Co-production process for extracting heparin, heparinoid, chondroitin, hyaluronic acid and protein from lung

Abstract

The invention belongs to the technical field of biology, and particularly relates to a co-production process for extracting heparin, heparinoid, chondroitin, hyaluronic acid and protein from lungs. According to the method, the FPA98 CL type strong base anion exchange resin suitable for glycosaminoglycan adsorption is adopted to adsorb heparin, heparinoid, chondroitin and hyaluronic acid, and sodium chloride solutions with different concentrations are adopted to perform stepwise elution on the heparin, the heparinoid, the chondroitin and the hyaluronic acid according to different combining capacities of the heparin, the heparinoid, the chondroitin and the hyaluronic acid with the resin; in order to improve the distinguishing capability of chondroitin and hyaluronic acid, sulfur trioxide ventilation treatment is carried out before enzymolysis. Through the process provided by the invention, co-production of heparin, heparinoid, chondroitin, hyaluronic acid and protein is successfully realized, and the process has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a co-production process for extracting heparin, heparinoids, chondroitin, hyaluronic acid and protein from lungs. Background Art

[0002] Heparin and heparinoids were discovered from dog livers in 1916 and are widely found in the liver, lungs, intestines and other organs of animals. Heparin and heparinoids have the function of preventing blood coagulation and dissolving blood clots, and are excellent natural anticoagulants. At present, heparin and heparinoids are the most effective and clinically used anticoagulants in the world. European and American countries first extracted heparin and heparinoids in large quantities from cattle lungs and cattle intestines. Due to the prevalence of mad cow disease, the extraction of heparin and heparinoids was stopped. Later, relevant experts found a large amount of heparin and heparinoids in pig small intestines. Since my country is a major pig-raising country, the raw materials for the extraction of heparin and heparinoids have gradually been replaced by pig small intestines. Most foreign countries rely on my country to import heparin and heparinoids.

[0003] At present, crude heparin and heparinoids in my country are mostly produced with pig small intestine mucosa as raw materials. Due to the simple extraction technology and small investment, there is almost one company in every county and city, competing for small intestine resources, forming vicious competition, so that heparin and heparinoid producers have meager profits. In addition, due to the foreign financial crisis, enterprises that use pig small intestine as raw material to produce heparin and heparinoids have difficulty in exporting casing products after removing heparin and heparinoids extracted from pig intestinal mucosa. Therefore, in order to improve economic benefits, it is necessary to select new raw materials for heparin and heparinoid extraction and develop new extraction technologies. According to expert analysis, although there are a large number of heparin and heparinoids in animal lungs, due to their tissue specificity, there are a large number of miscellaneous proteins that bind to heparin and heparinoids very firmly. How to effectively extract heparin and heparinoids from the lungs is an important research topic in the current biological community.

[0004] Although in recent years, a few domestic researchers have studied the extraction of heparin and heparinoids from pig lungs and have made some progress, these methods only produce heparin and heparinoids as a product, and do not solve the problem of comprehensive utilization of waste liquid after the extraction of heparin and heparinoids. In addition to heparin and heparinoids, animal lungs also contain a large amount of hyaluronic acid, chondroitin and other substances. How to maximize the extraction of beneficial substances in animal lungs is a technical problem that needs to be solved. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a co-production process for extracting heparin, heparinoids, chondroitin, hyaluronic acid and protein from the lung, which comprises the following steps: (1) Take fresh or frozen pig lung or cow lung, remove the fascia, wash, mince, grind, add an equal amount of deionized water, stir evenly to obtain a mixed solution; (2) introducing sulfur trioxide gas into the mixed solution until the pH value of the mixed solution reaches 4.0-5.0, and stirring for 2-4 hours to obtain a treated solution; (3) adjusting the pH of the treatment solution to 8.5-9, then adding a composite protease for enzymatic hydrolysis, killing the enzyme after the enzymatic hydrolysis, filtering out the residue, and obtaining an enzymatic hydrolyzate; (4) Cooling the enzymatic hydrolyzate to 45-55°C, adding a strong base anion exchange resin, and adsorbing the solution for 4-8 hours to obtain the adsorbed resin, and collecting the effluent I; (5) Wash the adsorbed resin with water until it is neutral, then mix it with a sodium chloride solution with a mass fraction of 1-2%, and wash it with stirring at 45-55°C for 2-4 hours to obtain the washed resin, and collect the effluent II; (6) Mix the washed resin with a 4-5% by mass sodium chloride solution, keep warm at 45-55°C and stir for 4-6 hours, separate the resin, and obtain eluent A; The resin separated in the previous step was mixed with a sodium chloride solution having a mass fraction of 12-15%, and stirred at 45-55°C for 4-6 hours to separate the resin to obtain an eluent B; The resin separated in the previous step is mixed with a sodium chloride solution with a mass fraction of 25-30%, and stirred at 45-55°C for 4-6 hours to separate the resin and obtain eluent C; The resin separated in the previous step was mixed with a sodium chloride solution having a mass fraction of 40-45%, and stirred at 45-55°C for 4-6 hours to separate the resin and obtain an eluent D; (7) adding 95% by volume ethanol to the eluents A, B, C and D for precipitation, standing for 2-4 hours, mixing the precipitate with ethanol for dehydration and drying to obtain hyaluronic acid, chondroitin, heparin and heparinoid, respectively; (8) Combine effluent I and effluent II, stir evenly, adjust the pH to 6.5-7.0, perform nanofiltration desalination and concentration using a 100-150Da nanofiltration membrane, collect the concentrate, and spray dry to obtain protein powder.

[0006] Furthermore, the composite protease is at least one of trypsin, 2709 alkaline protease and animal hydrolytic protease.

[0007] Furthermore, the added amount of the composite protease is 0.05-0.15 wt % of the treatment liquid.

[0008] Furthermore, the temperature of the enzymolysis is 48-52°C and the time is 4-6h.

[0009] Furthermore, the strong base anion exchange resin is FPA98 CL type resin.

[0010] Furthermore, in step (4), the amount of the strong base anion exchange resin added is 15-20% (m / v) of the volume of the enzymatic hydrolysate.

[0011] Furthermore, in step (5), the solid-liquid ratio of the resin to the sodium chloride solution is 1:2.2-2.6 g / mL.

[0012] Furthermore, in each elution step of step (6), the solid-liquid ratio of the resin to the sodium chloride solution is 1:1.5-1.8 g / mL.

[0013] Furthermore, the amount of ethanol added in the ethanol precipitation of step (7) is such that the ethanol concentration in the mixture of ethanol and the eluent reaches 40-60%.

[0014] Beneficial effects of the present invention In order to realize the co-production of heparin, heparinoid, chondroitin, hyaluronic acid, the present invention adopts FPA98 CL type strong base anion exchange resin suitable for glycosaminoglycan adsorption to adsorb heparin, heparinoid, chondroitin, hyaluronic acid, and utilizes heparin, heparinoid, chondroitin, hyaluronic acid and resin binding ability to adopt different concentrations of sodium chloride solution to carry out step-by-step elution. Further, in order to improve the ability to distinguish chondroitin and hyaluronic acid, sulfur trioxide ventilation treatment is carried out before enzymolysis, this one hand can make the protein denaturation in lung tissue fluid form disordered and relatively loose stretching structure, is conducive to subsequent enzymolysis, on the other hand can promote the sulfation of heparin, heparinoid side chain reaction site, effectively avoid the loss of side chain sulfate group in enzymolysis process, so as to ensure that the binding ability of heparin, heparinoid and strong base anion exchange resin is significantly stronger than the binding ability of chondroitin and strong base anion exchange resin, to be conducive to the difference elution of heparin, heparinoid and chondroitin. DETAILED DESCRIPTION

[0015] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0016] In the following examples, the FPA98 CL resin used was purchased from DuPont, and the particle size was 630-850 μm. It was activated as follows before use: the resin was soaked in warm water at 50°C for 20-24 hours, then rinsed with clean water, then added with a 10% NaOH solution and stirred for 1-2 hours, and finally rinsed with clean water until the pH of the washing solution was neutral.

[0017] The D-254 resin used (which is an anion exchange resin with a quaternary ammonium group on a macroporous styrene-divinylbenzene copolymer) was purchased from Shanghai Keraman Reagent Co., Ltd. with a catalog number of 154430.

[0018] Example 1 A co-production process for extracting heparin, heparinoids, chondroitin, hyaluronic acid, and protein from lungs, comprising the following steps: (1) Take fresh pig lungs, remove the fascia, wash, mince, grind, add an equal amount of deionized water, stir evenly, and obtain a mixed solution; (2) introducing sulfur trioxide gas into the mixed solution until the pH value of the mixed solution reaches 4.0, and stirring for 2 hours to obtain a treated solution; (3) The pH of the treatment solution was adjusted to 8.5, and then a composite protease consisting of equal amounts of trypsin, 2709 alkaline protease and animal hydrolyzing protease was added at 0.1% of the treatment solution mass, and the mixture was hydrolyzed at 50°C for 4 h. After the hydrolysis was completed, the mixture was heated to 85°C for 30 min to inactivate the enzyme, and the residue was filtered to obtain an enzymatic solution; (4) Cooling the enzymatic hydrolysate to 50°C, adding FPA98 CL type strong base anion exchange resin, the amount of resin added is 17% (m / v) of the volume of the enzymatic hydrolysate, and adsorbing at 50°C for 6 h to obtain the adsorbed resin, and collecting the effluent I; (5) The adsorbed resin was washed with water until neutral, and then mixed with a 1.5% sodium chloride solution at a solid-liquid ratio of 1:2.5 g / mL. The mixture was stirred and washed at 50°C for 2 h at a stirring speed of 500 rpm / min to obtain the washed resin, and the effluent II was collected; (6) The washed resin was mixed with a sodium chloride solution having a mass fraction of 4.5% at a solid-liquid ratio of 1:1.5 g / mL. The mixture was stirred at 50°C for 5 h at a stirring speed of 500 rpm / min. The resin was separated to obtain an eluent A. The resin separated in the previous step was mixed with a sodium chloride solution with a mass fraction of 13%, with a solid-liquid ratio of resin to brine of 1:1.5 g / mL, and stirred at 50°C for 5 h at a stirring speed of 500 rpm / min to separate the resin and obtain eluent B; The resin separated in the previous step was mixed with a sodium chloride solution with a mass fraction of 26%, with a solid-liquid ratio of resin to brine of 1:1.5 g / mL, and stirred at 50°C for 5 h at a stirring speed of 500 rpm / min to separate the resin and obtain eluent C; The resin separated in the previous step was mixed with a sodium chloride solution having a mass fraction of 40-45%, and stirred at 45-55°C for 4-6 hours to separate the resin and obtain an eluent D; (7) The eluents A, B and C were respectively mixed with 95% by volume ethanol to make the ethanol concentration in the mixture of ethanol and the eluent reach 45%, and the mixture was allowed to settle for 20 hours. The precipitate was collected, and the precipitate was mixed with 95% by volume ethanol for dehydration for 2 hours. After filtering, the precipitate was dried at 60° C. to obtain hyaluronic acid, chondroitin, heparin and heparinoid, respectively; (8) The effluent I and effluent II were combined, stirred evenly, the pH was adjusted to 6.8, and nanofiltration desalination and concentration were performed using a 150Da nanofiltration membrane. The concentrate was collected and spray-dried (the air inlet temperature was 210°C and the air outlet temperature was 85°C) to obtain protein powder.

[0019] Example 2 A co-production process for extracting heparin, heparinoids, chondroitin, hyaluronic acid, and protein from lungs, comprising the following steps: (1) Take frozen beef lung, remove the fascia, wash, mince, grind, add an equal amount of deionized water, stir evenly to obtain a mixed solution; (2) introducing sulfur trioxide gas into the mixed solution until the pH value of the mixed solution reaches 5.0, and stirring for 4 hours to obtain a treated solution; (3) Adjust the pH of the treatment solution to 9.0, then add 0.1% of the treatment solution mass of a composite protease consisting of equal amounts of trypsin, 2709 alkaline protease and animal hydrolyzing protease, perform enzymolysis at 50°C for 6 h, and after the enzymolysis is completed, heat the solution to 85°C to inactivate the enzyme for 30 min, filter out the residue, and obtain an enzymolysis solution; (4) Cooling the enzymatic hydrolysate to 50°C, adding FPA98 CL type strong base anion exchange resin, the amount of resin added is 20% (m / v) of the volume of the enzymatic hydrolysate, and adsorbing at 50°C for 6 h to obtain the adsorbed resin, and collecting the effluent I; (5) The adsorbed resin was washed with water until neutral, and then mixed with a 1.5% sodium chloride solution at a solid-liquid ratio of 1:2.5 g / mL. The mixture was stirred and washed at 50°C for 2 h at a stirring speed of 500 rpm / min to obtain the washed resin, and the effluent II was collected; (6) The washed resin was mixed with a sodium chloride solution having a mass fraction of 4.5% at a solid-liquid ratio of 1:1.5 g / mL. The mixture was stirred at 50°C for 5 h at a stirring speed of 500 rpm / min. The resin was separated to obtain an eluent A. The resin separated in the previous step was mixed with a sodium chloride solution with a mass fraction of 15%, with a solid-liquid ratio of resin to brine of 1:1.5 g / mL, and stirred at 50°C for 5 h at a stirring speed of 500 rpm / min to separate the resin and obtain eluent B; The resin separated in the previous step was mixed with a sodium chloride solution with a mass fraction of 30%, with a solid-liquid ratio of resin to brine of 1:1.5 g / mL, and stirred at 50°C for 5 h at a stirring speed of 500 rpm / min to separate the resin and obtain eluent C; The resin separated in the previous step was mixed with a sodium chloride solution having a mass fraction of 40-45%, and stirred at 45-55°C for 4-6 hours to separate the resin and obtain an eluent D; (7) The eluents A, B and C were respectively mixed with 95% by volume ethanol to make the ethanol concentration in the mixture of ethanol and the eluent reach 45%, and the mixture was allowed to settle for 20 hours. The precipitate was collected, and the precipitate was mixed with 95% by volume ethanol for dehydration for 2 hours. After filtering, the precipitate was dried at 60° C. to obtain hyaluronic acid, chondroitin, heparin and heparinoid, respectively; (8) The effluent I and effluent II were combined, stirred evenly, the pH was adjusted to 6.8, and nanofiltration desalination and concentration were performed using a 150Da nanofiltration membrane. The concentrate was collected and spray-dried (the air inlet temperature was 210°C and the air outlet temperature was 85°C) to obtain protein powder.

[0020] Comparative Example 1 The same process as in Example 1 was used to co-produce heparin, heparinoids, chondroitin, hyaluronic acid, and protein, except that the sulfur trioxide ventilation treatment in step (2) was omitted, and the mixed solution in step (1) was directly subjected to the enzymatic hydrolysis treatment in step (3). The results showed that the yield of heparin and heparinoids obtained from eluate C was low, and the chondroitin obtained from eluate B contained more heparin and heparinoid impurities.

[0021] Comparative Example 2 The same process as in Example 1 was used to co-produce heparin, heparinoids, chondroitin, hyaluronic acid, and protein, except that D-254 resin was used in place of FPA98 CL resin in step (4). It was found that heparin and heparinoids could not be obtained from eluent C, and the chondroitin obtained from eluent B contained a large amount of heparin and heparinoid impurities. It was found that the yield of heparin and heparinoids obtained from eluent C was very low, and the chondroitin obtained from eluent B contained a large amount of heparin and heparinoid impurities.

[0022] Comparative Example 3 The same process as in Example 1 was used to co-produce heparin, heparinoids, chondroitin, hyaluronic acid, and protein, except that in step (6), a 3% sodium chloride solution was used to elute the resin to obtain eluent A. The results showed that the yield of hyaluronic acid obtained from eluent A was low.

[0023] Comparative Example 4 The same process as in Example 1 was used to co-produce heparin, heparinoids, chondroitin, hyaluronic acid, and protein, except that in step (6), a 10% sodium chloride solution was used to elute the resin to obtain eluent B. The results showed that the yield of chondroitin obtained from eluent A was lower.

[0024] Comparative Example 5 The same process as in Example 1 was used to co-produce heparin, heparinoids, chondroitin, hyaluronic acid, and protein, except that in step (6), a 22% sodium chloride solution was used to elute the resin to obtain eluent C. The results showed that the yields of heparin and heparinoids obtained from eluent A were lower.

[0025] It should be noted that the preferred embodiments of the present invention are given in the specification of the present invention, but the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the present invention specification; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A process for extracting heparin, heparinoids, chondroitin, hyaluronic acid and protein from lungs, characterized in that: The following steps are involved: (1) Take fresh or frozen pig lung or cow lung, remove the fascia, wash, mince, grind, add an equal amount of deionized water, stir evenly to obtain a mixed solution; (2) introducing sulfur trioxide gas into the mixed solution until the pH value of the mixed solution reaches 4.0-5.0, and stirring for 2-4 hours to obtain a treated solution; (3) adjusting the pH of the treatment solution to 8.5-9, then adding a composite protease for enzymatic hydrolysis, killing the enzyme after the enzymatic hydrolysis, filtering out the residue, and obtaining an enzymatic hydrolyzate; (4) Cooling the enzymatic hydrolyzate to 45-55°C, adding a strong base anion exchange resin, and adsorbing the solution for 4-8 hours to obtain the adsorbed resin, and collecting the effluent I; (5) Wash the adsorbed resin with water until it is neutral, then mix it with a sodium chloride solution with a mass fraction of 1-2%, and wash it with stirring at 45-55°C for 2-4 hours to obtain the washed resin, and collect the effluent II; (6) Mix the washed resin with a 4-5% by mass sodium chloride solution, keep warm at 45-55°C and stir for 4-6 hours, separate the resin, and obtain eluent A; The resin separated in the previous step was mixed with a sodium chloride solution having a mass fraction of 12-15%, and stirred at 45-55°C for 4-6 hours to separate the resin to obtain an eluent B; The resin separated in the previous step is mixed with a sodium chloride solution with a mass fraction of 25-30%, and stirred at 45-55°C for 4-6 hours to separate the resin and obtain eluent C; The resin separated in the previous step was mixed with a sodium chloride solution having a mass fraction of 40-45%, and stirred at 45-55°C for 4-6 hours to separate the resin and obtain an eluent D; (7) adding a predetermined volume fraction of ethanol to each of the eluents A, B, C and D for precipitation, standing for 2-4 hours, mixing the precipitate with ethanol for dehydration, and drying to obtain hyaluronic acid, chondroitin, heparin and heparinoid, respectively; (8) Combine effluent I and effluent II, stir evenly, adjust the pH to 6.5-7.0, perform nanofiltration desalination and concentration using a 100-150Da nanofiltration membrane, collect the concentrate, and spray dry to obtain protein powder.

2. The co-production process according to claim 1, characterized in that: The composite protease is at least one of trypsin, 2709 alkaline protease and animal hydrolytic protease.

3. The co-production process according to claim 1, characterized in that: The added amount of the composite protease is 0.05-0.15wt% of the treatment liquid.

4. The co-production process according to claim 1, characterized in that: The temperature of the enzymolysis is 48-52° C. and the time is 4-6 hours.

5. The co-production process according to claim 1, characterized in that: The strong base anion exchange resin is FPA98 CL type resin.

6. The co-production process according to claim 1, characterized in that: In step (4), the amount of strong base anion exchange resin added is 15-20% (m / v) of the volume of the enzymatic hydrolyzate.

7. The co-production process according to claim 1, characterized in that: In step (5), the solid-liquid ratio of the resin to the sodium chloride solution is 1:2.2-2.6 g / mL.

8. The co-production process according to claim 1, characterized in that: In each elution step of step (6), the solid-to-liquid ratio of the resin to the sodium chloride solution is 1:1.5-1.8 g / mL.

9. The co-production process according to claim 1, characterized in that: The amount of ethanol added in the ethanol precipitation of step (7) is such that the ethanol concentration in the mixture of ethanol and the eluent reaches 40-60%.