CN108719607B - 一种包含溶菌酶的饲料添加剂 - Google Patents
一种包含溶菌酶的饲料添加剂 Download PDFInfo
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- CN108719607B CN108719607B CN201710243612.7A CN201710243612A CN108719607B CN 108719607 B CN108719607 B CN 108719607B CN 201710243612 A CN201710243612 A CN 201710243612A CN 108719607 B CN108719607 B CN 108719607B
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- feed additive
- lysozyme
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- streptomyces griseus
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Abstract
本发明涉及一种包含溶菌酶的饲料添加剂，其中所述溶菌酶采用发酵工艺制备，具体步骤如下：取液体培养基置于三角瓶中，按4％v/v的接种量接入灰色链霉菌的菌体种子，进行摇瓶发酵，发酵条件为20‑40℃，150‑300r/min，发酵培养24h后，将发酵液离心去除菌体，取上清液依次用硫酸铵盐析法、凝胶柱层析法和阳离子交换柱层析法纯化后，冷冻干燥即得。使用上述发酵工艺获得的溶菌酶饲料添加剂对仔猪腹泻具有良好的防治效果，配合中药提取物使用效果更佳。
Description
技术领域
本发明涉及饲料领域，具体涉及一种包含溶菌酶的饲料添加剂。
背景技术
近年来，随着畜牧产业的高度集约化生产，畜禽疾病的发生也日趋上升，由于疾病造成的死亡率逐年增加，给畜牧业造成重大的经济损失。腹泻是影响仔猪成活率和生长发育的主要因素之一，传统预防和治疗措施是使用抗生素。虽然抗生素在畜禽饲料中的使用对于有效控制疾病、提高饲料转化率、保障畜产品的稳定供应发挥了重要作用，但是由于饲料生产中抗生素的超范围、高剂量或多品种、低剂量的长期使用，以及养殖过程中抗生素滥用和乱用，带来的细菌耐药性问题显得尤为突出。随着欧盟2006年全面禁止在饲料中使用抗生素和美国公告2017年1月1日全面禁止在饲料中使用抗生素后，抗生素替代物研发一直是动物营养研究的热点。
溶菌酶(Lysozyme)，又称细胞壁水解酶，能专一性地作用于细菌细胞壁肽聚糖分子，能使N-乙酰胞壁酸与乙酰葡萄糖氨之间的β-1,4糖苷键断裂，使细菌细胞壁变得松驰而达到溶解细菌的效果。溶菌酶广泛分布于高等动植物组织及其分泌物、原生动物、昆虫、植物以及各种微生物中。
溶菌酶作为人体正常体液及组织中的非特异免疫因子之一，具有多种药理作用，参与机体多种免疫反应，有抗菌、抗病毒的功效，在机体正常防御功能和非特异免疫中，具有保持机体生理平衡的重要作用。它可以改善和增强巨噬细胞吞噬和消化功能，激活白细胞吞噬功能，并能改善细胞抑制剂所导致的白细胞减少，从而增强机体的抵抗力。溶菌酶能直接水解革兰氏阳性菌，在分泌型免疫球蛋白A、补体的参与下，还能水解革兰氏阴性菌如大肠杆菌等。对耐药性细菌同样具有溶菌作用，而且具有疗效显著和对人体副作用小的特点，因而是一种较为理想的药用酶。溶菌酶能与带负电荷的病毒蛋白直接作用，与DNA、RNA、脱辅基蛋白形成复盐，使病毒失活。溶菌酶作为酶类抗菌药，能参与粘多糖代谢，在配合内服和外用药的同时，可起到强力消炎作用，它还可与各种诱发炎症的酸性物质结合使其失活，并能够增强抗生素和其它药物的疗效，改善组织基质的粘多糖代谢，从而达到消炎、修复组织的目的。
溶菌酶是基因工程、细胞工程、发酵工程中必不可少的工具酶。在国外，溶菌酶多用于菌体内物质的提取。只要把对敏感的菌体悬液在适当缓冲液中，用溶菌酶处理，再结合使用超声波、冷冻离心等手段就可得到无细胞提取液，进一步精制可得到所需的菌体物质如蛋白质、核酸、酶及活性多肽等，因此生物工业的发展对溶菌酶制剂的需求将与日俱增。
目前，市场上见到最多的溶菌酶是蛋清溶菌酶，但其仅对革兰氏阳性菌有作用，限制了其应用范围。近年来国内外对多种来源的溶菌酶进行了广泛的研究，但有关发酵方法制备微生物溶菌酶的研究报道较少，导致此类非蛋清溶菌酶在实际工业化大生产上应用有限。
中药在抗菌同时具有改善和调节机体状态的特性，因此利用我国特有的中医理论和丰富的植物药资源开发控制仔猪腹泻制剂已经成为维持和发展养猪业的重要课题。
白头翁为多年生毛茛科植物，高达50cm，全株密被白色长柔毛，主根粗壮呈圆锥形，有时扭曲，外皮黄褐色。白头翁性寒，归大肠经，有清热解毒、凉血止泻之功效，常用于除肠胃湿热，为热毒血痢之良药。此外，也可用于治疗疟疾，或煎汤外洗用于外阴痒、阴肿的治疗，近年来用于治疗细菌性痢疾和阿米巴痢疾均有良效。白头翁抗菌性广泛，其提取物有较好的镇静、止痛作用，其中常用的有效成分为皂苷和白头翁素。
白头翁鲜汁、煎剂、乙醇提取物在体外均有明显的抗菌作用，对金黄色葡萄球菌、绿脓杆菌、痢疾杆菌较为敏感，对痢疾杆菌、枯草杆菌、沙门杆菌等也有显著抑制作用。另有研究表明，白头翁的抗菌有效成分为白头翁素及原白头翁素，二者均具有强烈的抗菌作用。
发明内容
本发明针对现有技术不足，通过筛选溶菌酶的发酵来源和发酵工艺，优化溶菌酶与中药提取物的配伍，得到了一种对仔猪腹泻防治具有良好效果的饲料添加剂。
本发明的技术方案是：一种包含溶菌酶的饲料添加剂，其中所述溶菌酶采用发酵工艺制备，具体步骤如下：取液体培养基置于三角瓶中，按4％v/v的接种量接入灰色链霉菌的菌体种子，进行摇瓶发酵，发酵条件为20-40℃，150-300r/min，发酵培养24h后，将发酵液离心去除菌体，取上清液依次用硫酸铵盐析法、凝胶柱层析法和阳离子交换柱层析法纯化后，冷冻干燥即得。
优选的，所述液体培养基包含葡萄糖和蛋白胨。
更优选的，所述葡萄糖的浓度为10g/L，所述蛋白胨的浓度为5g/L。
优选的，所述灰色链霉菌选自灰色链霉菌S-35。
优选的，所述发酵条件为30℃，200r/min。
优选的，所述凝胶柱层析法使用Sephadex G-100凝胶柱，所述阳离子交换柱层析法使用CM Sepharose Fast Flow层析柱。
优选的，所述硫酸铵盐析法的步骤如下：上清液加入硫酸铵至20％饱和度，搅拌1h后静置1h，10000r/min离心，沉淀用蒸馏水溶解，装入分子量为10000的透析袋中，置于10mmol/L PBS中充分透析；透析袋中上清液添加硫酸铵至70％饱和度，搅拌1h后静置1h，10000r/min离心，取沉淀透析，方法同上，得透析液待用。
优选的，所述饲料添加剂还包括中药提取物。
更优选的，所述中药提取物为白头翁提取物。
优选的，所述白头翁提取物的提取方法如下：将白头翁用5-20倍量水加热煮沸，沸腾后改文火再煮0.5-2小时，倒出药液后加2-5倍量水煎煮，重复1-3次，每次各0.5-2小时；合并药液后浓缩，静置过夜，以2500r/分钟的转速离心10分钟得上清液；取上清液用喷雾干燥机制成干粉，即得。
更优选的，所述白头翁提取物的提取方法如下：将白头翁用10倍量水加热煮沸，沸腾后改文火再煮1小时，倒出药液后加3倍量水煎煮，重复2次，每次各1小时；合并药液后浓缩，静置过夜，以2500r/分钟的转速离心10分钟得上清液；取上清液用喷雾干燥机制成干粉，即得。
最优选的，所述白头翁提取物与所述溶菌酶的重量比为1:1。
本发明的另一技术方案是，上述包含溶菌酶的饲料添加剂在制备防治仔猪腹泻的药物中的应用。
本发明的有益效果：
发明人通过筛选溶菌酶的发酵来源，优化发酵工艺参数，获得了对仔猪腹泻防治具有良好效果的溶菌酶，进一步发现将溶菌酶与中药提取物配伍使用防治效果更佳，证实本发明的饲料添加剂对无抗饲料添加剂的研发和仔猪腹泻等疾病的防治具有一定指导意义。
具体实施方式
一、发酵工艺制备溶菌酶
实施例1：
制备液体培养基，其中葡萄糖10g/L，蛋白胨5g/L。取25mL液体培养基置于三角瓶中，按4％(v/v)的接种量接入灰色链霉菌S-35的菌体种子，进行摇瓶发酵，发酵条件为30℃，200r/min，发酵培养24h后，将发酵液离心去除菌体，取上清液依次用硫酸铵盐析法(上清液加入硫酸铵至20％饱和度，搅拌1h后静置1h，10000r/min离心，沉淀用蒸馏水溶解，装入分子量为10000的透析袋中，置于10mmol/LPBS中充分透析；透析袋中上清液添加硫酸铵至70％饱和度，搅拌1h后静置1h，10000r/min离心，取沉淀透析，方法同上，得透析液)、Sephadex G-100凝胶柱层析法和CM Sepharose Fast Flow阳离子交换柱层析法纯化后，冷冻干燥，即得溶菌酶L1。
实施例2：
制备液体培养基，其中葡萄糖10g/L，蛋白胨5g/L。取25mL液体培养基置于三角瓶中，按4％(v/v)的接种量接入灰色链霉菌S-35的菌体种子，进行摇瓶发酵，发酵条件为20℃，150r/min，发酵培养24h后，将发酵液离心去除菌体，取上清液依次用硫酸铵盐析法(上清液加入硫酸铵至20％饱和度，搅拌1h后静置1h，10000r/min离心，沉淀用蒸馏水溶解，装入分子量为10000的透析袋中，置于10mmol/LPBS中充分透析；透析袋中上清液添加硫酸铵至70％饱和度，搅拌1h后静置1h，10000r/min离心，取沉淀透析，方法同上，得透析液)、Sephadex G-100凝胶柱层析法和CM Sepharose Fast Flow阳离子交换柱层析法纯化后，冷冻干燥，即得溶菌酶L2。
实施例3：
制备液体培养基，其中葡萄糖10g/L，蛋白胨5g/L。取25mL液体培养基置于三角瓶中，按4％(v/v)的接种量接入灰色链霉菌S-35的菌体种子，进行摇瓶发酵，发酵条件为40℃，300r/min，发酵培养24h后，将发酵液离心去除菌体，取上清液依次用硫酸铵盐析法(上清液加入硫酸铵至20％饱和度，搅拌1h后静置1h，10000r/min离心，沉淀用蒸馏水溶解，装入分子量为10000的透析袋中，置于10mmol/LPBS中充分透析；透析袋中上清液添加硫酸铵至70％饱和度，搅拌1h后静置1h，10000r/min离心，取沉淀透析，方法同上，得透析液)、Sephadex G-100凝胶柱层析法和CM Sepharose Fast Flow阳离子交换柱层析法纯化后，冷冻干燥，即得溶菌酶L3。
二、中药提取物的制备
实施例4：
将白头翁用10倍量水加热煮沸，沸腾后改文火再煮1小时，倒出药液后加3倍量水煎煮，重复2次，每次各1小时；合并药液后浓缩，静置过夜，以2500r/分钟的转速离心10分钟得上清液；取上清液用喷雾干燥机制成干粉，即得白头翁提取物P1。
实施例5：
将白头翁用20倍量水加热煮沸，沸腾后改文火再煮2小时，倒出药液后加5倍量水煎煮，重复3次，每次各2小时；合并药液后浓缩，静置过夜，以2500r/分钟的转速离心10分钟得上清液；取上清液用喷雾干燥机制成干粉，即得白头翁提取物P2。
实施例6：
将白头翁用5倍量水加热煮沸，沸腾后改文火再煮0.5小时，倒出药液后加2倍量水煎煮，重复1次，每次各0.5小时；合并药液后浓缩，静置过夜，以2500r/分钟的转速离心10分钟得上清液；取上清液用喷雾干燥机制成干粉，即得白头翁提取物P3。
三、本发明饲料添加剂对防治仔猪腹泻的影响
如无特殊说明，下述试验使用的蛋清溶菌酶购自上海户实医药科技有限公司，发酵溶菌酶为实施例1制备得到的溶菌酶L1，白头翁提取物为实施例4制备得到的白头翁提取物P1。
1.试验方案
试验在浙江省农科院畜牧兽医所海宁科技牧场进行。按照体重相近、公母各半的原则，随机选取100头断奶仔猪，随机分成5组(空白组、蛋清溶菌酶组、溶菌酶L1组、白头翁提取物P1组和混合组)，每组20头。试验过程中饲喂的基础日粮配方及营养水平见表1。
表1基础日粮配方及营养水平表
注：预混料中含有矿物质(钙、磷、食盐、铜、铁、锰、锌、硒、碘、钴)、多种维生素(维生素A、维生素D、维生素B1、维生素B2、维生素B12、生物素、叶酸、泛酸、烟酸、维生素K、维生素E)和部分氨基酸(赖氨酸、蛋氨酸、苏氨酸、色氨酸)，不含抗生素。
各试验组在基础日粮中添加饲料添加剂的方案如下：
(1)空白组：基础日粮；
(2)蛋清溶菌酶组：基础日粮+蛋清溶菌酶1.5kg/吨；
(3)溶菌酶L1组：基础日粮+溶菌酶L1 1.5kg/吨；
(4)白头翁提取物P1组：基础日粮+白头翁提取物P1 1.5kg/吨；
(5)混合组(溶菌酶L1与白头翁提取物P1按照重量比为1:1的比例混合)：基础日粮+溶菌酶L1 0.75kg/吨+白头翁提取物P10.75kg/吨。
2.饲养管理与测定指标
试验猪群自由采食和饮水，分餐饲喂，每天4次(时间分别为7:00、11:00、15:00、19:00)，免疫程序按照猪场饲养管理程序进行，出现腹泻立刻用硫酸新霉素和复合维生素配合口服补液盐进行治疗。整个试验期为40天(30日龄-70日龄)。
每天观察并详细记录各组仔猪腹泻发生情况，计算腹泻率。
腹泻率(％)＝试验期内仔猪腹泻的总头次/(试验天数×仔猪头数)×100％。
试验结束后，采集各组仔猪的粪样，进行粪便细菌基因组DNA的提取，引物为细菌16S rDNA V4区特异性引物，扩增片段测序，随后对高质量测序数据进行生物信息学分析，重点考察粪便中代表有害菌的Cyanobacteria(蓝藻门)含量。
3.试验结果
表2本发明饲料添加剂对防治仔猪腹泻的影响
试验结果表明(见表2)，与商业化蛋清溶菌酶相比，使用本发明工艺发酵生产的溶菌酶防治仔猪腹泻的效果更佳，证实本发明的发酵工艺对产生高药理活性的溶菌酶具有促进作用。此外，与白头翁提取物联用的混合组防治仔猪腹泻的作用更加明显，产生了难以预期的优异效果。
此外，仔猪粪样16S rDNA测序结果表明，与蛋清溶菌酶组相比，溶菌酶L1组和混合组中有害菌的Cyanobacteria(蓝藻门)含量分别降低了53.28％和68.29％，说明本发明的溶菌酶较市售溶菌酶更利于仔猪肠道有益菌群的建立。
综上所述，采用本发明饲料添加剂，特别是本发明制备的溶菌酶和中药提取物的组合物代替抗生素进行仔猪无抗养殖集成技术是可行的，且效果明显优于市售溶菌酶制剂。
上述实施例仅是本发明的优选实施方式，应当指出对于本领域的普通技术人员来说，在不脱离本发明的前提下，还可以做若干改进，这些也应视为本发明的保护范围之内。
Claims (8)
1.一种包含溶菌酶的饲料添加剂，其特征在于，所述溶菌酶采用发酵工艺制备，具体步骤如下：取液体培养基置于三角瓶中，按4％v/v的接种量接入灰色链霉菌的菌体种子，进行摇瓶发酵，发酵条件为30℃，200r/min，发酵培养24h后，将发酵液离心去除菌体，取上清液依次用硫酸铵盐析法、凝胶柱层析法和阳离子交换柱层析法纯化后，冷冻干燥即得；所述灰色链霉菌选自灰色链霉菌S-35。
2.根据权利要求1所述的饲料添加剂，其特征在于，所述液体培养基包含葡萄糖和蛋白胨。
3.根据权利要求2所述的饲料添加剂，其特征在于，所述葡萄糖的浓度为10g/L，所述蛋白胨的浓度为5g/L。
4.根据权利要求1-3任一项所述的饲料添加剂，其特征在于，所述饲料添加剂还包括中药提取物。
5.根据权利要求4所述的饲料添加剂，其特征在于，所述中药提取物为白头翁提取物。
6.根据权利要求5所述的饲料添加剂，其特征在于，所述白头翁提取物的提取方法如下：将白头翁用5-20倍量水加热煮沸，沸腾后改文火再煮0.5-2小时，倒出药液后加2-5倍量水煎煮，重复1-3次，每次各0.5-2小时；合并药液后浓缩，静置过夜，以2500r/分钟的转速离心10分钟得上清液；取上清液用喷雾干燥机制成干粉，即得。
7.根据权利要求6所述的饲料添加剂，其特征自在于，所述溶菌酶与所述白头翁提取物的重量比为1:1。
8.权利要求1-7任一项所述的饲料添加剂在制备防治仔猪腹泻的药物中的应用。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101912048A (zh) * | 2010-08-13 | 2010-12-15 | 中牧实业股份有限公司 | 一种兽用t4溶菌酶预混剂及其制备方法 |
| CN102847017A (zh) * | 2012-08-26 | 2013-01-02 | 裔群英 | 防治仔猪腹泻的口服液及其制备方法 |
| CN103333937A (zh) * | 2013-06-06 | 2013-10-02 | 徐州工程学院 | 一种利用枯草芽孢杆菌制备抗菌肽的工艺方法 |
| CN104920897A (zh) * | 2015-06-30 | 2015-09-23 | 江西瑞普康恩农牧发展有限公司 | 一种用于仔猪提高日增重减少下痢的饲料 |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03505676A (ja) * | 1989-04-24 | 1991-12-12 | ノボ ノルディスク アクティーゼルスカブ | バチルスパブリからの新規細胞壁溶菌酵素 |
| JP4276500B2 (ja) * | 2003-09-10 | 2009-06-10 | 焼津水産化学工業株式会社 | Ｎ−アセチルグルコサミンを含有する糖組成物の製造方法及び該方法を利用した飲食品の製造方法 |
| CN1308440C (zh) * | 2004-12-21 | 2007-04-04 | 中国水产科学研究院黄海水产研究所 | 一种新型海洋微生物溶菌酶及其高产溶菌酶的芽孢杆菌s－12 |
| CN102102095B (zh) * | 2010-11-30 | 2012-07-11 | 浙江大学 | 一种利用海洋链霉菌发酵制备溶菌酶的方法 |
| US11883443B2 (en) * | 2013-04-09 | 2024-01-30 | Novozymes A/S | Compositions and methods for improving the health of aquatic animals |
| CN105454695A (zh) * | 2016-01-25 | 2016-04-06 | 浙江艾杰斯生物科技有限公司 | 一种用于防治猪腹泻的饲料添加剂 |
| CN106421176A (zh) * | 2016-11-30 | 2017-02-22 | 李生涛 | 防治断奶仔猪腹泻的中药散剂 |
-
2017
- 2017-04-14 CN CN202110611966.9A patent/CN113180159B/zh active Active
- 2017-04-14 CN CN201710243612.7A patent/CN108719607B/zh active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101912048A (zh) * | 2010-08-13 | 2010-12-15 | 中牧实业股份有限公司 | 一种兽用t4溶菌酶预混剂及其制备方法 |
| CN102847017A (zh) * | 2012-08-26 | 2013-01-02 | 裔群英 | 防治仔猪腹泻的口服液及其制备方法 |
| CN103333937A (zh) * | 2013-06-06 | 2013-10-02 | 徐州工程学院 | 一种利用枯草芽孢杆菌制备抗菌肽的工艺方法 |
| CN104920897A (zh) * | 2015-06-30 | 2015-09-23 | 江西瑞普康恩农牧发展有限公司 | 一种用于仔猪提高日增重减少下痢的饲料 |
Non-Patent Citations (2)
| Title |
|---|
| 微生物溶菌酶的研究及应用;曹涛等;《中国调味品》;20110310(第03期);23-26 * |
| 链霉菌RX-17溶菌酶的产生条件及溶菌特异性研究;赵昕等;《药物生物技术》;20031230(第05期);282-285 * |
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