益生菌的制备及其对固始鸡生产性能、免疫性能和肠道微生态的影响

发布时间：2018-08-27 10:02

【摘要】：本研究旨在从健康家禽肠道中分离筛选出能耐受胃肠道低p H和高浓度胆盐环境及具有抗菌活性的唾液乳酸杆菌和蜡样芽孢杆菌,并与实验室保存的酿酒酵母菌按7:2:1(唾液乳酸杆菌9×108/g,蜡样芽孢杆菌2.5×108/g,酿酒酵母菌1.5×108/g)的比例复合后,按10%的接种量在30℃条件下72h发酵豆粕(豆粕:麸皮=7:3),并监测发酵过程中的一些常规指标的变化。继而用益生菌发酵豆粕和生豆粕制备的基础日粮分别饲养1日龄的固始鸡(公鸡),饲养期为28天,随后比较测定发酵豆粕对固始鸡生长、免疫、肠道微生态及肠道形态的影响。试验一耐酸耐胆盐及具有抗菌活性的唾液乳酸杆菌和蜡样芽孢杆菌的分离鉴定从健康家禽肠道中分离出具有产酸能力的乳酸杆菌和耐高温的芽孢杆菌,分别经过耐酸耐胆盐的初筛和复筛及乳酸菌抗菌活性的测定,得到对胃肠道环境具有较强耐受力的芽孢杆菌及耐酸耐胆盐且具有抗菌活性的乳酸杆菌。对筛选出的菌种进行16S r RNA序列鉴定,用MEGA5.0对鉴定出的菌株进行进化树的构建及分析。成功筛选出符合试验要求的唾液乳酸杆(Lactobacillus salivarius)和蜡样芽孢杆菌(Bacillus cereus)。试验二益生菌发酵豆粕过程中常规营养指标的动态监测用麦氏比浊仪分别对培养的唾液乳酸杆菌悬液、蜡样芽孢杆菌悬液和酿酒酵母菌悬液进行菌液浓度调整,按10%接种量以7:2:1(唾液乳酸杆菌9×108/g,蜡样芽孢杆菌2.5×108/g,酿酒酵母菌1.5×108/g)的比例添加到基础豆粕(豆粕:麸皮=7:3)中,在温度为30℃、湿度为70%-90%的环境中发酵72h,每隔12h于无菌环境中采样并通风换气,以检测发酵过程中一些常规指标的变化情况。结果表明:1)水分含量在整个发酵过程中基本维持在37%左右,无显著变化;2)蛋白质含量在整个发酵过程中的变化为先降低后显著升高(P0.05)的趋势,随后维持在相对较高的状态;3)p H值在发酵36h后显著降低(P0.05),随后都稳定在低p H范围内;4)活菌含量先显著降低(P0.05),36h显著升高(P0.05)后一直维持较高的活菌量。5)除苏氨酸外,各种氨基酸含量均呈现上升趋势,且到发酵72h时,各氨基酸含量均显著性提高(P0.05)。试验三发酵豆粕对固始鸡生长、免疫、肠道微生态及肠道形态的影响本试验选用1日龄固始鸡作为试验动物,共96只,分为2个处理,每个处理4个重复,分别用益生菌发酵豆粕和生豆粕制成的基础日粮饲喂,在实验的第1d、14d、28d测其生长性能后,每个处理随机取4只鸡放血致死,测其免疫器官指数,无菌分离各肠段并采集其内容物。结果表明:1)益生菌发酵豆粕显著提高了整个饲养期的平均日增重(p0.05);2)益生菌发酵豆粕对整个饲养期胸腺指数都有显著提高(p0.05),对饲养末期法氏囊指数也有显著提高(p0.05);3)益生菌发酵豆粕对14d十二指肠重量,28d回肠重量和28d空肠长度都有显著提高(p0.05);4)益生菌发酵豆粕显著增加了14d回肠、28d直肠乳酸菌数量(p0.05),显著降低了14d、28d盲肠大肠杆菌和28d盲肠沙门氏菌数量(p0.05);随着日龄的增长,十二指肠、空肠、回肠的微生物区系趋于平衡,且后期微生物种类逐渐增多,而盲肠和直肠后期微生态的多样性优于前期;回收条带以假单杆菌、芽孢杆菌和乳酸菌较多;5)益生菌发酵豆粕显著增加(p0.05)了28d固始鸡十二指肠、直肠的绒毛高度。实验结果表明,益生菌发酵豆粕能够促进固始鸡的生长,提高免疫器官指数,对肠道发育也有不同程度的促进作用,能够促进小肠微生物区系的平衡和大肠微生物的多样性,一定程度促进了肠道绒毛的发育。
[Abstract]:Salivary Lactobacillus and Bacillus cereus were isolated from the intestine of healthy poultry, which were able to tolerate low P H and high concentration of bile salts in the gastrointestinal tract and had antibacterial activity, and compared with the laboratory preserved Saccharomyces cerevisiae by 7:2:1 (salivary Lactobacillus 9 x108/g, Bacillus cereus 2.5 x108/g, Saccharomyces cerevisiae 1.5 x108/g). The soybean meal (soybean meal: bran = 7:3) was fermented at 30 C for 72 h at 10% inoculation rate, and the changes of some routine indexes during fermentation were monitored. Then, Gushi chickens (cocks) of 1 day old were fed with the basic diet of soybean meal fermented by probiotics and raw soybean meal respectively, and the feeding period was 28 days. Experiment 1 Isolation and identification of acid-tolerant and bile-salt-tolerant salivary Lactobacillus and Bacillus cereus with antibacterial activity from healthy poultry intestines, lactobacillus with acid-producing ability and Bacillus thermotolerant were screened and recombined. Bacillus spp. and Lactobacillus spp. with strong tolerance to gastrointestinal environment and acid and bile salt tolerance and antibacterial activity were obtained by screening and determination of antimicrobial activity of Lactobacillus spp. The strains were identified by 16S R RNA sequence analysis, and the strains were constructed and analyzed by MEGA 5.0. Lactobacillus salivarius and Bacillus cereus. Dynamic monitoring of routine nutritional indicators during soybean meal fermentation by probiotics in the second experiment. The concentration of salivary Lactobacillus suspension, Bacillus cerivarius suspension and Saccharomyces cerevisiae suspension were adjusted by McNephelometer. The concentration of salivary Lactobacillus suspension, Bacillus cerivarius suspension and Saccharomyces cerevisiae suspension The seed quantity was added to the base soybean meal (soybean meal: bran = 7:3) at a ratio of 7:2:1 (salivary Lactobacillus 9 x108 / g, Bacillus cereus 2.5 x108 / g, Saccharomyces cerevisiae 1.5 x108 / g), fermented for 72h in the environment of 30 C and 70% - 90% humidity, sampled every 12 hours in the aseptic environment and ventilated to detect some routine indexes in the fermentation process. The results showed that: 1) water content maintained at about 37% during the whole fermentation process, and there was no significant change; 2) protein content decreased at first and then increased significantly during the whole fermentation process (P 0.05), and then maintained a relatively high state; 3) P H value decreased significantly after 36 hours of fermentation (P 0.05), and then remained stable. In the low P H range, 4) the viable bacteria content decreased significantly (P 0.05), and increased significantly (P 0.05) for 36 hours, then maintained a high viable bacteria content. 5) except threonine, the contents of various amino acids increased significantly (P 0.05) at 72 hours of fermentation. Ninety-six one-day-old Gushi chickens were divided into two treatments. Each treatment had four replicates. The chickens were fed with the basic diet made of probiotic fermented soybean meal and soybean meal respectively. The growth performance was measured on the 1st, 14th and 28th day of the experiment. After each treatment, four chickens were randomly selected to bleed to death and their immune organs were measured. The results showed that: 1) probiotic fermented soybean meal significantly increased the average daily gain of the whole feeding period (p0.05); 2) probiotic fermented soybean meal significantly increased the thymus index of the whole feeding period (p0.05); 3) probiotic fermented soybean meal significantly increased the index of bursa of Fabricius at the end of feeding period (p0.05); The weight of duodenum, ileum and jejunum on day 14, 28 and 28 were significantly increased (p0.05). 4) The amount of rectal Lactobacillus on day 28 and ileum on day 14 and 28 were significantly decreased by probiotics fermented soybean meal (p0.05). The number of E. coli and Salmonella cecum on day 14, 28 and 28 was significantly decreased by probiotics fermented soybean meal (p0.05). The flora tended to be balanced, and the species of microorganisms increased gradually in the later stage, but the diversity of Microecology in the later stage of cecum and rectum was better than that in the earlier stage; the recovery bands were mainly Pseudomonas, Bacillus and Lactobacillus; 5) the fermentation of soybean meal by probiotics increased significantly (p0.05) the villus height of duodenum and rectum in Gushi chicken for 28 days. Fermented soybean meal can promote the growth of Gushi chickens, increase the index of immune organs, and promote the intestinal development in varying degrees. It can promote the balance of intestinal microflora and the diversity of intestinal microorganisms, and promote the development of intestinal villi to a certain extent.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831

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