CN112401071B - 一种胆汁酸复合菌剂在用于制备肉羊饲料添加剂中的应用 - Google Patents
一种胆汁酸复合菌剂在用于制备肉羊饲料添加剂中的应用 Download PDFInfo
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Abstract
本发明公开了一种胆汁酸复合菌剂在用于制备肉羊饲料添加剂中的应用。所述胆汁酸复合菌剂包括狄氏副拟杆菌菌悬液和胆汁酸，所述狄氏副拟杆菌菌悬液是由保藏编号为CGMCC No.20820的狄氏副拟杆菌LCG‑06培养发酵获得的。本发明的胆汁酸复合菌剂成分天然，无毒副作用，能够显著增加肉羊的生长速度，促进营养吸收，加快肉羊体内脂肪的分解，进而降低肉羊的脂肪率，并提高肉羊的屠宰体重和屠宰率，提高肉羊的养殖收益，因此具有广阔的应用前景。
Description
技术领域
本发明属于禽类养殖领域，具体涉及一种胆汁酸复合菌剂在用于制备肉羊饲料添加剂中的应用。
技术背景
肉羊是适应外界环境最强的家畜之一，而随着我国人民生活水平的提升，对肉羊的需求也逐渐增加。目前肉羊育肥多为异地育肥方式，牧繁农育方式居多，而牧区由于禁牧、限牧造成母羊饲养量降低，从而造成羊源紧张，而且由于没有饲养标准，有些育肥场饲料配比不科学，也会导致肉羊体积大小不同，缺乏营养，也会由于营养问题出现黄膘病，或者脂肪含量上升，这些都会影响肉羊的品质，影响养殖户的养殖收益，而且现在使用一些兽药或其他饲料添加剂，都可能会具有一定的副作用，并残留在肉羊体内，人们食用后会严重危害身体健康。因此，开发一种天然、高效，提高肉羊品质的天然菌剂是非常有必要的。
发明内容
为了解决现有技术的不足，本发明提供了一种胆汁酸复合菌剂在用于制备肉羊饲料添加剂中的应用。所述胆汁酸复合菌剂能够降低肉羊体脂以及出栏体重。
为实现上述发明目的，本发明采用以下技术方案予以实现：
本发明提供了一种胆汁酸复合菌剂在用于制备肉羊饲料添加剂中的应用。
进一步的，所述胆汁酸复合菌剂包括狄氏副拟杆菌菌悬液和胆汁酸。
进一步的，所述狄氏副拟杆菌菌悬液是由保藏编号为CGMCC No.20820的狄氏副拟杆菌LCG-06培养发酵获得的。
进一步的，所述胆汁酸复合菌剂的菌含量不少于5×108CFU/mL。
进一步的，所述胆汁酸复合菌剂的制备步骤具体如下：
(1)将所述狄氏副拟杆菌LCG-06于营养液体培养基中，37℃厌氧环境下活化40-48h；在活化菌液中加入质量浓度为20％蔗糖溶液，混合均匀后离心，沉淀物中添加质量浓度为10％的蔗糖溶液，重悬均匀后得到狄氏副拟杆菌LCG-06菌悬液；
(2)将所述狄氏副拟杆菌LCG-06菌悬液与胆汁酸混合均匀，得到胆汁酸复合菌剂。
进一步的，所述狄氏副拟杆菌LCG-06菌悬液与胆汁酸的体积质量比为8-10∶1。
进一步的，所述狄氏副拟杆菌LCG-06菌悬液的菌含量不少于2×109CFU/mL。
进一步的，所述胆汁酸包括不少于78％的猪胆酸和猪去氧胆酸以及不少于18％的鹅去氧胆酸。
进一步的，所述胆汁酸复合菌剂的使用方法为：所述胆汁酸复合菌剂的使用方法为：将胆汁酸复合菌剂以30-50mL/头/天的用量添加到肉羊饲料中，连续投喂20-40天。
进一步的，所述胆汁酸复合菌剂于每天中午添加到肉羊饲料中投喂肉羊。
进一步的，所述胆汁酸复合菌剂能够降低肉羊的脂肪率。
进一步的，所述胆汁酸复合菌剂能够增加肉羊的屠宰体重和屠宰率。
本发明与现有技术相比，具有以下优点和有益效果：
本发明从猪肠道中的粪便中筛选出一株狄氏副拟杆菌LCG-06，其不会对动物人体造成伤害，并且本发明利用蔗糖溶液将狄氏副拟杆菌LCG-06制备成菌悬液，再与胆汁酸以优化的比例复配制备得到复合菌剂，该复合菌剂成分天然，无毒副作用，能够显著增加肉羊的生长速度，促进营养吸收，加快肉羊体内脂肪的分解，进而降低肉羊的脂肪率，并提高肉羊的屠宰体重和屠宰率，提高肉羊的养殖收益，因此具有广阔的应用前景。
附图说明
图1为狄氏副拟杆菌LCG-06在TSA培养基上的菌落图片。
具体实施方式
以下结合具体实施例对本发明的技术方案做进一步详细的说明。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。
实施例1：
一、狄氏副拟杆菌LCG-06的筛选和鉴定
1、狄氏副拟杆菌LCG-06的筛选
从猪肠道内取肠内粪便，将10g粪便和100mL无菌水混匀，过滤去除杂质后制成粪便稀释液，然后继续向稀释液中添加无菌水，制备成10-1、10-2、10-3、10-4、10-5稀释液；再分别取10-2、10-3、10-4、10-5稀释液涂布至大豆酪蛋白琼脂培养基(TSA)上，在含有80％N2、10％CO2和10％H2的厌氧环境下，37℃培养4d，选取明显的菌落进行多次纯化后得到单菌落，并将其命名为LCG-06，然后置于大豆酪蛋白琼脂斜面培养基中冷藏保存。所述的TSA培养基的配方为：胰蛋白胨15.0g，大豆胨5.0g，氯化钠5.0g，琼脂15.0g，无菌水1000mL，pH 7.3±0.2。
如图1所示，所述菌株LCG-06在TSA培养基的菌落生物学特征为：圆形或类圆形，乳白色或米白色，扁平，直径0.2-1.8mm，表面光滑无光泽，半透明，边缘较整齐。
2、狄氏副拟杆菌LCG-06的鉴定
提取所述菌株LCG-06的基因组DNA，并以此为模板，使用16S rRNA通用引物进行PCR扩增，获得16S rRNA扩增序列后测序，然后将该序列进行Blast比对，结果显示菌株LCG-06与GenBank基因库中的狄氏副拟杆菌Parabacteroides distasonis同源性最高，因此判定菌株LCG-06为狄氏副拟杆菌。
将筛选到的狄氏副拟杆菌LCG-06进行菌种保藏，保藏单位：中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)；地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所；保藏日期：2020年09月25日；狄氏副拟杆菌Parabacteroides distasonis的保藏编号为CGMCC No.20820。
3、狄氏副拟杆菌LCG-06菌悬液的制备
(1)将保存的狄氏副拟杆菌LCG-06挑取至营养液体培养基(配方为胰蛋白胨15.0g，大豆胨5.0g，氯化钠5.0g，无菌水1000mL，pH 7.4)中，37℃厌氧环境下活化培养48h，得到狄氏副拟杆菌LCG-06菌液；
(2)在菌液中以1∶1的体积比加入质量浓度为20％的蔗糖溶液，混合均匀后10000rpm/min离心5min，得到菌株沉淀物；
(3)用质量浓度为10％的蔗糖溶液重新溶解上述菌株沉淀物，而蔗糖溶液的体积与菌株沉淀物的质量的比例为2∶1，混匀后得到狄氏副拟杆菌LCG-06菌悬液，该菌悬液的菌含量不少于2×109CFU/mL。
二、胆汁酸的制备
所述胆汁酸的制备工艺步骤如下：
(1)皂化：将粉碎过40目筛的1kg猪胆粉加入反应釜中，再加入质量浓度为10％的氢氧化钠溶液10L，加热搅拌至沸腾，并保持沸腾状态搅拌14h，然后将反应生成的皂化液冷却至固液分层，去除上清液，剩余固体即为胆汁酸皂化品；
(2)脱色：向胆汁酸皂化品中加6L水，加热至80℃使胆汁酸皂化品完全溶解，然后将其打入脱色罐中，加入300mL双氧水搅拌均匀后，常温反应24h，然后将反应溶液过滤至酸化槽中，得到滤液；
(3)酸化：将滤液冷却至室温，缓慢加入10％盐酸溶液，边加入边搅拌，待溶液pH值在3时停止加酸，离心过滤得到白色固形物；
(4)纯化：将白色固形物不断加水冲洗，将附着在白色固形物表面的水溶性杂质去除，待滤出的水检测呈中性后结束水洗；将离心过滤后的白色固形物放入烘箱中，100℃干燥至水分少于10％，得胆汁酸粗品；
(5)重结晶：将胆汁酸粗品投入萃取罐中，加入胆汁酸粗品重量10倍体积的乙酸乙酯搅拌至完全溶解，再加入0.08g无水硫酸钠脱水，过滤收集过滤液，将过滤液进行减压浓缩并回收乙酸乙酯，得到液体胆汁酸；
(6)干燥：将液体胆汁酸放入烘箱，在100℃下干燥至水分不超过1％时，得到成品胆汁酸。
成品胆汁酸中包含了78.6％的猪胆酸和猪去氧胆酸以及20.0％的鹅去氧胆酸。
三、胆汁酸复合菌剂的制备
将上述制得的成品胆汁酸以1∶10(g/mL)的比例与狄氏副拟杆菌LCG-06菌悬液混合均匀，即可得到胆汁酸复合菌剂，该复合菌剂中的菌含量不少于5×108CFU/mL。
实施例2
山东某养殖场，波尔山羊养殖规模500头，从其中随机选取20头作实验组，20头作对照组，40头羊均为4-5代羊。在出栏前1个月，实验组每天中午给山羊投喂含有胆汁酸复合菌剂的饲料，以每头羊30-50mL的添加量将胆汁酸复合菌剂均匀混合到饲料中，连续投喂25天，对照组的羊投喂等量的饲料，不添加胆汁酸复合菌剂。饲料选用混合比例相同的精饲料和草饲料，所有肉羊都按照常规养殖方法继续养殖。25天后屠宰，对照组的肉羊平均屠宰重量56.44kg，屠宰时可见较多黄白色脂肪；实验组的山羊平均屠宰重量54.47斤，黄白色脂肪较少，并且实验组的肉羊在25天的养殖过程中体重增加的更多，且平均屠宰率较对照组更高，说明本发明的胆汁酸复合菌剂能够有效的降低肉羊的脂肪率，提高肉羊的出栏体重和平均屠宰率。
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
Claims (8)
1.一种胆汁酸复合菌剂在用于制备肉羊饲料添加剂中的应用，其特征在于，所述胆汁酸复合菌剂包括狄氏副拟杆菌菌悬液和胆汁酸，所述狄氏副拟杆菌菌悬液是由保藏编号为CGMCC No. 20820的狄氏副拟杆菌LCG-06培养发酵获得的。
2.根据权利要求1所述的应用，其特征在于，所述胆汁酸复合菌剂的菌含量不少于5×108 CFU/mL。
3.根据权利要求1所述的应用，其特征在于，所述胆汁酸复合菌剂的制备步骤具体如下：
（1）将所述狄氏副拟杆菌LCG-06于营养液体培养基中，37℃厌氧环境下活化40-48 h；在活化菌液中加入质量浓度为20%蔗糖溶液，混合均匀后离心，沉淀物中添加质量浓度为10%的蔗糖溶液，重悬均匀后得到狄氏副拟杆菌LCG-06菌悬液；
（2）将所述狄氏副拟杆菌LCG-06菌悬液与胆汁酸混合均匀，得到胆汁酸复合菌剂。
4.根据权利要求3所述的应用，其特征在于，所述狄氏副拟杆菌LCG-06菌悬液与胆汁酸的体积质量比为8-10:1。
5.根据权利要求3所述的应用，其特征在于，所述胆汁酸包括不少于78%的猪胆酸和猪去氧胆酸以及不少于18%的鹅去氧胆酸。
6.根据权利要求1所述的应用，其特征在于，所述胆汁酸复合菌剂的使用方法为：将胆汁酸复合菌剂以30-50 mL/头/天的用量添加到肉羊饲料中，连续投喂20-40天。
7.根据权利要求6所述的应用，其特征在于，所述胆汁酸复合菌剂于每天中午添加到肉羊饲料中投喂肉羊。
8.根据权利要求1所述的应用，其特征在于，所述胆汁酸复合菌剂能够增加肉羊的屠宰体重和屠宰率。
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