Infrared analysis method of recombinant collagen gel dressing

Abstract

The invention discloses an infrared analysis method of a recombinant collagen gel dressing, and belongs to the technical field of analysis of recombinant collagen gel dressings. The method comprises the following steps: fully and uniformly mixing a recombinant collagen gel dressing and a salt solution, standing, centrifuging, removing a precipitate Carbomer, adding a protein precipitator into a supernatant, uniformly mixing, standing, centrifuging, adding water into the precipitate, redissolving, adding an absolute ethyl alcohol solution, centrifuging, drying at low temperature, and carrying out infrared detection. The recombinant collagen gel dressing is pretreated, so that the interference of auxiliary material components is reduced, the treated detection sample is subjected to infrared detection, and the result accuracy and stability are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis of recombinant collagen gel dressings, specifically an infrared analysis method for recombinant collagen gel dressings, and particularly relates to an infrared analysis method for a composition containing recombinant collagen, trehalose, and carbomer gel. Background Art

[0002] The recombinant collagen gel dressing is mainly composed of purified water, recombinant humanized collagen, trehalose, and carbomer. By forming a protective layer on the wound surface, it plays a physical barrier role. Recombinant humanized collagen repairs skin wounds, but scars are likely to form on the wound surface during the repair process. It is applicable to the care of non-chronic wounds.

[0003] Currently, the identification methods for proteins in recombinant collagen gel dressings mainly include those disclosed in pharmacopoeias or YY / T 1888-2023 / YYT 1849-2022. However, the formula of the recombinant collagen gel dressing is complex, and moisturizers, thickeners, or preservatives, etc. will have a great impact on the infrared analysis results of the sample, resulting in more miscellaneous peaks detected in the sample than the characteristic peaks of the standard product and unstable results. When using the above methods for the identification and analysis of recombinant collagen gel dressings, the sample is simply processed and then subjected to infrared detection. The detected spectrum is the same as the standard spectrum, with stable detection results and high repeatability; compared with the high-performance liquid chromatography method, it has the advantages of low detection cost and low detection difficulty, and can be used as a test method for the identification of recombinant collagen gel dressings.

[0004] Compared with the method of ethanol precipitation of recombinant collagen mentioned in the patent CN118443835A "Liquid Phase Peptide Map Analysis Method for Recombinant Collagen Liquid Dressing", after first precipitating with ethanol, 10% formic acid solution is added to the enzymatic hydrolysate obtained by enzymatic hydrolysis, and then reverse-phase high-performance liquid chromatography detection is carried out. This method has high requirements for personnel and equipment and is not suitable for popularization and application. Summary of the Invention

[0005] The purpose of the present invention is to provide an infrared analysis method for recombinant collagen gel dressings. This method first pre-treats the recombinant collagen gel dressing, reduces the interference of other auxiliary components in the gel dressing, and improves the accuracy and stability of the detection results.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows: An infrared analysis method for recombinant collagen gel dressings, comprising the following steps: (1) Mix the recombinant collagen gel dressing and calcium chloride solution / sodium chloride thoroughly and dissolve them evenly, then let them stand at room temperature for 0.5 - 2 h, and then centrifuge at 9000 - 10000 rpm for 5 - 8 min to remove the precipitate. The supernatant is the mixture containing recombinant collagen; (2) Take the supernatant and add anhydrous ethanol with a mass 5 - 10 times that of the supernatant to precipitate proteins. Let it stand at 4℃ - 8℃ for 1 - 2 hours, and then centrifuge at 9000 - 10000 rpm for 5 - 8 min; Remove the supernatant, take the precipitate for re - dissolution. After dissolution, add anhydrous ethanol with a mass 5 - 10 times that of the supernatant to precipitate proteins. Let it stand at 4℃ - 8℃ for 1 - 2 hours, and centrifuge at 9000 - 10000 rpm for 5 - 8 min; Remove the supernatant, take the precipitate and dry it at 36℃ for 24 - 48 h, and then perform infrared detection on the powder.

[0007] Further, in the step (1), the recombinant collagen gel dressing is a gel dressing that contains no other proteins except recombinant collagen.

[0008] Furthermore, the recombinant collagen gel dressing includes recombinant collagen and auxiliary components, and the auxiliary components are one or more of a humectant, a thickener, and a protein stabilizer.

[0009] The recombinant collagen described in the present invention is a common recombinant type of collagen in the art. In the specific embodiment of the present invention, the recombinant collagen gel dressing is composed of the following components by mass percentage: recombinant type III collagen ≥ 0.05%, trehalose 1% - 3%, carbomer 940 0.4% - 1.0%, glycerol 1% - 3%, butanediol 1% - 3%, triethanolamine 0.2% - 0.3%, and the balance is purified water. The recombinant type III collagen is recombinant humanized collagen produced by Pichia pastoris fermentation.

[0010] Further, in the step (1), the concentration of recombinant collagen in the recombinant collagen gel dressing is 0.5 mg - 1 mg / g.

[0011] Further, in the step (1), the mass concentration of the calcium chloride solution is 5%, and the mass ratio of the recombinant collagen gel dressing to the calcium chloride solution is 10∶1 - 2.

[0012] Further, in the step (1), the dosage of sodium chloride is 15% of the mass of the recombinant collagen gel dressing.

[0013] Further, in the step (2), when re - dissolving, 5 - 20 mL of purified water is added to the precipitate per 50 - 100 g of the recombinant collagen gel dressing.

[0014] Compared with the prior art, the beneficial effects of the present invention are: 1. The present invention uses calcium chloride solution or sodium chloride to pretreat the recombinant collagen gel dressing, and then uses ethanol reagent to precipitate the recombinant collagen. Such an operation can reduce the interference of moisturizers, thickeners and other auxiliary components (including carbomer and trehalose) in the test sample on the identification.

[0015] 2. This method is a simple method for processing infrared detection samples, which is simple and easy to operate. Description of the Drawings

[0016] Figure 1 It is the infrared spectrum of the recombinant type III collagen standard product.

[0017] Figure 2 It is the infrared spectrum of the sample after treatment in Example 1 of the recombinant collagen gel.

[0018] Figure 3 It is the infrared spectrum of the sample after treatment in Example 2 of the recombinant collagen gel. Detailed Description of the Invention

[0019] The present invention will be further described in detail below in conjunction with specific embodiments and drawings.

[0020] In the following embodiments, the recombinant type III collagen used is recombinant humanized collagen produced by Pichia pastoris, and is purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.

[0021] The recombinant collagen gel dressing used in the embodiments of the present invention is composed of the following components by mass percentage: 0.05% of recombinant type III collagen, 0.5% of carbomer 940, 2% of trehalose, 1.5% of glycerol, 1% of butanediol, 0.3% of triethanolamine, and the balance is purified water.

[0022] Example 1 The infrared analysis method of the recombinant collagen gel dressing in this example includes the following steps: Pretreatment: Using the recombinant collagen gel dressing as the test sample, weigh 50 g of the recombinant collagen gel dressing, place it in a 250 mL beaker, add 5 g of calcium chloride solution (5%), mix well, and let it stand at room temperature for 30 min, then centrifuge at 90000 rpm for 5 min to remove the precipitate. The supernatant is a mixture containing recombinant collagen; Protein precipitation with ethanol: Weigh the supernatant, add anhydrous ethanol five times the mass of the supernatant for precipitation; let it stand at 4°C for 90 min, then centrifuge at 9000 rpm for 5 min; remove the supernatant, add 10 mL of purified water to the precipitate for re-dissolution, and after complete dissolution, add anhydrous ethanol ten times the mass of the supernatant to precipitate the protein. Let it stand at 4°C for 60 min, then centrifuge at 10000 rpm for 5 min; remove the supernatant, dry the precipitate at 36°C for 48 hours, and then perform infrared detection on the powder.

[0023] Example 2 The infrared analysis method of the recombinant collagen gel dressing in this example includes the following steps: Pretreatment: Take the recombinant collagen gel dressing as the test sample, weigh 50 g of the recombinant collagen gel dressing, place it in a 250 mL beaker, add 15% sodium chloride of the gel mass and dissolve it evenly, then let it stand at room temperature for 30 min, and then centrifuge at 10000 rpm for 8 min to remove the precipitate. The supernatant is the mixture containing recombinant collagen; Protein precipitation with ethanol: Weigh the supernatant, add anhydrous ethanol ten times the mass of the supernatant for precipitation; let it stand at 4°C for 90 min, then centrifuge at 9000 rpm for 8 min; remove the supernatant, add 20 mL of purified water to the precipitate for re-dissolution, and after complete dissolution, add anhydrous ethanol ten times the mass of the supernatant to precipitate the protein. Let it stand at 8°C for 2 hours, then centrifuge at 10000 rpm for 8 min; remove the supernatant, dry the precipitate at 36°C for 48 hours, and then perform infrared detection on the powder.

[0024] Figure 1 The characteristic peaks of recombinant type III collagen can be obtained, amide A 3330 cm -1 、amide B 2942 cm -1 、amide I 1659 cm -1 、amide II 1527 cm -1 、amide III 1239 cm -1 、There are a total of 5 characteristic peaks. Compare Figure 2 and Figure 3 The spectra of the samples after treatment and detection show that the characteristic peaks are the same as those of the standard product, proving that the infrared identification results of the samples treated by this method are stable and can be used as a method for product identification.

[0025] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An infrared analysis method for recombinant collagen gel dressing, characterized in that: The following steps are involved: (1) After the recombinant collagen gel dressing and calcium chloride solution / sodium chloride are fully mixed / dissolved evenly, the mixture is allowed to stand at room temperature for 0.5-2 hours, and then centrifuged at 9000-10000 rpm for 5-8 minutes to remove the precipitate. The supernatant is a mixture containing recombinant collagen; (2) Take the supernatant and add 5 to 10 times the mass of anhydrous ethanol to precipitate the protein, let it stand at 4°C to 8°C for 1-2 hours, and then centrifuge at 9000-10000 rpm for 5-8 minutes; remove the supernatant, take the precipitate for redissolution, and after dissolution, add 5 to 10 times the mass of anhydrous ethanol to precipitate the protein, let it stand at 4°C to 8°C for 1-2 hours, and centrifuge at 9000-10000 rpm for 5-8 minutes; remove the supernatant, take the precipitate and dry it at 36°C for 24 to 48 hours, and then perform infrared detection on the powder.

2. The infrared analysis method of recombinant collagen gel dressing according to claim 1, characterized in that: In the step (1), the recombinant collagen gel dressing is a gel dressing that does not contain any other protein except recombinant collagen.

3. The infrared analysis method of recombinant collagen gel dressing according to claim 2, characterized in that: The recombinant collagen gel dressing comprises recombinant collagen and auxiliary material components, wherein the auxiliary material components are one or more of a moisturizer, a thickener and a protein stabilizer.

4. The infrared analysis method of the recombinant collagen gel dressing according to claim 3, characterized in that: The recombinant collagen gel dressing is composed of the following ingredients by mass percentage: recombinant type III collagen ≥ 0.05%, trehalose 1%-3%, carbomer 940 0.4%-1.0%, glycerol 1%-3%, butylene glycol 1%-3%, triethanolamine 0.2%-0.3%, and the balance is purified water. The recombinant type III collagen is a recombinant humanized collagen produced by Pichia pastoris fermentation.

5. The infrared analysis method of recombinant collagen gel dressing according to claim 1, characterized in that: In the step (1), the concentration of the recombinant collagen in the recombinant collagen gel dressing is 0.5 mg-1 mg / g.

6. The infrared analysis method of recombinant collagen gel dressing according to claim 1, characterized in that: In the step (1), the mass concentration of the calcium chloride solution is 5%, and the mass ratio of the recombinant collagen gel dressing to the calcium chloride solution is 10:1-2.

7. The infrared analysis method of recombinant collagen gel dressing according to claim 1, characterized in that: In the step (1), the amount of sodium chloride used is 15% of the mass of the recombinant collagen gel dressing.

8. The infrared analysis method of recombinant collagen gel dressing according to claim 1, characterized in that: In the step (2), 5-20 mL of purified water is added to the precipitate for every 50-100 g of recombinant collagen gel dressing during reconstitution.

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