CN113493570B - Method for reducing endotoxin in gelatin by adopting allantoin - Google Patents
Method for reducing endotoxin in gelatin by adopting allantoin Download PDFInfo
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Abstract
The invention discloses a method for reducing endotoxin in gelatin by adopting allantoin, which comprises the following steps: dissolving gelatin to be treated in water at 45-55 deg.C in a container, and adjusting pH of the gelatin solution to 4.5-6.0; adding sodium chloride, and stirring at 45-55 deg.C for 10-30min; adding allantoin to obtain a pretreatment solution; stirring and incubating at 45-55 deg.C for 15-60min, filtering, concentrating, sterilizing, drying, and pulverizing to obtain low endotoxin gelatin granule. The content of endotoxin in the gelatin treated by the method is lower than 200EU/g, the treatment process has no obvious influence on the gelatin property, the change degree of the freezing point and ash content index before and after treatment is within 5 percent, and the gelatin can be widely applied to the fields of preparing absorbable hemostatic sponges, renewable medicines, medical complementary foods and the like; the method has simple and mild process and can realize the quantitative preparation at the quasi-industrial level.
Description
Technical Field
The invention relates to the technical field of medicinal gelatin. More particularly, it relates to a method for reducing endotoxin in gelatin using allantoin.
Background
Gelatin is a product obtained by partially hydrolyzing collagen in animal skin and bone with acid, alkali and enzyme, and purifying. The conventional use of gelatin is very extensive and can be classified into food additive gelatin, pharmaceutical gelatin and industrial gelatin according to the field of application. The Chinese pharmacopoeia 2015 edition clearly stipulates the index requirements of medicinal gelatin and gelatin for capsules, but does not stipulate the content of endotoxin in the gelatin.
As a good protein source, the concentration, temperature and pH of the gelatin solution in the production process of gelatin are very suitable for the growth and reproduction of gram-negative bacteria. The chemical structure of Bacterial Endotoxins (BET) is Lipopolysaccharide (LPS), located in the outer membrane of gram-negative bacteria, which is constantly released into the environment by the bacteria. When gram-negative bacteria die and lyse, all lipopolysaccharides are released into the environment. When bacterial endotoxin is injected into mammals (including human beings), a certain threshold value is reached, the human defense system is activated, the body temperature is raised, the vomiting is not suitable, the skin vasoconstriction, the blood pressure is raised, the muscle ache is generated, and the serious patient can cause shock and death.
With the wide application of gelatin in the medical field, gelatin products such as gelatin plasma substitutes, gelatin hemostatic sponges, gelatin sutures, gelatin stents and the like which directly enter blood circulation are continuously put into use. However, the production process of low endotoxin gelatin is rarely reported in the literature. The raw material bone for producing the bone gelatin contains a certain amount of bacteria, and the bacteria are killed by high temperature, chemical reagents, sterilization and other methods in the production process, and a large amount of endotoxin is generated. The conventional gelatin production process has no endotoxin control and elimination process. The endotoxin of most gelatins is above 1000EU/g, and the endotoxin of partial gelatins exceeds 10000EU/g. When the bacterial endotoxin enters a human body in an oral form, the bacterial endotoxin is digested by stomach and intestine and does not generate toxicity to a healthy human body. Endotoxin levels therefore do not have to be taken into account when gelatin is administered orally, parenterally, in the form of capsule or tablet excipients. However, when gelatin is used as an injection or is in direct contact with blood and myelin sheath, endotoxin is the most important quality control index, and is required to be lower than a corresponding threshold value according to relevant documents such as pharmacopoeia of various countries. Endotoxin affects the adhesion, growth and differentiation of cells on the surface of a tissue engineering scaffold, so that the strict control of endotoxin indexes of implantable medical devices is the key for ensuring the safety of the devices.
Gelatin belongs to a macromolecular protein. In general, methods for removing endotoxin from proteins mainly include nonspecific removal and selective separation. The nonspecific removal method includes a dry heat method, a chemical method, an activated carbon adsorption method, an ultrafiltration method and an ion exchange method, and the dry heat method and the chemical method are not suitable because gelatin is denatured under high temperature, acidic and alkaline conditions, and the gelatin is crosslinked or degraded. The gelatin has a molecular weight distribution interval almost overlapping with endotoxin, and endotoxin cannot be separated from gelatin by ultrafiltration. A large number of anionic groups are contained in gelatin, and if an ion exchange method is used, a considerable portion of gelatin is adsorbed by the resin, resulting in a great decrease in the recovery rate of gelatin. The selective removal method mainly refers to affinity chromatography, utilizes the biological characteristics of substances to achieve the purpose of substance separation, is a theoretically ideal endotoxin removal method, has the difficulty that a proper ligand is developed and is immobilized on an affinity chromatography matrix to synthesize an affinity medium, and does not have the capacity of large-scale preparation at present. There is a literature on the use of Triton-X114 to isolate endotoxin from succinylated gelatin, but the residual Triton-X114 is toxic and difficult to completely remove.
Therefore, it is necessary to provide a method for effectively reducing endotoxin in gelatin, so as to expand the application range of gelatin.
Disclosure of Invention
The invention aims to provide a method for reducing endotoxin in gelatin by adopting allantoin, which takes the allantoin as an endotoxin adsorbent, can reduce the endotoxin content in the gelatin to be below 200EU/g, and can be applied in a large scale.
The invention also aims to provide the application of the low-endotoxin gelatin in preparing absorbable hemostatic sponges, renewable medicines, medical auxiliary foods and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a process for reducing endotoxin in gelatin using allantoin, comprising the steps of:
dissolving gelatin to be treated in water at 45-55 deg.C in a container, and adjusting pH of the gelatin solution to 4.5-6.0; adding sodium chloride, and stirring at 45-55 deg.C for 10-30min; adding allantoin to obtain a pretreatment solution; stirring and incubating at 45-55 deg.C for 15-60min, filtering, concentrating, sterilizing, drying, and pulverizing to obtain low endotoxin gelatin granule.
Gelatin belongs to protein and can be denatured or hydrolyzed under the conditions of high temperature and strong acid and alkali; gelatin contains a large number of groups of-COOH, -OH, -NH 2 and the like, and is easy to be combined with endotoxin in a hydrogen bonding mode. But compared with the common protein, the gelatin has unique sol-gel conversion property, the gel point of the gelatin sol with the concentration of 10 percent is between 25 and 30 ℃, and the gelatin with high freezing force mainly exists in a frozen state at room temperature when the weight concentration is more than 0.5 percent; the molecular weight of gelatin is between 5kDa and several thousand kDa, and almost covers the molecular weight distribution range of endotoxin, so that it is very difficult to separate endotoxin from gelatin.
The endotoxin content in the gelatin to be treated is usually more than 1000EU/g, even can exceed 10000EU/g, and can not be in direct contact with blood and myelin sheaths, thereby limiting the application of the gelatin. The invention provides a method for reducing endotoxin in gelatin, namely allantoin is adopted as an endotoxin adsorbent. The uremic toxin (2, 5-dioxy-4-imidazolidinyl urea) can adsorb endotoxin through hydrogen bonds and charge effects, has an excellent adsorption effect, is nontoxic, has physiological functions of promoting cell growth, promoting wound healing and the like, and is often used as a biological material additive.
In the present invention, the endotoxin concentration in gelatin is defined as endotoxin unit per gram of gelatin (EU), since the molecular weight and activity of endotoxin vary depending on the medium. The endotoxin concentration was determined with e.coli 0113: H10K was used as a reference for the endotoxin content of gelatin by limulus reagent color development.
Optionally, the gelatin to be treated is acid gelatin or alkali gelatin; when the gelatin to be treated is acid gelatin, adjusting the pH value of the gelatin solution to 5.5-6.0; when the gelatin to be processed is alkaline gelatin, adjusting the pH value of the gelatin solution to be 4.5-4.6.
According to the method for reducing endotoxin in gelatin by adopting allantoin, provided by the invention, the interaction between endotoxin and allantoin is enhanced and the separation efficiency is improved by adjusting the pH values of different types of gelatin solutions and adding the amount and the mode of sodium chloride. The invention combines the sol-gel conversion characteristic and the thermal degradation characteristic of the gelatin, controls the temperature at 45-55 ℃, can ensure that the gelatin solution is in a liquid state, and can avoid the further hydrolysis of the gelatin at high temperature; the solubility of allantoin is 0.5g/L, and excess allantoin will exist in the gelatin solution in a crystalline state, and adsorb negatively charged endotoxin through hydrogen bonds and electrostatic interaction. The isoelectric point of gelatin is an important factor affecting the adsorption efficiency and the gelatin yield, and in order to prevent the attraction of charges between gelatin and allantoin, it is necessary to adjust the pH of the gelatin solution according to the difference between the isoelectric points to positively charge the gelatin molecules, and since the isoelectric point of acid gelatin is 6 to 9, the pH is adjusted to 5.5 to 6.0. The isoelectric point of the alkali gelatin is 4.8-5.0, and the isoelectric point of the alkali gelatin solution is adjusted to be 4.5-4.6. The sodium chloride is added to reduce the competition between the gelatin and the allantoin for adsorption of endotoxin mainly by reducing the polarity of the gelatin. Optionally, the concentration of the gelatin solution is 2-10wt%. The appropriate concentration of gelatin solution is beneficial to improving the separation efficiency and reducing the endotoxin residue of the membrane filtration supplementing water. When the concentration of the gelatin is too low, the dissolving amount of the allantoin in the gelatin solution is increased, so that the dosage of the allantoin is increased, and the burden of removing the allantoin during the filtration of a post-treatment membrane is increased; when the concentration of the gelatin is too high, the viscosity of the solution is increased, and the difficulty in combination of allantoin and endotoxin is increased.
Optionally, the endotoxin content of the gelatin to be treated is 200-20000EU/g. The BET content of biological products in contact with blood is subject to very strict regulations by drug regulatory agencies in various countries. The Chinese pharmacopoeia 2015 edition, the United states pharmacopoeia USP <85> and the European pharmacopoeia Ph.Eur.2.6.14 stipulate that the endotoxin content of the parenteral preparation must be less than 5EU/Kg/h, the endotoxin limit of the radiopharmaceutical injection is 2.5EU/Kg/h, and the endotoxin threshold of the intrathecal injection is 0.2EU/Kg/h. The endotoxin content in the gelatin to be treated limits the application of the gelatin, and the endotoxin content in the gelatin treated by the method can be reduced to below 200 EU/g.
Optionally, the concentration of sodium chloride in the pretreatment solution is 5-50mmol/L; preferably, the concentration of allantoin in the pretreatment solution is in the range of 1 to 100g/L. The polarity of the gelatin solution is reduced by adopting sodium chloride, the optimal concentration is 25mmol/L, the adsorption efficiency of the allantoin to the endotoxin is highest, the adsorption efficiency cannot be improved by increasing the concentration of NaCl, the burden of post-treatment membrane filtration is increased, and when the concentration of the sodium chloride is lower than 5mmol/L, the concentration is too low, the polarity change of the gelatin is small, and the adsorption efficiency of the allantoin to the endotoxin is difficult to improve.
The solubility of allantoin in the gelatin aqueous solution is 0.5g/L, excessive allantoin is distributed in a crystal form in the solution, and endotoxin in the solution is adsorbed by stirring, most of the allantoin is dissolved when the allantoin is used excessively, the crystal allantoin is too small, and the adsorption of a large amount of endotoxin in the solution is difficult to satisfy. The use amount of allantoin is too large, so that the utilization rate of the allantoin is reduced, the waste of the allantoin is caused, and the burden of rough separation is increased.
Optionally, the water used in the method for reducing endotoxin in gelatin by using allantoin is secondary pure water, and the content of endotoxin is less than 0.05EU/mL.
Optionally, the container used in the method for reducing endotoxin in gelatin using allantoin is subjected to a dry heat method or a chemical method to remove endotoxin.
To avoid the influence of container and water on endotoxin content in gelatin, the endotoxin in gelatin is reducedDuring the course of purification, the endotoxin content in the vessel and water needs to be strictly defined. Wherein the container can be used for removing endotoxin by dry heat method or chemical method, the dry heat method is to maintain the container at 250 deg.C for 30min or 200 deg.C for 60min, the chemical method is to soak the container or equipment in contact with the glue solution in 0.2M NaOH solution for 30-60 min, or in 0.2M HCl solution for 30-60 min, or KOH or H with corresponding concentration can be selected2SO4。
Optionally, the filtration process comprises a coarse filtration process and a fine filtration process; the coarse filtration process uses cotton cake or filter paper as filter medium, and the fine filtration process uses tubular membrane as filter medium.
Optionally, the molecular weight cut-off of the tubular membrane is 5000g/mol, and the material of the tubular membrane is polyether sulfone.
The coarse filtration process can remove insoluble allantoin crystals, and the fine filtration process can retain and separate low molecular weight sodium chloride and allantoin, thereby realizing high-efficiency separation of impurities and gelatin.
In order to reduce the residual of allantoin and sodium chloride to the maximum extent, allantoin with a molecular weight of 158g/mol and sodium chloride with a molecular weight of 58g/mol are separated from gelatin with a molecular weight distribution interval of 5Kg/mol to thousands of Kg/mol by adopting a membrane filtration mode, and concentrated solution is diluted by adopting more than 3 original volumes, so that the residual of impurities does not influence the application of the gelatin.
Optionally, the sterilization mode is flash evaporation sterilization, and specifically is kept at 148 ℃ for 6-8s.
Optionally, the drying manner is freeze drying or hot air drying. The release preparation can be carried out by selecting a small fourdrinier wire for drying.
In another aspect, the invention provides the use of low endotoxin gelatin obtained by the above process. The content of endotoxin in the gelatin treated by the allantoin is lower than 200EU/g and lower than the threshold value specified by pharmacopoeia of various countries, and the gelatin can be used for preparing absorbable hemostatic sponges, renewable medicines, medical supplementary foods and the like.
The invention has the following beneficial effects:
the method for reducing endotoxin in gelatin by adopting allantoin provided by the invention takes the existing gelatin with high endotoxin content as a raw material, takes the allantoin as an endotoxin adsorbent, adsorbs the endotoxin in the gelatin by utilizing the action of hydrogen bonds and charges, enhances the interaction between the endotoxin and the allantoin by adjusting the pH value of different types of gelatin before adsorption, adding sodium chloride and other processes, and realizes the effect of efficiently removing the endotoxin in the gelatin. The endotoxin content in the gelatin treated by the method is lower than 200EU/g, the treated gelatin has no obvious change in properties, and the change degree of the freezing point and ash content index before and after treatment is within 5 percent, so that the gelatin can be widely applied to the fields of preparing absorbable hemostatic sponges, regenerative medicines, medical complementary foods and the like; the method has simple and mild process and can realize the quantitative preparation at the quasi-industrialization level.
Detailed Description
In order to make the technical solutions and advantages of the present invention more apparent, the following will describe embodiments of the present invention in further detail.
Examples
Example 1
Process for reducing endotoxin in alkaline gelatin (laboratory process):
1) All stainless steel containers and glass instruments in contact with the gelatin solution were kept at 250 ℃ for 30min by dry heating to remove endotoxin; removing endotoxin from plastic and silicone tube by chemical method, soaking in 0.2M NaOH solution for 30min, and washing with secondary pure water until pH is neutral;
2) 50g of alkaline bone gelatin to be treated with the endotoxin content of 5000EU/g magnitude is dissolved in 950mL of secondary pure water, the solution is heated and kept at 45 ℃, and the pH value of the solution is adjusted to 4.5;
3) Adding 1.46g NaCl into the gelatin solution, keeping the temperature at 45 ℃, and stirring for 15min;
4) Adding 20g of allantoin into the gelatin solution obtained in the step 3), and stirring and incubating for 30min at the temperature of 45 ℃;
5) Coarsely filtering the incubated gelatin solution by qualitative filter paper, and removing insoluble allantoin crystals to obtain coarse filtrate;
6) And (3) filtering the coarse filtrate by using an ultrafiltration membrane, wherein the molecular weight cut-off of the ultrafiltration membrane is larger than or equal to 5000g/mol, adding 1L of supplementary injection water into the concentrated solution when the concentration of the glue solution is concentrated to 30%, and adding 1L of injection water when the concentration of the glue solution is concentrated to 30%. And stopping concentrating when the concentration of the glue solution is 30% after water is added for the fourth time.
7) Carrying out flash evaporation sterilization on the concentrated glue solution to obtain a sterilized concentrated glue solution;
8) Freeze drying the sterilized concentrated glue solution to obtain low endotoxin gelatin;
the gelatin products before and after treatment are shown in table 1. The product can be used as absorbable hemostatic adjuvant, renewable medicinal raw material, and medical supplementary food material for patients in convalescent period of gastrointestinal diseases.
Table 1 example 1 key technical specifications of gelatin before and after treatment
Example 2
Process for reducing endotoxin in alkali gelatin (Industrial Process)
1) All stainless steel containers and glass instruments in contact with the gelatin solution were kept at 250 ℃ for 30min by dry heating to remove endotoxin; removing endotoxin from plastic or silicone tube by chemical method, soaking in 0.2M NaOH solution for 30min, and washing with secondary pure water until pH is neutral;
2) 50Kg of alkaline bone gelatin to be treated with the endotoxin content of 5000EU/g magnitude is dissolved in 950L of secondary pure water, heated and kept at 50 ℃, and the pH value of the gelatin solution is adjusted to 4.5;
3) Adding 1.46kg NaCl into the gelatin solution, keeping the temperature at 45-55 ℃, and stirring for 15min;
4) Adding 25Kg of allantoin into the gelatin solution obtained in the step 3, and stirring and incubating for 30min at the temperature of 45 ℃;
5) Coarsely filtering the incubated gelatin solution through cotton cakes, and removing insoluble allantoin crystals to obtain a coarse filtrate;
6) Filtering the coarse filtrate with ultrafiltration membrane, wherein the cutoff molecular weight of the ultrafiltration membrane is 5000g/mol, adding 1 ton of injection water into the concentrated solution when the concentration of the glue solution is concentrated to 30%, concentrating until the concentration of the glue solution reaches 30%, adding 1 ton of injection water, repeatedly diluting and concentrating, and stopping concentrating when the concentration of the concentrated solution reaches 30% after the fifth time of water addition;
7) Carrying out flash evaporation sterilization on the concentrated glue solution to obtain a sterilized concentrated glue solution;
8) Drying the sterilized concentrated glue solution by hot air to obtain low endotoxin gelatin;
the gelatin products before and after treatment are shown in table 2. The gelatin products before and after treatment are shown in table 1. The product can be used as absorbable hemostatic adjuvant, renewable medicinal raw material, and medical supplementary food material for patients in convalescent period of gastrointestinal diseases.
TABLE 2 Key technical indexes of gelatin before and after treatment in example 2
Example 3
Process for reducing endotoxin in acid gelatin (laboratory process):
1) All stainless steel containers and glass instruments in contact with the gelatin solution are kept at 250 ℃ for 30min by a dry heating method to remove endotoxin; removing endotoxin from plastic and silicone tube by chemical method, soaking in 0.2M NaOH solution for 30min, and washing with secondary pure water until pH is neutral;
2) Dissolving 50g of acid bone gelatin to be treated with the endotoxin content of 20000EU/g in 2.5L of second-grade pure water, heating and maintaining at 45 ℃, and adjusting the pH value of the solution to 5.8;
3) Adding 1.46g NaCl into the gelatin solution, keeping the temperature at 50 ℃, and stirring for 15min;
4) Adding 50g of allantoin into the gelatin solution obtained in the step (3), and stirring and incubating for 30min at the temperature of 50 ℃;
5) Coarsely filtering the incubated gelatin solution by qualitative filter paper, and removing insoluble allantoin crystals to obtain coarse filtrate;
6) And filtering the coarse filtrate with ultrafiltration membrane, wherein the cut-off molecular weight of the ultrafiltration membrane is greater than or equal to 5000g/mol, adding 1L of injection water into the concentrated solution when the concentration of the glue solution is concentrated to 30%, and adding 1L of injection water when the concentration of the glue solution reaches 30%. And stopping concentrating when the concentration of the glue solution is 30% after the fifth water addition.
7) Carrying out flash evaporation sterilization on the concentrated glue solution to obtain a sterilized concentrated glue solution;
8) Freeze drying the sterilized concentrated glue solution to obtain low endotoxin gelatin;
the gelatin products before and after treatment are shown in table 3. The product can be used as absorbable hemostatic adjuvant, renewable medicinal raw material, and medical supplementary food material for patients in convalescent period of gastrointestinal diseases.
TABLE 3 Key technical index of gelatin before and after treatment in example 3
| Index of gelatin | Before treatment | After treatment |
| Endotoxin (EU/g) | 1.84×104 | 175 |
| Freeze power (Bloom g) | 210 | 205 |
| Viscosity (mPa. S) | 4.78 | 4.69 |
| Viscosity reduction (%) | 2.8 | 2.5 |
| Gel point (. Degree.C.) (10% concentration) | 27.2 | 26.9 |
| Ash (%) | 0.7 | 0.5 |
| Moisture (%) | 10.8 | 4.7 |
Comparative example 1
The process for reducing endotoxin in alkali gelatin:
1) All stainless steel containers and glass instruments in contact with the gelatin solution are kept at 250 ℃ for 30min by a dry heating method to remove endotoxin; removing endotoxin from plastic and silicone tube by chemical method, soaking in 0.2M NaOH solution for 30min, and washing with secondary pure water until pH is neutral;
2) 50g of alkali-processed bone gelatin to be treated, the endotoxin content of which is 5000EU/g magnitude, is dissolved in 350mL of secondary pure water, the solution is heated and maintained at 65 ℃, and the pH value of the solution is adjusted to 4.5;
3) Adding 1.46g NaCl into the gelatin solution, keeping the temperature at 65 ℃, and stirring for 15min;
4) Adding 20g of allantoin into the gelatin solution obtained in the step 3), and stirring and incubating for 30min at the temperature of 65 ℃;
5) Coarsely filtering the incubated gelatin solution by qualitative filter paper, and removing insoluble allantoin crystals to obtain coarse filtrate;
6) And filtering the coarse filtrate with ultrafiltration membrane, wherein the cut-off molecular weight of the ultrafiltration membrane is greater than or equal to 5000g/mol, adding 1L of injection water into the concentrated solution when the concentration of the glue solution is concentrated to 30%, and adding 1L of injection water when the concentration of the glue solution is concentrated to 30%. And stopping concentrating when the concentration of the glue solution is 30% after water is added for the fourth time.
7) Carrying out flash evaporation sterilization on the concentrated glue solution to obtain a sterilized concentrated glue solution;
8) Freeze drying the sterilized concentrated glue solution to obtain low endotoxin gelatin;
the gelatin products before and after treatment are shown in table 4. The concentration of the gelatin solution in the treatment process is 12.5 percent, the endotoxin in the treated gelatin is 580EU/g which is far higher than that in example 1, and the freezing force and the viscosity of the gelatin are obviously reduced due to the high treatment temperature.
Table 4 key technical indices of gelatin before and after treatment in comparative example 1
| Index of gelatin | Before treatment | After treatment |
| Endotoxin (EU/g) | 5230 | 580 |
| Freeze power (Bloom g) | 230 | 195 |
| Viscosity (mPa. S) | 4.68 | 4.32 |
| Viscosity reduction (%) | 2.5 | 3.9 |
| Gel point (. Degree.C.) (10% concentration) | 27.5 | 26.2 |
| Ash (%) | 0.8 | 0.4 |
| Moisture (%) | 11.2 | 5.4 |
Comparative example 2
The process for reducing endotoxin in alkali gelatin comprises the following steps:
1) All stainless steel containers and glass instruments in contact with the gelatin solution were kept at 250 ℃ for 30min by dry heating to remove endotoxin; removing endotoxin from plastic and silicone tube by chemical method, soaking in 0.2M NaOH solution for 30min, and washing with secondary pure water until pH is neutral;
2) 50g of alkali-processed bone gelatin to be treated with the endotoxin content of 5000EU/g magnitude is dissolved in 950mL of secondary pure water, the solution is heated and maintained at 45 ℃, and the pH value of the solution is adjusted to 4.5;
3) Adding 1.46g NaCl into the gelatin solution, keeping the temperature at 45 ℃, and stirring for 15min;
4) Adding 0.7g of allantoin into the gelatin solution obtained in the step 3), and stirring and incubating for 30min at the temperature of 45 ℃;
5) Coarsely filtering the incubated gelatin solution by qualitative filter paper, and removing insoluble allantoin crystals to obtain coarse filtrate;
6) And (3) filtering the coarse filtrate by using an ultrafiltration membrane, wherein the molecular weight cut-off of the ultrafiltration membrane is larger than or equal to 5000g/mol, adding 1L of supplementary injection water into the concentrated solution when the concentration of the glue solution is concentrated to 30%, and adding 1L of injection water when the concentration of the glue solution is concentrated to 30%. And stopping concentrating when the concentration of the glue solution is 30% after water is added for the fourth time.
7) Carrying out flash evaporation sterilization on the concentrated glue solution to obtain a sterilized concentrated glue solution;
8) Freeze drying the sterilized concentrated glue solution to obtain low endotoxin gelatin;
the gelatin products before and after treatment are shown in table 4. The addition amount of allantoin during the treatment process is lower than 1g/L, and the endotoxin in the treated gelatin is 4120EU/g, which is much higher than that in example 1.
TABLE 5 Key technical index of gelatin before and after treatment of comparative example 2
| Index of gelatin | Before treatment | After treatment |
| Endotoxin (EU/g) | 5230 | 4120 |
| Freeze power (Bloom g) | 230 | 227 |
| Viscosity (mPa. S) | 4.68 | 4.60 |
| Viscosity reduction (%) | 2.5 | 2. |
| Gel point (. Degree.C.) (10% concentration) | 27.5 | 27.3 |
| Ash (%) | 0.8 | 0.4 |
| Moisture (%) | 11.2 | 5.5 |
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
Claims (7)
1. A method for reducing endotoxin in gelatin by using allantoin is characterized by comprising the following steps:
dissolving gelatin to be treated in water at 45-55 deg.C in a container, and adjusting pH of the gelatin solution; adding sodium chloride, and stirring at 45-55 deg.C for 10-30min; adding allantoin to obtain a pretreatment solution; the concentration of sodium chloride in the pretreatment solution is 5-50mmol/L, and the concentration of allantoin is 1-100g/L; then, stirring and incubating for 15-60min at 45-55 ℃, filtering, concentrating, sterilizing, drying and crushing to obtain low endotoxin gelatin particles;
the gelatin to be processed is acid gelatin or alkali gelatin; when the gelatin to be treated is acid gelatin, adjusting the pH value of the gelatin solution to be 5.5-6.0; when the gelatin to be processed is alkaline gelatin, adjusting the pH value of the gelatin solution to 4.5-4.6;
the concentration of the gelatin solution is 2-10wt%;
the endotoxin content of the gelatin to be treated is 200-20000EU/g;
the filtering process comprises a coarse filtering process and a fine filtering process; the coarse filtration process uses cotton cake or filter paper as filter medium, and the fine filtration process uses tubular membrane as filter medium.
2. The process of claim 1, wherein the water used in the process for reducing endotoxin in gelatin using allantoin is secondary purified water, and the endotoxin content is less than 0.05EU/mL.
3. The process of claim 1, wherein the endotoxin in gelatin is removed by dry heat or chemical treatment using a vessel used in the process for reducing endotoxin in gelatin using allantoin.
4. The method according to claim 1, wherein the tubular membrane has a molecular weight cut-off of 5000g/mol and is made of polyethersulfone.
5. The method according to claim 1, wherein the sterilization is flash sterilization, in particular maintained at 148 ℃ for 6-8s.
6. The method according to claim 1, wherein the drying is freeze drying or hot air drying.
7. Use of a low endotoxin gelatin obtained by the process of any one of claims 1 to 6 in the preparation of an absorbable hemostatic sponge, a renewable medical, or a medical supplement.
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| WO2013180650A1 (en) * | 2012-05-31 | 2013-12-05 | Agency For Science, Technology And Research | Methods for reducing levels of protein-contaminant complexes and aggregates in protein preparations by treatment with electropositive organic additives |
| CN104356211A (en) * | 2014-09-22 | 2015-02-18 | 山东信得科技股份有限公司 | Method for removing endotoxin in recombinant protein solution with application of Triton X-114 and allantoin |
| CN105001299A (en) * | 2015-07-14 | 2015-10-28 | 天津瑞普生物技术股份有限公司 | Method for removing endotoxin in protein |
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| WO2013180650A1 (en) * | 2012-05-31 | 2013-12-05 | Agency For Science, Technology And Research | Methods for reducing levels of protein-contaminant complexes and aggregates in protein preparations by treatment with electropositive organic additives |
| CN104507953A (en) * | 2012-05-31 | 2015-04-08 | 新加坡科技研究局 | Methods for reducing levels of protein-contaminant complexes and aggregates in protein preparations by treatment with electropositive organic additives |
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