CN113150069A - 一种透明质酸酶抑制剂及其制备方法 - Google Patents
一种透明质酸酶抑制剂及其制备方法 Download PDFInfo
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- CN113150069A CN113150069A CN202110213248.6A CN202110213248A CN113150069A CN 113150069 A CN113150069 A CN 113150069A CN 202110213248 A CN202110213248 A CN 202110213248A CN 113150069 A CN113150069 A CN 113150069A
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- heptapeptide
- adenosine
- hyaluronidase
- hyaluronidase inhibitor
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Abstract
本发明公开了一种透明质酸酶抑制剂及其制备方法。将腺苷七肽MATGNAD用于制备透明质酸酶抑制剂,腺苷七肽在透明质酸酶抑制剂中的质量分数为90%,制备工艺简单方便,效率高,环境污染小,可实现大规模产业化的生产。该透明质酸酶抑制剂对抑制透明质酸酶的抑制效率高,副作用小,耐高温、耐高湿、稳定性好,可用于制备透明质酸产品,在皮肤护理产品、饲料添加剂、生物兽药、食品添加剂等方面具有广阔的应用前景。
Description
技术领域
本发明涉及一种透明质酸酶抑制剂及其制备方法,属于饲料添加剂、生物兽药以及化妆品添加剂领域。
技术背景
透明质酸酶(hyaluronidase,HAase)亦称扩散因子,是透明质酸的水解酶,可以降解组织中的透明质酸,从而提高组织中液体渗透能力。透明质酸酶在人体分为内源性和外源性两种,内源性透明质酸酶主要是动物体自身产生。内源性透明质酸酶是过敏反应的参与者,透明质酸酶与炎症、过敏有强相关性,各种肥大细胞释放的组胺能调节透明质酸酶活性,从而增加组织通透性,引发炎症和过敏反应,故透明质酸酶是抗炎和抗过敏的重要研究对象(胡国胜,用体外炎症反应模型测定多种中草药成分的抗炎效果,中国化妆品学术研讨会,2002)。一些革兰氏阳性病原菌如链球菌、葡萄球菌、产气荚膜梭菌等也会产生毒力因子—外源性透明质酸酶,从而有利于细菌或者其它毒素在组织中扩散(刘勇,细菌来源透明质酸酶研究进展,国外医药抗生素分册,2010,31卷第2期)。一些动物毒液中存在高活性的透明质酸酶,降解血管周围基质中的透明质酸使毒素易于进入血管,是毒液从受伤部位进入体循环的关键(苏康,透明质酸酶的研究进展,生物技术通报,2014)。产气荚膜梭菌是引起家禽生产性能下降和坏死性肠炎的主要病原菌。许多研究表明,家禽肠道中产荚膜梭菌浓度104-105CUF/g食糜,可引起亚临床症状,导致料肉比下降,养殖效益下降,而浓度大于106CUF/g食糜时,可导致坏死性肠炎,死亡率增加。而透明质酸酶是产气荚膜梭菌的主要的毒力因子之一,其可以破坏组织,导致细菌和其他毒素进一步扩散,从而引起严重的家禽疾病。
透明质酸酶抑制剂具有广泛的用途,其可以添加于化妆品中,不但能增强制剂作用的持久性,还能缓减皮肤炎症和过敏反应(专利CN201210495829.4),还可以用于抗过敏和抗炎症的药物治疗,在医药领域具有重大的应用前景。目前的传统的透明质酸酶抑制剂主要有植物提取物(如生物碱、黄酮类化合物、萜类化合物等)、壳聚糖、和一些抗炎症药物。仙人草提取物、积雪草甙、藤茶黄酮和鸢尾苷是几种常见的透明质酸酶抑制剂,可以通过抑制透明质酸酶活性,减少透明质酸的降解,保证了真皮层透明质酸的含量,从而提高皮肤的保湿效果,并且对抗敏起相应的作用、在化妆品、保健品以及药品中具有重要的应用(胡国胜,用体外炎症反应模型测定多种中草药成分的抗炎效果,2002年中国化妆品学术研讨会论文集;孙培冬,刘超,王小洪,鸢尾苷对透明质酸酶抑制作用的研究,化学通报,2013,857~860;王小洪,孙培冬,杨柳青等,藤茶中粗提物对透明质酸酶的抑制研究,第九届中国化妆品学术研讨会论文集,2012;CN201710705705.7一种具有抗敏和保湿活性的射干鸢尾苷及制备方法和应用),但这些植物提取物工艺,由于受制于不同产地、不同季节采摘等因素,造成不同批次有效成分含量差异大,纯度不高,对透明质酸的抑制作用不强,效果不稳定。
腺苷七肽(又名杆菌七肽、microcin C7肽)是由肠杆菌属、乳杆菌属等微生物分泌的一种细菌素类抗菌肽,其由7个氨基酸残基组成的肽,C端通过N-酰磷酰胺键与5'-磷酸腺苷(AMP) 所结合形成小分子量抗菌肽,具有一定的抗菌能力(冉仁森,中国科学院大学,硕士学位论文, 2017)。腺苷七肽的N端的蛋氨酸(Met)残基和C端的天冬氨酸(Asp)为其保守序列,不同的微生物分泌的腺苷七肽中间的五个氨基酸残基有所差异(Olga Bantysh,mBio,2014,5 (3):e01059-14)。本单位从益生菌约氏乳杆菌(CGMCC.NO 19858)经过色谱法分离其不同发酵代谢产物成分,从中得到一种小肽,根据其结构,命名为腺苷七肽(专利CN202010720092.6一种约氏乳杆菌及其制备的腺苷七肽、制备方法),腺苷七肽具有抗菌和免疫调节的功能。
发明内容
本发明的目的是提供一种透明质酸酶抑制剂及其制备方法,以及透明质酸酶抑制剂的在制备透明质酸相关产品中的应用,本发明的目的是通过以下措施实现的:
本发明提供了腺苷七肽在制备透明质酸酶抑制剂中的应用。
上述腺苷七肽的氨基酸序列为MATGNAD。
本发明还提供了一种透明质酸酶抑制剂,以腺苷七肽作为活性成分,优选地,腺苷七肽在透明质酸酶抑制剂中的质量百分数为90%。
本发明还提供了一种透明质酸酶抑制剂的制备方法,包括以下步骤:
步骤1:将约氏乳杆菌(CGMCC.NO 19858)接种于50mL MRS肉汤培养基,培养11-12小时,再扩增于5L MRS肉汤培养基,继续培养10小时。
步骤2:将步骤1的5L约氏乳杆菌培养物接种于灭菌种子罐,加入50L种子培养基,5%NaOH调节pH=6.0~6.5,保持溶氧DO<10%,培养8~10小时,直至种子罐pH恒定不再下降。
步骤3:将种子罐发酵液泵如发酵罐中,加入500L发酵培养基,5%NaOH调节pH=6.0~6.5,保持溶氧DO<10%,并每隔4小时测定腺苷七肽的含量,培养10~12小时),直至发酵罐pH恒定不再下降,且腺苷七肽的含量达到4g/L,停止发酵;
步骤4:发酵液经过碟式离心机分离菌体和清液,分离因素12000G,进料压力0.1Mpa,清夜进入50nm陶瓷膜,通水量600~800L/m2·h-1进一步分离,清液通过1500Da分子量超滤膜和 300Da纳滤膜进行截留除杂,获得300~1500Da分子量的液体。
步骤5:将步骤4所获液体通过层析柱纯化分离,分离条件如下:A相:水,B相:乙腈,洗脱条件:0~50min:95%A,5%B;50~80min:85%A,15%B;80~180min:55%A,45%B;180~250min:5%A,95%B;收集140~160min液体,冷冻干燥,条件为-80~-45℃,压力 -0.05MPa,干燥1.5~2小时,即得到90%纯度的腺苷七肽。
上述制备方法中,所述种子培养基由蛋白胨、酵母提取液、葡萄糖、乙酸钠、柠檬酸二胺、磷酸氢二钾、七水硫酸镁组成,其中蛋白胨1%,酵母提取液1%,葡萄糖2%,乙酸钠0.5%,柠檬酸二胺0.2%,磷酸氢二钾0.2%,七水硫酸镁0.02%,以质量分数计算。
所述发酵培养基由蛋白胨、酵母提取液、葡萄糖、乙酸钠、柠檬酸二胺、磷酸氢二钾、七水硫酸镁组合,其中蛋白胨1.5%,酵母提取液1.5%,葡萄糖3%,乙酸钠0.5%,柠檬酸二胺0.2%,磷酸氢二钾0.4%,七水硫酸镁0.02%,以质量百分比计算。
本发明还提供了一种透明质酸产品,包含上述透明质酸酶抑制剂,优选地,透明质酸酶抑制剂中腺苷七肽的质量分数为90%。进一步地,上述透明质酸产品为透明质酸皮肤护理产品和生物兽药用品,透明质酸皮肤护理产品包括:保湿品、化妆水、精华素、洗面奶、沐浴液。
进一步地,上述透明质酸皮肤护理产品包还包括pH调节剂、防腐剂、功效成分和水。
优选地,一种透明质酸保湿品,含1%的透明质酸钠含量,0.6%的腺苷七肽,以质量百分比计算,保湿品的pH为6.8~7.0。
本发明还提供了一种饲料添加剂,其特征在于,含有上述透明质酸酶抑制剂。
有益效果:
1.本发明公开了一种透明质酸酶抑制剂,以腺苷七肽作为活性成分,具有抑制透明质酸酶的作用,抑制效率高,副作用小,且耐高温、耐高湿、稳定性好。
2.本发明提供了一种透明质酸酶抑制剂的制备方法,获得的透明质酸酶抑制剂中腺苷七肽的质量分数为90%,工艺简单方便,效率高,环境污染小,可实现大规模产业化的生产。
3.本发明提供了一种透明质酸酶抑制剂在制备透明质酸产品中的应用,及获得的透明质酸产品,在皮肤护理产品、饲料添加剂、生物兽药、食品添加剂方面具有广阔的应用前景。
附图说明:
图1为透明质酸抑制剂的的制备流程图
具体实施方案
一、透明质酸抑制剂的的制备方法:
1.在菌种室无菌操作进行接种约氏乳杆菌(CGMCC.NO 19858)于MRS肉汤培养基中(50mL),培养11~12小时,菌液浑浊,扩增于5L的MRS肉汤培养基,继续培养10小时,菌液浑浊,镜检。
2.镜检无杂菌,将5L培养好的约氏乳杆菌接种于灭菌种子罐中种子培养基为:蛋白胨 1%,酵母提取液1%,葡萄糖2%,乙酸钠0.5%,柠檬酸二胺0.2%,磷酸氢二钾0.2%,七水硫酸镁0.02%,通过补5%的NaOH溶液保持种子罐pH=6.0~6.5之间,通过调节搅拌速度保持溶氧DO<10%
3.当种子罐pH恒定不再下降(需8-10小时),将种子罐发酵液泵如发酵罐中(图1设备 1),发酵培养基为蛋白胨1.5%,酵母提取液1.5%,葡萄糖3%,乙酸钠0.5%,柠檬酸二胺0.2%,磷酸氢二钾0.4%,七水硫酸镁0.02%)通过补5%的NaOH溶液保持种子罐pH=6.0~ 6.5之间,通过调节搅拌速度保持溶氧DO<10%,并每隔4小时取样通过HPLC测定腺苷七肽的含量(测定方法参考:高效液相色谱法测定新型抗菌肽杆菌七肽的含量,中国饲料,2020 年15期)。
4.培养10~12小时,至发酵罐pH恒定不再下降,并且腺苷七肽的含量达到4g/L,发酵停止。
5.发酵也经过碟式离心机(图1设备2)进行菌体和上清液进行分离,分离因素12000G,进料压力0.1Mpa。
6.分离后清夜进入孔径50nm陶瓷膜,通水量600~800L/m22·h-,进一步分离(图1设备 3),清液通过1500Da分子量超滤膜和300Da纳滤膜进行截留除杂,获得300~1500Da分子量的液体。
7.将6获得的300~1500Da分子量的液体通过层析系统进行纯化分离(汉邦科技),液体经过层析柱(图1设备4)(江阴市创通层析设备有限公司,CXBG-8规格Ф108×1000mm,压力≤ 0.6MPa,容积8.4L;填料为C18硅胶,400目,青岛邦凯高新技术材料有限公司)进行分离,分离条件如下:A相:水,B相:乙腈,洗脱条件:0~50min:95%A,5%B;50~80min:85%A, 15%B;80~180min:55%A,45%B;180~250min:5%A,95%B;收集140~160min液体,冷冻干燥,条件为-80~-45℃,压力-0.05MPa,干燥1.5~2小时,得到含90%质量分数腺苷七肽的透明质酸酶抑制剂。
二、透明质酸酶抑制剂的活性检测
肝素钠是常见的透明质酸酶抑制剂,其具有抗炎,抗凝的作用,但长期在皮肤中使用,具有一定的副作用。本试验以肝素钠做阳性对照,用评价以腺苷七肽为活性成份的透明质酸酶抑制剂的活性,为皮肤护理产品的添加剂开发提供依据。
采用Elson-Morgan改良法测定不同浓度的腺苷七肽对透明质酸酶的作用。含0.3、0.6、1.2、 2.4、4.8、6.2mg/ml腺苷七肽的透明质酸酶抑制剂溶液0.5mL加入到0.5mL透明质酸酶(500 U/mL)液中,37℃保温20min;加入2.5mol/L的氯化钙溶液0.1mL,37℃保温20min;加入0.5 mg/mL的透明质酸钠0.5mL,37℃保温40min;室温放置10min,加入0.5mL乙酰丙酮溶液(3.5mL 乙酰丙酮溶于50mL 0.1mol/L碳酸钠溶液中)、5mol/L的NaOH溶液()0.1mL和蒸馏水0.5mL,放入沸水浴中15min;冷却5min,放入冰水浴10min;室温放置10min,加入1mLP-DAB显色剂(0.8g二甲氨基苯甲醛与15mL浓HCL以及75%无水乙醇混合均匀);充分振荡后,加入 3.5mL无水乙醇,室温放置30min;显色,于530nm处测定OD值。
计算透明质酸酶抑制率(%)=[(C-D)-(A-B)]/(C-D)×100%
试中:A—试样溶液的OD值
B—试样空白(各种提取液,酶钝化)的OD值
C—对照溶液(用去离子水代替试样溶液)的OD值
D—对照空白(用去离子水代替试样溶液、酶钝化)的OD值
腺苷七肽(表1)0.3mg/L、0.6mg/L、1.2mg/L、2.4mg/L、4.8mg/L、6.2mg/L的透明质酸酶抑制剂对透明质酸酶的抑制率分别为15.23%、29.74%、46.12%、67.85%、89.31%、93.87%,其6.2mg/mL的浓度下,与相同浓度肝素钠的抑制效果相近(表1)。
表1腺苷七肽对透明质酸酶抑制率
三、不同透明质酸酶抑制剂的抑制效果的比较
将常见的几种透明质酸酶抑制剂(仙人草提取物、积雪草甙提取物、藤茶黄酮和鸢尾苷提取物)以及腺苷七肽(90%)按照梯度稀释,配制成浓度为6.0、4.5、3.0、1.5、1.0、0.5mg/mL 的溶液,按照实施例2的方法测定透明质酸酶抑制率。
在有效活性成分浓度相同的情况下,腺苷七肽对透明质酸酶的抑制效果好于常见的几种植物来源的透明质酸酶抑制剂,在6mg/mL的浓度下,对透明质酸酶的抑制率可达到90%。以腺苷七肽为活性成分的透明质酸酶抑制剂,可成为潜在的化妆品的保湿剂和抗过敏制剂。
表2腺苷七肽与常见透明质酸酶抑制剂的抑制效果的比较
a,b,c同行数据肩标字母不相同者差异显著(P<0.05)。
四、透明质酸酶抑制剂的稳定性
透明质酸酶抑制剂样品(腺苷七肽质量分数为90%),内袋采用铝箔袋密封包装,外袋采用珠光膜编制袋。样品放置于培养皿,摊成≤5mm厚的薄层,进行如下试验:
1.高温试验
将样品置于密封洁净容器中,放到40℃培养箱中,放置10天,分别在0、5、10天,测定指标(腺苷七肽含量、水分、颗粒度)。
2.高湿试验
将样品置于密封洁净干燥器,放入饱和KNO3(湿度90±5%)中,在25℃条件下放置10 天,分别在0、5、10天,测定指标(腺苷七肽含量、水分、颗粒度)。
3.加速试验
将透明质酸酶抑制剂样品(粉末)放入干燥器中,里面加入NaCl饱和溶液(湿度为75 ±5%),在40℃隔水式培养箱放置6个月,分别于0、1、2、3、4、5、6月测定样品的外观形状、腺苷七肽含量、粒度。
高温试验发现,在40℃试验条件下透明质酸酶抑制剂中腺苷七肽的含量和水分基本不变,说明其在高温条件下透明质酸酶抑制剂的性状和性质是不会发生改变,这表明腺苷七肽对高温的耐受性较好,可以在高温下保存一定的时间(表3)。
高湿度试验发现,在75%湿度条件下,其增重为1.14%(<5%),在此湿度下,透明质酸酶抑制剂中的腺苷七肽含量没有发生明显变化。这表明腺苷七肽对高湿度的耐受性较好,可以在南方高湿度的条件下保存一定的时间。
加速试验发现,腺苷七肽在密封包装铝箔袋中,置于湿度75±5%,40℃,经过6个月的存放,三批次腺苷七肽的含量下降分别为1.2%,外观性状、水分、颗粒度未发生变化。
以上试验表明,腺苷七肽耐高温、耐高湿、稳定性好,可以作为皮肤护理产品添加剂、饲料添加剂、生物兽药、食品添加剂等。
表3高温(40℃条件)对腺苷七肽的影响
表4高湿(湿度95±5%,25℃)对腺苷七肽的影响
表5加速试验(湿度75±5%,40℃)
五、腺苷七肽保湿效果试验
年龄30~45岁的受试者30人,随机分为3组,每组10人,男女各半,在右手前臂内侧标记4×4cm2试验区域,按0.5ml样品/cm2的用量,将试样均匀涂布于试验区内。3组的试样分别为:
对照组:甘油7%,透明质酸钠1%,羟苯甲脂0.2%,NaOH或乳酸调节pH=6.8-7.0;
试验组1(鸢尾苷作为透明质酸酶抑制剂的活性成分):甘油7%,透明质酸钠1%,羟苯甲脂0.2%,鸢尾苷0.6%,NaOH或乳酸调节pH=6.8~7.0;
试验组2(腺苷七肽作为透明质酸酶抑制剂的活性成分):甘油7%,透明质酸钠1%,羟苯甲脂0.2%,腺苷七肽0.6%,NaOH或乳酸调节pH=6.8~7.0。
使用皮肤水分测试仪Corneometer CM825测量涂抹前及涂抹后1h、3h、6h时受试区域和空白对照区域的皮肤含水量。同一个受试者的测试由同一个测量人员完成,测定部位和室内温度、湿度保持一致。计算皮肤水增加量%=(涂抹后定值-涂抹前定值)/涂抹前定值×100%。试验数据以平均值±标准偏差(Mean±SD)表示,用SPSS STATISTICS软件及Excel处理工具进行数据分析处理,采用方差分析进行Duncan多重比较,P<0.05为差异显著。
可以看出,透明质酸本身具有保湿作用,添加抑制透明质酸酶活性的组合物后对透明质酸保湿作用的提升有促进作用,在使用6小时之后,与空白对照组相比,皮肤水分的增加量为3.1 倍,而与鸢尾苷相比,则提高了1.2倍(表6)。这可能是由于组合物抑制了透明质酸在皮肤表面的降解,进而提升保湿作用。
表6不同配方皮肤水增加量(%)
字母a,b,c为差异情况,字母不同表示差异显著(P<0.05)。
六、透明质酸酶抑制剂缓减产气荚膜梭菌对家禽肠道的损伤
选取210只21日龄健康爱拔益加(AA)肉公鸡,购于北京市爱拔益加家禽育种有限公司。试验采用完全随机设计。肉鸡于配有独立采食和饮水设施的鸡舍中饲养,自由采食和饮水。将 21日龄AA肉鸡随机分为5个处理,每个处理6个重复,每个重复7只鸡。肉鸡饲喂不含抗生素的玉米-豆粕型粉料。试验处理组分为:
空白对照(阴性对照):无任何处理,即正常饲喂,不攻毒,不饮药物;
试验组1:产气荚膜梭菌攻毒;
试验组2:产气荚膜梭菌攻毒,饮水腺苷七肽;
试验组3:产气荚膜梭菌攻毒,饮水杆菌肽;
试验组4:产气荚膜梭菌攻毒,饮水杆菌肽+腺苷七肽。
产气荚膜梭菌CVCC 2027接种于BHI培养基,37℃厌氧条件下培养18h;然后将发酵液接种于巯基乙酸盐培养基,37℃厌氧条件下培养24h。于21日龄(试验第1d),4个产气荚膜梭菌感染组的每只肉鸡经口灌服菌液1mL(2.0×108CFU/mL),每天1次,连续灌服7d,空白对照组的肉鸡灌服等体积的0.75%生理盐水。于试验7d,每个重复随机抽取1只鸡屠宰,即每个处理取6只肉鸡进行屠宰,取样。同时,第7d,三个试验组给予治疗,试验组一将腺苷七肽溶于水,终浓度分别为60mg/L,至于1L的饮水容器,饮水治疗7d;试验组二给予杆菌肽60mg/L,组合组为腺苷七肽30mg/L+杆菌肽30mg/L,治疗方式与试验组一相同。当盛有 1L含有腺苷七肽或杆菌肽的饮水被肉鸡喝完后,当天所剩下的时间给肉鸡饮用不含药物的饮用水。于产气荚膜梭菌攻毒期间和药物治疗期间测定肉鸡直肠温度。于试验14d,每个重复随机抽取1只鸡屠宰,取样。检测指标与测定方法如下:
(1)生长性能
分别于试验期第1、14、21和28d上午08:00以个体为单位空腹称重,以重复为单位记录 d 1~14、d 15~21和d 22~28阶段的采食量,计算平均日增重,平均日采食量和增重耗料比 (G:F)。计算G:F时,死亡鸡只的体重考虑在内。
(2)肠道损伤
每天观察肉鸡的病理学症状和死亡情况,对死亡鸡只进行尸体剖检,确定死亡原因。于试验28d,每个重复随机抽取2只鸡,处死后剖开腹腔,肉眼观察空肠和回肠的病理变化。根据肠道损伤的严重程度进行肠道病理损伤评分(0~4分),评分标准参考Dahiya等(2005)的方法。评分标准为六级,即0分(正常)、0.5分(小肠的浆膜和肠系膜严重充血)、1分(肠壁变薄、变脆,有红色淤点出现)、2分(肠壁出现针尖样坏死或溃疡点,肠腔内有少量气体)、 3分(肠壁出现成片状坏死或溃疡,肠腔内充满气体)和4分(肠壁出现弥散型坏死和出血,肠腔内充满大量气体)。取回肠肠道样品,冷冻研磨后,溶于水中,震荡,离心,测定透明质酸酶活性(方法同实施例2)
(3)回肠细胞因子
取3cm回肠肠段,0.75%生理盐水冲洗后,用0.1M PBS匀浆。将匀浆液4℃,3000g离心10min取上清液,用BCA蛋白质定量试剂盒(Thermo,美国)测定上清液中蛋白质浓度。回肠组织中IL-1β、IL-6和TNF-α的含量使用鸡源ELISA试剂盒(Cusabio Biotech Company,武汉)进行测定,测定步骤参考试剂盒说明书。
试验数据以平均值±标准偏差(Mean±SD)表示,用SPSS STATISTICS软件及Excel处理工具进行数据分析处理,采用方差分析进行Duncan多重比较,P<0.05为差异显著。
各个处理的肉鸡生长性能见表7。产气荚膜梭菌攻毒前(d 1至d 14),各处理之间的生长性能无显著差异(P>0.05)。产气荚膜梭菌攻毒,显著降低了攻毒期间(d 15至d 21)的平均日增重(P<0.05),降低了饲料转化效率(P<0.05)。治疗期间(d 22至d 28),与攻毒组比较,单独使用杆菌肽和杆菌肽+腺苷七肽能明显提高饲料转化率(P<0.05),而单独使用腺苷七肽没有作用(P>0.05)。同时发现,杆菌肽+腺苷七肽组能恢复到正常对照组的饲料转化率。说明杆菌肽+腺苷七肽效果优于其他攻毒组,能明显提高感染肉鸡的健康程度,恢复到正常状态。
空白对照组中的肉鸡没有发现小肠的坏死性损伤。而产气荚膜梭菌攻毒显著增加了小肠坏死指数(P<0.05),经过杆菌肽和腺苷七肽+杆菌肽治疗后,小肠坏死指数显著降低(P<0.05)。其中,联合杆菌肽和腺苷七肽+杆菌肽治疗效果最优。与攻毒组相比,不同治疗方式都能显著降低透明质酸酶活性,其中,联合用药组效果最优(P<0.05),明显由于单独治疗组(P<0.05)。
产气荚膜梭菌攻毒显著增加了回肠促炎性细胞因子IL-1β、IL-6和TNF-α表达量(P< 0.05),经过腺苷七肽、杆菌肽或联合治疗后,这些促炎性细胞因子表达量均有不同程度的下降。联合用药治疗后,回肠IL-6的水平可恢复到正常对照水平,IL-1β和TNF-α表达量明显下降(P<0.05)。
以上结果提示,本专利中以腺苷七肽为活性成分的透明质酸酶抑制剂能抑制透明质酸酶活性,降低产气荚膜梭菌通过分泌毒素扩散因子(透明质酸酶)来导致组织的损伤,而联合抗生素使用,可明显缓减组织损伤,减缓损伤,可作为饲料添加剂和生物兽药,与抗生素配伍使用,可减缓动物产气荚膜梭菌造成的坏死性肠炎。
表7腺苷七肽对产气荚膜梭菌攻毒对肉鸡生长性能的影响
a,b,c同行数据肩标字母不相同者差异显著(P<0.05)。
Claims (10)
1.腺苷七肽在制备透明质酸酶抑制剂中的应用。
2.如权利要求1所述的应用,其特征在于:所述腺苷七肽的氨基酸序列为MATGNAD。
3.一种透明质酸酶抑制剂,其特征在于:包含以质量百分数计90%的腺苷七肽。
4.如权利要求3所述透明质酸酶抑制剂的制备方法,包括以下步骤:
步骤1:将约氏乳杆菌(CGMCC.NO 19858)接种于50mL MRS肉汤培养基,培养11-12小时,再扩增于5L MRS肉汤培养基,继续培养10小时;
步骤2:将步骤1的5L约氏乳杆菌培养物接种于灭菌种子罐,加入50L 种子培养基,5%NaOH调节pH=6.0~6.5,保持溶氧DO<10%,培养8~10小时,直至种子罐pH恒定不再下降;
步骤3:将种子罐发酵液泵如发酵罐中,加入500L发酵培养基,5%NaOH调节pH=6.0~6.5,保持溶氧DO<10%,并每隔4小时测定腺苷七肽的含量,培养10~12小时,直至发酵罐pH恒定不再下降,且腺苷七肽的含量达到4g/L,停止发酵;
步骤4:发酵液经过碟式离心机分离菌体和清液,分离因素12000G,进料压力0.1Mpa,清夜进入50nm陶瓷膜,通水量600~800L/m²•h-¹进一步分离,清液通过1500Da分子量超滤膜和300Da纳滤膜进行截留除杂,获得300~1500Da分子量的液体;
步骤5:将步骤4所获液体通过层析柱纯化分离,分离条件如下:A相:水,B相:乙腈,洗脱条件:0~50min:95%A, 5%B;50~80min:85%A,15%B;80~180min:55%A,45%B;180~250min:5%A,95%B;收集140~160min液体,冷冻干燥,条件为-80~-45℃,压力-0.05MPa,干燥1.5~2小时,即得到90%纯度的腺苷七肽。
5.如权利要求4所述制备方法,其特征在于:种子培养基由蛋白胨、酵母提取液、葡萄糖、乙酸钠、柠檬酸二胺、磷酸氢二钾、七水硫酸镁组成,其中蛋白胨 1%,酵母提取液 1%,葡萄糖 2%,乙酸钠 0.5%,柠檬酸二胺 0.2%,磷酸氢二钾 0.2%,七水硫酸镁 0.02%,以质量分数计算;
发酵培养基由蛋白胨、酵母提取液、葡萄糖、乙酸钠、柠檬酸二胺、磷酸氢二钾、七水硫酸镁组合,其中蛋白胨 1.5 %,酵母提取液 1.5 %,葡萄糖 3%,乙酸钠 0.5%,柠檬酸二胺0.2%,磷酸氢二钾 0.4%,七水硫酸镁 0.02%,以质量百分比计算。
6.一种透明质酸产品,其特征在于:包含权利要求3所述透明质酸酶抑制剂,透明质酸酶抑制剂中腺苷七肽的质量分数为90%。
7.如权利要求6所述透明质酸产品,其特征在于:所述透明质酸产品为皮肤护理产品或生物兽药用品。
8.如权利要求7所述透明质酸产品,其特征在于:所述皮肤护理产品为保湿品、化妆水、精华素、洗面奶或沐浴液;所述皮肤护理产品包括pH调节剂、防腐剂、功效成分和水。
9.如权利要求8所述透明质酸产品,其特征在于,所述保湿品中透明质酸钠含量为1%,腺苷七肽含量为0.6%,保湿品pH为6.8-7.0。
10.一种饲料添加剂,其特征在于,含有如权利要求3所述透明质酸酶抑制剂。
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