鸡肠道乳酸菌分离鉴定及其功能研究
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摘要
随着养殖业的不断发展,微生态制剂的应用越来越广泛,对畜牧业的健康、可持续发展发挥了重要作用。本研究利用分离自鸡消化道的乳酸菌制成微生态制剂,对肉鸡进行了饲喂试验,通过增重、免疫器官指数及盲肠中微生物数量的变化检测,显示了鸡源乳酸菌微生态制剂对肉鸡的生产性能、免疫功能等均具有明显的增强作用。该研究结果为利用鸡源乳酸菌开发家禽用微生态制剂提供了理论依据和菌种保证。
本研究分三部分内容
一、鸡消化道乳酸菌的分离与鉴定本实验从健康肉鸡的消化道黏膜部位分离到4株(分别标记为SL-1、SL-2、SL-3、SL-4)产酸的菌株,通过染色镜检、生化实验及16S rRNA分析、鉴定,均属于乳酸菌属,4株菌分别为嗜酸乳杆菌(L.acidophilus)、卷曲乳杆菌(L.crispatus)、鼠李糖乳杆菌(L.rhamnosus)和唾液乳杆菌(L.salivarius)。
二、益生乳酸菌的筛选及微生态制剂的制备采用耐胆盐、耐酸试验,对分离的4株乳酸菌进行了筛选,结果表明,SL-3株在0.3%浓度胆盐培养基中可生长,培养48h后细菌数达3.47×109CFU/ml,在pH2.0环境下也能生长,培养2h后细菌数达1.38×103CFU/ml;利用SL-3株菌的培养上清液,通过双层平板法,对致病性大肠杆菌进行了抑菌试验,结果显示,该菌株可有效抑制大肠杆菌的生长,抑菌圈直径达21.2mm。采用常规厌氧培养方法,以SL-3分离株作为菌种制备成功了乳酸菌微生态制剂,经安全试验、储藏试验和细菌计数,该制剂达到质量标准,为开发利用乳酸菌微生态制剂应用于养禽业提供了保证。
三、乳酸菌微生态制剂的饲喂效果研究利用分离的SL-3株菌制备的乳酸菌微生态制剂对肉鸡进行了饲喂试验,通过检测鸡增重、料重比、免疫器官指数、抗体效价以及盲肠内容物的乳酸菌数量,探索SL-3株鸡源乳酸菌制剂和猪源CL-11株乳酸菌制剂对鸡生产性能及免疫功能等的影响。结果表明,35日龄时,0.3%浓度SL-3的微生态制剂组鸡增重较对照组和抗生素组分别提高了11.09%和7.18%(差异均显著P<0.05);相同浓度的猪源乳酸菌制剂较对照组和抗生素组分别提高了4.08%和0.42%;0.3%浓度组鸡的脾、法氏囊、胸腺器官指数较对照组分别增加了67.14%、16.47%和38.16%,较抗生素组分别提高了56%、22.02%和30.81%,相同浓度的猪源乳酸菌制剂较对照组和抗生素组分别提高了31.42%、6.82%、9.76%和22.66%、11.90%、3.92%;0.3%浓度鸡的抗体较对照组和抗生素组分别提高了2log2和1.75log2,差异均显著(P<0.05);饲喂0.3%浓度微生态制剂组鸡盲肠中的正常菌(乳酸菌)数量较对照组和抗生素组分别升高了1.86CFU/ml和3.09CFU/ml,而肠杆菌数量较对照组和抗生素组降低了7.24CFU/ml和2.45CFU/ml;相同浓度的猪源乳酸菌制剂较对照组和抗生素组分别提高了正常菌(乳酸菌)1.02CFU/ml和1.69CFU/ml。其他日龄段饲喂SL-3株微生态制剂组鸡也均具有促生长作用、增强免疫功能和调整肠道微生态平衡的作用。该试验结果表明,SL-3株乳酸菌制剂能够促进鸡体的免疫功能,也能促进鸡消化道的微生态平衡,但鸡源SL-3乳酸菌微生态制剂均比猪源乳酸菌制剂作用效果明显。该研究结果为开发利用SL-3株乳酸菌制剂提供了理论依据。
With the development of breeding, probiotics are widely used and it played an important role in the healthy and sustainable development of husbandry. This study focused on the function of lactobacilli isolated from the digestive tract of broilers. The isolated were used in the feeding test on broilers. Though the test of gaining weight, the index of immune organs and the count of microbes in content of cecum, we found that lactobacilli isolated from broilers have a strengthening function on the performance and immune function of the broilers. This study provided theory and strains for research and development of lactobacilli isolated from broilers
This study was divided into three parts:
Part one: The isolation and identification of lactobacilli from digestive tracts
In this study, 4 strains (named SL-1,SL-2,SL-3 and SL-4) were isolated from the mucosa of broilers’digestive tract, through dyeing, microscopic examination, biochemical test and the analysis of 16s rRNA, identification, they belong to genus of lactobacilli: L.acidophilus, L.crispatus, L.rhamnosus, L.salivarius.
Part two: screening of probiotic properties and preparation of probiotics
The test of bile salt resistance, tolerance to acidic pH and antimicrobial activity was conducted in the 4 isolated strains. The results showed SL-3 strain could grow in the 0.3% oxgall broth, after being cultivated for 48 h the count could reach to 3.47×109 CFU/ml. After being cultivated for 2h in pH2.0 broth, the count could reach to 1.38×103CFU/ml. The antimicrobial action of SL-3 supernatant against pathogenic E.coli was determined by the agar diffusion method. The result showed it could inhibit the growth of E.coli. The mean diameter of inhibition zone was 21.2mm.The preparation was made with SL-3 under anaerobic circumstances. Through test of safety, storage, and count, it conformed to the quality standard and it could be used to feed to broilers.
Part three: The effects of lactobacilli probiotics on broilers
Lactobacilli probiotics with SL-3 were fed to broilers. Through the test of gaining weight, feed/gain ratio, index of immune organs, antibody titer and count of lactobacilli in content of cecum, the effects of probiotics on performance and immune function was obtained. Two strains were used, SL-3 from broiler and CL-11 from swine. The results showed that on the 35th days the gaining weight of group fed on 0.3% SL-3 significantly higher than the control group and antibiotic group by 11.09% and 7.18%. The group fed on 0.3% lactobacilli from swine was higher than control group and antibiotic group by 4.08% and 0.42%; the group fed on 0.3% SL-3 strain increased the index of spleen, bursa, and thymus by 67.14%, 16.47%, 38.16% and 56%, 22.02%, 30.81%, respectively, compared to control group and antibiotic group. While the group fed on 0.3% lactobacilli from swine increased the above index by 31.42%, 6.82%, 9.76% and 22.66%, 11.90%, 3.92% respectively, compared to control group and antibiotic group. The group fed on 0.3% SL-3 strain increased the antibody titer by 2log2 and 1.75log2 respectively, compared to control group and antibiotic group ((P<0.05). And this group also increased the count of lactobacilli in the cecum by 1.86CFU/ml and3.09CDU/ml, decreased the count of enterobacter by 7.24CFU/ml and 2.45CFU/ml, in comparison with control group and antibiotic group. The group fed on swine lactobacilli also increased the count of lactobacilli of cecum by l.02CFU/ml and1.69CFU/ml. In other stages the group fed on SL-3 strain also improved the performance, strengthened immune function and balanced the count of normal flora of tract. The result of this study showed SL-3 strain can improve the performance and the normal flora, and it significantly better the strain from swine. The result provided theory for developing and using SL-3.
本研究分三部分内容
一、鸡消化道乳酸菌的分离与鉴定本实验从健康肉鸡的消化道黏膜部位分离到4株(分别标记为SL-1、SL-2、SL-3、SL-4)产酸的菌株,通过染色镜检、生化实验及16S rRNA分析、鉴定,均属于乳酸菌属,4株菌分别为嗜酸乳杆菌(L.acidophilus)、卷曲乳杆菌(L.crispatus)、鼠李糖乳杆菌(L.rhamnosus)和唾液乳杆菌(L.salivarius)。
二、益生乳酸菌的筛选及微生态制剂的制备采用耐胆盐、耐酸试验,对分离的4株乳酸菌进行了筛选,结果表明,SL-3株在0.3%浓度胆盐培养基中可生长,培养48h后细菌数达3.47×109CFU/ml,在pH2.0环境下也能生长,培养2h后细菌数达1.38×103CFU/ml;利用SL-3株菌的培养上清液,通过双层平板法,对致病性大肠杆菌进行了抑菌试验,结果显示,该菌株可有效抑制大肠杆菌的生长,抑菌圈直径达21.2mm。采用常规厌氧培养方法,以SL-3分离株作为菌种制备成功了乳酸菌微生态制剂,经安全试验、储藏试验和细菌计数,该制剂达到质量标准,为开发利用乳酸菌微生态制剂应用于养禽业提供了保证。
三、乳酸菌微生态制剂的饲喂效果研究利用分离的SL-3株菌制备的乳酸菌微生态制剂对肉鸡进行了饲喂试验,通过检测鸡增重、料重比、免疫器官指数、抗体效价以及盲肠内容物的乳酸菌数量,探索SL-3株鸡源乳酸菌制剂和猪源CL-11株乳酸菌制剂对鸡生产性能及免疫功能等的影响。结果表明,35日龄时,0.3%浓度SL-3的微生态制剂组鸡增重较对照组和抗生素组分别提高了11.09%和7.18%(差异均显著P<0.05);相同浓度的猪源乳酸菌制剂较对照组和抗生素组分别提高了4.08%和0.42%;0.3%浓度组鸡的脾、法氏囊、胸腺器官指数较对照组分别增加了67.14%、16.47%和38.16%,较抗生素组分别提高了56%、22.02%和30.81%,相同浓度的猪源乳酸菌制剂较对照组和抗生素组分别提高了31.42%、6.82%、9.76%和22.66%、11.90%、3.92%;0.3%浓度鸡的抗体较对照组和抗生素组分别提高了2log2和1.75log2,差异均显著(P<0.05);饲喂0.3%浓度微生态制剂组鸡盲肠中的正常菌(乳酸菌)数量较对照组和抗生素组分别升高了1.86CFU/ml和3.09CFU/ml,而肠杆菌数量较对照组和抗生素组降低了7.24CFU/ml和2.45CFU/ml;相同浓度的猪源乳酸菌制剂较对照组和抗生素组分别提高了正常菌(乳酸菌)1.02CFU/ml和1.69CFU/ml。其他日龄段饲喂SL-3株微生态制剂组鸡也均具有促生长作用、增强免疫功能和调整肠道微生态平衡的作用。该试验结果表明,SL-3株乳酸菌制剂能够促进鸡体的免疫功能,也能促进鸡消化道的微生态平衡,但鸡源SL-3乳酸菌微生态制剂均比猪源乳酸菌制剂作用效果明显。该研究结果为开发利用SL-3株乳酸菌制剂提供了理论依据。
With the development of breeding, probiotics are widely used and it played an important role in the healthy and sustainable development of husbandry. This study focused on the function of lactobacilli isolated from the digestive tract of broilers. The isolated were used in the feeding test on broilers. Though the test of gaining weight, the index of immune organs and the count of microbes in content of cecum, we found that lactobacilli isolated from broilers have a strengthening function on the performance and immune function of the broilers. This study provided theory and strains for research and development of lactobacilli isolated from broilers
This study was divided into three parts:
Part one: The isolation and identification of lactobacilli from digestive tracts
In this study, 4 strains (named SL-1,SL-2,SL-3 and SL-4) were isolated from the mucosa of broilers’digestive tract, through dyeing, microscopic examination, biochemical test and the analysis of 16s rRNA, identification, they belong to genus of lactobacilli: L.acidophilus, L.crispatus, L.rhamnosus, L.salivarius.
Part two: screening of probiotic properties and preparation of probiotics
The test of bile salt resistance, tolerance to acidic pH and antimicrobial activity was conducted in the 4 isolated strains. The results showed SL-3 strain could grow in the 0.3% oxgall broth, after being cultivated for 48 h the count could reach to 3.47×109 CFU/ml. After being cultivated for 2h in pH2.0 broth, the count could reach to 1.38×103CFU/ml. The antimicrobial action of SL-3 supernatant against pathogenic E.coli was determined by the agar diffusion method. The result showed it could inhibit the growth of E.coli. The mean diameter of inhibition zone was 21.2mm.The preparation was made with SL-3 under anaerobic circumstances. Through test of safety, storage, and count, it conformed to the quality standard and it could be used to feed to broilers.
Part three: The effects of lactobacilli probiotics on broilers
Lactobacilli probiotics with SL-3 were fed to broilers. Through the test of gaining weight, feed/gain ratio, index of immune organs, antibody titer and count of lactobacilli in content of cecum, the effects of probiotics on performance and immune function was obtained. Two strains were used, SL-3 from broiler and CL-11 from swine. The results showed that on the 35th days the gaining weight of group fed on 0.3% SL-3 significantly higher than the control group and antibiotic group by 11.09% and 7.18%. The group fed on 0.3% lactobacilli from swine was higher than control group and antibiotic group by 4.08% and 0.42%; the group fed on 0.3% SL-3 strain increased the index of spleen, bursa, and thymus by 67.14%, 16.47%, 38.16% and 56%, 22.02%, 30.81%, respectively, compared to control group and antibiotic group. While the group fed on 0.3% lactobacilli from swine increased the above index by 31.42%, 6.82%, 9.76% and 22.66%, 11.90%, 3.92% respectively, compared to control group and antibiotic group. The group fed on 0.3% SL-3 strain increased the antibody titer by 2log2 and 1.75log2 respectively, compared to control group and antibiotic group ((P<0.05). And this group also increased the count of lactobacilli in the cecum by 1.86CFU/ml and3.09CDU/ml, decreased the count of enterobacter by 7.24CFU/ml and 2.45CFU/ml, in comparison with control group and antibiotic group. The group fed on swine lactobacilli also increased the count of lactobacilli of cecum by l.02CFU/ml and1.69CFU/ml. In other stages the group fed on SL-3 strain also improved the performance, strengthened immune function and balanced the count of normal flora of tract. The result of this study showed SL-3 strain can improve the performance and the normal flora, and it significantly better the strain from swine. The result provided theory for developing and using SL-3.
引文
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