Researched On Bacillus Subtilis Producing Poly γ-glutamic Acid With Fermentation

y-poly glutamic acid (y-PGA) is a kind of homogeneous polyamino acid synthesized by many bacteria. It is a natural biodegradable polymer that is harmless to human and environment widely applied in industry of food, cosmetics, agricultural with the advantage of filmforming, bibering, antioxidant, plasticity, moisture retention, degradability and edibility stability.The paper mainly studied the best medium compasition and culture conditions of shaking flask and fermenter, the optimal extraction method,and analysis purification and Characterization of samples and application. The research showed:1. The single factors of medium composition and culture conditions were optimized. From this results the fermentation process was optimized by response surface method, and the suitable fermentation conditions were as follows:Flask content 50ml/250ml, pH6.5, temperatuer 37℃, rotation speed 150rpm, and fermentation time 84h; The optimized medium composition contained:L-glutamate7.34%, peptone 0.42%, corn saccharification liquid 6.07%, CaCl20.03%, NaCl1%, MnSO40.006%, KH2PO4 0.8%, MgSO40.15%. Under the conditions, the yield of y-PGA increased from 57.85 g/L to 68.3 g/L.2. This experiment were investigated fermentation rotational speed, ventilation and the pH in process of fermentation.The result shows that the fermentation process control pH in 6.5, ventilation for 8L/min, while in 100r/min speed, fermentation best effect. Under this conditions, the yield of y-PGA can get to 39 g/L.3. The extraction ofγ-PGA:a high viscosity fermentation broth was got by microbial fermentation,then organic solvent was used to precipitation and obtain y-PGA.The best extracted method was ethanol extraction.The concentration was 100%,the addition was 5 times; The fermented without bacterium body was boring under 100℃,we can get y-PGA about 27.28 g/L. With other temperature,the y-PGA we obtain from fermented less than 100℃.4. Sample purity analysis:Degradation products identified by thin-layer chromatography to prove that degradation products were only the glutamic acid; With 5300DV plasma tomic emission, we can analysis the samples contained metal ions about 70.1086 mg/g, namely 1g samples contain less than 0.1 g of metal ions; The best method to dry sample is freezing drying.5. Product identification:The product was detected by IR and UV, We could see that the polymer was polyamide and its largest ultraviolet absorption wavelength is located in 216 nm place;The molecular weight of y-PGA was 700kDa and 960kDa determined by SDS-PAGE and GFC; Observed the freeze dried samples of y-PGA by electron microscopy, photographs showing the freeze-drying without disrupting they-PGA internal structures, so the drying method is feasible.6. Part of the properties and application of y-PGA:y-PGA can occurs flocculation reaction with most metal ions, especially for Cr3+, Fe3+, Cu2+ the flocculation effect is particularly evident. In addition,it also occurs flocculation reaction with Zn2+,Mn2+,Ag+ and so on. Therefore, we can consider use it to sewage treatment. The different concentrations of y-PGA was mingle with lactobacillus as the cryoprotectants,5% of trehalose for comparison.The mixture was freeze-drying for 36h,and then plate counting.The powder with 5% ofγ-PGA contain living bacterium number equal to 5% of trehalose.The density is lower,the living bacterium number is less.