CN113243512A - 一种鲜味肽 - Google Patents
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- A—HUMAN NECESSITIES
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Abstract
本发明公开了一种鲜味肽，其氨基酸序列为Asp‑Glu‑Lys。本发明所提供的具有鲜味的生物活性肽Asp‑Glu‑Lys可与人类鲜味受体T1R1/T1R3活性位点结合，主要结合位点为位于受体T1R1/T1R3上的氨基酸残基Arg151和Asp147，能呈现鲜味，且具有安全无毒副作用、水溶性良好等特点，可作为调味品或食品添加剂在食品中应用。
Description
技术领域
本发明属于生物活性肽领域，具体涉及一种鲜味肽。
背景技术
“鲜味”一词源于日本词(うま味)这意味着好吃的味道。鲜味是指谷氨酸的味道，池田最先发现并从海藻汤中分离出来。2002年，鲜味被列为继甜、苦、酸、咸四种基本味之后的第五种基本味。鲜味可以通过减少饮食中钠的摄入量来降低成年人患慢性疾病的风险。除L-谷氨酸钠(MSG)外，还发现一些双功能酸、游离L-氨基酸、多肽及其衍生物或反应产物具有鲜味。特别是多肽及其衍生物是调味品的重要组成部分。因此，发现鲜味肽对于制备新的鲜味调味品或食品添加剂具有重要意义。
目前已报道的鲜味肽有CM、GCG、EDG、TESSSE和RGENESEEEGAIVT等100多种。目前主要利用多维色谱和超高效液相色谱-电喷雾电离-四极杆飞行时间质谱(UPLC-ESI-QTOF-MS/MS)鉴定蛋白水解物中的鲜味肽。然而，传统的分离纯化鲜味肽的方法通常耗时且昂贵。此外，在多种肽的复杂混合物中很难获得高纯度的鲜味肽。所以急需建立更简单高效的鲜味肽筛选方法。
计算机辅助筛选可应用于鲜味肽的筛选和鉴定，如虚拟酶解和分子对接等。利用分子对接技术可将小分子肽与鲜味受体结合，阐明鲜味作用机制。鲜味受体是G蛋白偶联受体家族(GPCRs)。每种味觉形态可能通过不同的机制影响受体细胞，因为受体的结构和相应的下游效应物是不同的。鲜味受体主要包括异二聚体1型味觉受体、成员1和3(T1R1/T1R3)以及味觉代谢谷氨酸受体mGluR4和mGluR1。研究表明，mGluRs信号主要发生在舌后，有助于区分鲜味等味觉物质的行为，而T1R1/T1R3信号主要发生在舌前，对偏好行为起着重要作用。鲜味受体T1R1/T1R3对谷氨酸和鲜味肽都敏感，因此，T1R1/T1R3被认为是最佳的鲜味受体。
本发明旨在公开一种鲜味肽，并将其应用于制备调味品或食品添加剂。
发明内容
本发明保护了一种鲜味肽，其氨基酸序列为Asp-Glu-Lys。本发明所提供的具有鲜味的生物活性肽Asp-Glu-Lys可与人类鲜味受体T1R1/T1R3活性位点结合，主要结合位点为位于受体T1R1/T1R3上的氨基酸残基Arg151和Asp147，能呈现鲜味，且具有安全无毒副作用、水溶性良好等特点。
本发明的一种具有鲜味的活性肽，有明显的呈鲜效果。当肽DEK的浓度为0.1％时，肽鲜味值为2.31，高于同浓度的味精溶液。
本发明公开的一种具有鲜味的活性肽，其获得可利用胃蛋白酶、胰蛋白酶和胰凝乳蛋白酶对卵转铁蛋白进行酶解并通过多维色谱纯化(凝胶过滤色谱、亲和色谱和半制备液相色谱)实现；也可通过固相化学合成方法实现。优选为通过固相化学合成方法实现。
本发明的目的通过以下技术方案实现：
(1)活性肽的筛选
本发明借助ExPASy PeptideCutter这一在线酶解程序对卵转铁蛋白序列(Accession of NCBI：CAA26040.1)进行虚拟酶解，通过Innovagen和ToxinPred网站对酶解得到的肽进行水溶性和毒性预测，筛选出水溶性好且无毒的33个三肽EAG，GTE，QGK，QCK，CAR，SGA，DGK，SDF，DSQ，DQL，ENR，DEK，CDR，DGG，AER，DEY，VEK，ADW，ECN，ANK，QEK，GDK和EGK。通过同源建模获得鲜味受体T1R1/T1R3的晶体结构，通过Discovery Studio(DS)2017client software中的CDOCKER程序进行分子对接来筛选能与鲜味受体T1R1/T1R3结合的肽。较低的对接分数意味着鲜味肽与鲜味受体T1R1/T1R3具有较高的结合能力。因此，选择对接得分最低的DEK(-99.50kcal/mol)进行电子舌分析。
(2)体外鲜味活性的测定
利用电子舌对筛选得到的活性肽DEK的味觉特性进行了验证。该味觉传感器由两个参考传感器和五个测试传感器组成。AAE、CT0、CA0、C00、AE1五个传感器分别检测鲜味、成味、酸味、苦味和涩味。
首先将80ml的肽溶液分为两部分，加入到两个50ml的样杯中进行检测。测试前，用正负极清洗液清洗传感器90s，之后在参考溶液中清洗120s，然后在另一参考溶液中重复。清洗后，在测量前对传感器平衡30秒，再测量30秒。然后将传感器清洗两次，并将其返回到参考溶液中测量余味值，每次测量完成一次。测量后，传感器自动清洗运行。每个样本测量4次，由于第一次测量值不稳定，将第一次数据删除。最后三次的平均值作为测试的结果。以相同浓度的味精为阳性对照，测量肽体外鲜味活性。
本发明与现有技术相比，具有如下有益效果：
本发明从卵转铁蛋白中筛选得到了一种具有鲜味的活性肽DEK。肽DEK具有安全无毒，水溶性良好等特点，可与人类鲜味受体T1R1/T1R3活性位点结合，主要结合位点为位于受体T1R1/T1R3上的氨基酸残基Arg151和Asp147。呈现鲜味，可将其应用于制备调味品或食品添加剂方面。
附图说明
本发明附图2幅，其中：
图1 DEK与T1R1/T1R3受体的对接结果2D图；
图2 MSG与T1R1/T1R3受体的对接结果2D图；
具体实施方式
下面以具体实施例的方式对本发明作进一步的阐述。
实施例1.具有鲜味活性的肽的筛选
本发明借助ExPASy PeptideCutter这一在线酶解程序对卵转铁蛋白序列进行虚拟酶解，通过Innovagen和ToxinPred网站对酶解得到的肽进行水溶性和毒性预测，筛选结果如表1所示。筛选出水溶性好且无毒的33个三肽EAG，GTE，QGK，QCK，CAR，SGA，DGK，SDF，DSQ，DQL，ENR，DEK，CDR，DGG，AER，DEY，VEK，ADW，ECN，ANK，QEK，GDK和EGK。通过同源建模获得鲜味受体T1R1/T1R3的晶体结构，通过Discovery Studio(DS)2017 client software中的CDOCKER程序进行分子对接来筛选能与鲜味受体T1R1/T1R3结合的肽。较低的对接分数意味着鲜味肽与鲜味受体T1R1/T1R3具有较高的结合能力。因此，选择对接得分最低的DEK(-99.50kcal/mol)进行电子舌验证。对接结果如表2所示。
表1酶解卵转铁蛋白释放三肽的溶解度和毒性筛选结果
表2卵转铁蛋白衍生的22个三肽与T1R1/T1R3分子对接结果
分子对接结果显示，分别分析了肽、MSG与鲜味受体T1R1/T1R3之间的相互作用，发现了5个重要的氨基酸残基Arg307，Asp147，Gln52，Arg151和Asn69在DEK与T1R1/T1R3的对接过程中起着至关重要的作用。图1显示DEK-T1R1复合物被两个碳氢键(Asp147和Gln52)，八个常规氢键(Arg307，Asn69，Ser148，Gln278，Gly72和Gln52)和一个盐桥(Glu301)稳定。此外，T1R1/T1R3的残基Arg151，Arg307和Asp147与DEK形成静电相互作用。图2显示，MSG-T1R1复合物被3个碳氢键(Asp147、Arg277和Gln52)、3个常规氢键(Arg151、His398和Asn69)和2个盐桥(Glu301和Asp147)稳定。此外，T1R1/T1R3中的残基Arg307与MSG形成静电相互作用，残基Try220与MSG形成了π-烷基相互作用。三肽DEK和MSG与T1R1/T1R3的对接结果表明，常规氢键在鲜味生成过程中发挥了重要作用。
实施例2.体外鲜味活性的测定
利用电子舌对筛选得到的活性肽DEK的味觉特性进行了验证。该味觉传感器由两个参考传感器和五个测试传感器组成。AAE、CT0、CA0、C00、AE1五个传感器分别检测鲜味、成味、酸味、苦味和涩味。
首先将80ml的肽溶液分为两部分，加入到两个50ml的样杯中进行检测。测试前，用正负极清洗液清洗传感器90s，之后在参考溶液中清洗120s，然后在另一参考溶液中重复。清洗后，在测量前对传感器平衡30秒，再测量30秒。然后将传感器清洗两次，并将其返回到参考溶液中测量余味值，每次测量完成一次。测量后，传感器自动清洗运行。为了减少系统误差，对每个样本进行了4次重复测量，消除了第一次测量结果。最后三次的平均值作为测试的结果。以相同浓度的味精为阳性对照，测量肽DEK体外鲜味活性。
结果表明，DEK浓度为0.10％时鲜味值为2.31，味精浓度为0.10％、0.20％和0.30％时鲜味值分别为0、0.56和1.28。三肽DEK的鲜味值明显高于浓度分别为0.10％、0.20％和0.30％的味精，表明DEK为鲜味肽。
实施例3.鲜味肽DEK的应用
在实际生产中，鲜味肽DEK可通过固相化学合成法制成，以粉末状、颗粒状或溶于水的液体状添加到调味品、食品以及保健品中，以满足以鲜味为主的口感需求。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
Claims (2)
1.一种鲜味肽，其特征在于，所述鲜味肽的氨基酸序列为Asp-Glu-Lys。
2.权利要求1所述的鲜味肽在制备调味品或食品添加剂中的应用。
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| US20090092582A1 (en) * | 2007-08-15 | 2009-04-09 | Oren Bogin | Compositions and methods for modifying properties of biologically active polypeptides |
| CN106279350A (zh) * | 2016-08-11 | 2017-01-04 | 西南大学 | 一种毛霉型豆豉苦味肽的制备方法及应用 |
| JP2017217005A (ja) * | 2017-08-24 | 2017-12-14 | 日清食品ホールディングス株式会社 | 塩味増強ペプチド |
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