A method for producing heparin sodium by microbial fermentation

Abstract

The application provides a method for producing heparin sodium through microbial fermentation and belongs to the technical field of polysaccharide preparation and is subordinated to IPC classification number C08B37.The method comprises the following steps: crushing pig small intestinal mucosa, high-pressure treatment, ultrasonic treatment, inoculating composite microorganisms for anaerobic fermentation, adsorbing the fermentation liquor through resin, eluting, alcohol precipitation, collecting the precipitate and obtaining heparin sodium.The pig small intestinal mucosa is crushed, high-pressure treated and ultrasonically treated, the dissolution of heparin sodium and nutrient components is promoted, the binding force between heparin sodium and other impurities is weakened, the composite microorganisms can not only decompose polysaccharide-protein compound and intercellular substance components in the mucosa but also degrade protein, fat and other impurity components in the mucosa, and the interference of the impurities on the extraction process of heparin sodium is reduced, and the resin can enrich heparin sodium and realize the separation of heparin sodium and the fermentation liquor.The various steps have a synergistic effect, and the titer and yield of heparin sodium are improved.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide preparation technology, belonging to IPC classification number C08B37, and specifically to a method for producing heparin sodium by microbial fermentation. Background Technology

[0002] Heparin sodium, as an important anticoagulant, is widely used clinically to prevent and treat thrombosis and embolic diseases, such as myocardial infarction and pulmonary embolism. It is also an indispensable anticoagulant in medical procedures such as hemodialysis and extracorporeal circulation. With the increasing aging of the global population and the rising incidence of cardiovascular diseases, the demand for heparin sodium continues to grow.

[0003] Currently, the production of heparin sodium mainly relies on the traditional method of extraction from the mucosa of the small intestine in pigs. However, this method has many limitations. On the one hand, the traditional extraction process consumes a large amount of chemical reagents, such as strong acids, strong alkalis, and organic solvents. These chemical reagents are not only expensive but also cause serious environmental pollution, and their subsequent treatment requires complex and costly environmental protection measures. On the other hand, the traditional process is cumbersome, involving multiple precipitation, centrifugation, and filtration operations. This not only results in low production efficiency but also makes heparin sodium susceptible to degradation or contamination by impurities during these multiple steps, leading to unstable product quality and difficulty in achieving the purity required for high-end pharmaceutical applications.

[0004] Given the shortcomings of traditional production methods, developing an efficient, environmentally friendly, and sustainable method for producing heparin sodium has become an urgent priority. Microbial fermentation technology, with its numerous advantages such as the fact that fermentation typically takes place under mild conditions, is environmentally friendly, aligns with the principles of green chemistry, and enables stable and sustainable production, offers a new technological approach and solution for heparin sodium production. It holds promise for overcoming the bottlenecks of traditional production methods and meeting the growing market demand. Summary of the Invention

[0005] The purpose of this invention is to provide a method for producing heparin sodium by microbial fermentation, which can improve the potency and efficiency of heparin sodium.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for producing heparin sodium by microbial fermentation, comprising: crushing the mucosa isolated from fresh pig small intestine, mixing it with water, treating it under high pressure and ultrasound, inoculating it with a composite microorganism for anaerobic fermentation, filtering it, adsorbing the fermentation broth with resin, eluting it with sodium chloride solution, precipitating the eluent with alcohol, collecting the precipitate, and obtaining heparin sodium; wherein the composite microorganism is composed of Akkermansia myxophilus and two Bifidobacteria in a weight ratio of 2-5:1.

[0008] Preferably, the crushing speed is 100-200 r / min, the temperature is 10-20℃, and the time is 10-20 min.

[0009] Preferably, the weight ratio of the mucosa to water is 1:5-10.

[0010] Preferably, the high pressure is 200-300 MPa, the temperature is 10-20℃, and the time is 2-5 min. Preferably, the ultrasonic power is 100-150 W, the temperature is 10-20℃, and the time is 2-5 min.

[0011] Preferably, the viability of the *Ackermania* var. *misotropy* is 1-2 billion CFU / g, and the viability of the *Bifidobacterium* var. *bifidobacterium* is 5-1 billion CFU / g; the inoculation amount of the composite microorganisms is 2‰-5‰ of the weight of the mucosa, and the fermentation temperature is 35-40℃ for 24-36 hours.

[0012] Preferably, the filter membrane used for filtration is a 0.22-0.45 μm filter membrane.

[0013] Preferably, the resin is a macroporous strong basic anion exchange resin, the amount of resin used is 5%-10% of the weight of the fermentation broth, and the adsorption time is 2-5 hours.

[0014] Preferably, the concentration of the sodium chloride solution is 1-2 mol / L, and the amount used is 2-5 times the weight of the resin; the elution speed is 100-200 r / min, and the time is 3-6 h.

[0015] Preferably, the alcohol precipitation comprises: mixing the eluent with an ethanol solution, allowing it to stand for 4-6 hours, and collecting the precipitate; wherein the volume percentage of the ethanol solution is 80%-90%.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention provides a method for producing heparin sodium through microbial fermentation, comprising: crushing the mucosa isolated from fresh pig small intestine, mixing it with water, treating it under high pressure and ultrasound, inoculating it with a complex microbial culture for anaerobic fermentation, filtering, adsorbing the fermentation broth with resin, eluting it with sodium chloride solution, precipitating the eluent with alcohol, collecting the precipitate, and obtaining heparin sodium. This invention, by crushing, treating, and ultrasounding the pig small intestine mucosa, can break down the mucosal cells, dissolving the heparin sodium and nutrients within the cells, weakening the binding force of heparin sodium with other impurities, and thus facilitating the extraction of heparin sodium and subsequent microbial fermentation. During fermentation, the complex microorganisms secrete various enzymes that not only decompose the heparin sodium-protein complex and intercellular matrix components in the mucosa, facilitating the extraction of heparin sodium, but also degrade proteins, fats, and other impurities in the mucosa, reducing interference from impurities in the heparin sodium extraction process and improving the extraction efficiency. The resin specifically binds to heparin sodium, adsorbing and enriching it from the relatively low concentration in the fermentation broth, achieving the separation of heparin sodium from the fermentation broth. The various steps in this invention exhibit synergistic effects, improving the potency and yield of heparin sodium. This method for producing pig casings using pig small intestinal mucosa significantly enhances the utilization value of pig casing processing waste, resulting in substantial economic and social benefits. Detailed Implementation

[0018] This invention provides a method for producing heparin sodium by microbial fermentation, comprising: crushing the mucosa isolated from fresh pig small intestine, mixing it with water, treating it under high pressure and ultrasound, inoculating it with a composite microorganism for anaerobic fermentation, filtering it, adsorbing the fermentation broth with resin, eluting it with sodium chloride solution, precipitating the eluent with alcohol, collecting the precipitate, and obtaining heparin sodium; wherein the composite microorganism is composed of Akkermansia myxophilus and two Bifidobacteria in a weight ratio of 2-5:1.

[0019] The preferred crushing speed of the present invention is 100-200 r / min, more preferably 150 r / min; the preferred temperature is 10-20℃, more preferably 15℃; and the preferred time is 10-20 min, more preferably 15 min. The preferred high pressure is 200-300 MPa, more preferably 250 MPa; the preferred temperature is 10-20℃, more preferably 15℃; and the preferred time is 2-5 min, more preferably 3 min. The preferred ultrasonic power is 100-150 W, more preferably 120 W; the preferred temperature is 10-20℃, more preferably 15℃; and the preferred time is 2-5 min, more preferably 3 min. This invention uses physical force to disrupt the integrity of mucosal cell membranes, allowing heparin sodium, which was originally encapsulated or tightly bound to the cells, to be released. High-pressure treatment enables the rapid dissolution of heparin sodium and nutrients from the cells. Ultrasonic treatment interferes with weak interactions such as van der Waals forces and hydrogen bonds, altering the arrangement of molecules and loosening the originally tightly bound molecular structure. This allows more internal bonds to be exposed, thereby weakening the binding force of heparin sodium with other impurities, which is more conducive to the extraction of heparin sodium and subsequent microbial fermentation.

[0020] The weight ratio of the mucosa to water in this invention is preferably 1:5-10, more preferably 1:8.

[0021] The Akkermansia muciniphila strain described in this invention is Akkermansia muciniphila. 24917, purchased from the China Industrial Microbial Culture Collection Center; the viability of the *Ackermania* strain is preferably 1-2 billion CFU / g, more preferably 1.5 billion CFU / g. The *Bifidobacterium* strain described in this invention is *Bifidobacterium bifidum*. 6168, purchased from the China Industrial Microbial Culture Collection Center, wherein the viability of the two Bifidobacteria is preferably 1-2 billion CFU / g, more preferably 1.5 billion CFU / g; the inoculation amount of the compound microorganism is preferably 2‰-5‰ of the mucosa weight, more preferably 3‰; the fermentation temperature is preferably 35-40℃, more preferably 37℃; and the fermentation time is preferably 24-36h, more preferably 30h.

[0022] This invention relates to *Ackermania myxophilus* and *Bifidobacterium*, which secrete different types of enzymes that complement each other in decomposing the contents of the porcine small intestinal mucosa. For example, the glycosidase secreted by *Ackermania myxophilus* preferentially acts on the glycan portion of the heparin sodium-protein complex in the mucosa, initially disrupting its structure. The protease secreted by *Bifidobacterium* further decomposes the protein bound to heparin sodium, making it easier for heparin sodium to be released from the complex. The combined action of these two enzyme systems more thoroughly disrupts the mucosal structure and releases more heparin sodium precursor polysaccharides than single-strain fermentation. Furthermore, the protease secreted by *Bifidobacterium* can degrade some protein impurities, while the lipases secreted by *Ackermania myxophilus* can decompose fatty impurities. Their synergistic effect can more comprehensively remove impurities from the porcine small intestinal mucosa, reducing their interference with the heparin sodium extraction process and improving the extraction efficiency of heparin sodium. The present invention utilizes the synergistic effect of Akkermansia mycotoxinophilus and two Bifidobacteria to remove impurities from the porcine small intestinal mucosa, thereby detaching heparin sodium from the complex structure and improving the extraction efficiency of heparin sodium.

[0023] The present invention also preferably includes enzyme inactivation after fermentation, wherein enzyme inactivation preferably includes treating the fermentation broth at 80-90°C for 10-20 minutes.

[0024] The filter membrane used in this invention is preferably a 0.22-0.45 μm membrane, more preferably a 0.22 μm membrane. This invention effectively retains bacteria in the fermentation broth and intercepts impurities such as microbial cell debris and metabolic solid particles, thereby improving the extraction efficiency of heparin sodium.

[0025] The resin described in this invention is a macroporous, strongly basic anion exchange resin, purchased from Kehaisi (Beijing) Technology Co., Ltd., model number: A-72MP has a main structure of polystyrene copolymer and type I quaternary ammonium functional groups. The resin dosage is preferably 5%-10% of the weight of the fermentation broth, more preferably 8%, and the adsorption time is preferably 2-5 hours, more preferably 3 hours. The macroporous strong-base anion exchange resin of this invention can specifically bind to heparin sodium, adsorbing and enriching it from the relatively low content of the fermentation broth, thereby achieving the separation of heparin sodium from the fermentation broth.

[0026] The concentration of the sodium chloride solution in this invention is preferably 1-2 mol / L, more preferably 1.5 mol / L, and the amount used is preferably 2-5 times the weight of the resin, more preferably 3 times; the elution speed is preferably 100-200 r / min, more preferably 150 r / min, and the time is preferably 3-6 h, more preferably 5 h.

[0027] The preferred method of alcohol precipitation in this invention includes: mixing the eluent with an ethanol solution, allowing it to stand for 4-6 hours, and collecting the precipitate; wherein the volume percentage of the ethanol solution is 80%-90%. This invention uses alcohol precipitation to precipitate heparin sodium, thereby further improving the purity of heparin sodium.

[0028] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0029] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0030] Unless otherwise specified, the following embodiments are all conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] Example 1

[0033] (1) Pretreatment of porcine small intestinal mucosa

[0034] The mucosa separated from fresh pig small intestine was placed in a homogenizer and homogenized for 15 minutes at 150 r / min and 15℃. After being mixed with deionized water (the weight ratio of mucosa to deionized water was 1:8), it was subjected to high pressure treatment at 250 MPa and 15℃ for 3 minutes, and then subjected to ultrasonic treatment at 120 W and 15℃ for 3 minutes to obtain a pretreatment solution.

[0035] (2) Compound microbial fermentation

[0036] The pretreatment solution was inoculated with a compound microorganism and fermented at 37°C for 30 h, then kept at 85°C for 15 min, and filtered through a 0.22 μm filter membrane to obtain the fermentation broth.

[0037] The complex microorganisms consist of Akkermansia muciniphila. 24917 and Bifidobacterium bifidum 6168 is composed of bacteria in a weight ratio of 3:1, with the bacterial activity of Akkermansia myxophilus at 1.5 billion CFU / g and the bacterial activity of Bifidobacterium bifidum at 1.5 billion CFU / g. The inoculation amount of the compound microorganisms is 3‰ of the weight of the mucosa.

[0038] (3) Resin adsorption and elution

[0039] After adding macroporous strong base anion exchange resin (8% of the weight of the fermentation broth) to the fermentation broth for adsorption for 3 hours, the mixture was filtered. The macroporous strong base anion exchange resin was then eluted with 1.5 mol / L sodium chloride solution (3 times the weight of the macroporous strong base anion exchange resin after adsorption) at 150 r / min for 5 hours to obtain the eluent.

[0040] (4) Alcohol precipitation

[0041] The eluent was mixed with an 85% ethanol solution at a volume ratio of 1:1, allowed to stand for 5 hours, the precipitate was collected, and dried at 35°C until the water content was 3% to obtain heparin sodium.

[0042] Example 2

[0043] (1) Pretreatment of porcine small intestinal mucosa

[0044] The mucosa separated from fresh pig small intestine was placed in a homogenizer and homogenized for 20 minutes at 100 r / min and 10℃. After being mixed with deionized water (the weight ratio of mucosa to deionized water was 1:5), it was subjected to high pressure treatment at 200 MPa and 10℃ for 5 minutes, and then subjected to ultrasonic treatment at 100 W and 10℃ for 5 minutes to obtain a pretreatment solution.

[0045] (2) Compound microbial fermentation

[0046] The pretreatment solution was inoculated with a compound microorganism, fermented at 35℃ for 36 h, kept at 80℃ for 20 min, and filtered through a 0.45 μm filter membrane to obtain the fermentation broth.

[0047] The complex microorganisms consist of Akkermansia muciniphila. 24917 and Bifidobacterium bifidum 6168 is composed of bacteria in a weight ratio of 2:1, with the bacterial activity of Akkermansia myxophilus at 1 billion CFU / g and the bacterial activity of Bifidobacterium bifidum at 1 billion CFU / g. The inoculation amount of the compound microorganisms is 2‰ of the weight of the mucosa.

[0048] (3) Resin adsorption and elution

[0049] Add macroporous strong base anion exchange resin (5% of the weight of the fermentation broth) to the fermentation broth for adsorption for 5 hours, filter, and then elute the macroporous strong base anion exchange resin with 1 mol / L sodium chloride solution (twice the weight of the macroporous strong base anion exchange resin after adsorption) at 100 r / min for 6 hours to obtain the eluent.

[0050] (4) Alcohol precipitation

[0051] The eluent was mixed with an 80% ethanol solution at a volume ratio of 1:1, allowed to stand for 4 hours, the precipitate was collected, and dried at 30°C until the water content was 5% to obtain heparin sodium.

[0052] Example 3

[0053] (1) Pretreatment of porcine small intestinal mucosa

[0054] The mucosa separated from fresh pig small intestine was placed in a homogenizer and homogenized for 10 minutes at 200 r / min and 20℃. After being mixed with deionized water (the weight ratio of mucosa to deionized water was 1:10), it was subjected to high pressure treatment at 300 MPa and 20℃ for 2 minutes, and then subjected to ultrasonic treatment at 150 W and 29℃ for 2 minutes to obtain a pretreatment solution.

[0055] (2) Compound microbial fermentation

[0056] The pretreatment solution was inoculated with a compound microorganism and fermented at 40℃ for 24 h, then kept at 90℃ for 10 min, and filtered through a 0.22 μm filter membrane to obtain the fermentation broth.

[0057] The complex microorganisms consist of Akkermansia muciniphila. 24917 and Bifidobacterium bifidum 6168 is composed of bacteria in a weight ratio of 5:1, with the bacterial activity of Akkermansia myxophilus at 2 billion CFU / g and the bacterial activity of Bifidobacterium bifidum at 2 billion CFU / g. The inoculation amount of the compound microorganisms is 5‰ of the weight of the mucosa.

[0058] (3) Resin adsorption and elution

[0059] Add macroporous strong base anion exchange resin (10% of the weight of the fermentation broth) to the fermentation broth for adsorption for 2 hours, filter, and then elute the macroporous strong base anion exchange resin with 2 mol / L sodium chloride solution (5 times the weight of the macroporous strong base anion exchange resin after adsorption) at 200 r / min for 3 hours to obtain the eluent.

[0060] (4) Alcohol precipitation

[0061] The eluent was mixed with a 90% ethanol solution at a volume ratio of 1:1, allowed to stand for 4 hours, the precipitate was collected, and dried at 40°C until the water content was 2% to obtain heparin sodium.

[0062] Comparative Example 1

[0063] The specific implementation method is the same as that in Example 1, except that the pressure is omitted and the ultrasonic treatment conditions are 120W and 15℃ for 6 minutes.

[0064] Comparative Example 2

[0065] The specific implementation method is the same as in Example 1, except that ultrasound is omitted and the pressure treatment conditions are high pressure treatment for 6 minutes at 250MPa and 15℃.

[0066] Comparative Example 3

[0067] The specific implementation method is the same as in Example 1, except that the fermentation microorganism is Akkermansia muciniphila. 24917, with a bacterial activity of 1.5 billion CFU / g, an inoculum size of 3‰ of the mucosa weight, and fermentation at 37℃ for 30 hours.

[0068] Comparative Example 4

[0069] The specific implementation method is the same as in Example 1, except that the fermentation microorganism is Bifidobacterium bifidum. The 6168 bacteria had a viability of 1.5 billion CFU / g, and the inoculum size was 3‰ of the mucosa weight. Fermentation was carried out at 37℃ for 30 hours.

[0070] Comparative Example 5

[0071] The specific implementation method is the same as in Example 1, except that the compound microorganisms consist of *Lactobacillus gasseri* GDM CC 60092 (purchased from Guangdong Provincial Microbial Culture Collection Center) and *Bifidobacterium longum*. 6202 (purchased from China Industrial Microbial Culture Collection Center) was composed of Lactobacillus gasseri with a cell count of 1.5 billion CFU / g and Bifidobacterium longum with a cell count of 1.5 billion CFU / g. The inoculum amount of the compound microorganisms was 3‰ of the weight of the mucosa. Fermentation was carried out at 37°C for 30 hours.

[0072] Comparative Example 6

[0073] The specific implementation method is the same as in Example 1, except that the resin is replaced with D301 macroporous weak base anion exchange resin (purchased from Tianjin Xijinna Environmental Protection Materials Technology Co., Ltd.).

[0074] Experimental Example 1

[0075] The potency and yield of heparin sodium in Examples 1-3 and Comparative Examples 1-6 of this invention were tested, and the specific results are shown in Table 1.

[0076] The heparin sodium potency was determined using the azurite A colorimetric method.

[0077] Heparin sodium yield (%) = mass of heparin sodium ÷ mass of mucosal powder × 100%.

[0078] Table 1. Potency and yield of heparin sodium in Examples 1-3 and Comparative Examples 1-6

[0079] Group Potency (U / mg) Yield (%) Example 1 182.8 4.33 Example 2 178.4 4.29 Example 3 175.8 4.25 Comparative Example 1 165.7 4.11 Comparative Example 2 160.3 3.95 Comparative Example 3 157.9 3.80 Comparative Example 4 150.5 3.67 Comparative Example 5 164.8 4.06 Comparative Example 6 162.2 4.02

[0080] As shown in Table 1, the heparin sodium produced by the bio-fermentation method of this invention has high potency and yield. Data from Example 1 and Comparative Examples 1-2 show that high-pressure and ultrasonic treatment of the porcine small intestinal mucosa promotes the dissolution of cell contents, weakens the binding force of heparin sodium with other impurities, and is more conducive to the extraction of heparin sodium. Data from Example 1 and Comparative Examples 3-5 show that the composite microorganisms *Ackermania myxophilus* and two *Bifidobacteria* exhibit a synergistic effect, and their combination significantly improves the potency and yield of heparin sodium. Data from Example 1 and Comparative Example 6 show that the macroporous strong-base anion exchange resin of this invention… Compared to other resins, A-72MP specifically adsorbs heparin sodium, improving its potency and yield. The various steps in this invention exhibit synergistic effects, achieving the desired improvement in heparin sodium potency and yield.

[0081] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for producing heparin sodium by microbial fermentation, characterized in that, The method is as follows: the mucosa separated from fresh pig small intestine is crushed, mixed with water, treated under high pressure and ultrasound, inoculated with compound microorganisms for anaerobic fermentation, filtered, the fermentation broth is adsorbed by resin, eluted with sodium chloride solution, the eluent is precipitated with alcohol, the precipitate is collected, and heparin sodium is obtained. The complex microorganism consists of Akkermansia myxophila and two Bifidobacteria in a weight ratio of 2-5:1; Akkermansia myxophilus and Bifidobacterium bifidum are specifically Akkermansia myxophilus. Akkermansia muciniphila CICC ® 24917 and Bifidobacterium bifidum Bifidobacterium bifidum CICC ® 6168; The resin is a macroporous, strongly basic anion exchange resin; The viability of Akkermansia myxophilus was 1-2 billion CFU / g, and the viability of Bifidobacterium bifidum was 5-1 billion CFU / g; the inoculum amount of the compound microorganisms was 2‰-5‰ of the weight of the mucosa, the fermentation temperature was 35-40℃, and the time was 24-36h.

2. The method according to claim 1, characterized in that, The crushing speed is 100-200 r / min, the temperature is 10-20℃, and the time is 10-20 min.

3. The method according to claim 1, characterized in that, The weight ratio of mucosa to water is 1:5-10.

4. The method according to claim 1, characterized in that, The high pressure is 200-300 MPa, the temperature is 10-20℃, and the time is 2-5 minutes.

5. The method according to claim 1, characterized in that, The ultrasound power is 100-150W, the temperature is 10-20℃, and the time is 2-5 minutes.

6. The method according to claim 1, characterized in that, The filter membrane used for filtration is a 0.22-0.45μm membrane.

7. The method according to claim 1, characterized in that, The amount of resin used is 5%-10% of the weight of the fermentation broth, and the adsorption time of the fermentation broth by the resin is 2-5 hours.

8. The method according to claim 1, characterized in that, The concentration of sodium chloride solution is 1-2 mol / L, and the amount used is 2-5 times the weight of the resin; the elution speed is 100-200 r / min, and the time is 3-6 h.

9. The method according to claim 1, characterized in that, Alcohol precipitation includes: Mix the eluent with an ethanol solution, let stand for 4-6 hours, and collect the precipitate; the volume percentage of the ethanol solution is 80%-90%.