CN115975876B - 一种预防雏鹅痛风的益生菌及其应用 - Google Patents
一种预防雏鹅痛风的益生菌及其应用 Download PDFInfo
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Abstract
本发明属于益生菌功能技术领域。本发明公开了一种预防雏鹅痛风的益生菌，该菌已于2022年5月12日保藏于中国微生物菌种保藏中心，地址为北京市阐扬去北辰西路1号院3号，保藏编号CGMCCNO.24882，分类命名副干酪乳杆菌Lactobac i l l us paracase i.本发明解决了预防雏鹅痛风相关问题。
Description
技术领域
本发明属于一种预防痛风的饲料添加剂，具体涉及一种预防痛风的益生菌的分离与功能鉴定。
背景技术
痛风是由于动物机体内长时间的嘌呤代谢和尿酸排泄异常而引起的一种营养代谢病，导致血液中的尿酸水平升高，尿酸盐在关节表面广泛沉积。痛风在鹅的饲养过程中较为常见，雏鹅患病死亡率高达50％，现在存在的有关于鹅痛风的病症基本都是以治疗为主，养殖者都是在鹅出现痛风的状况后才采取相应的治疗手段，且由于饲养条件的要求，在治疗的同时还在继续饲喂致病的高蛋白饲粮，这就导致很难达到对痛风的根治。
发明内容
本发明公开了一种预防痛风的益生菌，该菌已于2022年5月12日保藏于中国微生物菌种保藏中心，地址为北京市朝阳区北辰西路1号院3号，保藏编号CGMCC NO.24882，分类命名副干酪乳杆菌Lactobaci l l us paracasei.由于鹅的痛风是与在现在的养殖业中饲喂蛋白量过高，雏鹅不能依靠自身的代谢作用将其进行较好的代谢处理。本产品则是以益生菌的调节作用为出发点，通过益生菌在肠道菌群内的调节作用，促进肠道对嘌呤的吸收，减少尿酸的沉积，进而阻断痛风的发生。同时，由于肠道菌群的调节作用，可以在饲喂高蛋白日粮的同时进行处理，即保证了生产的需要，也解决了患病的问题。
本发明技术方案如下：1样品的采集与处理
出发前将所需的耗材准备齐全，取粪盒，镊子和手套。在泡沫箱中放入冰袋，防止采集后的样品细菌继续生长。以痛风恢复后的健康雏鹅为采样对象，用取粪盒扣取其棕色带有绿色纤维且成型的粪便，放入泡沫箱中带回实验室。
2细菌的分离
称取采回的雏鹅粪便1g，加入9ml生理盐水放入漩涡震荡仪中震荡混匀，将混匀后的粪液进行十倍稀释，取稀释液(10-6，10-7，10-8)的稀释液50μL置于到配好的NA琼脂培养基中，用涂布棒涂抹均匀，放入37℃恒温箱中培养24h。用接种环挑取培养基中的单一菌落进行划线，再次放入培养箱中培养，重复3-4次,直到纯化为单一菌落。
3细菌的筛选
通过革兰氏染色对所获得菌株进行形态上的初步筛选，取干净玻片，用接种环取一环无菌水于玻片上，而后挑取少量细菌，涂匀，待干燥后，固定。革兰氏染色：先用草酸铵结晶紫染液染色1mi n，用水冲洗，接着滴加卢氏碘液覆盖1mi n，用水冲洗，然后滴加95％乙醇至乙醇液不呈现紫色时停止约0.5mi n，最后用蕃红染液复染1mi n，水洗。油镜镜检(16×100)，选取革兰氏染色为阳性、形态为杆状或球状的菌株做后备菌株留用。
再通过生化特性试验筛选出产酸、产蛋白酶和淀粉酶的菌。
(1)产酸试验：将通过MRS培养基和革兰氏染色筛选出的细菌菌液按2％接种量接到LB培养基中，每隔2h测定一次培养液的pH值。
(2)产淀粉酶可检测试验：将各株菌点滴到淀粉培养基中，37℃恒温培养24h后滴加碘液，碘液范围要覆盖整个菌落，观察菌落周围有无透明圈。
(3)产蛋白酶试验：将各株菌接到酪蛋白培养基中，37℃恒温培养24h后，滴加酸性汞试剂，观察有无分解透明圈。
4.细菌的鉴定
利用天根生化有限公司的细菌基因提取试剂盒提取DNA，再利用通用引物343F(5′-TAC GGR AGG CAG CAG-3′)和798R(5′-AGG GTA TCT AAT CCT-3′)进行PCR扩增。反应体系25μL：2×premi xTaq12.5μL、上下游引物各1μL、模板DNA2μL、ddH2O8.5μL。PCR反应条件：94℃预变性5mi n；94℃变性30s、64℃退火30s、72℃延伸30s，共30个循环；72℃终延伸10mi n，4℃保存。将得到的PCR产物进行1％琼脂糖凝胶电泳试验测定DNA的大小以及浓度，若获得的信息在预期范围内，将剩余的PCR扩增产物送至上海生工公司进行16S rDNA测序。最后获得的DNA部分序列与NCB I基因库中进行比对，用MEGAⅪ进行发育树的构建，用MegAl i gn进行同源性比较。
本发明有益效果为：利用副干酪乳酸菌RV-M192在产肠道菌群内的调节作用，促进肠道对嘌呤的吸收，减少尿酸的沉积，进而达到预防痛风的作用。
附图说明
图1肾脏组织切片(200x)
具体实施例
益生菌效果验证实验
1.微生物制剂的制作
本试验采用冻干的方法制作益生菌制剂，将筛选鉴定后的细菌接种到200mL LB液体培养基中培养24h，扩培后的菌液与保护剂按1:1比例混合均匀后倒入灭菌的培养皿中，菌液厚度约为培养皿的1/3，放入-80℃预冻5h以上。预冻后用保鲜膜包裹培养皿，在上方用牙签扎孔，均匀密集分布，将处理好的培养皿放入冻干机中制成冻干粉菌制剂。收集冻干菌制剂称量计数后4℃保存。保存时间为两周，步骤计算细菌存活率。对冻干粉制剂进行复水，将复水得到的悬液用无菌生理盐水进行倍比稀释，吸取0.1mL合适浓度均匀涂布于固体培养基中，置于37℃培养，进行菌落计数，最后计算存活率，保证试验周期内冻干粉制剂中有效活菌量。
平板计数公式为：活菌量＝(菌落数/0.1mL)×稀释倍数×100％。
2.模型建立及分组饲喂
选用1日龄的雏鹅作为试验动物，24％蛋白作为痛风模型建立的最佳含量。试验分为5个组，分组1为痛风对照组，2-4为副干酪乳酸菌试验组.
试验雏鹅饲喂统一的24％蛋白含量日粮，每组六只雏鹅，试验1组为对照组灌服于试验组相同剂量的生理盐水，试验2-4组分别灌服1mL副干酪乳酸菌制剂(浓度分别为3x107CFU/mL，3x108CFU/mL，3x109CFU/mL)。雏鹅每天自由采食饮水。
实验用菌剂都是新鲜配制，每天准备好不同的菌液各1管，注意观察鹅的健康、存活等情况，并且做好记录。
3.血液生化指标检测
最后一天灌服菌液2h后，各组雏鹅颈静脉采血，每组采血时间不超过5分钟。在室温的条件下，血液凝固l h后，3500r/mi n离心l0mi n取血清，-20℃保存。用全自动生化分析仪检测血尿酸、尿素氮和肌酐水平，用试剂盒检测黄嘌呤氧化酶XOD含量。
4.HE染色检测肾脏损伤情况
动物处死之后，摘取一侧肾脏生理盐水冲洗血水和粪，4％对聚甲醛固定，石蜡包埋。全部石蜡标本均连续切片，石蜡切片二甲苯脱蜡，经各级乙醇至水洗，苏木素染色，自来水冲洗，盐酸乙醇酸化，水洗，伊红染色，常规脱水，透明中性树脂封片，电镜下扫描。
5.血液指标
表1
由表1可以看出，连续饲喂14d 24％蛋白饲料进行造模后，血液中尿素氮BUN、肌酐CRE、尿酸UA水平和黄嘌呤氧化酶XOD含量有明显的变化，与对照组相比，灌服副干酪乳杆菌RV-M192菌液，中剂量组和高剂量组使尿素氮分别降低10.8％和6.0％，差异显著水平分别为P<0.01和P<0.05；中、高剂量组使肌酐含量降低60％和28.7％，差异显著性水平均为P<0.01；低、中、高剂量组使尿酸水平降低6.4％、16.0％、10.8％，低剂量组差异显著性为P<0.05，中、高剂量组差异显著性为P<0.01；中剂量组使黄嘌呤氧化酶降低8.5％，P<0.01。
6HE染色
连续14d饲喂24％粗蛋白日粮并灌服副干酪乳杆菌RV-M192的益生菌制剂后对雏鹅肾脏的影响结果如图1所示。结果显示，模型组与空白相比，肾小管扩张、结构消失、有明显的空泡化，再次说明试验造模成功。另外，与模型组相比，灌服副干酪乳杆菌RV-M192，中剂量组肾小管上皮空泡化减少，甚至接近于空白组，低剂量组还有明显的空泡化，高剂量组有大量炎性细胞浸润。
Claims (2)
1.一株副干酪乳杆菌（Lactobacillus paracasei），其特征在于，该菌株于2022年5月12日保藏至中国微生物菌种保藏管理委员会普通微生物中心。地址为北京市朝阳区北辰西路1号院3号，保藏编号CGMCCNO.24882。
2.如权利要求1所述的副干酪乳杆菌在制备预防雏鹅痛风的饲料和饲料添加剂方面的应用。
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