复合微生物饲料添加剂的研究

发布时间：2018-04-02 00:31

本文选题：益生菌　切入点：发酵优化　出处：《兰州交通大学》2015年硕士论文

【摘要】：滥用抗生素严重地影响着畜产品的安全和质量,寻找一种抗生素替代品成为当前畜牧养殖急需解决的问题。复合微生物饲料添加剂来源于肠道正常菌群,能够通过优势菌群、生物拮抗等作用改善肠道微生物环境。调整并维持肠道微生物平衡,增强免疫能力,促进营养物质的消化吸收,起到促生长和防病的作用。因此,益生菌复合制剂已成为取代抗生素的首选开发研究对象。本研究采用耐酸和耐胆盐试验、选择性培养等方法,成功地从断奶羔羊新鲜粪便中分离纯化出一株具有良好耐受模拟胃酸和模拟胆盐环境的菌株,经鉴定为乳酸菌R1。通过单因素试验、正交优化试验、添加菌体促生长因子试验等对乳酸菌进行发酵培养基和培养条件的研究。同时对本实验室保藏的一株枯草芽孢杆菌与一株酿酒酵母进行了培养基和培养条件的优化。并对乳酸菌R1进行了微胶囊包埋,采用真空恒温干燥法将枯草芽孢杆菌发酵液制备干菌粉,采用自然风干法将酿酒酵母固体发酵物干燥研磨制备干菌粉。最后将乳酸菌R1微胶囊、枯草芽孢杆菌干菌粉和酿酒酵母干菌粉复配。制备了复合微生物饲料添加剂。利用此制剂在羔羊上进行了动物活体实验验证。实验结果如下:(1)乳酸菌R1在初始为培养条件为:pH6.0、温度34℃、静置培养12-14小时、接种量4%时生长状态最好。最适培养基为:蛋白胨0.5%,葡萄糖2%,牛肉膏1%,酵母膏0.5%,吐温80 0.1%,碳酸钙0.2%,乙酸钠0.5%,柠檬酸二铵0.2%,硫酸镁0.02%,硫酸锰0.005%;增菌因子配比为:番茄汁2%,土豆汁3%,胡萝卜汁6%,维生素C 0.2%;活菌数可以达到7.41×109 CFU/m L。乳酸菌R1微胶囊包覆率达到了90.5%同时具有较好的耐酸性、较好的肠溶性,在常温储藏90d存活率为79.5%,活菌数为3.2×109 CFU/g。(2)枯草芽孢杆菌在初始培养条件为:pH为7.0-7.5、温度35℃、装液量30mL/250、接种量7%、转速为200r/min、培养17h生长状态最好。最适培养基为葡萄糖3%、酵母膏1.5%、磷酸氢二钠0.2%、磷酸二氢钠0.1%;菌体生物量达到7.90×109 CFU/m L。木屑添加量为15g/25m L,芽孢产率接近95%,制备的干菌粉活菌数为5.71×1010CFU/g.(3)酿酒酵母在培养条件为:摇床转速150 r/min、温度25℃,接种量1%、装液量25m L/250m L、培养20h生长状态最好。最佳液体培养基为红糖5%、酵母膏2%、硫酸铵0.1%、氯化钾0.2%;酵母活菌数达到了2.38×108 CFU/mL固体培养基按麸皮∶糖蜜∶豆粕=7∶2∶1的比例配制,料水比1:0.6,用矿物质盐溶液拌料,培养时间60h,生物量最大值为8.73×109 CFU/g,比优化前提高了231.8倍。(4)动物活体试验表明:复合微生物饲料添加剂的使用,显著地提高了羔羊的日增重,降低肉料比,降低了断奶羔羊腹泻发生率,其中对照组腹泻发生率为26.7%,试验组腹泻发生率仅为6%,羔羊腹泻率降低了20.7%。本研究筛选得到较优良的微生物饲料添加剂菌株,对其进行发酵优化,制剂增重防病效果显著,为实际生产微生物饲料添加剂提供了一定的理论参考价值。
[Abstract]:The abuse of antibiotics seriously affect the quality and safety of animal products, to find a substitute for antibiotics has become the urgent problem to be solved. Animal husbandry compound microbial feed additives derived from normal intestinal flora, the dominant microflora, improve the intestinal microbial environment biological antagonistic action. Adjust and maintain the intestinal microflora balance, enhance immunity and promote digestion and absorption of nutrients, to promote growth and prevent disease. Therefore, probiotic combination has become a substitute for antibiotics preferred object of study. This study used acid and bile salts resistance test method of selective culture, successfully weaned lamb from fresh feces of isolated strains with good tolerance the simulation of gastric acid and bile salt environment simulation, and identified as Lactobacillus R1. by single factor test, orthogonal test, cell growth by adding Test of medium and culture conditions of the fermentation of lactic acid bacteria. Meanwhile a Bacillus subtilis strain of the yeast Saccharomyces cerevisiae and the optimization of medium and culture conditions of lactic acid bacteria. R1 was microencapsulated by vacuum drying, constant temperature method of Bacillus subtilis preparation of fermentation liquor dry bacterium powder, drying method of yeast fermentation solid preparation of dry grinding dry bacterium powder. The lactic acid bacteria R1 microcapsule, Bacillus subtilis bacteria and yeast powder dry dry bacterium powder compound. The composite microbial feed additives were prepared. The animal experiment in lamb by using this formulation. The experimental results are as follows: (1) R1 of lactic acid bacteria in the initial condition is: pH6.0, temperature 34 C, static culture for 12-14 hours, when the inoculation amount was 4%, the best growth state. The optimum medium was peptone 0.5%, glucose 2%, beef extract 1%, yeast extract 0.5%, Twain 800.1%, calcium carbonate 0.2%, 0.5% sodium acetate, ammonium citrate 0.2%, Magnesium Sulfate 0.02%, 0.005% manganese sulfate; enrichment factor ratio: Tomato Juice 2%, potato juice 3%, Fresh Carrot Juice 6%, vitamin C 0.2%; viable count can reach 7.41 * 109 CFU/m L. lactic acid bacteria R1 microencapsulation rate reached 90.5% and has good acid resistance, enteric and good storage in 90d survival rate was 79.5%, the living bacteria number is 3.2 * 109 CFU/g. (2) of Bacillus subtilis in initial culture conditions: pH 7.0-7.5, 35 degrees Celsius temperature, liquid volume 30mL/250. 7% inoculation quantity, speed 200r/min, 17h culture growth status of the best. The best medium was 3% glucose, 1.5% yeast extract, two sodium hydrogen phosphate 0.2%, 0.1% sodium dihydrogen phosphate; biomass reached 7.90 * 109 CFU/m L. sawdust was added into 15g/25m L, spore yield close to 95%, number of live bacteria was 5.71 bacteria powder preparation dry preparation * 1010CFU/g. (3) in yeast culture conditions: rotation speed of 150 r/min, temperature 25, inoculum size 1%, medium volume 25m L/250m L, 20h culture medium. The growth state of the best the best liquid is brown sugar 5%, yeast extract 2%, 0.1% ammonium sulfate, potassium chloride 0.2%; yeast bacteria number reached 2.38 x 108 CFU/mL solid medium by bran: molasses meal: =7: 2: 1 of the proportion, the ratio of material to water 1:0.6, with mineral salt solution mixing, fermentation time 60H, the maximum biomass was 8.73 * 109 CFU/g, 231.8 times more than that before optimization. (4) animal experiment table Ming: the use of composite microbial feed additives, and significantly improve the daily gain of lamb, decrease the ratio of feed to meat, reduce the incidence of diarrhea of weaned lamb, which controls the incidence of diarrhea was 26.7%, the incidence of diarrhea in experimental group was only 6%, lamb diarrhea rate decreased by 20.7%. in this study were obtained excellent. The fermentation of the feed additive strain has a significant effect on the weight gain and disease prevention. It provides a theoretical reference value for the actual production of microbial feed additives.

【学位授予单位】：兰州交通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816.7

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