An antioxidant peptide derived from deep-sea cold seeps and its applications

Abstract

This invention discloses an antioxidant peptide derived from deep-sea cold seeps and its applications, belonging to the fields of bioinformatics, development of microbial resources in extreme environments, and antioxidant peptide technology. The antioxidant peptide CSAOP4 obtained by this invention originates from a high-salt, low-temperature, high-pressure, oligotrophic environment, exhibiting excellent stability and activity. Adding 0.1 mg / mL of this antioxidant peptide resulted in an ABTS free radical scavenging rate of 44.45%, significantly superior to CSAOP1, CSAOP2, and CSAOP3 (ABTS free radical scavenging rates of 7.32%, 15.14%, and 27.71%, respectively). It is suitable for cosmetic production processes and can be mass-produced through solid-phase synthesis, offering simple preparation and high cost-effectiveness. This deep-sea cold seep antioxidant peptide is suitable for application in the cosmetic field, significantly extending the shelf life of cosmetics and reducing the risk of oxidative deterioration, showing broad application prospects.

Description

Technical Field

[0001] This invention relates to an antioxidant peptide derived from deep-sea cold seeps and its applications, belonging to the fields of bioinformatics, development of microbial resources in extreme environments, and antioxidant peptide technology. Background Technology

[0002] Excessive accumulation of free radicals caused by oxidative stress is closely related to various human diseases and food microbial spoilage. As key substances for free radical scavenging, antioxidants are experiencing continuous market demand growth, while consumers' health concerns are increasing. Traditional synthetic antioxidants such as BHT (butylated hydroxytoluene) and BHA (butylated hydroxyanisole) pose safety risks such as toxic residues and teratogenicity, while natural antioxidants such as tea polyphenols and vitamin C face problems such as insufficient activity, poor stability, and unclear mechanisms of action, limiting their applicable scenarios.

[0003] Deep-sea cold seeps, as extreme habitats (high pressure, high salinity, and low nutrient levels), have fostered unique metabolic systems in their microorganisms, which have evolved to adapt to these environments. The resulting bioactive peptides possess novel structures, strong antioxidant activity, and tolerance to extreme conditions, making them a high-quality resource of natural antioxidant peptides. However, current technologies face three major bottlenecks: first, the culturability of cold seep microorganisms is less than 0.1%, making it difficult to discover potential functional peptides using traditional isolation and culture methods; second, antioxidant peptide screening relies on in vitro experiments, which are time-consuming and costly, and high-throughput screening technologies are lacking; and third, there is a lack of targeted screening models for cold seep microbiome data, resulting in low screening efficiency and accuracy.

[0004] Machine learning technology, with its powerful feature extraction and prediction capabilities, has shown potential in the screening of bioactive peptides. However, there are currently no reports on combining machine learning with deep-sea cold seep microorganisms for targeted screening of highly active antioxidant peptides. Therefore, developing efficient screening technologies based on machine learning to discover novel antioxidant peptides from cold seep microorganisms is of significant industrial value for addressing the shortcomings of traditional antioxidants and promoting high-quality development in the antioxidant field. Summary of the Invention

[0005] This invention aims to overcome the limitations of existing antioxidant peptide screening methods, such as low efficiency, insufficient activity, and safety concerns. It provides a machine learning-based method for targeted screening of antioxidant peptides derived from deep-sea cold seep microorganisms, obtaining novel antioxidant peptides with high activity and high stability. The invention also clarifies the preparation methods and multi-scenario applications of these peptides, thereby achieving efficient research and development and industrialization of natural antioxidant products.

[0006] The first technical solution provided by the present invention is a deep-sea cold seep antioxidant peptide, the amino acid sequence of which is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0007] The aforementioned deep-sea cold seep antioxidant peptides are antimicrobial peptides (CSAOPs) selected from the gene resources of cold seep microbiomes in extreme deep-sea environments. These include CSAOP1, CSAOP2, CSAOP3, and CSAOP4, with amino acid sequences of Met-Glu-Gln-Val-Thr-Gly-His-Tyr-Tyr-Tyr-Tyr-His-Tyr-Tyr-Leu-Gly-Leu-His-Pro (MEQVTGHYYYYYHYYLGLHP, SEQ ID NO.1, named CSAOP1) and Ser-Ile-Glu-Tyr-Ala-Asp-Trp-His-Tyr-Gln-Asn-Pro-Trp-Phe-His (SIEYADWHYQNPWFH, SEQ ID NO.1). NO.2, named CSAOP2), Tyr-Ser-Val-Thr-Thr-Tyr-Gly-Tyr-Trp-Ile-Phe (YSVTTYGYWIF, SEQ ID NO.3, named CSAOP3), Glu-Asp-Trp-Val-Trp-Phe-Thr-Tyr-Thr-Ile-Tyr-Cys (EDWVWFTYTIYC, SEQ ID NO.4, named CSAOP4).

[0008] The second technical solution provided by the present invention is an antioxidant, which includes the deep-sea cold seep antioxidant peptides described in the first technical solution.

[0009] The third technical solution provided by the present invention is a product, which includes the deep-sea cold seep antioxidant peptide described in the first technical solution or the antioxidant described in the second technical solution.

[0010] In some embodiments, the product includes pharmaceuticals, cosmetics, health products, and food.

[0011] In some embodiments, the pharmaceutical product includes a drug carrier comprising microcapsules, microspheres, nanoparticles, and / or liposomes.

[0012] In some embodiments, the product includes additives, which contain solubilizers, co-solvents, latent solvents, and / or preservatives.

[0013] In some embodiments, the cosmetic also contains a base ingredient and / or conventional excipients.

[0014] In some embodiments, the matrix raw materials include oily raw materials, waxy raw materials, synthetic oily raw materials, powdery raw materials, gelling raw materials, coagulants, and surfactants.

[0015] In some embodiments, the conventional excipients include one or more of the following: humectants, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film-forming agents, emulsifiers, and cosmetic nutritional additives.

[0016] In some embodiments, the cosmetics include skin care products and sunscreens.

[0017] In some embodiments, the cosmetic product may be a toner, serum, lotion, or cream, etc.

[0018] In some embodiments, the dosage form of the product is a powder, emulsifier, oil, granule, microcapsule, tablet, pill, or oral liquid.

[0019] The fourth technical solution provided by this invention is the application of the deep-sea cold spring antioxidant peptide described in the first technical solution or the antioxidant described in the second technical solution in the anti-oxidation of cosmetics or food.

[0020] The fifth technical solution provided by the present invention is a method for inhibiting the oxidation of cosmetics, wherein the method involves adding the deep-sea cold spring antioxidant peptide described in the first technical solution or the antioxidant described in the second technical solution to the cosmetics.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The discovery and utilization of antioxidant peptides from deep-sea cold seep microorganisms are in the early stage. This invention combines machine learning technology with cold seep microorganism gene mining to construct an antioxidant peptide targeted screening system. The screening efficiency is significantly improved compared with traditional methods, reducing research and development time and breaking through the bottleneck of functional peptide mining of microorganisms in extreme environments, providing a new paradigm for high-throughput screening of antioxidant peptides.

[0022] (2) The antioxidant peptide CSAOP4 obtained by screening has excellent activity. When 0.1 mg / mL of this antioxidant peptide is added, the ABTS free radical scavenging rate is 44.45%, which is significantly better than CSAOP1, CSAOP2 and CSAOP3 (ABTS free radical scavenging rates are 7.32%, 15.14% and 27.71%, respectively). It can also be enhanced by activating the Keap1-Nrf2-ARE signaling pathway, indicating that it has good antioxidant activity and good application potential.

[0023] (3) Deep-sea cold spring antioxidant peptides are derived from high-salt, low-temperature, high-pressure, and oligotrophic environments, possess excellent stability, can be adapted to the production process of cosmetic products, and can be mass-produced through solid-phase synthesis. They are simple to prepare and cost-effective.

[0024] (4) Deep-sea cold spring antioxidant peptides are suitable for application in the cosmetics field. They can significantly extend the shelf life of cosmetics and reduce the risk of cosmetic oxidation and deterioration. They have broad application prospects. Attached Figure Description

[0025] Figure 1 High-performance liquid chromatography (HPLC) purification chromatogram and mass spectrum of CSAOP1, an antioxidant peptide from deep-sea cold seeps.

[0026] Figure 2 High-performance liquid chromatography (HPLC) purification chromatogram and mass spectrum of CSAOP2, an antioxidant peptide from deep-sea cold seeps.

[0027] Figure 3 High-performance liquid chromatography (HPLC) purification chromatogram and mass spectrum of deep-sea cold seep antioxidant peptide CSAOP3.

[0028] Figure 4 High-performance liquid chromatography (HPLC) purification chromatogram and mass spectrum of CSAOP4, an antioxidant peptide from deep-sea cold seeps.

[0029] Figure 5 This image shows the results of the antioxidant activity analysis of the deep-sea cold seep antioxidant peptide CSAOP, comparing the antioxidant activity of CSAOP-added and CSAOP-unadded peptides. The comparison includes (A) CSAOP1 vs. control group, (B) CSAOP2 vs. control group, (C) CSAOP3 vs. control group, (D) CSAOP4 vs. control group, and (E) comparison of the activities of different antioxidant peptides. The letters *, ***, and **** indicate no significant difference between groups, significant difference between groups, and significant difference, respectively. p<0.05 ), ( p<0.001 ), ( p<0.0001 ).

[0030] Figure 6 The results of molecular docking between the deep-sea cold seep antioxidant peptide CSAOP and the Keap1 protein (2FLU) are shown, including (A) CSAOP1, (B) CSAOP2, (C) CSAOP3, and (D) CSAOP4. Detailed Implementation

[0031] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0032] Experimental methods: Free radical scavenging assay using deep-sea cold seep antioxidant peptide ABTS: First, 4 mg of deep-sea cold seep antioxidant peptide was added to 2 mL of deionized water and vortexed to mix, thus obtaining the CSAOP stock solution. Second, ABTS working solution, reagent four working solution, reagent one working solution, and vitamin C working solution were prepared according to the ABTS kit instructions. Third, 50 μL of deionized water, 100 μL of reagent four working solution, and 850 μL of ABTS working solution were added to the blank group; 50 μL of the prepared CSAOP stock solution, 100 μL of reagent four working solution, and 850 μL of ABTS working solution were added to the assay group; 50 μL of the prepared CSAOP stock solution and 950 μL of reagent one working solution were added to the control group; and 50 μL of the prepared vitamin C working solution, 100 μL of reagent four working solution, and 850 μL of ABTS working solution were added to the positive control group. Vortex to mix, let stand at room temperature in the dark for 6 minutes, and measure the absorbance at 405 nm using an ELISA reader. Label them as A blank, A assay, A control and A positive control, respectively. The formula for calculating the ABTS free radical scavenging rate is: ABTS free radical scavenging rate D% = [A blank - (A assay - A control)] ÷ A blank × 100%. Each group has three biological replicates.

[0033] Raw materials used in the examples: This invention uses freeze-dried powder containing the main component of deep-sea cold seep antioxidant peptides with a purity greater than 95% as an example, but is not limited to solid, semi-solid, or liquid states. When applied in other forms such as mixtures, the amount used should be converted to the application amount disclosed in this invention.

[0034] Vitamin C, deionized water, centrifuge tubes, and 96-well plates were purchased from Guangzhou Shuoheng Biotechnology Co., Ltd. The Keap1 protein crystal structure (PDB ID: 2FLU) was obtained from the Protein Data Bank database (https: / / www.rcsb.org / ). The ABTS kit was purchased from Beijing Solarbio Technology Co., Ltd.

[0035] Example 1: Targeted Screening of Deep-Sea Cold Seep Antioxidant Peptide CSAOP Construction of Training Dataset: Using web crawling tools, 800 antioxidant peptide sequences, 200 non-antioxidant peptide sequences, and 600 random peptide sequences reported in the literature were collected. Physicochemical properties, including hydrophobicity, polarity, charge, and antioxidant activity such as ABTS scavenging rate, were integrated to construct a training dataset for later use. Machine Learning Model Construction and Training: Based on the classic ESM-2 protein language model, peptide sequence feature vectors were extracted, dataset label errors were cleaned, and a support vector machine was used to train the prediction model. Targeted Screening of Antioxidant Peptide Sequences from Deep-Sea Cold Seep Sediments: Metagenomic sequencing and assembly of microorganisms from deep-sea cold seep sediments were performed. Open reading frames were predicted using Prodigal, and candidate oligopeptide sequences of 2-20 amino acids were extracted to construct a candidate peptide library containing 927,856 sequences. The candidate peptide library was input into the trained model, and a weighted score combining molecular docking potential with the key antioxidant pathway protein Keap1 (PDB ID: 2FLU) and physicochemical properties was used to screen for high-potential antioxidant peptide sequences.

[0036] Preparation of deep-sea cold seep antioxidant peptides: CSAOP was synthesized by solid-phase synthesis and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) with acetonitrile as the mobile phase. The target peak was collected, and the purity was ≥95%. The molecular weight was verified by mass spectrometry (MALDI-TOF-MS). The theoretical value of CSAOP1 was 2597.85 Da, and the measured value was 2598.24 Da; the theoretical value of CSAOP2 was 1993.09 Da, and the measured value was 1993.26 Da; the theoretical value of CSAOP3 was 1399.54 Da, and the measured value was 1399.16 Da; the theoretical value of CSAOP4 was 1625.79 Da, and the measured value was 1625.52 Da.

[0037] We integrated metagenomic data of deep-sea cold seep microbial communities, assembled and annotated them using bioinformatics tools, extracted small peptide sequences of 2-20 amino acids, and combined them with reported antioxidant peptides and non-antioxidant peptide sequences downloaded from the UniProt database to construct a dataset. We used the classic ESM-2 protein language model to extract peptide features, identified and removed mislabeled data, and trained the model using the SVM algorithm. After multiple iterations, the model achieved a balanced accuracy of 80% and a Matthews correlation coefficient of 0.9. Using a training model to analyze deep-sea cold seep antioxidant peptides, candidate antioxidant peptide sequences with a predicted score ≥0.85 were screened. Combined with molecular docking and weighted scoring of peptide physicochemical properties, four high-potential CSAOP antioxidant peptide sequences were obtained. CSAOP was synthesized using a solid-phase synthesis method and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) with acetonitrile as the mobile phase. The target peak was collected, and the purity was ≥95%. The molecular weight was verified by mass spectrometry (MALDI-TOF-MS): CSAOP1 theoretical value was 2597.85 Da; CSAOP2 theoretical value was 1993.09 Da; CSAOP3 theoretical value was 1399.54 Da; and CSAOP4 theoretical value was 1625.79 Da.

[0038] Mass spectrometry analysis verified its molecular weight and peptide sequence, and the results are as follows: Figure 1 As shown, the purities of CSAOP1, CSAOP2, CSAOP3, and CSAOP4 are 95.17%, 95.66%, 95.12%, and 95.92%, respectively, and their actual molecular weights are 2598.24 ± 0.39 Da. Figure 1 ), 1993.26±0.17 Da ( Figure 2 ), 1399.16±0.38 Da ( Figure 3 ), 1625.52±0.27 Da ( Figure 4 Consistent with theoretical values, the three-dimensional structures are mostly random coils and α-helices. Figures 1-4 Their amino acid sequences are MEQVTGHYYYYYHYYLGLHP, SIEYADWHYQNPWFH, YSVTTYGYWIF, and EDWVWFTYTIYC (SEQ ID NO. 1~4).

[0039] Example 2: Effect of deep-sea cold seep antioxidant peptide CSAOP1 on ABTS free radical scavenging The inhibitory effect of CSAOP1 on ABTS free radicals was evaluated by an experiment on the scavenging rate of the deep-sea cold seep antioxidant peptide ABTS. The scavenging results of CSAOP1 after its addition are shown in Figure 5A. The experimental results show that CSAOP1 has a significant scavenging effect on ABTS free radicals. p<0.05After adding 0.1 mg / mL of CSAOP1, the ABTS radical scavenging rate was 7.32 ± 0.04%.

[0040] Example 3: Effect of deep-sea cold seep antioxidant peptide CSAOP2 on ABTS free radical scavenging The inhibitory effect of CSAOP2 on ABTS free radicals was evaluated by the scavenging rate experiment of the deep-sea cold seep antioxidant peptide ABTS. The scavenging results of free radicals after the addition of CSAOP2 are shown in Figure 5B. The experimental results show that CSAOP2 has a highly significant scavenging effect on ABTS free radicals. p<0.001 After adding 0.1 mg / mL of CSAOP2, the ABTS radical scavenging rate was 15.14 ± 0.07%.

[0041] Example 4: Effect of deep-sea cold seep antioxidant peptide CSAOP3 on ABTS free radical scavenging The inhibitory effect of CSAOP3 on ABTS free radicals was evaluated by the scavenging rate experiment of the deep-sea cold seep antioxidant peptide ABTS. The scavenging results of free radicals after the addition of CSAOP3 are shown in Figure 5C. The experimental results show that CSAOP3 has a highly significant scavenging effect on ABTS free radicals. p<0.001 After adding 0.1 mg / mL of CSAOP3, the ABTS radical scavenging rate was 27.71 ± 0.12%.

[0042] Example 5: Effect of deep-sea cold seep antioxidant peptide CSAOP4 on ABTS free radical scavenging The inhibitory effect of CSAOP4 on ABTS free radicals was evaluated by the scavenging rate experiment of the deep-sea cold seep antioxidant peptide ABTS. The scavenging results of free radicals after the addition of CSAOP4 are shown in Figure 5D. The experimental results show that CSAOP4 has a highly significant scavenging effect on ABTS free radicals. p<0.001 After adding 0.1 mg / mL of CSAOP4, the ABTS radical scavenging rate was 44.45 ± 0.19%.

[0043] Compared with CSAOP1, CSAOP2, CSAOP3, and CSAOP4, under the same conditions, showed significantly improved ability to scavenge ABTS radicals. Figure 5 The CSAOP4 group showed the highest activity, suggesting the potential of this antioxidant peptide in the cosmetics field.

[0044] Example 6: Molecular docking analysis of deep-sea cold seep antioxidant peptide CSAOP Molecular docking was performed between deep-sea cold seep antioxidant peptides and Keap1, a key protein in the antioxidant activity signaling pathway. The results are as follows: Figure 6As shown, CSAOP1, CSAOP2, CSAOP3, and CSAOP4 all bind tightly to the protein Keap1. CSAOP1 (… Figure 6 Compared to A), CSAOP2 ( Figure 6 B), CSAOP3 ( Figure 6 C) and CSAOP4 ( Figure 6 D) The number of amino acids bound to Keap1 increased significantly, with CSAOP4 binding to the most amino acids in Keap1, such as Arg and Phe, and exhibiting the highest binding energy. This finding is consistent with the wet experimental results of the deep-sea cold seep antioxidant peptide CSAOP scavenging ABTS free radicals, suggesting that the latter has higher antioxidant potential.

[0045] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Deep-sea cold seep antioxidant peptides, characterized in that, The amino acid sequence of the deep-sea cold seep antioxidant peptide is shown in SEQ ID NO.3 or SEQ ID NO.

4.

2. An antioxidant, characterized in that, The antioxidant includes the deep-sea cold spring antioxidant peptide as described in claim 1.

3. A product characterized in that, The product includes the deep-sea cold spring antioxidant peptide as described in claim 1 or the antioxidant as described in claim 2.

4. The product according to claim 3, characterized in that, The products include pharmaceuticals, cosmetics, health products, and food.

5. The product according to claim 4, characterized in that, The cosmetic also contains base ingredients and / or conventional excipients.

6. The product according to claim 5, characterized in that, The matrix raw materials include oil-based raw materials, wax-based raw materials, synthetic oil-based raw materials, powder-based raw materials, gelling agents, and surfactants; the conventional excipients include one or more of the following: moisturizers, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film-forming agents, emulsifiers, and cosmetic nutritional additives.

7. The product according to claim 4, characterized in that, The cosmetics mentioned include skin care products and sunscreens.

8. The product according to claim 3, characterized in that, The dosage form of the product is powder, emulsifier, oil, granules, microcapsules, tablets, pills, or oral liquid.

9. The application of the deep-sea cold spring antioxidant peptide of claim 1 or the antioxidant of claim 2 in the antioxidant properties of cosmetics or food.

10. A method for inhibiting the oxidation of cosmetics, characterized in that, The method involves adding the deep-sea cold spring antioxidant peptide of claim 1 or the antioxidant of claim 2 to cosmetics.

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