CN110655555A - 肌球蛋白来源活性肽及其应用 - Google Patents
肌球蛋白来源活性肽及其应用 Download PDFInfo
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Abstract
本发明涉及蛋白活性肽领域，具体涉及多个来源于肌球蛋白的活性肽，其氨基酸序列分别为Asp‑Lys(DK)、Glu‑Glu‑Lys(EEK)、Glu‑Asp‑Gln‑Lys(EDQK)、Ser‑Glu‑Gly‑Gly‑Arg(SEGGR)、Gln‑Asp‑Ser‑Ile‑Gly‑Ser(QDSIGS)。本发明所述的活性肽通过借助分子对接和虚拟筛选技术获得，均可以通过基因工程或化学方法合成，也可通过生物酶解技术结合纯化手段获得。本发明提供的五条活性肽，可作为基料或辅料来制作调味品，广泛应用于食品领域。
Description
技术领域
本发明属于蛋白活性肽领域，具体涉及肌球蛋白来源的生物活性肽及其应用。
背景技术
目前，生物活性肽的制备及其应用开发已经成为世界范围内研究的热点。鲜味肽因其可增强食物鲜味和醇厚味，且易于吸收，具有良好的加工特性及营养价值，而受到了越来越多的关注。并且，鲜味肽在食品中的应用也符合“天然、营养、安全”的食品发展趋势。鲜味肽不仅可以增强食物的鲜美口感，还可作为挥发性风味物质的前体物质与美拉德反应，进一步增强食品的特征香味；还能使糖更甜，使盐更咸，降低苦味和酸味，协同五种基本口味，使鲜味更具有质感。鲜味是继酸、甜、苦、咸之后的第五种基本味觉，可以给人带来欢愉感，是人体必不可少的味觉需求。在1908年，日本科学家首次在海带中分离出谷氨酸，并提出鲜味的概念，以umami命名。随后肌苷酸，鸟苷酸等核苷酸也被发现具有鲜味。1978年日本学者从牛肉的蛋白酶解液中分离、提取、纯化出一条美味肽-牛肉八肽(BMP)。随后，逐渐从肉类酶解物、水产品、火腿等食品中也提取出一些具有鲜味的多肽，并将其统一命名为鲜味肽。而鲜味肽是继MSG、IMP、GMP之后的一种理想的天然鲜味物质。
鲜味肽的制备方法主要有提取法和蛋白降解法，结合凝胶色谱、高效液相色谱等技术对其进行分离纯化得到更高纯度的鲜味肽。但是目前为止，已知食品中的鲜味肽种类有限，其对鲜味的贡献程度也有不同，同时鲜味肽作为一种新型天然鲜味剂也备受人们关注和研究，因此新型鲜味肽亟待开发。
发明内容
本发明目的在于提供：
一种生物活性肽DK，其包括由Asp-Lys组成的活性肽；
一种生物活性肽EEK，其包括由Glu-Glu-Lys组成的活性肽；
一种生物活性肽EDQK，其包括由Glu-Asp-Gln-Lys组成的活性肽；
一种生物活性肽SEGGR，其包括由Ser-Glu-Gly-Gly-Arg组成的活性肽；
一种生物活性肽QDSIGS，其包括由Gln-Asp-Ser-Ile-Gly-Ser组成的活性肽。
较优的，所述的活性肽均具有鲜味和浓厚感。可作为基料或辅料来制作调味品，广泛应用于食品加工中。选用味精作为阳性对照，将肽DK、EEK、EDQK、SEGGR、QDSIGS及味精(MSG)配制成0.1mg/ml的溶液。选用AAE、CT0、CA0、C00、AE1 5个测试传感器和2个参比传感器对样品进行检测。结果表明，肽DK、EEK、EDQK、SEGGR、QDSIGS的鲜味效果均要优于味精(MSG)。其中，肽EDQK的鲜味强度最高。
较优的，所述的活性肽DK、EEK、EDQK、SEGGR、QDSIGS均来自虾夷扇贝肌球蛋白，并且分别为肌球蛋白(GenBank：BAB40711.1)第595～597位、第342～345位、第1383～1387位、第1513～1518位、第1488～1494位的氨基酸残基。
本发明通过ToxinPred和peptide property calculator对活性肽DK、EEK、EDQK、SEGGR、QDSIGS进行毒性、水溶性性质的分析，利用鲜味肽的氨基酸组成、氨基酸的空间排列、分子量等进行多轮筛选，结合分子对接实验，活性肽DK、EEK、EDQK、SEGGR、QDSIGS与鲜味受体T1R1/T1R3结合紧密。
本发明的生物活性肽DK、EEK、EDQK、SEGGR、QDSIGS均可以通过基因工程的方法和化学方法人工合成，也可以从肌球蛋白中通过分离纯化、酶降解的方法获得。利用电子舌检测，结果表明肽段DK、EEK、EDQK、SEGGR、QDSIGS具有鲜味和浓厚感。
本发明相对于现有技术具有如下的优点及效果：
(1)本发明的鲜味肽是小分子量肽，易于被人体吸收。
(2)本发明开发的鲜味肽鲜味强度高，可以代替味精的使用，在食品中的应用意义较大。
(3)本发明获得的鲜味肽纯度高。
附图说明
本发明附图8幅，其中：
图1. 62种多肽氨基酸组成图；
图2.鲜味受体T1R1/T1R3模型的Ramachandran Plot(拉曼图谱)；
图3.鲜味受体T1R1/T1R3的胞外结构模型图；
图4.DK质谱图；
图5.EEK质谱图；
图6.EDQK质谱图；
图7.SEGGR质谱图；
图8.QDSIGS质谱图。
具体实施方式
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。
下面以具体实施例的方式对本发明作进一步阐述。
实施例1虾夷扇贝肌球蛋白的酶解筛选
选用胃蛋白酶(EC 3.4.23.1)，胰蛋白酶(EC 3.4.21.4)和胰凝乳蛋白酶(EC3.4.21.1)通过ExPASy PeptideCutter(http：//web.expasy.org/peptide_cutter/)用对肌球蛋白序列(GenBank：BAB40711.1)进行酶切，获得747条肽序列。
利用ToxinPred(http：//crdd.osdd.net/raghava//toxinpred/)对肽的毒性进行分析。通过peptide property calculator(http：//www.innovagen.com/)对肽的水溶性进行分析，筛选获得322条无毒、水溶性良好的活性肽。鲜味肽的呈味与与氨基酸组成、氨基酸的空间排列及肽的分子量有关，含有谷氨酸和天冬氨酸，以及含有一定的亲水性氨基酸，且肽链骨架长度为2-6时，具有较好的呈味效果，且二肽C端为带负电荷的酸性氨基酸，N端为带正电荷的碱性氨基酸，具有呈味特性。基于此，对322条肽进行筛选获得70条符合条件的肽序列。利用活性肽数据库BIOPEP(http：//www.uwm.edu.pl/biochemia/index.php/en/biopep)进行检索和比对，剔除已被报道过的鲜味肽。最终筛选得到62条未被报道的肽序列，结果见图1。
采用modeller 9.18构建鲜味受体T1R1/T1R3异源二聚体蛋白模型。选取metabotropic glutamate receptor(PDB ID：1ewk)作为建模模板。在gromacs2018.1软件包中对构建模型进行分子动力学优化。对优化后的鲜味受体模型进行拉氏图计算，结果如图2所示，数据分析表明该模型中有99.3％的氨基酸残基处于允许区内，根据90％的临界评价原则，表明鲜味受体T1R1/T1R3模型构型合理。鲜味受体T1R1/T1R3胞外结构模型如图3所示。
通过Discovery Studio(DS)2017客户端软件的CDOCKER程序进行分子对接。对接能量结果如表1所示，结果表示二肽DK、三肽EEK、四肽EDQK、五肽SEGGR和六肽QDSIGS具有最低的Docking Energy和Docking Interaction Energy得分，得分越低表示结合越紧密。因此对DK、EEK、EDQK、SEGGR和QDSIGS进行制备。
表1 多肽与鲜味受体T1R1/T1R3的对接能量
实施例2电子舌检测
本实施例选用味精(MSG)作为阳性对照。将肽DK、EEK、EDQK、SEGGR、QDSIGS及MSG配制成0.1mg/ml的溶液。选用AAE、CT0、CA0、C00、AE1 5个测试传感器和2个参比传感器对样品进行检测。采用插值法来分析样品的味道，数据处理结果见表2。
表2 基于电子舌的不同肽的Umami和Richness值
结果表明，活性肽DK、EEK、EDQK、SEGGR、QDSIGS的呈鲜效果均要优于味精(MSG)。其中，肽EDQK的鲜味强度最高。最终本发明提供了五条鲜味肽。
实施例3活性肽DK、EEK、EDQK、SEGGR、QDSIGS的化学合成
本实施例采用FMOC法固相合成肽DK、EEK、EDQK、SEGGR、QDSIGS。在温度为22℃～28℃和常压下，将Fmoc-Tyr(tBu)-Wang Resin加入到多肽固相合成反应器中，加入10mL 25％哌啶/DMF溶液脱除氨基保护基Fmoc，依次同2倍过量的下一个氨基酸的活化羟基反应延长肽链，缩合过程中采用茚三酮定性显色监测反应进程，直至得到合成所需的目标肽段。选择适当的切割条件将目标多肽从树脂裂解下来并除去侧链保护基，溶液经旋蒸后，加入无水乙醚使多肽沉淀析出，经离心、洗涤和真空干燥，即得到目标肽粗品。
采用高效液相色谱对目标肽的纯度进行分析，色谱条件：色谱柱为Gemini-NX C18(4.6×250mm，5μm)色谱柱，进样量20μL；流动相A液为0.1％的三氟乙酸乙腈溶液，流动相B液为0.1％的三氟乙酸水溶液；梯度洗脱方式为2％A+98％B在30min内降为100％A+0％B；流速为1.0mL/min；检测波长为220nm。自动收集洗脱峰。目标肽纯度分别为88％、95％、96％、91％、94％。
采用质谱法对样品进行鉴定。质谱条件为电喷雾电离源(ESI)，曲型脱溶剂装置(CDL)温度为250℃，加热块(Block)温度为200℃，雾化气流速为1.5L/min，探针(Probe)电压为+4.5kv。肽DK、EEK、EDQK、SEGGR、QDSIGS理论分子量分别为216.27Da、404.42Da、518.51Da、504.49Da、605.59Da。五个样品的质谱分析报告见图4、图5、图6、图7、图8，得到的分子离子峰分别为261.7、405.2、519.3、505.3、606.3，与肽DK、EEK、EDQK、SEGGR、QDSIGS理论分子量一致，确定样品为目标肽。
最后，还需注意的是，上述实施例是示例性的，不能理解为对本发明的限制，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、修改、替换和变型，其也应视为本发明的保护范围。
Claims (4)
1.一种生物活性肽DK，其氨基酸序列为Asp-Lys；
一种生物活性肽EEK，其氨基酸序列为Glu-Glu-Lys；
一种生物活性肽EDQK，其氨基酸序列为Glu-Asp-Gln-Lys；
一种生物活性肽SEGGR，其氨基酸序列为Ser-Glu-Gly-Gly-Arg；
一种生物活性肽QDSIGS，其氨基酸序列为Gln-Asp-Ser-Ile-Gly-Ser。
2.根据权利要求1所述的生物活性肽，其特征在于：源自虾夷扇贝中的肌球蛋白。
3.根据权利要求1所述的活性肽，配成0.1mg/ml溶液进行电子舌检测，Umami值分别为-1.5、-1.57、-0.97、-1.49和-2.27，Richness值分别为1.61、1.67、1.93、1.57和1.58。
4.权利要求1所述的生物活性肽DK、EEK、EDQK、SEGGR、QDSIGS在制备鲜味剂中的应用。
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