Haliotis discus hannai I type collagen peptide as well as screening method and application thereof

Abstract

The invention discloses haliotis discus hannai I-type collagen peptide as well as a screening method and application thereof, and belongs to the technical field of bioactive peptides. The peptide sequences of the haliotis discus hannai I type collagen peptide are GAAGDK and DSQSAR; the screening method specifically comprises the following steps: (1) virtual enzymolysis of the haliotis discus hannai I-type collagen; (2) screening the bioactive peptide; and (3) molecular docking. According to the invention, through rational design and a virtual screening strategy, a human gastrointestinal tract digestion process is simulated to carry out targeted virtual enzymolysis, a multi-target molecular docking technology is combined, brand-new bifunctional peptides GAAGDK and DSQSAR are accurately predicted and screened from a specific collagen sequence, and aiming at a dual-action mechanism of AGE-RAGE axis and oxidative stress, the application has the advantages of high sensitivity, high sensitivity and high stability. The composition has remarkable synergistic interaction potential in the aspects of preventing diabetic complications and delaying skin and body senescence, and a brand new core raw material is provided for developing next-generation anti-saccharification functional food and skin care products.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, and more specifically to a type I collagen peptide from *Abalone rotundifolia*, its screening method, and its application. Background Technology

[0002] The excessive accumulation of advanced glycation end products (AGEs) and the activation of their receptor RAGE are core pathological mechanisms driving the development of diabetic complications, skin aging, and various chronic diseases. AGE formation and oxidative stress mutually promote each other, creating a vicious cycle. Currently, strategies for intervening in the AGE-RAGE axis to combat glycation mainly focus on using chemical drugs or natural small-molecule inhibitors (such as aminoguanidine and quercetin), but these have limitations such as single-target therapy, low bioavailability, or potential side effects. Therefore, developing natural multi-target active ingredients that can simultaneously block the AGE-RAGE signaling pathway and scavenge reactive oxygen species is of great significance for the prevention and adjuvant improvement of glycation-related diseases.

[0003] Collagen, due to its unique amino acid composition (rich in glycine and proline) and biocompatibility, has become an important source of bioactive peptides. However, its application in the field of anti-glycation is mostly focused on improving skin appearance. The wrinkled abalone, as an important marine resource, possesses a unique type I collagen structure, potentially harboring undiscovered bioactive peptide sequences. Current techniques for extracting bioactive peptides from collagen often employ indiscriminate hydrolysis with broad-spectrum proteases (such as papain and neutral proteases) followed by activity-tracking separation. However, this method is inefficient, lacks clear targeting, and often yields peptides with known sequences or single activities, making it difficult to obtain novel bifunctional peptides with both potent anti-glycation and antioxidant activities.

[0004] More importantly, there are currently no reports in internationally recognized bioactive peptide databases of collagen-derived bioactive peptides that simultaneously target the AGE receptor (RAGE) and key antioxidant pathways, and have clearly defined sequences.

[0005] Therefore, how to extract bifunctional peptides with high activity, high safety, easy absorption and novel structure from type I collagen of abalone rotundifolia is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a wrinkled abalone type I collagen peptide and its screening method and application, so as to overcome the shortcomings of the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A type I collagen peptide from *Abalone fasciatus* with wrinkled discs, the peptide sequence being GAAGDK and DSQSAR.

[0008] The amino acid sequence of GAAGDK is shown in SEQ ID NO.1 of the sequence listing, specifically: Gly-Ala-Ala-Gly-Asp-Lys, with the Chinese name: glycine-alanine-alanine-glycine-aspartic acid-lysine.

[0009] The amino acid sequence of DSQSAR is shown in SEQ ID NO.2 of the sequence listing, specifically: Asp-Ser-Gln-Ser-Ala-Arg, with the Chinese name: Aspartic acid-serine-glutamine-serine-alanine-arginine.

[0010] This invention employs a rational design and virtual screening strategy to simulate the human gastrointestinal digestive process through targeted virtual enzymatic hydrolysis. Combined with multi-target molecular docking technology, it accurately predicts and screens novel bifunctional peptides from specific collagen sequences, effectively overcoming the blind spots of traditional methods. Based on this strategy, this invention has, for the first time, identified two novel peptides, GAAGDK and DSQSAR, with dual functionalities from type I collagen in *Abalone rotundifolia*, providing entirely new core ingredients for the development of next-generation anti-glycation functional foods and skincare products.

[0011] A method for screening the above-mentioned wrinkled abalone type I collagen peptides specifically includes the following steps: (1) Virtual enzymatic hydrolysis of type I collagen from wrinkled abalone The amino acid composition and proportion of type I collagen from *Abalone fasciatus* were analyzed and compared. Based on the amino acid composition, the potential of antioxidant and anti-glycation activities of type I collagen from *Abalone fasciatus* was preliminarily determined. Enzyme selection was used to perform virtual enzymatic digestion of type I collagen from *Abalone fasciatus*. (2) Screening of bioactive peptides The bioactive peptides obtained from enzymatic hydrolysis were sequentially subjected to activity prediction, hydrophilicity prediction, and toxicity prediction. (3) Molecular docking Molecular docking was performed on the target peptide library to screen out type I collagen peptides of *Abalone scabra* with potential activity and low receptor binding energy. The peptide sequences are GAAGDK and DSQSAR.

[0012] This invention employs trypsin, pepsin (pH>2), and pepsin (pH>1.3) to perform virtual enzymatic digestion of the protein sequence, simulating its digestion process in the gastrointestinal tract. Simultaneously, molecular docking technology is used to explore the binding affinity with different receptors (antioxidant receptors and advanced glycation end product receptors), providing a theoretical basis for the application of this protein in functional foods.

[0013] Furthermore, in step (1) above, the analysis and comparison used the ProtParam database.

[0014] Furthermore, in step (1) above, the enzymes used for virtual digestion are selected from the ExPASy Peptide Cutter and BIOPEP-UWM databases.

[0015] Furthermore, in step (2) above, the activity prediction uses the PeptideRanker online tool.

[0016] Furthermore, in step (2) above, the hydrophilicity prediction uses the Innovagen database.

[0017] Furthermore, in step (2) above, the toxicity prediction was performed using the ToxinPred software.

[0018] Furthermore, in step (3) above, the molecular docking was performed using AutoDock Vina software.

[0019] This invention also claims protection for the use of the above-mentioned wrinkled abalone type I collagen peptide in the preparation of products with anti-glycation, anti-oxidation, and anti-aging properties.

[0020] Furthermore, the aforementioned products include pharmaceuticals, food, and skincare products.

[0021] Furthermore, the aforementioned foods include functional foods, health foods, foods for special medical purposes, or dietary supplements.

[0022] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: 1. The peptide sequence is novel, and the intellectual property rights are clear. This invention identifies two novel bioactive peptide sequences, GAAGDK and DSQSAR, for the first time from type I collagen in *Abalone rotundifolia* (AYL88760.1). Rigorous comparison with the internationally authoritative bioactive peptide database (BIOPEP) confirms that these sequences are reported for the first time, possessing complete originality and independent intellectual property rights, providing a solid guarantee for the exclusive commercialization of the core ingredients.

[0023] 2. Excellent dual biological activity and synergistic mechanism of action Through systematic molecular docking verification, the two peptides of this invention can simultaneously bind efficiently to key antioxidant receptors (such as Keap1) and advanced glycation end product receptor RAGE (PDB: 6XQ5), demonstrating the unique advantage of "one peptide with two effects".

[0024] Anti-glycation (anti-AGE) activity: The peptide forms a stable hydrogen bond network with key residues (threonine THR, serine SER, valine VAL) in the active pocket of the RAGE receptor, which may competitively block the binding of AGEs to RAGE, thereby inhibiting the activation of downstream pro-inflammatory signaling pathways such as NF-κB and intervening in glycation damage at its source.

[0025] Antioxidant activity: By binding to key sites of antioxidant receptors (glycine GLY, threonine THR, valine VAL), peptides may activate the cell's own antioxidant defense system (such as the Nrf2 pathway), effectively scavenging free radicals and breaking the vicious cycle of glycation-oxidation.

[0026] This dual mechanism of action, targeting both the AGE-RAGE axis and oxidative stress, has significant synergistic potential in preventing diabetic complications and delaying skin and body aging.

[0027] 3. The structure fits the activity pattern, and the molecular basis of efficacy is solid. The selected peptides have a scientifically sound amino acid composition that aligns with the structure-activity relationship of highly active peptides.

[0028] 4. Rich in functional amino acids Both peptides contain acidic amino acid residues (aspartic acid D and glutamic acid E). Studies have shown that the carboxyl groups of acidic amino acids can directly scavenge free radicals by donating protons or indirectly exert antioxidant effects by chelating pro-oxidizing metal ions, providing a direct structure-activity relationship explanation for the antioxidant activity of the peptides. Meanwhile, the presence of small-molecule amino acids such as glycine (G) and alanine (A) in the peptides may facilitate their binding to the receptor's active pocket.

[0029] 5. Stable combination mode Molecular docking revealed that the peptide could form abundant hydrogen bonds with both target receptors, and the binding energy was low, suggesting a structural basis for its high activity and high stability in vivo.

[0030] 6. Excellent bioavailability and extremely high safety Easily absorbed: Both GAAGDK and DSQSAR are hexapeptides with small molecular weights, which aligns with the human body's direct absorption characteristics of short peptides. Both peptides are predicted to have good water solubility, ensuring their stability and bioavailability in functional food or skincare product formulations.

[0031] Safe and non-toxic: Virtual toxicity prediction indicates that all candidate peptides are non-toxic. Their source (abalone collagen) is a traditional edible marine ingredient, possessing extremely high safety for consumption and application.

[0032] 7. The preparation method is highly targeted and the process is reliable. The preparation method of this invention is based on virtual screening results and explicitly recommends using trypsin, pepsin (pH>2), or pepsin (pH>1.3) to enzymatically hydrolyze type I collagen of abalone with a specific sequence. This method simulates the human gastrointestinal digestive environment, efficiently and specifically releasing the target active peptide from the long chain of collagen. The process conditions are mature and easy for industrial production.

[0033] 8. It has broad application prospects and significant market value. The active peptides and enzymatic hydrolysates rich in these peptides of this invention can be widely used in the development of functional foods, health foods, special medical purpose formula foods, and high-end skin care cosmetics with functions such as anti-glycation, anti-oxidation, and delaying aging (especially skin glycation aging). Against the backdrop of an aging population and the growing market demand for "anti-aging" products, this natural dual-effect peptide raw material derived from marine collagen has enormous market potential and social benefits. Attached Figure Description

[0034] Figure 1 Visualize 3D plots of GAAGDK and DSQSAR with antioxidant and anti-glycation receptors; Figure 2 Visualize 2D plots of GAAGDK and DSQSAR with antioxidant and antiglycation receptors. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] In the following examples, the sequences of type I collagen from *Abalone fasciatus* (accession number: AYL88760.1) were obtained from the database of the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / ).

[0037]

[0038] Example 1: Virtual enzymatic hydrolysis of type I collagen from *Abalone fasciata* var. *wrinkledis* The amino acid composition and proportion of type I collagen from *Abalone fasciatus* were analyzed and compared using the ProtParam database (as shown in Table 1). Based on the amino acid composition, the potential of type I collagen from *Abalone fasciatus* for antioxidant and anti-glycation activities was preliminarily determined. Enzymes from the ExPASy Peptide Cutter (https: / / web.expasy.org / peptide_cutter / ) and BIOPEP-UWM (http: / / www.uwm.edu.pl / biochemia / index.php / pl / biopep) databases were selected for virtual enzymatic digestion of collagen from *Abalone fasciatus* (as shown in Table 2).

[0039] Table 1. Amino acid composition of type I collagen from *Abalone fasciatus*.

[0040] As shown in Table 1, the most abundant amino acids in the type I collagen of the wrinkled disc abalone are glycine, proline, and alanine.

[0041] The results of BIOPEP-UWM virtual enzymatic hydrolysis mostly yielded dipeptides, tripeptides, tetrapeptides, and pentapeptides, while ExPASy Peptide produced dipeptides and higher. Since short peptides are easily absorbed and digested by the human body, peptides of hexapeptides and below were selected for summarization. The summarized results are shown in Table 2.

[0042] Table 2. Results of virtual enzymatic hydrolysis of type I collagen from *Abalone fasciata* var. *wrinkledis*.

[0043] Example 2: Screening of bioactive peptides The bioactive peptides obtained from computer-simulated enzymatic hydrolysis were used to predict their activity using the PeptideRanker online tool, the Innovagen database was used to predict the hydrophilicity (solubility) of the peptides, and the ToxinPred software was used to predict their toxicity.

[0044] The results showed that 48 of the type I collagen peptides from the wrinkled abalone had a bioactivity score greater than 0.5; 44 had poor solubility, and 109 had good solubility; all peptides were predicted to be non-toxic.

[0045] Example 3: Molecular docking AutoDock Vina software was used to perform molecular docking on the target peptide library to screen for peptides with potential activity and low receptor binding energy. Subsequently, the sequences of the screened peptides were compared with the active peptides reported in the BIOPEP database. The reported peptides were removed, and finally, novel peptides that had not been reported were selected as the subjects of subsequent research.

[0046] To investigate the anti-glycation activity of type I collagen peptides from *Abalone rotundifolia*, the peptides were molecularly docked with the advanced glycation end products receptor (RAGE) (PDB: 6XQ5). Five candidate peptides with strong binding affinity were screened: WIPDR, DSQSAR, GAAGDK, LQGER, and LGPVGR. Simultaneously, five peptides with strong binding affinity to antioxidant receptors were also identified: GPAGAR, LGPVGR, DSAEGR, GAAGDK, and DSQSAR. The combined docking results indicate that the GAAGDK and DSQSAR peptides possess both good anti-glycation and antioxidant activities.

[0047] Further analysis of key residues in peptide-receptor interactions revealed that the core contributing residues for binding to antioxidant receptors in both peptides are glycine (GLY), threonine (THR), and valine (VAL), while the core contributing residues for binding to RAGE are threonine (THR), serine (SER), and valine (VAL). Furthermore, the stable binding of both types of peptide-receptors depends on hydrogen bonding. Figure 1-2 In addition, both peptides exhibit good solubility.

[0048] Observation of the amino acid composition of these two peptides revealed that both contain acidic amino acid residues. Research indicates that the carboxyl group (-COOH) of acidic amino acids can donate protons (H+). + ), scavenging hydroxyl radicals (OH) and superoxide anions (O2) - Reactive oxygen species (ROS) such as γ-rays and carnosine are also present. Furthermore, carnosine, as a bioactive peptide, possesses both antioxidant and anti-glycation activities and can disrupt AGEs cross-linking. This provides a theoretical basis for the research on GAAGDK and DSQSAR.

[0049] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A type I collagen peptide from *Abalone fasciatus*, characterized in that, The peptide sequences are GAAGDK and DSQSAR.

2. A method for screening type I collagen peptides of *Abalone fasciatus* as described in claim 1, characterized in that, Specifically, the following steps are included: (1) Virtual enzymatic hydrolysis of type I collagen from wrinkled abalone The amino acid composition and proportion of type I collagen from *Abalone fasciatus* were analyzed and compared. Based on the amino acid composition, the potential of antioxidant and anti-glycation activities of type I collagen from *Abalone fasciatus* was preliminarily determined. Enzyme selection was used to perform virtual enzymatic digestion of type I collagen from *Abalone fasciatus*. (2) Screening of bioactive peptides The bioactive peptides obtained from enzymatic hydrolysis were sequentially subjected to activity prediction, hydrophilicity prediction, and toxicity prediction. (3) Molecular docking Molecular docking was performed on the target peptide library to screen out type I collagen peptides of *Abalone scabra* with potential activity and low receptor binding energy. The peptide sequences are GAAGDK and DSQSAR.

3. The method for screening type I collagen peptides from *Abalone fasciatus* according to claim 2, characterized in that, In step (1), the analysis and comparison uses the ProtParam database.

4. The method for screening type I collagen peptides from *Abalone fasciatus* according to claim 2, characterized in that, In step (1), the enzyme used for virtual digestion is selected from the ExPASy Peptide Cutter and BIOPEP-UWM databases.

5. The method for screening type I collagen peptides from *Abalone fasciatus* according to claim 2, characterized in that, In step (2), the activity prediction uses the PeptideRanker online tool.

6. The method for screening type I collagen peptides from *Abalone fasciatus* according to claim 2, characterized in that, In step (2), the hydrophilicity prediction uses the Innovagen database.

7. The method for screening type I collagen peptides of *Abalone fasciatus* according to claim 2, characterized in that, In step (2), the toxicity prediction uses ToxinPred software.

8. The method for screening type I collagen peptides of *Abalone fasciatus* according to claim 2, characterized in that, In step (3), the molecular docking is performed using AutoDock Vina software.

9. The application of the wrinkle-reducing type I collagen peptide as described in claim 1 in the preparation of products with anti-glycation, anti-oxidation, and anti-aging properties.

10. The application according to claim 9, characterized in that, The products include pharmaceuticals, food, and skincare products.

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