A co-production process for extracting heparin, heparan, chondroitin, hyaluronic acid, proteins from the lungs

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a co-production process for extracting heparin, heparan, chondroitin, hyaluronic acid and protein from lung. The present application adopts FPA98 CL type strong base anion exchange resin suitable for adsorption of glycosaminoglycan to adsorb heparin, heparan, chondroitin and hyaluronic acid, and different concentrations of sodium chloride solution are used to stepwise elute heparin, heparan, chondroitin and hyaluronic acid due to different binding abilities of heparin, heparan, chondroitin and hyaluronic acid to the resin. In addition, in order to improve the distinguishing ability of chondroitin and hyaluronic acid, sulfur trioxide aeration treatment is carried out before enzymolysis. Through the process of the present application, the co-production of heparin, heparan, chondroitin, hyaluronic acid and protein is successfully realized, and has good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a co-production process for extracting heparin, heparan, chondroitin, hyaluronic acid and protein from lung. BACKGROUND

[0002] Heparin and heparan were discovered in 1916 from the liver of a dog, and widely exist in the liver, lung, intestine and other organs of animals. Heparin and heparan have the functions of preventing blood coagulation and dissolving thrombus, and are excellent natural anticoagulants. At present, heparin and heparan are the most effective and largest-dose anticoagulants in the world. The countries in Europe and America first extracted heparin and heparan from the lung and intestine of a cow in large quantities, and stopped the extraction of heparin and heparan due to the prevalence of mad cow disease. Later, relevant experts found that there is a large amount of heparin and heparan in the small intestine of a pig. Since China is a large country for raising pigs, the raw material for extracting heparin and heparan has been gradually replaced by the small intestine of a pig, and foreign countries mostly import heparin and heparan from China.

[0003] At present, the production of crude heparin and heparan in China mostly uses the mucosa of the small intestine of a pig as raw material. Since the extraction technology is simple and the investment is not large, almost every county and city has one factory, and the small intestine resources are fiercely competed for, resulting in a small profit of the producers of heparin and heparan. In addition, due to the financial crisis in foreign countries, the enterprises for producing heparin and heparan from the small intestine of a pig have difficulty in exporting the sausage products after extracting heparin and heparan from the mucosa of the small intestine. Therefore, in order to improve the economic benefit, it is necessary to select a new raw material for extracting heparin and heparan and research a new extraction technology. According to the analysis of experts, although there is a large amount of heparin and heparan in the lung of an animal, due to the tissue specificity, there are a large amount of impurities which are combined with heparin and heparan very firmly. How to effectively extract heparin and heparan from the lung is an important subject in the field of biology.

[0004] Although in recent years, a few scientific researchers in China have studied the extraction of heparin and heparan from the lung of a pig, and have made some progress, these methods only produce one product of heparin and heparan, and do not solve the problem of comprehensive utilization of the waste liquid after extracting heparin and heparan. In addition to heparin and heparan, the lung of an animal also contains a large amount of hyaluronic acid, chondroitin and other substances. How to maximize the extraction of beneficial substances from the lung of an animal is a technical problem to be solved. SUMMARY

[0005] In order to solve the above problems, the present application provides a co-production process for extracting heparin, heparan, chondroitin, hyaluronic acid and protein from lung, which comprises the following steps:

[0006] (1) taking fresh or frozen pig lung or cow lung, removing the fascia, washing, grinding, grinding into pulp, then adding an equal amount of deionized water, stirring uniformly to obtain a mixed liquid;

[0007] (2) passing sulfur trioxide gas into the mixed solution until the pH of the mixed solution reaches 4.0-5.0, stirring for 2-4 h, to obtain a treatment solution;

[0008] (3) adjusting the pH of the treatment solution to 8.5-9, then adding a complex protease for enzymolysis, inactivating the enzyme after the enzymolysis is completed, filtering to remove residues, and obtaining an enzymolysis solution;

[0009] (4) cooling the enzymolysis solution to 45-55℃, adding a strong base anion exchange resin, and incubating and adsorbing the treatment for 4-8 h to obtain the adsorbed resin and collect effluent I;

[0010] (5) washing the adsorbed resin with water until neutral, then mixing the resin with a sodium chloride solution with a mass fraction of 1-2%, incubating and stirring at 45-55℃ for 2-4 h to obtain the washed resin and collect effluent II;

[0011] (6) mixing the washed resin with a sodium chloride solution with a mass fraction of 4-5%, incubating and stirring at 45-55℃ for 4-6 h, separating the resin, and obtaining eluate A;

[0012] mixing the resin separated in the above step with a sodium chloride solution with a mass fraction of 12-15%, incubating and stirring at 45-55℃ for 4-6 h, separating the resin, and obtaining eluate B;

[0013] mixing the resin separated in the above step with a sodium chloride solution with a mass fraction of 25-30%, incubating and stirring at 45-55℃ for 4-6 h, separating the resin, and obtaining eluate C;

[0014] mixing the resin separated in the above step with a sodium chloride solution with a mass fraction of 40-45%, incubating and stirring at 45-55℃ for 4-6 h, separating the resin, and obtaining eluate D;

[0015] (7) precipitating eluates A, B, C, and D by adding ethanol with a volume fraction of 95%, standing for 2-4 h, dehydrating and drying the precipitates by mixing with ethanol, and respectively obtaining hyaluronic acid, chondroitin, heparin, and heparinoids;

[0016] (8) mixing effluents I and II, stirring uniformly, adjusting the pH to 6.5-7.0, performing nanofiltration desalination and concentration by using a nanofiltration membrane with a molecular weight cut-off of 100-150 Da, collecting the concentrated solution, and obtaining a protein powder after spray drying.

[0017] Further, the complex protease is at least one of trypsin, 2709 alkaline protease, and animal hydrolyzed proteinase.

[0018] Further, the added amount of the complex protease is 0.05-0.15wt% of the treatment solution.

[0019] Further, the enzymolysis temperature is 48-52℃, and the time is 4-6h.

[0020] Further, the strong base anion exchange resin is FPA98 CL resin.

[0021] Further, in step (4), the added amount of the strong base anion exchange resin is 15-20% (m / v) of the enzymolysis solution.

[0022] Further, in step (5), the solid-liquid ratio of the resin to the sodium chloride solution is 1:2.2-2.6g / mL.

[0023] Further, in each step of step (6), the solid-liquid ratio of the resin to the sodium chloride solution is 1:1.5-1.8g / mL.

[0024] Further, in the ethanol precipitation of step (7), the added amount of the ethanol is such that the ethanol concentration in the mixture of the ethanol and the eluent is 40-60%.

[0025] Advantages of the present application

[0026] In order to realize the co-production of heparin, heparan, chondroitin and hyaluronic acid, the present application uses FPA98 CL strong base anion exchange resin suitable for adsorption of glycosaminoglycans to adsorb heparin, heparan, chondroitin and hyaluronic acid, and uses different concentrations of sodium chloride solution to elute them in steps according to their different binding abilities to the resin. Further, in order to improve the differentiation ability of chondroitin and hyaluronic acid, the lung tissue fluid is subjected to sulfur trioxide aeration treatment before enzymolysis. This can denature the proteins in the lung tissue fluid into disordered and relatively loose stretched structures, which is conducive to subsequent enzymolysis, and can promote the sulfation of the side chain reaction sites of heparin and heparan, effectively avoiding the loss of side chain sulfate groups during enzymolysis, so as to ensure that the binding ability of heparin and heparan to the strong base anion exchange resin is significantly stronger than that of chondroitin and hyaluronic acid, thereby facilitating the differential elution of heparin, heparan and chondroitin. DETAILED DESCRIPTION

[0027] The present application will be further described below in combination with specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field.

[0028] In the following examples, the FPA98 CL resin used is purchased from DuPont Company, with a particle size of 630-850 μm. Before use, it is activated as follows: the resin is soaked in warm water at 50°C for 20-24 h, then washed with clean water, then added with a 10% NaOH solution by mass concentration and stirred for 1-2 h, and finally washed with clean water until the washing liquid is neutral.

[0029] The D-254 resin used (which is an anion exchange resin with quaternary ammonium groups on a macroporous structure of styrene-divinylbenzene copolymer) is purchased from Shanghai Kolamann Reagent Co., Ltd., with a product code of 154430.

[0030] Example 1

[0031] A process for co-production of heparin, heparan sulfate, chondroitin, hyaluronic acid and protein from lung, comprising the following steps:

[0032] (1) Take fresh pig lung, remove the fascia, wash, shred, grind, then add an equal amount of deionized water, stir to obtain a mixture;

[0033] (2) Pass sulfur trioxide gas into the mixture until the pH of the mixture reaches 4.0, stir for 2 h to obtain a treated liquid;

[0034] (3) Adjust the pH of the treated liquid to 8.5, then add a composite protease composed of equal amounts of trypsin, 2709 alkaline protease and animal hydrolyzed protease at 0.1% of the mass of the treated liquid, and carry out enzymatic hydrolysis at 50°C for 4 h, then raise the temperature to 85°C to inactivate the enzyme for 30 min, filter to remove the residue, and obtain an enzymatic hydrolysis liquid;

[0035] (4) Cool the enzymatic hydrolysis liquid to 50°C, add FPA98 CL strong base anion exchange resin, the amount of resin added is 17% (m / v) of the volume of the enzymatic hydrolysis liquid, and incubate at 50°C for 6 h to obtain the resin after adsorption, and collect the effluent I;

[0036] (5) Wash the resin after adsorption with water until neutral, then mix with a 1.5% sodium chloride solution by mass fraction, the solid-liquid ratio of the resin to the sodium chloride solution is 1:2.5 g / mL, incubate at 50°C and stir for 2 h, the stirring speed is 500 rpm / min, to obtain the washed resin, and collect the effluent II;

[0037] (6) Mix the washed resin with a 4.5% sodium chloride solution by mass fraction, the solid-liquid ratio of the resin to the brine is 1:1.5 g / mL, incubate at 50°C and stir for 5 h, the stirring speed is 500 rpm / min, separate the resin, and obtain the eluate A;

[0038] The resin separated in the previous step was mixed with a 13% by mass sodium chloride solution, the solid-liquid ratio of the resin to the brine was 1:1.5 g / mL, 50°C incubation and stirring for 5 h, the stirring speed was 500 rpm / min, the resin was separated, and eluate B was obtained;

[0039] The resin separated in the previous step was mixed with a 26% by mass sodium chloride solution, the solid-liquid ratio of the resin to the brine was 1:1.5 g / mL, 50°C incubation and stirring for 5 h, the stirring speed was 500 rpm / min, the resin was separated, and eluate C was obtained;

[0040] The resin separated in the previous step was mixed with a 40-45% by mass sodium chloride solution, 45-55°C incubation and stirring for 4-6 h, the resin was separated, and eluate D was obtained;

[0041] (7) Eluates A, B and C were each mixed with 95% by volume ethanol, so that the ethanol concentration in the mixture of ethanol and eluate was 45%, and the mixture was allowed to stand for 20 h, the precipitate was collected, the precipitate was mixed with 95% by volume ethanol for dehydration for 2 h, and then dried at 60°C after filtration, to obtain hyaluronic acid, chondroitin, and heparin and heparan sulfate, respectively;

[0042] (8) The effluent I and the effluent II were combined, stirred uniformly, the pH was adjusted to 6.8, and nanofiltration desalination and concentration were performed using a 150 Da nanofiltration membrane, the concentrated solution was collected, and protein powder was obtained after spray drying (the inlet temperature was 210°C, and the outlet temperature was 85°C).

[0043] Example 2

[0044] A co-production process for extracting heparin, heparan sulfate, chondroitin, hyaluronic acid and protein from lungs, comprising the following steps:

[0045] (1) A frozen bovine lung was taken, the fascia was removed, washed, minced, ground, and then an equal amount of deionized water was added, and the mixture was stirred uniformly to obtain a mixture;

[0046] (2) Sulfur trioxide gas was introduced into the mixture until the pH of the mixture reached 5.0, and the mixture was stirred for 4 h to obtain a treated solution;

[0047] (3) The pH of the treated solution was adjusted to 9.0, and then 0.1% of a composite protease composed of equal amounts of trypsin, 2709 alkaline protease and animal hydrolyzed protease was added to the treated solution, and the solution was enzymatically hydrolyzed at 50°C for 6 h, and then the temperature was increased to 85°C for 30 min to inactivate the enzyme, the residue was removed by filtration to obtain an enzymatic hydrolysate;

[0048] (4) The enzymatic solution is cooled to 50°C, and FPA98 CL type strong base anion exchange resin is added, the amount of resin added is 20% (m / v) of the volume of the enzymatic solution, and the adsorption treatment is carried out at 50°C for 6h, to obtain the adsorbed resin, and the effluent I is collected;

[0049] (5) The adsorbed resin is washed to neutral with water, and then mixed with a 1.5% sodium chloride solution, the solid-liquid ratio of the resin to the sodium chloride solution is 1:2.5 g / mL, and the washing is carried out at 50°C for 2h with stirring at a speed of 500 rpm / min, to obtain the washed resin, and the effluent II is collected;

[0050] (6) The washed resin is mixed with a 4.5% sodium chloride solution, the solid-liquid ratio of the resin to the brine is 1:1.5 g / mL, and the stirring is carried out at 50°C for 5h, to separate the resin and obtain the eluate A;

[0051] The resin separated in the above step is mixed with a 15% sodium chloride solution, the solid-liquid ratio of the resin to the brine is 1:1.5 g / mL, and the stirring is carried out at 50°C for 5h, to separate the resin and obtain the eluate B;

[0052] The resin separated in the above step is mixed with a 30% sodium chloride solution, the solid-liquid ratio of the resin to the brine is 1:1.5 g / mL, and the stirring is carried out at 50°C for 5h, to separate the resin and obtain the eluate C;

[0053] The resin separated in the above step is mixed with a 40-45% sodium chloride solution, the stirring is carried out at 45-55°C for 4-6h, to separate the resin and obtain the eluate D;

[0054] (7) The eluates A, B and C are each mixed with 95% ethanol by volume, so that the concentration of ethanol in the mixed solution of ethanol and eluate is 45%, and the mixture is allowed to stand for 20h, the precipitate is collected, the precipitate is mixed with 95% ethanol by volume for 2h of dehydration, and the precipitate is dried at 60°C after filtration, to obtain hyaluronic acid, chondroitin and heparin, heparinoids, respectively;

[0055] (8) The effluent I and the effluent II are mixed and stirred uniformly, the pH is adjusted to 6.8, and the mixture is subjected to nanofiltration desalination and concentration using a 150 Da nanofiltration membrane, the concentrated solution is collected, and the protein powder is obtained after spray drying (the inlet temperature is 210°C, and the outlet temperature is 85°C).

[0056] Comparative Example 1

[0057] The same process as in Example 1 was used to co-produce heparin, heparan sulfate, chondroitin, hyaluronic acid, and protein, except that the step (2) sulfur trioxide aeration was omitted and the step (3) enzymatic treatment was performed directly on the mixture from step (1). It was found that the heparin, heparan sulfate yield from eluate C was low and the chondroitin from eluate B contained a high amount of heparin, heparan sulfate impurities.

[0058] Comparative Example 2

[0059] The same process as in Example 1 was used to co-produce heparin, heparan sulfate, chondroitin, hyaluronic acid, and protein, except that the FPA98 CL resin was replaced with D-254 resin in step (4). It was found that no heparin, heparan sulfate was obtained from eluate C and the chondroitin from eluate B contained a high amount of heparin, heparan sulfate impurities. It was found that the heparin, heparan sulfate yield from eluate C was very low and the chondroitin from eluate B contained a very high amount of heparin, heparan sulfate impurities.

[0060] Comparative Example 3

[0061] The same process as in Example 1 was used to co-produce heparin, heparan sulfate, chondroitin, hyaluronic acid, and protein, except that in step (6), a 3% sodium chloride solution was used to elute the resin to obtain eluate A. It was found that the hyaluronic acid yield from eluate A was low.

[0062] Comparative Example 4

[0063] The same process as in Example 1 was used to co-produce heparin, heparan sulfate, chondroitin, hyaluronic acid, and protein, except that in step (6), a 10% sodium chloride solution was used to elute the resin to obtain eluate B. It was found that the chondroitin yield from eluate A was low.

[0064] Comparative Example 5

[0065] The same process as in Example 1 was used to co-produce heparin, heparan sulfate, chondroitin, hyaluronic acid, and protein, except that in step (6), a 22% sodium chloride solution was used to elute the resin to obtain eluate C. It was found that the heparin, heparan sulfate yield from eluate A was low.

[0066] It should be noted that the preferred embodiments of the present application are described in the specification, but the present application can be implemented in many different forms and is not limited to the embodiments described in the specification, which are not intended to be additional limitations on the content of the present application, and the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive. Furthermore, each of the above technical features continues to combine to form various embodiments not listed above, which are considered to be within the scope of the present application specification; further, for those skilled in the art, the above description can be improved or changed, and all these improvements and changes shall fall within the scope of the appended claims of the present application.

Claims

1. A process for co-production of heparin, heparan sulfate, chondroitin, hyaluronic acid and protein from lung, comprising the following steps: (1) taking fresh or frozen pig lung or bovine lung, removing fascia, washing, mincing, grinding, adding equal amount of deionized water, stirring to obtain a mixture; (2) passing sulfur trioxide gas into the mixture until the pH of the mixture reaches 4.0-5.0, stirring for 2-4 hours to obtain a treated solution; (3) adjusting the pH of the treated solution to 8.5-9, then adding a complex protease for enzymatic hydrolysis, inactivating the enzyme after the enzymatic hydrolysis, filtering to remove residues to obtain an enzymatic hydrolysate; (4) cooling the enzymatic hydrolysate to 45-55℃, adding a strong base anion exchange resin, and incubating and adsorbing for 4-8 hours to obtain the adsorbed resin and collect effluent I; the strong base anion exchange resin is FPA98 CL resin; (5) washing the adsorbed resin with water until neutral, then mixing with a 1-2% sodium chloride solution, incubating and stirring at 45-55℃ for 2-4 hours to obtain the washed resin and collect effluent II; (6) mixing the washed resin with a 4-5% sodium chloride solution, incubating and stirring at 45-55℃ for 4-6 hours, separating the resin to obtain eluate A; mixing the separated resin with a 12-15% sodium chloride solution, incubating and stirring at 45-55℃ for 4-6 hours, separating the resin to obtain eluate B; mixing the separated resin with a 25-30% sodium chloride solution, incubating and stirring at 45-55℃ for 4-6 hours, separating the resin to obtain eluate C; mixing the separated resin with a 40-45% sodium chloride solution, incubating and stirring at 45-55℃ for 4-6 hours, separating the resin to obtain eluate D; (7) precipitating eluates A, B, C and D with a predetermined volume fraction of ethanol, standing for 2-4 hours, dehydrating and drying the precipitates with ethanol to obtain hyaluronic acid, chondroitin, heparin and heparan sulfate, respectively; (8) mixing effluents I and II, stirring to obtain a uniform mixture, adjusting the pH to 6.5-7.0, desalting and concentrating by nanofiltration with a 100-150 Da nanofiltration membrane, collecting the concentrated solution, and spray drying to obtain a protein powder. 2.The process according to claim 1, wherein the complex protease is at least one of trypsin, 2709 alkaline protease and animal hydrolyzed proteinase. 3.The process according to claim 1, wherein the addition amount of the complex protease is 0.05-0.15 wt% of the treated solution. 4.The process according to claim 1, wherein the enzymatic hydrolysis is performed at a temperature of 48-52℃ for 4-6 hours. In step (4), the addition amount of the strong base anion exchange resin is 15-20% m / v of the volume of the enzymatic hydrolysate. 6.The process according to claim 1, wherein in step (5), the solid-liquid ratio of the resin to the sodium chloride solution is 1:2.2-2.6 g / mL. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 5. The co-production process of claim 1, wherein, ​ ​ ​ 7. The co-production process as claimed in claim 1, wherein, The solid-liquid ratio of the resin to the sodium chloride solution in each elution step of step (6) is 1:1.5-1.8 g / mL.

8. The co-production process as claimed in claim 1, wherein, The amount of ethanol added in the ethanol precipitation of step (7) is such that the ethanol concentration in the mixture of ethanol and eluate is 40-60%.