Andrias davidianus non-denatured collagen with anti-aging and skin repair effects, and preparation method and application thereof

Abstract

This invention discloses a non-denatured collagen from giant salamander skin with anti-aging and skin repair effects, its preparation method, and its applications. The collagen comprises: raw material pretreatment, low-temperature microfluidic degreasing, enzymatic extraction, purification, and collection. The preparation method combines degreasing with a weak alkaline solution with enzymatic extraction, effectively removing grease from the giant salamander skin and preventing interference from fat in the subsequent enzymatic hydrolysis process. Simultaneously, the weak alkaline solution, pulsed electric field, and low-frequency ultrasound synergistically assist in opening the tissue structure and improving enzymatic hydrolysis efficiency without causing denaturation damage to the collagen like strong alkalis. It also ensures efficient dissolution of the collagen at lower temperatures and suitable enzyme concentrations, while maximally protecting its natural triple helix structure. The method is mild, yields a high output, and the resulting non-denatured collagen from giant salamander skin exhibits good anti-aging and skin repair effects.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a non-denatured collagen from giant salamander skin with anti-aging and skin repair effects, its preparation method, and its application. Background Technology

[0002] Collagen is an important component of the extracellular matrix and possesses excellent biocompatibility and bioactivity. The skin of the giant salamander is rich in collagen, and its dense structure contains a large amount of fat and other proteins, posing a challenge to the extraction of high-purity, high-activity collagen.

[0003] Traditional collagen extraction methods mainly include: (1) Acid extraction: using low-concentration acid to dissolve collagen in tissues. This method is simple to operate, but it mainly extracts acid-soluble collagen, resulting in a low yield, and it is difficult to fully release collagen from dense tissues such as giant salamander skin. (2) Enzyme extraction: using proteases to selectively cleave the non-helical telomeres of collagen to improve its solubility. This method has a higher yield than the acid method and is currently the mainstream technology.

[0004] In the prior art, in order to obtain collagen with high purity, complex pretreatment is usually carried out before the enzyme, such as using NaOH to remove impurities, using organic solvents (such as isopropanol and chloroform) for defatting, and using acid solutions for pre-expansion. However, these methods have the following problems: (1) Complex process and high cost: The complex multi-step pretreatment (removal of impurities, defatting, and acid soaking) in the prior art not only increases the complexity of the process and the production cost, but also easily causes collagen loss when processing raw materials such as giant salamander skin, resulting in a decrease in yield. For example, conventional acid soaking treatment will significantly reduce the yield of collagen. (2) The structure is easily damaged: In order to pursue high purity, many existing processes adopt relatively harsh conditions (such as high temperature, strong alkali, and long-term enzymatic hydrolysis), which easily leads to the destruction of the natural triple helix structure of collagen, forming denatured gelatin, thereby losing the unique biological activities of collagen, such as cell adhesion, migration and proliferation. (3) Unclear product function positioning: At present, most collagen extraction technologies only focus on yield and purity indicators, and lack in-depth research and verification on the specific bioactive functions of the extracted products, resulting in unclear product functions and limited application value. (4) Specific problems of giant salamander skin: Giant salamander skin is rich in fat and has a dense structure. When processing this raw material, the existing general extraction methods often face problems such as incomplete defatting, difficulty in fully releasing collagen, or large loss of target products during pretreatment.

[0005] Therefore, it is of great significance to develop a method for preparing non-denatured collagen that can be efficiently extracted from giant salamander skin, retains the natural triple helix structure, and has clear biological efficacy. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, one of the objectives of this invention is to provide a method for preparing non-denatured collagen from giant salamander skin with anti-aging and skin repair effects. This method provides a preparation method that is simple, mild, and has a high yield, thereby solving the problems of low extraction efficiency, easy structural denaturation, complicated processes, and unclear product functions in the prior art.

[0007] The second objective of this invention is to provide a non-denatured collagen from giant salamander skin that has anti-aging and skin repair effects, obtained by the above preparation method. This collagen can retain the natural triple helix structure and its biological activity to the maximum extent.

[0008] The third objective of this invention is to provide an application of the above-mentioned non-denatured collagen from giant salamander skin in the preparation of anti-aging and / or skin repair products.

[0009] One of the objectives of this invention is achieved through the following technical solution: A method for preparing non-denatured collagen from giant salamander skin with anti-aging and skin repair effects includes the following steps: S1 Raw Material Pretreatment: Take giant salamander skin, wash and crush it, then soak the crushed giant salamander skin in a weak alkaline solution at a material-to-liquid ratio of 1:(15-25) (g / mL) for degreasing treatment to obtain pretreated giant salamander skin; This step does not involve acid soaking or protein removal in the raw material pretreatment process. Instead, it uses a weak alkaline solution for degreasing, which effectively removes the grease from the giant salamander skin, preventing fat from interfering with the subsequent enzymatic hydrolysis process. This avoids the use of acidic solutions or organic solvents that could damage its structure. Furthermore, the weak alkaline solution helps to open up the tissue structure and improve the efficiency of enzymatic hydrolysis, without causing collagen denaturation damage like strong alkalis. Therefore, a good balance is achieved between gentleness and effectiveness.

[0010] S2 Low-Temperature Microfluidic Degreasing: At a low temperature of 4℃, the remaining grease on the surface of the pretreated giant salamander skin is peeled off by microfluidic shear force to obtain the degreased giant salamander skin; In this step, after gentle alkaline degreasing, a low-temperature microfluidic degreasing process is combined to precisely remove the trace amounts of surface oil that still adhere to the alkaline treatment, thus completely preserving the active ingredients of the giant salamander skin.

[0011] S3 Enzymatic Extraction: The defatted giant salamander skin was pretreated with a 10 kV / cm pulsed electric field for 2-4 min to open the cell channels. Then, a protease solution was added under the assistance of 200W low-frequency ultrasound for gentle enzymatic extraction. The extraction conditions were: extraction time 5-6 h, extraction temperature 35℃-45℃, and enzyme dosage 800 U / mL-1000 U / mL, to obtain the enzymatically extracted solution. In this step, by employing mild enzymatic extraction conditions, we can ensure efficient dissolution of collagen at a lower temperature and a suitable enzyme concentration, while maximizing the protection of its natural triple helix structure from damage.

[0012] S4 Purification and Collection: The supernatant of the solution after enzymatic extraction is centrifuged and dialyzed to obtain the non-denatured collagen from the skin of the giant salamander.

[0013] Furthermore, in step S1, the crushed giant salamander skin is made into small pieces ranging from 1mm×1mm to 6mm×6mm.

[0014] Further, in step S1, the weak alkaline solution is one or more of sodium carbonate solution, sodium bicarbonate solution, potassium carbonate solution, sodium acetate solution, and sodium phosphate solution.

[0015] Furthermore, in step S1, the mass concentration of the weak alkaline solution is 4%-8%, such as 4%, 5%, 6%, 7%, 8%, etc. Preferably, the mass percentage concentration of the weak alkaline solution is 5%, at which the collagen yield is the highest.

[0016] Furthermore, in step S2, the microchannel size of the microfluidic system is 200-300 μm, the total flow rate is controlled at 60-80 mL / min, and the shear rate is controlled at 1000-2000 s⁻¹. -1 Repeat 2-3 times.

[0017] Further, in step S3, the protease solution is one or more of pepsin, neutral protease, trypsin, alkaline protease, and papain. Preferably, the protease solution is pepsin.

[0018] Furthermore, in step S3, the extraction conditions are: extraction time 5.4 h, extraction temperature 38 °C, and enzyme dosage 960 U / mL.

[0019] Furthermore, in step S4, the centrifugation speed is 7000 r / min-9000 r / min, and the centrifugation time is 10 min-20 min.

[0020] Further, in step S4, dialysis is performed using a dialysis bag with a capacity of 8000Da-14000Da.

[0021] The second objective of this invention is achieved by the following technical solution: A non-denatured collagen from giant salamander skin with anti-aging and skin repair effects is prepared using the preparation method described above.

[0022] The third objective of this invention is achieved through the following technical solution: An application of the above-mentioned undenatured collagen from giant salamander skin in the preparation of anti-aging and / or skin repair products.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The preparation method of this invention combines degreasing treatment with weak alkaline solution and low-temperature microfluidic control with enzymatic extraction, which can effectively remove the grease in the giant salamander skin and prevent the fat from interfering with the subsequent enzymatic hydrolysis process. At the same time, the weak alkaline solution, pulsed electric field and low-frequency ultrasound work together to help open the tissue structure and improve the efficiency of enzymatic hydrolysis, but without causing denaturation damage to collagen like strong alkali. It can also ensure that the collagen is efficiently dissolved at a lower temperature and a suitable enzyme concentration, while protecting its natural triple helix structure to the greatest extent. The conditions are mild and the yield is high. The obtained non-denatured collagen from giant salamander skin has good anti-aging and skin repair effects.

[0024] 2. Simplified Process for Giant Salamander Skin, Achieving "One Subtraction and One Addition": This invention, targeting giant salamander skin as a specific raw material, employs a weak alkaline solution and low-temperature microfluidic degreasing treatment, creatively eliminating the essential acid soaking and protein removal steps in traditional processes. This "subtraction" treatment not only simplifies the process and reduces costs but also unexpectedly achieves an "addition" effect—significantly increasing the final yield of non-denatured collagen. This indicates that cumbersome pretreatment is not necessary for the extraction of non-denatured collagen from giant salamander skin, and may even be detrimental. Mild Conditions and Optimized Parameters Synergistically Ensure "Non-Denatured" Structure: This invention uses pulsed electric field, low-frequency ultrasound synergistically assisted extraction with pepsin under mild conditions (38℃), and systematically optimizes the process parameters (5.4h, 960U / mL) using response surface methodology, resulting in a yield as high as 61.59%. More importantly, the synergistic effect of mild extraction conditions and precisely controlled enzymatic extraction parameters effectively protects the natural triple helix structure of collagen, yielding non-denatured collagen with high bioactivity. Causal verification of structure and function clarifies the dual efficacy of "anti-aging + repair": This invention not only pursues high yield but also prioritizes the preservation of bioactivity as the core objective of its process design. Zebrafish model experiments confirm that the non-denatured collagen prepared using this method exhibits significant anti-aging (reducing β-galactosidase activity and MDA content, and increasing GSH content and telomerase activity) and skin repair (significantly promoting tail fin regeneration) effects. This result conversely verifies the effective protection of collagen bioactivity by the mild extraction process of this invention, proving that the product is not ordinary denatured gelatin but active collagen with specific physiological functions, providing a solid scientific basis for its application in high-end functional foods, cosmetics, and biomedicine. Unique raw material source enables high-value utilization: Using giant salamander skin as raw material realizes the high-value utilization of giant salamander processing by-products, expanding the raw material sources for functional collagen and possessing both economic and environmental value. Attached Figure Description

[0025] Figure 1 Comparison of β-galactose-induced aging models in zebrafish from different control groups in the evaluation of anti-aging efficacy; Figure 2 A comparison of β-galactosidase staining intensities in a β-galactose-induced zebrafish aging model; Figure 3 Comparison of zebrafish tail fin damage models in different control groups during the evaluation of skin repair efficacy; Figure 4 A comparison of the regenerated area of ​​the caudal fin in a zebrafish caudal fin injury model. Detailed Implementation

[0026] The present invention will now be further described in conjunction with specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. In the following embodiments, non-denatured collagen refers to collagen whose natural triple helix structure is maximally preserved during extraction and preparation under mild conditions (such as low temperature, neutral pH, and specific enzymatic hydrolysis). The integrity of this structure is crucial for its biological activities, such as cell adhesion, migration, and proliferation.

[0027] Yield: refers to the percentage of collagen extracted from the raw material relative to the total mass of the raw material. It is an important indicator for measuring the efficiency of the extraction process.

[0028] β-galactosidase: an enzyme specifically expressed or with increased activity in senescent cells, often used as a biomarker of cellular senescence.

[0029] MDA (malondialdehyde): One of the main products of lipid peroxidation, its content can reflect the degree of lipid peroxidation in the body and indirectly reflect the degree of cell damage. It is a commonly used indicator for evaluating oxidative stress and aging.

[0030] GSH (glutathione): An important antioxidant and free radical scavenger in the body, its level reflects the body's antioxidant capacity.

[0031] Telomerase activity: Telomerase is an enzyme that maintains telomere length. Its activity is closely related to cell aging and lifespan, and decreased activity is one of the hallmarks of aging.

[0032] Example 1 A non-denatured collagen from giant salamander skin is prepared using the following steps: S1 Raw Material Pretreatment: Take giant salamander skin, clean it, and then mechanically crush it into small pieces of 1mm×1mm-2mm×2mm. Then, soak the crushed giant salamander skin in a 5% sodium carbonate solution at a material-to-liquid ratio of 1:15 (g / mL) for degreasing treatment to obtain pretreated giant salamander skin. S2 Low-Temperature Microfluidic Degreasing: At a low temperature of 4℃, residual grease on the surface of pretreated giant salamander skin is removed using microfluidic shear force. The microchannel size of the microfluidic system is 300μm, the total flow rate is controlled at 80 mL / min, and the shear rate is controlled at 2000 s. -1 The process was repeated twice to obtain defatted giant salamander skin; S3 enzymatic extraction: The defatted giant salamander skin was pretreated with a 10 kV / cm pulsed electric field for 3 min to open the cell channels, and then mild enzymatic extraction was carried out by adding pepsin solution under the assistance of 200W low-frequency ultrasound. The extraction conditions were: extraction time 5.4 h, extraction temperature 38℃, and enzyme dosage 960 U / mL to obtain the enzymatically extracted solution; S4 Purification and Collection: The supernatant of the solution after enzymatic extraction was centrifuged and dialyzed. The centrifugation speed was 7000 r / min and the centrifugation time was 10 min. Dialysis was performed using a 10000 Da dialysis bag to obtain non-denatured collagen from giant salamander skin.

[0033] Unlike Example 1, the changes and test comparisons of Examples 2-16 and Comparative Examples 1-22 are shown in Tables 1-7 below. Other steps and parameters are the same as in Example 1, and will not be repeated here.

[0034] Table 1 - Comparison of changes in sodium carbonate concentration: Table 2 - Comparison of changes in the feed-to-liquid ratio: Table 3 - Comparison of treatments containing protein removal and other alternative treatments: Table 4 - Comparison of different proteases: Table 5 - Comparison of Changes in Extraction Time: Table 6 - Comparison of changes in extraction temperature: Table 7 - Comparison of changes in enzyme dosage: Performance testing 1. Test subjects: There were normal control group, model control group and sample group. The normal control group was the experimental subjects that did not receive any intervention (representing the physiological level that an organism should have under ideal, healthy and undisturbed conditions). The model control group was the sample that used 0.25 mg / mL of Comparative Example 13 (i.e. the 0.25 mg / mL extract prepared by defatting with citric acid in Comparative Example 13). The sample group was the sample that used 0.25 mg / mL of Example 1.

[0035] 2. Evaluation of anti-aging efficacy (zebrafish experiment) A β-galactose-induced aging model was used in zebrafish to evaluate the anti-aging activity of the test subjects. Relevant indicators were measured 72 hours after sample treatment.

[0036] like Figure 1-2As shown in Table 8, the experimental results indicate that the 0.25 mg / mL collagen sample significantly reduced β-galactosidase activity and MDA content in the aging model zebrafish, while increasing GSH content and telomerase activity. Specifically, the MDA content recovered to a level similar to that of the normal control group, and the GSH content and telomerase activity were also significantly increased, demonstrating that the giant salamander skin collagen prepared in Example 1 of this invention possesses good anti-aging activity.

[0037] Table 8: 3. Evaluation of skin repair efficacy (zebrafish experiment) The skin repair efficacy of the test subjects was evaluated using a zebrafish tail fin injury model. The regenerated area of ​​the tail fin was measured 48 hours after sample treatment.

[0038] like Figure 3-4 As shown, the experimental results indicate that the collagen sample of 0.25 mg / mL can significantly promote the regeneration of the tail fin of damaged zebrafish, and the regeneration area is much larger than that of the model control group, indicating that the giant salamander skin collagen prepared in Example 1 of this invention has good skin repair effects.

[0039] In the efficacy verification applications described above, a β-galactose-induced zebrafish aging model and a caudal fin damage repair model were used to verify the anti-aging and skin repair activities of collagen. The results showed that the 0.25 mg / mL collagen sample from Example 1 significantly reduced β-galactosidase activity and MDA content in the aging model, while increasing GSH content and telomerase activity; simultaneously, it significantly promoted caudal fin regeneration area, demonstrating good skin repair function, further proving the effectiveness of the extraction process of this invention in protecting the bioactivity of collagen.

[0040] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing non-denatured collagen from giant salamander skin with anti-aging and skin repair effects, characterized in that, Includes the following steps: S1 Raw Material Pretreatment: Take giant salamander skin, wash and crush it, then soak the crushed giant salamander skin in a weak alkaline solution at a material-to-liquid ratio of 1:(15-25) for degreasing treatment to obtain pretreated giant salamander skin; S2 Low-Temperature Microfluidic Degreasing: At a low temperature of 4℃, the remaining grease on the surface of the pretreated giant salamander skin is peeled off by microfluidic shear force to obtain the degreased giant salamander skin; S3 Enzymatic Extraction: The defatted giant salamander skin was pretreated with a 10 kV / cm pulsed electric field for 2-4 min, and then a protease solution was added under the assistance of 200W low-frequency ultrasound for gentle enzymatic extraction. The extraction conditions were: extraction time 5h-6h, extraction temperature 35℃-45℃, and enzyme dosage 800U / mL-1000U / mL, to obtain the enzymatically extracted solution. S4 Purification and Collection: The supernatant of the solution after enzymatic extraction is centrifuged and dialyzed to obtain non-denatured collagen from giant salamander skin.

2. The method for preparing non-denatured collagen from giant salamander skin with anti-aging and skin repair effects according to claim 1, characterized in that, In step S1, the weak alkaline solution is one or more of sodium carbonate solution, sodium bicarbonate solution, potassium carbonate solution, sodium acetate solution, and sodium phosphate solution; in step S2, the microchannel size of the microfluidic system is 200-300 μm, the total flow rate is controlled at 60-80 mL / min, and the shear rate is controlled at 1000-2000 s. -1 Repeat 2-3 times.

3. The method for preparing non-denatured collagen from giant salamander skin with anti-aging and skin repair effects according to claim 1, characterized in that, In step S1, the mass percentage concentration of the weak alkaline solution is 4-8%.

4. The method for preparing non-denatured collagen from giant salamander skin with anti-aging and skin repair effects according to claim 3, characterized in that, The weak alkaline solution has a mass percentage concentration of 5%.

5. The method for preparing non-denatured collagen from giant salamander skin with anti-aging and skin repair effects according to claim 1, characterized in that, In step S3, the protease solution is one or more of pepsin, neutral protease, trypsin, alkaline protease, and papain.

6. The method for preparing non-denatured collagen from giant salamander skin with anti-aging and skin repair effects according to claim 5, characterized in that, The protease solution is pepsin.

7. The method for preparing non-denatured collagen from giant salamander skin with anti-aging and skin repair effects according to claim 1, characterized in that, In step S3, the extraction conditions are: extraction time 5.4 h, extraction temperature 38 °C, and enzyme dosage 960 U / mL.

8. The method for preparing non-denatured collagen from giant salamander skin with anti-aging and skin repair effects according to claim 1, characterized in that, In step S4, the centrifugation speed is 7000 r / min-9000 r / min, and the centrifugation time is 10 min-20 min; the dialysis is performed using a dialysis bag with a capacity of 8000 Da-14000 Da.

9. A non-denatured collagen from giant salamander skin with anti-aging and skin repair effects, characterized in that, The non-denatured collagen from giant salamander skin, which has anti-aging and skin repair effects, is prepared using the preparation method described in any one of claims 1-8.

10. An application characterized in that, The use of the non-denatured collagen from giant salamander skin as described in claim 9 in the preparation of anti-aging and / or skin repair products.

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