CN113801194B - 一种金枪鱼鱼卵的降血脂肽及其应用 - Google Patents
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Abstract
本发明提供一种金枪鱼鱼卵的降血脂肽，其氨基酸序列为：Pro‑Trp‑Gly‑Ile‑Glu‑Ala‑Leu‑Arg，ESI‑MS测定其分子量为941.1Da。本发明制备的高活性降血脂肽能减少OA(油酸)诱导的HepG2细胞内脂质堆积，下调成脂基因的表达水平，抑制成脂重要因子蛋白表达水平。本发明金枪鱼鱼卵的降血脂肽可作为药物或者辅助药物用于降血脂相关疾病的治疗，同时其可作为药品、保健食品和食品的安全添加剂。
Description
技术领域
本发明涉及多肽技术领域，具体涉及一种金枪鱼鱼卵的降血脂肽及其应用。
背景技术
金枪鱼是世界远洋渔业的重要作业鱼种之一，据联合国粮农组织统计，世界金枪鱼年产量超过600万吨，占公海渔业总产量70％以上。在金枪鱼加工过程中产生约占总重量50％～70％的下脚料，这些下脚料主要是金枪鱼内脏，碎肉，鱼头和鱼皮等。然而，我国金枪鱼下脚料的加工应用情况不尽如人意，90％以上仍然作为饲料原料或初级饲料利用，造成了金枪鱼资源的极大浪费，也给生态环境带来了较大压力。
发明内容
本发明以金枪鱼加工副产物-鱼卵为原料，利用酶解工艺和色谱制备技术制备得到降血脂肽，对综合、高效利用金枪鱼下脚料具有重要的意义。
一方面，本发明提供一种金枪鱼鱼卵的降血脂肽，其氨基酸序列为：Pro-Trp-Gly-Ile-Glu-Ala-Leu-Arg，ESI-MS测定其分子量为941.1Da。
另一方面，本发明提供了一种金枪鱼鱼卵降血脂肽的制备方法，其通过金枪鱼鱼卵脱脂、高温处理、酶解、超滤、凝胶柱层析及反相高效液相色谱纯化后得到。
具体地，一种金枪鱼鱼卵降血脂肽的制备方法，包括以下步骤：
1)金枪鱼鱼卵预处理：金枪鱼鱼卵匀浆至糊状后，加入乙酸乙酯，搅拌脱脂24h，过滤，干燥，得脱脂金枪鱼鱼卵粉末；取脱脂金枪鱼鱼卵粉末，加入蒸馏水，放于100℃水浴中2～3h，得脱脂金枪鱼鱼卵粉末溶液；
2)金枪鱼鱼卵的酶解：将步骤1)中脱脂金枪鱼鱼卵粉末溶液调温至35～40℃，调节pH值至1.5～2.0，加入胃蛋白酶，水解3～5h，于90～100℃、10min灭酶；样品降温至35～45℃时，调节pH值至7.0～8.0，加入胰蛋白酶，反应3～5h，于90～100℃、10min灭酶，冷却至常温，12000rmp离心20min，收集上清液，即金枪鱼鱼卵酶解液；
3)金枪鱼鱼卵降血脂寡肽的制备：将金枪鱼鱼卵酶解液经截留分子量为1kDa、3.5kDa、5kDa和10kDa的超滤膜进行分级，收集1kDa的组分，冻干，得金枪鱼鱼卵降血脂超滤酶解物，该超滤酶解物依次经凝胶柱层析和反相高效液相色谱(RP-HPLC)纯化，得到金枪鱼鱼卵降血脂寡肽Pro-Trp-Gly-Ile-Glu-Ala-Leu-Arg。
作为优选，所述步骤1)中的金枪鱼为鲣鱼Katsuwonus pelamis。
作为优选，所述步骤1)中匀浆至糊状后的金枪鱼鱼卵与乙酸乙酯的重量体积比g/ml为1:4～1:6。
作为优选，所述步骤1)中脱脂金枪鱼鱼卵粉末与蒸馏水的重量体积比g/ml为1:5。
作为优选，所述步骤2)中胃蛋白酶的添加量为脱脂金枪鱼鱼卵粉末溶液重量的0.8％～1.5％。
作为优选，所述步骤2)中胰蛋白酶的添加量为脱脂金枪鱼鱼卵粉末溶液重量的1.5％～2.0％。
作为优选，所述步骤3)的凝胶柱层析和RP-HPLC纯化的具体过程为：
凝胶柱层析：将上述金枪鱼鱼卵降血脂超滤酶解物溶于双蒸水配成浓度为20～25mg/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0.5～0.8mL/min，根据220nm下的吸光度制作凝胶层析色谱图，收集各色谱峰，测定个色谱峰组分对HepG2细胞模型中脂质堆积的影响效果，选择降低脂质堆积效果最好的色谱峰组分，冻干，得金枪鱼鱼卵降血脂凝胶层析酶解物；
RP-HPLC纯化：将上述金枪鱼鱼卵降血脂凝胶层析酶解物用双蒸水配成45～50μg/mL的溶液，利用RP-HPLC进行纯化，得金枪鱼鱼卵降血脂寡肽Pro-Trp-Gly-Ile-Glu-Ala-Leu-Arg。
进一步优选，所述RP-HPLC条件为：进样量5～10μL；色谱柱Kromasil C18(250mm×4.6mm，5μm)；流动相：80％乙腈；洗脱速度0.5～0.8mL/min；紫外检测波长220nm。
另一方面，本发明提供了一种金枪鱼鱼卵降血脂肽用于制备降血脂相关疾病的药物中的用途。
本发明制备的高活性降血脂肽Pro-Trp-Gly-Ile-Glu-Ala-Leu-Arg能减少OA(油酸)诱导的HepG2细胞内脂质堆积，下调成脂基因的表达水平，抑制成脂重要因子蛋白表达水平。因此，Pro-Trp-Gly-Ile-Glu-Ala-Leu-Arg在降脂机制方面既能下调成脂基因，又能促进脂肪酸β氧化，减少脂质的生成，降脂质水平大于脂质生成能力，具有良好的降脂活性，其在10μM浓度下可显著降低HepG2细胞脂质堆积模型中甘油三酯(TG)和总胆固醇(TC)含量，可作为药物或者辅助药物用于降血脂相关疾病的治疗，同时其可作为药品、保健食品和食品的安全添加剂。
附图说明
图1是实施例中不同超滤组分在10mg/mL浓度下对HepG2细胞脂质堆积模型中脂质含量的影响。
其中，空白组是指的没有加任何的正常细胞；模型组指的是加入油酸(100μM)导致脂肪堆积的细胞；阳性对照组为加“油酸(100μM)+辛伐他汀(10μM)”。
图2是实施例中不同组分的吸光度曲线图。
图3是实施例中葡聚糖凝胶柱层析分离不同组分在10mg/mL浓度下对HepG2细胞脂质堆积模型中脂质含量的影响。
其中，空白组是指的没有加任何的正常细胞；模型组指的是加入油酸(100μM)导致脂肪堆积的细胞；阳性对照组为加“油酸(100μM)+辛伐他汀(10μM)”。
图4是实施例中RP-HPLC分离组分的吸光度曲线。
图5是实施例中RP-HPLC分离组分在10mg/mL浓度下对HepG2细胞脂质堆积模型中脂质含量的影响。
其中，空白组是指的没有加任何的正常细胞；模型组指的是加入油酸(100μM)导致脂肪堆积的细胞；阳性对照组为加“油酸(100μM)+辛伐他汀(10μM)”。
图6Pro-Trp-Gly-Ile-Glu-Ala-Leu-Arg(PWGIEALR)的质谱图。
图7Pro-Trp-Gly-Ile-Glu-Ala-Leu-Arg(PWGIEALR)的结构。
图8Pro-Trp-Gly-Ile-Glu-Ala-Leu-Arg(PWGIEALR)对HepG2细胞脂质堆积模型内甘油三酯(TG)含量的影响。
其中，空白组是指的没有加任何的正常细胞；模型组指的是加入油酸(100μM)导致脂肪堆积的细胞；阳性对照组为加“油酸(100μM)+辛伐他汀(10μM)”。
图9Pro-Trp-Gly-Ile-Glu-Ala-Leu-Arg(PWGIEALR)对HepG2细胞脂质堆积模型内总胆固醇(TC)含量的影响。
其中，空白组是指的没有加任何的正常细胞；模型组指的是加入油酸(100μM)导致脂肪堆积的细胞；阳性对照组为加“油酸(100μM)+辛伐他汀(10μM)”。
具体实施方式
下列实施例用于进一步解释说明本发明，但是，它们并不构成对本发明范围的限制或限定。
实施例1
1)金枪鱼鱼卵预处理：取1000g鲣鱼Katsuwonus pelamis鱼卵匀浆至糊状后，按料液比1g:5ml加入乙酸乙酯，搅拌脱脂24h，过滤，干燥，得脱脂金枪鱼鱼卵粉末；取脱脂金枪鱼鱼卵粉末，按照料液比1:5(g/ml)加入蒸馏水，放于100℃水浴中3h，取出，备用；
2)金枪鱼鱼卵的酶解：将上述脂金枪鱼鱼卵粉末溶液调温至36℃，调节pH值至2.0，加入脱脂金枪鱼鱼卵粉末溶液重量1.0％胃蛋白酶，水解5h，于95℃、10min灭酶；样品降温至40℃时，调节pH值至7.5，加入脱脂金枪鱼鱼卵粉末溶液重量1.5％胰蛋白酶，反应3h，于95℃、10min灭酶，冷却至常温，12000rmp离心20min，收集上清液，即金枪鱼鱼卵酶解液(TRH)；
3)金枪鱼鱼卵降血脂寡肽的制备：将金枪鱼鱼卵酶解液经截留分子量为1kDa、3.5kDa、5kDa和10kDa的超滤膜进行分级，收集分级组分TRH1(MW<1kDa)、TRH2(1kDa<MW<3.5kDa)、TRH3(3.5kDa<MW<5kDa)、TRH4(5kDa<MW<10kDa)、和TRH5(MW>10kDa)，测定不同组分对在10mg/mL浓度下对HepG2细胞脂质堆积模型中脂质含量的影响(见图1)，TRH1降低脂质堆积效果最好，冻干；TRH1依次经凝胶柱层析和反相高效液相色谱(RP-HPLC)纯化，得到金枪鱼鱼卵降血脂寡肽，利用氨基酸序列分析仪和质谱测定其结构。
具体过程为：
①凝胶色谱层析：将上述TRH1用双蒸水配成浓度为20mg/mL的溶液，经过葡聚糖凝胶Sephadex G-15柱层析分离，用双蒸水进行洗脱，根据220nm下的吸光度曲线收集洗脱组分(见图2)和不同组分在10mg/mL浓度下对HepG2细胞脂质堆积模型中脂质含量的影响(见图3)，确定TRH1B为降血脂凝胶层析酶解物。
②高效液相色谱精制：将上述TRH1B用双蒸水配成浓度为50μg/mL的溶液，用RP-HPLC进行纯化(进样量20μL；色谱柱Kromasil C18(250mm×4.6mm，5μm)；流动相40％乙腈；紫外检测波长220nm)，测定分离组分在220nm下的吸光度曲线(见图4)和在10mg/mL浓度下对HepG2细胞脂质堆积模型中脂质含量的影响(见图5)，得到1个高活性降血脂寡肽(TRP5)。
③结构检测：收集降血脂活性最高的TRP5，经RP-HPLC检测达到测序要求，利用蛋白/多肽序列分析仪测定氨基酸序列为Pro-Trp-Gly-Ile-Glu-Ala-Leu-Arg(PWGIEALR)(见图7)，ESI-MS测定分子量为941.1Da(见图6)。
④功能评价：金枪鱼鱼卵降血脂肽Pro-Trp-Gly-Ile-Glu-Ala-Leu-Arg(PWGIEALR)能减少油酸(OA)诱导的HepG2细胞内脂质堆积；在10μM浓度下可显著降低HepG2细胞脂质堆积模型中甘油三酯(TG)含量(见图8)和总胆固醇(TC)(见图9)。综上，本发明制备得到的金枪鱼鱼卵降血脂肽Pro-Trp-Gly-Ile-Glu-Ala-Leu-Arg(PWGIEALR)显示显著的降脂活性。
Claims (2)
1.一种金枪鱼鱼卵降血脂肽，其特征在于所述降血脂肽的氨基酸序列为：Pro-Trp-Gly-Ile-Glu-Ala-Leu-Arg，ESI-MS测定其分子量为941.1 Da。
2.如权利要求1所述一种金枪鱼鱼卵降血脂肽用于制备降血脂相关疾病的药物中的用途。
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| CN105061558A (zh) * | 2015-02-03 | 2015-11-18 | 浙江海洋学院 | 一种金枪鱼蒸煮液活性肽及其制备方法和糖尿病治疗用途 |
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