KFTPAP, an antioxidant polypeptide derived from Haematococcus pluvialis, its preparation method and application

Abstract

This invention discloses an antioxidant polypeptide KFTPAP derived from Haematococcus pluvialis, its preparation method, and its applications, belonging to the field of antioxidant polypeptide technology. Using the residue from Haematococcus pluvialis after astaxanthin extraction as raw material, this invention isolates and purifies an antioxidant polypeptide KFTPAP with the amino acid sequence Lys-Phe-Thr-Pro-Ala-Pro, a molecular weight of 659.36 Da, high purity, good activity, and safety without toxicity. Testing shows that the antioxidant polypeptide derived from Haematococcus pluvialis of this invention can significantly improve the antioxidant capacity of *C. elegans*, inhibit fat biosynthesis, and has strong antioxidant and fat-reducing effects. Simultaneously, this invention facilitates the resource utilization of Haematococcus pluvialis residue.

Description

Technical Field

[0001] This invention belongs to the field of antioxidant peptide technology, specifically relating to an antioxidant peptide KFTPAP derived from Haematococcus pluvialis, its preparation method, and its application. Background Technology

[0002] Haematococcus pluvialis is a marine microalgae with extremely high nutritional value. Currently, the global annual production of Haematococcus pluvialis reaches 545 tons, and my country is one of the world's major producers of Haematococcus pluvialis astaxanthin. Astaxanthin can be extracted from Haematococcus pluvialis; however, the large-scale production of astaxanthin from Haematococcus pluvialis leaves behind a large amount of Haematococcus pluvialis residue. Currently, the residue is either used as animal feed or discarded directly. However, Haematococcus pluvialis residue contains rich nutritional value, with a protein content of 20.8%–22.2%, indicating that there is still high added value in the residue worth exploring. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides an antioxidant polypeptide, KFTPAP, derived from Haematococcus pluvialis, which can be obtained from Haematococcus pluvialis residue. The invention involves first extracting proteins from the Haematococcus pluvialis residue, then performing enzymatic hydrolysis. Using ABTS free radical scavenging capacity as an activity tracking indicator, the polypeptide is separated and purified using ultrafiltration, preparative liquid chromatography, and analytical liquid chromatography. The resulting polypeptide, KFTPAP, exhibits very high antioxidant properties, which are verified through biological experiments. This invention can be used as an antioxidant or lipid-lowering drug, facilitating the resource utilization of Haematococcus pluvialis residue.

[0004] This invention is achieved using the following technical solution:

[0005] The amino acid sequence of KFTPAP, an antioxidant polypeptide derived from Haematococcus pluvialis, is: Lys-Phe-Thr-Pro-Ala-Pro(KFTPAP).

[0006] The antioxidant polypeptide KFTPAP of this invention is obtained by extracting protein from Haematococcus pluvialis residue, followed by enzymatic hydrolysis and purification of the protein.

[0007] The antioxidant polypeptide KFTPAP derived from Haematococcus pluvialis of this invention can be used as an antioxidant or lipid-lowering drug.

[0008] Compared with the prior art, the present invention has the following beneficial effects:

[0009] The antioxidant polypeptide KFTPAP derived from Haematococcus pluvialis of this invention has a molecular weight of 659.36 Da, high purity, good activity, and is safe and non-toxic. Testing showed that the antioxidant polypeptide derived from Haematococcus pluvialis of this invention can significantly improve the antioxidant capacity of *C. elegans*, inhibit lipid biosynthesis, and has strong antioxidant and fat-reducing effects. At the same time, this invention facilitates the resource utilization of Haematococcus pluvialis residue. Attached Figure Description

[0010] Figure 1 The ABTS scavenging activity of ultrafiltration components F1, F2 and F3;

[0011] Figure 2 Liquid chromatograms (A) and ABTS scavenging activity (B) of components I, II, III, IV, V and VI;

[0012] Figure 3 Liquid chromatograms (A) and ABTS scavenging activity (B) of components i, ii and iii;

[0013] Figure 4 The effect of different concentrations of HPp on SOD enzyme activity in Caenorhabditis elegans;

[0014] Figure 5 The effect of different concentrations of HPp on the CAT enzyme activity of Caenorhabditis elegans;

[0015] Figure 6 The effect of different concentrations of HPp on the MDA content of Caenorhabditis elegans;

[0016] Figure 7 The effect of different concentrations of HPp on lipid content in Caenorhabditis elegans;

[0017] Figure 8 The effect of different concentrations of HPp on the TG content of Caenorhabditis elegans. Detailed Implementation

[0018] To make the objectives, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of the present invention will be thorough and complete.

[0019] Materials: The substrate remaining after extracting astaxanthin from Haematococcus pluvialis by Jingzhou Natural Astaxanthin Co., Ltd. (Haematococcus pluvialis residue). Other experimental materials are shown in Table 1.

[0020] Table 1 Reagent List

[0021]

[0022]

[0023] Example 1

[0024] A method for obtaining antioxidant peptides from Haematococcus pluvialis includes the following steps:

[0025] (1) Enzymatic hydrolysis

[0026] Haematococcus pluvialis protein extracted from Haematococcus pluvialis residue was diluted with water to prepare a 9% (w:v) protein solution. 0.7% (w:w) alkaline protease (subtilisin) was added, and the pH of the solution was adjusted to 11.5. The enzymatic hydrolysis temperature was 40℃, and the hydrolysis time was 3 hours. After hydrolysis, the solution was centrifuged, the supernatant was collected, and freeze-dried to obtain the Haematococcus pluvialis hydrolysate. The extraction method of Haematococcus pluvialis protein is as follows:

[0027] Astaxanthin-extracted Haematococcus pluvialis residue was mixed with 0.05 mol / L PBS buffer at a ratio of 1 g: 20 mL. The pH was then adjusted to 11.5, and the mixture was heated in a water bath at 56 °C with stirring for 30 min. After centrifugation and filtration, the supernatant was collected, and the pH of the supernatant was adjusted to 4.2. The mixture was then allowed to stand overnight at 4 °C. After centrifugation, the solid precipitate was collected (10000 rpm, 4 °C, 15 min). The solid precipitate was reconstituted with water, the pH was adjusted to neutral, and then freeze-dried to obtain Haematococcus pluvialis protein.

[0028] (2) Ultrafiltration

[0029] The Haematococcus pluvialis enzymatic hydrolysate was dispersed in water, then purified by ultrafiltration, and freeze-dried to obtain F1 (molecular weight > 10 kDa), F2 (molecular weight 3-10 kDa), and F3 (molecular weight < 3 kDa) Haematococcus pluvialis enzymatic hydrolysates. The ABTS scavenging ability was then tested. Figure 1 As shown;

[0030] (3) Preparative liquid chromatography separation and purification

[0031] The ultrafiltration-treated Haematococcus pluvialis hydrolysate F3 was dissolved in water to prepare a 1 mg / mL solution. After filtration, the solution was purified using preparative liquid chromatography (HPLC). A 5 mL sample was loaded, and six products (I-VI) with retention times of 8.6-8.9 min, 9.2-9.5 min, 10-10.4 min, 12.8-13 min, 13.4-13.7 min, and 13.7-14.7 min were collected. The chromatograms are shown below. Figure 2 A is displayed; then the ABTS scavenging ability of the above six products is tested, such as... Figure 2 As shown in Figure B, product VI exhibits the highest antioxidant properties.

[0032] The preparative liquid chromatograph used in this instrument employs a SunFire Prep C18 OBD column with dimensions of 19×250 mm and a diameter of 5 μm.

[0033] The chromatographic conditions are as follows:

[0034] Mobile phase A is a 0.1% (v / v) trifluoroacetic acid solution, and mobile phase B is acetonitrile;

[0035] Linear gradient elution program: 0-18 min, phase B concentration 10-32.5%;

[0036] Flow rate 5 mL / min;

[0037] The detection wavelength is 214nm;

[0038] (4) Analytical liquid chromatography separation and purification

[0039] The product VI collected in step (3) was reconstituted with water to prepare a 0.5 mg / mL solution. After filtration, it was purified a second time using an analytical liquid chromatograph with a sample loading volume of 20 μL. Products i, ii, and iii with retention times of 4.75–5.2 min, 6–6.75 min, and 7.25–7.75 min were collected. Figure 3 As shown in Figure A, the ABTS scavenging ability of the three products was then tested. Figure 3 As shown in Figure B, product i exhibits the highest antioxidant properties.

[0040] The analytical liquid chromatograph used a Diamonsil C18 column with dimensions of 4.6 × 250 mm and 5 μm. The chromatographic conditions for the secondary purification were as follows:

[0041] Mobile phase A is a 0.1% (v / v) trifluoroacetic acid solution, and mobile phase B is acetonitrile;

[0042] Linear gradient elution program: 0-10 min, B phase concentration 10%, 10-20 min, B phase concentration 10-20%, 20-22 min, B phase concentration 20-10%;

[0043] Flow rate: 0.5 mL / min;

[0044] The detection wavelength is 214nm.

[0045] The present invention performs ABTS scavenging activity analysis on the protease hydrolysate obtained in step (1) and the components F3, VI and i obtained after ultrafiltration, as shown in Table 2.

[0046] Table 2. ABTS scavenging activity of different purified polypeptide fractions derived from Haematococcus pluvialis.

[0047]

[0048] Note: The clearance rate in Table 2 is the activity of the substance measured at a concentration of 0.25 mg / mL; the activity factor refers to the increase in ABTS clearance rate of each purified component compared to the protease hydrolysate.

[0049] Example 2

[0050] The composition and sequence of antioxidant peptide component i from Haematococcus pluvialis were determined by HPLC-MS / MS.

[0051] Chromatographic conditions: Liquid chromatography was performed using an Easy nLC 1200 nanoliter liquid chromatography system (Thermo Fisher, USA), with a C18 reversed-phase column (Acclaim PepMap RSLC, 75μm×25cm C18-2μm). Mobile phase A: 0.1% formic acid / water; Mobile phase B: 80% acetonitrile, 0.1% formic acid; Gradient elution program: 0-60 min, 5-38% B.

[0052] Mass spectrometry conditions: The ThermoFisher Q Exactive system (ThermoFisher, USA) was used in combination with a nano-spray Nano Flex ion source (ThermoFisher, USA), with a spray voltage of 1.9 kV and an ion transfer tube heating temperature of 275 °C.

[0053] The detection method of this invention has a scanning range of 100-1500 m / z, and data processing and de novo analysis are performed using PEAKS Studio software. Ten polypeptide sequences, named i-1 to i-10, were obtained after searching the Uniprot database; their peptide information is shown in Table 3.

[0054] Table 3

[0055]

[0056]

[0057] The PeptideRanker system (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to perform preliminary bioactivity prediction on 10 peptides. Table 4 shows that the activity prediction scores of i-1 to i-10 were all higher than 50, indicating that these peptides had a greater than 50% probability of possessing physiological activity, meeting the preliminary screening criteria. Then, ABTS free radicals were identified as ligands, and the binding between peptides and ligands was simulated using Autodock software, and the minimum binding free energies between each peptide were compared (Table 4). The results showed that the binding free energies of i-1 to i-8 with ABTS were less than 0, indicating that they could spontaneously interact with the ligands under normal conditions; among them, i-1 had the lowest minimum binding energy with ABTS, at -5.01 kcal / mol, indicating that this peptide was relatively most likely to bind to ABTS free radicals and exert a scavenging effect, meaning that i-1 had the strongest antioxidant capacity. Finally, the hexapeptide i-1 was identified as an active peptide of Haematococcus pluvialis and named HPp; and this is the first time that the bioactive peptide KFTPAP has been isolated from a food-derived natural product.

[0058] Table 4

[0059]

[0060] Example 3

[0061] To validate the activity of the bioactive peptide HPp, high-purity HPp (KFTPAP, purity >98%) was synthesized by Shanghai Aminolink Biotechnology Co., Ltd. The antioxidant activity of product i-1 (KFTPAP, i.e., HPp) was evaluated using Caenorhabditis elegans. The specific methods are as follows:

[0062] *C. elegans* was grown on NGM agar plates and incubated at 20°C. Eggs at the same developmental stage were obtained using a lysis method. Synchronization was completed when the fertilized eggs of *C. elegans* reached the L4 stage. 2 mg of the peptide product KFTPAP was dissolved in 378 μL of distilled water and mixed with *E. coli* OP50 to prepare bacterial suspensions with final concentrations of 0, 50, 100, and 200 μM. These suspensions were then inoculated into NGM plates as food for *C. elegans*.

[0063] The preparation parameters for NGM agar medium are as follows (based on 1L):

[0064]

[0065] After shaking well, sterilize at 121℃ for 30 minutes, keep at 80℃ for 15 minutes, and then add the following solution (cholesterol filtration sterilization, the rest high temperature sterilization).

[0066]

[0067] Activity evaluation: After 4 days of treatment with the active ingredient, *C. elegans* were washed with M9 buffer into centrifuge tubes, then ground at low temperature for 10 min, and centrifuged to obtain the nematode homogenate (5000 rpm, 10 min). Three parallel experiments were set up for each treatment method. Following the instructions, the protein content of *C. elegans* in each group was determined using a BCA kit, and the MDA content and SOD and CAT enzyme activities were determined using corresponding kits. Figure 4-6 As shown.

[0068] from Figure 4-6 It can be seen that treatment with three concentrations of HPp resulted in an enhanced trend in the SOD and CAT enzyme activities of *C. elegans*. Specifically, the SOD enzyme activity in the 100 μM HPp treatment group was significantly higher than that in the control group, increasing by 8.04% (P<0.05); the CAT enzyme activity in nematodes treated with 100 and 200 μM HPp concentrations was significantly higher than that in the control group, increasing by 1.56 and 1.13 times, respectively (P<0.05). In summary, HPp can effectively improve the antioxidant enzyme system of *C. elegans* and exert its antioxidant effect.

[0069] C. elegans cultured with low and medium concentrations of HPp can reduce MDA levels in the nematodes to some extent. Notably, culturing C. elegans with 100 μM HPp significantly reduced MDA levels by 38.8% (P<0.05). These results indicate that HPp effectively reduces MDA levels in C. elegans. Comparatively, at lower concentrations, HPp enhanced CAT enzyme activity and inhibited MDA levels more effectively than the previously reported yak bone collagen peptide UU1 (CAT enzyme activity enhancement: 0.33-fold, MDA inhibition rate: 18.78%, see the paper "Novel antioxidant peptides from Yak bones collagen enhanced the capacities of antiaging and antioxidant in Caenorhabditis elegans"). This may be due to HPp's smaller molecular weight, making it more readily absorbed by the human body or more readily interacting with target free radicals, thus exerting stronger antioxidant activity.

[0070] Therefore, it is evident that the Haematococcus pluvialis antioxidant peptide HPp can significantly improve the antioxidant capacity of *C. elegans*, exhibiting a strong antioxidant effect, with the optimal effect observed at a concentration of 100 μM. This invention lays the foundation for the future application of antioxidant peptides derived from Haematococcus pluvialis.

[0071] The method for determining the ABTS scavenging activity of this invention is as follows:

[0072] Prepare ABTS stock solution (final concentration of 7 mmol / L ABTS and 2.45 mmol / L potassium persulfate) and let it stand overnight at room temperature in the dark. Before measurement, dilute with 0.01 mol / L PBS buffer (pH 7.4) to prepare ABTS working solution, so that the absorbance of the solution at 734 nm is 0.70 ± 0.05. For measurement, add 100 μL of ABTS working solution and 100 μL of the test sample to a 96-well plate, and let it stand at room temperature in the dark for 10 min, then measure the absorbance at 734 nm (A1); at the same time, the blank group value (A0) is obtained by reacting 100 μL of ABTS working solution with 100 μL of tertiary water, and the control group (A2) is obtained by mixing 100 μL of the test sample with 100 μL of PBS buffer.

[0073]

[0074] In the formula: A0 represents the absorbance of the blank; A1 represents the absorbance of the sample; A2 represents the absorbance of the control.

[0075] Example 4

[0076] The lipid-lowering activity of the antioxidant peptide HPp of this invention was determined using Caenorhabditis elegans. The specific experiments are as follows:

[0077] (1) Activity evaluation

[0078] Lysed eggs were cultured on high-glucose NGM medium (containing 10 mM glucose). *E. coli* OP50 bacterial suspensions containing 0, 50, 100, and 200 μM HPp were added to the medium. Nematodes treated with the drug for 3 days (approximately 1000 nematodes, 1 mL suspension) were washed with M9 buffer and anesthetized for 20 min with 1% NaN3 solution added at 10% v / v. The precipitate was fixed with 1 mL of 4% paraformaldehyde solution, followed by three freeze-thaw cycles. The fixative was washed with PBS buffer, reserving 1 mL of the sample solution. An equal volume of 60% isopropanol was added, and the mixture was centrifuged (1500 rpm, 4°C, 20 min), retaining the precipitate. The isopropanol was washed with PBS buffer, reserving 500 μL of the sample solution. 1 mL of Oil Red O staining working solution was added, and the mixture was stained with shaking in the dark for 12 h (400 rpm, 25°C, 12 h). After staining, the staining solution and isopropanol were washed away sequentially with 60% isopropanol and three-stage water (3000 rpm, 20 s) until the supernatant was a clear, pale red. Images of the nematodes (bright field, 10× objective) were captured using an electron microscope CX41 (Guangzhou Mingmei Optoelectronic Technology Co., Ltd.). ImageJ software was used to analyze the staining intensity in the images to quantify the degree of lipid deposition in the nematodes. Three parallel experiments were conducted for each treatment method, with at least 15 nematodes photographed in each experiment.

[0079] Following the instructions, the protein content of each group of *C. elegans* was determined using the BCA kit, and the TG content in the nematodes was determined using the TG kit.

[0080] (2) Preparation of staining working solution

[0081] To prepare a 5 mg / mL Oil Red O stock solution, add 10 mL of isopropanol to 0.05 g of Oil Red O dye (CAS: 1320-06-5) and dissolve it in the dark at 50-60 °C for 20 min. Mix the Oil Red O stock solution and primary water at a ratio of 3:2 (v:v) (5000 rpm, 4 °C, 1 min). Filter the supernatant through a 0.25 μm syringe filter to obtain the Oil Red O working solution. Finally, mix the Oil Red O working solution with 2% Triton X-100 at a ratio of 1:1 to obtain the Oil Red O staining working solution.

[0082] (3) Results Analysis

[0083] 1. HPp reduces the fat content of high-lipid nematodes.

[0084] Lipids in nematodes are synthesized in the intestinal epithelial cells. Oil Red O dye can specifically interact with and stain neutral lipid molecules, allowing for direct observation and quantitative analysis of lipid content in *C. elegans*. Therefore, high-lipid nematodes were cultured in a high-glucose medium, and the drug-treated nematodes were fed different concentrations of *HPp*. The effect of *HPp* on lipid content in the nematodes was then observed using Oil Red O staining.

[0085] The staining intensity of nematodes was quantified using Imagej software. Figure 7 B) This was used to determine the fat content of each treatment group. The results showed that the fat content of the high-fat nematodes was significantly higher than that of the normal group (P<0.05), indicating that the high-fat model was successfully established. After ingesting 50, 100, and 200 μM HPp, the fat content of the high-fat nematodes was significantly lower than that of the groups that did not ingest the drug (P<0.05). Figure 7 A); significant differences were also found among the drug groups, with the 100 μM group having the lowest fat content (P<0.05), which was 12.97% lower than the high-fat group. The results indicate that all three concentrations of HPp treatment effectively reduced the fat content of nematodes in the high-fat group.

[0086] 2. HPp reduces the triglyceride content of high-lipid nematodes.

[0087] Triglycerides (TG) are the main storage form of fat in nematodes, and their content is strongly correlated with the results of Oil Red O staining. Therefore, by measuring the TG content of each group of nematodes, we further verified the effect of HPp on fat accumulation in nematodes.

[0088] like Figure 8As shown, the TG content of nematodes in the high-fat group was significantly higher than that in the normal group (P<0.05), indicating that the high-fat model was successfully established. After ingesting 50 and 100 μM HPp, the TG content in the high-fat nematodes was significantly lower than that in the groups without drug ingestion (P<0.05), decreasing by 18.48% and 19.62%, respectively; while the 200 μM treatment group showed no significant change compared to the model group (P>0.05). The results indicate that 50 and 100 μM HPp treatment can effectively reduce the TG content of nematodes in the high-fat group. The TG experiment results were largely consistent with the Oil Red O staining results, showing a high correlation.

[0089] In summary, HPp can effectively reduce lipid deposition in high-lipid nematodes and exert a lipid-lowering effect, with the best effect observed at a concentration of 100 μM.

[0090] The effects of isorhamnetin and caffeol on TG content in nematodes were compared in "Fat-lowering effects of isorhamnetin are via NHR-49-dependent pathway in Caenorhabditis elegans" and "Kahweol Reduces Food Intake of Caenorhabditis elegans", as shown in Table 5.

[0091] Table 5

[0092] HPp 100μM 12.97％ 19.62％ Rhamnetin 100μM Undetermined 17％ coffee alcohol 120μM Undetermined 17％

[0093] Table 5 shows that HPp reduced the TG content of nematodes by a higher rate than isorhamnetin (17%) and caffeol (17%), indicating that HPp has a stronger fat-reducing effect. This invention lays the foundation for the future application of fat-reducing peptides derived from Haematococcus pluvialis.

[0094] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. An antioxidant polypeptide KFTPAP derived from Haematococcus pluvialis, characterized in that, The amino acid sequence of the antioxidant polypeptide KFTPAP is: Lys-Phe-Thr-Pro-Ala-Pro.

2. The use of the antioxidant polypeptide KFTPAP derived from Haematococcus pluvialis according to claim 1, characterized in that, As an antioxidant.

3. The use of the antioxidant polypeptide KFTPAP derived from Haematococcus pluvialis according to claim 1 for the preparation of lipid-lowering drugs.

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