CN112521449B - 两种具有苦味抑制作用的活性肽 - Google Patents
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Abstract
本发明公开了两种具有苦味抑制作用的活性肽，其氨基酸序列分别为Ala‑Asp‑Met和Ala‑Asp‑Trp。本发明所提供的两种具有苦味抑制作用的活性肽能与苦味受体T2R14有效结合，对苦味物质具有持续稳定的抑制效果，且具有安全无毒副作用、水溶性良好等特点，可用于具有苦味的食品、药品以及保健品中，具有广泛的应用前景和十分重要的意义。
Description
技术领域
本发明属于生物活性肽领域，具体涉及两种具有苦味抑制作用的活性肽。
背景技术
人类能够感知五种基本味道，即酸味、甜味、苦味、咸味和鲜味。苦味被认为是一种不愉快的味觉感受，它可以在食品蛋白水解和药物加工过程中形成。由于苦味会对食物的摄取产生消极作用，而且某些药物的苦味很难让患者吞咽，因此抑制苦味是非常重要的，这是食品和制药行业长期存在的一个挑战。
苦味感知由苦味受体TAS2Rs介导，这些TAS2Rs受体在人类舌头上表达，属于G蛋白偶联受体家族。迄今为止，几乎所有25种人类苦味受体已经被鉴定出来，但大多数TAS2Rs仍然没有已知的配体。这些TAS2Rs受体具有单个配体结合口袋，这种结合口袋能够在保持高选择性的同时识别许多结构不同的苦味物质。TAS2R14(T2R14)是对各种苦味化合物调谐最广的苦味受体之一，它能识别多种多样的天然或合成的苦味化合物，包括许多药物。因此，T2R14可作为抑制苦味的有效靶点。
传统的脱苦方法主要包括活性炭吸附、大孔树脂吸附、超滤等选择性分离法以及掩盖法。但是，选择性分离法对多肽的损失比较大，影响多肽的营养性和活性，特别是对于苦味肽作为主要活性组分的物质影响更大；掩盖法需要添加环糊精、改性淀粉等苦味掩盖剂或其他风味物质，其附加成分不符合健康天然的新型饮食观念。例如，发明专利：一种吡格列酮的苦味抑制剂和包含吡格列酮的口服制剂，专利号为：201410473474.8。该专利涉及一种吡格列酮的苦味抑制剂和包含吡格列酮的口服制剂，所述吡格列酮的苦味抑制剂以氯化钙作为有效成分。而食源性生物活性肽以其安全无毒副作用、功能多样和容易修饰等优点，受到食品科学、化学以及生物学等领域的广泛关注。因此，发现食源性TAS2Rs抑制肽成为当今的研究热点。海洋鱼类是优质蛋白质的巨大来源宝库。虹鳟被誉为“水中人参”，营养价值丰富，需求量大，已从北美西部引殖到很多国家，在我国实现人工养殖。因此，从虹鳟伴肌动蛋白中筛选出水溶性好、具有生物活性、无毒且能与苦味受体T2R14结合的苦味抑制肽，不仅可以解决食品、药品及保健品的不良苦味，还提高了虹鳟鱼的附加价值。
本发明旨在发现两种具有苦味抑制作用的活性肽，并将其应用于克服食品苦味而不损害食品本身的风味和营养价值的抑制剂。
发明内容
本发明公开了两种具有苦味抑制作用的活性肽，其氨基酸序列分别为Ala-Asp-Met(ADM)和Ala-Asp-Trp(ADW)。
本发明的两种具有苦味抑制作用的活性肽，作用于苦味受体T2R14这个靶点，竞争性抑制苦味物质与苦味受体T2R14的结合，对苦味物质具有持续稳定的抑制效果，且具有安全无毒副作用、水溶性良好等特点。
本发明的两种具有苦味抑制作用的活性肽，对苦味物质具有显著的抑制作用。作为具有苦味的食材，例如苦瓜、杏仁、咖啡、茶、可可、啤酒花等；作为具有苦味的药材，例如黄连、黄柏、当药、苍术、大黄等，均能显示苦味抑制效果。其中，当肽ADM、ADW的浓度为0.25mg/mL时，肽对奎宁的抑制率可分别达到82.56％和75.34％。
本发明的两种具有苦味抑制作用的活性肽，可以应用于食品、药品以及保健品领域。对于本发明的一个实施方式的苦味抑制肽作为对象的苦味，可列举出源自食材的苦味或源自药材的苦味。作为具有苦味的食材，例如可列举出苦瓜、杏仁、咖啡、茶、可可、啤酒花等；作为具有苦味的药材，例如可列举出黄连、黄柏、当药、苍术、大黄等。但是，并不限定于此。
本发明的两种具有苦味抑制作用的活性肽，其抑制苦味物质活性的半抑制浓度(IC50)分别为420.32μM和403.29μM。
本发明的两种具有苦味抑制作用的活性肽，其获得可利用胃蛋白酶、胰蛋白酶和胰凝乳蛋白酶对虹鳟伴肌动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。
本发明的两种具有苦味抑制作用的活性肽，其形态没有特别限定。例如，通过溶于水制成液剂，通过喷雾干燥制成粉末状或颗粒状，通过冷冻干燥或加热干燥制成固形剂来使用。优选的，当制成液剂使用时，相对于100ml的液剂，活性肽的含量为7.5-25mg。
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这一在线酶切工具对虹鳟伴肌动蛋白序列进行虚拟酶切，得到687个二肽及其以上的活性肽。通过peptide property calculator、PeptideRanker和Discovery Studio(DS)2017 R2 Client软件的ADMET程序对687个活性肽进行水溶性、生物活性和毒性的预测，筛选得到水溶性好、活性评分高于0.5且无毒的22个肽GKP、LPPDAPEL、PASDNPVL、DW、SM、DM、DAM、GSG、NDPQF、CSPVDM、PDSM、ADW、DGM、PADAPEF、PADSPQF、SDW、SPVDM、MPDSM、ADM、IPPDM、SIPDRPEF和PDIM。从BitterDB数据库中获得苦味受体T2R14的晶体结构，并将其作为蛋白靶标。通过DS中的CDOCKER程序进行分子对接来筛选能与苦味受体T2R14紧密结合的肽，并明确活性位点。以‘-CDOCKER_ENERGY’得分为指标，将筛选得到的肽序列与BIOPEP数据库中已报道的苦味抑制肽进行比较，获得四个理论上能竞争性抑制苦味物质与T2R14受体的结合且未经报道的肽ADM、ADW、SPVDM和CSPVDM，这四个肽的‘-CDOCKER_ENERGY’得分均低于阳性对照肽LEGSLE的得分(66.2088kcal/mol)。
(2)体外苦味抑制活性的测定
通过电子舌对一定浓度的活性肽ADM、ADW、SPVDM和CSPVDM的苦味抑制活性进行测定。分别取一定质量的肽ADM、ADW、SPVDM和CSPVDM，加入到30mL水中混匀，加入50mL奎宁(1mM)配制成待测溶液，以奎宁作为空白对照组，以肽LEGSLE作为阳性对照组。设置好循环次数、数据文件名称、样品数量、样品名称和测试方法后，安装好电极，开始测量。
测量过程如下：(1)在正负极溶液中清洗传感器90s，然后在两种基准液中清洗传感器120s；(2)在调节溶液中平衡传感器30s；(3)测量每个样品30s；(4)两次各清洗传感器3s后，将其浸入基准液中30s以测量回味值。每个样品重复测量四次。测量完成后，处理数据。
本发明与现有技术相比，具有如下有益效果：
本发明从虹鳟伴肌动蛋白中筛选得到了两种具有苦味抑制作用的活性肽ADM、ADW。肽ADM、ADW具有安全无毒、具有生物活性、水溶性良好等特点，竞争性抑制苦味物质与苦味受体T2R14的结合，对苦味物质具有持续稳定的抑制效果，而不损害食品本身的风味和营养价值。相比于其他糖类、盐类等苦味掩盖剂，肽的添加更加符合低糖低盐、健康天然的新型饮食观念。因此可用于具有苦味的食品、药品以及保健品中，具有广泛的应用前景。
附图说明
本发明附图4幅，其中：
图1 ADM与T2R14受体的对接结果2D图；
图2 ADM与T2R14受体的对接结果3D图；
图3 ADW与T2R14受体的对接结果2D图；
图4 ADW与T2R14受体的对接结果3D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.具有苦味抑制活性的肽的筛选
本发明借助ExPASy PeptideCutter(http：//web.expasy.org/peptide_cutter/)这一在线酶切工具对虹鳟伴肌动蛋白序列进行虚拟酶切，得到687个二肽及其以上的活性肽。通过在线工具peptide property calculator(http：//www.innovagen.com/proteomics-tools)、PeptideRanker(http：//distilldeep.ucd.ie/PeptideRanker/)和Discovery Studio(DS)2017 R2 Client软件的ADMET程序对687个活性肽进行水溶性、生物活性和毒性的预测，筛选得到水溶性好、活性评分高于0.5且无毒的22个肽GKP、LPPDAPEL、PASDNPVL、DW、SM、DM、DAM、GSG、NDPQF、CSPVDM、PDSM、ADW、DGM、PADAPEF、PADSPQF、SDW、SPVDM、MPDSM、ADM、IPPDM、SIPDRPEF和PDIM。从BitterDB数据库(http：//bitterdb.agri.huji.ac.il/dbbitter.php)中获得苦味受体T2R14的晶体结构，并将其作为蛋白靶标。通过DS中的CDOCKER程序进行分子对接来筛选能与苦味受体T2R14紧密结合的肽，并明确活性位点。以‘-CDOCKER_ENERGY’得分为指标，将筛选得到的肽序列与BIOPEP数据库(http：//www.uwm.edu.pl/biochemia/index.php/en/biopep)中已报道的苦味抑制肽进行比较，获得四个理论上能竞争性抑制苦味物质与T2R14受体的结合且未经报道的肽ADM、ADW、SPVDM和CSPVDM，这四个肽的‘-CDOCKER_ENERGY’得分均低于阳性对照肽LEGSLE的得分(66.2088kcal/mol)。
分子对接结果显示，肽ADM可以与T2R14的残基ASP168、THR86形成氢键相互作用，与残基PHE247形成π-烷基相互作用(图1、图2)；肽ADW可以与T2R14的残基ASP168、THR86形成氢键相互作用，与残基TRP89、PH243、PHE247形成π-π相互作用(图3、图4)。
实施例2.活性肽对苦味物质的抑制活性的测定
通过电子舌对一定浓度的肽ADM、ADW、SPVDM和CSPVDM的苦味抑制活性进行测定。分别取一定质量的肽ADM、ADW、SPVDM和CSPVDM，加入到30mL水中混匀，加入50mL奎宁(1mM)配制成待测溶液，以奎宁作为空白对照组，以肽LEGSLE作为阳性对照组。设置好循环次数、数据文件名称、样品数量、样品名称和测试方法后，安装好电极，开始测量。
测量过程如下：(1)在正负极溶液中清洗传感器90s，然后在两种基准液中清洗传感器120s；(2)在调节溶液中平衡传感器30s；(3)测量每个样品30s；(4)两次各清洗传感器3s后，将其浸入基准液中30s以测量回味值。每个样品重复测量四次。测量完成后，处理数据。
结果表明，肽ADM、ADW可有效抑制苦味受体T2R14的活性，其IC50值分别为420.32μM和403.29μM；而肽SPVDM和CSPVDM的苦味抑制作用并不显著，即使肽的添加量达到0.15mg/mL时，对应的苦味抑制率也仅达到35.83％和34.13％。
实施例3.苦味抑制活性肽ADM、ADW的应用
在实际生产中，苦味抑制活性肽ADM、ADW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具有苦味的食品、药品以及保健品中。例如，将活性肽制成料包加入茶、咖啡中以抑制饮品中的苦味，同时保留饮品中的独特风味；将活性肽添加到苦瓜中抑制其苦味从而制成苦瓜干、苦瓜茶等苦瓜加工品，消除人们对苦瓜的抵触感，提高苦瓜清热解毒、养血滋肝、降血糖等食用和药用价值；将活性肽添加到药品尤其是中草药中，黄连、黄柏、当药、苍术等药品成分的苦味得以抑制，有助于人们更加愉快地服下药物；将活性肽添加到阿胶糕等保健食品中，在抑制其自身苦味的同时又能提高保健食品的营养价值，为保健品行业提供了一个解决苦味难题的新思路。
以上所述的实施例对本发明的技术方案进行了详细说明，应理解的是以上所述仅为本发明的具体实施例，并不用于限制本发明，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围的前提下，还可以做出各种变化和变型，这些变化和变型也应视为本发明的保护范围。
Claims (1)
1.一种具有苦味抑制作用的活性肽在制备苦味掩盖剂中的应用，所述具有苦味抑制作用的活性肽的氨基酸序列为Ala-Asp-Met或Ala-Asp-Trp。
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| CN105358983A (zh) * | 2013-04-24 | 2016-02-24 | 卓莫赛尔公司 | 用于鉴定调节苦味的化合物的测定 |
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| 基于分子对接技术从虾夷扇贝中筛选抗SARS-Cov-2活性肽研究;步营等;《食品科学技术学报》;20200707;第38卷(第4期);摘要，表1、4、5 * |
| 鸡蛋蛋白ACE抑制肽的筛选、鉴定及其作用机制;于志鹏等;《食品科学》;20190909;第41卷(第12期);摘要 * |
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