Method 1. Extraction method
Soaking, alkali refining, acidification Put about 2-3 tons of tap water in the algae washing tank, put in 120kg of seaweed, and after the algae body expands, carefully wash the Mannitol Pulver on the seaweed into the water, and the washed seaweed is used for extracting sodium alginate . Wash the second batch of seaweed with the washing liquid, and wash about four batches like this. Add 300g/L (30%) sodium hydroxide solution to the above washing solution, Ph10-11, and let it stand for 8 hours until the fucoidan solution, starch and other organic viscous substances are fully coagulated and precipitated. Siphon the supernatant, acidify it with sulfuric acid (1:1), adjust it to pH 6-7, further remove the jelly, and obtain a neutral supernatant.
Seaweed or kelp [tap water] → lotion [NaOH] → [pH10-11, 8h] supernatant
[H2SO4]→[pH6-7] Concentrate the neutral clear liquid and wash with alcohol. Heat the above neutral clear liquid with direct fire or steam until boiling and evaporation. The temperature is 110-115°C. A large amount of sodium chloride is precipitated. Take out the glue dirt until it becomes a concentrated solution, take a small sample and pour it on the ground, let it solidify after a little cooling, and then discharge the material, containing more than 30% of Mannitol Pulver and about 10% of water. Cool the concentrated solution to 60-70°C, add 95% ethanol (2:1) while it is hot, keep stirring, and gradually cool to room temperature, then centrifuge to dry to remove gum, and obtain off-white loose matter.
Neutral clear liquid [110-115°C] → concentrate [ethanol] → [60-70°C] loose matter extraction Weigh the loose matter, put it into an extraction pot equipped with a reflux condenser, add 94% of 8 times the amount Ethanol, stirred, heated slowly, boiled and refluxed for 30 minutes to discharge, cooled in running water for 8 hours, left for a day and night, centrifuged and dried to obtain white loose Mannitol Pulver crude product, containing 70%-80% mannitol. The same operation as above, ethanol recrystallization once, to obtain industrial mannitol, the content is more than 90%, and the Cl- content is <0.5%.
Loose matter [94% ethanol]→[reflux for 30min, cooling for 8h] refine the crude Mannitol Pulver, take industrial mannitol and add distilled water to dissolve it, then add 1/10-1/8 of medicinal activated carbon, keep stirring, keep warm at 80℃ for half hours, filter while hot, wash the activated carbon twice with a little water, combine the washed filtrate, and concentrate until the mannitol reaches 70%, if there is turbidity, re-filter. Cool to room temperature under stirring, crystallize, filter with suction, wash the crystals, and filter with suction to obtain crystalline Mannitol Pulver.
Crude mannitol [distilled water, activated carbon] → [80°C, 30min] concentrate the filtrate → crystallize mannitol and dry the above crystallized mannitol, after the Cl- is qualified (Cl-<0.007%), dry it with steam at 105-110°C , turn it frequently, take it out in 4 hours, and it is the finished product of medicinal Mannitol Pulver. Content 98%-102%, melting point 166-168°C, [α]20D+23°-+24°
Crystalline mannitol [105-110°C, 4h] → finished medicinal mannitol
Preparation of Injection The finished product of mannitol is dissolved in water for injection according to the dose to form a solution, and the mannitol injection is obtained after sterilization.
Mannitol Pulver Finished Product [Water for Injection] → Mannitol Injection
Method two, direct fermentation method
Breeding strains Aspergillus Oryzae3.409 was transplanted into a test tube filled with 10ml slant medium (2.1% agar was added to the wort) and cultured at 31°C for 4 days. Get slant bacteria.
The strain preservation system adopts the low-temperature preservation method, put the strain on a slant in a refrigerator at 4°C, and store it for 2-3 months for passage once. Before use, it needs to be re-transplanted and activated to ensure fermentation.
Amitramycin [strain slant medium] → [31°C, 4d] slant strain
Seed culture Take 2 wort slant planes of strains that have been cultured for 4 days, and transplant them into 17.5L seed medium. Preparation of seed medium: sodium nitrate 0.3%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, potassium chloride 0.05%, ferrous sulfate 0.001%, corn steep liquor 0.5%, starch hydrolyzed sugar 2%, corn flour 2% , initial pH6-7. Ventilation 1: 0.5V/(V·min), temperature 31°C±1°C, stirring at 350r/min, tank pressure 98.1kPa (1kgf/cm2) and culture for 20-24h to obtain seed culture solution.
Incline strains [seed medium] → [pH6-7, 31°C, 20-24h] seed culture solution
Preparation of fermentation medium: sodium nitrate 0.3%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, ferrous sulfate 0.001%, corn steep liquor 3%, corn flour 3.5%, starch hydrolyzed sugar 12.5%, initial pH 6-7 .
Take 350L of fermentation medium and put it into a 600L fermentation tank, steam sterilize it at 146.15kPa (1.5kgf/cm2) for 30min, transfer it into the seed culture solution, the inoculum size is 5%, the tank pressure is 98.1kPa (1kgf/cm2), the ventilation volume 1: 0.3V/(V min), after 20 hours of fermentation, change to 1: 0.4V/(V min), temperature 30-32°C, stirring 230r/min, fermentation cycle 4-5d. In order to prevent foaming, add 200ml soybean oil when making ingredients. The obtained fermented liquid contains more than 5% of mannitol, and the conversion rate is 40%.
Seed culture liquid [steam] → [pH6-7, 30-32℃, 4-5d] fermentation liquid
Coagulation, concentration, crystallization, decolorization, desalination Heat the fermentation broth at 100°C for 5 minutes, add 1% activated carbon at 80-85°C for 30 minutes, filter or shake dry to obtain a clear liquid. Then the clarified filtrate was concentrated in vacuum at 50-60°C to 31°Be, left at room temperature for 24h, and dried to obtain mannitol crystals. Dissolve the crystals in 0.7 times water, add 2% activated carbon, heat at 70°C for 30 minutes to decolorize, and filter. The filtrate is desalted by 717 strong basic anion exchange resin (OH-type) and strong acidic cation exchange resin (H+ type) 2:1 mixed bed resin, and the effluent is checked for no chloride ion reaction.
Fermentation broth [1% activated carbon] → [100°C, 5min; 80-85°C, 30min] clarified filtrate [55-60°C] → [decompression] crude product [water, 2% activated carbon] → [70°C, 30min] Filtrate [yin and yang mixed bed resin] → effluent
Concentrate, crystallize, and dry the effluent with steam at 55-60°C, 80kPa (600mmHg) under reduced pressure to 25°Be, let the concentrated solution cool to room temperature for 24h to crystallize, shake dry, dry at 105-110°C, and pulverize to obtain Medicinal finished product, yield 60%, content 98.83%-100.9%.
Effluent [55-60°C] → [Decompression] Concentrate [Room Temperature] → [24h] Crystallization [105-110°C] → API
For the preparation, take an appropriate amount of water for injection, heat it to about 90°C, weigh Mannitol Pulver at a content of 20%, dissolve it, add activated carbon in a water bath and heat for 5 minutes to decolorize, filter through a Buchner funnel, and then add water for injection to the full amount. After the determination of content and pH is qualified, filter it with G3 vertically fused glass filter ball until it is clear, dispense it into 50ml, 100ml or 250ml ampoules or infusion bottles, and sterilize it with 98.1kPa (1kgf/cm2) hot-pressed steam for 40min to obtain Mannitol Pulver injection.
