【摘要】：本文對本實驗室所保藏的一株具有耐高溫、耐酸、耐膽鹽能力,且已被證明能提高仔豬生長性能的凝結芽孢桿菌BCRC11592的工業(yè)生產(chǎn)條件進行研究。首先對其工業(yè)培養(yǎng)基成分、接種種齡和發(fā)酵培養(yǎng)基裝液量進行優(yōu)化,并在較系統(tǒng)的培養(yǎng)基和發(fā)酵條件的基礎上,利用全自動液體發(fā)酵罐進行小試生產(chǎn)試驗,分析發(fā)酵罐的機械攪拌速度、通氣量和p H恒定等條件對菌體生長和芽孢形成的影響,確定最佳發(fā)酵工藝條件;再對發(fā)酵液進行濃縮、吸附制備凝結芽孢桿菌粉制劑。此后,對該產(chǎn)品進行體外模擬制粒溫度、體外消化、藥敏試驗和產(chǎn)品貯存試驗,對產(chǎn)品性能指標進行初步評判。為凝結芽孢桿菌制劑的開發(fā)和工業(yè)生產(chǎn)提供一定的理論依據(jù)和數(shù)據(jù)基礎。試驗一凝結芽孢桿菌工業(yè)培養(yǎng)基的優(yōu)化將保藏的凝結芽孢桿菌BCRC11592的菌種進行活化后,以菌種數(shù)和芽孢數(shù)為測定指標,以接種種齡、碳源、氮源和無機鹽為因素進行單因素試驗,在此基礎上,再利用正交設計原理以兩種碳源比例、兩種氮源比例、碳氮比和培養(yǎng)基物質(zhì)總量為因素,選擇四因素三水平的正交試驗,對這種復合培養(yǎng)基再進行進一步優(yōu)化,最后確定實驗室中的最適裝液量。優(yōu)化后的培養(yǎng)基組成為:玉米粉5 g/L,豆粕7.5 g/L,NH4Cl 7.5 g/L,麩皮10 g/L,K2HPO4?3H2O 1 g/L,Mn SO4?H2O 0.3 g/L。在前期試驗中已經(jīng)優(yōu)化過的發(fā)酵培養(yǎng)條件下,此種培養(yǎng)基的發(fā)酵液菌種數(shù)可以達到2.20×109 cfu/m L,芽孢數(shù)為2.02×109 cfu/m L。試驗二凝結芽孢桿菌制劑的制備工藝前期優(yōu)化過的培養(yǎng)基首先經(jīng)蒸煮、過濾制成適合液體發(fā)酵罐的培養(yǎng)基,將發(fā)酵罐和加入的培養(yǎng)基共同滅菌處理后,添加1%的菌體種子液完成接種。選取單因素實驗方式對發(fā)酵罐的機械攪拌速度和通氣量進行優(yōu)化,最適攪拌速度為200r/min,最適通氣量為350L/h,發(fā)酵過程中不需要保持p H恒定。發(fā)酵完成的發(fā)酵液采用微濾濃縮工藝:操作壓力0.14~0.16MPa,溫度37℃~40℃,濃縮時間40min,發(fā)酵液濃縮2.86倍。用礦物質(zhì)吸附劑吸附發(fā)酵濃縮液,最適吸附比例為1:1,吸附后直接獲得凝結芽孢桿菌制劑產(chǎn)品。試驗三凝結芽孢桿菌制劑產(chǎn)品評價體外模擬制粒溫度和胃腸道環(huán)境,對產(chǎn)品的耐熱性、耐膽鹽性、耐酸性進行評測,得出產(chǎn)品在85℃處理10min存活率為71%,經(jīng)胃液消化后存活率為86.8%,過腸液后存活率為69%;藥敏試驗證實了產(chǎn)品對阿莫西林不敏感可以聯(lián)合使用,對新霉素和氟苯尼考較為敏感,建議避免同時使用;產(chǎn)品在25℃和35℃儲存30天后仍保持50%作用的存活率,儲存60天后活菌數(shù)依舊可以到達108數(shù)量級。
[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，

本文編號：2467780