A chlorella pyrenoidosa polypeptide and application thereof
Patent Information
- Application Number
- CN202611346875.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-01
- Publication Date
- 2026-11-03
AI Technical Summary
The existing preparation process of Chlorella peptides is not optimized, and the functions and applications are not clearly defined by existing technologies. This limits the separation, purification, and structural-activity relationship of their mechanisms of action. The problem that existing technologies have not solved is how to achieve in-depth analysis of their mechanisms of action and their application in functional foods and efficient utilization.
Protein-nucleated Chlorella peptides were prepared using a complex enzymatic hydrolysis method, which included homogenization, complex enzymatic hydrolysis, enzyme inactivation, centrifugation, freeze drying, dialysis, and reversed-phase high-performance liquid chromatography. Highly immunologically active peptides were identified by combining HPLC-MS/MS and PeptideRanker tools, and the core peptides were determined by AutoDock molecular docking.
The preparation of high-purity Chlorella protein peptides was achieved, which significantly promoted the proliferation of immune cells, especially the VEFYGPDR and RAPGAPGAVP peptides, which showed significant immunomodulatory functions in splenic lymphocytes, T lymphocytes and B lymphocytes stimulated by ConA/LPS.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of polypeptide preparation technology, specifically relating to a protein-nucleated Chlorella polypeptide and its applications. Background Technology
[0002] In recent years, bioactive peptides derived from natural proteins have gradually become a hot topic in the research of immunonutrition and functional foods due to their low toxicity, high biocompatibility, and multi-target immunomodulatory properties. These bioactive peptides can regulate the homeostasis of the body's immune system through multiple levels and pathways, showing significant application potential in the prevention and control of chronic diseases, the repair of immune dysfunction, and adjuvant intervention in tumor treatment. Chlorella proteoglycans, as a microalgae resource rich in high-quality protein, provides an ideal raw material for the development of natural immunomodulatory peptides. Studies have shown that its enzymatic hydrolysis product, Chlorella proteoglycans polypeptide, has significant immunomodulatory effects, enhancing the body's defense function through mechanisms such as regulating immune cell activity, influencing cytokine secretion, and improving immune response pathways, demonstrating application prospects with both nutritional support and immune repair benefits.
[0003] However, current research and technological applications of Chlorella proteoglycans peptides still face significant limitations. First, the preparation process has not yet been precisely optimized. Existing studies mostly use single proteases (such as alkaline proteases or trypsin) for hydrolysis, resulting in low degrees of hydrolysis. Second, the structure-activity relationship remains unclear. Current research lacks systematic isolation, purification, and structural identification of key immunomodulatory fragments in Chlorella proteoglycans peptides, and the immunoregulatory functions of characteristic peptides have not yet been clarified, limiting the in-depth analysis of their mechanisms of action and their efficient utilization. Summary of the Invention
[0004] This invention provides a Chlorella proteinoid polypeptide and its application. The provided Chlorella proteinoid polypeptide has high immunomodulatory activity and can be used to prepare products with immune function and to promote recovery.
[0005] This invention first provides a protein-nucleated Chlorella polypeptide, the preparation method of which includes the following steps:
[0006] 1) Preparation of Chlorella proteinensis powder solution:
[0007] A Chlorella proteinaceae powder solution was prepared by dissolving Chlorella proteinaceae powder in distilled water at a material-to-liquid ratio of 1:4 (mass-volume ratio g / ml).
[0008] 2) Homogenization:
[0009] The prepared Chlorella protein powder solution was homogenized. The homogenizer speed was set to 15,000 rpm and the homogenization was continued for 5 minutes to break the cell wall structure of Chlorella and obtain a uniformly dispersed Chlorella suspension.
[0010] 3) Complex enzyme hydrolysis:
[0011] Adjust the pH of the Chlorella suspension prepared in step 2) to 6.5-7.0; then add papain and flavor protease complex enzyme at 1%-2% of the mass of Chlorella pyrenoidosa powder for enzymatic hydrolysis; set the temperature to 55℃ and the shaking speed to 150 rpm, and maintain the enzymatic hydrolysis reaction for 4 hours;
[0012] The enzyme is a complex of papain and flavor protease, with the mass ratio of papain to flavor protease being 1:1 to 2:1.
[0013] 4) Enzyme inactivation treatment:
[0014] After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolysis system is quickly transferred to a boiling water bath and heated for 10 minutes to denature and inactivate the complex enzyme, thus terminating the enzymatic hydrolysis reaction.
[0015] 5) Centrifugal separation:
[0016] The inactivated enzymatic hydrolysis system was transferred to a high-speed refrigerated centrifuge, and the temperature was set at 4℃ and the speed at 9000 rpm. After centrifugation for 10 min, the supernatant was obtained.
[0017] 6) Supernatant collection and pretreatment:
[0018] The supernatant after centrifugation was filtered using a 0.22 μm aqueous filter membrane; the filtered supernatant was then transferred to a vacuum freeze dryer and freeze-dried for 12 h to obtain crude Chlorella peptide powder.
[0019] 7) Sephadex G-25 gel column chromatography purification:
[0020] Take the crude Chlorella peptide powder obtained in step 6), dissolve it in ultrapure water and prepare a peptide solution of 10 mg / mL, filter it through a 0.22 μm filter membrane and load it onto a Sephadex G-25 gel column after equilibration; continue to use ultrapure water as the mobile phase, set the flow rate to 0.5 mL / min and collect the eluent in the tube;
[0021] 8) Dialysis:
[0022] Desalination was performed using ultrapure water dialysis at 4°C for 24 hours, with the ultrapure water being replaced every 8 hours to remove salt from the system. After dialysis, the product was freeze-dried again to obtain preliminarily purified Chlorella peptide powder.
[0023] 9) Purification by reversed-phase high-performance liquid chromatography (RP-HPLC):
[0024] The preliminarily purified peptide powder obtained in step 8) was dissolved in ultrapure water to prepare a peptide solution of 5 mg / mL. After filtration through a 0.22 μm aqueous filter membrane, 20 μL was injected into the HPLC injection valve. A gradient elution program was used: 0-5 min, 5% B; 5-25 min, 5%-20% B; 25-40 min, 20% B; 40-45 min, 20%-5% B; 45-50 min, 5% B. The corresponding fractions were collected. The solution was then concentrated using a rotary evaporator to remove acetonitrile. The concentrated solution was freeze-dried to obtain high-purity Chlorella peptides.
[0025] Furthermore, the amino acid sequence of the aforementioned Chlorella protein is VEFYGPDR (SEQ ID NO:1) and RAPGAPGAVP (SEQ ID NO:2).
[0026] The present invention also provides the use of the aforementioned Chlorella peptide in the preparation of products for enhancing immunity.
[0027] In another aspect, the present invention provides an article for enhancing immunity, comprising a pharmacologically effective concentration of the aforementioned Chlorella protein polypeptide.
[0028] This invention uses HPLC-MS / MS to identify the sequence of Chlorella proteoglycans polypeptides, and combines PeptideRanker immunoreactivity prediction and AutoDock molecular docking to identify the core highly immunomodulatory peptide. By isolating splenic lymphocytes from SPF-grade ICR mice, the MTT assay was used to detect the effect of the highly immunomodulatory peptide on the proliferation activity of splenic lymphocytes, T lymphocytes, and B lymphocytes under ConA / LPS stimulation. The peptide significantly promoted the proliferation of immune cells, demonstrating its immunomodulatory function. Attached Figure Description
[0029] Figure 1 Figure 1 shows the molecular docking diagrams of the immune peptide and TLR-4 (ID: 5IJD). Figure 2 shows the TLR4 molecular docking diagram of the peptide VEFYGPDR and Figure 3 shows the TLR4 molecular docking diagram of the peptide RAPGAPGAVP. In the figures, purple in (a) and (b) represents the peptide ligand, gray represents the protein residues attached to the peptide ligand, indicating the bonding effect, and (c) represents the type of force of binding between the peptide and TLR4.
[0030] Figure 2 These are mass spectra of immune peptides, where Figure A is the mass spectrum of immune peptide VEFYGPDR and Figure B is the mass spectrum of immune peptide RAPGAPGAVP.
[0031] Figure 3Figure 1 shows the structure of an immune peptide, where Figure A shows the structure of the immune peptide VEFYGPDR and Figure B shows the structure of the immune peptide RAPGAPGAVP.
[0032] Figure 4 The figure shows the effect of immune peptides on the proliferative activity of splenic lymphocytes. Different letters indicate significant differences between different components, P < 0.05.
[0033] Figure 5 Figure A shows the effect of immune peptides on the proliferative activity of T / B lymphocytes, while Figure B shows the effect of immune peptides on the proliferative activity of B lymphocytes. Different letters indicate significant differences between different components, P < 0.05. Detailed Implementation
[0034] The present invention will now be described in detail with reference to specific embodiments.
[0035] Example 1: Preparation of Chlorella peptides with protein nucleus
[0036] Frozen Chlorella powder with a protein nucleus was thawed at -20°C, and visible impurities were removed. The powder was dissolved in distilled water at a material-to-liquid ratio of 1:4 (m / v) and homogenized at 15,000 rpm for 5 minutes in a high-speed homogenizer to break down cell walls and disperse clumps, resulting in a homogeneous Chlorella suspension. A complex enzyme was then prepared by combining papain and flavor protease at a mass ratio of 1:1 to 2:1. The pH of the suspension was adjusted to 6.5-7.0 using 0.01-0.05 mol / L sodium hydroxide solution or 0.01-0.035 mol / L hydrochloric acid solution. The complex enzyme was added at 1%-2% of the substrate mass. The system was then placed in a 55°C water bath and shaker for 4 hours for enzymatic hydrolysis. After the reaction, the system was heated in a boiling water bath for 10 minutes to inactivate the enzyme and terminate the reaction. The system was then transferred to a centrifuge and centrifuged at 4°C and 9,000 rpm for 10 minutes, and the supernatant was collected. The supernatant was filtered through a 0.22 μm aqueous filter membrane and then freeze-dried in a vacuum freeze dryer for 12 h to obtain crude Chlorella peptide powder.
[0037] Crude peptide powder was dissolved in ultrapure water, filtered through a 0.22 μm filter membrane, and loaded onto a Sephadex G-25 gel column. Ultrapure water was used as the mobile phase. The eluent was collected, desalted, and freeze-dried to obtain a preliminarily purified peptide. The preliminarily purified peptide was then dissolved in ultrapure water, filtered through a 0.22 μm filter membrane, and loaded onto a Zorbax SB-C18 column. Gradient elution was performed using ultrapure water as mobile phase A and 100% acetonitrile as mobile phase B. The eluent was collected, transferred to a rotary evaporator for concentration to remove acetonitrile, and finally freeze-dried to obtain high-purity Chlorella protein peptides.
[0038] Example 2: Identification of polypeptide sequences from Chlorella vulgaris
[0039] Sequence identification of *Chlorella vulgaris* peptides was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). A mobile phase of 0.1% formic acid-water / acetonitrile was used, with elution following a specific gradient (2%–80% B, 50 min). Data acquisition was conducted in positive ion mode (scan range 50–1200 m / z, spray voltage 3.60 kV, capillary temperature 320 °C). Subsequently, candidate immunomodulatory peptides with an activity score >0.45 were screened using the PeptideRanker tool, and molecular docking with the TLR4 receptor (PDBID: 5IJD) was performed using AutoDock. Peptides with a binding energy <-7.0 kcal / mol were ultimately selected as potential active peptides.
[0040] Mass spectrometry was used to analyze the mass spectra of the detected peptides. Based on database matching and the characteristics of their molecular weight and amino acid sequence, six candidate immunomodulatory peptides were further screened: QDDVTLR, RAPGAPGAVP, SADPETFAR, VDALELGVR, VEFYGPDR, and VESLDLQLR. Molecular docking results (Table 1) showed that VEFYGPDR and RAPGAPGAVP had the highest immunomodulatory activity scores, 0.4798 and 0.4672, respectively. Their binding energies to TLR4 were -8.8 kcal / mol and -7.4 kcal / mol, respectively, both forming stable complexes. The molecular docking of VEFYGPDR and RAPGAPGAVP with TLR4 (ID: 5IJD) is shown in the figure below. Figure 1 As shown. The binding sites of VEFYGPDR and TLR4 are VAL61, TRP23, ILE46, PHE151, PHE76, PHE121, CYS133, ILE153, LEU54, PHE126, TRY131, and GLU92. Hydrogen bonding is indicated in green, and hydrophobicity is as follows. Figure 1 As shown in (b) of A. Figure 1Image A(c) shows that the binding of VEFYGPDR to TLR4 is primarily dependent on van der Waals forces. The main difference between RAPGAPGAVP and TLR4 is that they bind stably via five hydrogen bonds, with the peptide residues binding to the receptor proteins GLU507, ASN529, THR575, ASN552, ASN484, SER480, GLN505, ASN456, LYS433, ARG434, LEU432, VAL435, LYS458, HIS429, TYR454, and LYS503. Mass spectra of VEFYGPDR and RAPGAPGAVP are shown below. Figure 2 As shown, their molecular compositions are 982.46 Da and 892.50 Da, respectively, and their structures are as follows: Figure 3 As shown, the sequence identification of the Chlorella peptide lays the foundation for further verification of its immunomodulatory function.
[0041] Table 1: Binding Energy of Immunomodulatory Peptides
[0042] sequence TLR4 binding energy (kcal / mol) Activity score QDDVTLR -5.8 0.258047 RAPGAPGAVP -7.4 0.467158 SADPETFAR -6.8 0.567462 VDALELGVR -6.7 0.222769 VEFYGPDR -8.8 0.47976 VESLDLQLR -7.1 0.305191
[0043] Example 3: Immunomodulatory function of Chlorella peptides
[0044] SPF-grade male ICR mice were selected, and cells were aseptically isolated from the spleen. Splenic lymphocyte suspensions were obtained through grinding, centrifugation, erythrocyte lysis, and adhesion purification. T and B lymphocytes were then stimulated with ConA and LPS, respectively, and different immune peptide treatment groups (VEFYGPDR, RAPGAPGAVP, and a mixed peptide of Chlorella protein and nucleus) were established. After 36 hours of culture, the absorbance at 570 nm was measured using the MTT assay to evaluate the effects of each peptide on lymphocyte proliferation activity.
[0045] Depend on Figure 4 It can be seen that the two highly active immunopeptides, VEFYGPDR and RAPGAPGAVP, and the mixed peptide of Chlorella nucleatum (CHP) all significantly promoted the proliferation of splenic lymphocytes, and this effect was concentration-dependent. When the concentration of the immunopeptides increased from 100 μg / mL to 1000 μg / mL, the proliferation rate of splenic lymphocytes induced by VEFYGPDR increased from 177.7% to 243.75%, the proliferation rate induced by CHP increased from 174.78% to 241.15%, and the proliferation rate of RAPGAPGAVP increased from 147.86% to 197.39% within the same concentration range. However, their proliferation activities at all concentrations were significantly lower than those of VEFYGPDR and CHP (P<0.05).
[0046] In the assay of T / B lymphocyte proliferation activity, Figure 5The results showed that all three immunopeptides promoted the proliferation of both T and B lymphocytes in a concentration-dependent manner. At a concentration of 1000 μg / mL, VEFYGPDR promoted the proliferation of T lymphocytes by 121.88% and B lymphocytes by 161.01%; RAPGAPGAVP promoted the proliferation of T lymphocytes by 116.73% and B lymphocytes by 159.88%; and CHP promoted the proliferation of T lymphocytes by 120.60% and B lymphocytes by 160.45%.
[0047] These results confirm the potential of immunopeptides derived from Chlorella proteoglycans, especially two highly active immunopeptides, VEFYGPDR and RAPGAPGAVP, in regulating the body's adaptive immune response, providing key experimental evidence for their application as immunomodulators in the development of functional foods.
Claims
1. A protein-nucleated Chlorella polypeptide, characterized in that, The preparation method of the protein-nucleated Chlorella polypeptide includes the following steps: 1) Preparation of Chlorella proteinensis powder solution: Chlorella pyrenoidosa powder is dissolved in distilled water to prepare Chlorella pyrenoidosa powder solution; 2) Homogenization: The prepared Chlorella powder solution was homogenized to obtain a uniformly dispersed Chlorella suspension. 3) Complex enzyme hydrolysis: Adjust the pH of the Chlorella suspension prepared in step 2) to 6.5-7.0; then add papain and flavor protease complex enzyme at 1%-2% of the mass of Chlorella pyrenoidosa powder for enzymatic hydrolysis. 4) Enzyme inactivation treatment: After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolysis system is quickly transferred to a boiling water bath and heated for 10 minutes to denature and inactivate the complex enzyme, thus terminating the enzymatic hydrolysis reaction. 5) Centrifugal separation: The inactivated enzymatic hydrolysis system was transferred to a high-speed refrigerated centrifuge, and the temperature was set at 4℃ and the speed at 9000 rpm. After centrifugation for 10 min, the supernatant was obtained. 6) Supernatant collection and pretreatment: The supernatant after centrifugation was filtered using a 0.22 μm aqueous filter membrane; the filtered supernatant was then transferred to a vacuum freeze dryer and freeze-dried to obtain crude Chlorella peptide powder. 7) Sephadex G-25 gel column chromatography purification: Take the crude Chlorella peptide powder obtained in step 6), dissolve it in ultrapure water and prepare a peptide solution of 10 mg / mL. After filtering through a 0.22 μm filter membrane, load the solution onto a Sephadex G-25 gel column after equilibration. Continue to use ultrapure water as the mobile phase and set the flow rate to 0.5 mL / min to collect the eluent in the tube. 8) Dialysis: Desalination was performed using ultrapure water dialysis at 4°C for 24 hours, with the ultrapure water being replaced every 8 hours to remove salt from the system; after dialysis, the product was freeze-dried again to obtain preliminarily purified Chlorella peptide powder. 9) C18 reversed-phase high-performance liquid chromatography purification: Take the preliminarily purified peptide powder obtained in step 8), dissolve it in ultrapure water to prepare a peptide solution of 5 mg / mL, filter it through a 0.22 μm aqueous filter membrane, and inject 20 μL into the HPLC injection valve; use a gradient elution program: 0-5 min, 5% B; 5-25 min, 5%-20% B; 25-40 min, 20% B; 40-45 min, 20%-5% B; 45-50 min, 5% B; collect the corresponding components; then concentrate using a rotary evaporator to remove acetonitrile; the concentrated solution is freeze-dried to obtain high-purity Chlorella protein peptides.
2. The Chlorella polypeptide according to claim 1, characterized in that, In step 1), the Chlorella proteinensis powder is dissolved in distilled water at a mass-to-volume ratio of 1:4 (g / ml) to prepare a Chlorella proteinensis powder solution.
3. The Chlorella polypeptide according to claim 1, characterized in that, In step 3), the pH value is adjusted using a 0.01-0.05 mol / L sodium hydroxide solution or a 0.01-0.035 mol / L hydrochloric acid solution.
4. The Chlorella polypeptide according to claim 1, characterized in that, In step 3), the enzymatic hydrolysis temperature is 55°C, the shaking speed is 150 rpm, and the enzymatic hydrolysis reaction is maintained for 4 hours.
5. The Chlorella polypeptide according to claim 1, characterized in that, In step 3), the papain and flavor protease complex enzyme is used, wherein the mass ratio of papain to flavor protease is 1:1 to 2:
1.
6. The Chlorella polypeptide according to claim 1, characterized in that, The amino acid sequence of the polypeptide is SEQ ID NO:1 or SEQ ID NO:
2.
7. The use of the Chlorella peptide according to any one of claims 1-6 in the preparation of articles for enhancing immunity.
8. A product for enhancing immunity, characterized in that, The product contains a pharmacologically effective concentration of the Chlorella protein-nucleated polypeptide as described in any one of claims 1-6.