聚谷氨酸高产菌株选育及其发酵产物应用

发布时间：2018-04-10 20:47

本文选题：γ-PGA + 菌株选育　；参考：《河南农业大学》2015年硕士论文

【摘要】：本试验进行了产聚谷氨酸(γ-PGA)新菌株的选育,并对菌株进行了形态学、分子生物学上的鉴定,对发酵产物进行定性定量的分析,探索了其适宜的发酵培养条件及发酵底物,利用豆渣进行固体发酵,对其发酵产物γ-PGA的保水性进行研究并探索适宜的施用量,为进一步开发作为农业保水剂提供研究基础。结果如下:(1)从豆腐乳中筛选出一株产γ-PGA菌株pga-1,该菌株生长快、表面粘稠,经革兰氏染色表明其为革兰氏阳性菌。提取细菌DNA,基于16S r RNA完成测序,鉴定此菌株为Bacillus subtilis。其发酵产物纯化水解后,利用HPLC进行液相色谱分析,其水解单一,产物为谷氨酸,与纸层析结果一致。(2)筛选出其适宜的扩繁培养基,葡萄糖30.0 g/L,酵母膏8.0 g/L,K2HPO4·3H2O 2.0g/L,Mg SO4·7H2O 0.25 g/L,谷氨酸钠30.0 g/L,p H=7.0-7.5。经摇床培养扩繁15 h菌体数量最多,最适宜作为发酵种子液。(3)该菌株10次传代培养中,发酵物γ-PGA产量从9.13 g/L增加到18.15 g/L,增幅达98.80%,产能稳定性良好。在以柠檬酸、甘油为碳源的初始发酵培养基中,37℃恒温、200 r/min摇床培养60 h,γ-PGA产量可达18.41 g/L。(4)初始培养基为:柠檬酸10 g/L,甘油60 g/L,NH4Cl 7.0,谷氨酸钠20.0 g/L,K2HPO4·3H2O 0.5 g/L,Fe Cl3·6H2O 0.05 g/L,Ca Cl2·2H2O 0.15 g/L,Mg SO4·7H2O 0.5 g/L,Mn SO4·H2O 0.1 g/L,p H=7.0-7.5。优化后菌株最适宜发酵条件为甘油80 g/L,柠檬酸12g/L,谷氨酸钠30 g/L,接种量为5%,其他条件不变情况下γ-PGA最高产量可达28.9g/L。(5)在含γ-PGA产物的发酵液土壤保水试验中,发酵产物2倍、5倍稀释液处理土壤最终含水量分别为3.09%和1.37%,分别高于CK 3.5倍、1.5倍,保水效果显著。(6)豆渣固体发酵产物γ-PGA保水性能及与化学保水剂二者处理土壤保水试验中,在室温培养15 d后,其中化学保水剂处理土壤含水量为6.39%,约为CK处理1.17倍,豆渣固体发酵γ-PGA处理土壤含水量最高为9.20%,约为CK处理1.68倍。因此,豆渣固体发酵γ-PGA相对于化学保水剂保水效果更显著。(7)在豆渣固体发酵产物γ-PGA不同浓度配比保水试验中,虽然施用量越大保水效果越显著,但也明显抑制了种子的发芽率及幼苗生长。在1:20、1:80、1:100、1:150和1:300五个配比梯度中,发芽率最高的是1:150处理,为95.33%,小麦幼苗芽长1:150处理的也较长,为11.07 cm,因此推荐施用发酵产物最佳配比浓度为1:150,即γ-PGA与土壤质量比为1:600(g/g)时更有利于种子萌发及生长。综上所述,此次筛选出的Bacillus subtilis产能突出,经优化后更具开发研究潜力,其液体及豆渣固体发酵产物γ-PGA保水性能显著,固体发酵产物初步保水剂与土壤按照1:150质量比施用不仅能提升土壤保水性,也能促进小麦种子发芽及幼苗生长。
[Abstract]:In this experiment, a new strain producing polyglutamic acid (纬 -PGA) was selected, and its morphology and molecular biology were identified. The fermentation products were analyzed qualitatively and quantitatively, and the suitable fermentation conditions and substrate were explored.Using soybean dregs for solid fermentation, the water retention of 纬 -PGA was studied and the appropriate application amount was explored, which provided the basis for further development as agricultural water-retaining agent.The results were as follows: (1) A 纬 -PGA producing strain pga-1 was screened from tofu milk. The strain grew rapidly and became viscous on the surface. The strain was Gram-positive by Gram staining.The bacterial DNA was extracted and sequenced based on 16s r RNA. The strain was identified as Bacillus subtilis.After purification and hydrolysis of the fermentation product, HPLC was used for liquid chromatographic analysis. The hydrolyzed product was single and the product was glutamic acid, which was consistent with the result of paper chromatography.Glucose 30.0 g / L, yeast extract 8.0 g / L, K2HPO 4 3H2O 2.0 g / L, mg SO4 7H2O 0.25 g / L, sodium glutamate 30.0 g / L H0 7.0-7.5.In 10 passages of the strain, the yield of 纬 -PGA increased from 9.13 g / L to 18.15 g / L, an increase of 98.80 g / L, with a good productivity stability.The optimum fermentation conditions are glycerin 80 g / L, citric acid 12 g / L, sodium glutamate 30 g / L, inoculation volume 5, and the highest yield of 纬 -PGA can reach 28.9 g / L 路L ~ (5) under other conditions.The final water content of soil treated with 2 times and 5 times dilution solution was 3.09% and 1.37, respectively, which was higher than that of CK 3.5 times and 1.5 times, respectively, and the water retention effect was remarkable. 6) the water retention of soybean dregs by solid fermentation product 纬 -PGA and chemical water-retaining agent was significant.After 15 days of culture at room temperature, the soil water content of chemical water-retaining agent treatment was 6.39, about 1.17 times that of CK treatment, and the highest soil moisture content of soybean residue solid fermentation 纬 -PGA treatment was 9.20 and about 1.68 times of CK treatment.Therefore, the water retention effect of solid fermentation of soybean dregs 纬 -PGA was more significant than that of chemical water-retaining agent.But it also inhibited seed germination rate and seedling growth.Of the five gradients of 1: 20, 1: 80, 1: 100, 1: 150 and 1: 300, the highest germination rate was 1: 150, which was 95.33. The wheat seedling bud length of 1: 150 was also longer.The optimum concentration of fermentation product is 1: 150, that is, the ratio of 纬 -PGA to soil mass is 1: 600 g / g) is more favorable for seed germination and growth.To sum up, the selected Bacillus subtilis has outstanding production capacity, and has more potential for development and research after optimization, and its liquid and soybean dregs solid fermentation product 纬 -PGA has remarkable water retention performance.The application of the primary water-retaining agent to the soil at the mass ratio of 1: 150 could not only improve the soil moisture retention, but also promote the germination of wheat seeds and the growth of wheat seedlings.
【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】：S182;S482.99

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