醋糟发酵生产饲用微生物产品的研究

发布时间：2018-05-07 10:07

本文选题：醋糟 + 地衣芽孢杆菌　；参考：《河北大学》2015年硕士论文

【摘要】：地衣芽胞杆菌(Bacillus licheniformis)和枯草芽孢杆菌(Bacillus subtilis)是两种常用的饲料添加剂生产用菌株。目前,地衣芽孢杆菌和枯草芽孢杆菌发酵生产的原料主要取材于玉米粉、豆粕以及麸皮等等,随着人口压力增大,粮食供给越来越紧张的情况下,材料取材越来越难,难以实现长期生产发展。醋糟作为一种纤维素原料,取材方便且储备量较大,对其进行开发利用,生产各类益生菌,不失为一种长远之策。本实验首先将醋糟进行预先处理工艺以及利用纤维素酶酶解醋糟的工艺优化,其次,探讨了采用醋糟酶解液作为碳源代替传统培养基发酵生产地衣芽孢杆菌和枯草芽孢杆菌的可行性,然后通过正交实验、不同醋糟初始糖浓度实验,优化了醋糟酶解液发酵生产枯草芽孢杆菌的发酵培养基,最后,在优化培养基的基础上,进一步考察了芽孢杆菌的7 L发酵罐补料分批发酵实验,并且初步考察了其发酵参数。进一步分析了利用醋糟酶解液发酵生产丙酸的可行性,为丙酸的生产提供了一定技术支持。本论文的主要研究成果如下:(1)首先,我们采用水热处理技术对醋糟进行初步的预处理,经机械粉碎,纤维素酶酶解,得到葡萄糖含量为20.00 g/L的醋糟酶解液;其次,对酶解醋糟的纤维素酶酶解工艺进行了探索,主要研究了固液比、纤维素酶酶量、p H以及酶解时间对醋糟酶解的影响,最终确定了酶解醋糟的最佳条件为:固液比为2.0:10、460 U/g醋糟、p H4.8、纤维素酶酶解时间为48 h,最后获得葡萄糖浓度为27.00 g/L的醋糟酶解。(2)通过与葡萄糖发酵培养基的对比,对醋糟酶解液发酵生产芽孢杆菌的可行性进行了探讨,结果显示,醋糟酶解液可以作为碳源,替代传统发酵培养基发酵生产芽孢杆菌。在葡萄糖浓度相当的情况,醋糟酶解液发酵周期40 h,枯草芽孢杆菌的活菌数可达到4.64×1010 cfu/m L,与传统葡萄糖培养基(3.56×1010 cfu/m L)相比,提高了30.34%。发酵周期28 h,地衣芽孢杆菌的活菌数达到2.03×109 cfu/m L,与传统葡萄糖培养基(8.07×108 cfu/m L)相比,提高了151.55%。(3)采用正交实验L9(43)对枯草芽孢杆菌醋糟酶解液发酵培养基进行了优化,结果表明,醋糟酶解液初始糖浓度为29.30 g/L,最佳发酵培养基配方为玉米浆20.0 g/L、磷酸盐1.0 g/L、硫酸铵5.0 g/L,枯草芽孢杆菌活菌数可达到5.26×1010 cfu/m L。然后考察了不同初始糖浓度的醋糟酶解液对枯草芽孢杆菌发酵的影响,结果表明,初始糖浓度为50.00 g/L时,枯草芽孢杆菌TS-02活菌数可达到5.76×1010 cfu/m L,同时芽孢生成率在85%以上。7 L发酵罐结果表明,发酵周期28 h,枯草芽孢杆菌的活菌数可达到6.16×1010cfu/m L,比摇瓶发酵培养提高了32.76%。7 L发酵罐分批补料发酵结果显示,发酵周期为18 h,活菌数达到8.25×109 cfu/m L,芽孢产率为83%以上,比摇瓶培养提高了2.23%。(4)初步探讨了醋糟酶解液发酵生产丙酸的可行性,结果显示,醋糟酶解液可以作为碳源发酵生产丙酸,发酵周期132 h时,丙酸产量达24.35 g/L。考察了醋糟浓缩液发酵生产丙酸的情况,初步探索高糖浓缩液对丙酸发酵的影响情况。结果表明,发酵周期132 h,丙酸产量达18.8 g/L。丙酸产量较酶解液低,可能是由于高糖浓度醋糟酶解液抑制了产酸丙酸杆菌的生长或其中产生了某些抑制性的物质。
[Abstract]:Bacillus licheniformis (Bacillus licheniformis) and Bacillus subtilis (Bacillus subtilis) are two commonly used production strains for feed additives. At present, the raw materials produced by Bacillus licheniformis and Bacillus subtilis are mainly derived from corn flour, soybean meal and bran and so on. With the increase of population pressure, the supply of grain is getting tighter and tighter. It is difficult to obtain material for long term production. Vinegar Grains, as a kind of cellulose material, can be used as a kind of cellulose material, which is convenient and has a large reserve. It is a long-term strategy to develop and utilize all kinds of probiotics. In this experiment, vinegar grains were first treated and cellulase was used to hydrolyse vinegar grains. Process optimization, secondly, the feasibility of producing Bacillus licheniformis and Bacillus subtilis by using vinegar residue as carbon source instead of traditional culture medium was discussed. Then through orthogonal experiment, the experiment of the initial sugar concentration of different vinegar grains was carried out, and the fermentation medium of Bacillus subtilis fermentation was optimized by fermentation of Vinegar Grains. Finally, the optimization culture was carried out. On the basis of the nutrient base, the experiment of feed batch fermentation in the 7 L fermenting tank of bacillus was further investigated, and its fermentation parameters were preliminarily investigated. The feasibility of producing propionic acid by fermentation of vinegar grains was further analyzed, and some technical support was provided for the production of propionic acid. The main research results of this article are as follows: (1) first of all, we pick up the production of propionic acid. The process of enzymatic hydrolysis of Vinegar Grains with glucose content of 20 g/L was obtained by mechanical pulverization and enzymatic hydrolysis. Secondly, the enzymatic hydrolysis process of enzymatic hydrolysis of vinegar grains was explored. The effects of solid-liquid ratio, cellulose enzyme amount, P H and enzyme hydrolysis time on the hydrolysis of vinegar grains were mainly studied. The optimum conditions for enzymatic hydrolysis of vinegar residue were as follows: the ratio of solid to liquid was 2.0:10460 U/g Vinegar Grains, P H4.8, the enzymatic hydrolysis time of cellulase was 48 h, and the enzymatic hydrolysis of Vinegar Grains with glucose concentration of 27 g/L was obtained. (2) the feasibility of producing Bacillus by fermentation with glucose fermentation medium was discussed by comparison with glucose fermentation medium. The enzymatic hydrolysate of vinegar grains can be used as a carbon source instead of the traditional fermentation medium to produce Bacillus spore. In the case of equal glucose concentration, the fermentation period of acetic acid hydrolysate is 40 h, and the number of Bacillus subtilis can reach 4.64 x 1010 cfu/m L. Compared with the traditional glucose medium (3.56 x 1010 cfu/m L), the fermentation period of 30.34%. is increased by 28. H, the living bacteria of Bacillus licheniformis reached 2.03 * 109 cfu/m L, compared with the traditional glucose medium (8.07 x 108 cfu/m L), improved 151.55%. (3) using orthogonal experiment L9 (43) to optimize the fermentation medium of Bacillus subtilis vinegar residue enzyme solution fermentation medium. The results showed that the initial sugar concentration of the hydrolysate of vinegar grains was 29.30 g/L and the optimum fermentation medium. The formula was corn pulp 20 g/L, phosphate 1 g/L, ammonium sulfate 5 g/L, the number of Bacillus subtilis viable bacteria can reach 5.26 x 1010 cfu/m L., and then the effect of different initial sugar concentration of acetic acid hydrolysate on Bacillus subtilis fermentation was investigated. The results showed that the number of Bacillus subtilis TS-02 live bacteria could reach 5.76 * 1 when the initial sugar concentration was 50 g /L. 010 cfu/m L, and the result of the spore formation rate of more than 85%.7 L fermentor showed that the fermentation period was 28 h, the living bacteria of Bacillus subtilis could reach 6.16 x 1010cfu/m L, which showed that the fermentation period of the 32.76%.7 L fermenting tank was 18 h and the number of living bacteria reached 8.25 x 109 cfu/m L, and the spore yield was 83% more than that of the shake flask fermentation. The feasibility of producing propionic acid by enzymatic hydrolysis of vinegar grains was preliminarily studied by 2.23%. (4). The results showed that the hydrolysate of vinegar grains could produce propionic acid as a carbon source. When the fermentation period was 132 h, the yield of propionic acid was 24.35 g/L. and the condition of propionic acid produced by the fermentation of Vinegar Grains concentrated solution was investigated and the high sugar concentrate was initially explored to propionic acid. The results showed that the fermentation period was 132 h, the yield of propionic acid was 18.8 g/L. and the yield of propionic acid was lower than that of the hydrolysate, which may be due to the inhibition of the growth of propionic acid by the high glucose concentration of acetic acid enzyme solution or some inhibitory substances.

【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816.3;TQ920.6

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