CN113073124A - 鸢乌贼酮体分离蛋白酶解物作为抗氧化剂的应用 - Google Patents
鸢乌贼酮体分离蛋白酶解物作为抗氧化剂的应用 Download PDFInfo
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Abstract
本发明提供了一种鸢乌贼酮体分离蛋白酶解物作为抗氧化剂的应用，该鸢乌贼酮体分离蛋白酶解物的制备包括在鸢乌贼酮体分离蛋白中加入去离子水，调节温度和pH至酶反应条件，并加入蛋白酶进行酶解，其中，温度为40±1℃，pH值为2.0‑10.5，酶解时间为4±0.1h；本发明首次以鸢乌贼为原料的海洋生物提取胴体分离蛋白，其蛋白纯度超过85％，获得1000Da以下的蛋白酶解物含量超过60％，并用绿色可控的酶解工艺得到鸢乌贼胴体分离蛋白酶解物，通过DPPH自由基清除率为指标评价鸢乌贼胴体分离蛋白酶解物的抗氧化活性，从而填补了关于海洋生物抗氧化活性肽的制备的研究空白。
Description
技术领域
本发明属于生物提取技术领域，具体涉及一种鸢乌贼酮体分离蛋白酶解物作为抗氧化剂的应用，进一步制备海洋生物抗氧化肽。
背景技术
海洋生物胴体分离蛋白含人体所必需的多种氨基酸，是一种待开发的优质蛋白资源，具有较高的研究价值。胴体分离蛋白经限制性内切蛋白酶水解后的酶解产物中氨基酸之间的肽键产生不同分子质量大小的肽和游离氨基酸，该酶解产物的优点是易于吸收、利用率高、功效活性高，具有抗氧化、降血压、滋阴血、益肺气等活性，可作为天然抗氧化剂用来开发具有强抗氧化活性的功能性食品来改善机体健康状况。
发明内容
针对现有技术中的不足，本发明的第一个目的是提供一种鸢乌贼酮体分离蛋白酶解物的应用。
本发明的第二个目的是提供上述鸢乌贼胴体分离蛋白酶解物的制备方法。
为达到上述第一个目的，本发明的解决方案是：
一种鸢乌贼酮体分离蛋白酶解物(Isolated protein hydrolysates fromsymplectoteuthis oualaniensis,LPHFSO)作为抗氧化剂的应用。
为达到上述第二个目的，本发明的解决方案是：
一种上述的鸢乌贼酮体分离蛋白酶解物的制备方法，其包括：
在鸢乌贼酮体分离蛋白中加入去离子水，调节温度和pH至酶反应条件，并加入蛋白酶进行酶解。
作为本发明的优选实施例，温度为40±1℃，pH值为2.0-10.5；酶解时间为4±0.1h。
作为本发明的优选实施例，蛋白酶包括胃蛋白酶、酸性蛋白酶、碱性蛋白酶和木瓜蛋白酶。
作为本发明的优选实施例，鸢乌贼酮体分离蛋白酶解物由鸢乌贼酮分离蛋白酶解得到。
作为本发明的优选实施例，鸢乌贼酮体分离蛋白的制备方法包括如下步骤：
(1)将鸢乌贼分割头足，保留酮体，加入酸液进行清洗，并切割成小块；
(2)将切割的小块在碱液中浸渍，之后在3000±10r/min离心10±0.1min；
(3)得到的上清液中加入酸液，并调至等电点为5.5，3000±10r/min离心10±0.1min，沉淀为鸢乌贼酮体分离蛋白。
作为本发明的优选实施例，步骤(1)中，酸液为柠檬酸。
作为本发明的优选实施例，步骤(2)中，碱液为氢氧化钠。
作为本发明的优选实施例，步骤(3)中，酸液为盐酸。
作为本发明的优选实施例，鸢乌贼胴体分离蛋白中分子量240-200kDa占比为40％，分子量175kDa占比为10％，分子量99kDa占比为5％，分子量60kDa占比为15％，分子量55kDa占比为10％，分子量44kDa占比为5％，分子量36kDa占比为15％。
由于采用上述方案，本发明的有益效果是：
本发明首次以鸢乌贼为原料的海洋生物提取胴体分离蛋白，其蛋白纯度超过85％，获得1000Da以下的蛋白酶解物含量超过60％，并用绿色可控的酶解工艺得到鸢乌贼胴体分离蛋白酶解物，通过DPPH自由基清除率为指标评价鸢乌贼胴体分离蛋白酶解物的抗氧化活性，从而填补了关于海洋生物抗氧化活性肽的制备的研究空白。
附图说明
图1为本发明中实施例1的鸢乌贼胴体分离蛋白制备流程图。
图2为本发明中实施例1的鸢乌贼胴体分离蛋白样品的SDS-PAGE图。
图3为本发明中实施例2不同浓度的鸢乌贼胴体分离蛋白酶解物对DPPH自由基清除率的影响图。
具体实施方式
本发明提供了一种鸢乌贼酮体分离蛋白酶解物作为抗氧化剂的应用。
以下实施例中所用鸢乌贼为上海海洋大学水产学院捕捞，并放置-20℃冷冻保存。
实施例1
如图1所示，本实施例的鸢乌贼胴体分离蛋白具体提取步骤如下：
(1)印度洋鸢乌贼常温解冻，饮用水清洗2次。
(2)分割头足，保留胴体；剥皮，加入0.1mol/L柠檬酸水溶液清洗2次，切割成长度和宽度均为2cm小块，称重。
(3)原料放入0.1mol/L的NaOH水溶液浸制8h，料液比为1∶3。
(4)除去油脂后的原料加入0.1mol/L的NaOH水溶液(料液比1∶3)在均质机中进行均质2次，匀浆液3000r/min离心10min。
(5)上清液加入6mol/L的HCl，并将上清液调至等电点即pH为5.5后3000r/min离心10min，获得鸢乌贼胴体分离蛋白。
实施例2
采用实施例1中鸢乌贼胴体分离蛋白制备鸢乌贼胴体分离蛋白酶解物，具体步骤如下：
(1)称取鸢乌贼胴体分离蛋白1g，按底物质量浓度3％加入去离子水。
(2)调节温度和pH至酶反应最适条件(见表1)。
(3)混匀后，按酶底比9000加入蛋白酶(见表1)酶解时间4h。
(4)获得鸢乌贼胴体分离蛋白酶解物。
表1酶解反应条件
效果例1：SDS-PAGE分析
运用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测实施例1的鸢乌贼胴体分离蛋白的分子质量，如图2所示，其中，M为高分子质量Marker，a为鸢乌贼胴体分离蛋白。由图2可以看出，鸢乌贼分离蛋白为7个条带，分子量分别为240-200kDa、175kDa、99kDa、60kDa、55kDa、44kDa、36kDa，其中240kDa左右蛋白是鸢乌贼肌球蛋白重链(Myosin heavy chain，MHC)，99kDa蛋白是鸢乌贼副肌球蛋白(Paramyosin，PM)，分子量接近44kDa的是肌动蛋白(Actin，A)，分子量相对较小的是原肌球蛋白(Tromyosin，TM)约36kDa。其中MHC条带的条带较宽、颜色较深，这部分蛋白相近分子质量较多含量高，出现模糊的蛋白条带，与文献结果基本一致。蛋白条带清晰，没有因酸碱处理，而导致大分子蛋白的明显聚集和降解。经过碱溶酸沉提取的鸢乌贼胴体分离蛋白经冷冻干燥后通过凯氏定氮测得其蛋白含量为86％。
效果例2：鸢乌贼胴体分离蛋白氨基酸分析
实施例1的鸢乌贼胴体分离蛋白经HCl水解后，通过日立LA8080超高速氨基酸自动分析仪测定16种氨基酸，另取样用NaOH水解后采用同机测定色氨酸。
采用上述氨基酸自动分析法，共检测了鸢乌贼胴体分离蛋白的17种氨基酸，结果如表2。由表2可以看出，鸢乌贼胴体分离蛋白的8种必需氨基酸中亮氨酸、赖氨酸、缬氨酸、异亮氨酸含量较高，分别为5.59g/100g、5.31g/100g、3.04g/100g、3.27g/100g。其中必需氨基酸占总氨基酸达42.56％，必需氨基酸占非必需氨基酸达74.09％。根据FAO/WHO世界卫生组织提出的理想模式，蛋白质其组成氨基酸的TEAA/TAA为40％左右，TEAA/TNEAA在60％以上时则认为它是一种质量较好的蛋白质。与挪威三文鱼相比，鸢乌贼胴体分离蛋白有着类似的氨基酸构成结构，必需氨基酸和非必需氨基酸的比例适宜，营养结构合理，都属于优质蛋白质。通常抗氧化肽含有的氨基酸有Met、Arg、Leu、Ala和Glu，在鸢乌贼胴体分离蛋白中该类氨基酸占比高达42.19％。从这方面很好的显示出来应该具有制备抗氧化肽的可行性。
表2鸢乌贼胴体分离蛋白氨基酸构成及含量
以上结果为三次重复测定的平均值。
效果例3：鸢乌贼胴体分离蛋白酶解物DPPH自由基清除能力结果分析
不同浓度鸢乌贼胴体分离蛋白酶解物对比抗坏血酸Vc和DPPH自由基清除率的影响结果如图3。由图3可知，1mg/ml的LPHFSO DPPH自由基清除率为22.7％，2mg/ml的LPHFSODPPH自由基清除率为32.1％，3mg/ml的LPHFSO DPPH自由基清除率为43.9％，4mg/ml的LPHFSO DPPH自由基清除率为52.5％，5mg/ml的LPHFSO DPPH自由基清除率为65.6％，在0-5mg/ml浓度范围内，随着浓度的增加，LPHFSO DPPH自由基清除率有着显著的提升，和抗坏血酸Vc清除率的对比来看，虽然清除能力不及Vc，但不失为一种良好的抗氧化剂。而且以后进一步分离纯化后的抗氧化活性也会较大有提升，为以后从鸢乌贼胴体制备抗氧化活性强的海洋功能蛋白肽提供基础。
上述对实施例的描述是为了便于该技术领域的普通技术人员能理解和使用本发明。熟悉本领域技术人员显然可以容易的对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中，而不必经过创造性的劳动。因此，本发明不限于上述实施例。本领域技术人员根据本发明的原理，不脱离本发明的范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (9)
1.一种鸢乌贼酮体分离蛋白酶解物作为抗氧化剂的应用。
2.一种如权利要求1所述的鸢乌贼酮体分离蛋白酶解物的制备方法，其特征在于：其包括：
在鸢乌贼酮体分离蛋白中加入去离子水，调节温度和pH至酶反应条件，并加入蛋白酶进行酶解。
3.根据权利要求2所述的制备方法，其特征在于：所述温度为40±1℃，所述pH值为2.0-10.5；所述酶解时间为4±0.1h。
4.根据权利要求2所述的制备方法，其特征在于：所述蛋白酶包括胃蛋白酶、酸性蛋白酶、碱性蛋白酶和木瓜蛋白酶。
5.根据权利要求2所述的制备方法，其特征在于：所述鸢乌贼酮体分离蛋白的制备方法包括如下步骤：
(1)将鸢乌贼分割头足，保留酮体，加入酸液进行清洗，并切割成小块；
(2)将切割的小块在碱液中浸渍，之后在3000±10r/min离心10±0.1min；
(3)得到的上清液中加入酸液，并调至等电点为5.5，3000±10r/min离心10±0.1min，沉淀为鸢乌贼酮体分离蛋白。
6.根据权利要求5所述的制备方法，其特征在于：步骤(1)中，所述酸液为柠檬酸。
7.根据权利要求5所述的制备方法，其特征在于：步骤(2)中，所述碱液为氢氧化钠。
8.根据权利要求5所述的制备方法，其特征在于：步骤(3)中，所述酸液为盐酸。
9.根据权利要求5所述的制备方法，其特征在于：所述鸢乌贼胴体分离蛋白中分子量240-200kDa占比为40％，分子量175kDa占比为10％，分子量99kDa占比为5％，分子量60kDa占比为15％，分子量55kDa占比为10％，分子量44kDa占比为5％，分子量36kDa占比为15％。
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