【作者】 董晓丽；

【导师】 刁其玉；

【作者基本信息】 中国农业科学院 ， 动物营养与饲料科学， 2013， 博士

【摘要】 本文从土壤中分离筛选出能抗大肠杆菌、金黄色葡萄球菌等病原菌的两株菌种，采用16SrRNA基因分子技术进行菌种鉴定，并应用体外法评价其作为益生菌的效果。研究分离的两株菌种在断奶仔猪的作用效果和对仔猪肠道内微生物的影响；以犊牛为试验动物，研究分离两株菌种对断奶前后犊牛的生长性能和瘤胃发酵及微生物区系变化的影响。围绕益生菌的筛选和对幼龄动物的作用影响，完成6个试验，主要内容如下：试验一、乳酸菌GF103的分离鉴定及体外益生效果评价从北京市大兴种猪场附近土壤中分离得到一株能抑制病原菌大肠杆菌和金黄色葡萄球菌生长的乳酸菌GF103，经16S rRNA基因序列分析鉴定为植物乳杆菌(Lactobacillus plantarum)，GenBank登录号为JN560899。体外益生效果评价表明，乳酸菌GF103在pH=3的条件下活菌数较高，能够耐受0.3%胆盐。乳酸菌GF103在人工模拟胃液中培养0.5h活菌数不受影响，培养3h活菌数降低1log值；在人工模拟肠液中培养3h活菌数不变。结果表明，乳酸菌GF103具备益生菌特性，可进一步研究其作为微生物添加剂在动物营养和日粮中的应用效果。试验二、枯草芽孢杆菌B27筛选鉴定及体外益生效果评价从土壤中分离出36株芽孢杆菌，经过抗病原菌试验筛选一株能够抑制大肠杆菌、金黄色葡萄球菌和鼠伤寒沙门氏菌的菌株，其编号为B27。经16S rRNA基因分子技术鉴定为枯草芽孢杆菌(Bacillus subtilis)，GenBank登录号为JQ673431。模拟体外胃肠道环境，枯草芽孢杆菌B27在pH=3的条件下存活率为72%，能够耐受0.3%胆盐，在人工模拟胃肠液中培养3h存活率分别是87.7%和96.8%。结果表明，枯草芽孢杆菌B27具备作为益生菌的潜力，可作为益生菌添加剂进一步研究其在动物营养和日粮中的作用效果。试验三、饲喂益生菌对断奶仔猪生长性能、粪便微生物和血清指标的影响采用单因素完全随机试验设计，选用144头35-37日龄的断奶仔猪(大×长)，平均体重9.7±0.88kg，随机分为4组，每组分为6栏(6重复)，每栏6头仔猪。试验期35d，试验分为4个处理：1)基础日粮(对照，CT组)；2)基础日粮+植物乳杆菌(LB组)；3)基础日粮+枯草芽孢杆菌(BS组)；4)基础日粮+植物乳杆菌和枯草芽孢杆菌的混合物(LBS组)。结果表明，试验14d，饲喂益生菌的仔猪日增重具有增加的趋势，与对照组相比饲料转化率得到显著改善(P<0.05)；添加不同益生菌的仔猪平均日采食量和饲料转化率差异不显著(P>0.05)。整个试验期，饲喂植物乳杆菌的仔猪粪中大肠杆菌的数量显著低于对照组(P<0.05)。与对照组相比，试验14d仔猪日粮添加植物乳杆菌和枯草芽孢杆菌复合物显著提高血清总蛋白、球蛋白和肌酐水平(P<0.05)，并显著降低血清白蛋白与球蛋白的比值(P<0.05)。整个试验期，饲喂益生菌的仔猪血清IgM浓度显著提高(P<0.05)；试验35d，日粮添加枯草芽孢杆菌或乳酸菌和枯草芽孢杆菌的复合物能提高仔猪血清IgA的浓度。结果表明，日粮添加植物乳杆菌，枯草芽孢杆菌或其复合菌能改善断奶仔猪早期的生长性能，并能提高免疫力。试验四、饲喂益生菌对断奶仔猪肠道微生物区系的影响采用单因素完全随机试验设计，试验设计同试验三。试验期35d，分别于试验14d和35d，每个处理随机取4头仔猪(每个重复1头)进行屠宰试验，分析胃肠道组织和内容物微生物的变化。结果显示，断奶仔猪饲喂益生菌14d时胃内食糜pH显著低于对照组(P<0.01)；复合菌组仔猪十二指肠食糜pH显著低于对照组(P<0.01)。小肠各段组织形态与对照组相比差异不显著(P>0.05)。随着肠道向后延伸，回肠微生物种类多于空肠，盲肠最多；与断奶前期比较，断奶后期仔猪肠道微生物种类增多。仔猪断奶前期日粮中添加益生菌对肠道内微生物的作用影响明显，断奶后期益生菌对肠道内微生物作用减弱。与对照组相比，日粮中添加益生菌对断奶仔猪在肠道各段中细菌总数、乳酸菌和大肠杆菌的数量差异不显著(P>0.05)，但是饲喂益生菌具有降低肠道大肠杆菌的趋势。结果表明，饲喂益生菌对断奶仔猪初期阶段作用效果较明显，能够改善肠道微生物区系；断奶后期仔猪肠道微生物种类增多，仔猪的个体差异大于益生菌的作用影响。试验五、益生菌对哺乳期犊牛生长性能、血清指标、瘤胃发酵及瘤胃微生物区系的影响采用单因素的随机化设计，选取24头自然分娩的新生荷斯坦犊牛(公母各半)，按照出生胎次、初生重和出生时间相近的原则分为3组，每组8头(公母各半)。试验处理为：CT组为对照组，饲喂基础日粮(不添加抗生素和益生菌)；LB组为植物乳杆菌组，饲喂基础日粮+植物乳杆菌GF103(1.7×1010CFU/h.d)；LBS组为复合菌组，饲喂基础日粮+植物乳杆菌GF103(8.6×109CFU/h.d)+枯草芽孢杆菌B27(2.0×108CFU/h.d)。试验期共8周。试验中测定犊牛的生长性能、粪便评分、血清指标、瘤胃发酵和瘤胃微生物的变化。结果显示，益生菌对哺乳期犊牛的日增重、采食量和饲料转化率等生长性能指标影响差异不显著(P>0.05)，但是整个试验期犊牛的饲料转化率具有改善的趋势。通过对犊牛粪便评分发现，益生菌能够改善粪便评分状况，减少腹泻发生。饲喂益生菌的犊牛血清生化指标差异不显著(P>0.05)。饲喂复合菌后犊牛瘤胃戊酸含量显著高于其它处理组(P<0.05)，但是其他VFA含量差异不显著(P>0.05)。应用PCR/DGGE和RT-PCR对瘤胃微生物进行分析发现，哺乳期犊牛饲喂益生菌对犊牛瘤胃微生物区系影响不显著，其个体差异大于组间处理的影响；瘤胃内未检测到饲喂的益生菌。这些结果表明，饲喂哺乳期犊牛益生菌具有改善犊牛饲料转化率的趋势，并能减轻腹泻发生率。试验六、益生菌对断奶后犊牛生长性能、血清指标、瘤胃发酵及瘤胃微生物的影响本试验采用单因素的随机化试验设计，选取24头刚断奶荷斯坦犊牛(公母各半)，按照出生胎次、断奶重相近的原则分为3组，每组8头(公母各半)。试验处理为：CT组为对照组，饲喂基础日粮(不添加抗生素和益生菌)；LB组为植物乳杆菌组，饲喂基础日粮+植物乳杆菌GF103制剂(1.7×1010CFU/h.d)；LBS组为复合菌组，饲喂基础日粮+植物乳杆菌GF103制剂(8.6×109CFU/h.d)+枯草芽孢杆菌B27制剂(2.0×108CFU/h.d)。试验期共4周。结果表明，饲喂益生菌0-14d断奶犊牛的饲料转化率显著高于对照组(P<0.05)；整个试验期饲喂复合菌的犊牛饲料转化率显著高于对照组(P<0.05)；但是日增重、采食量均无显著差异(P>0.05)。断奶犊牛饲喂益生菌后的血清指标和瘤胃发酵参数均无显著影响(P>0.05)。通过PCR/DGGE分析发现，瘤胃中未检测出日粮中添加的益生菌的条带，但是相同处理的不同犊牛瘤胃微生物区系相似性较高，能够聚合在一起，影响瘤胃微生物区系。应用RT-PCR技术检测瘤胃常见微生物数量，复合菌组的黄色瘤胃球菌和白色瘤胃球菌的数量均显著低于对照组(P<0.05)。结果证明，益生菌在犊牛断奶后的2周作用效果明显，能够改善饲料转化率，影响瘤胃微生物区系。更多还原

【Abstract】 In this study, two different strains of microorganisms were isolated from soil and identified by16SrRNA molecular technology, and the properties of these strains were evaluated in vitro. The in vivoeffects of these microbes as probiotics in weaned piglets and pre-and post-weaning calves wereconducted. The present study included6trials which described as follows.Experiment1: Isolation and identification of Lactobacillus sp. GF103and assessment of it asa potential probioticA Lactobacillus sp. strain GF103which inhibited the growth of pathogenic bacteria E.coli andStaphylococcus aureus was isolated from soil nearby Beijing Daxing Breeding pig farm. It wasidentified by16S rRNA sequence analysis as Lactobacillus plantarum (GenBank accession number:JN560899). In vitro assessments showed that a large amount of the strain GF103survived under thecondition of pH=3. The strain GF103had tolerated0.3%of bile salt. The viable counts of strain GF103did not change after0.5h of growing in simulated gastric fluid and decreased1log value after3h. Theviable counts of strain GF103did not decrease in simulated small intestinal fluid after3h. The strainlikely survived and propagated in the animal’s intestinal tract. These results suggested that the strainGF103possessed probiotic properties and should be further studied its application in animals as feedadditives.Experiment2: Isolation and identification of Bacillus subtilis B27and assessment it aspotential probioticsThirty-six Bacillus sp. were isolated from soil and the strain B27inhibited the growth ofpathogenic bacteria E. coli, staphylococcus aureus and salmonella. By16S rRNA sequence analysis itwas identified as Bacillus subtilis (GenBank accession number: JQ673431). In vitro assessment showedthat72%of the strain B27survived under the condition of pH=3. It tolerated0.3%of bile salt. Theviable counts of strain GF103in simulated gastric and small intestinal fluid after3h were87.7and96.7%, respectively. This strain likely survived and propagated in the animal’s intestinal tract. Theseresults indicated that Bacillus subtilis B27had probiotic properties and could be used as feed additives.Experiment3: Effects of dietary probiotics on growth performance, fecal microbiota andserum profiles in weaned pigletsOne hundred and forty-four piglets of Large White×Landrace weaned at35-37days of age wereselected and divided into four groups, and the piglets from each group were assigned randomly to sixpens (replicates) with6animals each. Each group was fed one of four diets for5weeks: a basal dietwithout antibiotics and probiotics (control), or the basal diet supplemented with Lactobacillusplantarum GF103, Bacillus subtilis B27, or a mixture of Lactobacillus plantarum GF103and Bacillussubtilis B27. During the first two weeks of the trial, the piglets supplemented with probiotics had lower(P<0.05) average daily feed intake (ADFI) than control. The feed conversion ratio (FCR) was improved(P<0.05) in probiotic-supplemented groups when compared with that of control. The counts of E. coli in feces of the piglets supplemented with Lactobacillus plantarum GF103was lower (P<0.05) than that ofcontrol. On day14, dietary supplementation of the combination of Lactobacillus plantarum GF103andBacillus subtilis B27increased (P<0.05) the serum concentration of total protein, globulin, andcreatinine, but decreased (P<0.05) the ratio of serum albumin to serum globulin when compared withcontrol. On the same day, probiotic-supplemented piglets had increased (P<0.05) serum IgMconcentrations compared with control animals. Supplementation of Bacillus subtilis B27or thecombination of Lactobacillus plantarum GF103and Bacillus subtilis B27increased (P<0.05) the serumIgA concentrations at the end of the trial. These results indicated that dietary probiotics improvedgrowth performance and enhanced immune responses at the early stage of the post-weaning period inpiglets.Experiment4: Changes of intestinal bacteria in weaned piglets supplemented with probioticsThe experiment design was the same as experiment3. The trial period lasted for35d. On14and35d,4piglets from each treatment (one from each pen, totally16piglets in each period) wereslaughtered for sample collection. The intestinal tissues were taken for histological masurements and thecontents of intestinal were collected for analysis of bacteria. The results showed that, during the firsttwo weeks after weaning, the piglets supplemented with probiotics had lower pH in gastral fluids thanthat in control (P<0.05), the piglets supplemented with complex probiotics also had lower pH induodenal fluids (P<0.05). There were no significant difference between on intestinal samples of pigletssupplemented with probiotics and control group (P>0.05). Compared with the first two weeks, thespecies of intestinal microbiota became more diverse in the last three weeks. The dietary supplementedwith probiotics was more effective at the early stage of the post-weaning period than that at the laterstage in piglets. Although the number of total bacteria, lactic acid bacteria and E.coli in the digestivetract of piglets was not influenced by probiotics, the piglets supplemented with probiotics tended tohave a decreased population of E.coli in the intestinal tract. These results indicated that the probioticsimproved intestinal bacterial community at the early stage. The species of intestinal bacteria becamemore diverse, and the microbiota in the intestinal tract became balanced.Experiment5: Effects of probiotics on growth performance, serum pararmeter,rumenfermentation and ruminal microflora in the pre-weaned calvesTwenty-four newborn Holstein calves were randomly divided into3groups (with4males and4females each). A basal diet without antibiotics and probiotics was used as control (CT group), and theother two diets were supplemented with Lactobacillus plantarum GF103(1.7×1010CFU/h.d) as LBgroup or with a complex of Lactobacillus plantarum GF103(8.6×109CFU/h.d) and Bacillus subtilisB27(2.0×108CFU/h.d) as LBS group. The trial lasted for8weeks. The results showed that there wereno significant difference on growth performance (P>0.05), but feed conversion efficiency was improvedby probiotics (P<0.05). The fecal scores of calves supplemented with probiotics were improved and thediarrhea incidence was decreased. Dietary probiotics did not affect serum parameters of calves (P>0.05).The calves supplemented with a complex of Lactobacillus plantarum and Bacillus subtilis had highervalerate in rumen fluids (P>0.05), but the other VFA were not affected by probiotics (P>0.05). The rumen microbial community of calves supplemented with probiotics was not affected as indicated byPCR/DGGE or RT-PCR analysis. Besides, the probiotics bacteria were not found in the rumen. Theseresults suggested that supplementation with probiotics decreased the incidence of diarrhea, but theprobiotics did not colonize in the rumen.Experiment6: Effects of probiotics on growth performance, serum pararmeter,rumenfermentation and ruminal microflora in the post-weaned calvesTwenty-four post-weaned calves were randomly divided into3groups with4males and4femaleseach. A basal diet without antibiotics or probiotics was used as control, and the other two group werefed the control diet supplemented with Lactobacillus plantarum GF103(1.7×1010CFU/h.d) as LBgroup or with a complex of Lactobacillus plantarum GF103(8.6×109CFU/h.d) and Bacillus subtilisB27(2.0×108CFU/h.d) as LBS group. The trial lasted for4weeks. The results showed that the feedconversion ratio (FCR) was improved (P<0.05) in probiotic-supplemented groups when compared withthat of control during the first two weeks and the entire trial. But average daily gains (ADG) and feedintake (FI) were not affected by probiotics (P>0.05). There were no significant differences in serumparameters and rumen fermentation (P>0.05) among the treatments. The probiotics bacteria were notfound in rumen fluids, but the microflora polymerized together with the same treatments and formed abranch by DGGE analysis. The counts of Ruminococcus flavefaciens and Ruminobacter albus in therumen of calves supplemented with the complex probiotics were lower than those in control. Theseresults showed that probiotics improved performance and FCR in post-weaned calves at the early stage.更多还原