CN105795129A - 一种防治猪流行性腹泻的饲料添加剂及其制备、使用方法 - Google Patents
一种防治猪流行性腹泻的饲料添加剂及其制备、使用方法 Download PDFInfo
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Abstract
本发明的目的是提供一种防治猪流行性腹泻的饲料添加剂及其制备、使用方法，该防治猪流行性腹泻的饲料添加剂以茶茎和/或茶梗作为原料，粉碎成粉末或制备提取物而制成。本发明利用冬季修剪茶树获得的茶茎、茶梗等为质量稳定、易获得的原料来制备防治猪流行性腹泻的饲料添加剂，利用了茶茎、茶梗富含茶多酚等防霉和抗氧化活性成分，且咖啡因等生物碱含量明显减少而不具备中枢兴奋作用等特点，避免了抗生素药物的使用，具有成本低、无药物残留、无耐药性、无毒副作用的优点，制备时工艺简单、耗能低、无污染，非常容易实现产业化，为茶农的茶园废料提供了新的用途，具有很好的经济和社会效益。
Description
技术领域
本发明属于畜禽类饲料添加剂领域，具体涉及到一种防治猪流行性腹泻的饲料添加剂及其制备、使用方法。
背景技术
近年来，随着人民生活水平的提高，猪肉消费量也逐年增加，进而带动了国内养殖业的快速发展。然而猪病频繁爆发严重危害养猪业，其中猪腹泻占猪病总量的35～45％，是一年四季均可能发生的高发病率疾病，同时也可引起其它病毒或细菌并发或继发感染。至今为止，人们发现在引起猪腹泻病的各种病因中，以细菌和病毒引起的传染性疾病危害是最为严重，常见的有猪流行性腹泻病毒、轮状病毒病、致病性大肠杆菌等、也存在混合感，且呈上升趋势，尤其危害于大型集约化的养猪场。病猪主要表现为腹泻，同时生长缓慢，饲料报酬降低，生产性能下降，甚至引起猪死亡。
目前对于猪腹泻的防治主要采用庆大霉素、氟哌酸、恩诺沙星、阿莫西林、硫酸粘菌素、环丙沙星和氟苯尼考等抗生素药物。由于长期使用或滥用抗生素，其负面效应也日益显露，其结果不仅引起猪免疫功能低下，破坏胃肠道微生物生态平衡，干扰其正常的消化吸收机能，也显著影响了饲料的生物利用，导致细菌产生耐药性和抗生素残留等问题。而且，人们长期食用有抗生素残留的猪肉制品，容易引起过敏反应、毒性反应等，危害人们的健康。此外，金刚烷胺、γ干扰素及达菲等抗病毒药物，因成本高等原因难以被养殖户接受。尽管疫苗具有一定的作用，但预防单一，且效果并不稳定，难以对应病毒的快速变异。因此，一种能够高效、无残留、无耐药、抗菌、抗病毒及抗感染作用的预防和治疗猪腹泻的药物及组成物无疑是养殖业的迫切需求，且具有着巨大的市场空间。
发明内容
本发明的目的是提出一种具有抗菌、抗病毒和抗感染作用的防治猪流行性腹泻的饲料添加剂及其制备、使用方法，以促进集约化猪养殖业的发展。
本发明的防治猪流行性腹泻的饲料添加剂以茶茎和/或茶梗作为原料，粉碎成粉末或制备提取物而制成。
在本发明中，上述粉末的粒径小于0.85mm，以便于牲畜消化吸收，并减少对牲畜消化系统的损害。
进一步地，为降低成本，所述茶茎、茶梗是在冬季修剪茶树时获得的。目前冬季修剪茶树时，所修剪下来的茶茎、茶梗均当做废物处理，将其用于防治猪流行性腹泻的饲料添加剂，属于废物利用，成本接近于零，大大提高了市场竞争力。
本发明的防治猪流行性腹泻的饲料添加剂的机理如下：
近年来，随着机械化采茶和制茶的广泛普及，使得茶叶的采集和生产过程中产生大量茶茎、茶梗等茶叶废弃物。至今为止的研究证明，茶茎、茶梗等富含茶多酚类化合物，其中表儿茶素(EC)、表儿茶素没食子酸酯(ECG)、表没食子儿茶素(EGC)和表没食子儿茶素没食子酸酯(EGCG)占儿茶素总量的70％左右，具有显著的抗菌、抗病毒、抗炎、抗氧化及抗突变等多种生物活性。此外，茶叶中的咖啡因等嘌呤类生物碱虽然具有促进消化等作用，但由于该类化合物具有较强的中枢神经兴奋作用而不适合用于饲料添加剂。与茶叶相比茶梗和茶茎中的咖啡因等生物碱含有少，而富含茶多酚和纤维，因此有益于制备用于防治猪腹泻的饲料添加剂。
下面为茶茎、茶梗及茶树叶提取物的制备与成分分析：
茶茎、茶梗及芽茶由贵州宏鑫茶业开发有限公司提供。实验准确称取干燥茶茎、茶梗及芽茶各500g，经粉碎机粉碎后回收细粉，加入10倍量的水溶液，加热提取30分钟，提取液经纱布过滤后，滤饼再用相同条件提取一次，纱布过滤，合并两次滤液。滤液浓缩后，经冷冻干燥得提取物用于本研究。鉴于茶茎、茶梗及茶提取物是以饲料添加剂作为最终产品开发，故从安全性角度考虑提取溶剂尽量选择水、乙醇或含水乙醇为宜。提取时茶茎、茶梗及茶与溶剂的比率没有特定限制，但一般茶茎、茶梗及茶与溶剂的比例5:1～50范围为宜。本发明对提取温度没有特定限制，从室温到溶剂沸点范围都可适用。当然室温、常压及在溶剂沸点范围内提取最为理想。提取方法可以采用加热提取、超声提取、微波提取等，也可多种方法结合起来应用。提取时间从30分钟到24小时范围均可以。
本实验采用的分析方法为高效液相色谱法。DIONEX液相分析仪为Ultimate3000型，电子天平为SartoriusBP211D十万分之一型，恒温干燥箱为德国Memmert型，超纯水仪为Merckmillipore公司，小型粉碎机为杭州熊火科技有限公司，乙腈为Fisher(色谱纯)，甲酸为广州化学试剂厂(分析纯)。对照品咖啡因(Caffeine，Caff)，可可碱(Theobromine，Tb)，没食子儿茶素没食子酸酯((-)-Gallocatechingallate，GCG)，表没食子儿茶素没食子酸酯((-)-Epigallocatechingallate，EGCG)购自日本和光纯药株式会社；没食子酸(Gallicacid，GA)，儿茶素((+)-Catechin，C)购自美国Sigma公司；没食子儿茶素((-)-Gallocatechin，GC)，表没食子儿茶素(Epigallocatechin，EGC)，表儿茶素(Epicatechin，EC)购自日本栗田工业株式会社；表儿茶素没食子酸酯((-)-Epicatechin-3-gallate，ECG)购自上海晶纯生化科技股份有限公司。
色谱条件，色谱柱为COSMOSIL5C18-AR-II(4.6*250mm，5μm，Nacalaitesque.Inc.，Japan)，流速为1ml/min，定量检测波长为231nm，柱温为35℃，进样量为20μl。流动相A：H2O(含0.1％甲酸)；流动相B：CH3CN(含0.1％甲酸)。供试样品经蒸馏水溶解后离心，经0.45μm水膜过滤后进HPLC分析，样品中各成分含量见表1。
表1.茶茎、茶梗及芽茶提取物中生物碱及多酚类化合物含量(％)
| 提取物成分 | 芽茶 | 茶茎 | 茶梗 |
| GA | 0.14 | 0.15 | 0.19 |
| Tb | 0.28 | 0.08 | 0.04 |
| GC | 0.74 | 0.16 | 0.04 |
| EGC | 2.56 | 0.51 | □ |
| C | 0.26 | 0.23 | 0.14 |
| Caff | 3.27 | 0.93 | 0.28 |
| EC | 0.87 | 0.56 | 0.14 |
| EGCG | 4.53 | 1.22 | □ |
| GCG | 1.52 | 0.06 | □ |
| ECG | 1.78 | 1.02 | 0.04 |
□:未检测出。
表1为茶茎、茶梗及茶叶提取物中的儿茶素和咖啡因的含量，茶茎、茶梗提取物中的咖啡因含量显著低于茶叶提取物，能够明显减少对畜禽的中枢兴奋作用，同时其富含的儿茶素等多酚类抗菌、抗病毒活性成分，有利于饲料在饲料添加剂上的应用。
下面继续测试及分析茶茎、茶梗提取物的抗病原菌作用：
活性检测采用纸片-琼脂扩散法敏感试验(Kirby-Bauer法)，其原理是根据抑菌环直径大小与最低抑菌浓度的相关性进行活性评价。实验使用Mueller-Hinton琼脂，制备厚为4mm及pH7.2～7.4的琼脂平板。琼脂凝固后，放置于4℃环境中保存，并提供实验使用。实验前，复活冻干菌株以1.5×l08/m1接种琼脂管，实验时制备细菌悬液，悬浮于生理盐水中，振荡混匀后校正悬液浓度为107CFU/ml。接种平板时，用灭菌的棉拭子蘸取菌液均匀涂布整个培养基表面。实验样品携载纸片直径为6mm，每片吸水量约20μl，用无菌镊子将药敏纸片贴在涂好菌液的平板上，纸片间中心间隔30mm，平皿置于37℃恒温箱内培养24小时后，检测抑菌圈直径，每种样品设6个样本取平均值。
表2.茶茎、茶梗提取物的最小抑菌浓度(MICmg/ml)
从表2中可以看出，茶茎、茶梗的提取物对金黄色葡萄球菌、白色念珠菌、绿脓杆菌、大肠杆菌、痢疾杆菌、肠杆菌等均显示较强的抑菌活性，因此可用由于上述病菌造成的猪腹泻的防治。
下面测试及分析茶茎、茶梗提取物的体外抗病毒作用：
实验使用DMEM(Dulbecco′smodifiedeaglemedium)细胞培养液含4500mg/LD-葡萄糖、300mg/LL-谷氨酰胺，110mg/L丙酮酸钠，5％胎牛血清，1％青霉素，1％链霉素)。将实验Vero细胞用0.25％的胰酶消化后，吹打使细胞分散，用DMEM培养液稀释成1×106细胞/ml，加至96孔细胞培养板中，置5％CO2，37℃培养箱中培养，待细胞长成单层细胞后，进行体外抗病毒试验。细胞染色采用四甲基偶氮唑盐比色法(MTT法)，酶标仪测定490nm处的吸光度值(OD)，计算抑制率。
(一)茶茎及茶梗提取物的Vero细胞毒性作用：将茶茎及茶梗提取物用PBS溶解后以0.22μm滤膜除菌，用细胞培养液连续梯度稀释后，分别添加到Vero细胞培养板中，每一浓度提取物设4个培养孔，置5％CO2，37℃条件下培养72小时，倒置显微镜下观察细胞形态，用MTT法检测细胞毒性，并确定药效作用浓度。细胞毒性测定结果显示，茶茎及茶梗提取物在50μg/ml以上的作用浓度对细胞形态有不同程度的影响，而在50μg/ml以下无明显细胞毒性。
(二)茶茎及茶梗提取物对PEDV致细胞病变的抑制作用：实验将猪流行性腹泻病毒(porcineepidemicdiarrheavirus，PEDV)接种于Vero细胞单层，置5％CO2，37℃培养72小时，收获感染细胞后超声粉碎，病毒液分装后-40℃保存，PEDV株滴度为5x107PFU/ml。10倍梯度稀释后接种Vero细胞，用于体外抗PEDV病毒活性评价。对照药病毒唑注射液100g/L(江苏兴化制药厂)。
(1)茶茎及茶梗提取物对病毒的直接作用：将不同浓度的提取物与PEDV病毒混合，室温作用4小时后，分别添加到Vero细胞培养板中，每一浓度提取物设4个培养孔，置5％CO2，37℃条件下培养30分钟后，除去培养液，用PBS缓冲液洗涤三次，并添加细胞培养液在5％CO2，37℃条件下培养72小时，用MTT法检测提取物的抗病毒活性。
(2)茶茎及茶梗提取物对病毒增殖的抑制活性：实验将PEDV病毒接种至Vero细胞培养板中，每一浓度提取物设4个培养孔，5％CO2，37℃条件下作用30分钟后，除去培养液，用PBS缓冲液洗涤三次，并添加细胞培养液和不同浓度的提取物及药物在5％CO2，37℃条件下培养72小时，用MTT法检测提取物的抗病毒活性。
(3)茶茎及茶梗提取物对病毒增殖的预防活性：实验将不同浓度的提取物添加到Vero细胞培养板中，5％CO2，37℃条件下作用8小时后，除去培养液，用PBS缓冲液洗涤三次，每一浓度提取物设4个培养孔，接种PEDV病毒，在37℃条件下作用30分钟后，除去培养液，用PBS缓冲液洗涤三次，并添加细胞培养液在5％CO2，37℃条件下培养72小时，用MTT法检测提取物对病毒增殖的预防活性。
实验结果如表3所示，茶茎及茶梗提取物在体外三种作用方式中均显示的较好的
抗病毒活性。
表3.茶茎及茶梗提取物对PEDV致细胞病变的抑制作用(OD值)，对照病毒唑：
下面测试及分析茶茎/茶梗饲料添加剂对猪腹泻的防治作用。
猪饲料实验选用玉米/豆粕/膨化大豆型猪饲料，玉米、豆粕均为1.8mm筛片粉碎。其组成成分为：玉米51.0％，膨化大豆16.2％，豆粕13.0％，乳清粉6.4％，鱼粉6.2％，小麦麸3.0％，石粉1.5％，磷酸二氢钙1.0％，食盐0.35％，赖氨酸盐酸盐0.22％，DL-蛋白酸0.13％，维生素微量元素混合物1.0％。其中，每1千克饲料中维生素微量元素混合物含Fe150.0mg，Zn180.0mg，Cu150.0mg，Mn50.0mg，I0.3mg，Se0.3mg，VA6500IU，VD20IU，VB12.8mg，VB26.2mg，VB63.8mg，VB1235mg，VB61.1mg，VK33.5mg，叶酸0.83mg，烟酸33.0mg，泛酸18.0mg，
(一)茶茎/茶梗饲料添加剂对仔猪生产性能的影响：饲料添加剂在仔猪中的使用效果选用信高养殖场健康断奶仔猪180头，经统计分析体重无显著差异，按雄雌随机分组。实验在漏缝地板仔猪保育舍进行，温度控制为25～28℃，相对湿度控制在50-70℃。仔猪在每天8:0，12:00，16:00及20:00定时饲喂。饲料采用粉料拌湿的喂料方式。且每次饲料量为料槽略有余料为宜。实验期间自由摄水。每天清扫舍室一次。每隔4天喷雾消毒一次。其他按猪场饲养管理进行。记录实验开始与结束期间，投料量与剩余量，计算体重和采食量。如表4所示，实验结果表明，茶茎/茶梗饲料添加剂能够提高仔猪日增重，降低饲料比分，促进养殖质量和效益。
表4.茶茎/茶梗饲料添加剂对仔猪生产性能的影响
| 指标 | 对照 | 茶茎/茶梗提取物 | 茶茎/茶梗微分 |
| 始体重 | 6.82±0.03 | 6.84±0.02 | 6.79±0.01 |
| 末体重 | 15.23±0.08 | 16.91±0.30 | 17.11±0.06 |
| 1-5周平均日增重 | 0.240 | 0.288 | 0.295 |
| 1-5周平均日采食量 | 0.42±0.04 | 0.46±0.07 | 0.46±0.06 |
| 1-5周料/重比 | 1.75 | 1.59 | 1.56 |
(二)茶茎/茶梗饲料添加剂对猪腹泻的防治作用：自然感染仔猪176头，食欲低下，有黄白色稀粪。通过每天早7:30到晚21:30连续观察仔猪的排粪情况。粪便评分标准为：(1)0分：条形或粒形，(2)1分：软粪/成形，(3)2分：稠状/成形/粪水无分离现象，(4)3分：液状/不成形/粪水有分离现象。将2分与3分确认为腹泻。统计仔猪腹泻数目，计算腹泻率。通过实验室诊断，确诊为大肠杆菌引起的仔猪黄痢和仔猪白痢。实验将病猪随机分为3组，观察各组的采食，饮水，体重，粪便，及死亡等现象。疗效判断标准为，(1)治愈：病猪腹泻消失，食欲正常，停药二周后无复发。(2)有效：腹泻减轻或消失，粪便性状好转，水分明显减少，病程缩短。(3)无效：腹泻次数与性状无改变，或出现死亡现象。如表5所示，实验结果表明，茶茎/茶梗饲料添加剂在防治仔猪黄白痢疾病上具有显著的治疗效果。
表5.茶茎/茶梗饲料添加剂对猪腹泻的治疗效果
| 组别 | 头数 | 治愈 | 有效 | 死亡 | 总有效率(％) |
| 茶茎/茶梗提取物 | 58 | 52 | 57 | 0 | 98.2 |
| 茶茎/茶梗微粉 | 58 | 49 | 55 | 0 | 94.8 |
| 对照组 | 60 | 31 | 46 | 5 | 76.6 |
上述实验证明，茶茎、茶梗微粉或提取物添加入饲料后，对猪腹泻具有显著的治疗效果。
本发明中提到的茶茎、茶梗提取物的制备方法有以下两种：
1、将原料粉碎成粉末并投入到溶剂中形成溶液，将溶液通过加热提取、微波提取或者超声提取得到提取物；所述粉末与溶剂的重量比为5:1～50；所述溶剂为水或者乙醇或者含水乙醇。
2、将原料粉碎成粉末并投入到水中形成一次溶液，将一次溶液加热预定时间后，将一次溶液进行过滤得到一次滤渣和一次滤液；将一次滤渣再次投入水中形成二次溶液，将二次溶液加热预定时间后，将二次溶液进行过滤得到二次滤渣和二次滤液，将一次滤液、二次滤液混合后，进行浓缩、冷冻、干燥得到提取物。
上述两种提取物的制备方法均可将茶茎、茶梗中的有效成分析出。
上述防治猪流行性腹泻的饲料添加剂的使用方法如下：当防治猪流行性腹泻的饲料添加剂为粉末状时，将防治猪流行性腹泻的饲料添加剂混合入饲料中并搅拌均匀，所述防治猪流行性腹泻的饲料添加剂与饲料的重量比为1～5：1000；当防治猪流行性腹泻的饲料添加剂为液态提取物时，先将防治猪流行性腹泻的饲料添加剂稀释到体积倍数的40倍后，再洒入饲料中并搅拌均匀，所述防治猪流行性腹泻的饲料添加剂与饲料的重量比为0.5～2：1000。
本发明利用冬季修剪茶树获得的茶茎、茶梗等为质量稳定、易获得的原料来制备防治猪腹泻饲料添加剂，利用了茶茎、茶梗富含茶多酚等抗菌抗病毒活性成分，且咖啡因等生物碱含量明显减少的特点，避免了抗生素药物的使用，具有成本低、无药物残留、无耐药性、无毒副作用的优点，制备时工艺简单、耗能低、无污染，非常容易实现产业化，为茶农的茶园废料提供了新的用途，具有很好的经济和社会效益。
具体实施方式
下面通过对实施实例的描述，对本发明的具体实施方式如所涉及的各构件的形状、构造、各部分之间的相互位置及连接关系、各部分的作用及工作原理等作进一步的详细说明。
实施例1：
本实施例的防治猪腹泻饲料添加剂以在冬季修剪茶树时获得的茶茎和茶梗作为原料，经过粉碎成粉末而制成，粉末的粒径小于0.85mm(20目)，以便于牲畜消化吸收，并减少对牲畜消化系统的损害。
在使用时，将粉末状的防治猪流行性腹泻的饲料添加剂混合入饲料中并搅拌均匀，所述防治猪腹泻饲料添加剂与饲料的重量比为1～5：1000W/W。
实施例2：
本实施例的防治猪流行性腹泻的饲料添加剂以在冬季修剪茶树时获得的茶茎和茶梗作为原料，制备提取物而制成，具体的制备方法有以下两种：
1、将原料粉碎成粉末并投入到溶剂中形成溶液，将溶液通过加热提取、微波提取或者超声提取得到提取物；所述粉末与溶剂的重量比为5:1～50；所述溶剂为水或者乙醇或者含水乙醇。本专利实施具体例为，将原料粉碎成粗粉，取10kg茶茎和茶梗的粗粉，置提取罐中，加10倍量水回流提取，保持沸腾2小时，过滤，滤渣再加入10倍量水回流提取，保持沸腾1小时，过滤，合并两次滤液。滤液置浓缩罐中60℃减压浓缩至相对密度为1.2左右的流浸膏，流浸膏取出装入托盘，保持1cm左右厚度，置烘箱中60℃减压干燥，制成提取物2.28kg。
2、将原料粉碎成粉末并投入到水或含水乙醇溶液中形成一次溶液，将一次溶液加热预定时间后，将一次溶液进行过滤得到一次滤渣和一次滤液；将一次滤渣再次投入水或含水乙醇溶液中形成二次溶液，将二次溶液加热预定时间后，将二次溶液进行过滤得到二次滤渣和二次滤液，将一次滤液、二次滤液混合后，进行浓缩、干燥得到提取物。本实施具体例为，防治猪流行性腹泻的饲料添加剂以在冬季修剪茶树时获得的茶茎和茶梗作为原料，将原料粉碎成粗粉，取10kg茶茎和茶梗的粗粉，置提取罐中，加10倍量50％乙醇回流提取，保持沸腾2小时，过滤，滤渣再加入10倍量50％乙醇回流提取，保持沸腾1小时，过滤，合并两次滤液。滤液置浓缩罐中60℃减压浓缩至相对密度为1.0左右的流浸膏，流浸膏直接喷雾干燥，制成提取物2.13kg。
在使用时，先将防治猪腹泻饲料添加剂稀释到体积倍数的40倍后，再洒入饲料中并搅拌均匀，所述防治猪腹泻饲料添加剂与饲料的重量比为0.5～2：1000W/W。
Claims (6)
1.一种防治猪流行性腹泻的饲料添加剂，其特征在于以茶茎和/或茶梗作为原料，粉碎成粉末或制备提取物而制成。
2.根据权利要求1所述的防治猪流行性腹泻的饲料添加剂，其特征在于所述粉末的粒径小于0.85mm。
3.根据权利要求1所述的防治猪流行性腹泻的饲料添加剂，其特征在于所述茶茎、茶梗是在冬季修剪茶树时获得的。
4.根据权利要求1所述的防治猪流行性腹泻的饲料添加剂的制备方法，其特征在于所述提取物的制备过程如下：将原料粉碎成粉末并投入到溶剂中形成溶液，将溶液通过加热提取、微波提取或者超声提取得到提取物；所述粉末与溶剂的重量比为5:1～50；所述溶剂为水或者乙醇或者含水乙醇。
5.根据权利要求1所述的防治猪流行性腹泻的饲料添加剂的制备方法，其特征在于所述提取物的制备过程如下：将原料粉碎成粉末并投入到水中形成一次溶液，将一次溶液加热预定时间后，将一次溶液进行过滤得到一次滤渣和一次滤液；将一次滤渣再次投入水中形成二次溶液，将二次溶液加热预定时间后，将二次溶液进行过滤得到二次滤渣和二次滤液，将一次滤液、二次滤液混合后，进行浓缩、冷冻、干燥得到提取物。
6.根据权利要求1所述的防治猪流行性腹泻的饲料添加剂的使用方法，其特征在于当防治猪流行性腹泻的饲料添加剂为粉末状时，将防治猪流行性腹泻的饲料添加剂混合入饲料中并搅拌均匀，所述防治猪流行性腹泻的饲料添加剂与饲料的重量比为1～5：1000；当防治猪流行性腹泻的饲料添加剂为液态提取物时，先将防治猪流行性腹泻的饲料添加剂稀释到体积倍数的40倍后，再洒入饲料中并搅拌均匀，所述防治猪流行性腹泻的饲料添加剂与饲料的重量比为0.5～2：1000。
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