CN113875896B - 用于治疗和预防猪肠道损伤的饲料添加剂 - Google Patents
用于治疗和预防猪肠道损伤的饲料添加剂 Download PDFInfo
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Abstract
本发明利用蒙脱石的吸水膨胀性，依靠离子键作用将pGLP‑2插入蒙脱石中，利用蒙脱石易与肠黏膜结合，承载pGLP‑2到达靶器官发挥肠道损伤修复功能，从而解决pGLP‑2的胃肠道给药途径。本发明的第一个目的是提供一种饲料添加剂，该饲料添加剂中蒙脱石(MMT)作为靶向控释载体材料，可携带猪胰高血糖素样肽‑2(pGLP‑2)通过饲喂途径到达靶向作用部位，发挥其肠道修复功能。本发明的第二个目的是提供上述的饲料添加剂的制备方法。本发明的本发明的第三个目的是提供一种上述饲料添加剂的用途。
Description
技术领域
本发明涉及仔猪饲料添加剂技术领域，具体涉及一种pGLP-2/MMT插层复合物及其制备方法和应用。
背景技术
在生猪养殖中，仔猪断奶，病原体或霉菌毒素的侵染，抗营养因子的摄入，环境的改变等各种因素的应激均会导致肠道功能损伤，进而引发腹泻、生长受阻和死亡等，严重影响养猪生产，研发具有肠道保护与修复的制剂具有紧迫的需求。
猪胰高血糖素样肽-2(pGLP-2)能够通过特异性促进肠上皮细胞增殖、抑制肠上皮细胞凋亡、抑制胃酸分泌、降低肠道渗透性等促进损伤肠黏膜的结构恢复以及吸收和屏障功能的改善，且效果强于以往发现的其他非特异的生长因子。2012年，美国食品和药物管理局(FDA)批准hGLP-2类似物——替度鲁肽(皮下注射给药)为罕见病药物，用于靶向治疗成人肠易激综合征和短肠综合征等。畜用的pGLP-2制剂，本发明人已通过酶作用位点氨基酸改变及修饰剂偶联阻抗酶解而使其长效化，注射途径试验表明稳定有效。但胃肠道途径添加剂的方式，因胃酸及消化酶等降解，以及胃肠道黏液层和上皮层阻挡，有效成分难以进入损伤组织细胞，目前尚未有成功的制剂。
CN112998147A公开了一种壳聚糖-载铜蒙脱石散插层复合材料，所述插层复合材料采用载铜蒙脱石散作为插层的主体材料，使用壳聚糖进行插层复合，其中，插层主体材料为铜蒙脱石，是利用Cu2+的抗菌作用，将Cu2+与蒙脱石中Na+、K+等阳离子交换后得到的金属无机抗菌剂。插层材料为壳聚糖，是甲壳素脱乙酰基得到的一种带正电荷的直链多糖，在动物饲料中的主要功能是中和胃酸，增强动物的免疫力，抑制病原菌的繁殖；其中，对比文件中将蒙脱石分散到含Cu2+溶液中，存在潜在的环境污染风险。
发明内容
针对上述问题，本发明利用蒙脱石的吸水膨胀性，依靠离子键作用将pGLP-2插入蒙脱石中，利用蒙脱石易与肠黏膜结合，承载pGLP-2到达靶器官发挥肠道损伤修复功能，从而解决pGLP-2的胃肠道给药途径。
本发明的第一个目的是提供一种饲料添加剂，该饲料添加剂中蒙脱石(MMT)作为靶向控释载体材料，可携带猪胰高血糖素样肽-2(pGLP-2)通过饲喂途径到达靶向作用部位，发挥其肠道修复功能。本发明的第二个目的是提供上述的饲料添加剂的制备方法。本发明的本发明的第三个目的是提供一种上述饲料添加剂的用途。
为了实现上述的第一个目的，本发明采用了以下的技术方案：
提供一种用于治疗和/或预防猪肠道损伤的饲料添加剂，该饲料添加剂包括猪胰高血糖素样肽-2(pGLP-2)以及与该多肽通过氢键结合的蒙脱石(MMT)。
本发明利用了MMT作为靶向控释载体材料，承载pGLP-2到达靶器官发挥肠道损伤修复功能。具体地，利用蒙脱石强的胶体性和吸附性，将吸附有效成分pGLP-2与肠道黏膜结合，靶向输送到达肠黏膜表面；pGLP-2通过特异性促进肠上皮细胞增殖、抑制肠上皮细胞凋亡、抑制胃酸分泌、降低肠道渗透性等促进损伤肠黏膜的结构恢复以及吸收和屏障功能的改善，且作用效果强于以往发现的其他非特异的生长因子，应用价值大。
作为优选，所述猪胰高血糖素样肽-2的氨基酸序列为His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Val-Leu-Asp-Asn-Leu-Ala-Thr-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Leu-His-Thr-Lys-Ile-Thr-Asp-Ser-Leu-NH2。
作为优选，所述蒙脱石体积平均径为1.53～6.52μm。
本发明提供的饲料添加剂能够在酸性环境下基本不释放pGLP-2，而在碱性环境中释放量达到最大，而饲料经过仔猪的胃部到达肠道，众所周知，胃部呈酸性环境而肠道呈碱性环境，如此使得该饲料添加剂在经过胃部时不发生释放而到达肠道后最大量释放。且pGLP-2的特异性受体分布在肠道上皮，本发明实现了将该饲料添加剂能够通过饲喂途径靶向送达作用部位，解决了直接采用pGLP-2饲喂时无法到达靶向作用部位的技术问题。
为了实现上述的第二个目的，本发明采用了以下的技术方案：
提供上述的前述饲料添加剂的制备方法，包括以下的步骤：
1)将pGLP-2制备成pH小于4.4的溶液；
2)将MMT缓慢加入步骤1)制备的pGLP-2溶液中，室温下搅拌反应；
3)烘干、粉碎，得到pGLP-2/MMT插层复合物。
步骤1)将pGLP-2溶解于0.2mol/L pH3.4的甘氨酸-盐酸缓冲溶液，终浓度为5mg/L。
本发明将pGLP-2制备成pH小于4.4的溶液，如此在，pGLP-2处于小于其等电点pH4.4的溶液中时，其自身带有正电荷，能够与带有负电荷的蒙脱石发生吸附作用，提高其二者的吸附能力；在本发明中，蒙脱石对将酸性环境下的pGLP-2吸附达到了99％；在一些具体实施例中，所述步骤1)将pGLP-2溶解于0.2mol/L pH3.4的甘氨酸-盐酸缓冲溶液，终浓度为5mg/L。
作为优选，步骤2)所述pGLP-2与MMT的质量比为1:10000，反应时间为30～60分钟。
作为优选，步骤2)室温控制在25℃，搅拌速度为300～600转/分钟。
作为优选，所述步骤3)中在55℃烘干。
为了实现上述的第三个目的，本发明采用了以下的技术方案：
本发明将前述所述的饲料添加剂用于制备治疗和预防猪肠道损伤的饲料。在一些具体实施方式中，所述饲料添加剂占饲料总量的1‰。
与现有技术相比，本发明具有以下有益效果：
本发明将pGLP-2/MMT通过氢键作用发生结合，pGLP-2复合在该MMT的层间，利用其在碱性环境下最大释放以及在酸性环境下基本不释放的释放特性，可以将其通过饲喂途径送达靶向作用部位；同时该添加剂结合了pGLP-2和MMT促进肠上皮细胞生长、收敛、修复黏膜和止泻的特点，两者相互配合，协同作用，能够促进断奶仔猪肠道损伤的修复和提高仔猪免疫功能。
本发明进一步通过试验验证，将该饲料添加剂用于饲喂断奶仔猪，能够促进断奶仔猪肠道损伤的修复和提高仔猪免疫功能，可以广泛用于猪饲料添加剂领域；此外，所用原料价格低廉，无残留、无抗药性和无毒副作用。
附图说明
图1为pGLP-2、MMT以及pGLP-2/MMT插层复合物的FT-IR谱图；
具体实施方式
为了便于理解本发明，下面将参照实施例对本发明进行更全面的描述。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
实施例1 pGLP-2/MMT复合物的制备和体外释放性能检测
本实施例中，pGLP-2的结构式为：pGLP-2(1-35)，序列如下：His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Val-Leu-Asp-Asn-Leu-Ala-Thr-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Leu-His-Thr-Lys-Ile-Thr-Asp-Ser-Leu-NH2。MMT的粒径为3.80μm。
称取10mg pGLP-2粉末，加入到10mL pH3.4的甘氨酸－盐酸缓冲溶液中，充分溶解后分别加入200mg蒙脱石，室温下搅拌30min后，4度10000rpm离心3min。
使用BCA法检测加入MMT前后pGLP-2溶液在562nm处的吸光度，计算出吸附量为98.67％。
离心后的沉淀，重悬于pH值分别为9(Tris-盐酸缓冲液)、7.3(Tris-盐酸缓冲液)、5(乙酸-乙酸钠缓冲液)和2(甘氨酸-盐酸缓冲液)的释放介质中，同时设置缓冲介质的空白对照，37℃恒温振荡器中0.5h、2h和3h后取样，同时向体系中补充等量的新鲜释放介质，使用BCA法检测562nm处的吸光度，计算得出释放介质中pGLP-2的浓度和累计释放量。结果见表1，说明pGLP-2/MMT复合物在碱性环境中释放量最大，在中性环境中释放量次之，在酸性环境中基本上不释放pGLP-2；pGLP-2/MMT复合物在碱性和中性环境中0.5h即可达释放平衡状态；这样，饲料添加剂能够在酸性环境下基本不释放pGLP-2，而在碱性环境中释放量达到最大，而饲料经过仔猪的胃部到达肠道，众所周知，胃部呈酸性环境而肠道呈碱性环境，如此使得该饲料添加剂在经过胃部时不发生释放而到达肠道后最大量释放。且pGLP-2的特异性受体分布在肠道上皮，本发明实现了将该饲料添加剂能够通过饲喂途径靶向送达作用部位，解决了直接采用pGLP-2饲喂时无法到达靶向作用部位的技术问题。
表1 pGLP-2/MMT复合物的体外累计释放量和释放比例，μg/mL(％)
制备的pGLP-2/MMT插层复合物，其FT-IR检测结果见图1，3200～3550cm-1为分子间氢键-OH的伸缩振动峰，在pGLP-2/MMT复合物中变宽，证明了羟基与pGLP-2的官能团发生作用形成了氢键。
实施例2 MMT/pGLP-2插层复合物调控断奶仔猪肠道的研究
材料和方法
48只断奶仔猪，按体重随机分为2组，每组4个重复，每个重复6头猪。对照组饲喂基础日粮，试验组每吨饲料中添加1‰的MMT/pGLP-2插层复合物。试验期28天，试验第28早上6:30，每组选6头注射生理盐水，选6头按100微克/kg体重注射LPS，9:30处死试验猪，采集肠道样品供检测。
采用SPSS软件中ANOVA分析试验数据，并用DUNCAN氏方法进行多重比较，P≤0.05为差异显著。
试验结果
由表2可知，与对照组相比，添加MMT/pGLP-2复合物的仔猪空回肠长度(P＝0.09)显著增加，空回肠的重量比对照组增加14.34％。表明添加MMT/pGLP-2复合物有促进断奶仔猪小肠生长的作用。
表2仔猪空回肠长度、重量和占体重的比例
由表3可知，与对照组相比，添加MMT/pGLP-2复合物仔猪空肠黏膜的麦芽糖酶、蔗糖酶、乳糖酶、ATP酶活力和SIgA含量；以及回肠黏膜的麦芽糖酶、乳糖酶、ATP酶活力和SIgA含量极显著(P<0.001)增加。与注射LPS组相比，饲喂MMT/pGLP-2复合物的仔猪注射LPS后，其空肠黏膜的麦芽糖酶、蔗糖酶、乳糖酶和ATP酶活力；以及回肠黏膜的麦芽糖酶、蔗糖酶和ATP酶活力极显著(P<0.01)增加。表明，饲喂MMT/pGLP-2复合物(1)不仅可以显著增加断奶仔猪肠道刷状缘二糖酶酶活，且可以显著抑制LPS应激对仔猪肠道刷状缘二糖酶酶活性的影响。(2)显著提高断奶仔猪肠道的黏膜免疫屏障功能。
表3肠道刷状缘二糖酶酶活和分泌型免疫球蛋白A(SIgA)含量
说明添加MMT/pGLP-2复合物不仅显著增加断奶仔猪肠道刷状缘二糖酶酶活，且显著抑制LPS应激对肠道二糖酶活性的影响；提高断奶仔猪肠道的黏膜免疫屏障功能，增加空回肠长度。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。
Claims (10)
1.用于治疗和/或预防猪肠道损伤的饲料添加剂，其特征在于，该饲料添加剂包括猪胰高血糖素样肽-2(pGLP-2)以及与该多肽通过氢键结合的蒙脱石(MMT)。
2.根据权利要求1所述的饲料添加剂，其特征在于，所述猪pGLP-2的氨基酸序列为His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Val-Leu-Asp-Asn-Leu-Ala-Thr-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Leu-His-Thr-Lys-Ile-Thr-Asp-Ser-Leu-NH2。
3.根据权利要求1所述的饲料添加剂，其特征在于，所述MMT体积平均径为1.53～6.52
μm。
4.一种权利要求1～3任一项所述的饲料添加剂的制备方法，其特征在于，该方法包括以下的步骤：
1)将pGLP-2制备成pH小于4.4的溶液；
2)将MMT缓慢加入步骤1)制备的pGLP-2溶液中，室温下搅拌反应；
3)烘干、粉碎，得到pGLP-2/MMT插层复合物。
5.根据权利要求4所述的pGLP-2/MMT饲料添加剂的制备方法，其特征在于，所述步骤1)将pGLP-2溶解于0.2mol/L pH3.4的甘氨酸-盐酸缓冲溶液，终浓度为5mg/L。
6.根据权利要求4所述的pGLP-2/MMT饲料添加剂的制备方法，其特征在于，所述步骤2)所述pGLP-2与MMT的质量比为1:10000，反应时间为30～60分钟。
7.根据权利要求4所述的pGLP-2/MMT饲料添加剂的制备方法，其特征在于，所述步骤2)室温控制在25℃，搅拌速度为300～600转/分钟。
8.根据权利要求4所述的pGLP-2/MMT饲料添加剂的制备方法，其特征在于，所述步骤3)中在55℃烘干。
9.权利要求书1～3中任意一项所述的饲料添加剂在制备用于治疗和预防猪肠道损伤饲料中的应用。
10.根据权利要求9所述的应用，其特征在于，所述饲料添加剂占饲料总量的1‰。
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