猪用乳酸杆菌、双歧杆菌、纳豆芽孢杆菌复合活菌制剂的研制
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摘要
随着我国工厂化养猪的发展,猪群的数量和规模越来越大,疾病传染的机会增加,仔猪腹泻所造成的损失越来越突出。目前,防治仔猪腹泻以使用抗生素为主,但长期、大量使用不仅造成药物残留,促进耐药菌株的产生,而且会打破宿主菌群平衡。微生态制剂以其天然、无残留、安全可靠及不产生抗药性等优点而成为抗生素的替代品,是国内外研究的热点。本研究通过筛选健康仔猪肠道乳酸杆菌,配合其他有益生菌研制猪用复合活菌制剂,对于防治仔猪腹泻将具有重要的实践价值和理论意义。
本研究选择黑龙江省不同地区猪场,采集12～60日龄健康仔猪肠道粪样,分离乳酸杆菌,通过细菌形态、培养特性、生化试验和PCR种属分析进行鉴定。然后对双歧杆菌、纳豆芽孢杆菌及分离的乳酸杆菌进行抑菌试验、耐酸试验、耐胆盐试验、耐消化酶试验及温度敏感性等试验,筛选对仔猪消化道内胃酸及消化酶等因素耐受力强的菌株。应用二次正交旋转组合设计方案,进行筛选菌株发酵培养基及冷冻干燥保护剂各组分的优化试验;并通过响应面法进行分析。制备活菌制剂,通过仔猪饲养试验,确定复合微生态制剂的临床应用效果。
结果从仔猪肠道粪便分离到革兰氏阳性,厌氧,无芽孢杆菌48株。生化试验和PCR种属分析,确定18株为乳酸杆菌。其中,罗伊氏乳杆菌7株,嗜酸乳杆菌5株,约氏乳杆菌2株,短乳杆菌1株,干酪乳杆菌假植物亚种1株,植物乳杆菌1株,詹氏乳杆菌1株。通过耐受力筛选试验确定双歧杆菌、纳豆杆菌及6株乳酸杆菌对猪源大肠杆菌和沙门氏菌具有较好抑制作用;双歧杆菌、纳豆杆菌及3株乳酸杆菌对温度、pH、胆盐及消化酶具有较好的耐受性。通过单因素实验,确定乳酸杆菌适宜的发酵培养条件为:温度37℃,pH6.5,接种量3%,装液量3/5,静止培养时间16h。通过二次正交旋转组合设计试验和各因素响应面法分析,确定乳酸杆菌发酵培养基各组分比例为:大豆蛋白胨5%、葡萄糖1%、酵母浸粉1.7%、低聚糖0.3%,发酵液16h的实际最大OD620值为1.073。冷冻干燥保护剂各组分比例为:蔗糖15%、甘油3%、山梨醇15%、脱脂乳15%;按菌悬液(v)/保护剂(v)为1:1,加入冷冻干燥保护制剂,菌体冻干存活率的平均值为71.6%。仔猪饲养试验结果表明:以罗伊氏乳杆菌、双歧杆菌和纳豆杆菌制备的复合活菌制剂可提高仔猪日增重和饲料效率,降低仔猪腹泻率。
综上所述,健康仔猪肠道乳酸杆菌种类较多,其中嗜酸乳杆菌、罗伊氏乳杆菌、干酪乳杆菌与有益菌双歧杆菌、纳豆芽孢杆菌对胃肠道环境因素具有较强的耐受力。以此为基础制备的活菌制剂可用于防治仔猪腹泻。
With the development of industrial pig farming, the number and size of herd growing Increasingly, contacts become more frequently between the individual, opportunities for the spread of disease are increasing, the losses caused by diarrhea has become increasingly prominent. At present, Piglet Diarrhea Prevention mainly to the use of antibiotics, Antibiotics have the advantages of fast cure, but long-term and a great quantity use of antibiotics will cause residual contamination and break the balance of the host bacteria, Probiotics is natural, non-toxic, no side effects, non-polluting, non-residue, secure, reliable, and not resistance, and is regarded as a substitute for antibiotics, is a hot research at home and abroad. For the prevention and treatment of piglets diarrhea and Reducing the incidence of piglets diarrhea, this study is to develop a compound probiotics with Lactobacillus, Bifidobacterium, Bacillus natto, whice promote the growth of pigs ,improve feed efficiency and provide a theoretical basis and technical support for industrial production of compound probiotics .
In this study, 64 faecal samples were obtained in piglets of 12～60 age from different pig farms in Province. The strains isolated from the faecal samples were analyzed by morphology、biochemical identification and PCR assay; The bionomics of Lactobacillus, Bifidobacterium, Bacillus natto was studied and researched by antimicrobial activity、pH tolerance、bile-salt resistance、digestive enzyme resistance and heat resistance,et al, thereby Strong resistance strains were screened; The ingredients for fermentative medium and Cryoprotectants were optimized by using the Quadratic Orthogonal Rotation Combination Design; and Response Surface Methodology was applied to analyze test data. The effect on application of compound probiotics was researched by Feeding trial of piglets.
48 strains are identified as Gram-Positive,anaerobic and nonspore-bearing bacillus through the observation of colonial morphology and cultural character. Then, 18 strains are defined eventually as Lactobacillus through the physiological and biochemical identification and PCR assay. 7 strains were defined as L.reuteri,4 strains as L.acidophilus,2 strains as L.johnsonnii,1 strain as L. brevis, 1 strain as L.casei subsp.pseudoplantarum,1 strain as L. plantarum and 1 strain as L. jensenii.It is can be sure that Bifidobacterium, Bacillus natto and 6 strains as Lactobacillus can inhibit E.coli and Salmonella of swine and Bifidobacterium, Bacillus natto, strains DYF5、DYF1 and DYF8 have better tolerance on On the temperature, pH, bile-salt and digestive enzymes by tolerance screening test. By the single factor experiment, it is sured that The optimal growth condition of Lactobacillus is as follows: 37℃, pH6.5, inoculum amount 3%, medium volume 3/5, stationary culture 16h, then by using the Quadratic Orthogonal Rotation Combination Design and Response Surface Methodology, the optimum conditions of Lactobacillus fermentative medium are as follows: 5% soy peptone, 1% glucose, 1% yeast powder and 0.3% oligosaccharide, the average 16h OD620 reached a higher level of 1.073 and the optimum conditions of Cryoprotectant are as follows: 15% Sucrose, 3% glycerine, 15% Sorbitol and 15% skimmed milk. the Average survival rate reached a higher level of 71.6%, The ratio is 1:1(bacteria suspension (v):Cryoprotectant (v) ).The results of feeding trial of piglets showed that compound probiotics can remarkably raise piglets daily weight gain and feed efficiency, reduce the rate of piglets diarrhea, enhance economic efficiency
In short, Lactobacillus has more types in Healthy piglets intestine,and L.acidophilus、L. reuteri、L.casei、Bifidobacterium and Bacillus Natto has strong tolerance with environmental factors of the gastrointestinal tract. Then the probiotics that can be use to prevention and cure piglet diarrhea was completed base on the results.
本研究选择黑龙江省不同地区猪场,采集12～60日龄健康仔猪肠道粪样,分离乳酸杆菌,通过细菌形态、培养特性、生化试验和PCR种属分析进行鉴定。然后对双歧杆菌、纳豆芽孢杆菌及分离的乳酸杆菌进行抑菌试验、耐酸试验、耐胆盐试验、耐消化酶试验及温度敏感性等试验,筛选对仔猪消化道内胃酸及消化酶等因素耐受力强的菌株。应用二次正交旋转组合设计方案,进行筛选菌株发酵培养基及冷冻干燥保护剂各组分的优化试验;并通过响应面法进行分析。制备活菌制剂,通过仔猪饲养试验,确定复合微生态制剂的临床应用效果。
结果从仔猪肠道粪便分离到革兰氏阳性,厌氧,无芽孢杆菌48株。生化试验和PCR种属分析,确定18株为乳酸杆菌。其中,罗伊氏乳杆菌7株,嗜酸乳杆菌5株,约氏乳杆菌2株,短乳杆菌1株,干酪乳杆菌假植物亚种1株,植物乳杆菌1株,詹氏乳杆菌1株。通过耐受力筛选试验确定双歧杆菌、纳豆杆菌及6株乳酸杆菌对猪源大肠杆菌和沙门氏菌具有较好抑制作用;双歧杆菌、纳豆杆菌及3株乳酸杆菌对温度、pH、胆盐及消化酶具有较好的耐受性。通过单因素实验,确定乳酸杆菌适宜的发酵培养条件为:温度37℃,pH6.5,接种量3%,装液量3/5,静止培养时间16h。通过二次正交旋转组合设计试验和各因素响应面法分析,确定乳酸杆菌发酵培养基各组分比例为:大豆蛋白胨5%、葡萄糖1%、酵母浸粉1.7%、低聚糖0.3%,发酵液16h的实际最大OD620值为1.073。冷冻干燥保护剂各组分比例为:蔗糖15%、甘油3%、山梨醇15%、脱脂乳15%;按菌悬液(v)/保护剂(v)为1:1,加入冷冻干燥保护制剂,菌体冻干存活率的平均值为71.6%。仔猪饲养试验结果表明:以罗伊氏乳杆菌、双歧杆菌和纳豆杆菌制备的复合活菌制剂可提高仔猪日增重和饲料效率,降低仔猪腹泻率。
综上所述,健康仔猪肠道乳酸杆菌种类较多,其中嗜酸乳杆菌、罗伊氏乳杆菌、干酪乳杆菌与有益菌双歧杆菌、纳豆芽孢杆菌对胃肠道环境因素具有较强的耐受力。以此为基础制备的活菌制剂可用于防治仔猪腹泻。
With the development of industrial pig farming, the number and size of herd growing Increasingly, contacts become more frequently between the individual, opportunities for the spread of disease are increasing, the losses caused by diarrhea has become increasingly prominent. At present, Piglet Diarrhea Prevention mainly to the use of antibiotics, Antibiotics have the advantages of fast cure, but long-term and a great quantity use of antibiotics will cause residual contamination and break the balance of the host bacteria, Probiotics is natural, non-toxic, no side effects, non-polluting, non-residue, secure, reliable, and not resistance, and is regarded as a substitute for antibiotics, is a hot research at home and abroad. For the prevention and treatment of piglets diarrhea and Reducing the incidence of piglets diarrhea, this study is to develop a compound probiotics with Lactobacillus, Bifidobacterium, Bacillus natto, whice promote the growth of pigs ,improve feed efficiency and provide a theoretical basis and technical support for industrial production of compound probiotics .
In this study, 64 faecal samples were obtained in piglets of 12～60 age from different pig farms in Province. The strains isolated from the faecal samples were analyzed by morphology、biochemical identification and PCR assay; The bionomics of Lactobacillus, Bifidobacterium, Bacillus natto was studied and researched by antimicrobial activity、pH tolerance、bile-salt resistance、digestive enzyme resistance and heat resistance,et al, thereby Strong resistance strains were screened; The ingredients for fermentative medium and Cryoprotectants were optimized by using the Quadratic Orthogonal Rotation Combination Design; and Response Surface Methodology was applied to analyze test data. The effect on application of compound probiotics was researched by Feeding trial of piglets.
48 strains are identified as Gram-Positive,anaerobic and nonspore-bearing bacillus through the observation of colonial morphology and cultural character. Then, 18 strains are defined eventually as Lactobacillus through the physiological and biochemical identification and PCR assay. 7 strains were defined as L.reuteri,4 strains as L.acidophilus,2 strains as L.johnsonnii,1 strain as L. brevis, 1 strain as L.casei subsp.pseudoplantarum,1 strain as L. plantarum and 1 strain as L. jensenii.It is can be sure that Bifidobacterium, Bacillus natto and 6 strains as Lactobacillus can inhibit E.coli and Salmonella of swine and Bifidobacterium, Bacillus natto, strains DYF5、DYF1 and DYF8 have better tolerance on On the temperature, pH, bile-salt and digestive enzymes by tolerance screening test. By the single factor experiment, it is sured that The optimal growth condition of Lactobacillus is as follows: 37℃, pH6.5, inoculum amount 3%, medium volume 3/5, stationary culture 16h, then by using the Quadratic Orthogonal Rotation Combination Design and Response Surface Methodology, the optimum conditions of Lactobacillus fermentative medium are as follows: 5% soy peptone, 1% glucose, 1% yeast powder and 0.3% oligosaccharide, the average 16h OD620 reached a higher level of 1.073 and the optimum conditions of Cryoprotectant are as follows: 15% Sucrose, 3% glycerine, 15% Sorbitol and 15% skimmed milk. the Average survival rate reached a higher level of 71.6%, The ratio is 1:1(bacteria suspension (v):Cryoprotectant (v) ).The results of feeding trial of piglets showed that compound probiotics can remarkably raise piglets daily weight gain and feed efficiency, reduce the rate of piglets diarrhea, enhance economic efficiency
In short, Lactobacillus has more types in Healthy piglets intestine,and L.acidophilus、L. reuteri、L.casei、Bifidobacterium and Bacillus Natto has strong tolerance with environmental factors of the gastrointestinal tract. Then the probiotics that can be use to prevention and cure piglet diarrhea was completed base on the results.
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