【摘要】 为筛选用于鸡源生物制剂研发的优质乳酸乳球菌，本研究将采自健康鸡的粪便样品涂布MRS琼脂培养基，观察菌落形态并经革兰氏染色镜检、鉴定其生理生化特性。采用PCR扩增分离菌16S rRNA基因序列、测序，采用BLAST比对分析分离菌16S rRNA基因序列的同源性，利用MEGA 11软件构建分离菌该基因的系统发育树。结果显示，获得一株革兰氏染色呈蓝紫色球状、生理生化鉴定结果与标准乳酸乳球菌基本一致的优势菌。经PCR扩增并测序获得的分离菌与乳酸乳球菌亚种（LC483568/NR116443）16S rRNA基因序列的同源性达99%，且二者位于同一进化分支，表明分离到一株乳酸乳球菌PG-1株。将分离菌分别培养于不同pH值的培养液中至不同时间点，检测OD_600nm值并绘制生长曲线与产酸曲线。通过平板计数法统计活菌数并计算分离菌的存活率。分别检测分离菌对胆盐、人工胃液、人工肠液及高温的耐受性。通过测分离菌的OD_630nm值计算分离菌的疏水率和自凝聚率，评估分离菌PG-1株的疏水性和自凝聚力。结果显示：PG-1株在pH5.0～pH9.0均生长良好；在含0.1%与0.3%胆盐中的存活率分别达96.7%与81.45%；在人工胃液、人工肠液中的存活率分别为65.20%、93.16%；经60℃处理15 min后细菌的存活率仍能达46.83%，不耐受70℃及以上高温；分离菌在培养8 h～24 h的自凝聚率为22.59%～36.5%，表面疏水率为50%。采用纸片扩散法检测分离菌对9类共23种抗菌药物的敏感性，利用PCR检测分离菌5类共15种耐药基因。确定分离菌在哥伦比亚绵羊血平板中无溶血现象后将90只健康1日龄AA肉仔鸡随机均分为分离菌组（PG-1）、金霉素组（AGP）与阴性对照组（Con）。PG-1组鸡在基础日粮中按质量比添加0.1%的分离菌，AGP组鸡在基础日粮中添加45 mg/kg金霉素，Con组鸡饲喂基础日粮。试验共持续42 d，观察并记录各组鸡的健康与死亡状况，计算各组鸡的平均日增重（ADG）、平均日采食量（ADFI）和料肉比（F/G）。评价PG-1菌株的安全性和益生性。结果显示，PG-1株对4类抗生素（磺胺类、氨基糖苷类、四环素类和大环内酯类）均耐药，检测到β-内酰胺类的bla_TEM、磺胺类的Su1、Su2、氨基糖苷类的aph（3’）-IIa、ant（3’’）-Ia以及大环内酯类的MefE、MefA 7种耐药基因，PG-1株的耐药表型与其耐药基因结果基本相符。各组鸡在实验期间均无死亡。在试验1 d～28 d,PG-1组鸡的ADG显著高于Con组和APG组鸡（P<0.05）;3组鸡的ADFI均无显著差异；PG-1组鸡的F/G显著低于Con组和AGP组鸡（P<0.05）。在试验28 d～42 d,3组鸡的上述各生长性能均无显著差异。综上所述，本研究分离的乳酸乳球菌生长性能良好，具有较强的环境耐受力、自凝聚力和表面疏水性，且对鸡的安全性与益生性均较好，可作为潜在的益生菌候选菌株应用于肉鸡及别的动物的日常饲养。更多还原

【Abstract】 To screen high-quality Lactococcus lactis strains for chicken-derived biologics development, fecal samples from healthy chickens were collected and streaked on MRS agar medium. The colonies were observed for morphological characteristics,examined under Gram staining microscopy, and characterized physiologically and biochemically. The 16S rRNA gene fragments of the isolated strains were amplified via PCR and sequenced, and BLAST analysis was carried out to determine genetic homology,resulting in a phylogenetic tree using MEGA 11 software. The results showed that one dominant strain was obtained, which was bluepurple spherical in Gram staining and its physiological and biochemical identification results were basically consistent with the standard Lactococcus lactis. The homology of the 16S r RNA gene sequence of the isolated strain was 99% with that of Lactococcus lactis subsp.（LC483568/NR116443） and they located in the same evolutionary branch, which indicated that a strain of Lactococcus lactis named PG-1 was isolated. The isolate PG-1 was cultivated inculture mediumwith different pH values, and the OD_{600 nm}values were detected at different time points and the growth curves and acidogenic curves were plotted. The survival rate of the isolate was calculated using the plate counting method to detectits tolerance to bile salt, artificial gastric fluid, artificial intestinal fluid and high temperature. The hydrophobicity and self-agglutination of the isolate PG-1 were evaluated by calculating the hydrophobic rate and self-aggregation rate. The results showed that PG-1 strain grew well in the pH range of 5.0 to 9.0. Its survival rates in 0.1% and 0.3%bile salts were 96.7% and 81.45%, respectively. The survival rates were 65.20% in artificial gastric and 93.16% in intestinal fluids.After treatmentat 60℃ for 15 minutes, the survival rate remained at 46.83%, but the bacteria showed no heat-resistance to 70℃ or higher. After 8 hours-24 hours of culture, the self-aggregation rates of the isolate were 22.59%-36.5%, with a surface hydrophobic rate of 50%. Disk diffusion method was used to detect the sensitivity of the isolate to 23 kinds of antibiotics in 9 categories. A total of15 drug resistance genes in 5 categories of isolate were detected by PCR. The isolate showed no hemolytic ring on the Colombian sheep blood plate which confirmed that the isolate had no hemolytic activity. Ninety healthy 1-day-old AA broilers were randomly divided into three groups: PG-1（basal diet supplemented with 0.1% isolated bacteria）, AGP（basal diet supplemented with 45g/kg chlortetracycline）, and Con（negative control）. The experiment lasted for 42 days, the health status and mortality of each group of chickens were observed and recorded, and the average daily gain（ADG）, average daily feed intake（ADFI） and feed to weight ratio（F/G） were calculated to evaluate the safety and probiotic properties of PG-1 strain. The results showed that PG-1 strain was resistant to 4 kinds of antibiotics（sulfonamides, aminoglycosides, tetracyclines and macrolides）, and contained 7 resistance genes including bla_TEMof β-lactam, Su1 and Su2 of sulfonamides, aph（3’）-IIa and ant（3’’）-Ia of aminoglycosides, MefE and MefA of macrolides. The resistance phenotype of PG-1 strain was consistent with the existence of resistance genes. During the experiment, no chickens died in any group. The average daily gain（ADG） of PG-1 group was significantly higher than that of Con group and chlortetracycline（APG）group（P<0.05） from day 1 to day 28. The average daily feed intake（ADFI） among the three groups showed no significant difference.The feed-to-gain ratio（F/G） of PG-1 group was significantly lower than that of Con group and APG group（P<0.05）. In the later stage of 28 days-42 days, there was no significant difference in the growth performance of the three groups. In conclusion, the isolated Lactococcus lactis has good growth performance, strong environmental tolerance, self-aggregation and surface hydro-phobicity, and good safety and probiotic properties for chickens, which can be used as a potential probiotic candidate for daily feeding of broilers and other animals.更多还原