【作者】 王娜；

【导师】 赵敏；

【作者基本信息】 东北林业大学 ， 微生物学， 2014， 博士

【摘要】 微生物制剂以其无毒、无残留、无耐药性及低成本、可有效补充消化道有益微生物等诸多优点,成为替代抗生素的理想制品之一,而被广泛应用。本文从仔猪的粪便中分离鉴定的益生菌-蜡样芽孢杆菌(Bacillus cereus),又外购了2个益生菌：鼠李糖乳杆菌(lactobacillus rhamnose)、戊糖乳杆菌(lactobacillus pentose)。以这3株细菌为研究对象,对3株益生菌分别进行培养和发酵条件优化,以期用廉价培养基成分获得到较高的活菌数；然后对3株益生菌的稳定性、抗逆性、菌种间的最佳配比及制得的微生物制剂的饲料酶活性进行研究,研究了复合微生物制剂对断奶仔猪生产性能、肠道定植和肠道菌群等的影响。获得如下研究结果：从待分离样品中,经分离、纯化,生理生化鉴定和16S rDNA测序分析,分离到一株肠道益生菌-蜡样芽孢杆菌。经过安全性检验可知,该菌株安全、无毒副作用,可作为制备微生物制剂的备用菌株。采用单因素试验及正交试验的方法对分离筛选到的蜡样芽孢杆菌(Bacillus cereus)及外购的鼠李糖乳杆菌(lactobacillus rhamnose)、戊糖乳杆菌(lactobacillus pentose)分别进行培养基组成和培养条件优化,获得lactobacillus pentose最佳培养基配方为0.5%乳糖+0.5%废糖蜜,0.5%棉粕粉,0.15%磷酸二氢钠+0.15%磷酸氢二钠；Bacillus cereus最佳培养基配方为0.5%蔗糖+0.5%废糖蜜,1%棉粕粉,0.1%氯化钠；鼠李糖乳杆菌的最佳培养基配方为0.5%乳糖+0.5%废糖蜜,0.5%棉粕粉,0.3%磷酸二氢钠+磷酸氢二钠。最优发酵条件,即lactobacillus rhamnose最佳发酵条件为：发酵时间24h,起始pH为6.5,温度为35℃,接种量5%,装液量20%；蜡样芽孢杆菌最佳发酵条件为：发酵时间28h,起始pH为7.5,温度37℃,接种量7%,装液量40%；鼠李糖乳杆菌最佳发酵条件为：发酵时间28h,起始pH为7.0,温度37℃,接种量7%,装液量40%。最后,以各自优化后的最佳培养基配方和发酵条件,活菌数分别达1.32×109cfu/ml、1.15×109cfu/ml和166×109cfu/ml。对3株益生菌的耐热性、肠道耐受性及贮存稳定性进行研究。结果表明,在65℃的条件下,蜡样芽孢杆菌、鼠李糖乳杆菌和戊糖乳杆菌的存活率分别为92.54%、90.43%和87.01%,说明3株益生菌均具有较好的耐高温性质。体外模拟动物肠道耐受性试验结果显示,3株益生菌均能在pH为1.5的胃液中处理3h后保持106活性以上达,说明3株益生菌均能够在正常仔猪胃肠道中耐受较低pH；并且,3株益生菌均能在胰蛋白酶溶液处理3h后维持活菌数为108,在0.40%的胆汁溶液中维持活菌数在106以上,说明,3株益生菌均能够耐受胰蛋白酶溶液和胰蛋白酶溶液等消化酶的作用,完全可以耐受仔猪的胃液并进入小肠发挥益生作用。贮存稳定性试验结果表明,加有保护剂(5%葡萄糖溶液)、-7℃(冷冻)条件下贮存的菌粉稳定性明显高于其他组,90d后活菌数量仍保持在109cfu/g水平。通过急性毒理试验表明,以1;1:1比例混合的3株益生菌对小鼠一般体征、行为、活动、毛发、色泽、饮食、粪便均未有明显不良反应,小鼠未出现死亡现象；各组体重持续增长,与对照组相比,试验组体重变化无显著性差异(P>0.05)；肝脏指数、脾脏指数和肾脏指数无显著性变化,可初步判定混合益生菌无毒性。通过将1：1：1混合的3株益生菌分别与大肠杆菌(Escherichia coli)和沙门氏菌(salmonella)共培养,并采用3株接种时间(益生菌与有害菌同时接种、先接种益生菌后接种有害菌和先接种有害菌再接种益生菌)来验证3株益生菌对大肠杆菌和沙门氏菌的抑菌能力,结果表明,混合益生菌在三个接种时间均具有一定的抑菌效果。但混合益生菌先24h接种的效果要明显好的多。而晚24h接种,仍然具有一定的拮抗效果,只是拮抗的时间要到48h效果明显。并且,进一步研究混合益生菌发酵液、发酵滤液和菌体悬液抑菌功效表明,混合益生菌发酵液、发酵滤液和菌体悬液均可抑菌大肠杆菌和沙门氏菌,并且发酵液和发酵滤液的抑菌效果明显优于菌体悬液。以1：1：1、1：2：1、2：1：2、1：1：2(依次为蜡样芽孢杆菌、鼠李糖乳杆菌和戊糖乳杆菌)的比例混合3株益生菌,制得4种混合微生物制剂饲喂断奶仔猪,研究各微生物制剂对仔猪生长性能的影响。结果表明,饲粮中添加1：1：1的比例混合的微生态活菌制剂,在提高仔猪的生长性能效果最显著,并显著降低了腹泻率。并且,添加混合微生态菌制剂的试验组与对照组相比,试验组的粗蛋白质、粗脂肪、钙和磷均有提高,并且,每头猪的经济效益还提升了16.194元。对3株益生菌的产饲料酶-蛋白酶、淀粉酶、纤维素酶和β-甘露聚糖酶进行了定性检测。再以此为依据,对3株益生菌和1：1：1的比例混合的微生物制剂的产酶活性做了进一步研究。结果表明,蜡样芽孢杆菌的蛋白酶和淀粉酶的活性最高分别达到70.52U/ml和25.47U/ml,且只有蜡样芽孢杆菌具有产甘露聚糖酶能力,酶活力为5.35U/ml；鼠李糖乳杆菌的纤维素酶活性最高,达4.06U/ml。将3株益生菌混合制成的微生物制剂,制剂具有4种饲料酶的活性,虽然4种饲料酶活力均未达到单一菌株能够达到的最高值,但各饲料酶均能检测到活性,且活力较高,蛋白酶、淀粉酶、纤维素酶和甘露聚糖酶分别达到62.35U/ml、21.67U/ml、3.83U/ml和5.21U/ml。应用Real-time PCR方法准确地对试验期间仔猪粪便标本中的3株特定益生菌进行定量检测。结果表明,喂食微生物制剂期间(28天),试验组中3株益生菌的数量多高于对照组(P<0.05)；在停喂微生物制剂两周后,于42天时试验组仔猪粪样中检测出3株益生菌存在,且3株益生菌数量均超过断奶当日水平,表明3株益生菌均已定植在仔猪肠道内。试验期间粪样的pH研究表明,在对照组中,由于断奶的影响,仔猪粪便pH呈现先上升后下降趋势,直到35d后才逐渐恢复；试验组仔猪粪样pH始终较对照组低,且基本保持在断奶水平。说明通过饲喂微生物制剂仔猪消化道保持偏酸性环境,可利于有益微生物的生长和定植。利用DGGE技术对断奶仔猪粪样中微生物菌群进行跟踪研究。结果显示,饲喂微生物制剂可以通过在仔猪消化道内定植来促进肠道益生菌(双歧杆菌、嗜酸乳杆菌)的生长繁殖,抑制有害菌(沙门氏菌、金黄色葡萄球菌)的生长,从而达到调节肠道菌群的效果。并且,相似性结果显示,对照组中,断奶0和7d之间相似性最低,为38%,以后逐渐增加,7和28d间相似性为50%；而试验组仔猪断奶0和7d相似性达67%,以后相似性迅速降低,7和28d间相似性为50%；多样性结果显示,实验期内试验组多样性多高于对照组,并且在整个试验期间保持相对稳定,说明断奶后日粮中添加微生物制剂使仔猪肠道菌群趋向复杂化、多样化,试验组仔猪粪样菌群的相似性、多样性均高于对照组。更多还原

【Abstract】 Probioties have become the ideal alternative to antibiotics for its non-toxic, no residue, no drug resistance as well as its low cost.In order to screen fine strains for probioties, we detected the stability, resistance to reverse, the optimal proportion between, engraftment and feed enzyme activity of strains. One strain of probiotic(Bacillus cereus) were separated from the raw material. We optimized the culture medium and culture conditions of Bacillus cereus, lactobacillus rhamnose and lactobacillus pentose that were purchased, using the method of single factor experiment and orthogonal analysis. This study examined the weaned pig production performance and the change of intestinal flora to verify the efficacy of probiotics. After a security test in mice, we can get to know, the Bacillus cereus is safe and non-toxic. This study selected a security available strains for preparation of probiotics. Contents of each experiment as follows:From separation of samples, separation, purification, physiological and biochemical identification and16s rDNA sequencing analysis, a strain isolated intestinal probiotics-waxy bacillus. Through security inspection shows that the strain is safe, non-toxic side effects, preparation of microbial agents can be used as a backup.The ingredients of optimum culture medium of lactobacillus pentose as follow:0.5%lactose+0.5%molasses,0.5%cotton pulp powder, sodium dihydrogen phosphate0.15%+0.15%sodium hydrogen phosphate. The ingredients of optimum culture medium of Bacillus cereus as follow:0.5%sucrose+0.5%molasses,1%cotton pulp powder,0.1%sodium chloride. The ingredients of optimum culture medium of lactobacillus rhamnose as follow:0.5%lactose+0.5%molasses,0.5%cotton pulp powder, sodium dihydrogen phosphate0.15%+0.15%sodium hydrogen phosphate. The best fermentation conditions of lactobacillus pentose were asa follows:fermentation time24h, initial pH6.5, temperature35℃, Inoculation Amount5%and liquid volume20%. The best fermentation conditions of Bacillus cereus: fermentation time28h, initial pH7.5, temperature37℃, Inoculation Amount7%and liquid volume40%. Following the best fermentation conditions of lactobacillus rhamnosus were asa follows:fermentation time28h, initial pH7.0, temperature37℃, Inoculation Amount7%and liquid volume40%. Finally, the number of living bacterium with their best culture medium formula and the optimized fermentation conditions were all impressive,1.32×109cfu/ml,1.15×109cfu/mland1.66×109cfu/ml.The results showed that under the condition of65℃, the survival rates of Bacillus cereus, lactobacillus rhamnose and lactobacillus pentose are92.54%,90.43%and92.54%respectively, shows that the three strains probiotics has good high temperature resistant properties. In vitro animal intestinal tolerance test, according to the results of3strains of probiotics are handled in gastric juice pH1.5for3h, they still could keep active more than106, so we think the3strains are able to show in normal piglets gastrointestinal tolerance low pH. And the3strains can in trypsin solution treatment for3h maintain living bacterium number is108, and in0.40%of bile in the solution to maintain living bacterium number above106, shows that the three strains of probiotics are able to tolerate trypsin solution and the effect of digestive enzymes, such as trypsin solution can tolerate gastric juice and into the small intestine of piglets play a good role. Anyhow, the results of storage stability showed that the stability of bacteria powder with protectant glucose solution (5%) and in frozen7℃is significantly higher than other groups, the number of living bacterium remains at109cfu/g level after90days.Through acute toxicity test showed that the probiotics have no significant adverse reactions of mice in general physical characteristics, behaviors, activities, hair, colour and lustre, diet, and excrement and urine, and does not appear death phenomenon in mice. The weight of each group keep sustained growth trend. Compared with control group, there was no significant difference (P>0.05) on the weight in experimental group. And there was no significant change on the liver index, the index of spleen and kidney index, so we can initially determined mixed probiotic non-toxic. The inhibition ability test in different inoculation time showed that whether inoculated simultaneously or successively, the mixed probiotic all have certain bacteriostatic effect. But there was better effect if the mixed probiotics inoculation earilier24h. There still has a certain antagonism24h after inoculation, just time to48h of antagonism effect obviously. And further studies show that mixed probiotic fermented liquid, the fermentation filtrate and bacteria suspension are antibacterial escherichia coli and salmonella, and obviously better in fermentation medium and fermentation filtrate bacteria suspension.Four kinds of mixed probiotics were made of three kinds of probiotics(followed by Bacillus cereus, Lactobacillus rhamnosus and Lactobacillus pentosus) in the proportion of1:1:1,1:2:1,2:1:2,1:1:2. Feeding weaned piglets to research the effect of probiotics on growth performance in piglet. The results show that, the combination of probiotics in1:1:1could significantly enhance the growth performance of piglet, and significantly reduce the diarrhea rate. Compared with control group, crude protein, crude fat, calcium and phosphorus were increased, and the economic benefits of each pig increased to16.194yuan.Production of3strains feed enzyme(protease, amylase, cellulose enzyme and beta mannose) has carried on the qualitative detection. Then on this basis, the three strains of probiotics and:the proportion of mixed microbial agents produce enzymes to do the further research. The results showed that the protease and amylase in Bacillus cereus were the highest reached70.52U/ml and70.52U/ml, and only Bacillus cereus has the capacity of mannase, was 5.35U/ml. The cellulose enzyme of Lactobacillus rhamnose was the highest-4.06U/ml. Mix3strains of probiotics made of microbial preparation, preparation have four kinds of feed enzyme activity, although the four kinds of feed enzyme activity failed to meet a single strain can reach the highest level, but the feed enzyme activities, can detect and high activity protease, amylase, cellulose enzyme and mannase reached62.35U/ml,21.67U/ml,3.83U/ml and5.21U/ml.Three specific probiotics in fecal samples of piglets were accurately quantitative analysis by real-time PCR. The results show that, during feeding the probiotics (28days), the numbers of three specific probiotics in probiotics group were much higher than these in the control group (P<0.05). Stop feeding probiotics two weeks later, detecting the existential state of the three kinds of probiotics in fecal samples of the probiotic piglets on the42day, we got to know, the three kinds of probiotics still exist in fecal samples, and the numbers of the three kinds of probiotics are more than the number of weaning. The results indicate that the three kinds of probiotics have been engraftment in the gastrointestinal tract of piglets, during the research, we detect the pH value in the fecal samples showed a trend of decline after rising first for weaned, it was not until after35d gradually restored. Fecal samples pH of piglet in probiotic group is always lower than the control group, and basic remain at the level of weaning. The results show that feeding the probiotics could keep the acid environment in digestive tract of piglets, it can conducive to the growth of beneficial microbes.Using DGGE technology of weaned piglets tracking study on microbial flora in fecal samples. According to the results of feeding probiotics can colonize the piglet in the digestive tract to promote intestinal probiotics(Bifidobacterium and Lactobacillus acidophilus) grow, restrain harmful bacteria(Salmonella, Staphylococcus aureus) growth, so as to adjust the effect of intestinal flora. And similarity, according to the results of the control group, the lowest similarity between0and7d, weaning was38%, and later gradually increase, the similarity between7and28d is50%. Weaned piglets and test group0and7d similarity was67%, and the similarity after falling rapidly, the similarity between7and28d is50%. Diversity, according to the results of the experiment period, much higher than the control group, experimental group diversity and relatively stable during the whole test, shows that adding microbial agents after weaning diet complicate the piglets intestinal trend, diversification, experimental piglets fecal samples the similarity and diversity of bacteria were higher than the control group.更多还原