Research On Production Methods Of Sisal Sapogenin By Biotransformation

Natural sapogenin has rich pharmacological properties, which are anti-inflammatory, antibacterial, analgesic, hemostatic, anti-aging and fall blood sugar and so on. Furthermore, as an important drug intermediate, sapogenin is widely used in manufacturing adrenal cortical hormone, sex hormone and anabolic hormones the three categories which have more than 200 kinds of drugs. In some areas, it even can replace scarce and precious ginseng sapogenin, which showed a strong advantages. However, the traditional waste acid and alkali chemical technology has been far from enough to satisfy the demand of the current industrialized production, and traditional technology caused environmental pollution. In addition,5% sisal blade is utilized, the remaining 95% of the components are not being used when sisal spinning. Based on this, we put forward the way of production sapogenin by biotransformation.In this paper, macroporous resin method and silica gel column chromatography method was used in the extraction and purification of sisal saponins and sapogenin; Flow injection chemiluminescence method was used as our on-line detection method to detect the content of sisal saponins and sapogenin; and HPLC-ELSD method was used to detect the content of sapogenin. In the biotransformation of ZG-21, a sisal sapogenin production bacteria strain, morphological and molecular biological characteristics were identified. And the conditions of sisal produced fermentation were optimized, including the concentrations of different trace metal ions in the process of fermentation; In the enzymatic properties of the strain ZG-21, the enzymatic reaction conditions between the crude extract of degrading enzymes and sisal saponins were optimized. This all provide a theoretical basis to a cleaner production processes of sisal sapogenin.The main research results of this paper are as follows:1. On the extraction of sisal saponins, the article investigated seven kinds macroporous resin on the property of static adsorption and desorption of sisal saponins, respectively; such as D-101, D-101C, DA201-H, LX-22, LX-68M, LXA-8 and LX-762 so on. The D-101C is the optimal among this resins, the adsorption rate and the resolution was 75.1% and 84.8%; In addition,70% of ethanol is the best concentration to desorption the resin to extract the saponins.2. On the purification of sisal sapogenin, this paper choosed silica as immobile phase, chloroform:methanol:water=80:20:5(V/V/V), the ternary system as the mobile phase, flow velocity was 1 mL/min, collecting every two minutes,21-25 tube were samples that purificated sisal sapogenin through analyzing elution curve. The result was further confirmed by reverse high performance liquid chromatography/evaporative light-scattering detector chromatography, the method which separated and purified sisal sapogenin could obtain the relative standard of above 95 %.3. On the end detection method, this paper established a reverse high performance liquid chromatography/evaporative light-scattering detector to detect sapogenin, the method of standard curve equation(y=6688.8x-77788, R2=0.9996) was acquired, the linear range for sisal sapogenin was 50-2000 μg/mL, a minimum detection limit of 5 μg/mL.4. On-line detection methods, this article chooses higher sensitivity of luminal-potassium ferricyanide chemiluminescence system, through the optimization of detection reagent concentration condition, we established the method of flow injection chemiluminescence detection of sisal saponins and sapogenin.(1) When the concentration of luminal was 4.0 X 10-5 mol/L, the concentration of potassium ferricyanide was 3.0×10-5 mol/L, the optimal conditions for detection of sisal saponins was to preparation luminal contained sodium hydroxide with concentration of 0.1 mol/L. The standard curve equation was y=1576.7x+20093, R2=0.9973, x standed for mg/mL, the concentration has a good linear relationship within the scope of 0.1 to 5.0 mg/mL.(2) When prepared luminol that the concentration of 1.0 X 10-5 mol/L and potassium ferricyanide that the concentration of 1.6 X 10-5 mol/L with 0.1 mol/L sodium hydroxide solution, sapogenin was dissolved in anhydrous ethanol, the above statement was the optimal conditions of detection of sisal sapogenin. The standard curve equation was y=721.41x+25.242, R2=0.9996, x standed for mg/mL, the linear range for sisal sapogenin was 0.1-1.0 mg/mL.5. About filtrating the strain to convert the sisalagenin, this paper collected 384 soil samples around karst landscape of cap rock in guilin,22 strains were obtained through the initial filtration, that can grow in culture medium with sisal saponins as the carbon source,22 strains including 17 strains of bacillus and 5 strains of coccus. Then, four strains of bacillus ZG-04, ZG-14, ZG-19, ZG-21 and one of coccus ZG-15 were obtained through filtrate again, their degradation rate is relatively high in the 22 strains. After identified the fermentation product of 5 strains, found that only ZG-21 can convert the sisalagenin and the obain rate is relatively high.6. About direct fermentation on the sisalagenin strains ZG-21 for filtrating, With orthogonal experiment to optimize the fermentation conditions of temperature, time and pH value, get the optimal fermentation technology as follows:temperature 32 ℃,5 days time, pH value of 6; Trace metal ions concentration interference effect of fermentation experiment found that a certain concentration of Na+, K+ and Cu2+ has promoting effect to fermentation, Mg2+, Al3+and NH4C1 has inhibitory effect on fermentation, and Fe3+ has little effect on fermentation.7. About the sisalagenin strains ZG-21 for filtrating in the enzymology characteristics, through extraction of saponins degradation enzyme from of the fermented liquid of the sisalagenin strains ZG-21, and studied the effect of the temperature, time and pH for the enzymatic reaction, obtained the optimal enzymatic reaction conditions:temperature 33 ℃, time of 12 h, the substrate pH value is 6. The experimental results of metal ion disruption enzymatic reaction show that Cu2+, Zn2+ and Ca2+ has promoting effect to the enzymatic reaction, Al3+ and Ag+ has inhibitory effect on the enzymatic reaction, while the rest of the Na+, K+, Mg2+, Fe3+, Co2+ has little influence on the enzymatic reaction; Glucan gel column chromatography was used to rough ZG-21 strains saponins degradation enzyme for further purification, after purification, the fermentation effect of saponin degradation enzyme has improved.8. About identification of ZG-21 strains on the molecular biology, through the morphologic observation of the strain, and measured the growth curve. Based on 16S rRNA molecular identification and physiological and biochemical characteristics analysis of Strains ZG-21, the strain was identified as bacillus genus.