Use of a recombinant collagen type I-antioxidant peptide composition having antioxidant function

Abstract

The application discloses application of a recombinant collagen type I-antioxidative peptide composition with an antioxidative function, and belongs to the technical field of biotechnology.The application provides a recombinant collagen type I-antioxidative peptide composition prepared by compounding a recombinant collagen type I and a schisandra antioxidant peptide.The composition has high free radical scavenging activity, and has stability and biological safety;and the antioxidative effect is better than that of single recombinant collagen type I or the schisandra antioxidant peptide.The amino acid sequence of the schisandra antioxidant peptide with antioxidative activity is FYPF, FLPW, FYLPF, ANWLPF, IFSPWL, FPFTYAMMLM, GPSFPIFI or GISWGFL.The application provides a scientific basis and technical support for development of antioxidative products in the fields of medical dressings, functional foods, skin care cosmetics and the like.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of a recombinant type I collagen-antioxidant peptide composition with antioxidant function. Background Technology

[0002] The balance between oxidation and antioxidation is crucial for human health. Under environmental stress and metabolic disorders, the body is prone to excessive accumulation of reactive oxygen species, leading to oxidative problems such as lipid peroxidation and DNA damage, accelerating cell aging, which is particularly prominent in skin tissue. Long-term air pollution can induce sebum peroxidation in the skin, damaging the barrier function and moisturizing ability, resulting in sensitivity and inflammation. UVB radiation and internal imbalances can exacerbate free radical disorder, causing aging problems such as fine lines and pigmentation. Therefore, effectively scavenging free radicals, restoring the oxidation-antioxidant balance, and repairing oxidative damage to the skin have become core needs in the food, cosmetics, and pharmaceutical industries.

[0003] Current antioxidants all have limitations. Synthetic antioxidants have potential toxicity, limiting their application; natural small-molecule antioxidants have good biocompatibility, but poor water solubility and easy metabolism make it difficult to reach deep target tissues; natural peptide antioxidants, while possessing advantages such as high biocompatibility and easy absorption, generally suffer from poor stability, insufficient safety verification, and unclear mechanisms of action, hindering industrialization. Schisandra chinensis-derived peptides possess both antioxidant activity and biocompatibility, showing significant advantages among natural antioxidant peptides. Type I collagen, as a core component of the skin, can maintain structural integrity, enhance elasticity, and assist in antioxidant activity, but recombinant expression faces bottlenecks such as triple-helix instability and low expression efficiency. Currently, there are no reports on the combined use of Schisandra chinensis-derived peptides and type I collagen. Therefore, establishing a stable combination of Schisandra chinensis antioxidant peptides and recombinant type I collagen can simultaneously solve the problems of low efficiency of single antioxidants and limited collagen function, providing a new direction for the development of functional foods, skin repair cosmetics, and medical antioxidants. Summary of the Invention

[0004] This invention relates to the application of a recombinant type I collagen-antioxidant peptide composition with antioxidant function. Addressing the problems of poor stability and unclear mechanisms of action of existing natural antioxidant peptides, low expression efficiency and poor stability of recombinant collagen, and the lack of integrated "antioxidant-repair" compositions for the skin, this invention provides novel Schisandra chinensis antioxidant peptides, optimized expression of recombinant type I collagen, and the construction of a synergistic composition between the two. Through analysis and verification using cell models, peptidomics, and molecular docking techniques, this invention provides a safe and efficient technical solution for the development of antioxidant functional foods, skin repair cosmetics, and other products.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a recombinant type I collagen-antioxidant peptide composition, comprising recombinant type I collagen and Schisandra chinensis antioxidant peptides. The recombinant type I collagen comprises peptide chains as shown in SEQ ID NO.2 and SEQ ID NO.4, and the amino acid sequence of the Schisandra chinensis antioxidant peptides comprises FYPF, FLPW, FYLPF, ANWLPF, IFSPWL, FPFTYAMMLM, GPSFPIFI, or GISWGFL.

[0007] In the above scheme, the recombinant type I collagen is obtained by the following method: constructing a recombinant engineered bacterium expressing the coding regions of proline hydroxylase, human COL1A1, and COL1A2 mature peptides;

[0008] The genetically engineered bacteria were cultured, and the resulting culture medium was used to separate and disrupt the bacterial cells using ultrasound. The supernatant was obtained by centrifugation, and the supernatant was subjected to ultrafiltration and salting out, followed by ion exchange chromatography to obtain the recombinant type I human collagen.

[0009] Furthermore, the nucleotide sequences encoding the proline hydroxylase, human COL1A1, and COL1A2 mature peptide regions are shown in SEQ ID NO.5, SEQ ID NO.1, and SEQ ID NO.3, respectively. Ultrafiltration was performed using a 30 kb ultrafiltration tube; salting out was performed using a gradient elution with 20%-60% ammonium sulfate; and ion exchange chromatography was performed using a gradient elution with a buffer containing 0.5-0.8 mol / L NaCl.

[0010] Preferably, the amino acid sequence of the Schisandra chinensis antioxidant peptide is ANWLPF.

[0011] Preferably, the mass ratio of the recombinant type I collagen to the Schisandra chinensis antioxidant peptide is 1:(1-5).

[0012] Preferably, the mass ratio of the recombinant type I collagen to the Schisandra chinensis antioxidant peptide is 1:2.

[0013] The present invention also provides a Schisandra chinensis antioxidant peptide with antioxidant activity, wherein the amino acid sequence of the Schisandra chinensis antioxidant peptide is FYPF, FLPW, FYLPF, ANWLPF, IFSPWL, FPFTYAMMLM, GPSFPIFI, or GISWGFL.

[0014] The present invention also provides the application of the recombinant type I collagen-antioxidant peptide composition in the preparation of antioxidant products.

[0015] The present invention also provides the use of the recombinant type I collagen-antioxidant peptide composition in the preparation of products that alleviate UVB-induced cellular oxidative damage.

[0016] The present invention also provides the application of the aforementioned Schisandra chinensis antioxidant peptide in the preparation of antioxidant products.

[0017] The present invention also provides an antioxidant product comprising the recombinant type I collagen-antioxidant peptide composition or the Schisandra chinensis antioxidant peptide.

[0018] The aforementioned antioxidant products include, but are not limited to, antioxidant products used in medical dressings, functional foods, skin repair cosmetics, and other fields.

[0019] The present invention discloses the following beneficial effects:

[0020] This invention confirms that Schisandra chinensis peptides exhibit scavenging activity exceeding 80% against various free radicals and demonstrate excellent stability and biosafety. Specifically, they exhibit DPPH scavenging rates higher than in normal environments, even in extreme conditions such as sodium chloride concentrations of 2-8 mg / mL, temperatures ranging from 40-100°C, acidic environments with pH 3 or 5, and in the presence of pancreatic and gastric juices. They retain their original antioxidant activity even after 7 days at room temperature, and show no cytotoxicity or hemolytic activity. Furthermore, the peptides demonstrate strong antioxidant capacity against UVB-induced HaCaT cells, with SOD and CAT activities recovering to 48.70% and 68.96% of the control group, respectively, while MDA levels showed no significant difference compared to the control group. Further screening yielded eight novel Schisandra chinensis antioxidant peptide sequences with excellent antioxidant activity. Simultaneously, this invention provides an expression and purification process for recombinant type I collagen, achieving successful expression of the target protein and increasing the protein content by more than five times after purification. Based on the above results, this invention further prepares a compound composition of recombinant type I collagen and novel Schisandra chinensis polypeptide. This compound composition exhibits superior antioxidant activity compared to the single components while ensuring good biocompatibility. This invention provides a scientific basis and technical support for the development of antioxidant products in the fields of medical dressings, functional foods, and skin repair cosmetics. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The scavenging rates of Schisandra chinensis antioxidant peptide (SCP) against DPPH (A), ABTS (B), hydroxyl radicals (C) and superoxide anions (D) are given.

[0023] Figure 2A is the molecular weight distribution of SCP; B is the standard curve between elution time and the logarithm of molecular weight (lg(M)); C is the chromatogram of SCP; D is the molecular weight distribution of SCP.

[0024] Figure 3 The following parameters represent the stability and biocompatibility of SCP: A) Effect of different salt ion concentrations on SCP DPPH clearance rate; B) Effect of different pH values ​​on SCP DPPH clearance rate; C) Effect of different digestive enzymes on SCP DPPH clearance rate; D) Effect of different temperatures on SCP DPPH clearance rate; E) Effect of storage time on SCP DPPH clearance rate; F) Cytotoxicity of SCP against HaCaT; G) Hemolytic activity of SCP against mouse erythrocytes; Different letters indicate significant differences between groups (P<0.05); "*" indicates P<0.05.

[0025] Figure 4 SCP was used to alleviate UVB-induced oxidative damage in HaCaT cells; A and D represent the effects of different concentrations of SCP on UVB-induced cell viability (A), SOD activity (B), CAT activity (C), and MDA content (D) of HaCaT cells, respectively; different letters indicate significant differences between groups (P<0.05).

[0026] Figure 5 Peptide sequence identification and screening for SCP; A represents the statistics of active peptides in SCP; B and E represent the distribution ratio, length distribution, molecular weight distribution, and number distribution of hydrophobic amino acids of peptide PeptideRanker activity score, respectively.

[0027] Figure 6 The structural features and structure-activity relationships of the best antioxidant peptides in SCP (FYPF, FLPW, FYLPF, ANWLPF, IFSPWL, FPFTYAMMLM, GPSFPIFI, GISWGFL) are analyzed.

[0028] Figure 7 The results of the screening for the best antioxidant peptide sequences; the DPPH free radical scavenging activities of FYPF, FLPW, FYLPF, ANWLPF, IFSPWL, and FPFTYAMMLM, with different letters indicating significant differences between groups (P<0.05).

[0029] Figure 8 PCR positive clone colony identification results; M: DL5000 DNA Marker; 1~20: PCR results of different clones; -: negative control (sterile water); +: positive control;

[0030] Figure 9The SDS-PAGE expression results of recombinant type I collagen are shown; the two lanes from left to right represent standard protein molecules and recombinant type I collagen, respectively.

[0031] Figure 10 The results of Western Blot expression of recombinant type I collagen are shown; the three lanes from left to right represent the standard protein molecule, the expression level of unpurified (cell lysed only) protein, and the purified protein, respectively.

[0032] Figure 11 The effects of different compositions on UVB-induced oxidative damage in HaCaT cells are shown in Figure A. The effect of different compositions on the viability of UVB-induced HaCaT cells relative to the control group is shown in Figure B. The effect of different compositions on the SOD activity of UVB-induced HaCaT cells relative to the control group is shown in Figure B. * P<0.05 ** P<0.01 *** P<0.001: Statistically different from the control group; # P < 0.05 ## P < 0.01 ### P < 0.001: Statistically different compared to the UVB damage group;

[0033] Figure 12 A represents the antioxidant capacity of ANWLPF, recombinant type I collagen, and composition 3; B represents the DPPH free radical scavenging rate of different components; C represents the effect of different components on UVB-induced SOD activity in HaCaT cells relative to the control group; D represents the effect of different components on UVB-induced CAT activity in HaCaT cells relative to the control group; and D represents the effect of different components on UVB-induced MDA content in HaCaT cells relative to the control group. Different letters indicate significant differences between groups (P<0.05). * P<0.05 ** P<0.01 *** P<0.001: Statistically different from the control group; # P<0.05 ## P<0.01 ### P<0.001: Statistically different compared to the UVB damage group;

[0034] Figure 13 The results are the hemolytic activity assay of mouse erythrocytes of composition 3. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] 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.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] Example 1

[0041] I. Obtaining the Novel Schisandra Antioxidant Peptide Sequence

[0042] 1. Identification of the antioxidant activity of Schisandra chinensis polypeptides

[0043] Schisandra chinensis polypeptide (SCP) was purchased from Shandong Dashu Co., Ltd., and solutions of different concentrations were prepared. The scavenging rates of four different free radicals—DPPH, ABTS, hydroxyl radical, and superoxide anion—were determined according to the kit instructions, with glutathione (GSH) as a positive control.

[0044] The results are as follows Figure 1 As shown, SCP exhibits dose-dependent scavenging activity against DPPH, ABTS, and hydroxyl radicals, while its activity against superoxide anion radicals is relatively weak. Notably, at a concentration of 8 mg / mL, SCP's DPPH scavenging activity is comparable to that of GSH (P>0.05). Furthermore, the half-maximal inhibitory concentration (IC50) of SCP against DPPH and hydroxyl radicals is also shown.50 The concentrations were only 3.89 mg / mL and 1.27 mg / mL, respectively. Overall, SCP exhibited strong antioxidant activity.

[0045] 2. Determination of the molecular weight of Schisandra chinensis polypeptides

[0046] Separation of SCP was performed using a high-performance liquid chromatography (HPLC) system (Waters, USA, MA) via gel filtration column. The mobile phase was acetonitrile-water-trifluoroacetic acid (40:60:0.1 v / v), with a flow rate of 0.5 mL / min. Detection was performed at 220 nm UV. A standard curve was established based on the correlation between retention time and the logarithm of relative molecular weight (lg(M)) to determine the molecular weight distribution of SCP. The standard curve confirmed a linear relationship between these two parameters. Figure 2 (A). Further chromatographic analysis showed that SCP had multiple elution peaks, indicating that it is composed of components with different molecular weights (A). Figure 2 (B) Specifically, 97.49% of the peptides in SCP have a molecular weight less than 1 kDa, while peptides with a molecular weight greater than 5 kDa account for only 0.43% ( Figure 2 The presence of C indicates that SCP is primarily composed of low-molecular-weight peptides with high antioxidant activity.

[0047] 3. Biosafety and stability of Schisandra chinensis polypeptides

[0048] 3.1 Effect of sodium chloride on the antioxidant properties of Schisandra chinensis peptides

[0049] A 10 mg / mL SCP solution was prepared using different concentrations of NaCl solution (0, 2, 4, 6, 8, and 10 mg / mL) as solvents. The DPPH scavenging rate was measured after the solution was allowed to stand at room temperature for 1 h. The results showed that the DPPH scavenging rate of SCP increased to over 73.63% with increasing sodium chloride concentration. Figure 3 (A)

[0050] 3.2 Effects of acidity and alkalinity on the antioxidant properties of Schisandra chinensis peptides

[0051] A 10 mg / mL SCP solution was divided into six equal portions, and the pH values ​​were adjusted to 3, 5, 7, 9, and 11, respectively. After shaking at room temperature for 2 hours, the pH of each group was adjusted back to 7, and the DPPH scavenging rate was measured. The results showed that the DPPH scavenging rate of SCP was 10.64% higher in an acidic environment (pH=3) than in a neutral environment (pH=7). Figure 3 (B)

[0052] 3.3 Effects of simulated pancreatic and gastric juices on the antioxidant properties of Schisandra chinensis peptides

[0053] Simulated gastric juice was prepared by dissolving pepsin in hydrochloric acid solution at pH 2.0 to a final activity of 2000 U / mL, and simulated pancreatic juice was prepared by dissolving trypsin in phosphate buffer solution at pH 7.0 to a final activity of 100 U / mL. Both simulated solutions were refrigerated at 4°C and sterilized by filtration through a 0.22 μm filter before use, and were prepared fresh each time. SCP was prepared into 10 mg / mL solutions using both simulated solutions as solvents and incubated at 37°C and 100 rpm for 2 h. After incubation, the enzyme was inactivated by heating in a 95°C water bath for 10 min, and the DPPH clearance rate was measured. The results showed that the antioxidant capacity of the simulated gastric juice and simulated pancreatic juice digestion groups was higher than that of the undigested group. Figure 3 (C)

[0054] 3.4 Effect of temperature on the antioxidant properties of Schisandra chinensis peptides

[0055] A 10 mg / mL SCP solution was incubated at different temperatures (25, 40, 60, 80, 100 °C) for 2 h, then rapidly cooled to room temperature on ice, and the DPPH scavenging rate was measured. The results showed that the DPPH scavenging rate of SCP increased continuously with increasing temperature. Figure 3 (D).

[0056] 3.5 Effect of storage time on the antioxidant properties of Schisandra chinensis peptides

[0057] SCP was prepared into a 10 mg / mL solution using distilled water and stored at room temperature. Samples were taken and measured on days 0, 1, 3, 5, and 7. After 7 days of storage, the DPPH clearance rate of Schisandra chinensis polypeptide showed no significant difference compared to the initial value (P > 0.05). Figure 3 (E).

[0058] 3.6 Cytotoxicity Detection

[0059] HaCaT cells were stored at a density of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 96-well plates and cultured for 24 h in DMEM medium containing 10% fetal bovine serum. Subsequently, they were cultured for another 24 h in DMEM medium containing different concentrations (50, 75, 100, 125, 250, and 500 μg / mL) of SCP. Finally, cell viability was determined using the CCK-8 assay. The results confirmed that Schisandra chinensis polypeptide not only had no cytotoxicity but also promoted cell proliferation. Figure 3 (Middle F).

[0060] 3.7 Hemolytic performance test

[0061] Fresh mouse red blood cells were washed three times with PBS, centrifuged at 1000 rpm for 5 min at 4°C, and the supernatant was discarded. The precipitate was diluted with PBS to obtain a 1% red blood cell dilution. SCP solutions at concentrations of 1, 2, 4, and 8 mg / mL were mixed with an equal volume of red blood cell dilution and incubated at 37°C and 200 rpm for 1 h. After incubation, the mixture was centrifuged, and the supernatant was measured at 570 nm to calculate the hemolysis rate. 0.1% Triton X-100 (PC group) and PBS-treated group (NC group) were used as positive and negative controls, respectively.

[0062] The formula for calculating the hemolysis rate is: Hemolysis rate (%) = (A-A0) / (A1-A0) × 100.

[0063] In the formula, A, A0, and A1 represent the OD values ​​of the experimental group, negative control, and positive control group, respectively.

[0064] The results showed that the hemolytic rate of Schisandra chinensis peptides was much lower than that of 0.1% Triton X-100 buffer, and its hemolytic activity remained below 0.8% within a concentration range of 1-8 mg / mL, which confirms that SCP has good biocompatibility. Figure 3 (G).

[0065] 4. Schisandra chinensis polypeptide alleviates UVB-induced oxidative damage in HaCaT cells.

[0066] 4.1 Effects of different concentrations of Schisandra chinensis polypeptide on UVB-induced HaCaT cell viability

[0067] HaCaT cells were stored at a density of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 mJ / cm² in 96-well plates and cultured for 24 h. Then, 40 mJ / cm² was used as a buffer. 2 Cells were irradiated with UVB. The culture medium was then changed to contain different concentrations of SCP (0, 50, 75, 100, and 125 μg / mL), and the cells were incubated for another 24 h. Finally, the cell viability of HaCaT cells treated with different methods was calculated using the CCK-8 assay. The results showed that at concentrations of 75 μg / mL and 125 μg / mL, SCP increased the average cell viability from 55.59% to 114.67% and 124.46%, respectively, even higher than the control group (…). Figure 4 (A)

[0068] 4.2 Effects of different concentrations of Schisandra chinensis polypeptide on UVB-induced SOD, CAT, and MDA activities in HaCaT cells

[0069] HaCaT cells were stored at 3 × 10⁶ cells per well. 5Cells were seeded at a density in 6-well plates and cultured for 24 h. After UVB treatment as described in 4.1, cells were cultured for another 24 h in medium containing SCP (50, 75, and 100 μg / mL). The levels of SOD, CAT, and MDA in the cells were determined using a quantitative assay kit. The results showed that SOD and CAT activities recovered in a dose-dependent manner after SCP treatment, reaching 48.70% and 68.96% of the control group, respectively, at a concentration of 100 μg / mL. The level of MDA, a marker of lipid peroxidation, also decreased in a dose-dependent manner after SCP treatment, with no significant difference compared to the control group at a concentration of 100 μg / mL (P>0.05). Figure 4 (BD).

[0070] 5. Identification and screening of peptide sequences

[0071] 5.1 Identification and Screening Procedure for Peptide Sequences

[0072] The sequence composition of SCP was identified using LC-MS / MS. The experimental instrument was a VANQUISH NEO nano-liquid chromatography system (Thermo Scientific, USA, MA), with the column temperature precisely controlled at 55°C and the flow rate set at 0.3 μL / min. During the detection process, mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an 80% acetonitrile solution containing 0.1% formic acid. After the experiment, the LC-MS / MS results of SCP were analyzed using Proteome Discoverer software. Simultaneously, a FASTA database containing Schisandra chinensis protein sequences was constructed. Through database searching, all peptide sequence information of SCP was finally obtained. A total of 8451 peptides were identified in Schisandra chinensis, including 2650 potentially active peptides and 5801 inactive peptides. Figure 5 (A). Of these peptides, 250 peptides had a PeptideRanker score higher than 0.95, accounting for 2.96% of the total peptides. Further analysis of these 250 peptides showed that most peptides contained 2-6 amino acid residues, while peptides containing more than 7 amino acid residues accounted for only 4%. Figure 5 (Middle BC). Furthermore, peptides with a molecular weight less than 1000 Daltons account for 96.8%, exhibiting characteristics of highly antioxidant peptides. Additionally, the peptides contain over 600 hydrophobic amino acids, including F (phenylalanine), L (leucine), I (isoleucine), and A (alanine). Figure 5 middle DE).

[0073] 5.2 Screening for bioactive peptides

[0074] Antioxidant peptide sequences in SCP were screened using the PeptideRanker database (http: / / distilldeep.ucd.ie / PeptideRanker / ) and the Bio-UWM database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php). In short, PeptideRanker was used to predict the probability that the identified peptide sequences were bioactive peptides, with a screening threshold set to 0.9. Bio-UWM was then used to predict the bioactivity of the bioactive peptides, confirming 63 peptides with potential antioxidant activity.

[0075] 5.3 Screening for the best antioxidant peptides

[0076] Structural data for DPPH (CID: 2735032) and ABTS (CID: 5360881) were obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). Peptides were modeled using an online platform (https: / / cloud.yinfotek.com). Molecular docking was performed using Aurodock vina with DPPH and ABTS as ligands and the peptides as acceptors, and the binding energies between the peptides and ligands were obtained. The docking results were visualized and analyzed using PyMOL 2.3.0 and Discovery Studio 2019 (Tables 1-2). The results showed that all 63 peptides exhibited complete binding capacity for DPPH and ABTS radicals. FYPF and FYLPF showed relatively low docking binding energies in both radical systems: -4.5 kcal / mol and -4.3 kcal / mol for DPPH, and -4.6 kcal / mol and -4.2 kcal / mol for ABTS, respectively. In addition, intermolecular interactions such as hydrogen bonding and hydrophobic interactions also play a crucial role. Specifically, amino acid residues Trp4 and Phe6 in GISWGFL form two hydrogen bonds with DPPH, while residues Ser3 and Trp5 in IFSPWL bind to ABTS through hydrophobic interactions. Moreover, in the interaction of all peptides with these two free radicals, phenylalanine (Phe), tryptophan (Trp), and leucine (Leu) appear more frequently than other amino acid residues, suggesting that these residues may be key active sites for SCP free radical scavenging. Based on the binding energy of Schisandra chinensis peptides to DPPH / ABTS free radicals, FYPF (SEQ ID NO.7), FLPW (SEQ ID NO.8), FYLPF (SEQ ID NO.9), ANWLPF (SEQ ID NO.10), IFSPWL (SEQ ID NO.11), FPFTYAMMLM (SEQ ID NO.12), GPSFPIFI (SEQ ID NO.13), and GISWGFL (SEQ ID NO.14) were screened as the optimal antioxidant peptides. Figure 6 ).

[0077] Table 1. Molecular docking analysis results of Schisandra chinensis peptides with DPPH / ABTS free radicals

[0078]

[0079] Table 2. Molecular docking analysis results of Schisandra chinensis peptides with DPPH / ABTS free radicals (see Table 1)

[0080]

[0081] 6. Optimal synthesis and screening of antioxidant peptides

[0082] Based on the identified amino acid sequences, antioxidant peptides FYPF, FLPW, FYLPF, ANWLPF, IFSPWL, and FPFTYAMMLM (synthesized by Nanjing Jietai Biotechnology Co., Ltd.) were further synthesized using solid-phase synthesis. The DPPH scavenging rate was then determined according to the method described in "1. Identification of the Antioxidant Activity of Schisandra Peptides". The results showed that ANWLPF achieved a DPPH scavenging rate of 54%, which was higher than that of the other five antioxidant peptides. Figure 7 Therefore, ANWLPF was identified as the optimal novel Schisandra chinensis antioxidant peptide sequence, which will be subsequently used to compose collagen antioxidant compositions.

[0083] II. Recombinant Type I Collagen Expression

[0084] 1. Design and screening of recombinant human type I collagen and proline hydroxylase gene sequences

[0085] 1.1 Based on the structural and functional characteristics of type I collagen, the codon-optimized Col I A1 and Col I A2 sequences were synthesized respectively. AlphaFold3 was then used to predict the protein structure, and the recombinant human type I collagen sequence with a triple helix conformation was determined.

[0086] The Col I A1 nucleotide sequence (SEQ ID NO.1) is as follows:

[0087]

[0088] The amino acid sequence of the Col I A1 protein (SEQ ID NO.2) is as follows:

[0089]

[0090] The Col I A2 nucleotide sequence (SEQ ID NO.3) is as follows:

[0091]

[0092] The amino acid sequence of the Col I A2 protein (SEQ ID NO.4) is as follows:

[0093]

[0094] 1.2 Human proline hydroxylase P4H (NC_000003.12) was co-expressed with recombinant human type I collagen to achieve synergistic effects. This enzyme can exert hydroxylation in yeast.

[0095] The P4H nucleotide sequence (SEQ ID NO.5) is as follows:

[0096] .

[0097] The amino acid sequence of the P4H protein (SEQ ID NO.6) is as follows:

[0098] MEGFETSDRPGVCDGKYYEKIDGFLSDIECDVLINAAIKKGLIKSEVGGATENDPIKLDPKSRNSEQTWFMPGEHEVIDKIQKKTREFLNSKKHCIDKYNFEDVQVARY KPGQYYYHHYDGDDCDDACPKDQRLATLMVYLKAPEEGGGGETDFPTLKTKIKPKKGTSIFFWVADPVTRKLYKETLHAGLPVKSGEKIIANQWIRAVKWSHPQFEK*.

[0099] 2. Obtain yeast strains that stably express recombinant human type I collagen.

[0100] 2.1 The codon-optimized Col I A1 and Col I A2 gene sequences (SEQ ID NO.1 and SEQ ID NO.3) were synthesized, cloned into the pPIC3.5k expression vector, and the recombinant expression plasmid Col I A1 / 2-pPIC3.5k-Dual was constructed.

[0101] 2.2 The human P4H gene sequence (SEQ ID NO.5) was synthesized and cloned into the PICZA expression vector to construct the recombinant expression plasmid P4H-PICZA.

[0102] 2.3 The Col I A1 / 2-pPIC3.5k-Dual and P4H-PICZA plasmids were linearized using restriction endonucleases, and the linearization results were confirmed by agarose gel electrophoresis. The linearized plasmids were then electroporated into Pichia pastoris GS115 competent cells and plated on MD medium (containing 100 μg / mL bleomycin) and incubated upside down at 30°C for approximately 2-3 days.

[0103] 2.4 After single-clone colonies formed, the recombinant strains were identified using PCR technology. Primers are shown in Table 3. Primer pair 5194-F (SEQ ID NO.15) and 3'AOX (SEQ ID NO.16) were used to identify successful integration of the Col I A1 / 2 gene, and primer pair P4H-F (SEQ ID NO.17) and 3'AOX (SEQ ID NO.16) were used to identify successful integration of the P4H gene. Agarose gel electrophoresis results showed that a large number of positive clones were obtained through screening. Figure 8 ).

[0104] Table 3 Primer Information

[0105]

[0106] 2.5 Ten PCR-verified positive colonies were selected and inoculated into BMGY medium, and cultured at 28.5°C until OD500. 600 After 2-6 hours, replace the medium with BMMY medium (1% methanol) for induction, and then incubate the resuspended bacterial solution at 28.5℃ for 48 hours.

[0107] 2.6 After centrifuging 100 μL of the bacterial culture from step 2.3 at 12000 rpm for 5 min, 80 μL of the supernatant was transferred to a 1.5 mL centrifuge tube. The cell sample was then resuspended in 80 μL of PBS in the same 1.5 mL centrifuge tube. 20 μL of 5× Loading Buffer was added, and the mixture was incubated in a boiling water bath for 30 min. Protein expression was detected using SDS-PAGE and Western blot. The results are as follows: Figure 8 and Figure 9 As shown, this indicates that recombinant human type I collagen was successfully expressed.

[0108] 3. Establish a purification process for recombinant human type I collagen.

[0109] 3.1 Collect the fermentation broth of the engineered bacteria, separate and collect the bacterial cells, disrupt the cells using ultrasound, resuspend the cells in PBS, and centrifuge to obtain a supernatant containing type I collagen. (The buffer is PBS).

[0110] 3.2 The supernatant containing type I collagen was concentrated using a 30 kb ultrafiltration tube to remove low molecular weight proteins, and the retained concentrate was collected.

[0111] 3.3 The above-mentioned retained concentrate was further purified by salting out with a gradient concentration of ammonium sulfate (20%, 30%, 40%, 50%, 60%).

[0112] 3.4 While ultrafiltration concentration and ammonium sulfate precipitation can remove impurities and increase protein concentration, they cannot separate the target protein from protein degradation products and other contaminating proteins. Therefore, further protein purification is required. Thus, ion exchange chromatography is used to separate the target protein.

[0113] The sample obtained from 60% saturated ammonium sulfate precipitation was pretreated and loaded onto a Hi-Trap SPFF 5 mL cation exchange pre-packed column. A Cytiva AKTA protein purification system was used for linear gradient elution with buffer containing 0–1 mol / L NaCl. Elution fractions corresponding to 0–0.3 mol / L, 0.3–0.5 mol / L, 0.5–0.8 mol / L, and 0.8–1 mol / L NaCl were collected. Western blot analysis identified the 0.5–0.8 mol / L NaCl range as the target elution peak for recombinant type I collagen. Western blot analysis was performed on the purified sample and the unpurified crude sample within this range. The results showed that, under the same protein loading conditions, the content of type I collagen was significantly increased after purification. Figure 10 ).

[0114] III. Establishment of a Schisandra chinensis polypeptide-collagen composition with antioxidant function

[0115] 1. Composition of the antioxidant composition

[0116] As shown in Table 4, compositions with antioxidant functions were prepared by using recombinant type I collagen and novel Schisandra chinensis antioxidant peptide (ANWLPF) in different proportions. 100 μg of recombinant type I collagen and 500 μg of novel Schisandra chinensis antioxidant peptide (ANWLPF) were dissolved in sterile water or serum-free culture medium, and the volume was adjusted to 1 mL. The mixture was thoroughly mixed to obtain composition 1. Compositions 2 and 3 were prepared using the same method.

[0117] Table 4. Methods for constructing the composition of Schisandra chinensis antioxidant peptides and recombinant type I collagen.

[0118]

[0119] In Table 4 above, the recombinant type I collagen is the type I collagen and P4H co-expression product obtained by expression and purification through the method shown in "II. Expression of Recombinant Type I Collagen".

[0120] 2. Effects of different compositions on UVB-induced oxidative damage in HaCaT cells

[0121] 2.1 Cell proliferation assay

[0122] HaCaT cells were stored at a density of 1 × 10⁶ cells per well. 4Cells were seeded at a density of [number] cells per well in 96-well plates and cultured for 24 h. Following the method described in "4.1 Effect of Different Concentrations of Schisandra chinensis Polypeptide on UVB-Induced HaCaT Cell Viability," cells were treated with UVB, then the culture medium was replaced with different compositions, and the cells were incubated for another 24 h. The cell viability of HaCaT cells treated with different compositions was calculated using the CCK-8 assay. The results showed that cell viability recovered to 66.38% after treatment with composition 3. Figure 11 (A)

[0123] 2.2 SOD Activity Assay

[0124] Following the method described in "4.1 Effect of Different Concentrations of Schisandra chinensis Polypeptides on UVB-Induced HaCaT Cell Viability," cells were treated with UVB and then cultured for 24 h in medium containing different compositions. SOD activity in the cells was measured using a quantitative assay kit. The results showed that after treatment with composition 3, SOD activity recovered to 93.29% of the control group, significantly higher than that of the damaged group (…). Figure 11 (B)

[0125] In summary, composition 3 has been preliminarily verified to have the best antioxidant effect. Further comprehensive determination of the composition's in vitro free radical scavenging ability and UVB-induced cellular oxidative damage repair level will be carried out.

[0126] 3. Identification of the antioxidant activity of the optimal composition

[0127] 3.1 In vitro free radical scavenging ability of the composition

[0128] The DPPH free radical scavenging activity of composition 3 was determined according to the kit instructions, with antioxidant peptides and recombinant type I collagen at the same concentration as controls. The results showed that the free radical scavenging activity of composition 3 was 11.22% and 32.76% higher than that of antioxidant peptides and recombinant type I collagen alone, respectively. Figure 12 (A)

[0129] 3.2 The composition alleviates UVB-induced oxidative damage in HaCaT cells

[0130] HaCaT cells were used at a rate of 2 × 10⁶ cells per well. 5Cells were seeded at a density of 1,000 cells per well in 6-well plates and treated with UVB according to the method described in "4.1 Effect of different concentrations of Schisandra chinensis peptides on UVB-induced HaCaT cell viability". After treatment with culture medium containing composition 3 and the same amount of antioxidant peptides and recombinant type I collagen for 24 h, the levels of SOD, CAT, and MDA in the cells were measured using a quantitative assay kit. The results showed that after treatment with composition 3, the activities of SOD and CAT recovered to 85.06% and 77.91% of the control group, respectively, which was higher than that of the single treatment (…). Figure 12 (B and C). Even more significantly, MDA levels recovered to a level not significantly different from the control group, but were lower than those treated with a single antioxidant peptide and single recombinant type I collagen by 23.97% and 55.36%, respectively. Figure 12 (D).

[0131] Overall, composition 3 exhibits stronger antioxidant activity than single antioxidant peptides or recombinant type I collagen.

[0132] 4. Biocompatibility determination of the optimal composition

[0133] A 1% erythrocyte diluent was prepared according to the method described in "3.7 Hemolytic Performance Test". Composition 3 was mixed with an equal volume of erythrocyte diluent and incubated at 37°C and 200 rpm for 1 h. After incubation, the mixture was centrifuged, and the supernatant was used to determine its OD value at 570 nm. 0.1% Triton X-100 and PBS treatment groups were used as positive and negative controls, respectively. The hemolytic activity of Composition 3 and the control group was calculated. The results showed that the hemolysis rate of Composition 3 was much lower than that of 0.1% Triton X-100 buffer, and its hemolytic activity remained below 10%. Figure 13 Composition 3 was confirmed to have good biocompatibility.

[0134] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A recombinant type I collagen-antioxidant peptide composition, characterized in that, It includes recombinant type I collagen and Schisandra chinensis antioxidant peptide, wherein the recombinant type I collagen includes peptide chains as shown in SEQ ID NO.2 and SEQ ID NO.4, and the amino acid sequence of the Schisandra chinensis antioxidant peptide is ANWLPF.

2. The recombinant type I collagen-antioxidant peptide composition according to claim 1, characterized in that, The mass ratio of the recombinant type I collagen to the Schisandra chinensis antioxidant peptide is 1:(1-5).

3. The recombinant type I collagen-antioxidant peptide composition as described in claim 2, characterized in that, The mass ratio of the recombinant type I collagen to the Schisandra chinensis antioxidant peptide is 1:

2.

4. A Schisandra chinensis antioxidant peptide with antioxidant activity, characterized in that, The amino acid sequence of the Schisandra chinensis antioxidant peptide is ANWLPF.

5. The use of the recombinant type I collagen-antioxidant peptide composition according to any one of claims 1-3 in the preparation of antioxidant products.

6. The use of the recombinant type I collagen-antioxidant peptide composition according to any one of claims 1-3 in the preparation of functional foods that help with antioxidation or skin repair cosmetics that alleviate UVB-induced cellular oxidative damage.

7. The application of the Schisandra chinensis antioxidant peptide as described in claim 4 in the preparation of antioxidant products.

8. An antioxidant product, characterized in that, Includes the recombinant type I collagen-antioxidant peptide composition according to any one of claims 1-3 or the Schisandra chinensis antioxidant peptide according to claim 4.

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