Non-destructive sterilization method for liquid collagen and liquid collagen

Abstract

This application provides a non-destructive sterilization method for liquid collagen, comprising: step S1, obtaining a liquid collagen solution; step S2, adding sodium chloride particles to the liquid collagen solution and stirring evenly to achieve a sodium chloride concentration range of 3%-36% in the liquid collagen solution; step S3, incubating the liquid collagen solution containing sodium chloride; and step S4, subjecting the incubated liquid collagen solution to ultra-high pressure treatment to complete the non-destructive sterilization of the liquid collagen. The method provided by this application improves sterilization efficiency by adding sodium chloride particles to the liquid collagen without causing collagen denaturation, effectively preserving the triple helix structure of collagen and avoiding loss of collagen activity. Compared with other sterilization methods, the ultra-high pressure process is safe and convenient, and is particularly suitable for the sterilization of liquid collagen raw materials.

Description

Technical Field

[0001] This application relates to the field of medical biomaterial sterilization technology, specifically to a non-destructive sterilization method for liquid collagen and liquid collagen. Background Technology

[0002] Collagen possesses excellent biocompatibility, biodegradability, bioactivity, and processability, making it an important material in regenerative medicine, cosmetics, and health foods. However, quality control systems for collagen-based products still face some challenges, primarily due to the conflict between collagen's bioactivity, structure, and sterilization methods. Collagen has a three-dimensional biological structure similar to natural tissues and organs, providing cells with a microenvironment closer to their in vivo survival. However, these biological structures are easily affected, or even destroyed, by conventional sterilization methods.

[0003] Sterilization is a crucial and challenging aspect of controlling the quality of liquid collagen. Common sterilization methods (including moist heat sterilization, dry heat sterilization, gas sterilization, and radiation sterilization) all have limitations and can easily adversely affect the final product. Heat sterilization significantly impacts collagen structure. Heating causes the triple helix domains of collagen to unwind and gradually transform into randomly coiled peptide chains. Therefore, common moist heat and dry heat sterilization methods are unsuitable for collagen sterilization. Ethylene oxide (ETO) and plasma gases are important options for gas sterilization, but ETO can leave toxic residues in collagen products and affect their mechanical and physical properties. Plasma gases can only be used on thinner materials, and the introduced free charge carriers (electrons and ions) can damage the material's biological environment. Radiation sterilization methods include ultraviolet (UV) sterilization, ionizing radiation, and cobalt-60 rays. UV sterilization has limited penetration and can only be used for surface or shallow liquid sterilization. Prolonged UV radiation can also reduce the mechanical properties of collagen. Ionizing radiation and cobalt-60 rays can affect the cross-linking degree of collagen products to varying degrees, and may even lead to denaturation and destruction of the ordered structure of collagen. Summary of the Invention

[0004] To at least partially solve the above problems and address the shortcomings of existing collagen sterilization technologies, this application provides a method for non-destructive sterilization of liquid collagen using salt-assisted ultra-high pressure technology. This method can effectively kill Escherichia coli in liquid collagen while preserving the triple helix structure of the collagen. The application method is simple, safe, and effective.

[0005] According to one aspect of this application, embodiments of this application provide a non-destructive sterilization method for liquid collagen, comprising:

[0006] Step S1: Obtain a liquid collagen solution;

[0007] Step S2: Add sodium chloride granules to the liquid collagen solution and stir until homogeneous, so that the concentration of sodium chloride in the liquid collagen solution is in the range of 3%-36%;

[0008] Step S3: Incubate the liquid collagen solution containing sodium chloride;

[0009] Step S4: The incubated liquid collagen solution is subjected to ultra-high pressure treatment to complete the non-destructive sterilization of the liquid collagen.

[0010] Preferably, in step S2, the concentration range of sodium chloride is 5%-36%; preferably, the concentration range of sodium chloride is 8%-33%; preferably, the concentration range of sodium chloride is 10%-30%; preferably, the concentration range of sodium chloride is 15%-25%.

[0011] Preferably, in step S3, the incubation time is 0.5h-10h; preferably, the incubation time is 1h-9h; preferably, the incubation time is 2h-8h; preferably, the incubation time is 3h-6h.

[0012] Preferably, in step S4, the pressure of the ultra-high pressure treatment is 100MPa-500MPa, and the time of the ultra-high pressure treatment is 5min-20min.

[0013] Preferably, in step S1, active collagen is dissolved in glacial acetic acid solution to obtain a liquid collagen solution.

[0014] Preferably, the active collagen is fresh collagen extracted from tissue or collagen reconstituted after freeze-drying of tissue collagen.

[0015] Preferably, the mass concentration percentage of active collagen is 0.01%-0.05%.

[0016] Preferably, the method further includes:

[0017] Step S5: After ultra-high pressure treatment, the liquid collagen solution is centrifuged at high speed, and the supernatant is dialyzed to obtain liquid collagen that has undergone non-destructive sterilization.

[0018] Preferably, during high-speed centrifugation, the rotation speed is 6000 rpm / min-10000 rpm / min.

[0019] According to another aspect of this application, an embodiment of this application provides a liquid collagen protein, which is prepared by the method described above.

[0020] The beneficial effects of this invention are as follows:

[0021] (1) The present invention uses salt-assisted ultra-high pressure technology to sterilize liquid collagen contaminated by Escherichia coli. The addition of sodium chloride particles to the liquid collagen improves the sterilization efficiency without causing collagen denaturation. It can effectively preserve the triple helix structure of collagen and avoid the loss of collagen activity.

[0022] (2) Compared with other sterilization methods, the ultra-high pressure process is safe and convenient, and is especially suitable for the sterilization of liquid adhesive raw materials. Attached Figure Description

[0023] Figure 1 This is a morphological observation image of *E. coli* in Example 1 of the present invention, wherein... Figure 1 The image in the middle left shows the morphological observation of E. coli before ultra-high pressure treatment after incubation. Figure 1 The right image shows the morphology of Escherichia coli after ultra-high pressure treatment;

[0024] Figure 2 This is a morphological observation image of Escherichia coli after ultra-high pressure treatment in Example 2 of the present invention;

[0025] Figure 3 This is a morphological observation of Escherichia coli after ultra-high pressure treatment in Example 3 of the present invention;

[0026] Figure 4 This is a morphological observation of Escherichia coli after ultra-high pressure treatment in Example 4 of the present invention;

[0027] Figure 5 This is a morphological observation of Escherichia coli after ultra-high pressure treatment in Example 5 of the present invention;

[0028] Figure 6 This is a morphological observation image of *E. coli* in Comparative Example 1 of the present invention, wherein... Figure 6 The image in the middle left shows the morphological observation of E. coli before ultra-high pressure treatment after incubation. Figure 6 The right image shows the morphology of Escherichia coli after ultra-high pressure treatment;

[0029] Figure 7 This is a morphological observation of Escherichia coli after ultra-high pressure treatment in Comparative Example 2 of the present invention;

[0030] Figure 8 This is a morphological observation of Escherichia coli after ultra-high pressure treatment in Comparative Example 3 of the present invention;

[0031] Figure 9 For the morphological observation of Escherichia coli in Comparative Example 4 of this invention, wherein, Figure 9 The image in the middle left shows the morphological observation of E. coli before ultra-high pressure treatment after incubation. Figure 9 The right image shows the morphology of Escherichia coli after ultra-high pressure treatment;

[0032] Figure 10This is a morphological observation of Escherichia coli after ultra-high pressure treatment in Comparative Example 5 of the present invention;

[0033] Figure 11 This is a morphological observation of Escherichia coli after ultra-high pressure treatment in Comparative Example 6 of the present invention;

[0034] Figure 12 The CD spectrum of liquid collagen after ultra-high pressure treatment in Examples 1-5 of this invention is shown. Example 1 corresponds to the 100 MPa curve, Example 2 corresponds to the 200 MPa curve, Example 3 corresponds to the 300 MPa curve, Example 4 corresponds to the 400 MPa curve, and Example 5 corresponds to the 500 MPa curve. The control curve is the control group curve, and the horizontal axis is the wavelength.

[0035] Figure 13 The CD spectra of liquid collagen after ultra-high pressure treatment in Comparative Examples 4-6 of this invention are shown. Comparative Example 4 corresponds to the 300 MPa curve, Comparative Example 5 corresponds to the 400 MPa curve, Comparative Example 6 corresponds to the 500 MPa curve, and control is the control group curve. The horizontal axis represents the wavelength. Detailed Implementation

[0036] The preferred embodiments of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection claimed in this application. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the materials used are common commercially available products. The equipment and materials used in each embodiment are the same.

[0037] Example 1

[0038] A non-destructive sterilization method for liquid collagen includes the following steps:

[0039] (1) Sodium chloride particles were added to a 0.5 mg / mL liquid collagen solution contaminated with Escherichia coli to make the sodium chloride concentration 3%, and the sample was placed at room temperature for 6 h for incubation.

[0040] (2) Place the above solution into an ultra-high pressure device and apply an ultra-high pressure of 100 MPa for 10 min.

[0041] After applying pressure, the E. coli precipitate was collected by centrifugation and the sample was prepared for morphological observation.

[0042] Example 2

[0043] A non-destructive sterilization method for liquid collagen includes the following steps:

[0044] (1) Sodium chloride particles were added to a 0.5 mg / mL liquid collagen solution contaminated with Escherichia coli to make the sodium chloride concentration 3%, and the sample was placed at room temperature for 6 h for incubation.

[0045] (2) Place the above solution into an ultra-high pressure device and apply an ultra-high pressure of 200 MPa for 10 min.

[0046] After applying pressure, the E. coli precipitate was collected by centrifugation and the sample was prepared for morphological observation.

[0047] Example 3

[0048] A non-destructive sterilization method for liquid collagen includes the following steps:

[0049] (1) Sodium chloride particles were added to a 0.5 mg / mL liquid collagen solution contaminated with Escherichia coli to make the sodium chloride concentration 3%, and the sample was placed at room temperature for 6 h for incubation.

[0050] (2) Place the above solution into an ultra-high pressure device and apply an ultra-high pressure of 300 MPa for 10 min.

[0051] After applying pressure, the E. coli precipitate was collected by centrifugation and the sample was prepared for morphological observation.

[0052] Example 4

[0053] A non-destructive sterilization method for liquid collagen includes the following steps:

[0054] (1) Sodium chloride particles were added to a 0.5 mg / mL liquid collagen solution contaminated with Escherichia coli to make the sodium chloride concentration 3%, and the sample was placed at room temperature for 6 h for incubation.

[0055] (2) Place the above solution into an ultra-high pressure device and apply an ultra-high pressure of 400 MPa for 10 min.

[0056] After applying pressure, the E. coli precipitate was collected by centrifugation and the sample was prepared for morphological observation.

[0057] Example 5

[0058] A non-destructive sterilization method for liquid collagen includes the following steps:

[0059] (1) Sodium chloride particles were added to a 0.5 mg / mL liquid collagen solution contaminated with Escherichia coli to make the sodium chloride concentration 3%, and the sample was placed at room temperature for 6 h for incubation.

[0060] (2) Place the above solution into an ultra-high pressure device and apply an ultra-high pressure of 500 MPa for 10 min.

[0061] After applying pressure, the E. coli precipitate was collected by centrifugation and the sample was prepared for morphological observation.

[0062] Comparative Example 1

[0063] A sterilization method for liquid collagen includes the following steps:

[0064] (1) Sodium chloride particles were added to a 0.5 mg / mL liquid collagen solution contaminated with Escherichia coli to make the sodium chloride concentration 0.9%, and the sample was placed at room temperature for 0.5 h for incubation;

[0065] (2) Place the above solution into an ultra-high pressure device and apply an ultra-high pressure of 300 MPa for 10 min.

[0066] After applying pressure, E. coli were collected and samples were prepared for morphological observation.

[0067] Comparative Example 2

[0068] A sterilization method for liquid collagen includes the following steps:

[0069] (1) Sodium chloride particles were added to a 0.5 mg / mL liquid collagen solution contaminated with Escherichia coli to make the sodium chloride concentration 0.9%, and the sample was placed at room temperature for 0.5 h for incubation;

[0070] (2) Place the above solution into an ultra-high pressure device and apply an ultra-high pressure of 400 MPa for 10 min.

[0071] After applying pressure, E. coli were collected and samples were prepared for morphological observation.

[0072] Comparative Example 3

[0073] A sterilization method for liquid collagen includes the following steps:

[0074] (1) Sodium chloride particles were added to a 0.5 mg / mL liquid collagen solution contaminated with Escherichia coli to make the sodium chloride concentration 0.9%, and the sample was placed at room temperature for 0.5 h for incubation;

[0075] (2) Place the above solution into an ultra-high pressure device and apply an ultra-high pressure of 500 MPa for 10 min.

[0076] After applying pressure, E. coli were collected and samples were prepared for morphological observation.

[0077] Comparative Example 4

[0078] A sterilization method for liquid collagen includes the following steps:

[0079] (1) Sodium chloride particles were added to a 0.5 mg / mL liquid collagen solution contaminated with Escherichia coli to make the sodium chloride concentration 0.9%, and the sample was placed at room temperature for 6 h for incubation.

[0080] (2) Place the above solution into an ultra-high pressure device and apply an ultra-high pressure of 300 MPa for 10 min.

[0081] After applying pressure, the E. coli precipitate was collected by centrifugation and the sample was prepared for morphological observation.

[0082] Comparative Example 5

[0083] A sterilization method for liquid collagen includes the following steps:

[0084] (1) Sodium chloride particles were added to a 0.5 mg / mL liquid collagen solution contaminated with Escherichia coli to make the sodium chloride concentration 0.9%, and the sample was placed at room temperature for 6 h for incubation.

[0085] (2) Place the above solution into an ultra-high pressure device and apply an ultra-high pressure of 400 MPa for 10 min.

[0086] After applying pressure, the E. coli precipitate was collected by centrifugation and the sample was prepared for morphological observation.

[0087] Comparative Example 6

[0088] A sterilization method for liquid collagen includes the following steps:

[0089] (1) Sodium chloride particles were added to a 0.5 mg / mL liquid collagen solution contaminated with Escherichia coli to make the sodium chloride concentration 0.9%, and the sample was placed at room temperature for 6 h for incubation.

[0090] (2) Place the above solution into an ultra-high pressure device and apply an ultra-high pressure of 500 MPa for 10 min.

[0091] After applying pressure, the E. coli precipitate was collected by centrifugation and the sample was prepared for morphological observation.

[0092] The comparison results of Examples 1-5 and Comparative Examples 1-6 show that when the sodium chloride concentration is increased to 3.0%, with sufficient incubation time, the ultra-high pressure sterilization effect is significantly improved compared to the sodium chloride concentration of 0.9% (the basic culture system maintains a sodium chloride concentration of 0.9% to maintain the osmotic pressure of the culture system).

[0093] The comparative results of Examples 1-5 show that under ultra-high pressure treatment of 100 MPa, some E. coli showed obvious pores on their surface. As the pressure increased, the number of pores on the surface of E. coli increased significantly. At 500 MPa, E. coli was almost completely destroyed.

[0094] The results of the comparison of Examples 1-3 show that when the sodium chloride concentration in the basal culture system is 0.9%, ultra-high pressure treatment alone has no destructive effect on Escherichia coli without sufficient incubation time.

[0095] The results of the comparison of Examples 4-6 show that when the sodium chloride concentration in the basal culture system is 0.9%, after sufficient incubation and ultra-high pressure treatment, it has a slight destructive effect on Escherichia coli. The number of pores increases with the increase of pressure, but even when the pressure is increased to a high level (500 MPa), a large number of E. coli are still not destroyed, and its sterilization effect is difficult to meet the sterilization requirements of collagen.

[0096] Depend on Figure 1 and Figure 9 It can be seen that without ultra-high pressure treatment, simply using 3% sodium chloride or 0.9% sodium chloride with sufficient incubation time will not achieve sterilization.

[0097] Depend on Figure 12 and Figure 13 It can be seen that the positive peak of liquid collagen after salt-assisted ultra-high pressure sterilization is stable around 220 nm with almost no change in intensity, proving that the triple helix structure of liquid collagen remains stable. Due to the influence of salt, the negative peak is unstable, and after dialysis treatment, a significant negative peak appears around 197 nm.

[0098] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0099] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application. The scope of this application is defined by the appended claims.

Claims

1. A method for the non-destructive sterilization of liquid collagen, characterized in that, include: Step S1: Obtain a liquid collagen solution; Step S2: Add sodium chloride granules to the liquid collagen solution and stir until homogeneous, so that the concentration of sodium chloride in the liquid collagen solution is in the range of 3%-36%; Step S3: Incubate the liquid collagen solution containing sodium chloride; Step S4: The incubated liquid collagen solution is subjected to ultra-high pressure treatment to complete the non-destructive sterilization of the liquid collagen.

2. The method according to claim 1, characterized in that, In step S2, the concentration of sodium chloride ranges from 5% to 36%; preferably, the concentration ranges from 8% to 33%; preferably, the concentration ranges from 10% to 30%; preferably, the concentration ranges from 15% to 25%.

3. The method according to claim 1, characterized in that, In step S3, the incubation time is 0.5h-10h; preferably, the incubation time is 1h-9h; preferably, the incubation time is 2h-8h; preferably, the incubation time is 3h-6h.

4. The method according to claim 1, characterized in that, In step S4, the pressure of the ultra-high pressure treatment is 100MPa-500MPa, and the treatment time is 5min-20min.

5. The method according to claim 1, characterized in that, In step S1, active collagen is dissolved in glacial acetic acid solution to obtain liquid collagen solution.

6. The method according to claim 5, characterized in that, The active collagen is fresh collagen extracted from tissue or collagen obtained by lyophilizing tissue collagen and then rehydrating it.

7. The method according to claim 5, characterized in that, The mass concentration percentage of active collagen is 0.01%-0.05%.

8. The method according to claim 1, characterized in that, Also includes: Step S5: After ultra-high pressure treatment, the liquid collagen solution is centrifuged at high speed, and the supernatant is dialyzed to obtain liquid collagen that has undergone non-destructive sterilization.

9. The method according to claim 8, characterized in that, During high-speed centrifugation, the rotation speed is 6000 rpm / min-10000 rpm / min.

10. A liquid collagen, characterized in that, The liquid collagen is prepared by the method according to any one of claims 1-9.

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