CN117143850A

CN117143850A - Pepsin preparation method for removing blood group A substances

Info

Publication number: CN117143850A
Application number: CN202311266174.8A
Authority: CN
Inventors: 张革; 周后杰; 张忠元; 武梦霞; 吴东
Original assignee: Sichuan Deebio Pharmaceutical Co ltd
Current assignee: Sichuan Deebio Pharmaceutical Co ltd
Priority date: 2023-09-27
Filing date: 2023-09-27
Publication date: 2023-12-01

Abstract

The application relates to the technical field of biology, and particularly discloses a pepsin preparation method for removing blood group A substances, which comprises the following steps: s1, taking pig mucous membrane, crushing, and adding purified water and concentrated hydrochloric acid; s2, carrying out acidolysis treatment on the crushed material to obtain acidolysis solution; s3, adding an aqueous solution of hydrochloric acid into acidolysis solution, stirring and extracting, filtering, and collecting filtrate; s4, carrying out chromatographic filtration on the filtrate by using a Sephadex G200 chromatographic column, and collecting and filtering; s5, filtering the filtrate through a sterilizing filter membrane, and collecting the sterilizing filtrate; s6, adding cold dilute acetone into the degerming filtrate until the specific gravity is 0.85-0.89, standing, removing the solution, and collecting the precipitate; s7, dehydrating the precipitate obtained in the step S6 by using cold fresh acetone until the specific weight is 0.81-0.82, and removing the solution; collecting a product; s8, carrying out gradient drying on the product at 40-70 ℃ to obtain pepsin. The application can effectively remove the blood group A-like substances in the pepsin and prepare the pepsin with strong activity and high safety.

Description

Pepsin preparation method for removing blood group A substances

Technical Field

The application relates to the technical field of biology, in particular to a pepsin preparation method for removing blood group A substances.

Background

Pepsin is white or light yellow powder extracted from pig, cattle and sheep stomach, is the only proteolytic enzyme in the stomach, has the optimal pH of 1.5-2.0, acts on peptide bonds to decompose protein into peptone or amino acids such as tyrosine and phenylalanine, is the primary component of western medicines such as pepsin tablets, multienzyme tablets and pepsin oral solution, and is used for treating gastrointestinal diseases such as pepsin deficiency caused by dyspepsia due to excessive eating of protein food and reduced digestive function in recovery period after illness, such as chronic atrophic gastritis, chronic gastric distention and pernicious anemia.

Most people have poor eating habits such as irregular three meals, heavy and spicy oil preference, alternating cold and hot, and the like, so that the gastrointestinal tract function is reduced, and the demand for pepsin is increased. In addition pepsin is an irreplaceable tool enzyme in the production process of vaccine, antiserum and antitoxin preparation, but pepsin extracted from animal belly membrane contains a large amount of blood group A substances (polysaccharide and polypeptide), and injection of antiviral containing the blood group A substances can increase the incidence of hemolytic diseases.

In the traditional extraction method of pepsin, processes such as salting out, recrystallization and the like cannot remove the blood group-A substances, so that a pepsin preparation method for removing the blood group-A substances is developed, the purity of the product is improved, the pepsin activity is enhanced, and the comprehensive medication safety is improved.

Disclosure of Invention

The application aims to provide a pepsin preparation method for removing blood group A substances, which can effectively remove the blood group A substances in pepsin and prepare pepsin with strong activity and high safety.

The application is realized by the following technical scheme:

a pepsin preparation method for removing blood group A substances comprises the following steps:

s1, taking pig mucous membrane, crushing, and adding purified water and concentrated hydrochloric acid;

s2, carrying out acidolysis treatment on the crushed material obtained in the step S1 to obtain acidolysis solution;

s3, adding an aqueous solution of hydrochloric acid into the acidolysis solution obtained in the step S2, stirring and extracting, filtering, and collecting filtrate;

s4, subjecting the filtrate obtained in the step S3 to chromatographic filtration through a Sephadex G200 (Sephadex G200) chromatographic column, and collecting and filtering;

s5, filtering the filtrate obtained in the step S4 through a sterilizing filter membrane, and collecting the sterilizing filtrate;

s6, adding cold dilute acetone into the degerming filtrate obtained in the step S5 until the specific gravity is 0.91-0.95, standing, removing sediment, and collecting filtrate; adding a salt solution into the filtrate, standing, and removing the precipitate; adding cold fresh acetone into the filtrate until the specific gravity is 0.85-0.89, standing, removing the solution, and collecting the precipitate;

s7, dehydrating the precipitate obtained in the step S6 by using cold fresh acetone until the specific weight is 0.81-0.82, and removing the solution; collecting a product;

s8, carrying out gradient drying on the product obtained in the step S7 at 40-70 ℃ to obtain pepsin.

The sterilizing filter membrane is an ultrafiltration membrane.

Before extraction, the crushed material is subjected to acidolysis treatment, so that the activity of pepsin can be stimulated to the greatest extent, and the enzyme efficiency is improved.

According to the application, after extraction, sephadex G200 is adopted to carry out column chromatography separation to carry out chromatographic separation on the extracting solution, so that blood group substances like A can be effectively removed, and then filtering and degerming are carried out through a degerming filter membrane, so that the prepared high-purity pepsin meets medical requirements.

According to the application, after column chromatography separation, diluted acetone and salt solution pepsin are used for purification and protection, and compared with the traditional precipitation process, the purity and the enzyme activity of the prepared product are greatly improved.

Compared with the conventional drying process, the gradient drying temperature is lower than that of the conventional process, so that the energy consumption is reduced, the water activity of the product is reduced to the maximum extent, and the product stability is improved.

In conclusion, according to the pepsin preparation method for removing the blood group A substances, the extraction liquid is subjected to column chromatography separation by using Sephadex G200, so that the blood group A substances in pepsin can be effectively removed. And the method comprises the steps of carrying out proper acidolysis treatment before extraction, exciting the activity of pepsin with maximum force, improving the enzyme efficiency, adopting a sterilization filter membrane to further filter and sterilize, improving the safety of the prepared pepsin, and purifying and protecting the pepsin by using dilute acetone and salt solution after column chromatography separation.

Further, in the step S1, the consumption of the purified water is 10% -15% based on the weight of the broken porcine mucosa; the consumption of the concentrated hydrochloric acid is 2.0 to 2.5 percent.

Further, in step S2, the specific process of acidolysis is:

stirring and acidolysis for 2-4 hours at 45-55 ℃ and pH of 2.5-3.0, and cooling to 10 ℃ after acidolysis.

Further, in step S3, the pH of the aqueous hydrochloric acid solution is 2.5 to 3.0.

In step S3, the amount of the aqueous solution of hydrochloric acid is 8-10 times the volume of the acidolysis solution.

Further, sephadex G200 is adopted for separating pepsin from blood group A substances by column chromatography; the separation condition is pH 4-6

Further, in step S5, filtration is performed using a 0.22 μm sterilization filter.

The ratio of the dilute acetone is 0.90, the ratio of the new acetone is 0.79, the temperatures of the cold dilute acetone and the cold new acetone are between-20 ℃ and-15 ℃, so in the step S6, the first precipitation is carried out from the cold dilute acetone (0.90 lighter than the dilute acetone) at the temperature of-20 ℃ to-15 ℃ to the solution ratio of 0.91-0.95, the second precipitation is carried out from the filtrate after filtration by adopting the cold new acetone (0.79 lighter than the dilute acetone) at the temperature of-20 ℃ to-15 ℃ to the solution ratio of 0.85-0.89.

Further, in step S6, after the first precipitation, a calcium chloride solution and a sodium chloride solution are added into the filtrate to be stirred, and then the temperature is reduced to 10 ℃, and the mixture is kept stand for 3 to 4 hours. The addition amounts of the calcium chloride solution and the sodium chloride solution are 2% of the volume of the solution, the concentration of the calcium chloride solution is 10%, and the concentration of the sodium chloride solution is 25%.

In the purification process, firstly, cold dilute acetone (0.90) is selected for precipitation to remove impurities, so that the purity of the product is greatly improved, and meanwhile, the loss of pepsin in the precipitation process is reduced. Then a certain salt solution is added, so that the impurity protein is further removed, and the activity of pepsin in the solution is protected. Finally, adding cold fresh acetone for precipitation, thereby improving the product yield.

In conclusion, according to the pepsin preparation method for removing the blood group A substances, the extraction liquid is subjected to column chromatography separation by using Sephadex G200, so that the blood group A substances in pepsin can be effectively removed. And the proper acidolysis treatment is carried out before extraction, so that the activity of pepsin can be stimulated with the greatest force, the enzyme efficiency is improved, the sterilization is further carried out by adopting a sterilization filter membrane, and the safety of the prepared pepsin is improved. In the purification process, firstly, cold dilute acetone (lighter than 0.90) is selected for precipitation to remove impurities, so that the purity of the product is greatly improved, and meanwhile, the loss of pepsin in the precipitation process is reduced. Then a certain salt solution is added, so that the impurity protein is further removed, and meanwhile, calcium ions are added to protect the activity of pepsin in the solution. Finally, cold fresh acetone (lighter than 0.79) is added for precipitation, so that the product yield is improved. The pepsin prepared by the preparation method disclosed by the application does not contain blood group A substances, and is strong in activity and high in safety.

Further, in step S8, the gradient drying process is as follows:

drying at 40-55 deg.c for 8.5 hr; drying at 60+ -2deg.C for 12 hr; drying at 65+ -2deg.C for 6 hr; drying at 70+ -2deg.C for 2 hr.

Compared with the conventional drying process, the gradient drying temperature is lower than that of the conventional process, so that the energy consumption is reduced, the water activity of the product is reduced to the maximum extent, and the stability is improved.

Further, the specific process of drying for 8.5 hours at the temperature of 40-55 ℃ is as follows:

drying at 40 ℃ for 2 hours; drying at 45 ℃ for 2 hours; drying at 50 ℃ for 2 hours; drying at 55℃for 2.5 hours.

The application further reduces the energy consumption by carrying out further gradient drying within the range of 40-55 ℃, and simultaneously further reduces the water activity of the product and improves the stability.

Compared with the prior art, the application has the following advantages and beneficial effects:

1. according to the application, the extraction liquid is subjected to column chromatography separation by using Sephadex G200, the blood group-like substances in pepsin are removed, and acidolysis treatment is performed before extraction, so that the activity of the pepsin can be stimulated with maximum strength, the enzyme efficiency is improved, ultrafiltration is adopted to further filter and sterilize, and the safety of the prepared pepsin is improved.

2. The reagent used in the application has low risk coefficient, can be recycled, does not cause environmental pollution, and is easy for industrial operation.

Drawings

The accompanying drawings, which are included to provide a further understanding of embodiments of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the principles of the application. In the drawings:

FIG. 1 is a flow chart of the pepsin preparation method of the present application.

Detailed Description

For the purpose of making apparent the objects, technical solutions and advantages of the present application, the present application will be further described in detail with reference to the following examples and the accompanying drawings, wherein the exemplary embodiments of the present application and the descriptions thereof are for illustrating the present application only and are not to be construed as limiting the present application.

Examples:

the pepsin prepared by the traditional method has low purity and poor activity, and the processes such as salting out, recrystallization and the like can not remove blood group A substances, and the product can not reach the medical grade, so the preparation of the medical pepsin with high safety coefficient is a problem which needs to be solved at present.

The application aims to provide a pepsin preparation method for removing blood group A substances, which is characterized in that the pepsin extracting solution is further purified by column chromatography, so that the blood group A substances in pepsin can be effectively removed, and the pepsin with strong activity and high safety is prepared.

As shown in figure 1, the pepsin preparation method for removing blood group A substances specifically comprises the following steps:

s1, taking pig mucous membrane, crushing, and adding purified water and concentrated hydrochloric acid; the consumption of the purified water is 10 to 15 percent based on the weight of the broken porcine mucosa; the consumption of the concentrated hydrochloric acid is 2.0 to 2.5 percent.

S2, carrying out acidolysis treatment on the crushed material obtained in the step S1 to obtain acidolysis solution; the acidolysis process comprises the following steps:

stirring and acidolysis for 2-4 hours at 45-55 ℃ and pH of 2.5-3.0, and cooling to 10 ℃ after acidolysis. Acidolysis is carried out on the crushed materials before extraction, so that the activity of pepsin can be stimulated to the greatest extent, and the enzyme efficiency is improved.

S3, adding 8-10 times of hydrochloric acid aqueous solution with pH value of 2.5-3.0 into acidolysis solution, stirring and extracting for 2-4 h, filtering to remove insoluble substances, and collecting filtered clarified liquid.

S4, subjecting the filtrate obtained in the step S3 to chromatographic filtration through a Sephadex G200 chromatographic column, collecting and filtering, and specifically, separating pepsin from blood group A substances through column chromatography by adopting Sephadex G200; the separation condition is pH 4-6; the Sephadex G200 chromatographic column can thoroughly remove blood group A substances.

S5, filtering the filtrate obtained in the step S4 through a 0.22 mu m sterilization filter membrane, and collecting the sterilization filtrate.

S6, adding cold dilute acetone at the temperature of minus 20 ℃ into the degerming filtrate obtained in the step S5 until the specific weight is 0.85-0.89, standing for 2-3 h, removing the solution, and collecting the precipitate.

In a preferred case, in the step S6, cold dilute acetone (lighter than 0.90) with the temperature of minus 10 ℃ to minus 20 ℃ is firstly adopted to carry out first precipitation until the specific weight of the solution is 0.91 to 0.95, and then the filtrate after filtration is adopted to carry out second precipitation after the cold fresh acetone (lighter than 0.79) with the temperature of minus 20 ℃ is adopted to carry out second precipitation until the specific weight of the solution is 0.85 to 0.89.

In a preferred case, in step S6, after the first precipitation, a calcium chloride solution and a sodium chloride solution are added to the filtrate and stirred, and then cooled to 10℃and allowed to stand for 3-4 hours. The addition amounts of the calcium chloride solution and the sodium chloride solution are 2% of the volume of the solution, the concentration of the calcium chloride solution is 10%, and the concentration of the sodium chloride solution is 25%.

S7, dehydrating the precipitate obtained in the step S6 by using cold fresh acetone at the temperature of minus 20 ℃ until the specific weight is 0.81-0.82, and removing the solution; and collecting the product.

S8, carrying out gradient drying on the product obtained in the step S7 at 40-70 ℃ to obtain pepsin, and carrying out gradient drying at 40-70 ℃, wherein compared with the conventional drying process, the gradient drying temperature is lower than that of the conventional process, so that the energy consumption is reduced, the water activity of the product is reduced to the maximum extent, and the stability is improved. The gradient drying process comprises the following steps:

In a preferred embodiment, the drying at 40-55deg.C for 8.5 hours comprises the following steps:

According to the method, the acid hydrolysis liquid is subjected to chromatographic filtration by using a Sephadex G200 chromatographic column, so that the blood group A-like substances in pepsin can be effectively removed, and the acid hydrolysis treatment is carried out before the extraction, so that the activity of the pepsin can be stimulated with the greatest force, the enzyme efficiency is improved, the prepared pepsin is further filtered and sterilized by ultrafiltration, and the safety of the prepared pepsin is improved, namely the pepsin prepared by the preparation method does not contain the blood group A-like substances or contains a small amount of medical allowed blood group A-like substances, and the activity is strong and the safety is high; the vitality is more than 11000U/g.

In order to better understand the technical scheme provided by the application, the following specific processes for preparing pepsin by applying the preparation method provided by the embodiment of the application and effects thereof are respectively described in a plurality of specific examples.

The overall description is shown in table 1:

TABLE 1

Example 1	Control process
		Example 2	Based on example 1, a one-step Sephadex G200 column chromatography (PH 6) separation was added,
example 3	Based on example 2, the column chromatography separation pH4.0 was changed
		Example 4	In the process of the precipitation based on the example 3, dilute acetone is firstly used for precipitation once
Example 5	On the basis of example 4, a further addition of salt solution was added during the precipitation.
		Example 6	On the basis of example 5, new acetone was used for precipitation to a specific weight of 0.85 to 0.87

Example 1:

the pepsin preparation method for removing blood group A substances specifically comprises the following steps:

s1, taking 100kg of pig mucous membrane and crushing; adding 10% of purified water and 2.0% of concentrated hydrochloric acid by weight of the pig mucosa crushed material;

s2, stirring and acidolysis for 2 hours at the temperature of 45 ℃ and the pH value of 2.5, and cooling to 10 ℃ for standby after acidolysis is completed;

s3, adding 8 times of hydrochloric acid aqueous solution with the pH of 2.5 into acidolysis solution, stirring and extracting for 2 hours, filtering to remove insoluble substances, and collecting filtered clear solution;

s4, filtering the filtrate obtained in the step S3 through a 0.22 mu m sterilization filter membrane, and collecting the sterilization filtrate;

s5, adding cold dilute acetone at the temperature of minus 20 ℃ into the degerming filtrate obtained in the step S4 until the specific weight is 0.89, standing for 2 hours, separating the supernatant, and collecting the precipitate;

s6, dehydrating the precipitate obtained in the step S5 by using cold fresh acetone at the temperature of minus 20 ℃ to the specific weight of 0.81/10 ℃ and removing the solution; collecting a product;

s7, carrying out gradient drying on the product obtained in the step S6 at 40-70 ℃ to obtain pepsin, wherein the specific process of gradient drying is as follows:

drying at 40 ℃ for 2h to 45 ℃ for 2h to 50 ℃ for 2h to 55 ℃ for 2.5h, drying at 60+/-2 ℃ for 12 hours, drying at 65+/-2 ℃ for 6 hours, and drying at 70+/-2 ℃ for 2 hours.

The pepsin activity 9864U/g prepared in the embodiment detects the content of the blood group substance A, and each 1mg/ml protein contains 0.016mg/ml of the blood group substance A, the drying loss is 1.8%, and the product yield is 10.2 per mill.

Example 2:

this example was based on example 1, after S4, followed by a one-step Sephadex G200 column chromatography (PH 6).

s1, taking 1kg of pig mucous membrane and crushing; adding 10% of purified water and 2.0% of concentrated hydrochloric acid by weight of the pig mucosa crushed material;

s5, separating the filtrate by column chromatography through Sephadex G200, and collecting the flow-through liquid at pH of 6.0;

s6, adding cold dilute acetone at the temperature of minus 20 ℃ into the flow-through liquid obtained in the step S5 until the specific weight is 0.89, standing for 2 hours, separating the supernatant, and collecting the precipitate;

The pepsin activity 11846U/g prepared in the embodiment, the content of the blood group substance A is detected, each 1mg/ml protein contains 0.002mg/ml of the blood group substance A, and the product yield is 9.25%.

Example 3:

based on the embodiment example 2, the column chromatography separation pH4.0 was changed.

s5, separating the filtrate by column chromatography through Sephadex G200, and collecting the flow-through liquid at pH of 4.0;

The pepsin activity 11464U/g prepared in the embodiment, the content of the blood group substance A is detected, each 1mg/ml protein contains 0.002mg/ml of the blood group substance A, and the product yield is 9.38%.

Example 4:

on the basis of example 3, in the precipitation process of step S6, dilute acetone is first used for precipitation once.

s6, adding cold dilute acetone (lighter than 0.90) at the temperature of-20 ℃ to the flow-through liquid obtained in the step S5 until the temperature is 0.95/10 ℃ lighter than the temperature, and filtering to remove filter residues; adding cold fresh acetone (lighter than 0.79) at-20deg.C to lighter than 0.89, standing for 2 hr, separating supernatant, and collecting precipitate;

The pepsin activity 18726U/g prepared in the embodiment, the content of the blood group substance A is detected, the blood group substance A is not detected in every 1mg/ml protein, and the product yield is 5.84%.

Example 5:

on the basis of the embodiment example 4, a salt solution is added in one step during the precipitation in the step S6.

s5, adding cold dilute acetone (lighter than 0.90) at the temperature of-20 ℃ to the flow-through liquid obtained in the step S5 to the temperature of 0.95/10 ℃ lighter than the temperature, and filtering to remove filter residues. Adding sodium chloride solution (25%) and calcium chloride solution (10%) with volume of 2% into the filtrate, mixing, standing for 4 hr, filtering to remove precipitate, adding cold fresh acetone (0.79) with a specific weight of 0.89 at-20deg.C into the filtrate, standing for 2 hr, separating supernatant, and collecting precipitate;

The pepsin activity 20846U/g prepared in the embodiment is detected, the blood group substance of the class A is not detected in every 1mg/ml protein, and the product yield is 5.99%.

Example 6:

on the basis of example 5, in step S6, new acetone was used for precipitation to a weight of 0.85.

s5, adding cold dilute acetone (lighter than 0.90) at the temperature of-20 ℃ to the flow-through liquid obtained in the step S5 to the temperature of 0.95/10 ℃ lighter than the temperature, and filtering to remove filter residues. Adding sodium chloride solution (25%) and calcium chloride solution (10%) with volume of 2% into the filtrate, mixing, standing for 4 hr, filtering to remove precipitate, adding cold fresh acetone at-20deg.C to specific weight of 0.85, standing for 2 hr, separating supernatant, and collecting precipitate;

The pepsin activity 20574U/g prepared in the embodiment, the content of the blood group substance A is detected, the blood group substance A is not detected in every 1mg/ml protein, and the product yield is 6.15%.

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the application, and is not meant to limit the scope of the application, but to limit the application to the particular embodiments, and any modifications, equivalents, alternatives, improvements, etc. that fall within the spirit and principles of the application are intended to be included within the scope of the application.

Claims

1. The pepsin preparation method for removing blood group A substances is characterized by comprising the following steps:

s4, subjecting the filtrate obtained in the step S3 to chromatographic filtration through a Sephadex G200 chromatographic column, and collecting and filtering;

2. The method for preparing pepsin for removing blood group A substances according to claim 1, wherein in the step S1, the amount of purified water is 10% -15% by weight of the broken porcine mucosa; the consumption of the concentrated hydrochloric acid is 2.0 to 2.5 percent.

3. The method for preparing pepsin from blood group a according to claim 1, wherein in step S2, the specific process of acidolysis is:

4. The method for preparing pepsin according to claim 1, wherein in step S3, the pH of the aqueous hydrochloric acid solution is 2.5-3.0.

5. The method for preparing pepsin according to claim 1, wherein in step S3, the amount of the aqueous hydrochloric acid solution is 8-10 times the volume of the acidolysis solution.

6. The method for preparing pepsin from blood group a according to claim 1, wherein in step S5, sephadex G200 is used for separating pepsin from blood group a by column chromatography; the separation condition is pH 4-6.

7. The method for preparing pepsin for removing blood group A substances according to claim 1, wherein in the step S6, dilute acetone with a specific gravity of 0.91-0.95 at-20 ℃ to-15 ℃ is adopted for the first precipitation, and cold fresh acetone with a specific gravity of 0.85-0.89 at-20 ℃ to-15 ℃ is adopted for the filtrate after filtration for the second precipitation.

8. The method for preparing pepsin for removing blood group A material according to claim 7, wherein in step S6, after the first precipitation, a calcium chloride solution and a sodium chloride solution are added to the filtrate and stirred, then cooled to 10 ℃, and the mixture is left for 3 to 4 hours, and the filtrate is collected by filtration.

9. The method for preparing pepsin according to claim 8, wherein in step S6, after the first precipitation, the concentration of the calcium chloride solution is 10% and the volume of the solution is 2%; the sodium chloride solution had a concentration of 25% and the added volume was 2% of the liquid volume.

10. The method for preparing pepsin according to claim 1, wherein in step S8, the gradient drying process is as follows: