本文選題：γ-PGA + 菌株選育�。� 參考：《河南農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：本試驗(yàn)進(jìn)行了產(chǎn)聚谷氨酸(γ-PGA)新菌株的選育,并對(duì)菌株進(jìn)行了形態(tài)學(xué)、分子生物學(xué)上的鑒定,對(duì)發(fā)酵產(chǎn)物進(jìn)行定性定量的分析,探索了其適宜的發(fā)酵培養(yǎng)條件及發(fā)酵底物,利用豆渣進(jìn)行固體發(fā)酵,對(duì)其發(fā)酵產(chǎn)物γ-PGA的保水性進(jìn)行研究并探索適宜的施用量,為進(jìn)一步開(kāi)發(fā)作為農(nóng)業(yè)保水劑提供研究基礎(chǔ)。結(jié)果如下:(1)從豆腐乳中篩選出一株產(chǎn)γ-PGA菌株pga-1,該菌株生長(zhǎng)快、表面粘稠,經(jīng)革蘭氏染色表明其為革蘭氏陽(yáng)性菌。提取細(xì)菌DNA,基于16S r RNA完成測(cè)序,鑒定此菌株為Bacillus subtilis。其發(fā)酵產(chǎn)物純化水解后,利用HPLC進(jìn)行液相色譜分析,其水解單一,產(chǎn)物為谷氨酸,與紙層析結(jié)果一致。(2)篩選出其適宜的擴(kuò)繁培養(yǎng)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,谷氨酸鈉30.0 g/L,p H=7.0-7.5。經(jīng)搖床培養(yǎng)擴(kuò)繁15 h菌體數(shù)量最多,最適宜作為發(fā)酵種子液。(3)該菌株10次傳代培養(yǎng)中,發(fā)酵物γ-PGA產(chǎn)量從9.13 g/L增加到18.15 g/L,增幅達(dá)98.80%,產(chǎn)能穩(wěn)定性良好。在以檸檬酸、甘油為碳源的初始發(fā)酵培養(yǎng)基中,37℃恒溫、200 r/min搖床培養(yǎng)60 h,γ-PGA產(chǎn)量可達(dá)18.41 g/L。(4)初始培養(yǎng)基為:檸檬酸10 g/L,甘油60 g/L,NH4Cl 7.0,谷氨酸鈉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。優(yōu)化后菌株最適宜發(fā)酵條件為甘油80 g/L,檸檬酸12g/L,谷氨酸鈉30 g/L,接種量為5%,其他條件不變情況下γ-PGA最高產(chǎn)量可達(dá)28.9g/L。(5)在含γ-PGA產(chǎn)物的發(fā)酵液土壤保水試驗(yàn)中,發(fā)酵產(chǎn)物2倍、5倍稀釋液處理土壤最終含水量分別為3.09%和1.37%,分別高于CK 3.5倍、1.5倍,保水效果顯著。(6)豆渣固體發(fā)酵產(chǎn)物γ-PGA保水性能及與化學(xué)保水劑二者處理土壤保水試驗(yàn)中,在室溫培養(yǎng)15 d后,其中化學(xué)保水劑處理土壤含水量為6.39%,約為CK處理1.17倍,豆渣固體發(fā)酵γ-PGA處理土壤含水量最高為9.20%,約為CK處理1.68倍。因此,豆渣固體發(fā)酵γ-PGA相對(duì)于化學(xué)保水劑保水效果更顯著。(7)在豆渣固體發(fā)酵產(chǎn)物γ-PGA不同濃度配比保水試驗(yàn)中,雖然施用量越大保水效果越顯著,但也明顯抑制了種子的發(fā)芽率及幼苗生長(zhǎng)。在1:20、1:80、1:100、1:150和1:300五個(gè)配比梯度中,發(fā)芽率最高的是1:150處理,為95.33%,小麥幼苗芽長(zhǎng)1:150處理的也較長(zhǎng),為11.07 cm,因此推薦施用發(fā)酵產(chǎn)物最佳配比濃度為1:150,即γ-PGA與土壤質(zhì)量比為1:600(g/g)時(shí)更有利于種子萌發(fā)及生長(zhǎng)。綜上所述,此次篩選出的Bacillus subtilis產(chǎn)能突出,經(jīng)優(yōu)化后更具開(kāi)發(fā)研究潛力,其液體及豆渣固體發(fā)酵產(chǎn)物γ-PGA保水性能顯著,固體發(fā)酵產(chǎn)物初步保水劑與土壤按照1:150質(zhì)量比施用不僅能提升土壤保水性,也能促進(jìn)小麥種子發(fā)芽及幼苗生長(zhǎng)。
[Abstract]:In this experiment, a new strain producing polyglutamic acid (緯 -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 緯 -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 緯 -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 緯 -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 緯 -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 緯 -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 緯 -PGA treatment was 9.20 and about 1.68 times of CK treatment.Therefore, the water retention effect of solid fermentation of soybean dregs 緯 -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 緯 -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 緯 -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.
【學(xué)位授予單位】：河南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S182;S482.99

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