Preparation method of cobamamide crude product
阅读说明：本技术 一种腺苷钴胺粗品的制备方法 (Preparation method of cobamamide crude product ) 是由 户鹏东 裴立忠 高宏伟 沈毅 马琼 于 2020-07-23 设计创作，主要内容包括：本发明涉及一种腺苷钴胺粗品的制备方法,其工艺为包括将脱氮假单孢杆菌发酵液经喷雾干燥制成腺苷钴胺菌丝粉,然后采用丙酮复合溶媒浸提、离心分离、减压浓缩得到腺苷钴胺浓缩液Ⅰ,再经大孔树脂吸附、丙酮水溶液解析、减压浓缩得到腺苷钴胺浓缩液Ⅱ,最后经絮凝过滤、丙酮结晶,抽滤、真空干燥,得到腺苷钴胺粗品。本发明将脱氮假单孢杆菌发酵液进行喷雾干燥,得到腺苷钴胺菌丝粉,再用丙酮复合溶媒进行浸提,避免脱氮假单孢杆菌发酵液异常时,经板框过滤困难状况,最大限度回收腺苷钴胺,有利于提高产品质量和收率,具有工艺操作更为简捷,生产进度易保障,生产操作连贯性强,产生废水量少等特点。(The invention relates to a process for preparing adenosyl cobalamin crude product, which comprises, spray drying the denitrified pseudomonas fermentation liquor to prepare adenosyl cobalamin hypha powder, then leaching with acetone composite solvent, centrifugal separation, decompression concentration to obtain adenosyl cobalamin concentrated solution I, then absorbing with macroporous resin, resolving with acetone water solution, decompression concentration to obtain adenosyl cobalamin concentrated solution II, finally flocculating filtering, crystallizing with acetone, suction filtering, vacuum drying to obtain the adenosyl cobalamin crude product. The invention carries out spray drying on the pseudomonas denitrificans fermentation liquor to obtain adenosylcobalamine hypha powder, and then uses acetone composite solvent to carry out extraction, thereby avoiding the difficult condition of filtering through a plate frame when the pseudomonas denitrificans fermentation liquor is abnormal, recovering the adenosylcobalamine to the maximum extent, being beneficial to improving the product quality and yield, and having the characteristics of simpler process operation, easy guarantee of production progress, strong continuity of production operation, less waste water production and the like.)
1. a preparation method of a cobamamide crude product is characterized by comprising the following process steps: spray drying the pseudomonas denitrificans fermentation liquor to prepare cobamamide hypha powder, then leaching by adopting an acetone composite solvent, centrifugally separating, concentrating under reduced pressure to obtain cobamamide concentrated solution I, adsorbing by macroporous resin, resolving an acetone aqueous solution, concentrating under reduced pressure to obtain cobamamide concentrated solution II, and finally obtaining a crude cobamamide product by flocculation filtration, acetone crystallization, suction filtration and vacuum drying;
the acetone composite solvent is a mixed solution of acetone, isopropanol and water, the volume ratio of the acetone composite solvent to the water is 3.5-4.5: 1.5-2.0: 5.5-6.5, and the feeding ratio is as follows: wMycelium powder：WAcetone composite solvent=1:2.5～3。
2. The process for the preparation of a crude adenosylcobalamin according to claim 1, characterized in that the spray drying conditions are: the air inlet temperature is 110-140 ℃, and the air outlet temperature is 65-70 ℃.
3. The method for preparing a crude cobamamide product according to claim 1, wherein said acetone complex solvent is leached 2-3 times for 3-6 hours each time.
4. The method for preparing a crude adenosyl cobalamin according to claim 1, wherein the macroporous resin adsorption, acetone aqueous solution desorption and reduced pressure concentration are that the adenosyl cobalamin concentrated solution I is adsorbed by macroporous resin, then is desorbed by 30-50% acetone aqueous solution, and is reduced pressure concentrated by a thin film evaporator to obtain the adenosyl cobalamin concentrated solution II.
5. The method for preparing the crude adenosylcobalamin according to claim 1, wherein the flocculation filtration is that 0.15 to 0.3 percent of polymeric aluminum ferric silicate is added into a concentrated adenosylcobalamin solution II, stirred for 15 to 20min, and then sequentially filtered by a plate-and-frame filter and a liquid filter.
6. The method for producing a crude adenosylcobalamin according to claim 1, wherein the acetone crystallization is a crystallization of adenosylcobalamin by adding acetone to the obtained filtrate in an amount of 8 to 10 times the volume thereof.
Technical Field
The invention belongs to the technical field of biological fermentation and extraction, and particularly relates to a preparation method of a cobamamide crude product.
Background
Cobamamide (adenosylcobalamin) is vitamin B12One of them, commonly known as coenzyme vitamin B12(5, 6-Dimethylimidazolyl-5-deoxyadenylylcobalamin).
Cobamamide participates in methyl conversion and folic acid metabolism in vivo, and promotes the reduction of methyl folic acid into tetrahydrofolic acid; it is also involved in tricarboxylic acid cycle, is very important for the formation of lipoprotein in the myelin sheath of nerve, and can make mercaptoenzyme be in active state, so that it is involved in extensive protein and fat metabolism, and can promote the development and maturation of erythrocyte, and is the necessary factor for completely forming nerve sheath spinal cord fiber and maintaining the function of epithelial cell of digestive system. The cobamamide can be directly absorbed by human body without transformation, has strong affinity with tissue cells and can be preserved in vivo for a long time.
Cobamamide is a substance necessary for cell growth and proliferation and maintenance of nerve myelin, is an important coenzyme for cell synthesis of nucleotide, has better clinical value in aspects of nourishing and repairing nerves, improving anemia and the like, and is clinically used for treating polyneuritis, radiculitis, trigeminal neuralgia, sciatica, nerve palsy, megaloblastic anemia, dystrophic anemia and anemia in pregnancy, and can also be used for treating liver diseases, long-term atrophic gastritis and the like. The preparation formulation comprises tablets and freeze-dried powder injection.
The molecular formula of cobamamide (adenosylcobalamin) is C72H100CoN18O17P, molecular weight 1579.57, is yellow orange hexagonal crystal, presents dark red crystal form powder when exposed to air, is dissolved in water (1: 80), is slightly soluble in ethanol and phenol, is insoluble in acetone, diethyl ether, dichloroethane, etc., and is odorless and tasteless. The cobamamide aqueous solution is neutral, has a solubility in water of 16.4mmol/L (24 ℃), and has a pH of<3.5 the aqueous solution is yellow, pH>The 3.5 aqueous solution is red, but is most stable at pH = 4.5-5, and does not break when heated at 120 ℃ for 20 min. Cobalt in the cobamamide is in trivalent oxidation state and is slowly oxidized into hydrocobamamide in the air. Can be used as raw material for producing cyanocobalamin and mecobalamin.
In industrial production, the extraction process of cobamamide includes adding polyaluminium chloride or aluminium sulfate into vitamin B12 fermented liquid (denitrifying pseudomonas fermentation liquid), flocculating the fermented liquid to precipitate protein, improving the property of the fermented liquid, filtering with sharp plate frame to obtain filter residue, adding water to regulate into slurry, wall breaking and separating in tubular centrifuge, mixing the centrifugal filtrate with the plate frame filtrate, adsorbing with macroporous resin, desorbing with dilute acetone or dilute ethanol, decompressing and concentrating the desorbed liquid, and adding acetone to crystallize to obtain coarse cobamamide product.
The production process has the following problems:
1 the fermentation liquor of pseudomonas denitrificans contains a large amount of fermentation metabolites such as protein, amino acid and the like, and saccharides and oils which are not metabolized, so that more sticky fine substances are contained in the fermentation liquor, a flocculating agent is added to flocculate the sticky fine substances, the blockage of pores of filter cloth of a plate filter cloth can be relieved, and a certain filtration assisting effect is achieved.
2 the cobamamide is fermented in the cell, the cobamamide is mainly existed in the mycelium, during the acidification and filtration, a part of cobamamide enters the filtrate, but a part of cobamamide is existed in the filter residue, the filter residue needs to be added with water and mixed into pulp, and the pulp is rotated at high speed by a centrifuge to break the wall, so as to extract the cobamamide. In order to improve the yield, water is added for multiple times, pulp mixing and leaching are needed, a centrifugal machine is easy to pollute, the times of unpicking and washing are multiple, the continuity operation is influenced, the amount of generated wastewater is large, and the environment-friendly treatment pressure is increased.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a preparation method of a cobamamide crude product, which effectively improves the product quality, improves the yield, is convenient and fast in process operation, strong in production operation continuity and less in waste water generation.
The technical scheme adopted for realizing the purpose is as follows:
a preparation method of a cobamamide crude product is characterized by comprising the following process steps: spray drying the pseudomonas denitrificans fermentation liquor to prepare cobamamide hypha powder, then leaching by adopting an acetone composite solvent, centrifugally separating, concentrating under reduced pressure to obtain cobamamide concentrated solution I, adsorbing by macroporous resin, resolving an acetone aqueous solution, concentrating under reduced pressure to obtain cobamamide concentrated solution II, and finally obtaining a crude cobamamide product by flocculation filtration, acetone crystallization, suction filtration and vacuum drying;
the acetone composite solvent is a mixed solution of acetone, isopropanol and water, the volume ratio of the acetone composite solvent to the water is 3.5-4.5: 1.5-2.0: 5.5-6.5, and the feeding ratio is as follows: wMycelium powder：WAcetone composite solvent=1:2.5～3。
The spray drying conditions were: the air inlet temperature is 110-140 ℃, and the air outlet temperature is 65-70 ℃.
And leaching the acetone composite solvent for 2-3 times, and each time lasts for 3-6 hours.
The macroporous resin adsorption, acetone aqueous solution analysis and reduced pressure concentration are characterized in that the cobamamide concentrated solution I is adsorbed by macroporous resin, then is analyzed by 30-50% acetone aqueous solution, and is reduced pressure concentrated by a film evaporator to obtain cobamamide concentrated solution II.
The flocculation filtration is to add 0.15-0.3% (w/v) of polymeric aluminum ferric silicate into the cobamamide concentrated solution II, stir for 15-20 min, and then sequentially filter through a plate-and-frame filter and a liquid filter.
And the acetone crystallization is to add acetone with the volume of 8-10 times of the filtrate to separate out the cobamamide crystals.
Compared with the traditional cobamamide extraction process, the invention has the technical advantages that:
1, the fermentation liquor of the pseudomonas denitrificans is subjected to spray drying to obtain mycelium powder containing cobamamide, and the cobamamide is extracted and prepared by the mycelium powder containing cobamamide, so that the situations that the filtration of the pseudomonas denitrificans fermentation liquor is easily blocked by plate-and-frame filtration, and the filtration progress and the yield are difficult to guarantee are avoided, the whole process is simpler and more convenient to operate, the production progress is easy to guarantee, and the improvement of the product quality and the yield are facilitated.
2 the invention adopts the mixed solution composite solvent composed of acetone, isopropanol and water to leach the cobamamide, thereby improving the ester solubility, increasing the affinity of hypha cell walls and being beneficial to entering hypha cells to extract the cobamamide. Compared with a simple water soaking and centrifuge high-speed wall breaking extraction mode, the yield is 3-5%, the production operation continuity is strong, and the amount of generated wastewater is small.
Detailed Description
The invention is illustrated below by way of examples, which are to be understood as being illustrative and not limiting. The scope and core content of the invention are to be determined by the claims.
The following experiments were carried out on a Pseudomonas denitrificans fermentation broth (cell concentration 15% (w/v), fermentation titer 290. mu.g/ml) respectively, wherein:
500L of pseudomonas denitrificans fermentation liquor is pumped into a spray drying device, the air inlet temperature is controlled to be 110-140 ℃, the air outlet temperature is controlled to be 65-70 ℃, 75kg of cobamamide hypha powder is obtained and is divided into 5 parts, and each part is 15kg (containing 0.029kg of cobamamide), and the experiments of the embodiment are respectively carried out.
A100L of Pseudomonas denitrificans fermentation broth was taken for comparative experiment.