Collagen tripeptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to collagen tripeptide as well as a preparation method and application thereof. The method comprises the following steps: carrying out depolymerization treatment on a raw material containing collagen to construct a magnetic immobilized enzyme; by utilizing the magnetic immobilized enzyme, raw materials subjected to depolymerization treatment are subjected to enzymolysis at a certain temperature, pH and reaction time, and with the assistance of a magnetic field, rapid separation and cyclic utilization of the immobilized enzyme can be realized. And finally, carrying out enzyme deactivation, solid-liquid separation and ultrafiltration to obtain the collagen tripeptide with GPH content of more than or equal to 59wt.%, and compared with the existing collagen tripeptide, the collagen tripeptide has higher GPH content. The preparation method is simple in process, easy and convenient to operate, low in cost, capable of achieving mass production and suitable for industrialization, and has remarkable practicability and economic benefits.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular relates to a collagen tripeptide and a preparation method and application thereof. Background Art

[0002] Collagen tripeptide (CTP) is the smallest active unit of collagen, consisting of three amino acids. Tripeptides rich in the glycine-proline-hydroxyproline (Gly-Pro-Hyp, GPH) sequence have been shown to exhibit significant biological activity, including promoting skin fibroblast proliferation, enhancing skin elasticity, and exhibiting antioxidant and anti-aging benefits. Fish skin, a major byproduct of aquatic product processing, is rich in type I collagen and is an ideal raw material for the production of collagen tripeptides. However, while traditional methods for producing collagen tripeptides include acid extraction, alkaline extraction, hot water extraction, enzymatic hydrolysis, or fermentation, they suffer from low GPH yields (typically <3.6%), imprecise process control, and low enzyme utilization. Therefore, there is an urgent need to develop new and efficient collagen tripeptide production technologies to increase GPH content. Summary of the Invention

[0003] The present invention aims to provide a collagen tripeptide and a preparation method thereof, and to increase the content of GPH in the collagen tripeptide.

[0004] The present invention provides a method for preparing collagen tripeptide, comprising the following steps: Depolymerizing the raw material containing collagen to obtain a depolymerized raw material; The composite enzyme is immobilized using magnetic Fe3O4 nanoparticles to obtain a magnetic immobilized enzyme; the composite enzyme includes proline endoprotease, alkaline protease and papain; The depolymerized raw material is mixed with a magnetic immobilized enzyme, and magnetic field-assisted enzymolysis is performed to obtain an enzymatic hydrolyzate; performing enzyme inactivation treatment and solid-liquid separation on the enzymatic hydrolysate in sequence, and collecting the supernatant; Ultrafiltration is performed on the supernatant, and the filtrate is collected to obtain collagen tripeptide; The particle size of the magnetic Fe3O4 nanoparticles is 50-300 nm; The mass ratio of the complex enzyme to the magnetic Fe3O4 nanoparticles is 1:(1-10); The temperature of the immobilization treatment is 20-50°C and the time is 1-6 hours; The mass ratio of the magnetic immobilized enzyme to the depolymerized raw material is (1-5):100; The magnetic field intensity of the magnetic field-assisted enzymolysis is 0.5-2.0 T, the enzymolysis temperature is 40-60° C., the enzymolysis pH is 6.0-9.0, and the enzymolysis time is 5-24 h.

[0005] Preferably, the particle size of the magnetic Fe3O4 nanoparticles is 80-200 nm; the temperature of the immobilization treatment is 25-40°C, and the time is 2-5 hours.

[0006] Preferably, the mass ratio of the complex enzyme to the magnetic Fe3O4 nanoparticles is 1:(2-8); the mass ratio of proline endoprotease, alkaline protease and papain in the complex enzyme is (1-3):(2-4):(2-4).

[0007] Preferably, the mass ratio of the magnetic immobilized enzyme to the depolymerized raw material is (1.5-3):100.

[0008] Preferably, the magnetic field strength of the magnetic field-assisted enzymatic hydrolysis is 1.0-1.5T.

[0009] Preferably, the enzymatic hydrolysis temperature of the magnetic field-assisted enzymatic hydrolysis is 50-55° C., the enzymatic hydrolysis pH is 6.5-8.5, and the enzymatic hydrolysis time is 8-20 h.

[0010] Preferably, the collagen content in the raw material containing collagen is ≥30wt.%; the raw material containing collagen includes fresh fish skin; The depolymerization treatment temperature is 80-100°C and the time is 30 minutes; The temperature of the enzyme inactivation treatment is 80-100°C and the time is 30 minutes; The enzyme inactivation treatment is performed at a temperature of 85-95°C for 10-20 minutes. The solid-liquid separation method includes centrifugation, the centrifugal speed is 6000~12000r / min, and the time is 10~30min; The molecular weight cut-off of the ultrafiltration was <10 kDa.

[0011] Preferably, after obtaining the collagen tripeptide, the collagen tripeptide is freeze-dried to obtain collagen tripeptide powder.

[0012] The present invention also provides a collagen tripeptide obtained by the preparation method described in the above technical solution, wherein the content of GPH tripeptide in the collagen tripeptide is ≥59wt.%.

[0013] The present invention also provides the use of the collagen tripeptide described in the above technical solution in the preparation of a product having one or more functions of increasing skin collagen content, enhancing skin elasticity, and soothing and reducing redness.

[0014] Beneficial effects: The present invention depolymerizes collagen-containing raw materials to construct a magnetic immobilized enzyme. The magnetic immobilized enzyme is then used to enzymatically hydrolyze the depolymerized raw materials under a specific temperature, pH, and reaction time. With the assistance of a magnetic field, the immobilized enzyme can be rapidly separated and recycled. Finally, enzyme inactivation, solid-liquid separation, and ultrafiltration are performed to obtain a collagen tripeptide with a GPH content of ≥59 wt.%. This collagen tripeptide has a higher GPH content than existing collagen tripeptides. Furthermore, the preparation method of the present invention is simple, easy to operate, low-cost, and mass-producible, making it suitable for industrialization and possessing significant practicality and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0016] Figure 1 This is a physical picture of the collagen tripeptide powder obtained in Example 1. DETAILED DESCRIPTION

[0017] The present invention provides a method for preparing collagen tripeptide, comprising the following steps: Depolymerizing the raw material containing collagen to obtain a depolymerized raw material; The composite enzyme is immobilized using magnetic Fe3O4 nanoparticles to obtain a magnetic immobilized enzyme; the composite enzyme includes proline endoprotease, alkaline protease and papain; The depolymerized raw material is mixed with a magnetic immobilized enzyme, and magnetic field-assisted enzymolysis is performed to obtain an enzymatic hydrolyzate; performing enzyme inactivation treatment and solid-liquid separation on the enzymatic hydrolysate in sequence, and collecting the supernatant; Ultrafiltration is performed on the supernatant, and the filtrate is collected to obtain collagen tripeptide; The particle size of the magnetic Fe3O4 nanoparticles is 50-300 nm; The mass ratio of the complex enzyme to the magnetic Fe3O4 nanoparticles is 1:(1-10); The temperature of the immobilization treatment is 20-50°C and the time is 1-6 hours; The mass ratio of the magnetic immobilized enzyme to the depolymerized raw material is (1-5):100; The magnetic field intensity of the magnetic field-assisted enzymolysis is 0.5-2.0 T, the enzymolysis temperature is 40-60° C., the enzymolysis pH is 6.0-9.0, and the enzymolysis time is 5-24 h.

[0018] The present invention performs depolymerization treatment on raw materials containing collagen to obtain depolymerized raw materials.

[0019] In one embodiment, the collagen content in the collagen-containing raw material of the present invention is ≥30 wt.%. In one embodiment, the collagen-containing raw material of the present invention comprises fresh fish skin. In one embodiment, the fish skin of the present invention comprises tilapia skin and / or cod skin.

[0020] In one embodiment, the temperature of the depolymerization treatment of the present invention is 80-100° C.; in another embodiment, the temperature of the depolymerization treatment of the present invention is 100° C. In one embodiment, the time of the depolymerization treatment of the present invention is 30 minutes.

[0021] The invention utilizes magnetic Fe3O4 nanoparticles to perform immobilization treatment on a composite enzyme to obtain a magnetic immobilized enzyme; the composite enzyme comprises proline endoprotease, alkaline protease and papain.

[0022] The magnetic Fe3O4 nanoparticles described in the present invention have a particle size of 50 to 300 nm. In one embodiment, the magnetic Fe3O4 nanoparticles described in the present invention have a particle size of 80 to 200 nm; in another embodiment, the magnetic Fe3O4 nanoparticles described in the present invention have a particle size of 100 nm. The particle size-to-surface area (total surface area per unit mass) of magnetic Fe3O4 nanoparticles is inversely proportional to their particle size. The smaller the particle size, the greater the total surface area for the same mass of nanoparticles. Due to their extremely small size, nanoscale carriers have a relatively small shielding effect on the spatial structure, conformation, and active sites of enzyme proteins. Compared to larger magnetic particles, the magnetic Fe3O4 nanoparticles defined in the present invention exhibit less loss of enzyme activity, better preserve their natural catalytic ability, and offer the advantages of high loading, high efficiency, easy recovery, and reusability.

[0023] The complex enzyme of the present invention comprises a proline endoprotease, an alkaline protease, and a papain. In one embodiment, the mass ratio of the proline endoprotease, alkaline protease, and papain is (1-3):(2-4):(2-4); in another embodiment, the mass ratio of the proline endoprotease, alkaline protease, and papain is 3:4:3. In one embodiment, the alkaline protease of the present invention is alkaline protease 37071. The present invention does not strictly require the source of the enzymes; commonly purchased enzymes can be used. The complex enzyme of the present invention, comprising the proline endoprotease, alkaline protease, and papain, exhibits a synergistic effect. The complex enzyme is capable of specifically recognizing and cleaving the Gly-XY (X: approximately 90% or more of the second position is proline (Pro) or hydroxyproline (Hyp); Y: the third position can be any amino acid, such as hydroxyproline, alanine, serine, or glutamic acid) repeating sequence in collagen.

[0024] The mass ratio of the complex enzyme to the magnetic Fe₃O₄ nanoparticles described herein is 1:(1-10). In one embodiment, the mass ratio of the complex enzyme to the magnetic Fe₃O₄ nanoparticles described herein is 1:(2-8); in another embodiment, the mass ratio of the complex enzyme to the magnetic Fe₃O₄ nanoparticles described herein is 1:(3-5). Excessive enzyme loading (excessive mass ratio of complex enzyme to magnetic Fe₃O₄ nanoparticles) can cause enzyme molecules to accumulate on the limited surface area of ​​the nanoparticles, shielding each other's active sites and ultimately reducing overall catalytic efficiency. An overly dense enzyme layer can hinder the access of large collagen peptide chains to the encapsulated enzyme molecules. By limiting the mass ratio of the complex enzyme to the magnetic Fe₃O₄ nanoparticles described herein, each enzyme is immobilized on the carrier at an optimal density, allowing for sufficient proximity to achieve synergistic action while preventing interference. This ensures maximum active site availability, improves the final yield of collagen tripeptides, and reduces the use of magnetic Fe₃O₄ nanoparticles, resulting in the most cost-effective single-use immobilized enzyme.

[0025] The temperature of the immobilization treatment of the present invention is 20-50°C. As an embodiment, the temperature of the immobilization treatment of the present invention is 25-40°C; as another embodiment, the temperature of the immobilization treatment of the present invention is 30°C. The time of the immobilization treatment of the present invention is 1-6 hours. As an embodiment, the time of the immobilization treatment of the present invention is 2-5 hours; as another embodiment, the time of the immobilization treatment of the present invention is 4 hours. As an embodiment, the immobilization treatment of the present invention is a shaking treatment. The present invention uses magnetic Fe3O4 nanoparticles to immobilize the complex enzyme, and the complex enzyme is fixed on the magnetic Fe3O4 nanoparticles, which can achieve rapid recovery and reuse of the complex enzyme, and still maintain more than 80% activity after repeated use 10 times; in addition, after the complex enzyme is immobilized using magnetic Fe3O4 nanoparticles, the reaction conditions are mild, which can avoid GPH degradation caused by excessive hydrolysis of the free enzyme and improve the GPH retention rate.

[0026] After obtaining the depolymerized raw material and the magnetic immobilized enzyme, the present invention mixes the depolymerized raw material and the magnetic immobilized enzyme, and performs magnetic field-assisted enzymolysis to obtain an enzymolysis solution.

[0027] The mass ratio of the magnetically immobilized enzyme to the depolymerized raw material described herein is (1-5):100. In one embodiment, the mass ratio of the magnetically immobilized enzyme to the depolymerized raw material described herein is (1.5-3):100; in another embodiment, the mass ratio of the magnetically immobilized enzyme to the depolymerized raw material described herein is 2:100. This specified mass ratio of the magnetically immobilized enzyme to the depolymerized raw material of the present invention has the advantage of achieving efficient hydrolysis in a single batch reaction while ensuring that the enzyme can be recovered magnetically after the reaction, allowing for reuse dozens or even hundreds of times.

[0028] The magnetic field strength of the magnetic field-assisted enzymatic hydrolysis of the present invention is 0.5 to 2.0 T. As an embodiment, the magnetic field strength of the magnetic field-assisted enzymatic hydrolysis of the present invention is 1.0 to 1.5 T. The present invention limits the magnetic field strength and utilizes the magnetic field to assist in timely separation of the product and enzyme, thereby reducing further hydrolysis of GPH.

[0029] The enzymatic hydrolysis temperature of the present invention is 40~60°C. As an embodiment, the enzymatic hydrolysis temperature of the present invention is 50~55°C. The enzymatic hydrolysis pH of the present invention is 6.0~9.0. As an embodiment, the pH of the present invention is 6.5~8.5; as another embodiment, the pH of the present invention is 7.3~8.0. The enzymatic hydrolysis time of the present invention is 5~24h. As an embodiment, the enzymatic hydrolysis time of the present invention is 8~20h; as another embodiment, the enzymatic hydrolysis time of the present invention is 10~15h. The enzymatic hydrolysis temperature, pH and time limited by the present invention have the advantages of ensuring the peak activity of the complex enzyme, improving the binding ability of the complex enzyme with the substrate, and thus generating more collagen tripeptides.

[0030] After obtaining the enzymatic hydrolysate, the present invention sequentially performs enzyme inactivation treatment and solid-liquid separation on the enzymatic hydrolysate, and collects the supernatant.

[0031] As one embodiment, the temperature of the enzyme inactivation treatment of the present invention is 85~95℃; as another embodiment, the temperature of the enzyme inactivation treatment of the present invention is 90℃. As one embodiment, the time of the enzyme inactivation treatment of the present invention is 10~20min; as another embodiment, the time of the enzyme inactivation treatment of the present invention is 20min. As one embodiment, the solid-liquid separation method of the present invention includes centrifugation. As one embodiment, the rotation speed of the centrifugation of the present invention is 6000~12000rpm; as another embodiment, the rotation speed of the centrifugation of the present invention is 8000r / min. As one embodiment, the time of the centrifugation of the present invention is 10~30min; as another embodiment, the time of the centrifugation of the present invention is 15min.

[0032] After obtaining the supernatant, the present invention performs ultrafiltration on the supernatant, collects the filtrate, and obtains collagen tripeptide.

[0033] In one embodiment, the molecular weight cutoff of the ultrafiltration of the present invention is <10 kDa; in another embodiment, the molecular weight cutoff of the ultrafiltration of the present invention is 3 kDa.

[0034] In one embodiment, the present invention freeze-dries the collagen tripeptide to obtain a collagen tripeptide powder. In one embodiment, the freeze-drying temperature is between -60°C and -30°C; in another embodiment, the freeze-drying temperature is between -50°C. In one embodiment, the freeze-drying time is between 24 and 36 hours; in another embodiment, the freeze-drying time is 48 hours.

[0035] The preparation method provided by the present invention features a simple process, easy operation, low cost, and mass production capability. The resulting collagen tripeptide has a GPH content of ≥59 wt.%, a higher GPH content than existing collagen tripeptides. Furthermore, the preparation method completely avoids the pollution associated with traditional alkaline collagen extraction (saving 50% of water resources and eliminating alkali liquid discharge), operates under mild reaction conditions (45-50°C), reduces energy consumption, and utilizes a non-toxic and biodegradable carrier material. The preparation method provided by the present invention is suitable for industrialization and offers significant practicality and economic benefits.

[0036] In view of the advantages of the present invention, the collagen tripeptide obtained by the preparation method of the present invention also falls within the scope of protection of the present invention. The content of GPH tripeptide in the collagen tripeptide of the present invention is ≥59 wt.%.

[0037] The present invention also provides the use of the collagen tripeptide described in the above technical solution in the preparation of a product having one or more of the following effects: increasing skin collagen content, enhancing skin elasticity, and soothing and reducing redness.

[0038] As an embodiment, the collagen of the present invention includes type I collagen and / or type III collagen.

[0039] To further illustrate the present invention, a collagen tripeptide provided by the present invention, its preparation method and application are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1 A method for preparing collagen tripeptide comprises the following steps: 1. Raw material pretreatment: Mix fresh tilapia skin or cod skin (collagen content ≥30%) and water in a mass-to-volume ratio of 1 g:5 mL, and heat at 100°C for 30 min to denature and depolymerize the collagen to obtain the enzymatic substrate.

[0041] 2. Construction of magnetic immobilized enzyme system (1) Carrier selection: carboxyl-modified superparamagnetic Fe3O4 nanoparticles with a particle size of 50 nm; (2) Preparation of compound enzyme: Proline endoprotease, alkaline protease 37071 and papain were mixed in a mass ratio of 3:4:3; (3) Immobilization conditions: The complex enzyme and the carrier were mixed in a pH 7.0 phosphate buffer at a mass ratio of 1:2, and shaken at 25°C and 100-200 rpm for 2 h to obtain the magnetic immobilized enzyme.

[0042] 3. The magnetic immobilized enzyme obtained in step 2 and the substrate obtained in step 1 were mixed in a mass ratio of 2:100 (2% addition amount), the pH was adjusted to 7.3, and enzymatic hydrolysis was carried out at 50°C for 10 h under a magnetic field strength of 0.1 T.

[0043] 4. After the enzymatic hydrolysis in step 3 is completed, the enzymatic hydrolyzate is sequentially subjected to enzyme inactivation treatment and centrifugation treatment, the supernatant is collected, and the unhydrolyzed residue is removed; wherein, the enzyme inactivation temperature is 90°C, the time is 20 minutes, and the centrifugal speed is 8000 r / min, and the time is 15 minutes; 5. The supernatant obtained in step 4 was ultrafiltered with a molecular weight cut-off of 3 kDa to obtain the target collagen tripeptide (molecular weight of about 300 Da), and freeze-dried at -50°C for 48 h to obtain collagen tripeptide powder ( Figure 1 ).

[0044] Examples 2 to 8 Collagen tripeptide was prepared according to the steps of Example 1. The differences between Examples 2 to 8 and Example 1 are as follows: the particle size of Fe3O4 nanoparticles in step 2 (A), the mass ratio of the complex enzyme to the carrier in step 2 (B), the shaking temperature in step 2 (C), the shaking time in step 2 (D), the enzymatic hydrolysis temperature in step 3 (E), the enzymatic hydrolysis pH in step 3 (F), the enzymatic hydrolysis time in step 3 (G), the mass ratio of the magnetic immobilized enzyme to the substrate in step 3 (H), and the magnetic field intensity in step 3 (I), which are specifically listed in Table 1.

[0045] Table 1 Relevant parameters during the preparation of Examples 1 to 8

[0046] Comparative Examples 1 to 6 Collagen tripeptide was prepared according to the steps of Example 1. The differences between Examples 2 to 8 and Example 1 are as follows: the particle size of Fe3O4 nanoparticles in step 2 (A), the mass ratio of the complex enzyme to the carrier in step 2 (B), the shaking temperature in step 2 (C), the shaking time in step 2 (D), the enzymatic hydrolysis temperature in step 3 (E), the enzymatic hydrolysis pH in step 3 (F), the enzymatic hydrolysis time in step 3 (G), the mass ratio of the magnetic immobilized enzyme to the substrate in step 3 (H), and the magnetic field intensity in step 3 (I), which are specifically listed in Table 2.

[0047] Table 2 Relevant parameters during the preparation of Example 1 and Comparative Examples 1 to 6

[0048] Test Example 1 GPH and total tripeptide content test The GPH standard is a short peptide composed of three amino acids with a fixed sequence of Gly-Pro-Hyp, with a purity of ≥98%. It can be purchased directly (Beijing Shengmeino, Sigma SML2420, Bachem 4016908.0001) and kept dry at -20°C and away from light for 24 months. The GPT standard is a short peptide composed of three amino acids with a fixed sequence of Gly-Pro-Thr, with a purity of ≥98%. It can be purchased directly (Beijing Shengmeino, Sigma SML2420, Bachem 4016908.0001) and kept dry at -20°C and away from light.

[0049] The amino acid sequence of the tripeptide is: small collagen formed by three amino acids linked (glycine-XY), which is also the basic sequence unit of collagen (X: more than 90% of the second position is proline (Pro) or hydroxyproline (Hyp); Y: the third position can be any amino acid such as hydroxyproline, alanine, serine, glutamic acid, etc.). The GPH standard and the GPT standard were mixed at a mass ratio of 4:1 to obtain a mixed standard. The collagen tripeptides prepared in Examples 1 to 8 and Comparative Examples 1 to 6 were used as test samples, and the GPH content and the total content of the tripeptides in the test samples were determined by HPLC-UV method, wherein the relevant parameter settings are shown in Table 3.

[0050] Table 3 GPH test method related parameters

[0051] Total tripeptide content (%) = (ΣA_3 peptide / A_total) × 100; where, • ΣA_3 peptide: the sum of the peak areas of all 3 peptides within the retention time of 8–14 min; • A_total: sum of the peak areas at 220 nm for the entire chromatogram; • Conversion factor: Corrected by the slope of the GPH / GPT mixed calibration curve.

[0052] The test results are shown in Tables 4 and 5. Table 4 GPH and total tripeptide content in collagen tripeptides of Examples 1 to 8 (%)

[0053] Note:* P <0.05,** P <0.01, the same below.

[0054] Table 5 GPH and total tripeptide contents in collagen tripeptides of Example 1 and Comparative Examples 1-6 (%)

[0055] Note:*** P <0.001, the same below.

[0056] According to Tables 4 and 5, it can be seen that the GPH content and the total tripeptide content in the collagen tripeptides obtained in Comparative Examples 1 to 6 are significantly lower than those in the collagen tripeptides obtained in Examples 1 to 8, respectively. This may be because the extreme environment leads to a lower GPH content and the appearance of more peptides in other forms. Compared with Examples 2 to 8, the GPH content and the total tripeptide content in the collagen tripeptide obtained in Example 1 are the highest, reaching 4.8% and 65%.

[0057] Test Example 2 Molecular weight distribution test Test samples: collagen tripeptides prepared in Examples 1 to 8 and Comparative Examples 1 to 6; Reference standard: GB 31645-2018 Appendix A; Test method: High performance liquid chromatography-size exclusion chromatography (HPLC-SEC); Chromatographic column: gel permeation chromatography column (TSK-Gel G2000SWXL, 7.8 × 300 mm); Mobile phase: 0.1 mol / L phosphate buffer (pH 6.8); Flow rate: 0.8 mL / min; Detection wavelength: 220 nm; Standards: Peptide standards with known molecular weight (such as cytochrome C, aprotinin, etc.); Molecular weight range: <3000 Da (collagen tripeptides are usually located in this range); Data processing: draw a standard curve based on the retention time of the standard and calculate the sample molecular weight; Test indicators: weight average molecular weight and molecular weight distribution range; The test results are shown in Tables 6 and 7.

[0058] Table 6 Molecular weight distribution test results of collagen tripeptides of Examples 1 to 8

[0059] Table 7 Molecular weight distribution test results of collagen tripeptides in Example 1 and Comparative Examples 1 to 6

[0060] According to Tables 6 and 7, it can be seen that the total average molecular weight of the collagen tripeptides obtained in Comparative Examples 1 to 6 is significantly higher than that of the collagen tripeptides obtained in Examples 1 to 8. The total average molecular weight of Example 1 is 479 Da, which is between the collagen tripeptides (200 to 500 Da). According to the relative molecular weight distribution, most of about 65.96% is distributed between 189 and 500 Da, which belongs to the molecular weight range of the tripeptide.

[0061] Test Example 3 Fibroblast qPCR test: ColI , ColIII and ELAN Gene 1. Principle: Skin fibroblasts are the primary cell type in the dermis and are responsible for synthesizing and maintaining the extracellular matrix (ECM), including important components such as collagen and elastin. The ColI (collagen type I), ColIII (collagen type III), and ELAN (elastin) genes play key roles in the structure and function of the skin. The primer sequences for genetic testing are shown in Table 8.

[0062] Table 8 Primer sequences of each gene

[0063] 2. Experimental steps: (1) Samples to be tested: collagen tripeptides prepared according to Examples 1 to 8 and Comparative Examples 1 to 6; (2) Cell treatment and RNA extraction Cell culture: HSF (human skin fibroblasts, mainly derived from the dermis of the skin) were seeded in 6-well plates (density 5×10 5 / well), and after the degree of confluence reaches 80%, proceed as follows: Experimental group: Cultured in DMEM medium containing 0.1wt.% of the test sample for 24 hours; 0.1wt.% is the maximum safe concentration for HSF fibroblasts, with a fibroblast survival rate of >90%; Positive control: cultured in DMEM medium containing 10 ng / mL TGF-β1 (to promote collagen synthesis) for 24 h; Negative control: cultured in DMEM medium for 24 h After the incubation period, the culture medium was discarded and the cells were washed twice with PBS. 1 mL of TRIzol was added to each well to lyse the cells and transfer them to EP tubes. Total RNA was extracted using the chloroform method, washed with 75% ethanol, and dissolved in DEPC-free water. RNA purity was determined by Nanodrop assay (A260 / A280 ratio should be 1.8-2.0).

[0064] (3) cDNA synthesis Perform reverse transcription using a reverse transcription kit (e.g., Takara PrimeScript RT Master Mix). The reverse transcription system is as follows: 1 μg RNA + 4 μL 5× Master Mix + RNase-free water to 20 μL. The reverse transcription procedure is: 37°C for 15 min → 85°C for 5 sec (to inactivate the reverse transcriptase).

[0065] (4) qPCR reaction The cDNA obtained in step (3) was used as a template and PCR amplification was performed using the primers in Table 8. The reaction system for PCR amplification (20 μL) consisted of 10 μL of SYBR Green Mix, 0.4 μL of each forward and reverse primer (final concentration 0.2 μM), 2 μL of cDNA template, and 7.2 μL of RNase-free water. The PCR amplification procedure was as follows: pre-denaturation at 95°C for 30 seconds; denaturation at 95°C for 5 seconds, annealing at 60°C for 30 seconds, for 40 cycles; and heating from 65°C to 95°C, with a temperature increase of 0.5°C every 5 seconds.

[0066] (5) Data analysis Ct value acquisition: Automatic analysis by instrument software (such as Bio-Rad CFX Manager).

[0067] Relative gene expression was calculated using the relative quantification (2-ΔΔCt) method. The results are shown in Tables 9 and 10. ΔCt = Ct (target gene) - Ct (internal reference GAPDH); ΔΔCt = ΔCt (experimental group) - ΔCt (negative control group). Fold change in gene expression = 2-ΔΔCt.

[0068] Table 9 Effects of Examples 1 to 8 Collagen Tripeptides on Gene Expression

[0069] Table 10 Effects of collagen tripeptides in Example 1 and Comparative Examples 1-6 on gene expression

[0070] According to Table 9 and Table 10, it can be seen that compared with the collagen tripeptides obtained in Comparative Examples 1 to 6, the collagen tripeptides obtained in Examples 1 to 8 Col1A1 、 Col3A1 and ELAN The gene expression levels were significantly increased in all groups, with the collagen tripeptide obtained in Example 1 showing a more significant increase, increasing by 148%, 201%, and 87%, respectively, demonstrating a significant effect in enhancing gene expression. This indicates that the efficacy of collagen tripeptide is related to the GPH and tripeptide content; the higher the content, the better the effect.

[0071] Test Example 4 Soothing and redness-removing effect test 1. Test model: methyl nicotinate irritated skin model; 2. Test Material: Methyl nicotinate is an ester derivative of niacin. It activates prostaglandin pathways and releases histamine, causing dilation of skin capillaries and the formation of reversible erythema (redness). Features include: Rapid onset of action: Visible erythema is visible 5-15 minutes after application. Repeatability: After the erythema subsides (approximately 1-2 hours), thermal stimulation (warm water / hot air) can be reapplied to observe the degree of "redness" and assess skin irritability. Controllability: The intensity of the erythema can be controlled by adjusting the concentration (0.1%-1%).

[0072] 3. Test methods and procedures: (1) Subject screening Inclusion criteria: Healthy adults (18-60 years old) with no history of skin disease. No lesions, dermatitis, or allergies at the test site (forearm / face).

[0073] Exclusion criteria: Pregnant or breastfeeding women, those who have recently used antihistamines, NSAIDs (such as aspirin), or glucocorticoids.

[0074] (2) Model building steps Skin pretreatment: Clean the test area (inner forearm) and let it sit at room temperature (20-25°C) for 30 minutes.

[0075] Methyl nicotinate stimulation: Use 0.5%-1% methyl nicotinate solution (dissolved in ethanol / propylene glycol) and apply evenly to a circular area with a diameter of 2 cm.

[0076] Observation and recording of erythema: Time points: 5, 15, 30, and 60 minutes.

[0077] Assessment method: Visual scoring (0-4 points): 0 = no erythema; 1 = mild redness; 2 = moderate redness; 3 = marked redness; 4 = severe redness with edema.

[0078] Instrument detection: Chromameter: measure a* value and erythema value.

[0079] (3) Relief test Test samples: collagen tripeptides prepared in Examples 1 to 8 and Comparative Examples 1 to 6; Intervention plan: add the test sample (from the example and comparative example) to the base emulsion to a concentration of 0.5 wt.%, to obtain the corresponding emulsion, and immediately apply the obtained emulsion after erythema is induced by methyl nicotinate; Control group: after methyl nicotinate induced erythema, a basic lotion without the test sample was immediately applied; The preparation method of the base emulsion is as follows: 7.0% glycerin, 3.0% butylene glycol, 5.0% 1% sodium hyaluronate aqueous solution, 0.5% panthenol, 0.1% disodium EDTA and the balance of phase A are mixed to obtain phase B; 0.8% of a mixture of phenoxyethanol and caprylyl glycol (the mass ratio of phenoxyethanol to caprylyl glycol is 1:1) and 0.2% essence are added to phase B, and the pH is adjusted to 5.5-6.0 using a 10% citric acid aqueous solution; wherein the composition of phase A is: 10.0% caprylic / capric triglyceride, 2.5% cetearyl olivate / sorbitan olivate, 1.5% cetearyl alcohol, 0.3% vitamin E (tocopherol) and the balance deionized water.

[0080] Evaluation indicators: a. Erythema fading time: observe the fading of redness after 10 minutes, 30 minutes and 1 hour; b. Test indicators: a* value and erythema value. The results are shown in Tables 11 and 12.

[0081] Table 11 Soothing and redness-removing effects of collagen tripeptides in Examples 1 to 8

[0082] Table 12 Soothing and redness-removing effects of collagen tripeptides in Example 1 and Comparative Examples 1 to 6

[0083] According to Tables 11 and 12, compared with the collagen tripeptides obtained in Comparative Examples 1 to 6, at 5 minutes, the erythema value of Example 1 was reduced by 5.08%, and the a value was reduced by 7.03%. At 30 minutes, the erythema value of the control group was reduced by 5.15%, and the a value was reduced by 15.27%, while that of Example 1 was reduced by 16.36%, and the a value was reduced by 23.46%. At 1 hour, the erythema value and a value of the control group were reduced by 14.96% and 27.23%, respectively, while those of Example 1 were reduced by 29.02% and 34.51%, respectively, indicating that the sample has the effect of soothing and fading redness. And this effect is related to the content of GPH and tripeptide, and the higher the content, the better the effect.

[0084] It can be seen from the above content that the technical solution provided by the present invention can increase the GPH content in collagen tripeptide.

[0085] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for preparing collagen tripeptide, characterized in that: The steps include: Depolymerizing the raw material containing collagen to obtain a depolymerized raw material; The composite enzyme is immobilized using magnetic Fe3O4 nanoparticles to obtain a magnetic immobilized enzyme; the composite enzyme includes proline endoprotease, alkaline protease and papain; The depolymerized raw material is mixed with a magnetic immobilized enzyme, and magnetic field-assisted enzymolysis is performed to obtain an enzymatic hydrolyzate; performing enzyme inactivation treatment and solid-liquid separation on the enzymatic hydrolysate in sequence, and collecting the supernatant; Ultrafiltration is performed on the supernatant, and the filtrate is collected to obtain collagen tripeptide; The particle size of the magnetic Fe3O4 nanoparticles is 50-300 nm; The mass ratio of the complex enzyme to the magnetic Fe3O4 nanoparticles is 1:(1-10); The temperature of the immobilization treatment is 20-50°C and the time is 1-6 hours; The mass ratio of the magnetic immobilized enzyme to the depolymerized raw material is (1-5):100; The magnetic field intensity of the magnetic field-assisted enzymolysis is 0.5-2.0 T, the enzymolysis temperature is 40-60° C., the enzymolysis pH is 6.0-9.0, and the enzymolysis time is 5-24 h.

2. The preparation method according to claim 1, characterized in that The particle size of the magnetic Fe3O4 nanoparticles is 80-200 nm; the temperature of the immobilization treatment is 25-40° C., and the time is 2-5 hours.

3. The preparation method according to claim 1, characterized in that The mass ratio of the complex enzyme to the magnetic Fe3O4 nanoparticles is 1:(2-8); the mass ratio of proline endoprotease, alkaline protease and papain in the complex enzyme is (1-3):(2-4):(2-4).

4. The preparation method according to claim 1, characterized in that The mass ratio of the magnetic immobilized enzyme to the depolymerized raw material is (1.5-3):

100.

5. The preparation method according to claim 1, characterized in that The magnetic field intensity of the magnetic field-assisted enzymatic hydrolysis is 1.0-1.5T.

6. The preparation method according to claim 1, characterized in that The magnetic field-assisted enzymatic hydrolysis has an enzymatic hydrolysis temperature of 50-55° C., an enzymatic hydrolysis pH of 6.5-8.5, and an enzymatic hydrolysis time of 8-20 h.

7. The preparation method according to claim 1, characterized in that: The collagen content in the raw material containing collagen is ≥30wt.%; the raw material containing collagen includes fresh fish skin; The depolymerization treatment temperature is 80-100°C and the time is 30 minutes; The enzyme inactivation treatment is performed at a temperature of 85-95°C for 10-20 minutes. The solid-liquid separation method includes centrifugation, the centrifugal speed is 6000~12000r / min, and the time is 10~30min; The molecular weight cut-off of the ultrafiltration was <10 kDa.

8. The preparation method according to any one of claims 1 to 7, characterized in that After obtaining the collagen tripeptide, the collagen tripeptide is freeze-dried to obtain collagen tripeptide powder.

9. The collagen tripeptide obtained by the preparation method according to any one of claims 1 to 8, characterized in that: The content of GPH tripeptide in the collagen tripeptide is ≥59 wt.%.

10. Use of the collagen tripeptide according to claim 9 in the preparation of a product having one or more functions of increasing skin collagen content, enhancing skin elasticity, and soothing and reducing redness.

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