| γ-poly glutamic acid is a biodegradative polypeptide synthesized by microorganism.γ-PGA produced by microbes usually consists of thousands of glutamate monomers and its molecular weight (Mw) has 200-1000KDa.γ-PGA produced by different bacterium strains has different molecular weight.γ-PGA can be degradated to glutamate that is endogenic material,so it doesn't have cumulative toxic. Inγ-PGA,α-amino groups are joined withγ-carboxyl groups by amide linkages.γ-PGA has good water solubility and is prone to be modified.Basing on these characteristics,γ-PGA is fit for being used to drug deliverysystem. By traditional liquid fermention the concentration of theγ-PGA is lower, the purification for products is difficult ,production cost is higher and because of the fermentation production cost factor, the domestic has not really form industrial production.Berberine,because of the low solubility,is absorbed into the body difficultly .Thus, it cause the low esitsbioavailability (usually 10%, <25%) tolerability poor, poor efficacy,thesegreatly reduce its application. We should improved greatly the bioavailability, efficacy, and reduce its negative effectsThe main researches we did are:(l) Production ofγ-PGA with high effieiency:(2)Separation and purification ofγ-PGA; determination and low molecular weightγ-PGA preparation(3) the traditional berberine dosing preparations for increasing solubilityOptimizeing the fermentation conditions for poly glutamine by Bacillus subtilis Natto with response surface methodology. Firstly, plackett-burman design was used to evaluate the effects of related factors on poly glutamine .It was found that glucose concentration isthemostsignificant positive, followed by concentrations of soybean power and glutamate .And then ,the concentrations of glucose ,soybean power and glutamate were further optimized using response surface analysis .Results revealed that the optimal medium constituents are as follows: glucose 8%,soybean power3.2%,glutamate4.8% , MgSO40.06% , CaCl20.06% , K2HPO4·0.39% ,KH2PO40.3%, pH7.5(before sterilization). The optimize conditions allow poly-glutamine production to increase from 15.4g/L to32g/L.The fermentation liquid with high viseosity containingyγ-PGA was centrifugated eliminate the thalli.4 times volume industrial ecohol with adjusting pH was addedto the supernatant,And theγ-PGA precipitated.Theγ-PGA was dissolved in deionized water,dialyzedEliminate the little molecules and frozendried in vacuum to get the white crystal.The TLC, electrophoresis method, GPC spectrometry determinedγ-PGA ,Purity with about 75%,The relative molecular weight was 426 KDaWhen with acerbity degradation method and controlling method of fermentation time,the molecular weight reduced respectively ,getting40-100KDa section research the fermentation process the strain was fermented for 56 h in theoptimum conditions, the yield ofγ-PGA reached most. The activity of aminotransferase dropped significantly away spin of the enzyme activity has a certain degree of improvement D/L ratio configuration ofγ-PGA will gradually be close to1:4 as the fermentationThe concentration of the berberine hydrochloride was measured by spectrophotometry Standard curve was Y =70464X, linear fitting coefficients reach 0.9996.The experimental results showed that the interference of solubilizer could be neglected; The solubility of berberine hydrochloric increased with temperature, at 80℃had a maximum; When acidic solution neutral and alkaline solubility could decline; Takingγ-PGA,β-cyclodextrins, hydroxypropyl cyclodextrins as the closure of berberine hydrochloride, their maximums of solubilization could reach 1.80 and 2.15 times as the aqueous solubility. Polyethylene glycol 4,000 had a worse dissolved effection. |
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