CN113616715B - 治疗仔猪腹泻及改善其肠道菌群的发酵中药口服液 - Google Patents
治疗仔猪腹泻及改善其肠道菌群的发酵中药口服液 Download PDFInfo
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Abstract
本发明公开了一种治疗仔猪腹泻及改善其肠道菌群的发酵中药口服液，其是将1~1.5kg中药组合物、0.8~1.2L复合益生菌菌液、0.5~1kg红糖、0.3~0.6kg杨树花混合均匀，调节含水量为55~65%，在37℃密封发酵6~8天完成中药发酵，制作中药提取液时，向发酵中药中继续添加0.8~1.2L复合益生菌菌液、0.5~0.7L去离子水，混匀后在23~26℃静置浸泡提取24~30h，静置浸泡期间不定期搅拌，再用滤网过滤除去滤渣即得发酵中药口服液；本发明发酵中药口服液充分发挥了益生菌、中药及其发酵产物的功效，能显著治疗仔猪腹泻，改善仔猪肠道菌群，减少饲用抗生素滥用导致的细菌耐药性、抗生素药物残留，保障食品生产安全；本发明发酵中药口服液制备方法简单，适于工业化生产和市场推广应用。
Description
技术领域
本发明属于复合益生菌制剂技术领域，具体涉及一种治疗仔猪腹泻及改善其肠道菌群的发酵中药口服液。
背景技术
仔猪是现代化养猪生产的核心，其健康状况对后期的生长发育产生极其重要的影响。由于仔猪自身的免疫系统发育还不完全，有害微生物的侵入容易引起仔猪腹泻。仔猪出生一个月内，因消化腺体和体温调节中枢发育功能不完善、不健全，如温度发生变化、乳脂率过高，极易引起消化不良，导致腹泻。腹泻是猪场养殖中的常见疾病，可导致仔猪成活率下降，影响肠道菌群构成，肠道菌群异常也容易引起腹泻，给养殖业带来巨大的损失。
为减少这一风险，抗生素作为生长促进因子被大量用于畜禽养殖业，为养殖业做出了巨大贡献。但是，抗生素的长期大量使用，破坏了机体的微生态平衡，导致耐药菌的产生和细菌的变异。严重影响仔猪成活率，并容易造成动物内源性感染或二次感染。抗生素在养殖业的淡出及全面禁止是大势所趋。因此，亟需研究开发一种新型制剂，有效的抗生素替代品控制仔猪腹泻。
益生菌有多种益生性能，益生菌可产生抗菌物质并竞争性与病原性微生物黏附于肠上皮细胞来阻止病原体在肠道的黏附和定植；益生菌可提高病毒感染时肠上皮细胞的存活率和保护肠道的正常生理结构并刺激保护性免疫反应来调节机体免疫；益生菌可通过调节肠道微生态平衡来保护和抵御病原微生物的侵袭；益生菌代谢产物有机酸、消化酶、抗菌物质等具有拮抗、抑制病原微生物的生长,促进或提高宿主免疫功能的作用。且复合益生菌在畜禽的肠道形态、抗病性、机体免疫方面表现出更好的有益作用。
中草药是天然植物，不仅具有无毒副作用、无耐药性、无污染及无药物残留等特点，还具有增强动物免疫功能、提高动物抗应激能力、有效调节机体代谢、改善畜禽品质等功能，深受广大养殖户的青睐。
益生菌和中草药是目前公认的绿色饲料添加剂，本发明发酵中药口服液益生菌活菌数≥108CFU/mL，包含益生菌有机酸、消化酶、抗菌物质等代谢产物、复合中草药活性成分发酵转化及溶出。经益生菌发酵后，导致某些中药成分分解转化成新的药物成分，特别是益生菌产生的多种酶能促进中草药有效成分被动物机体充分吸收和利用，而中草药中则含有丰富的营养能促进益生菌的大量增殖，两者相辅相成，更好地发挥中草药这一天然药物的药效作用。经复合益生菌发酵的中草药既具备中草药的效果，也具备微生物的益生辅助功能。从而调整仔猪肠道内菌群平衡，进而使得初生仔猪具有良好的健康状况。微生物菌液可通过改善动物肠道菌群，增强动物对有害微生物的抑制作用，提高机体免疫力等。
发明内容
本发明提供了一种治疗仔猪腹泻及改善其肠道菌群发酵中药口服液，该发酵中药口服液是将1~1.5kg中药组合物、0.8~1.2L复合益生菌菌液、0.5~1kg红糖、0.3~0.6kg杨树花混合均匀，调节含水量为55~65%，在37℃密封发酵6~8天完成中药发酵；制作中药提取液时，向发酵中药中继续添加0.8~1.2L复合益生菌菌液、0.5~0.7L去离子水，混匀后在23~26℃静置浸泡提取24~30h静置浸泡期间不定期搅拌以利于物质溶出，再用80目滤网过滤除去滤渣即得发酵中药口服液，此发酵中药口服液中有效活菌数≥108CFU/mL。
本发明复合益生菌菌液是将有效活菌数108~109CFU/mL的唾液乳杆菌(Lactobacillus salivarius)菌液、有效活菌数108~109CFU/mL乳酸乳球菌(Lactococcus lactis) 菌液、有效活菌数108~109CFU/mL植物乳杆菌（Lactobacillus plantarum）菌液按体积比（0.9~1.2）：（0.9~1.1）：（0.9~1.1）的比例混合制得混合菌液，然后按混合菌液与有效活菌数108~109CFU/mL的地衣芽孢杆菌（Bacillus licheniformis）菌液的体积比为4~6:1的比例，将混合菌液与地衣芽孢杆菌菌液混匀制得。
其中唾液乳杆菌(Lactobacillus salivarius) 保藏编号为CGMCC No.20700（202011523897.8）、乳酸乳球菌(Lactococcus lactis) 保藏编号为CGMCC No.20699（202011337808.0）、植物乳杆菌（Lactobacillus plantarum）保藏号为CGMCC No.21266（202110637585.8）、地衣芽孢杆菌（Bacillus licheniformis）保藏编号为CGMCC No.21267（202110289676.7），这些菌种均在其他专利申请文献中公开过。
本发明所述中药组合物的组成物及重量份为甘草4-6份、黄连4-6份、青蒿5-7份、枳实5-7份、山楂5-7份、桔梗5-7份、忍冬藤5-7份、柴胡5-7份、蒲公英5-7份、大青叶5-7份、鱼腥草5-7份、黄芩5-7份、栀子5-7份、夏枯草5-7份、苍术5-7份、黄柏9-11份、泽泻20-25份、茯苓30-35份、黄芪30-35份。
本发明方法具有如下特点：
（1）本发明发酵中药口服液充分发挥了益生菌、中药及其发酵产物的功效，能显著治疗仔猪腹泻，改善仔猪肠道菌群；在仔猪日龄第6-20天，将发酵中药口服液灌服至仔猪胃中，灌服剂量每次为15mL，日服一次，治疗三天即可；
（2）本发明发酵中药口服液益生菌活菌数≥108CFU/mL，包含益生菌有机酸、消化酶、抗菌物质等代谢产物、复合中草药活性成分发酵转化及溶出；经益生菌发酵后，导致某些中药成分分解转化成新的药物成分，特别是益生菌产生的多种酶能促进中草药有效成分被动物机体充分吸收和利用，而中草药中则含有丰富的营养能促进益生菌的大量增殖，两者相辅相成，更好地发挥中草药这一天然药物的药效作用；经复合益生菌发酵的中草药既具备中草药的效果，也具备微生物的益生辅助功能；
（3）本发明所述中药组合物含较丰富的营养物、益生元和抑制致病菌的中药有效成分，能通过改善肠道菌群，提高动物的生长性能，增强机体对病原菌的抵抗力，刺激机体免疫应答的产生，并维持机体较高水平的免疫应答，在致病性细菌感染的情况下能够显著改善机体肠道微生态环境，提高机体对病原菌的抵抗力；
（4）本发明发酵中药口服液能够抑制有害病原菌的繁殖，减少抗生素作为预防和治疗动物疾病的制剂在饲料中长期亚治疗剂量和超高剂量添加，避免饲用抗生素滥用导致的细菌耐药性、抗生素药物残留和食品安全等方面的危害。本发明发酵中药口服液能够显著提高肠粘膜免疫水平，减少或避免肠道疾病的发生，维持肠道的微生态平衡。
附图说明
图1为实施例3肠道菌群门水平组成；
图2为实施例3肠道菌群属水平组成。
具体实施方式
下面通过实施例对本发明的技术方案作进一步详细说明，但本发明的内容并不局限于此，本实施例中方法如无特殊说明均为常规方法，所用材料、试剂等如无特殊说明均从商业途径获得或按常规方法配制。
实施例1：本治疗仔猪腹泻及改善其肠道菌群发酵中药口服液的制备
（1）将按照体积比50%甘油:菌液=7:3比例保存的菌种在37℃水浴1min进行复苏，复苏后的唾液乳杆菌、乳酸乳球菌、植物乳杆菌用接种环在MRS培养基上划线，地衣芽孢杆菌菌种用接种环在LB培养基上划线，然后置于37℃恒温培养箱中倒置培养12h；分别挑取单菌落接种至无菌液体培养基中，将其置于摇床培养至OD600值0.6~0.8，将菌液以1%的接种量传代培养，然后再将传代菌液接种到所需用量的无菌液体培养基中37℃培养，获得有效活菌数108~109CFU/mL的唾液乳杆菌菌液、有效活菌数108~109CFU/mL乳酸乳球菌菌液、有效活菌数108~109CFU/mL植物乳杆菌、有效活菌数108~109CFU/mL的地衣芽孢杆菌菌液；
（2）按体积比1:1:1的比例将唾液乳杆菌、乳酸乳球菌、植物乳杆菌混合制得混合菌液，然后体积比为4:1的比例，将混合菌液与地衣芽孢杆菌菌液混匀制得复合益生菌菌液；
（3）中药组合物的组成物及重量份为甘草4份、黄连6份、青蒿5份、枳实5份、山楂6份、桔梗6份、忍冬藤5份、柴胡5份、蒲公英6份、大青叶6份、鱼腥草5份、黄芩5份、栀子6份、夏枯草7份、苍术7份、黄柏9份、泽泻20份、茯苓35份、黄芪35份；
（4）将1kg中药组合物、0.8L复合益生菌菌液、0.5kg红糖、0.6kg杨树花混合均匀，调节含水量为60%，在37℃密封发酵6~8天完成中药发酵，制作中药提取液时，向发酵中药中继续添加0.8L复合益生菌菌液、0.5L去离子水，混匀后在23℃静置浸泡提取30h（期间不定期搅拌以利于物质溶出），再以80目滤网过滤除去滤渣即得发酵中药口服液。
实施例2：本治疗仔猪腹泻及改善其肠道菌群发酵中药口服液的制备
（1）将按照体积比50%甘油:菌液=7:3比例保存的菌种在37℃水浴1min进行复苏，复苏后的用接种环将唾液乳杆菌、乳酸乳球菌、植物乳杆菌菌种在MRS培养基上划线，用接种环将地衣芽孢杆菌菌种在LB培养基上划线，然后置于37℃恒温培养箱中倒置培养12h；分别挑取单菌落至无菌液体培养基摇床培养至OD600值0.6~0.8，将菌液以1%的接种量传代培养，然后再将传代菌液接种到无菌液体培养基中37℃培养，获得有效活菌数108~109CFU/mL的唾液乳杆菌菌液、有效活菌数108~109CFU/mL乳酸乳球菌菌液、有效活菌数108~109CFU/mL植物乳杆菌、有效活菌数108~109CFU/mL的地衣芽孢杆菌菌液；
（2）按体积比0.9:1:1.1的比例将唾液乳杆菌、乳酸乳球菌、植物乳杆菌混合制得混合菌液，然后体积比为5:1的比例，将混合菌液与地衣芽孢杆菌菌液混匀制得复合益生菌菌液；
（3）中药组合物的组成物及重量份为甘草6份、黄连4份、青蒿6份、枳实6份、山楂7份、桔梗7份、忍冬藤7份、柴胡7份、蒲公英7份、大青叶7份、鱼腥草6份、黄芩7份、栀子6份、夏枯草6份、苍术6份、黄柏11份、泽泻25份、茯苓30份、黄芪30份；
（4）将1.5kg中药组合物、1.0L复合益生菌菌液、0.8kg红糖、0.3kg杨树花混合均匀，调节含水量为65%， 在37℃密封发酵7天后，向发酵物料中继续添加1.0L复合益生菌菌液、0.7L去离子水，混匀后在25℃下密封静置提取24h（期间不定期搅拌以利于物质溶出），过滤除去滤渣即得发酵中药口服液。
实施例3：复合益生菌菌液中的4个菌种对模拟胃肠液的耐受性
人工胃液配制：取1mol/L稀盐酸1.64mL于容器中，加水80mL，加入1g胃蛋白酶，调节pH3.0，混匀定容至100mL，用0.22μm滤膜除菌；相同方法配制pH4.0人工胃液，置于4℃冰箱备用；
人工肠液配制：取磷酸二氢钾0.68g，加水80mL溶解，加入1g胰蛋白酶，用0.lmol/L氢氧化钠溶液调节pH6.8；加水适量稀释至100mL，过0.22μm 滤膜除菌，制成模拟肠液，置于4℃冰箱备用；
将按照体积比50%甘油:菌液=7:3比例保存的菌种在37℃水浴一分钟进行复苏，复苏后的唾液乳杆菌、乳酸乳球菌、植物乳杆菌用接种环在MRS培养基上划线，地衣芽孢杆菌菌种用接种环在LB培养基上划线，然后置于37℃恒温培养箱中倒置培养12h；分别挑取单菌落至无菌液体培养基摇床培养至OD600值0.6~0.8，将菌液以1%的接种量传代培养，唾液乳杆菌、乳酸乳球菌、植物乳杆菌接种至同一个MRS液体培养基混合培养，地衣芽孢杆接种至LB液体培养基培养基。37℃、150rpm摇床培养至OD600值0.6~0.8；各取10mL活化后的混合菌、地衣芽孢杆菌菌悬液，13000r/min离心10min后收集菌体，分别加入10mL模拟胃肠液，涡旋混匀后37℃静置培养4h，混合菌于0h、4h用MRS琼脂培养基采用平板计数法测定人工胃液及人工肠液处理前后活菌数量，地衣芽孢杆菌于0h、4h用LB琼脂培养基采用平板计数法测定人工胃液及人工肠液处理前后活菌数量，实验结果见表1；
存活率＝[N1/N0]×100％(N0：0h活菌数；N1：经模拟胃肠液消化后的活菌数)
表1 混合乳酸菌和芽孢人工模拟胃肠液的存活率
发酵中药口服液抑菌实验：
用双层平板牛津杯扩散法测定发酵中药口服液的抑菌效果，将灭菌的LB琼脂固体培养基倒于培养皿中冷却后，将灭菌冷却至50℃左右的LB琼脂半固体培养基4mL分别与100μL大肠杆菌、志贺氏菌、金黄色葡萄球菌、沙门氏菌均匀混合并倒至LB固体培养基上，使混合液在LB固体培养基上均匀分布。待混合液凝固后用无菌镊子将已灭菌牛津杯置于混合液上，并向牛津杯中加入用0.22μm滤膜过滤后的发酵中药口服液200μL；于4℃冰箱中扩散4h后37℃培养恒温12h，观测抑菌圈的大小；抑菌圈直径大小使用游标卡尺进行测量(保留两位有效数字)，独立重复三次实验，牛津杯直径为8mm，实验结果见表2；
表2 治疗仔猪腹泻及改善其肠道菌群发酵中药口服液的抑菌特性
由表1、表2实验结果可知，发酵中药口服液具有较好的胃肠液耐受性，同时具有广谱抑制致病菌的优良特性。
实施例3：发酵中药口服液对腹泻仔猪的治疗过程粪便形态观察及菌群多样性分析
腹泻仔猪粪便形态：水样，无纹理，扁平摊开；
健康仔猪粪便形态：较干燥，明显成团，粘稠不易分开；
根据腹泻仔猪、健康仔猪粪便形态，确定腹泻仔猪为实验对象并取其粪便样品，命名为disease组，根据仔猪粪便形态确定健康仔猪并取其粪便样品，命名为health组，对腹泻仔猪饲喂发酵中药口服液，饲喂方式为灌服15mL/d，饲喂两天发现粪便形态：非常潮湿，有明显的纹理，捡拾后地面有残留且粪便不能维持形状；能明显区别于腹泻仔猪粪便形态，继续取粪便样品，命名为treatment组，相同饲喂方式继续饲喂第三天，发现仔猪粪便形态与健康仔猪粪便形态相似，取粪便样品，命名为recure组，每组三个平行编号送至上海美吉生物医药科技有限公司进行16Sr RNA微生物多样性测序。
采用Illumina PE300测序平台对样品进行测序，采用美吉生物云平台对肠道菌群可操作分类单元( operational taxonomic units，OTUs)数量，alpha与beta多样性以及差异菌门与菌属进行综合分析。
对4组仔猪肠道菌群进行alpha与beta多样性分析，各组覆盖率达到0.99以上，说明数据覆盖度高，有很好的可靠性，alpha多样性指数shannon指数、chao指数采用平均值±标准差的方式表示。
Shannon指数Student T检验分析显示treatment组仔猪肠道菌群多样性显著高于disease组（P <0.05），recure组仔猪肠道菌群多样性极显著低于treatment组（P <0.001）；
chao指数Student T检验分析显示disease组仔猪肠道菌群丰富度极显著低于health组（P <0.01），recure组仔肠道菌群丰富度显著低于health组（P <0.05），treatment组仔猪肠道菌群丰富度极显著高于disease组（P <0.01），treatment组仔猪肠道菌群丰富度极显著高于recure组（P <0.001），分析结果见表3，其中每一个指数值后字母相同表示没有显著差异，字母不同则差异性显著。
表3 仔猪肠道菌群的alpha与beta多样性分析
Shannon指数为菌群多样性的统计，指数值越大表明多样性越高；Chao指数为菌群丰富度的统计，指数值越大表示物种越多，Coverage用于计算覆盖率，值越大表示覆盖越好。
菌群结构分析：本研究分为四个组，每组三个平行取平均值，根据测序结果，门分类物种排名前7的相对丰度分布见图1，云南黑猪肠道菌群主要由厚壁菌门Firmicutes、拟杆菌门Bacteroidota、放线菌门Actinobacteriota、变形菌门Proteobacteria、梭杆菌门Fusobacteriota、脱硫杆菌Desulfobacterota、Synergistota组成；各组在7个门水平的相对丰度见表4，用平均值加减标准差表示；单因素方差分析（One-way ANOVA）显示在放线菌门Actinobacteriota、脱硫杆菌Desulfobacterota、Synergistota组间两两比较存在显著差异（P <0.05）。
表4 各组仔猪肠道菌群在门水平的相对丰度值
注：同一门水平相对丰度值后有相同字母表示组间没有显著差异，不同字母代表组间有显著差异
在属水平中，云南黑猪仔猪肠道的优势菌属为乳杆菌属Lactobacillus、拟杆菌属Bacteroides、norank_f__Muribaculaceae、norank_f__Eubacterium_coprostanoligenes_ group、Olsenella、unclassified_f__Lachnospiraceae、双歧杆菌属Bifidobacterium、Christensenellaceae_R-7_group、Escherichia-Shigella、肠球菌属Enterococcus。不同组优势属水平的相对丰度分布见图2；相对丰度值见表5，单因素方差分析（One-way ANOVA）显示在乳杆菌属Lactobacillus、norank_f__Muribaculaceae、Olsenella、unclassified_ f__Lachnospiraceae、Bifidobacterium不同组的细菌相对丰度差异性显著（P <0.05）。
表5 各组仔猪肠道菌群在属水平的相对丰度值
注：同一属水平相对丰度值后有相同字母表示组间没有显著差异，不同字母代表组间有显著差异
在优势门、属水平中，相对丰度最高的是厚壁菌门Firmicutes、乳杆菌属Lactobacillus且在厚壁菌门Firmicutes、乳杆菌属Lactobacillus水平上recure组细菌相对丰度高于treatment组高于disease组，recure组与health组之间比较无显著差异。
仔猪腹泻为猪场养殖的常见疾病，肠道菌群平衡对于机体维持健康状态具有重要的意义。肠道内细菌能够参与营养物质的代谢，厚壁菌门Firmicute细菌与能量吸收有关，实验结果显示：disease组厚壁菌门细菌相对丰度为58.73%，treatment组为78.24%，recure组为87.44%，health组为89.93%；说明本发明制得的发酵中药口服液饲喂云南黑猪能提高厚壁菌门Firmicute细菌的相对丰度，且经过治疗，recure组和health组厚壁菌门Firmicute细菌的相对丰度无显著差异。发酵中药口服液饲喂后recure组放线菌门Actinobacteriota、脱硫杆菌Desulfobacterota相对丰度显著高于health组（P <0.001）、recure组Synergistota细菌相对丰度显著高于health组（P <0.05）。
recure组乳杆菌属Lactobacillus细菌相对丰度显著高于disease组（P <0.05），乳杆菌属细菌在不同组的相对丰度分布很好的解释了不同组间云南黑猪肠道菌群的多样性及丰富度差异。云南黑猪肠道菌群多样性及丰富度treatment组高于disease组是因为treatment组饲喂了两天发酵中药口服液，云南黑猪肠道菌群多样性recure组低于treatment组是因为在治疗过程中，发酵中药口服液能抑制多种致病性细菌生长。云南黑猪肠道菌群丰富度disease组低于health组是因为health组黑猪肠道微生态更加丰富；云南黑猪肠道菌群丰富度recure组低于health组可能是因为发酵中药口服液在治疗过程中改变了黑猪肠道菌群结构，虽然recure组的丰富度更低，但是益生性更强；
recure组双歧杆菌属Bifidobacterium细菌相对丰度显著高于health组（P <0.001）。Escherichia-Shigella是引起细菌性腹泻的常见细菌，经过发酵中药口服液饲喂其相对丰度由disease组的5.59%降至recure组的0.26%，低于health组的1.16%。
综上，本发明治疗仔猪腹泻及改善其肠道菌群的发酵中药口服液能显著提高仔猪肠道益生性细菌的相对丰度，同时提高与碳水化合物的消化吸收能力、肠道抗炎和黏膜免疫能力相关的肠道微生物的相对丰度，降低引起腹泻的条件致病菌相对丰度，起到很好的止泻效果且治疗周期短。
Claims (1)
1.一种治疗仔猪腹泻及改善其肠道菌群的发酵中药口服液，其特征在于：将1~1.5kg中药组合物、0.8~1.2L复合益生菌菌液、0.5~1kg红糖、0.3~0.6kg杨树花混合均匀，调节含水量为55~65%，在37℃密封发酵6~8天完成中药发酵，制作中药提取液时，向发酵中药中继续添加0.8~1.2L复合益生菌菌液、0.5~0.7L去离子水，混匀后在23~26℃静置浸泡提取24~30h，静置浸泡期间不定期搅拌，再用滤网过滤除去滤渣即得发酵中药口服液；
所述复合益生菌菌液是将有效活菌数108~109CFU/mL的唾液乳杆菌(Lactobacillus salivarius)菌液、有效活菌数108~109CFU/mL乳酸乳球菌(Lactococcus lactis) 菌液、有效活菌数108~109CFU/mL植物乳杆菌（Lactobacillus plantarum）菌液按体积比0.9~1.2:0.9~1.1:0.9~1.1的比例混合制得混合菌液，然后按混合菌液与有效活菌数108~109CFU/mL的地衣芽孢杆菌（Bacillus licheniformis）菌液的体积比为4~6:1的比例，将混合菌液与地衣芽孢杆菌菌液混匀制得；其中唾液乳杆菌(Lactobacillus salivarius) 保藏编号为CGMCC No.20700、乳酸乳球菌(Lactococcus lactis) 保藏编号为CGMCC No.20699、植物乳杆菌（Lactobacillus plantarum）保藏号为CGMCC No.21266、地衣芽孢杆菌（Bacillus licheniformis）保藏编号为CGMCC No.21267；
所述中药组合物的组成物及重量份为甘草4-6份、黄连4-6份、青蒿5-7份、枳实5-7份、山楂5-7份、桔梗5-7份、忍冬藤5-7份、柴胡5-7份、蒲公英5-7份、大青叶5-7份、鱼腥草5-7份、黄芩5-7份、栀子5-7份、夏枯草5-7份、苍术5-7份、黄柏9-11份、泽泻20-25份、茯苓30-35份、黄芪30-35份。
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