CN116178492A - 藜麦胰脂肪酶抑制肽及其在制备减肥用食品中的应用 - Google Patents
藜麦胰脂肪酶抑制肽及其在制备减肥用食品中的应用 Download PDFInfo
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Abstract
本申请提供了一种藜麦胰脂肪酶抑制肽NVEGSALLPA及其在制备减肥用食品中的应用。本申请的藜麦胰脂肪酶抑制肽NVEGSALLPA来源于藜麦蛋白的食品用酶酶解产物，胰脂肪酶抑制效果显著且具有良好的安全性。
Description
发明领域
本申请属于食品和代谢疾病治疗领域，具体的，本申请提供了藜麦胰脂肪酶抑制肽及其在制备减肥用食品中的应用。
背景技术
随着营养水平的提高和生活方式的变化，肥胖日益成为影响全球大量人口健康的重要问题。除少数遗传和内分泌原因之外，肥胖的主要起因在于日常饮食中摄入过多的能量，如甘油三酯。因此，抑制脂质吸收和代谢的相关酶是抑制肥胖的常见策略之一，其中胰脂肪酶是最受关注的靶点之一。
现有的市售胰脂肪酶抑制剂仅有奥利司他一种，其虽然具有确切的减肥效果，但也有油性粪便、腹泻、头痛、直肠不适、疲乏等不良反应。因此，寻找新的胰脂肪酶抑制剂，特别是天然来源的、安全的胰脂肪酶抑制剂，是肥胖治疗的研究方向之一。
藜麦（Chenopodium quinoa Willd.）虽然名为“麦”，但实际为一种原产南美洲的藜科藜属植物。藜麦营养丰富，蛋白和脂肪含量高于一般粮食作物而葡萄糖果糖含量低于一般粮食作物，是一种调整糖脂代谢的理想食品。目前对藜麦的开发基本集中在其作为粮食作物的加工方面，尚没有对其中的高含量成分藜麦蛋白进行深入研究以提供藜麦胰脂肪酶抑制肽的先例。
2～10个氨基酸的小肽。溶解性较好、稳定、容易能避开胃肠道消化，克服蛋白质被消化酶破坏从而不能口服的弊端，口服或注射效果都较理想，并且相对分子质量小、易于修饰调控、合成成本低、可大量制备，是一类具有良好应用潜力的胰脂肪酶抑制剂选择。
发明内容
针对上述情况，申请人利用多种食品用酶酶解藜麦蛋白，从胰脂肪酶抑制效果较好的酶解物中分离到了一种胰脂肪酶抑制肽NVEGSALLPA，其稳定、安全且胰脂肪酶抑制效果优良，可用于制备多种减肥食品，特别是饮料类食品。
一方面，本申请提供了来源于一种藜麦胰脂肪酶抑制肽，其氨基酸序列为NVEGSALLPA。
进一步地，上述藜麦胰脂肪酶抑制肽由藜麦蛋白经胃蛋白酶和无花果蛋白酶酶解制备获得。
进一步地，所述酶解的酶解体系中包含5000U/mL的胃蛋白酶和5000U/mL无花果蛋白酶，以及4% w/v的乳酸钙或者2% w/v的焦磷酸钠。
进一步地，所述酶解在37摄氏度下，酶解4小时。
进一步地，所述藜麦胰脂肪酶抑制肽使用Fmoc固相合成法制备。
另一方面，本申请提供了上述藜麦胰脂肪酶抑制肽在制备减肥食品或者保健品中的应用。
进一步地，所述减肥食品或者保健品为液体饮料或者固体饮料。
另一方面，本申请提供了减肥食品或者保健品，其包含上述藜麦胰脂肪酶抑制肽。
进一步地，所述减肥食品或者保健品为液体饮料或者固体饮料。
在已知序列的情况下，本领域技术人员可以通过现有知识制备所需纯度的肽，这些方法包括但不限于酶解后提取，或者固相合成的方法，本领域技术人员可以通过控制参数、使用适合的试剂和纯化方法来保证肽产品中不包含有害物质，保证其安全性。
附图说明
图1为不同酶酶解藜麦蛋白产物的胰脂肪酶抑制活性检测结果。
图2为固相合成肽产品的质谱图。
具体实施方式
下面结合具体实施例详述本发明，但本发明的保护范围不局限于以下实施例。
材料和基本方法
藜麦品种：
实验中使用我国北方常见的陇藜1号藜麦（红色）。
上述藜麦产地为山西和内蒙古。
酶制剂：
食品级胃蛋白酶（20万U/g）、胰蛋白酶（10万U/g）、木瓜蛋白酶（10万U/g）、糜蛋白酶（10万U/g）、无花果蛋白酶（10万U/g）、、菠萝蛋白酶（5万U/g）均购自东恒华道生物科技有限公司。
胰脂肪酶抑制活性的检测：
使用pH 7.0的PBS配制2.0mg/mL胰脂肪酶溶液（SIGMA L3126），离心取上清。
使用pH 7.0的PBS配制10mM的对硝基苯基丁酸酯溶液。
将40微升待测溶液（5mg/mL水解物样本溶液或蒸馏水对照）、40微升上述胰脂肪酶溶液和20微升上述对硝基苯基丁酸酯溶液混合后，在37摄氏度水浴中孵育30分钟，使用酶标仪记录孵育后体系以及样本和对照的空白405nm吸光度。
使用以下公式计算胰脂肪酶活性抑制率（%）：
胰脂肪酶活性抑制率（以%表示）=1-（样本吸光度-样本空白吸光度）/（对照吸光度-对照空白吸光度）
如无特殊说明，所列抑制率为三个平行实验的平均抑制率
LC-MS/MS分析：
使用离心超滤管离心分离得到藜麦蛋白酶解物中小于3 kDa的级份，将所得级分过柱除盐后使用LC-MS/MS（安捷伦StreamSelect，在线纳喷离子源）分析。参数：进样量：5 μL，柱流量：500 nL/min，柱温：40°C，电喷雾电压：2 kV。流动相A相：0.1%甲酸水溶液，B相：0.1%甲酸的80%的ACN溶液。梯度从5%的B相起始，平衡2分钟，以非线性梯度升高到95%；MS：扫描范围（m/z）：350-1550，分辨率：120000，最大注入时间：100毫秒；HCD-MS/MS：分辨率：30000，动态排除时间：30秒，最大注入时间：54毫秒。PEAKS DB对uniprot-Setaria_italica数据库搜库。
肽的性能预测：
生物活性预测：PeptideRanker
胃蛋白酶和胰蛋白酶消化预测：PeptideCutter
毒性预测：ToxinPrep（SVM）
过敏性预测：AllerTOP
亲水性预测：ExPasy
不稳定性预测：ExPasy
肽的安全性实验
一般饲喂实验使用Wistar大鼠，180-240g体重，每日灌胃给予被试肽（8mg/kg）或等量蒸馏水（每组10只，雌雄各半），连续10日，正常饮水和饲喂，观察期为3周。观察和检验项目包括日常活动、饮食、行为；谷丙转氨酶、谷草转氨酶、肌酐（每周检测）；以及观察期结束时解剖后的肝肾重量和器官情况观察。
大剂量实验使用Wistar大鼠，200-220g体重，一次性灌胃被试肽100mg/kg，200mg/kg（每个剂量10只，雌雄各半），观察期为7日。观察项目为动物死亡情况、日常活动、饮食、行为。
实施例1藜麦蛋白的提取
参照本领域中的现有做法和前期摸索，藜麦蛋白的提取方法如下：
将藜麦种子清洗、烘干、磨粉后过100目筛；室温下以1:30料液比（g/mL）95%的食用乙醇搅拌1小时，静置8小时；弃上清，烘干得脱脂藜麦粉；按照1:30料液比（g/mL）向脱脂藜麦粉中加入纯净水；使用1M NaOH将pH调至9.5，40摄氏度水浴中下提取3小时；离心取上清，以1M HCl调节pH至4.5，静置30分钟，离心取沉淀；纯净水洗涤沉淀，冻干得藜麦蛋白粉。
各批次的提取率为70%左右，蛋白纯度在69%以上。
实施例2藜麦蛋白的酶解
基本酶解实验方法
将实施例1方法制备的藜麦蛋白粉按照5% w/v的比例溶解于纯净水中；调整pH后加入酶；150rpm的摇床上在37摄氏度下酶解4小时；沸水浴灭酶10分钟；冷却后离心，取上清液即为酶解物，可根据需要以液体或者固体状态保存。
经过前期预实验，几种蛋白酶中，仅有胃蛋白酶酶解产物能提供较为明显的胰脂肪酶抑制活性，因此，后续实验中使用胃蛋白酶为基础配置复合酶解体系，并适当选择胃蛋白酶和其他蛋白酶可以共同作用的pH（具体筛选实验未展示，由于酶解实验牵扯的变量较多，酶、用量、pH、温度、辅助试剂等，以下仅展示部分代表性方案和结果）：
胃蛋白酶 （5000U/mL）pH 5.0；
胃蛋白酶（5000U/mL）、胰蛋白酶（5000U/mL），pH 5.5；
胃蛋白酶（5000U/mL）、木瓜蛋白酶（2000U/mL）pH 5.0；
胃蛋白酶（5000U/mL）、糜蛋白酶（3000U/mL）pH 5.5；
胃蛋白酶（5000U/mL）、无花果蛋白酶（5000U/mL）pH 5.0；
胃蛋白酶（5000U/mL）、菠萝蛋白酶（3000U/mL）pH 5.5；
酶解物的胰脂肪酶抑制率结果如图1所示，相比单用胃蛋白酶，各种复合酶解方案都能提供更好的胰脂肪酶抑制活性，其中胃蛋白酶和无花果蛋白酶、胃蛋白酶和木瓜蛋白酶的组合能提供明显优于其他组合的抑制效果（40%以上的抑制率）。
实施例3 乳化剂对酶解效果的影响
为了进一步提高酶解效果，申请人验证了更多的复合酶方案，结果表明，更多种的蛋白酶组合并不能提供更好的抑制效果。后续申请人考虑藜麦脂肪含量较高，明显超过其他粮食作物，而且其中部分脂肪以脂蛋白的形式存在，通过一般有机溶剂脱脂并不能除去，在后续碱提和酶解过程中这些脂质的阻隔可能会影响酶解的过程，因此考虑在酶解体系中加入乳化剂来提高酶解效果。部分乳化剂和螯合剂的结果如表1所示（由于种类、浓度因素较多，仅展示部分代表性结果）：
表1 酶解体系中加入乳化剂后的胰脂肪酶抑制率（%）
| 胃蛋白酶（5000U/mL）、无花果蛋白酶（5000U/mL）pH 5.0 | 胃蛋白酶（5000U/mL）、木瓜蛋白酶（2000U/mL）pH 5.0 | |
| 乳酸钙（2% w/v） | 50.2% | 44.6% |
| 乳酸钙（4% w/v） | 54.1% | 46.8% |
| 蔗糖脂肪酸酯（4% w/v） | 46.8% | 48.8% |
| 蔗糖脂肪酸酯（6% w/v） | 48.2% | 50.3% |
| 焦磷酸钠（2% w/v） | 56.9% | 46.6%% |
| 焦磷酸钠（4% w/v） | 51.5% | 49.5% |
结果表明，多种乳化剂乳酸钙、蔗糖脂肪酸酯、焦磷酸钠均能提高水解物的胰脂肪酶抑制效果，其中4% w/v的乳酸钙和2% w/v的焦磷酸钠对胃蛋白酶加无花果蛋白酶的效果尤为明显。
实施例4 酶解物的肽分析
将实施例3中效果最好的胃蛋白酶（5000U/mL）、无花果蛋白酶（5000U/mL）pH 5.0、焦磷酸钠（2% w/v）酶解组的酶解物按照“材料和基本方法”部分的方法鉴定其中的肽，并以“肽的性能预测”部分中所列软件预测性质，鉴定出的预测生物活性大于0.6的部分肽如下表2所示：
表2 鉴定的部分肽及其性质
预期生物活性较高的几种肽的亲水性均一般，但考虑大多数食品和保健品制备中亲水性问题并无大的影响，因此选择预测生物活性最高，且稳定性较好的NVEGSALLPA进行进一步研究。
为进一步明确肽的性质和安全性，我们委托南京金斯瑞生物科技有限公司使用采用Fmoc固相合成法制备肽纯品NVEGSALLPA（纯度在96%以上，质谱图见图2；纯品乙醚沉淀后经过多次乙醚洗涤，纯净水溶解，HPLC脱盐后冻干）。
按照“材料和基本方法”记载的方法检测胰脂肪酶抑制率，5mg/mL的肽NVEGSALLPA抑制率可以达到69.2%。
实施例5 肽的安全性实验
按照“材料和基本方法”部分中肽的安全性实验部分所述进行一般饲喂实验和大剂量实验。
一般饲喂实验中，两组的日常活动、饮食、行为无明显异常；解剖后器官情况无明显异常；其他参数如下表3所述
表3 一般饲喂实验中的血生化结果（平均值）
无论是从饲喂肽后的变化趋势还是从与对照的比较来看，肽NVEGSALLPA并无明显的肝肾毒性，对动物的日常行为和脏器情况也没有显著影响。
大剂量实验中，所有被试大鼠均没有发生死亡，从日常活动看，也无明显异常。可以预期该肽的半数致死剂量在500mg/kg以上，达到了一般食品原料的水平。
肽NVEGSALLPA来源于常见粮食藜麦的食用酶酶解，加之以上实验又详细验证了肽NVEGSALLPA的安全性。可以认为肽NVEGSALLPA可以用于制备食品和保健品，特别适合液体或者固体饮料以及口含片、糖果等食品和保健品的制备。
实施例6 食品制备实例
减肥茶（茶包）：绿茶粉5份，乌龙茶粉2份、山楂粉5份，荷叶粉3份，紫苏叶粉1份、肽NVEGSALLPA 0.05份。
减肥饮料：绿茶粉5份，山楂粉5份，橘皮粉3份，薏苡仁粉2粉，代糖适量，肽NVEGSALLPA 0.05份，加水50-70份配制。
减肥茶糖：卡拉胶2份、山梨糖醇24份、麦芽糖醇40份、低聚异麦芽糖18份、乌龙茶粉30粉，肽NVEGSALLPA 0.5份，水80份；熬糖浆后混入茶水，待温度低于50摄氏度后混入肽，倒模成型。
Claims (10)
1.一种藜麦胰脂肪酶抑制肽，其特征在于，所述藜麦胰脂肪酶抑制肽的氨基酸序列为NVEGSALLPA。
2.根据权利要求1所述的藜麦胰脂肪酶抑制肽，其中所述藜麦胰脂肪酶抑制肽由藜麦蛋白经胃蛋白酶和无花果蛋白酶酶解制备获得。
3. 根据权利要求2所述的藜麦胰脂肪酶抑制肽，其中所述酶解的酶解体系中包含5000U/mL的胃蛋白酶和5000U/mL无花果蛋白酶，以及4% w/v的乳酸钙或者2% w/v的焦磷酸钠。
4.根据权利要求3所述的藜麦胰脂肪酶抑制肽，其中所述酶解在37摄氏度下，酶解4小时。
5.根据权利要求1所述的藜麦胰脂肪酶抑制肽，其中所述藜麦胰脂肪酶抑制肽使用Fmoc固相合成法制备。
6.根据权利要求1-5任一项所述的藜麦胰脂肪酶抑制肽在制备减肥食品或者保健品中的应用。
7.根据权利要求6所述的应用，其中所述减肥食品或者保健品为液体饮料或者固体饮料。
8.减肥食品或者保健品，其特征在，所述减肥食品或者保健品包含根据权利要求1-5任一项所述的藜麦胰脂肪酶抑制肽。
9.根据权利要求8所述的减肥食品或者保健品。
10.其中所述减肥食品或者保健品为液体饮料或者固体饮料。
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