Antioxidant collagen polypeptide, preparation method and application thereof

Abstract

This invention provides an antioxidant collagen polypeptide, its preparation method, and its application. The amino acid sequence of the collagen polypeptide is IDGRPGPIGPA or ISGPHypGPHypGPA. The collagen polypeptide of this invention possesses antioxidant activity; it can bind to Keap1 and inhibit the Nrf2-Keap1 protein interaction, effectively protecting cells from oxidative damage.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. Specifically, this invention relates to a collagen polypeptide, its preparation method, and its application. Background Technology

[0002] Oxidative stress is a pathological state caused by an imbalance between the production and clearance of free radicals within cells, leading to dysfunction of cells, tissues, and organs. It is considered one of the important inducing factors for various diseases. External factors such as environmental pollution, irregular diet, and lifestyle can all lead to excessive accumulation of free radicals in the body, thus causing oxidative stress. Increasing research shows that oxidative stress gradually increases with age, further contributing to the occurrence and exacerbation of age-related diseases.

[0003] Collagen peptides are small-molecule active substances obtained by hydrolyzing collagen under suitable conditions. They possess excellent biological activities, such as anti-cancer, blood pressure lowering, anti-inflammatory, and promoting mineral absorption. Furthermore, collagen peptides have been found to have good antioxidant activity, exerting their antioxidant effects through effective scavenging of different types of free radicals or by promoting the activation of antioxidant enzymes and increasing the levels of non-enzymatic antioxidants in the body. Studies have shown that dietary antioxidant intake helps prevent and alleviate oxidative stress in the body, thereby reducing the incidence of oxidative stress-related diseases to some extent.

[0004] Chinese patent CN111334551B discloses a production process for bovine hide collagen peptides, including S1 cleaning, S2 denaturation, S3 enzymatic hydrolysis, S4 centrifugation, S5 enzyme inactivation, S6 impurity removal, and S7 finished product. This invention has the following advantages and effects: The application utilizes a combination of ultrasonic, denaturation, and enzymatic hydrolysis processes, making it easily adaptable to bovine hides from various origins; the collagen peptide product produced by the process provided in this application has good solubility and dispersibility, excellent sensory quality, and high yield; the large amount of active peptides in the product gives it strong antioxidant capacity. However, this product contains a large number of components with weak antioxidant capacity, exhibiting weak DPPH free radical scavenging ability, with a half-inhibitory concentration above 30 mg / mL, resulting in limited overall antioxidant activity.

[0005] Furthermore, Chinese patent CN108250291B discloses an antioxidant collagen polypeptide, characterized in that: the amino acid sequence of the antioxidant collagen polypeptide is SSGPPVPGPMGPMGPR, and the polypeptide chain contains two methionine residues. However, this patent only discloses that the polypeptide has antioxidant activity, but does not disclose its ability to bind to Keap1 and inhibit the Nrf2-Keap1 protein interaction.

[0006] Therefore, the purpose of this invention is to provide a small molecule collagen polypeptide that can bind to Keap1 and inhibit the Nrf2-Keap1 protein interaction to exert antioxidant activity. Summary of the Invention

[0007] The purpose of this invention is to provide a small molecule peptide that can bind to Keap1 and inhibit the Nrf2-Keap1 protein interaction to exert antioxidant activity, its manufacturing process, and its application. To achieve the purpose of this invention, the following technical solution is proposed:

[0008] One aspect of this invention relates to a collagen polypeptide, wherein the amino acid sequence of the collagen polypeptide is IDGRPGPIGPA or ISGPHypGPHypGPA, wherein Hyp is an abbreviation for hydroxyproline.

[0009] This invention also relates to a food product containing the aforementioned collagen polypeptide. There is no particular limitation on the amount of the collagen polypeptide used in the food product; for example, it can be between 0.5 wt% and 5 wt%.

[0010] Another aspect of the present invention relates to a cosmetic product containing the aforementioned collagen polypeptide. There is no particular limitation on the amount of the collagen polypeptide used in the cosmetic product; for example, it can be between 0.1 wt% and 5.0 wt%.

[0011] Another aspect of the present invention relates to a method for preparing the above-mentioned collagen polypeptide, wherein the collagen polypeptide is subjected to enzymatic hydrolysis of bovine bone collagen by alkaline papain. The enzymatic hydrolysis conditions are: pH 7-8, temperature 55-65℃, hydrolysis time 80-160 min, substrate concentration 20-30% (w / v), total enzyme dosage 6000-10000 U / g, and the ratio of alkaline protease to papain is 1:2-4. The prepared collagen hydrolysate is separated by ion chromatography and reversed-phase high-performance liquid chromatography.

[0012] Preferably, the collagen polypeptide is subjected to enzymatic hydrolysis of bovine bone collagen using alkaline papain. The hydrolysis conditions are: pH 7.7, temperature 59.6℃, hydrolysis time 120 min, substrate concentration 25% (w / v), total enzyme dosage 8396 U / g, and alkaline protease to papain ratio 1:3. The prepared collagen hydrolysate is separated by ion chromatography and reversed-phase high-performance liquid chromatography.

[0013] It should be noted that although the collagen polypeptide of the present invention can be prepared by sea cucumber, the collagen polypeptide of the present invention can also be prepared by solid-phase synthesis.

[0014] Another aspect of the present invention relates to the application of the above-mentioned collagen peptides, which are used to prepare antioxidants or cosmetics with antioxidant effects.

[0015] The beneficial technical effects of this invention are as follows:

[0016] This invention discloses an antioxidant collagen polypeptide (IDGRPGPIGPA affinity = -9.1 kcal / mol, ISGPHypGPHypGPA affinity = -9.2 kcal / mol) capable of binding to Keap1 and inhibiting the Nrf2-Keap1 protein interaction, and its preparation method. The method involves enzymatic hydrolysis of bovine bone collagen using alkaline papain, followed by separation, identification, molecular docking simulation, and chemical synthesis to obtain the antioxidant collagen polypeptide capable of binding to Keap1 and inhibiting the Nrf2-Keap1 protein interaction, effectively protecting cells from oxidative damage. The collagen polypeptide of this invention uses bovine bone collagen as its raw material, which has the advantages of being widely available and having a high protein content. Attached Figure Description

[0017] Figure 1 The scavenging ability of antioxidant collagen peptides against DPPH and ABTS free radicals;

[0018] Figure 2 The protective effect of antioxidant collagen peptides against H2O2-induced oxidative damage in cells. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0020] Example 1: Preparation of collagen peptides

[0021] The preparation method provided by this invention is as follows:

[0022] (1) Extraction of bovine bone collagen hydrolysate

[0023] Degreased and decalcified bovine bone was mixed with deionized water to achieve a substrate concentration of 25% (w / v). The pH of the system was adjusted to 7.7, the reaction temperature was 59.6℃, the enzymatic hydrolysis time was 120 min, the total enzyme dosage was 8396 U / g, and the ratio of alkaline protease to papain was 1:3. After the enzymatic hydrolysis reaction was completed, the supernatant was collected by centrifugation, filtered, and freeze-dried to obtain bovine bone collagen hydrolysate.

[0024] (2) Separation, purification and identification of enzymatic hydrolysis products

[0025] Bovine bone collagen hydrolysate was initially separated using DEAE52 cellulose anion exchange chromatography. Gradient elution was performed using buffers containing different concentrations (0, 0.1, 0.2, 0.3, 0.4, and 1.0 M) of NaCl at a flow rate of 1 mL / min. Separates from the same peak were collected under 280 nm monitoring and then dialyzed for desalting. The optimal antioxidant active component was then separated using reversed-phase high-performance liquid chromatography (RP-HPLC). A C18 column (10 mm × 250 mm) was used, with a detection wavelength of 280 nm, an injection volume of 50 μL, a column temperature of 30 °C, and a flow rate of 1.0 mL / min. Mobile phase A consisted of ultrafiltered water containing 0.05% TFA, and mobile phase B consisted of acetonitrile containing 0.05% TFA. The peptide elution program was: 5-55% mobile phase B for the first 35 min; 55-95% mobile phase B for 35-40 min. The antioxidant activity of different elution fractions was collected and measured, and the peptide sequence of the fraction with the best antioxidant activity was determined by LC-MS / MS.

[0026] (3) Molecular docking screening and activity verification of antioxidant active peptides

[0027] Based on the crystal structure of Keap1 (PDB ID: 4IQK), the binding affinity of peptides to Keap1 was predicted using Autodock Vina. Active collagen peptides with high affinity for Keap1 were screened, with peptide sequences of IDGRPGPIGPA or ISGPHypGPHypGPA (IDGRPGPIGPA affinity = -9.1 kcal / mol, ISGPHypGPHypGPA affinity = -9.2 kcal / mol). The affinity of the Keap1 ligand IQK is -11.3 kcal / mol. The active peptides screened using this method showed high affinity for Keap1. The antioxidant activity of the collagen peptides was then verified through in vitro chemical synthesis.

[0028] from Figure 1 As can be seen, IDGRPGPIGPA (P1) at a concentration of 5 mM exhibited scavenging rates of 66.58±2.87% and 73.78±2.04% for DPPH and ABTS free radicals, respectively, while ISGPHypGPHypGPA (P2) at a concentration of 5 mM showed scavenging rates of 59.50±1.88% and 64.33±3.45% for DPPH and ABTS free radicals, respectively. Figure 2 As can be seen, IDGRPGPIGPA (P1) or ISGPHypGPHypGPA (P2) at a concentration of 50 μM can effectively improve the resistance of cells to H2O2-induced oxidative damage and significantly increase cell activity.

[0029] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An application of a collagen polypeptide, wherein the collagen polypeptide is used to prepare an antioxidant that improves cellular oxidative damage, or to prepare a cosmetic with antioxidant activity, wherein the amino acid sequence of the collagen polypeptide is IDGRPGPIGPA.

2. The application according to claim 1, wherein the collagen polypeptide is prepared by solid-phase synthesis.

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