Phycocyanin peptide as well as screening method and application thereof
Four novel peptides, ASEIAG, LDSVNR, AEQPQL, and AGDASV, were precisely extracted from phycocyanin using virtual enzymatic hydrolysis and molecular docking technology. This solved the problems of single peptide activity and poor targeting in existing phycocyanin technologies, and enabled the efficient extraction of peptides with ACE inhibitory and antioxidant activities, which are suitable for the development of functional foods.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGDONG LABORATORY OF SOUTHERN OCEAN SCIENCE AND ENGINEERING (GUANGZHOU)
- Filing Date
- 2025-12-26
- Publication Date
- 2026-04-21
AI Technical Summary
Existing technologies make it difficult to extract peptides from phycocyanin that are highly active, safe, easily absorbed, and have novel structures, while also possessing dual functions of ACE inhibition and antioxidation. Furthermore, traditional enzymatic hydrolysis methods suffer from problems of blindness and poor targeting.
Using a rational design approach, combined with gastrointestinal digestion simulation and multi-target computer-aided screening, four novel peptides—ASEIAG, LDSVNR, AEQPQL, and AGDASV—were precisely extracted from phycocyanin through virtual enzymatic digestion and molecular docking technology. This process simulated the human digestive process and screened out peptides with clear activity and stable binding ability.
It has achieved efficient and targeted extraction of four novel peptides from phycocyanin, which have clear ACE inhibitory and antioxidant activities, making them suitable for the development of functional foods. It provides a new core material basis and has excellent dual bioactivity and good bioavailability.
Smart Images
Figure CN121895438A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioactive peptide technology, and more specifically to a phycocyanin peptide, its screening method, and its application. Background Technology
[0002] Hypertension and oxidative stress are core risk factors for various chronic diseases such as cardiovascular and cerebrovascular diseases and diabetes. Currently, while commonly used angiotensin-converting enzyme (ACE) inhibitors (such as captopril and enalapril) have proven efficacy, they often have side effects such as dry cough and angioedema. Furthermore, synthetic antioxidants (such as BHT and BHA) also pose potential safety risks. Therefore, identifying bioactive peptides with both antihypertensive and antioxidant functions from natural food sources has become a research hotspot in the fields of functional foods and preventive medicine.
[0003] Phycocyanin, a natural pigment protein derived from cyanobacteria such as Spirulina, is widely used for food coloring and nutritional fortification due to its good water solubility, safety, and proven health benefits such as anti-inflammatory and immunomodulatory effects. Current technologies have involved hydrolyzing phycocyanin using chemical methods or commercial proteases (such as alkaline proteases and flavor proteases) to obtain crude extracts or mixed peptides with single antioxidant or ACE inhibitory activities. However, these methods suffer from drawbacks such as high enzymatic indiscriminate hydrolysis, unclear active peptide sequences, and poor targeting, and the resulting peptides often exhibit single activity and low efficiency. More importantly, novel single peptide sequences with both clearly defined ACE inhibitory and antioxidant activities identified from phycocyanin are extremely rare.
[0004] Therefore, how to extract bifunctional peptides with high activity, high safety, easy absorption and novel structure from phycocyanin is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0005] In view of this, the purpose of the present invention is to provide a phycocyanin peptide and its screening method and application, so as to overcome the shortcomings of the prior art.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: A phycocyanin peptide with the peptide sequences ASEIAG, LDSVNR, AEQPQL, and AGDASV.
[0007] The amino acid sequence of ASEIAG is shown in SEQ ID NO.1 of the sequence listing, specifically: Ala-Ser-Glu-Ile-Ala-Gly, with the Chinese name: Alanine-Serine-Glutamic Acid-Isoleucine-Alanine-Glycine.
[0008] The amino acid sequence of LDSVNR is shown in SEQ ID NO.2 of the sequence listing, specifically: Leu-Asp-Ser-Val-Asn-Arg, and its Chinese name is: Leucine-Aspartic acid-Serine-Valine-Asparagine-Arginine.
[0009] The amino acid sequence of AEQPQL is shown in SEQ ID NO.3 of the sequence listing, specifically: Ala-Glu-Gln-Pro-Gln-Leu, and its Chinese name is: Alanine-Glutamic Acid-Glutamine-Proline-Glutamine-Leucine.
[0010] The amino acid sequence of AGDASV is shown in SEQ ID NO.4 of the sequence listing, specifically: Ala-Gly-Asp-Ala-Ser-Val, and its Chinese name is: Alanine-Glycine-Aspartic Alanine-Serine-Valine.
[0011] This invention, through a rationally designed approach combining gastrointestinal digestion simulation (virtual enzymatic hydrolysis) and multi-target computer-aided screening (molecular docking), precisely extracted four novel, well-defined, and easily absorbed bifunctional peptides—ASEIAG, LDSVNR, AEQPQL, and AGDASV—from the specific amino acid sequence of phycocyanin. This not only breaks through the limitations of traditional enzymatic hydrolysis technology but also provides a novel core material basis for developing next-generation functional foods or ingredients based on precision nutrition for lowering blood pressure and providing antioxidant benefits.
[0012] A method for screening the above-mentioned phycocyanin peptides specifically includes the following steps: (1) Virtual enzymatic hydrolysis of phycocyanin The amino acid composition and proportion of phycocyanin were analyzed and compared. Based on the amino acid composition, the antioxidant activity and ACE inhibition potential of phycocyanin were preliminarily determined. Enzymes were selected to perform virtual enzymatic digestion of phycocyanin. (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 for phycocyanin peptides with potential activity and low receptor binding energy. The peptide sequences were ASEIAG, LDSVNR, AEQPQL, and AGDASV.
[0013] 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 its binding affinity to different receptors (antioxidant receptors and ACE inhibitory receptors), providing a theoretical basis for the application of this protein in functional foods.
[0014] Furthermore, in step (1) above, the analysis and comparison used the ProtParam database.
[0015] Furthermore, in step (1) above, the enzymes used for virtual digestion are selected from the ExPASy Peptide Cutter and BIOPEP-UWM databases.
[0016] Furthermore, in step (2) above, the activity prediction uses the PeptideRanker online tool.
[0017] Furthermore, in step (2) above, the hydrophilicity prediction uses the Innovagen database.
[0018] Furthermore, in step (2) above, the toxicity prediction was performed using the ToxinPred software.
[0019] Furthermore, in step (3) above, the molecular docking was performed using AutoDock Vina software.
[0020] This invention also claims protection for the use of the above-mentioned phycocyanin peptide in the preparation of products that help lower blood pressure, provide antioxidant effects, and delay aging.
[0021] Furthermore, the aforementioned products include pharmaceuticals, food, and skincare products.
[0022] Furthermore, the aforementioned foods include functional foods, health foods, foods for special medical purposes, or dietary supplements.
[0023] 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 four novel bioactive peptide sequences from phycocyanin (AMW27916.1): ASEIAG, LDSVNR, AEQPQL, and AGDASV. Comparison with the internationally authoritative bioactive peptide database (BIOPEP) confirms that these sequences are all reported for the first time, possessing complete independent intellectual property rights, and providing a fundamental guarantee for the exclusive protection of the core components and product development.
[0024] 2. Excellent dual biological activity and a clear synergistic mechanism of action Through systematic molecular docking verification, the four peptides of this invention simultaneously exhibit strong binding affinity with the ACE receptor (PDB: 1O8A) and the key antioxidant receptor Keap1 (both binding energies are in the dominant range).
[0025] ACE inhibition potential: Peptides competitively occupy substrate binding sites by forming multiple hydrogen bonds with key residues in the ACE active pocket (such as tyrosine TYR, glutamic acid GLU, and serine SER), thereby exhibiting potential ACE inhibitory activity and providing a molecular basis for assisting in the regulation of blood pressure.
[0026] Antioxidant potential: The peptide may interfere with the Keap1-Nrf2 interaction and activate the cell’s own antioxidant defense system by binding to key binding sites of the Keap1 protein (such as ARG-415 and ALA-556), especially through the contribution of hydrophobic amino acids (leucine LEU, alanine ALA, and valine VAL).
[0027] This "one peptide, two targets" characteristic allows a single component to simultaneously target two interrelated pathological processes: hypertension and oxidative stress, giving it a unique advantage in synergistic effects in preventing cardiovascular and cerebrovascular diseases.
[0028] 3. It has outstanding structural advantages and possesses the basis for highly active molecules. The selected peptides have a scientifically sound amino acid composition that aligns with the structure-activity relationship of highly active peptides.
[0029] 4. Rich in active amino acids The peptides commonly contain hydrophobic amino acids (such as Leu, Val, and Ile) closely related to ACE inhibitory activity, as well as acidic amino acids (Asp and Glu) related to metal ion chelation and antioxidant activity. LDSVNR also contains the basic amino acid arginine (Arg), further enhancing its binding potential to the ACE receptor.
[0030] 5. Stable combination mode Molecular docking revealed that the peptide could form a rich network of hydrogen bonds with both receptors, and the binding energy was low, suggesting a structural basis for its high activity in vivo.
[0031] 6. Good bioavailability and safety Easily absorbed: All four peptide segments are hexapeptides with small molecular weights, which aligns with the direct absorption characteristics of short peptides in the human gastrointestinal tract. All peptide segments are predicted to have good water solubility, ensuring stable dispersion in functional food systems and effective delivery within the body.
[0032] High safety: Virtual toxicity predictions indicate that these peptides are non-toxic, derived from recognized safe food-grade raw materials (spirulina), and are safe for long-term consumption.
[0033] 7. The preparation method is highly efficient and has strong targeting. 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 phycocyanin with specific sequences. This method simulates the human digestive process, efficiently and specifically releasing the target active peptide from the raw protein, with mild process conditions and easy industrialization.
[0034] 8. It has broad application prospects and high market value. The active peptides and enzymatic hydrolysates rich in these peptides of the present invention can be directly used to develop functional foods, health foods, special medical purpose formula foods, or dietary supplements with functions such as assisting in lowering blood pressure, anti-oxidation, and delaying aging. Derived from natural phycocyanin and possessing dual effects, they highly align with current consumer demands for "medicine and food from the same source" and "multi-functional" health products, possessing enormous market potential and social benefits. Attached Figure Description
[0035] Figure 1 Visualize 3D plots of SEIAG, LDSVNR, AEQPQL, AGDASV with ACE receptors and antioxidant receptors; Figure 2 Visualize SEIAG, LDSVNR, AEQPQL, AGDASV with ACE receptors and antioxidant receptors in 2D. Detailed Implementation 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, sequences such as phycocyanin (accession number: AMW27916.1) were obtained from the database of the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / ).
[0037] The amino acid sequence of phycocyanin is shown in SEQ ID NO.5 of the sequence listing, specifically: MFDAFTKVVSQADTRGEMLSTAQIDALSQMVAESNKRLDSVNRITSNASTIVSNAARSLFAEQPQLIAPGGNAYTSRRMAACLRDMEIILRYVTYAVFAGDASVLEDRCLNGLRETYLALGTPGSSVAVGVGKMKEAALAIVNDPAGITPGDCSALASEIAGYFDRAAAAVS.
[0038] Example 1: Virtual enzymatic hydrolysis of phycocyanin The amino acid composition and proportion of phycocyanin were analyzed and compared using the ProtParam database (as shown in Table 1). Based on the amino acid composition, the antioxidant activity and ACE inhibition potential of phycocyanin were preliminarily determined. Enzymes from the ExPASyPeptide Cutter (https: / / web.expasy.org / peptide_cutter / ) and BIOPEP-UWM (http: / / www.uwm.edu.pl / biochemia / index.php / pl / biopep) databases were selected to perform virtual enzyme digestion of phycocyanin (as shown in Table 2).
[0039] Table 1 Amino acid composition of phycocyanin
[0040] As shown in Table 1, the most abundant amino acids in phycocyanin are alanine, serine, and leucine. The content of hydrophobic amino acids (alanine and leucine) and branched-chain amino acids (leucine) are positively correlated with ACE inhibition rate and antioxidant activity (FRAP and DPPH scavenging).
[0041] The results of BIOPEP-UWM virtual enzymatic hydrolysis were mostly dipeptides, tripeptides, tetrapeptides, and pentapeptides, while ExPASy Peptide yielded dipeptides and above. 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 phycocyanin
[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 hydrophilicity (solubility) of the peptides was predicted using the Innovagen database, and their toxicity was predicted using the ToxinPred software.
[0044] The results showed that only 6 peptides obtained from phycocyanin had a bioactivity score greater than 0.5; 21 had poor solubility and 23 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 those of active peptides reported in the BIOPEP database. The reported peptides were removed, and novel peptides that had not been reported were selected as the subjects of subsequent research.
[0046] To further verify the binding ability of the obtained peptides to different receptors, all peptides were docked with receptors. The results showed that the binding energies of all peptides to both receptors were in the range of -4 to -8 kcal / mol. The top five peptides with the highest binding energy to the ACE receptor (PDB: 1O8A) were ASEIAG, LDSVNR, AEQPQL, AGDASV, and DMEII; the top five peptides with the highest binding energy to the antioxidant receptor were ASEIAG, LDSVNR, AEQPQL, AGDASV, and AAAAVS. Based on the combined binding energy data, it is clear that ASEIAG, LDSVNR, AEQPQL, and AGDASV exhibit outstanding binding ability to both receptors, making them superior candidate peptides with dual receptor binding activity. Furthermore, all four peptides showed good solubility.
[0047] Further analysis of the interaction mechanisms revealed that when the four dominant peptides bind to the ACE receptor, tyrosine (TYR), glutamic acid (GLU), and serine (SER) are the main contributing amino acids, and the interaction is primarily through hydrogen bonding. However, when binding to the antioxidant receptor, leucine (LEU), alanine (ALA), and valine (VAL) are the main contributing amino acids, and stable binding also relies on hydrogen bonding. Figure 1-2 The biological activity of peptides is closely related to their amino acid sequence and structural conformation.
[0048] Amino acid composition analysis showed that all four dominant peptides contained acidic amino acids (D / E) and hydrophobic amino acids (Ala, Leu, Val, Ile, Pro), while only LDSVNR contained the basic amino acid Arg(R). Combined with related studies, it is known that acidic amino acids can bind to Zn. 2+ Fe 2+ Antioxidant metal ions exert their antioxidant effects. ACE inhibitory activity is mainly related to the content of hydrophobic amino acids (such as Leu and Val) and basic amino acids (Lys and Arg) in the peptide. Furthermore, molecular docking studies indicate that peptides containing hydrophobic amino acids (Leu and Val) and aromatic amino acids (Tyr and Phe) are more likely to bind to ACE or Keap1; the more hydrogen bonds and the lower the binding energy, the stronger the peptide activity generally is. ARG-415 and ALA-556 are key binding residues when peptides bind to the Keap1 receptor.
[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 phycocyanin peptide, characterized in that, The peptide sequences are ASEIAG, LDSVNR, AEQPQL, and AGDASV.
2. A method for screening phycocyanin peptides as described in claim 1, characterized in that, Specifically, the following steps are included: (1) Virtual enzymatic hydrolysis of phycocyanin The amino acid composition and proportion of phycocyanin were analyzed and compared. Based on the amino acid composition, the antioxidant activity and ACE inhibition potential of phycocyanin were preliminarily determined. Enzymes were selected to perform virtual enzymatic digestion of phycocyanin. (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 for phycocyanin peptides with potential activity and low receptor binding energy. The peptide sequences were ASEIAG, LDSVNR, AEQPQL, and AGDASV.
3. The method for screening phycocyanin peptides according to claim 2, characterized in that, In step (1), the analysis and comparison uses the ProtParam database.
4. The method for screening phycocyanin peptides 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 phycocyanin peptides according to claim 2, characterized in that, In step (2), the activity prediction uses the PeptideRanker online tool.
6. The method for screening phycocyanin peptides according to claim 2, characterized in that, In step (2), the hydrophilicity prediction uses the Innovagen database.
7. The method for screening phycocyanin peptides according to claim 2, characterized in that, In step (2), the toxicity prediction uses ToxinPred software.
8. The method for screening phycocyanin peptides according to claim 2, characterized in that, In step (3), the molecular docking is performed using AutoDock Vina software.
9. The application of the phycocyanin peptide as described in claim 1 in the preparation of products that assist in lowering blood pressure, anti-oxidation, and delaying aging.
10. The application according to claim 9, characterized in that, The products include pharmaceuticals, food, and skincare products.