CN112608365B - 一种减轻体重的发酵型小分子肽及其制备方法和应用 - Google Patents
一种减轻体重的发酵型小分子肽及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种减轻体重的发酵型小分子肽及其制备方法和应用，所述发酵型小分子肽包括RKVP和EQFL。本发明通过采用合适的水解条件，制备得到了富含本发明小分子肽的水解物，本发明的小分子肽具有较高的抗氧化活性，对于减轻体重具有重要意义。
Description
技术领域
本发明属于生物制药技术领域，具体涉及一种减轻体重的发酵型小分子肽及其制备方法和应用。
背景技术
包括人在内的需氧型生物为了生存而通过呼吸摄入氧，此时摄入体内的氧的一部分在能量代谢时转变为过氧化物自由基、过氧化氢、羟基自由基等活性氧物种。这样的活性氧物种原本参与以细菌或病毒感染时的巨噬细胞的病原体除去机制为代表的生物体防御等，在维持健康方面发挥重要的功能。但是，认为由于大气污染、紫外线等环境因素、或吸烟等生活习惯、精神压力等，生物体内的平衡被破坏，活性氧物种一旦过剩，就会与生物体中的蛋白质、脂质、DNA等发生反应，引起蛋白质变性、脂质过氧化物生成、遗传损伤等，从而导致发生生活习惯病、促进衰老。因此，逐渐认识到除了生物体所具有的机制以外，来自饮食的抗氧化活性成分的摄取对于维持健康也是非常重要的，并推荐积极地摄取抗氧化物质作为第7种营养素。
人工合成抗氧化剂价格低、产量高等优点而广泛地应用于食品、护肤品、化妆品工业。但是，随着经济发展，人们对健康越来越重视，致使人们更加青睐安全、纯天然、无毒副作用的天然抗氧化剂，如茶多酚、抗坏血酸及蛋白肽等。食源性抗氧化肽的种类有很多，按照来源分类可分为植物源、动物源、乳源和蛋源等抗氧化肽。植物源主要有玉米、大豆、小麦谷、米糠、油菜籽、花生、豆鹰嘴、大米、米渣、花椒籽、核桃、黑豆、麦胚等。
采用酶水解蛋白制备抗氧化肽是主流的方法，但由于许多蛋白酶有特异性的酶切位点及蛋白质氨基酸序列的独特性，因此选用不同的蛋白及酶组合对于生成具有独特功能性的肽化合物具有重要意义。就现有的酶解方法而言，有些使用了无特异性或广泛特异性的蛋白酶，这样的生产重复性低并且会产生游离氨基酸，或是肽链会被水解的过小导致功能性降低，最后产物的收率也较低，总体而言还缺乏稳定、高效制备抗氧化肽的方法。
本申请的发明人在先申请CN110024950A公开了一种黑色植物小分子肽全营养代餐粉，其在满足人体每日营养需求的基础上，通过蛋白比例的最优组合达到减缓蛋白消耗速率、控制饮食、减轻体重、改善睡眠、缓解眼部干涩、抗疲劳等效果，其制备方法有效简化了生产流程，保持了产品营养值，增加了蛋白体系稳定性。但是，由于该代餐粉中活性成分含量较低，其降低体重的效果有待进一步改进。
发明内容
本发明的目的是提供一种减轻体重的发酵型小分子肽及其制备方法和应用。具体而言，为了实现本发明的目的，本发明拟采用如下的技术方案：
本发明一方面涉及一种减轻体重的发酵型小分子肽，其包括RKVP和EQFL。
在本发明的一个优选实施方式中，所述RKVP的含量为0.8wt％以上，EQFL的含量为0.6wt％以上。
本发明另一方面还涉及含有上述发酵型小分子肽的水解物，其中包含50wt％以上的短肽、20wt％以上的多糖、2.5wt％以上的花青素。
本发明另一方面还涉及上述水解物的制备方法，其特征在于通过如下步骤制备得到：
(1)将重量份为黑芝麻50-100份、黑米50-100份、黑豆50-100份、黑枸杞50-100份、黑花生50-100份、黑木耳50-100份和黑桑葚50-100份粉碎，溶解在水中；
(2)加入纤维素酶和果胶酶的混合物，调节pH至4-5，加热反应液至50-60℃，放入搅拌器中，酶促水解1-3小时；
(3)水解之后加入木瓜蛋白酶和菠萝蛋白酶，搅拌均匀得到反应液，调节反应液的pH至7-8，加热反应液至50-60℃，放入搅拌器中，酶促水解时间为2-4小时；
(4)水解之后加入丝氨酸蛋白酶，在pH6.5-7.5和50-60℃温度下进行酶促水解3-5小时；
(5)在热处理使酶变性；
(6)离心并回收液相；
(7)先通过4-6kDa膜对液相进行超滤和渗滤并回收渗透物；
(8)再通过150-300Da膜对来自超滤的渗透物进行纳米过滤并回收渗余物；
(9)进行冷冻干燥，得到水解物。
本发明另一方面还涉及一种代餐粉，其特征在于包括上述水解物30-50份、浓缩牛奶蛋白60～100份、结晶果糖60～100份、混合果蔬粉60～100份、复合维生素6～10份、复合矿物质6～10份、鸡肉粉10～50份、牛肉粉10～50份、魔芋粉100～200份。
本发明另一方面涉及上述小分子肽作为抗氧化剂和/或减轻体重中的应用。
本发明另一方面还涉及上述代餐粉在减轻体重中的应用。
有益效果
本发明通过采用合适的水解条件，制备得到了富含本发明小分子肽的水解物，本发明的小分子肽具有较高的抗氧化活性，对于减轻体重具有重要意义。
具体实施方式
为了进一步理解本发明，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
如无特殊说明，本发明实施例中所涉及的试剂均为市售产品，均可以通过商业渠道购买获得。
实施例1：
(1)将重量份为黑芝麻100份、黑米100份、黑豆100份、黑枸杞100份、黑花生100份、黑木耳100份和黑桑葚100份粉碎，以10％干物质(DM)溶解在水中(成为糊状)；
(2)加入纤维素酶(2.0％/DM)和果胶酶(1.0％/DM)的混合物，调节pH至4.5，加热反应液至55℃，放入搅拌器中，在40rpm的条件下反应，酶促水解2小时；
(3)水解2小时之后加入木瓜蛋白酶(1.5％/DM)和菠萝蛋白酶(2.0％/DM)，搅拌均匀得到反应液，调节反应液的pH至7.5，加热反应液至55℃，放入搅拌器中，在40rpm的条件下反应，酶促水解时间为3h；
(4)水解之后加入丝氨酸蛋白酶(1.5％DM)，在pH 7.0和55℃温度下进行酶促水解4小时；
(5)在85℃下热处理45分钟以使酶变性；
(6)离心并回收液相；
(7)先通过5kDa膜对液相进行超滤和渗滤并回收渗透物；
(8)再通过200Da膜对来自超滤的渗透物进行纳米过滤并回收渗余物。
(9)进行冷冻干燥，得到水解物，经过测试，所述水解物中包含57％重量的短肽、25％的多糖、3％的花青素以及15％的其它物质。
比较例1：
参照实施例1，其区别在于步骤(3)中不加入木瓜蛋白酶。由此得到的得到水解物，经过测试，所述水解物中包含37％重量的短肽、35％的多糖、2％的花青素以及26％的其它物质。
比较例2：
参照实施例1，其区别在于步骤(3)中不加入菠萝蛋白酶。由此得到的得到水解物，经过测试，所述水解物中包含32％重量的短肽、32％的多糖、1％的花青素以及35％的其它物质。
比较例3：
参照实施例1，其区别在于先进行步骤(4)，再进行步骤(3)。由此得到的得到水解物，经过测试，所述水解物中包含43％重量的短肽、37％的多糖、2％的花青素以及18％的其它物质。
对于上述水解产物，通过测定ORAC值(μmol TE/mmol)和DPPH值(μmol TE/mmol)来比较其抗氧化活性，实验结果如表1所示。
表1：水解物抗氧化活性测试结果
从上述比较结果来看，实施例1的抗氧化活性高于比较例1-3，由此说明实施例1的水解产物具有较高的抗氧化活性。
对于由实施例1和比较例1-3所得到的水解物，分析其包含的短肽的含量，通过检测发现，实施例1中丰度最高的短肽为RKVP和EQFL，其含量达到水解物的0.83％和0.67％，而在比较例中RKVP和EQFL的含量最高的是比较例3，仅为0.45％和0.36％，比较例3中含量较高的是ERRQ和FTSE，分别为0.69％和0.59％。
为了进一步确定是否是由于RKVP和EQFL的存在使得水解物的抗氧化活性较高，对于检测出的丰度较高的短肽，使用通常的肽的化学合成方法即Fmoc固相合成法来合成，作为抗氧化活性测定用的样品。对于合成的短肽，测定ORAC值(μmol TE/mmol)DPPH值(μmolTE/mmol)，将各肽序列的分析结果汇总于表2。
表2：短肽抗氧化活性测试结果
基于上表可以看出，通过同时包含表现出高ORAC值和表现出高DPPH值的RKVP和EQFL，从而使得水解物的抗氧化性能全面优异。
代餐减肥实施效果:
取实施例1的水解物40份，与浓缩牛奶蛋白60份、结晶果糖60份、混合果蔬粉60份、复合维生素6份、复合矿物质6份、鸡肉粉10份、牛肉粉10份、魔芋粉200份，干混均匀，设定干混机转速为1500r/min，均匀混合10min，将混合后的干粉进行过筛，控制固体粉末颗粒直径在600μm以下，以保证产品有较佳的溶解效果。将过筛后干粉置于铝箔复合袋中，充入氮气后封口，即可得所述全营养代餐粉。
冲饮方法：取所述全营养代餐粉20g，加入100-300ml、40-80℃的水,搅拌均匀，即可食用,每日3次。
作为比较，按照CN 110024950 A实施例2所制备的代餐粉作为比较。
实验对象：选取80名体重相当的20-50周岁成年人作为考察对象(男女各一半)，随机分为2组，每组40人(男女各一半)；
统计上述代餐人群一周的体重测量，记录代餐前后的体重变化，结果如表3所示。表3中可以看出使用本发明全营养代餐粉后减肥效果明显好于现有技术代餐粉的减肥效果，通过减少进餐时摄入过多的淀粉和脂肪，提高了减肥效果。
表3减肥效果测试结果
以上描述了本发明优选实施方式，然其并非用以限定本发明。本领域技术人员对在此公开的实施方案可进行并不偏离本发明范畴和精神的改进和变化。
序列表
<110> 吉林省特医食品生物科技有限公司
中国科学院长春应用化学研究所
<120> 一种减轻体重的发酵型小分子肽及其制备方法和应用
<130> CP20787
<141> 2020-12-22
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Claims (2)
1.一种减轻体重的发酵型小分子肽组合物，所述发酵型小分子肽组合物由RKVP和EQFL组成。
2.权利要求1所述的小分子肽组合物在制备抗氧化剂和/或体重减轻剂中的应用。
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