CN114369143A - 一种生物活性多肽及其应用 - Google Patents
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Abstract
本发明属于多肽开发应用技术领域，具体涉及一种生物活性多肽及其应用。本发明提供的多肽，其氨基酸序列如SEQ ID NO.1所示，N端进行乙酰化修饰，提高抗菌肽的稳定性；C端进行酰胺化修饰，增大C端疏水性；同时，本发明提供的多肽中含有天冬氨酸，提高了抗菌活性；通过残基替换，减弱抗菌肽毒性。同时，经进一步证明，本发明提供的多肽具有较强的抗菌和免疫调节活性，且稳定性高，抗菌活性强，毒性低，可用于畜禽饲料的制备，增强机体免疫力，提高饲料利用率。
Description
技术领域
本发明属于多肽开发应用技术领域，具体涉及一种生物活性多肽及其应用。
背景技术
随着各种致病基因的不断变异，常规抗生素都出现了相应的抗药性致病株系，致病菌的抗药性问题已经日益严重地威胁着动物健康。抗菌肽因其抗菌杀菌、抗菌谱广、种类多、相对分子量较小、靶菌株不易产生抗性突变等优点，具着广阔的应用前景，这对寻找抗生素替代品具有重要意义。
天然抗菌肽通常是由30多个氨基酸残基组成的碱性小分子多肽，水溶性好，分子量大约为4000道尔顿左右。大部分抗菌肽具有热稳定性，在l00℃下加热10～15min仍能保持其活性。多数抗菌肽的等电点大于7，表现出较强的阳离子特征。同时，抗菌肽对较大的离子强度和较高或较低的pH值均具有较强的抗性。此外，部分抗菌肽尚具备抵抗胰蛋白酶或胃蛋白酶水解的能力。
然而，大量天然的抗菌肽活性并不高，或者对真核细胞存在较高的细胞毒性，因此在作为抗生素替代品研制之前需要通过分子改良进一步提高其活性，或降低细胞毒性。此外，细菌感染已成为影响畜牧业生产的重要因素，尤其是耐药菌株的出现，更增加了动物疾病的治疗难度。因此，迫切需要研究一种新型活性多肽，增强机体免疫力，有效替代抗生素作为饲料添加剂的使用，从而提高畜产品安全性。
发明内容
针对现有技术普遍存在的缺陷，本发明提供了一种生物活性多肽及其应用。本发明提供的多肽具有较强的抗菌和免疫调节活性，且稳定性高，抗菌活性强，毒性低，可用于畜禽饲料的制备，增强机体免疫力，提高饲料利用率。
为了达到上述目的，本发明采用的技术方案为：
一种生物活性多肽，所述生物活性多肽的氨基酸序列如SEQ ID NO.1所示。
所述化学修饰为在氨基酸序列的N端进行乙酰化修饰，C端进行酰胺化修饰。
KADCADCGKKKKKKKKADCADCPPPPPPPN(SEQ ID NO.1)；
本发明还提供了一种所述的生物活性多肽在制备饲料添加剂中的应用。
优选地，所述饲料添加剂包含如下组分及其重量百分数：所述化学修饰后的生物活性多肽3～5％，卵磷脂1～2％，大豆蛋白粉0.8～1.5％，黄芪1～2％，党参1～1.5％，槲皮素0.5～0.8％，维生素A 1～1.5％，维生素C 0.8～1.2％，豆粕25～35％，鱼粉25～30％，膨化玉米粉19.5～40.9％。
优选地，所述饲料添加剂包含如下组分及其重量百分数：所述饲料添加剂包含如下组分及其重量百分数：权利要求2所述化学修饰后的生物活性多肽4％，卵磷脂1.5％，大豆蛋白粉1.2％，黄芪1.5％，党参1.3％，槲皮素0.7％，维生素A1.2％，维生素C1.0％，豆粕30％，鱼粉25％，膨化玉米粉32.6％。
优选地，在畜禽饲料中，所述饲料添加剂的添加量为0.5～2.0％。
优选地，所述修饰后的生物活性多肽用于增强机体免疫力，提高饲料利用率。
本发明中，通过对抗菌肽进行化学修饰：N端乙酰化，提高了抗菌肽稳定性；C端酰胺化，增大C端疏水性；含有天冬氨酸，提高抗菌活性；通过残基替换，减弱了抗菌肽本身的毒性。本发明经修饰后的抗菌肽，具有较强的抗菌和免疫调节活性，高稳定性、高抗菌活性、低毒性；用于饲养畜禽，能显著增加肠道乳酸菌的数量，并可有效降低肠道致病菌大肠杆菌、沙门氏菌数量，增强机体免疫力，提高饲料利用率，减少对动物细胞具有毒副作用或会导致溶血等缺陷。
进一步地，本申请还加入了黄芪、党参、槲皮素等具有一定要用功效的物质，在提高动物饲料利用率的基础上，可以进一步提高其机体免疫力，防止各种疾病的发生，而且，申请人还以为发现，槲皮素与具有一定抗氧化功能的维生素A、维生素C共同使用，可以进一步提高饲料利用率，减少食物浪费，有利于节约型社会的和谐发展。
与现有技术相比，本发明提供的生物活性多肽具有如下优势：
(1)本发明提供的生物活性多肽，通过化学修饰，有效提高了抗菌肽的稳定性和抗菌活性，减少了对动物细胞具有毒副作用或导致溶血等缺陷；
(2)本发明提供的生物活性多肽，具有较强的抗菌和免疫调节活性，可用于畜禽饲料的制备，有效降低肠道致病菌的数量，增强机体免疫力，提高饲料利用率，替代抗生素使用，从而提高畜产品安全性。
具体实施方式
下面结合具体实施例对本发明作进一步解释，但是应当注意的是，以下实施例仅用以解释本发明，而不能用来限制本发明，所有与本发明相同或相近的技术方案均在本发明的保护范围之内。本实施例中未注明具体技术或条件者，按照本领域常规技术方法和仪器说明书内容进行操作；所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
所述卵磷脂可购自上海易恩化学技术有限公司，货号：R002341-1g；所述大豆蛋白粉可购自河南冉升化工产品有限公司。
实施例1生物活性多肽的修饰过程
N端进行乙酰化修饰，提高抗菌肽的稳定性；C端进行酰胺化修饰，增大C端疏水性；同时含有天冬氨酸，提高抗菌活性；通过残基替换，减弱抗菌肽毒性。
实施例2一种饲料添加剂
所述饲料添加剂包含如下组分及其重量百分数：所述饲料添加剂包含如下组分及其重量百分数：实施例1所述化学修饰后的生物活性多肽5％，卵磷脂1％，大豆蛋白粉0.8％，黄芪1％，党参1％，槲皮素0.5％，维生素A1％，维生素C 0.8％，豆粕25％，鱼粉25％，膨化玉米粉19.5％。
实施例3一种饲料添加剂
所述饲料添加剂包含如下组分及其重量百分数：实施例1所述化学修饰后的生物活性多肽5％，卵磷脂2％，大豆蛋白粉1.5％，黄芪2％，党参1.5％，槲皮素0.8％，维生素A1.5％，维生素C 1.2％，豆粕35％，鱼粉30％，膨化玉米粉19.5％。
实施例4一种饲料添加剂
所述饲料添加剂包含如下组分及其重量百分数：实施例1所述化学修饰后的生物活性多肽4％，卵磷脂1.5％，大豆蛋白粉1.2％，黄芪1.5％，党参1.3％，槲皮素0.7％，维生素A 1.2％，维生素C 1.0％，豆粕30％，鱼粉25％，膨化玉米粉32.6％。
对比例1一种饲料添加剂
所述饲料添加剂剂与实施例4类似；
与实施例4的区别在于，对比例1中的饲料添加剂采用的生物活性多肽不进行修饰。
对比例2一种饲料添加剂
所述饲料添加剂与实施例4类似；
与实施例4的区别在于对比例2中的饲料添加剂中不包含生物活性多肽。
对比例3一种饲料添加剂
所述饲料添加剂与实施例4类似；
与实施例4的区别在于对比例2中的饲料添加剂中不包含槲皮素，将膨化玉米粉的重量百分数增加至33.3％。
对比例4一种饲料添加剂
所述饲料添加剂与实施例4类似；
与实施例4的区别在于对比例2中的饲料添加剂中不包含维生素A，将膨化玉米粉的重量百分数增加至33.8％。
对比例5一种饲料添加剂
所述饲料添加剂与实施例4类似；
与实施例4的区别在于对比例2中的饲料添加剂中不包含维生素C，将膨化玉米粉的重量百分数增加至33.6％。
试验例1抑菌实验
1.试验样品：实施例1经化学修饰的生物活性多肽；不经化学修饰的生物活性多肽；
2.试验菌种：大肠杆菌，沙门氏菌，乳酸菌；
3.试验方法：将试验菌种大肠杆菌、沙门氏菌及乳酸菌分别置于一个LB液体培养基中进行培养24h，然后取出培养菌种，用纯化水稀释至1.0×106CFU/mL，然后各取1mL置于培养皿中，向其中LB固体培养基，放冷后制成平板，然后将试验样品分别加纯化水稀释成0.1g/L的溶液，用加样器各取20μL溶液，采用打孔法加入到平板中，继续培养12h，观察抑菌圈直径大小；同时以不加多肽的菌种培养组为空白对照组。
4.试验结果：具体试验结果参见表1。
表1不同试验样品的抑菌圈直径大小对比
由表1可知，与不经修饰的多肽组相比，采用本发明实施例1经修饰的生物活性多肽，其对大肠杆菌、沙门氏菌的抑菌活性显著，而对乳酸菌的增殖无抑制作用。
在培养12h后，乳酸菌不产生抑菌圈的基础上，将本发明经修饰的生物活性多肽与空白对照组的乳酸菌进行含量测定，发现添加本发明经修饰的生物活性多肽组的乳酸菌含量为2.5×108CFU/mL，而空白对照组中乳酸菌含量仅为9.2×107CFU/mL，表明本发明经修饰的生物活性多肽对乳酸菌增殖具有促进作用。
试验例2饲料利用率试验
1.试验样品：分别将本发明实施例2-4组及对比例1-5组制得的饲料添加剂加入基础日粮(参照美国NRC(2012)猪营养需要)中，各组中饲料添加剂的加入量分别为：实施例2组0.5％，实施例3组2.0％，实施例4组1.3％，对比例1组1.3％，对比例2组1.3％，对比例3组1.3％，对比例4组1.3％，对比例5组1.3％。同时以不添加饲料添加剂的普通饲料为空白对照组；
2.试验对象：选择21日龄、体重相近(7.3±0.2)kg健康的“杜×长×大”断奶仔猪共90头，按照单因素随机分组试验原则，随机分成9组，每组10头；
3.试验方法：将上述试验样品分别投喂给各组试验对象，每天更新饲料，每天自由饮水，试验周期为14天，每天测量2次体重和采食量，试验结束时计算每组猪的初重、末重、平均日增重、平均日采食量及料肉比，均取每组动物中10头的平均值来统计。
4.试验结果：具体试验结果参见表2。
表2不同试验样品的试验结果对比
| 试验样品 | 初重/kg | 末重/kg | 平均日增重/kg | 平均日采食量/kg | 料肉比 |
| 实施例2组 | 7.32 | 15.05 | 0.55 | 0.58 | 1.05 |
| 实施例3组 | 7.36 | 15.09 | 0.55 | 0.58 | 1.05 |
| 实施例4组 | 7.24 | 15.18 | 0.57 | 0.59 | 1.04 |
| 对比例1组 | 7.31 | 14.57 | 0.52 | 0.56 | 1.08 |
| 对比例2组 | 7.28 | 14.34 | 0.50 | 0.55 | 1.09 |
| 对比例3组 | 7.25 | 14.71 | 0.53 | 0.57 | 1.07 |
| 对比例4组 | 7.29 | 14.82 | 0.54 | 0.57 | 1.06 |
| 对比例5组 | 7.30 | 14.83 | 0.54 | 0.57 | 1.06 |
| 空白对照组 | 7.32 | 14.07 | 0.48 | 0.53 | 1.10 |
由表2可知，与对照组相比，采用本发明制成的饲料1、2、3组断奶仔猪的饲料利用率明显增高，而与对比例1及对比例2组的饲料相比，由于改变了抗菌肽化学修饰，或者去掉抗菌肽，导致料肉比提高、即饲料利用率有所降低，证明了本发明各组分之间的相互协同促进作用；而对比例3-5组由于改变了槲皮素与维生素的协同关系，导致饲料利用率同样降低，但是其降低率低于对比例1-2组，证明了本申请修饰后的生物多肽在本发明中发挥重要作用。
最后应当说明的是，上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (8)
1.一种生物活性多肽，其特征在于，所述生物活性多肽的氨基酸序列如SEQ ID NO.1所示。
2.一种化学修饰后的生物活性多肽，其特征在于，所述生物活性多肽为权利要求1所述的生物活性多肽。
3.如权利要求2所述化学修饰后的生物活性多肽，其特征在于，所述化学修饰为在氨基酸序列的N端进行乙酰化修饰，C端进行酰胺化修饰。
4.一种如权利要求2所述化学修饰后的生物活性多肽在制备饲料添加剂中的应用。
5.如权利要求4所述的应用，其特征在于，所述饲料添加剂包含如下组分及其重量百分数：权利要求2所述化学修饰后的生物活性多肽3～5％，卵磷脂1～2％，大豆蛋白粉0.8～1.5％，黄芪1～2％，党参1～1.5％，槲皮素0.5～0.8％，维生素A 1～1.5％，维生素C 0.8～1.2％，豆粕25～35％，鱼粉25～30％，膨化玉米粉19.5～40.9％。
6.如权利要求5所述的应用，其特征在于，所述饲料添加剂包含如下组分及其重量百分数：权利要求2所述化学修饰后的生物活性多肽4％，卵磷脂1.5％，大豆蛋白粉1.2％，黄芪1.5％，党参1.3％，槲皮素0.7％，维生素A 1.2％，维生素C 1.0％，豆粕30％，鱼粉25％，膨化玉米粉32.6％。
7.如权利要求5所述的应用，其特征在于，在畜禽饲料中，所述饲料添加剂的添加量为0.5～2.0％。
8.如权利要求4所述的应用，其特征在于，所述化学修饰后的生物活性多肽用于增强机体免疫力，提高饲料利用率。
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