凝结芽孢杆菌培养基的优化及其制备工艺的研究
发布时间：2019-04-28 16:21
【摘要】：本文对本实验室所保藏的一株具有耐高温、耐酸、耐胆盐能力,且已被证明能提高仔猪生长性能的凝结芽孢杆菌BCRC11592的工业生产条件进行研究。首先对其工业培养基成分、接种种龄和发酵培养基装液量进行优化,并在较系统的培养基和发酵条件的基础上,利用全自动液体发酵罐进行小试生产试验,分析发酵罐的机械搅拌速度、通气量和p H恒定等条件对菌体生长和芽孢形成的影响,确定最佳发酵工艺条件;再对发酵液进行浓缩、吸附制备凝结芽孢杆菌粉制剂。此后,对该产品进行体外模拟制粒温度、体外消化、药敏试验和产品贮存试验,对产品性能指标进行初步评判。为凝结芽孢杆菌制剂的开发和工业生产提供一定的理论依据和数据基础。试验一凝结芽孢杆菌工业培养基的优化将保藏的凝结芽孢杆菌BCRC11592的菌种进行活化后,以菌种数和芽孢数为测定指标,以接种种龄、碳源、氮源和无机盐为因素进行单因素试验,在此基础上,再利用正交设计原理以两种碳源比例、两种氮源比例、碳氮比和培养基物质总量为因素,选择四因素三水平的正交试验,对这种复合培养基再进行进一步优化,最后确定实验室中的最适装液量。优化后的培养基组成为:玉米粉5 g/L,豆粕7.5 g/L,NH4Cl 7.5 g/L,麸皮10 g/L,K2HPO4?3H2O 1 g/L,Mn SO4?H2O 0.3 g/L。在前期试验中已经优化过的发酵培养条件下,此种培养基的发酵液菌种数可以达到2.20×109 cfu/m L,芽孢数为2.02×109 cfu/m L。试验二凝结芽孢杆菌制剂的制备工艺前期优化过的培养基首先经蒸煮、过滤制成适合液体发酵罐的培养基,将发酵罐和加入的培养基共同灭菌处理后,添加1%的菌体种子液完成接种。选取单因素实验方式对发酵罐的机械搅拌速度和通气量进行优化,最适搅拌速度为200r/min,最适通气量为350L/h,发酵过程中不需要保持p H恒定。发酵完成的发酵液采用微滤浓缩工艺:操作压力0.14~0.16MPa,温度37℃~40℃,浓缩时间40min,发酵液浓缩2.86倍。用矿物质吸附剂吸附发酵浓缩液,最适吸附比例为1:1,吸附后直接获得凝结芽孢杆菌制剂产品。试验三凝结芽孢杆菌制剂产品评价体外模拟制粒温度和胃肠道环境,对产品的耐热性、耐胆盐性、耐酸性进行评测,得出产品在85℃处理10min存活率为71%,经胃液消化后存活率为86.8%,过肠液后存活率为69%;药敏试验证实了产品对阿莫西林不敏感可以联合使用,对新霉素和氟苯尼考较为敏感,建议避免同时使用;产品在25℃和35℃储存30天后仍保持50%作用的存活率,储存60天后活菌数依旧可以到达108数量级。
[Abstract]:The industrial production conditions of Bacillus coagulans BCRC11592, which has been proved to be able to improve the growth performance of piglets, have been studied in this paper, which is a strain of Bacillus coagulans stored in our laboratory, which is resistant to high temperature, acid and bile salt and has been proved to improve the growth performance of piglets. First of all, the composition of industrial medium, the age of inoculation and the quantity of fermentation medium were optimized. On the basis of more systematic culture medium and fermentation condition, the pilot-scale production experiment was carried out in full-automatic liquid fermentor. The effects of mechanical stirring speed, aeration rate and pH constant on cell growth and spore formation were analyzed, and the optimum fermentation conditions were determined. Then the fermentation broth was concentrated and the powder preparation of Bacillus coagulans was prepared by adsorption. After that, simulated granulation temperature in vitro, digestion in vitro, drug sensitivity test and product storage test were carried out to evaluate the properties of the product. It provides a theoretical basis and data basis for the development and industrial production of Bacillus coagulants. Experiment 1 the optimization of industrial culture medium for Bacillus coagulans activated the bacteria of Bacillus coagulans BCRC11592, and tested the number of bacteria and spores, and the factors of inoculating age, carbon source, nitrogen source and inorganic salt as single factor test. On this basis, the orthogonal design principle was used to select four factors and three levels of orthogonal experiment to further optimize the composite medium with the proportion of two carbon sources, the ratio of two nitrogen sources, the ratio of carbon to nitrogen and the total amount of medium material. Finally, the optimal volume of liquid in the laboratory is determined. The optimized culture medium was composed of corn meal 5 g / L, soybean meal 7.5 g / L, NH _ 4Cl _ 7.5 g / L, bran 10 g / L, K _ 2HPO _ 4 / 3H _ 2O _ 1 g / L, mn SO4?H2O 0.3 g / L. Under the optimized fermentation culture conditions, the fermentation broth species and spores were 2.20 脳 10 ~ 9 cfu/m / L and 2.02 脳 10 ~ 9 cfu/m 路L ~ (- 1) 路L ~ (- 1) and 2.02 脳 10 ~ 9 cfu/m 路L ~ (- 1) respectively. The culture medium optimized for the preparation of two clotting Bacillus preparations was firstly boiled and filtered to make the medium suitable for the liquid fermentor. After the fermentation pot and the added medium were sterilized together, the culture medium was treated by sterilizing the fermentor and the added medium together. 1% of the cell seed liquid was added to complete the inoculation. Single factor experiment was used to optimize the mechanical stirring speed and aeration rate of the fermentor. The optimum stirring speed was 200 rmin and the optimum aeration rate was 350 L / h. It was not necessary to keep pH constant in the fermentation process. The fermentation broth was concentrated by microfiltration: the operating pressure was 0.14 脳 0.16 MPA, the temperature was 37 鈩，
本文编号：2467780
[Abstract]:The industrial production conditions of Bacillus coagulans BCRC11592, which has been proved to be able to improve the growth performance of piglets, have been studied in this paper, which is a strain of Bacillus coagulans stored in our laboratory, which is resistant to high temperature, acid and bile salt and has been proved to improve the growth performance of piglets. First of all, the composition of industrial medium, the age of inoculation and the quantity of fermentation medium were optimized. On the basis of more systematic culture medium and fermentation condition, the pilot-scale production experiment was carried out in full-automatic liquid fermentor. The effects of mechanical stirring speed, aeration rate and pH constant on cell growth and spore formation were analyzed, and the optimum fermentation conditions were determined. Then the fermentation broth was concentrated and the powder preparation of Bacillus coagulans was prepared by adsorption. After that, simulated granulation temperature in vitro, digestion in vitro, drug sensitivity test and product storage test were carried out to evaluate the properties of the product. It provides a theoretical basis and data basis for the development and industrial production of Bacillus coagulants. Experiment 1 the optimization of industrial culture medium for Bacillus coagulans activated the bacteria of Bacillus coagulans BCRC11592, and tested the number of bacteria and spores, and the factors of inoculating age, carbon source, nitrogen source and inorganic salt as single factor test. On this basis, the orthogonal design principle was used to select four factors and three levels of orthogonal experiment to further optimize the composite medium with the proportion of two carbon sources, the ratio of two nitrogen sources, the ratio of carbon to nitrogen and the total amount of medium material. Finally, the optimal volume of liquid in the laboratory is determined. The optimized culture medium was composed of corn meal 5 g / L, soybean meal 7.5 g / L, NH _ 4Cl _ 7.5 g / L, bran 10 g / L, K _ 2HPO _ 4 / 3H _ 2O _ 1 g / L, mn SO4?H2O 0.3 g / L. Under the optimized fermentation culture conditions, the fermentation broth species and spores were 2.20 脳 10 ~ 9 cfu/m / L and 2.02 脳 10 ~ 9 cfu/m 路L ~ (- 1) 路L ~ (- 1) and 2.02 脳 10 ~ 9 cfu/m 路L ~ (- 1) respectively. The culture medium optimized for the preparation of two clotting Bacillus preparations was firstly boiled and filtered to make the medium suitable for the liquid fermentor. After the fermentation pot and the added medium were sterilized together, the culture medium was treated by sterilizing the fermentor and the added medium together. 1% of the cell seed liquid was added to complete the inoculation. Single factor experiment was used to optimize the mechanical stirring speed and aeration rate of the fermentor. The optimum stirring speed was 200 rmin and the optimum aeration rate was 350 L / h. It was not necessary to keep pH constant in the fermentation process. The fermentation broth was concentrated by microfiltration: the operating pressure was 0.14 脳 0.16 MPA, the temperature was 37 鈩，
本文编号：2467780
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