高血压是一种全球性的公共健康问题,是中风、心肌梗塞、心力衰竭、动脉瘤等许多疾病的诱因,极大的影响着人们的健康。血管紧张素转换酶(angiotensin convertingenzyme,ACE)是一种二肽缩肽酶,通过肾素-血管紧张素***和激肽释放酶-激肽***(kallikrein-kinin system,KKS)对血压进行调节。ACE能够催化血管紧张素I生成导致血管收缩的血管紧张素II,同时令具有血管舒张作用的缓激肽失活,从而导致血压升高。因此,抑制ACE的活性能够有效降低血压、预防和治疗高血压及相关疾病。常见的ACE抑制剂药物有卡托普利、苯那普利、依那普利、赖诺普利等,这些药物长期服用会有味觉失调、咳嗽、皮疹、头痛等副作用。相比于化学合成药物,食源性蛋白获得的ACE抑制肽具有更加安全、温和、易吸收、无副作用的特点。
人体代谢会产生很多活性氧和自由基,氧化应激损伤和自由基代谢失调会造成细胞损伤,引起一些疾病如动脉硬化、糖尿病、哮喘、皮肤老化、机体衰老等。一些人工合成的抗氧化剂被用于食品防腐等,相比于天然的抗氧化剂其活性更强,但是也存在一定的副作用,如对DNA的损伤和毒性。生物活性肽已被证明具备良好的抗氧化活性,而且安全无毒,对维护人体健康、预防疾病意义重大。
现阶段使用的胶原蛋白肽产品多为陆生动物来源,如猪、牛等。与陆生动物来源的胶原蛋白肽相比,深海鱼皮来源的胶原蛋白肽更具优势,如提取容易、重金属和毒素含量低、免疫原性低、无人畜共患病风险以及不受宗教风俗约束等。深海鱼加工过程产生的下脚料鱼皮,胶原蛋白含量占蛋白总量的70~80%,来源丰富,价格低廉,为开发新型胶原蛋白肽产品提供了新思路。另外,从海洋加工下脚料中提取高附加值的胶原蛋白肽,能够合理利用资源,避免环境污染问题,有利于我国蓝色海洋经济和绿色可持续发展。
目前国内已有多种生物活性的胶原蛋白肽产品,但是普遍存在分子量偏大,分子量分布指数宽宽的问题,导致有效活性成分含量低,产品纯度不高。并且,胶原蛋白本身具备一定的免疫原性,会对机体产生刺激,这主要来自于非螺旋结构的端肽序列,包括N端和C端的氨基酸序列。此外,生产过程中残留的杂蛋白、细菌、内毒素等也会引发生物体内的免疫反应,这都限制了胶原蛋白肽产品在生物体中的应用。目前,具备低免疫原性的深海鱼类来源的抗氧化、抗高血压的小分子胶原蛋白肽产品还未见报道。
发明内容
针对现有技术存在的问题,本发明提供了一种具备抗高血压和抗氧化活性的低免疫原性深海鱼皮小分子胶原蛋白肽及制备方法,可应用于食品、保健品和医学相关用途。
为实现上述发明目的,本发明的技术方案包括以下步骤:
(1)鱼皮预处理:将深海鱼皮用钢丝刷洗净背部的鱼肉组织,在软化水中浸泡3小时,用软化水进行反复多次高压冲洗后沥水。然后将处理干净的鱼皮进行剪碎,获得大小合适的碎鱼皮。向鱼皮中加入氢氧化钠碱溶液处理12~24小时,去除鱼皮中的脂肪和杂蛋白,用软化水将鱼皮洗至中性;
(2)端肽去除:将处理后的鱼皮按照料液比1:15~1:20加入到0.5M乙酸中,并加入浓度为60~100U/mL的胃蛋白酶,磁力搅拌处理12~72h。调整pH值为中性,反应液采用100kDa透析袋透析;
(3)复合酶解-超滤耦合工艺获得胶原蛋白肽:将上述液体调整pH值为中性,加入鱼皮质量1%~3%的复合蛋白酶,于40~60℃酶解2~24h,得到胶原蛋白肽粗提液。将粗提液在95~100℃保温10~20min,灭酶。并通过截留分子量为1kDa的超滤膜,保留滤过组分,冷冻干燥为粉状;
(4)生物活性小分子胶原蛋白肽的纯化:用超纯水将上一步得到的胶原蛋白肽粉末溶解,配置成10~20mg/mL的溶液,使用葡聚糖凝胶Sephedax G25分子筛层析对其进行分离纯化,收集合并各个峰组分。选取活性最高的组分,使用反相色谱C18柱进行进一步精制纯化,收集各峰组分。最终得到具有抑制ACE活性和抗氧化活性的胶原蛋白肽组分,冷冻干燥。
(5)细菌和内毒素去除:冷冻干燥后得到活性小分子胶原蛋白肽粉末使用60Co辐照处理,灭活内毒素,灭菌包装后得的具备降血压和抗氧化活性的低免疫原性深海鱼皮小分子胶原蛋白肽成品。
优选地,步骤(1)中的碱溶液使用氢氧化钠溶液,浓度为0.01~0.02M,鱼皮与碱溶液料液比为1:10~1:20,处理时间为12~24小时。
优选地,步骤(2)中的乙酸溶液使用0.5M浓度,料液比1:15~1:20,胃蛋白酶浓度使用60~100IU/mL。
优选地,步骤(3)中的复合蛋白酶使用中性蛋白酶和菠萝蛋白酶,两者质量比为1:2~1:4,酶解温度为40~60℃,酶解时间2~24h。超滤膜使用截留分子量为1kDa的膜,保留滤过组分。
优选地,步骤(4)中分子筛层析使用葡聚糖凝胶Sephedax G25填料,超纯水作为流动相,线性流速0.8ml/min,220nm波长处检测。反相色谱使用XTerra Prep MS C18制备柱,流动相A液为含0.1%TFA的5%乙腈水溶液,B液为含0.1% TFA的90%乙腈水溶液,0-5min使用100%的A液平衡柱子,5-20min,线性上升到100% B液洗脱,220nm波长处检测洗脱峰。
优选地,步骤(6)中采取60Co辐照处理灭活内毒素。
本发明还提供所述制备方法所制得的低免疫原性深海鱼皮小分子胶原蛋白肽。
本发明提供的低免疫原性胶原蛋白肽分子量小,分布范围窄,集中在300~600Da之间,平均分子量500Da。
本发明还提供所述低免疫原性胶原蛋白肽在降血压和抗氧化方面的应用。
与现有技术相比,本发明所达到的有益效果是:
本发明在制备小分子深海鱼皮胶原蛋白肽时,采取了多种降低免疫原性的方法,如氢氧化钠的预处理,去除鱼皮中的杂质颗粒、脂质、非胶原蛋白质;端肽序列是最容易引发机体免疫反应的因素,使用胃蛋白酶处理去除胶原蛋白端肽,降低酶解产物可能引起的免疫反应;灭菌和辐射处理灭活细菌和内毒素,进一步降低胶原蛋白肽的免疫原性。
本发明获得的小分子胶原蛋白肽分子量更小,分布范围更加集中。复合酶解—超滤耦合工艺,使本发明获得的胶原蛋白肽分子量范围集中在300-600Da,远低于市面上的产品,其生物活性更高,机体吸收更充分,也避免了生物大分子进入体内可能引发的免疫反应。
分子筛层析和反向色谱层析的精制纯化,得到具备最高活性的降血压和抗氧化小分子胶原蛋白肽。市面上的胶原蛋白肽产品多为直接酶解后的产物,未经进一步纯化,分子量分布宽,是成分复杂,杂质多,活性低,可能会引起副作用。一些根据已知序列合成的纯肽产品虽然纯度高,活性强,但是只能由化学合成获得,成本高,污染大,无法量产。本产品经纯化后得到的胶原蛋白肽组分含有生物活性最高的组分,制备方便,价格低廉,纯化步骤简单,易于放大,可充分降低成本,利用海洋废弃物资源。
本发明制备了低免疫原性的深海鱼皮小分子胶原蛋白肽,具备高效的抗高血压和抗氧化活性,具有工艺流程简单、成本低廉、反应温和、污染小、环境友好的优点,可应用于特医原料和医疗保障领域。
附图说明
图1不同分子量胶原蛋白肽抗氧化能力;
图中:a.DPPH自由基;b.ABTS自由基;c.超氧阴离子自由基;d.脂质过氧化物
图2不同分子量胶原蛋白肽对ACE的抑制效果;
图3胶原蛋白肽经Sephadex G-25分子筛纯化洗脱峰;
图4胶原蛋白肽经XTerra Prep MS C18柱反向高效液相色谱纯化洗脱峰;
图5胃肠消化后小分子胶原蛋白肽对ACE抑制和抗氧化效果;
图6小分子胶原蛋白肽的细胞毒性测试。
图中:a.生理盐水组72h细胞生长情况b.胶原蛋白肽组24h细胞生长情况c.胶原蛋白肽组72h细胞生长情况
具体实施方式
下述实施方式更好地说明本发明内容。但本发明不限于下述实施例。
实施例1:深海鱼皮胶原蛋白肽的制备
(1)鱼皮预处理:将深海鱼皮用钢丝刷洗净背部鱼肉组织,在软化水中浸泡3小时,用软化水进行反复多次高压冲洗后沥水。然后将处理干净的鱼皮进行剪碎,获得大小合适的碎鱼皮。按照鱼皮与碱溶液料液比为1:10~1:20加入0.01~0.02M的氢氧化钠溶液,处理12~24小时,去除鱼皮中的脂肪和杂蛋白,用软化水将鱼皮洗至中性;
(2)端肽去除:将处理后的鱼皮按照料液比1:15~1:20加入到0.5M乙酸中,并加入浓度为60~100U/mL的胃蛋白酶,磁力搅拌处理12~72h,去除端肽,反应液经过100KD透析袋透析,保留截留液体;
(3)复合酶解-超滤膜耦合工艺:将上述液体调整pH值为中性,继续加入鱼皮质量1%~3%的复合蛋白酶,于40~60℃酶解2~24h,得到胶原蛋白肽粗提液。将粗提液在95~100℃保温10~20min,灭酶。将粗提液通过截留分子量为1K的超滤膜,离心收集滤过液,冷冻干燥。通过调控酶解的时间控制水解度,并通过不同分子量大小的超滤膜,可得到平均分子量从500D到15000D不等的胶原蛋白肽产品。
(4)生物活性小分子胶原蛋白肽的纯化:用超纯水将上一步得到的胶原蛋白肽粉末溶解,配置成10~20mg/mL的溶液,使用葡聚糖凝胶SephedaxG25分子筛层析对其进行分离纯化,收集合并各个峰组分。测定生物活性后,选取活性最高的组分进行冷冻干燥。并使用反相色谱XTerra Prep MS C18柱柱进行进一步精制纯化,收集各峰组分。最终得到具有ACE抑制和抗氧化活性的胶原蛋白肽组分。
(5)细菌和内毒素去除:冷冻干燥后得到胶原蛋白肽粉末使用60Co辐照处理,灭活内毒素,灭菌包装,得的具备降血压和抗氧化活性的低免疫原性小分子胶原蛋白多肽成品。
实施例2:不同平均分子量鳕鱼皮胶原蛋白肽抗氧化活性的比较
根据文献报道,小分子量胶原蛋白肽具有较好的抗机体和细胞过氧化等功能,在食品保健和药品开发领域有着较好的发展预期。本发明得到的胶原蛋白肽分子量范围在300-600Da之间,平均分子量500Da,与其它更大平均分子量的胶原蛋白肽相比,具备更优的抗氧化活性。对通过调控水解程度得到的平均分子量分别为500Da、1000Da、3000Da、7000Da、15000Da的胶原蛋白肽产品进行抗氧化活性的测定。
(1)DPPH自由基清除活性
将上述不同分子量的胶原蛋白肽分别溶解于2mL去离子水。向各样品中加入2mL用甲醇配制好的DPPH溶液,烘箱37℃保温30min后,分别测量其吸光度。用2mL乙醇溶液替代同体积样品溶液作为阴性对照。DPPH自由基的特征吸收峰为517nm,检测517nm处吸光值的变化,并按照下式计算DPPH清除率。
A0为没有添加样品时的吸光度,A为添加样品后的吸光度。
不同分子量胶原蛋白肽样品在1mg/ml浓度时的DPPH自由基清除能力如图1a所示。可以看出,不同分子量大小的胶原蛋白肽都拥有着较好的自由基清除能力,以平均分子量为500Da的小分子胶原蛋白肽最优。
(2)ABTS自由基清除活性
将上述不同平均分子量的胶原蛋白肽分别溶解于2ml去离子水。向各样品管中各自加入2mL使用二硫酸钾配制好的ABTS溶液,同时设置一组空白组。用2ml去离子水替代样品作为阴性对照。ABTS自由基的特征吸收峰为728nm,本实验选取728nm处的吸光值,并按照下式计算ABTS清除率。
A0为没有添加样品时的吸光度,A为添加样品后的吸光度。
不同分子量胶原蛋白肽样品在1mg/ml浓度时的ABTS自由基清除能力如图1b所示。可以看出,全部胶原蛋白肽都拥有着较好的ABTS自由基清除能力,其中平均分子量为500Da的小分子胶原蛋白肽效果最佳。
(3)超氧阴离子自由基清除活性
将上述不同分子量的胶原蛋白肽分别溶解于2mL去离子水,采用NADH法测量超氧阴离子自由基清除能力。取1ml预先配置好的氯化硝基四唑蓝(NBT)、1ml NADH混匀,加入1mL不同平均分子量大小的胶原蛋白肽溶液,然后向反应混合物中加入1ml吩嗪甲基硫酸盐(PMS)溶液来引发反应。25℃下孵育5分钟后,在560nm处测量相应的空白测量吸光度。
不同分子量胶原蛋白肽样品在1mg/ml时的抗氧化能力如图1c所示,可以看出,与未加胶原蛋白肽的样品相比,不同分子量大小的胶原蛋白肽都有着较好的超氧阴离子自由基清除能力,其中平均分子量为500Da的小分子肽在自由基清除方面有着最好的效果。
(4)脂质过氧化自由基清除活性
将上述不同分子量的胶原蛋白肽分别溶解于2mL去离子水,向870μL PBS溶液中加入2mL胶原蛋白肽溶液、30μL亚油酸、2mL无水乙醇和100μL吐温20。配制好后,用锡纸包裹试管,提供避光环境,放入37℃恒温水浴锅,孵育12h。移取100μL反应液,依次加入4.75mL75%乙醇、100μL 30%硫氰酸铵溶液、100μL 20mmol/L氯化亚铁溶液(使用3.5%HCl配制)。加完试剂使用旋涡震荡仪混匀,每隔24h采用500nm波长处测量数据。抗坏血酸作为阳性对照,去离子水作为阴性对照。
不同分子量胶原蛋白肽样品在1mg/ml时的抗氧化能力如图1d所示,可见与未加胶原蛋白肽的样品相比,不同分子量大小的胶原蛋白肽都有着较好的脂质过氧化物自由基清除能力,其效果与抗坏血酸相当。
综合以上各种类型的自由基清除实验结果,平均分子量为500Da的小分子肽比其它大小的胶原蛋白肽更具优势。
实施例2:不同平均分子量鳕鱼皮胶原蛋白肽ACE酶抑制效果的比较
为了验证胶原蛋白肽对ACE酶活性的抑制效果,采用体外FAPGG(N-[3-(2-呋喃基)丙烯酰]-L-苯丙氨酰-甘氨酰-甘氨酸)底物法验证胶原蛋白肽对ACE酶活性抑制效果。向100μL的0.1U ACE溶液中加入90μL FAPGG溶液,40μL胶原蛋白肽溶液,立即用酶标仪测定340nm处吸光值。37℃孵育30min后,再次测定340nm处吸光值,评价ACE抑制效果。结果如图2所示,可见,分子量为500Da的小分子胶原蛋白肽具有最佳的ACE酶抑制效果。
实施例3:层析纯化前后胶原蛋白肽的生物活性
(1)将经过复合酶解-超滤耦合工艺得到的小分子胶原蛋白肽经过分子筛Sephadex G-25凝胶分离。样品配置成20mg/mL浓度,以超纯水为洗脱缓冲液,线性流速0.8mL/min,在220nm处检测(图3)。收集各洗脱峰,进行ACE抑制和ABTS自由基清除能力测试,结果显示最后一个洗脱峰G活性最强,比纯化前样品都有明显提高(表1)。
表1分子筛纯化后胶原蛋白肽组分ACE抑制和ABTS自由基清除能力
(2)使用反相高效液相色谱对胶原蛋白肽进行进一步的纯化。冷冻干燥后的胶原蛋白肽分子筛分离组分重新溶解于超纯水,使用Waters公司的XTerra Prep MS C18柱进行分离。流速2mL/min,流动相A为5%乙腈水溶液,含0.1% TFA,流动相B为90%乙腈水溶液,含0.1%TFA。0-5min使用100%的A液平衡柱子,5-20min,线性上升到100% B液,220nm处检测洗脱峰(图4)。收集各组分,经过ACE抑制和ABTS自由基清除实验测试,组分3的生物活性最高(表2)。
表2C18反相色谱纯化后胶原蛋白肽组分ACE抑制和ABTS自由基清除能力
实施例4:小分子胶原蛋白肽胃肠消化对生物活性的影响
将上述纯化后的小分子胶原蛋白肽溶解于10mL去离子水,加入预先配置好的1M的HCl调节pH=2,加入胃蛋白酶(5%w/w),37℃震荡水浴锅内孵育1h,后将反应液加热至100℃,保持30分钟,将酶灭活。放置冷却至室温以后,加入1M的NaHCO3,调节pH为5.3,加入胰蛋白酶(5%w/w),再加入2M的NaOH调节pH=7.5,37℃震荡水浴锅内孵育2h,将反应液加热至100℃灭活20min,待冷却至室温,离心5000rpm/min,取上清液,检测胶原蛋白肽胃肠消化前后ACE抑制和ABTS自由基清除能力变化。
由图5可知,纯化前和纯化后的胶原蛋白肽在经过模拟胃肠消化后,ACE抑制和自由基清除能力较之前变化不大,或有所上升。推测在经过胃肠消化液处理后的胶原蛋白肽活性成分未被降解,或者降解的小分子量的寡肽仍具有生物活性。
实施例4:小分子胶原蛋白肽免疫原性测试
(1)小分子胶原蛋白肽免疫原性的测试
参考中华人民共和国医药行业标准,医疗器械免疫原性评价方法YY/T 1465.1-2016,使用体外T淋巴细胞转化实验对纯化和处理后的胶原蛋白肽进行免疫原性分析。取新鲜的无菌脾细胞悬液,调整细胞浓度至2×106cell/mL。使用酶标仪检测胶原蛋白肽对淋巴细胞增值的影响。200μL细胞悬液加入终浓度为5μg/mL的胶原蛋白肽,37℃下二氧化碳培养箱中培养3天,离心弃上清,加入浓度为1mg/mL的噻唑蓝(MTT)50μL,继续培养6h,加入150μLDMSO,震荡平板,在570nm波长处检测吸光值,参照波长为630nm。阳性对照组使用5μg/mL刀豆蛋白A。
经试验验证,小分子胶原蛋白肽未对淋巴细胞产生特异性或者非特异性免疫应答,细胞增殖率不高于阴性对照组,说明制备得到的小分子胶原蛋白肽不会引起机体的淋巴细胞特异性或者非特异性免疫应答。
(2)小分子胶原蛋白肽细胞毒性的测试
使用小鼠成纤维细胞NIH 3T3进行小分子胶原蛋白肽细胞毒性的测试。结果表明,小分子胶原蛋白肽对细胞无毒性,可以显著促进成纤维细胞增殖,72h时细胞增值率可达20%(图6)。
(3)小分子胶原蛋白肽皮内刺激试验
取小分子胶原蛋白肽冻干粉,溶于0.9%氯化钠注射液中,震荡5分钟。健康兔固定后,剪去背部脊柱两侧被毛,用推子小心的把被毛去除干净。使用不同浓度的小分子胶原蛋白肽溶液,在每只兔脊柱左侧用注射器皮内注射0.2mL,在右侧注射0.9%生理盐水和0.2mL植物油制备的阳性对照液。注射后24h、48h和72h后观察记录各注射部位情况。结果显示,胶原蛋白肽注射部位无红斑和水肿出现,说明低免疫原性小分子胶原蛋白肽对皮肤的刺激符合试验要求。
(4)小分子胶原蛋白肽急性全身毒性试验
准备健康小鼠,标准饮食,将小鼠随机分为试验和对照组,每组至少5只。使用0.9%氯化钠注射液准备小分子胶原蛋白肽溶液,用0.22μm的无菌滤膜过滤除菌。经鼠尾静脉注射50mL/kg不同浓度的小分子胶原蛋白肽溶液,注射速度不超过0.1mL/s。无菌生理盐水作为对照,植物油作为阳性对照,并于市售胶原蛋白肽产品进行对比。注射后4h、24h、48h和72h观察和记录所有动物的状态、毒性表现和死亡数。结果发现,在72h观察期内,阳性对照组出现明显毒性反应,胶原蛋白肽注射组的小鼠反应不大于阴性对照组,优于市售胶原蛋白肽产品组,可以认为去端肽小分子胶原蛋白肽不会产生急性全身毒性。
实施例5:小分子胶原蛋白肽抗高血压动物实验
构建肾动脉狭窄型高血压模型小鼠,28~30天后取造模成功的小鼠进行试验。使用0.9%氯化钠注射液准备小分子胶原蛋白肽溶液,用0.22μm的无菌滤膜过滤除菌。小鼠随机分组,经尾静脉注射小分子胶原蛋白肽溶液,0.9%生理盐水作为阴性对照,卡托普利作为阳性对照。实时动态观察和记录血压波形,收缩压、舒张压、平均压、心率。结果如表3所示,小分子胶原蛋白肽组和卡托普利组都对小鼠有明显的降血压效果,小分子胶原蛋白肽降压效果高于卡托普利组。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (2)
1.一种低免疫原性、降血压、抗氧化的深海鱼皮小分子胶原蛋白肽的制备方法,包括以下步骤:
(1)鱼皮预处理:将深海鱼皮用钢丝刷洗净背部的鱼肉组织,在软化水中浸泡3小时,用软化水进行反复多次高压冲洗后沥水。然后将处理干净的鱼皮进行剪碎,获得大小合适的碎鱼皮。向鱼皮中加入氢氧化钠碱溶液处理12~24小时,去除鱼皮中的脂肪和杂蛋白,用软化水将鱼皮洗至中性;
(2)端肽去除:将处理后的鱼皮按照料液比1:15~1:20加入到0.5M乙酸中,并加入浓度为60~100U/mL的胃蛋白酶,磁力搅拌处理12~72h。调整pH值为中性,反应液采用100kDa透析袋透析;
(3)复合酶解-超滤耦合工艺获得胶原蛋白肽:将上述液体调整pH值为中性,加入鱼皮质量1%~3%的复合蛋白酶,于40~60℃酶解2~24h,得到胶原蛋白肽粗提液。将粗提液在95~100℃保温10~20min,灭酶。并通过截留分子量为1kDa的超滤膜,保留滤过组分,冷冻干燥为粉状;
(4)生物活性小分子胶原蛋白肽的纯化:用超纯水将上一步得到的胶原蛋白肽粉末溶解,配置成10~20mg/mL的溶液,使用葡聚糖凝胶Sephedax G25分子筛层析对其进行分离纯化,收集合并各个峰组分。选取活性最高的组分,使用反相色谱C18柱进行进一步精制纯化,收集各峰组分。最终得到具有抑制ACE活性和抗氧化活性的胶原蛋白肽组分,冷冻干燥。
(5)细菌和内毒素去除:冷冻干燥后得到活性小分子胶原蛋白肽粉末使用60Co辐照处理,灭活内毒素,灭菌包装后得的具备降血压和抗氧化活性的低免疫原性深海鱼皮小分子胶原蛋白肽成品。
其中,步骤(1)中的碱溶液使用氢氧化钠溶液,浓度为0.01~0.02M,鱼皮与碱溶液料液比为1:10~1:20,处理时间为12~24小时。
步骤(2)中的乙酸溶液使用0.5M浓度,料液比1:15~1:20,胃蛋白酶浓度使用60~100IU/mL。
步骤(3)中的复合蛋白酶使用中性蛋白酶和菠萝蛋白酶,两者质量比为1:2~1:4,酶解温度为40~60℃,酶解时间2~24h。超滤膜使用截留分子量为1kDa的膜,保留滤过组分。
步骤(4)中分子筛层析使用葡聚糖凝胶Sephedax G25填料,超纯水作为流动相,线性流速0.8ml/min,220nm波长处检测。反相色谱使用XTerra Prep MS C18制备柱,流动相A液为含0.1%TFA的5%乙腈水溶液,B液为含0.1%TFA的90%乙腈水溶液,0-5min使用100%的A液平衡柱子,5-20min,线性上升到100%B液洗脱,220nm波长处检测洗脱峰。
步骤(6)中采取60Co辐照处理灭活内毒素。
2.根据权利要求1所述的制备方法制得的低免疫原性小分子胶原蛋白肽,以及在食品、保健品及医用领域中的应用。
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CN117653562A (zh) * | 2023-11-27 | 2024-03-08 | 广州亚丽化妆品有限公司 | 一种具有抗衰老及提高免疫力功效的胶原蛋白肽组合物及其制备方法 |
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