Microbial fermentation
Breeding strain Leyconostoc meseteroides. Preparation of medium: According to the ratio of mass/volume, 10% sucrose, 0.25% peptone, 0.08% disodium hydrogen phosphate, add water to 100%, boil to dissolve, filter with filter paper, take 3ml of filtrate and dispense it into test tubes, use Tighten gauze cotton, place it in a grid, pressurize 117.72kPa (1.2kgf/cm2) at 120°C for 30min, let it cool, and put it in an incubator to obtain a clear and transparent liquid medium for inoculation. According to the above liquid medium ratio, add 1.5%-2% agar, boil to dissolve, take 5ml and divide it into test tubes, plug tightly with gauze cotton, 117.72kPa (1.2kgf/cm2), sterilize under pressure for 30min, cool, That is solid medium.
Purification and cultivation of strains Take 5 solid mediums, heat and melt them, place them on a water bath at 50-60°C for incubation, and number them respectively. Select a test tube of bacterial species in a sterile cabinet, dip one ear of white gold fungus and inoculate it into the No. 1 test tube containing solid medium, shake well, and then dip the No. 1 test tube and inoculate it into the No. 2 test tube, shake well, and in turn Inoculated into the No. 5 test tube. Then, while it is still hot (about 50°C), pour it into the correspondingly numbered petri dish one by one, lay it flat, cool it, and place it upside down in an incubator. Convex, transparent, sticky colonies. Select bacterial species in the petri dish No. 3-5, and the normal number of colonies should be 5-20. Use a crayon to draw a circle on the outer wall of the petri dish to select typical colonies (obvious features and suitable size). In a sterile cabinet, dip the delineated colonies with white gold ear and inoculate them in a test tube containing a liquid medium, and incubate at 25°C for 24h. After passage for 2-3 generations, carry out experiments such as fermentation, hydrolysis, and division of small samples, select strains with high yield, good composition, and high yield, and store them in a refrigerator at 2-4 °C for expanded production use.
Leuconostoc membranae [medium]→[25℃, 24h] colony
For seed culture, take 400ml (medium bottle) and 4000ml (large bottle) of liquid culture medium, inoculate a bacterial test tube (3ml) in the middle bottle, and then inoculate about 100ml of the seed liquid cultured in the middle bottle into the large bottle. Cultivated at 25°C for 20-24h, the end point pH is 3.8-4.3, and those with good fermentation can be used for production.
Colony [liquid medium] → [25℃, 20-24h] seed culture medium
Fermentation and precipitation are 15% sucrose, 0.25% peptone, 0.15% disodium hydrogen phosphate, add normal water to 100%, make a fermentation medium and put it in the fermenter, the inoculum amount is 2.5%, stir for 10-15min, control pH7- 7.4, about 25℃, fermented for 20-24h, and the final pH of the fermentation broth was 4.2-5, reaching the end point. Add 85%±5% ethanol to precipitate, and then wash the precipitate with 60%-70% ethanol to obtain the crude Dextran Obat polymer for hydrolysis with a yield of 85%.
Seed culture medium [fermentation medium]→[25℃, 20-24h, pH7-7.4] fermentation broth [85% ethanol, 60%-70% ethanol]→[pH4.2-5] crude polymer dextran
Hydrolysis, Neutralization and Purification The crude polymer Dextran Obat is heated and dissolved in distilled water. Calculated by the mass of the hydrolyzate, add 0.1% hydrochloric acid, keep the temperature at 95-100°C, hydrolyze, add distilled water to dilute the concentration to 11%, and control the endpoint viscosity to 2.7- 2.9, slowly neutralize to pH 6-6.5 with 6mol/L NaOH, add anhydrous calcium chloride 2.4g/L (0.24%), and finally add 766 type coarse-grained activated carbon 8g/L (0.8%), decolorize under stirring, Filtration to obtain the hydrolyzate for division.
Crude product [HCl, NaOH, CaCl2, 766 activated carbon] → [95-100℃, pH6-6.5] Division with the hydrolyzate in one step Take the hydrolyzate and add ethanol to make the concentration 40%-40.5%, keep at 40℃ for 22h Then, the supernatant was collected and divided into two stages, and the precipitates were impurities and macromolecular dextran.
Second-level division The supernatant after the first-level division was added with ethanol to make the concentration reach 45%-45.5%, stirred for 15 minutes, kept at 40°C for 22 hours, and the precipitate was medium molecular Dextran Obat.
The supernatant after the three-level division and the second-level division was added with ethanol to make its concentration reach 48%-48.5%, stirred for 15 minutes, kept at 40°C for 22 hours, and precipitated as low-molecular-weight Dextran Obat.
Add ethanol to the supernatant after the four-level division and the three-level division to make the concentration reach 55.5%-56.5%, stir for 15 minutes, stand at room temperature for 6 hours, and precipitate as small molecule Dextran Obat.
The obtained Dextran Obat is dried and divided, dehydrated with more than 90% ethanol, and the impurities are removed to prepare a loose alcohol-containing powder, which is centrifuged and dried to obtain the finished dextran with a total yield of 60%.
Dextran finished product [more than 90% ethanol] → alcohol-containing powder [centrifugation, drying] → preparation of finished dextran injection Dextran Obat finished product
Make sterile aqueous solution with glucose or sodium chloride respectively according to injection requirements.
Dextran Obat finished product [glucose, water] → glucose injection
Dextran Obat finished product [sodium chloride, water] → [sterilized] sodium chloride injection.
