A short peptide, a polypeptide extract of Chlorella pyrenoidosa and its preparation method and application

Abstract

The present invention provides a short peptide, a polypeptide extract of Chlorella pyrenoidosa and its preparation method and application, belonging to the technical field of functional polypeptides. The present invention can overcome the problem that the performance of microalgae polypeptide lipid-lowering products in the prior art needs to be improved. The short peptide FLGPF provided by the present invention has a small molecular weight and a high inhibitory rate on the activity of pancreatic lipase. The content of the short peptide FLGPF in the polypeptide extract of Chlorella pyrenoidosa obtained by the preparation method of the present invention can reach 7.1-8.6%, and has good application prospects. The polypeptide extract of Chlorella pyrenoidosa provided by the present invention can significantly reduce the contents of triglyceride and cholesterol in Caenorhabditis elegans, indicating that it has good application prospects in the development of weight loss functional foods or health products. The present invention provides technical support for the development of safe and effective pancreatic lipase inhibitors, and also has important significance for promoting the development of the Chlorella industry.

Description

Technical Field

[0001] The present invention relates to the technical field of functional polypeptides, and particularly relates to a short peptide, a polypeptide extract of Chlorella pyrenoidosa and a preparation method and application thereof. Background Art

[0002] Chlorella belongs to Chlorophyta, Chlorophyceae, Chlorococcales, Oocystaceae, Chlorella, and is a kind of ubiquitous unicellular green algae with extremely wide ecological distribution, fast growth rate, easy cultivation and high application value. Chlorella pyrenoidosa contains a large amount of protein and chlorophyll, with contents as high as 42-58% and 3-5% of dry weight respectively. Its amino acid composition is higher than the protein standard for human nutrition promulgated by the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO), and it is a high-quality protein resource with safe edible property. Many biological activities of Chlorella protein and its hydrolyzed polypeptides have been reported, mainly concentrated in antioxidant activity, anti-tumor, hypoglycemic, hypotensive, mineral chelation and other aspects.

[0003] With the continuous improvement of people's material life, unhealthy high-fat diets and bad living habits induce the occurrence of various chronic diseases. Investigations show that the proportion of adults with lipid metabolism disorders in China is as high as 30%. Currently, commonly used lipid-lowering drugs have great hepatotoxicity and unsatisfactory curative effects on secondary lipid metabolism disorders. Searching for natural biological resources with the effect of improving lipid metabolism and developing nutritional health foods and biopharmaceuticals with high efficiency and low toxicity for regulating blood lipid have become the current global focus and research hotspots.

[0004] In recent years, many studies have shown that bioactive peptides derived from natural proteins have significant blood lipid-lowering effects, with long-lasting drug effects and small side effects. Moreover, the active peptide has a small molecular weight, good cell permeability and is easily digested and absorbed by the human body. As a natural active substance that can effectively improve the blood lipid balance in the body, it has attracted the attention of many scholars and also shows great potential in high-tech industries such as functional foods, health products and drugs. At present, the existing microalgae-derived polypeptides have relatively large molecular weights, and their performance in terms of bioavailability still needs to be improved. Summary of the Invention

[0005] The purpose of the present invention is to provide a short peptide, a polypeptide extract of Chlorella pyrenoidosa and a preparation method and application thereof, so as to solve the problem that the performance of existing polypeptide lipid-lowering products needs to be improved.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a short peptide, and the amino acid sequence of the short peptide is shown in SEQ ID NO.1.

[0008] The present invention also provides the use of the above-mentioned short peptide in the preparation of a product for inhibiting pancreatic lipase activity.

[0009] The present invention also provides a polypeptide extract of Chlorella pyrenoidosa, which contains the above-mentioned short peptide.

[0010] The present invention also provides a preparation method of the above-mentioned polypeptide extract of Chlorella pyrenoidosa, which comprises the following steps:

[0011] Subjecting the Chlorella protein solution to enzymatic hydrolysis, enzyme inactivation, and centrifugation in sequence to obtain a supernatant;

[0012] Subjecting the supernatant to freeze-drying and ultrafiltration in sequence to obtain an ultrafiltration product;

[0013] Subjecting the ultrafiltration product to vacuum concentration, freeze-drying, and purification by Sephadex gel chromatography in sequence to obtain the polypeptide extract of Chlorella pyrenoidosa.

[0014] Preferably, the Chlorella protein solution uses water as a solvent, and the concentration of the Chlorella protein solution is 10-20 g / kg;

[0015] The enzymatic hydrolysis uses papain;

[0016] Calculated by the mass of Chlorella protein, the dosage of papain is 4000-8000 U / g;

[0017] The temperature of the enzymatic hydrolysis is 40-60 °C;

[0018] The time of the enzymatic hydrolysis is 3-6 h.

[0019] Preferably, the temperature of the enzyme inactivation is 90-100 °C;

[0020] The time of the enzyme inactivation is 5-15 min;

[0021] The rotation speed of the centrifugation is 6000-10000 r / min;

[0022] The time of the centrifugation is 10-20 min.

[0023] Preferably, the cold trap temperature for the freeze-drying is -50 to -60 °C, and the vacuum degree for the freeze-drying is 60-100 Pa;

[0024] The molecular weight of the ultrafiltration product is <5 kDa;

[0025] The temperature of the vacuum concentration is 50-60 °C;

[0026] The pressure of the vacuum concentration is 0.08-0.09 MPa.

[0027] Preferably, the main peak F2 is collected after purification and separation by Sephadex gel chromatography, and the polypeptide extract of Chlorella pyrenoidosa is obtained after freeze-drying.

[0028] Preferably, the Sephadex gel used for Sephadex gel chromatography purification has a specification of Sephadex G-25;

[0029] The sample injection volume for Sephadex gel chromatography purification is 1-5% of the column volume, the sample injection concentration is 10-30 mg / mL, the flow rate is 0.5-1.5 mL / min, and the detection wavelength is 220 nm.

[0030] The present invention also provides the application of the above-mentioned polypeptide extract of Chlorella pyrenoidosa in the preparation of lipid-lowering products.

[0031] Technical effects and advantages of the present invention:

[0032] When the concentration of the short peptide FLGPF provided by the present invention is 8 mg / mL, the inhibition rate of pancreatic lipase activity reaches 50.12%. The inhibition type of pancreatic lipase activity is reversible inhibition and is non-competitive inhibition. It can interact with 3 amino acid residues on the human PTL protein, mainly forming π-hydrogen bonds, π-cations and hydrogen bond interactions, and inhibits the activity of pancreatic lipase by occupying the catalytic or substrate binding site. The content of the short peptide FLGPF in the polypeptide extract of Chlorella pyrenoidosa obtained by the preparation method of the present invention can reach 7.1-8.6%. Compared with other ultrafiltration fractions and other main peaks obtained by purification, it can achieve a better inhibitory effect on pancreatic lipase. When the concentration of the polypeptide extract of Chlorella pyrenoidosa provided by the present invention is 8 mg / mL, the inhibition rate of pancreatic lipase activity reaches 42.33%. The inhibition type is reversible inhibition and is non-competitive inhibition. The polypeptide extract of Chlorella pyrenoidosa of the present invention shows a certain lipid-lowering effect on the high-fat model Caenorhabditis elegans, and can significantly reduce the triglyceride and cholesterol contents in the nematodes. It shows that the polypeptide extract of Chlorella pyrenoidosa described in the present invention has good application prospects in the development of weight loss functional foods or health products. The present invention provides technical support for the development of safe and effective pancreatic lipase inhibitors, and also has important significance for promoting the development of the Chlorella industry. Description of the drawings

[0033] Figure 1 It is a Sephadex G-25 gel chromatogram;

[0034] Figure 2 It is the secondary mass spectrum of the short peptide FLGPF (579.3057 Da) obtained by LC-MS / MS identification;

[0035] Figure 3 It is the chromatogram of the synthesized peptide of FLGPF;

[0036] Figure 4 The inhibitory effect of the FLGPF synthetic peptide on pancreatic lipase at different concentrations;

[0037] Figure 5 The type of inhibitory effect of the FLGPF synthetic peptide on pancreatic lipase;

[0038] Figure 6 The double-reciprocal curve of the reversible inhibition of pancreatic lipase by the FLGPF synthetic peptide;

[0039] Figure 7 The two-dimensional (A), surface (B), and three-dimensional (C) binding mode diagrams of FLGPF with human pancreatic lipase (PTL);

[0040] Figure 8 The comparison results of the inhibitory effects of different ultrafiltration fractions on pancreatic lipase;

[0041] Figure 9 The inhibitory effect of the peptide segment purified by Sephadex G-25 gel chromatography on pancreatic lipase;

[0042] Figure 10 The inhibitory effect of the polypeptide extract of Chlorella pyrenoidosa on pancreatic lipase at different concentrations;

[0043] Figure 11 The type of inhibitory effect of the polypeptide extract of Chlorella pyrenoidosa on pancreatic lipase;

[0044] Figure 12 The double-reciprocal curve of the reversible inhibition of pancreatic lipase by the polypeptide extract of Chlorella pyrenoidosa. Detailed implementation mode

[0045] The present invention provides a short peptide, the sequence of the short peptide is FLGPF, that is, Phe-Leu-Gly-Pro-Phe, as shown in SEQ ID NO.1, and the short peptide can be artificially synthesized or naturally formed.

[0046] The present invention also provides the application of the above short peptide in the preparation of a product for inhibiting the activity of pancreatic lipase. The product is preferably a health product, a functional food, and a drug; the product preferably uses the short peptide of the present invention as the sole active ingredient; the product preferably further includes excipients, and the types of the excipients can be fillers, sweeteners or other taste-adjusting excipients, disintegrants, lubricants, binders, coatings, colorants, preservatives, etc.; the pharmaceutical dosage form of the present invention is preferably powder, tablet, granule, capsule, solution, emulsion, suspension or injection, etc., and the functional food is preferably functional beverage, jelly product, candy product, flour product, etc.

[0047] The present invention also provides a polypeptide extract of Chlorella pyrenoidosa, which contains the above-mentioned short peptide, and the content of the above-mentioned short peptide in the polypeptide extract of Chlorella pyrenoidosa is more than 6%.

[0048] The present invention also provides a preparation method of the above-mentioned polypeptide extract of Chlorella pyrenoidosa, which comprises the following steps:

[0049] Enzymatically hydrolyze, inactivate the enzyme, and centrifuge the Chlorella protein solution in sequence to obtain a supernatant;

[0050] Freeze-dry and ultrafilter the supernatant in sequence to obtain an ultrafiltration product;

[0051] Vacuum concentrate, freeze-dry, and purify the ultrafiltration product in sequence to obtain the polypeptide extract of Chlorella pyrenoidosa.

[0052] In the present invention, the Chlorella protein solution preferably uses water as a solvent, the water is preferably pure water, the concentration of the Chlorella protein solution is preferably 10-20 g / kg, more preferably 12-18 g / kg, and the Chlorella protein in the Chlorella protein solution is preferably extracted by the following steps: using Chlorella pyrenoidosa as a raw material, adding pure water according to a mass-volume ratio of 1:48-52, soaking at room temperature for 1-3 h, fully stirring and swelling, then performing repeated freeze-thawing. The repeated freeze-thawing is preferably carried out 4-6 times between -20°C and 37°C. After repeated freeze-thawing, adjust the pH to 11.5-12.5 with 0.5-1.5 mol / L NaOH solution, perform constant-temperature water bath extraction at 60-80°C for 1-3 h, centrifuge at 5000-7000 r / min for 10-20 min, take the supernatant, filter it, adjust the pH to 3-4 with 0.5-1.5 mol / L HCl solution, let it stand at room temperature for 1-3 h, then centrifuge at 7000-9000 r / min for 10-20 min, discard the supernatant, wash the precipitate with pure water until neutral, and perform freeze-drying under the conditions of a cold trap temperature of -50 to -60°C and a vacuum degree of 60-100 Pa to obtain Chlorella protein.

[0053] In the present invention, the chlorella protein solution is enzymatically hydrolyzed. The enzymatic hydrolysis preferably uses papain; the dosage of the papain is preferably 4000 - 8000 U / g, and the dosage refers to the enzyme - substrate ratio [E] / [S], more preferably 5000 - 7000 U / g; the temperature of the enzymatic hydrolysis is preferably 40 - 60 °C, more preferably 45 - 55 °C; the time of the enzymatic hydrolysis is preferably 3 - 6 h, more preferably 4.5 - 5.5 h; the pH of the enzymatic hydrolysis is preferably 5.5 - 6.5; after the enzymatic hydrolysis in the present invention, enzyme inactivation is carried out. The temperature of the enzyme inactivation is preferably 90 - 100 °C, more preferably 95 - 100 °C; the time of the enzyme inactivation is preferably 5 - 15 min, more preferably 8 - 12 min; after the enzyme inactivation, it is preferably cooled to room temperature. The room temperature is preferably 24 - 30 °C. After cooling, centrifugation is carried out. The rotation speed of the centrifugation is preferably 6000 - 10000 r / min, more preferably 7000 - 9000 r / min; the time of the centrifugation is preferably 10 - 20 min, more preferably 13 - 17 min; the supernatant is obtained by centrifugation, and the supernatant is freeze - dried. The cold trap temperature used for the freeze - drying is preferably - 50 - - 60 °C, more preferably - 54 - - 58 °C, and the vacuum degree of the freeze - drying is preferably 60 - 100 Pa, more preferably 70 - 90 Pa; the product of the freeze - drying is ultrafiltered to obtain an ultrafiltered product. The molecular weight of the ultrafiltered product is preferably <5 kDa. The collected ultrafiltered product is vacuum - concentrated. The temperature of the vacuum concentration is preferably 50 - 60 °C, more preferably 53 - 57 °C; the pressure of the vacuum concentration is preferably 0.08 - 0.09 MPa; after the vacuum concentration, freeze - drying is carried out again. The cold trap temperature used for the freeze - drying is preferably - 50 - - 60 °C, more preferably - 54 - - 58 °C, and the vacuum degree of the freeze - drying is preferably 60 - 100 Pa, more preferably 70 - 90 Pa; after the freeze - drying, purification is carried out. The purification preferably uses Sephadex gel chromatography. The main peak F2 is collected after separation and freeze - dried to obtain the polypeptide extract of chlorella pyrenoidosa. In this process, the Sephadex gel specification preferably used is Sephadex G - 25, the injection volume preferably used is 1 - 5% of the column volume, more preferably 2 - 4%, the injection concentration is preferably 10 - 30 mg / mL, more preferably 15 - 25 mg / mL, the flow rate is 0.5 - 1.5 mL / min, more preferably 0.8 - 1.2 mL / min, the detection wavelength is preferably 220 nm, the cold trap temperature used for the freeze - drying is preferably - 50 - - 60 °C, more preferably - 54 - - 58 °C, and the vacuum degree of the freeze - drying is preferably 60 - 100 Pa, more preferably 70 - 90 Pa.

[0054] The present invention also provides the application of the above-mentioned Chlorella pyrenoidosa polypeptide extract in the preparation of lipid-lowering products. The products are preferably health products, functional foods and drugs. The products preferably use the Chlorella pyrenoidosa polypeptide extract of the present invention as the sole active ingredient. The products preferably further include excipients, and the types of the excipients can be fillers, sweeteners or other excipients for adjusting taste, disintegrants, lubricants, binders, coatings, colorants, preservatives, etc. The pharmaceutical dosage forms of the present invention are preferably powders, tablets, granules, capsules, solutions, emulsions, suspensions or injections, etc. The functional foods are preferably functional beverages, jelly products, confectionery products, flour products, etc.

[0055] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0056] Example 1

[0057] (1) Using Chlorella pyrenoidosa as the raw material, pure water was added at a mass-to-volume ratio of 1:50, soaked at room temperature for 2 h, fully stirred and swollen, and then subjected to repeated freezing and thawing. Repeated freezing and thawing was carried out 5 times between -20 °C and 37 °C. The pH was adjusted to 12.0 with 1 mol / L NaOH solution, and the mixture was extracted in a constant temperature water bath at 70 °C for 2 h, centrifuged at 6000 r / min for 15 min. The supernatant was filtered and the pH was adjusted to 3.5 with 1 mol / L HCl solution. After standing at room temperature for 2 h, it was centrifuged at 8000 r / min for 15 min. The supernatant was discarded, and the precipitate was washed with pure water until neutral. Freeze-drying was carried out under the conditions of a cold trap temperature of -50 °C and a vacuum degree of 90 Pa to obtain Chlorella protein.

[0058] (2) Weigh an appropriate amount of Chlorella protein, add pure water to prepare a 15 g / kg Chlorella protein solution, adjust the pH to 6.0, add papain, and the enzyme-substrate ratio [E] / [S] is 6000 U / g. The enzymatic hydrolysis temperature is 50 °C, and the enzymatic hydrolysis time is 5 h. After the enzymatic hydrolysis is completed, the temperature is raised to 100 °C to inactivate the enzyme for 10 min, cooled to room temperature, centrifuged at 8000 r / min for 15 min, and the supernatant is taken to obtain the Chlorella protein hydrolysate. Freeze-drying was carried out under the conditions of a cold trap temperature of -50 °C and a vacuum degree of 90 Pa to obtain the Chlorella protein hydrolysate.

[0059] (3) Ultrafiltration was carried out on the Chlorella protein hydrolysate collected in step (2) with ultrafiltration membranes with a molecular weight cut-off of 10 kDa and 5 kDa to obtain different fractions (>10 kDa, 5-10 kDa, and <5 kDa). The <5 kDa fraction was collected, vacuum concentrated and then freeze-dried. The vacuum concentration parameters were: temperature 55 °C, pressure 0.09 MPa, and the freeze-drying parameters were: cold trap temperature -50 °C, vacuum degree 90 Pa, to obtain the <5 kDa fraction.

[0060] (4) The <5 kDa fraction was further purified by Sephadex gel chromatography. The Sephadex gel was Sephadex G-25, the eluent was ultrapure water, the injection volume was 3% of the column volume, the injection concentration was 20 mg / mL, the flow rate was 1 mL / min, and the detection wavelength was 220 nm. The main peak F2 was collected (the Sephadex G-25 gel chromatogram is as shown in Figure 1 ), and freeze-dried. The freeze-drying parameters were: cold trap temperature -50 °C, vacuum degree 90 Pa, to obtain the Chlorella pyrenoidosa polypeptide extract.

[0061] Example 2

[0062] (1) Using Chlorella pyrenoidosa as the raw material, pure water was added at a mass-volume ratio of 1:30, and it was soaked at room temperature for 2 h and stirred thoroughly to swell, then subjected to repeated freeze-thaw cycles between -20 °C and 37 °C for 4 times. The pH was adjusted to 10.0 with 1 mol / L NaOH solution, and it was extracted in a constant temperature water bath at 50 °C for 4 h, centrifuged at 6000 r / min for 15 min. The supernatant was taken, filtered, and the pH was adjusted to 3.0 with 1 mol / L HCl solution, left standing at room temperature for 2 h, and then centrifuged at 8000 r / min for 15 min. The supernatant was discarded, and the precipitate was washed with pure water until neutral, and freeze-dried under the conditions of cold trap temperature -56 °C and vacuum degree 80 Pa to obtain Chlorella protein.

[0063] (2) An appropriate amount of Chlorella protein was weighed, and a 20 g / kg Chlorella protein solution was prepared by adding pure water. The pH was adjusted to 5.5, papain was added, and the enzyme-substrate ratio [E] / [S] was 5000 U / g. The enzymatic hydrolysis temperature was 55 °C, and the enzymatic hydrolysis time was 4 h. After the enzymatic hydrolysis was completed, the temperature was raised to 100 °C to inactivate the enzyme for 10 min, cooled to room temperature, and centrifuged at 7000 r / min for 15 min. The supernatant was taken to obtain the Chlorella protein hydrolysate, and freeze-dried under the conditions of cold trap temperature -56 °C and vacuum degree 80 Pa to obtain the Chlorella protein hydrolysate.

[0064] (3) The Chlorella protein hydrolysate collected in step (2) was ultrafiltered with ultrafiltration membranes with a molecular weight cut-off of 10 kDa and 5 kDa to obtain different fractions (>10 kDa, 5 - 10 kDa, and <5 kDa). The <5 kDa fraction was collected, vacuum concentrated and then freeze-dried. The vacuum concentration parameters were: temperature 60 °C, pressure 0.09 MPa, and the freeze-drying parameters were: cold trap temperature -56 °C, vacuum degree 80 Pa, to obtain the <5 kDa fraction.

[0065] (4) Further purify the <5 kDa fraction by Sephadex gel chromatography. The Sephadex gel specification is Sephadex G-25, the eluent is ultrapure water, the injection volume is 2% of the column volume, the injection concentration is 25 mg / mL, the flow rate is 1 mL / min, and the detection wavelength is 220 nm. Collect the main peak F2 (the Sephadex G-25 gel chromatogram is as shown in Figure 1 ), freeze-dry it, and the freeze-drying parameters are: cold trap temperature -56 °C, vacuum degree 80 Pa, to obtain the Chlorella pyrenoidosa polypeptide extract.

[0066] Example 3

[0067] (1) Use Chlorella pyrenoidosa as the raw material, add pure water according to the mass-volume ratio of 1:40, soak at room temperature for 2 h, fully stir and swell, then carry out repeated freeze-thawing between -20 °C and 37 °C for 6 times, adjust the pH to 11.0 with 1 mol / L NaOH solution, carry out constant temperature water bath extraction at 60 °C for 3 h, centrifuge at 6000 r / min for 15 min, take the supernatant, filter it, adjust the pH to 4.0 with 1 mol / L HCl solution, let it stand at room temperature for 2 h, then centrifuge at 8000 r / min for 15 min, discard the supernatant, wash the precipitate with pure water until neutral, and freeze-dry it under the conditions of cold trap temperature -58 °C and vacuum degree 70 Pa to obtain Chlorella protein.

[0068] (2) Weigh an appropriate amount of Chlorella protein, add pure water to prepare a 10 g / kg Chlorella protein solution, adjust the pH to 6.5, add papain, the enzyme-substrate ratio [E] / [S] is 4000 U / g, the enzymatic hydrolysis temperature is 60 °C, the enzymatic hydrolysis time is 3 h, after the enzymatic hydrolysis is completed, raise the temperature to 100 °C to inactivate the enzyme for 10 min, cool to room temperature, centrifuge at 6000 r / min for 20 min, take the supernatant to obtain the Chlorella protein hydrolysate, and freeze-dry it under the conditions of cold trap temperature -58 °C and vacuum degree 70 Pa to obtain the Chlorella protein hydrolysate.

[0069] (3) Use ultrafiltration membranes with a molecular weight cut-off of 10 kDa and 5 kDa to ultrafilter the Chlorella protein hydrolysate collected in step (2) to obtain different fractions (>10 kDa, 5 - 10 kDa, and <5 kDa). Collect the <5 kDa fraction, vacuum concentrate it and then freeze-dry it. The vacuum concentration parameters are: temperature 50 °C, pressure 0.09 MPa, and the freeze-drying parameters are: cold trap temperature -58 °C, vacuum degree 70 Pa, to obtain the <5 kDa fraction.

[0070] (4) The <5 kDa fraction was further purified by Sephadex gel chromatography. The Sephadex gel was Sephadex G-25, the eluent was ultrapure water, the sample injection volume was 1% of the column volume, the sample injection concentration was 30 mg / mL, the flow rate was 1 mL / min, and the detection wavelength was 220 nm. The main peak F2 was collected (the Sephadex G-25 gel chromatogram is as shown in Figure 1 ), and freeze-dried. The freeze-drying parameters were: cold trap temperature -58 °C, vacuum degree 70 Pa, to obtain the Chlorella pyrenoidosa polypeptide extract.

[0071] Example 4

[0072] The FLGPF synthetic peptide (FLGPF, as shown in SEQ ID NO.1) was prepared by the Fmoc solid-phase synthesis method and entrusted to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0073] Comparative Example 1

[0074] The difference from Example 1 was only that the Chlorella pyrenoidosa protease hydrolysate collected in step (2) was ultrafiltered with ultrafiltration membranes with a molecular weight cut-off of 10 kDa and 5 kDa to obtain different fractions (>10 kDa, 5-10 kDa, and <5 kDa). The >10 kDa fraction was collected.

[0075] Comparative Example 2

[0076] The difference from Example 1 was only that the Chlorella pyrenoidosa protease hydrolysate collected in step (2) was ultrafiltered with ultrafiltration membranes with a molecular weight cut-off of 10 kDa and 5 kDa to obtain different fractions (>10 kDa, 5-10 kDa, and <5 kDa). The 5-10 kDa fraction was collected.

[0077] Comparative Example 3

[0078] The difference from Example 1 was only that the main peak F1 was collected after further purification by Sephadex gel chromatography.

[0079] Experimental Example 1

[0080] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to detect the content of the FLGPF short peptide in the Chlorella pyrenoidosa polypeptide extracts prepared in Examples 1-3.

[0081] After desalting, the polypeptide sample was centrifuged and dried, then redissolved in 100 μL of Nano-LC mobile phase A (0.1% formic acid / water), bottled and loaded for online LC-MS / MS analysis. The dissolved sample was loaded onto a nanoViper C18 pre-column (3 μm, 100 Å) with a volume of 2 μL, and then rinsed with 20 μL for desalting. The liquid phase was an Easy nLC 1200 nano-liquid phase system (ThermoFisher, USA). After the sample was desalted and retained on the pre-column, it was separated by the analytical column. The specifications of the analytical column were C18 reversed-phase chromatography column (Acclaim PepMap RSLC, 75 μm × 25 cm C18-2 μm 100 Å). The gradient used in the experiment was that the mobile phase B (80% acetonitrile, 0.1% formic acid) increased from 5% to 38% within 30 min. The mass spectrometry was a ThermoFisher Q Exactive system (ThermoFisher, USA) combined with a nano-spray Nano Flex ion source (ThermoFisher, USA). The spray voltage was 1.9 kV, and the heating temperature of the ion transfer tube was 275 °C. The original raw spectrum files collected by mass spectrometry were processed and retrieved using PEAKS Studio 8.5 (Bioinformatics Solutions Inc., Waterloo, Canada) software. The database was the Chlorella pyrenoidosa species protein database downloaded from Uniprot. The retrieval parameters were set as follows: the mass tolerance of the first-order mass spectrometry was 10 ppm, and that of the second-order mass spectrometry was 0.05 Da.

[0082] A total of 999 polypeptides were identified by LC-MS / MS. The secondary mass spectrometry diagram of the identified short peptide FLGPF (579.3057 Da) is as Figure 2 shown. It was obtained by identification that the proportion of the short peptide FLGPF in the total amount of polypeptides in the Chlorella pyrenoidosa polypeptide extract in Example 1 was 7.9%, in Example 2 was 8.6%, and in Example 3 was 7.1%.

[0083] Performance verification of the short peptide FLGPF in Experimental Example 2

[0084] Take the synthetic peptide FLGPF in Example 4. Through high-performance liquid chromatography and mass spectrometry analysis, it was determined that the purity of the synthetic peptide FLGPF was greater than 99%, as Figure 3 shown.

[0085] Determine the pancreatic lipase inhibition rate of the synthetic peptide FLGPF at concentrations of 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 mg / ml. The method is as follows:

[0086] Mix 80 μL of pH 8.0 Tris-HCl buffer, 40 μL of sample solution, and 120 μL of 10 mg / mL pancreatic lipase solution. After mixing evenly, incubate at 37 °C for 10 min. Then immediately add 160 μL of 0.8 mmol / L p-nitrophenyl palmitate (pNPP) as the substrate, and place it at 37 °C for reaction for another 20 min. After the reaction is completed, immediately terminate the reaction by heating in a water bath at 100 °C for 5 min. The terminated reaction solution is centrifuged at 10,000 r / min for 10 min to remove the precipitate. Take 200 μL of the supernatant and transfer it to a 96-well microplate. The microplate reader records the absorbance value at 405 nm. Use orlistat (8 μg / mL) as a positive control and calculate according to the following formula:

[0087]

[0088] In the formula: A: absorbance value of the control test group; B: absorbance value of the sample test group;

[0089] a: absorbance value of the control blank group; b: absorbance value of the sample blank group.

[0090] The results showed that when the concentration of the FLGPF synthetic peptide was 8 mg / mL, the inhibition rate of pancreatic lipase activity reached 50.12%, as Figure 4 shown.

[0091] The inhibition mechanism of the FLGPF synthetic peptide on pancreatic lipase activity was determined as follows:

[0092] 4-Nitrophenol (pNP) standard curve: Prepare a series of pNP solutions with different concentrations (0.01, 0.02, 0.03, 0.04, 0.05 mmol / L), and measure the absorbance values of pNP with different concentrations at 405 nm. With the pNP concentration as the abscissa and the absorbance value as the ordinate, obtain the 4-nitrophenol (pNP) standard curve.

[0093] Prepare solutions of the FLGPF synthetic peptide at 0 mg / mL and 1 mg / mL. Fix the concentration of p-nitrophenyl palmitate (pNPP) at 0.8 mmol / L, and measure the initial velocity of the enzymatic reaction when the mass concentration of pancreatic lipase is 0, 5, 10, 15, 20 mg / mL respectively. The reaction system is the same as the above method for testing the inhibition rate of pancreatic lipase activity. According to the 4-nitrophenol (pNP) standard curve, obtain the content (C: mmol / L) of the corresponding reaction product (pNP), and then calculate the reaction rate of each reaction. The reaction rate V = C / t, where C refers to the content of 4-nitrophenol (pNP) after the reaction, and t refers to the reaction time (min) after adding the FLGPF synthetic peptide solution to p-nitrophenyl palmitate (pNPP). Plot the initial velocity of the enzymatic reaction against the enzyme mass concentration, asFigure 5 As shown, the FLGPF synthetic peptide was formulated into solutions with concentrations of 0 mg / mL, 1 mg / mL, and 2 mg / mL. The concentration of pancreatic lipase was fixed at 10 mg / mL, and the initial velocity of the enzymatic reaction was measured when the concentration of p-nitrophenyl palmitate (pNPP) was 8, 4, 2, and 1 mmol / L. The reaction system was the same as the above-mentioned test method for the inhibition rate of pancreatic lipase activity. A Lineweaver-Burk double-reciprocal curve was plotted with the reciprocal of the reaction rate (1 / v) against the reciprocal of the substrate concentration (1 / [S]), as shown in Figure 6 shown.

[0094] The results showed that the inhibition type of the FLGPF short peptide on pancreatic lipase activity was reversible inhibition and non-competitive inhibition.

[0095] Using the crystal structure of human pancreatic lipase (PTL) as the receptor (PDB: 1LPB), the short peptide FLGPF was flexibly docked with the receptor using MOE software to determine the key amino acid residues and interaction forces between FLGPF and the receptor, and a binding mode diagram was drawn using PyMOL, as shown in Figure 7 shown, where FLGPF is represented as a yellow stick model, the residues around the binding pocket are represented as blue stick models, and the backbone of the receptor is represented as a blue cartoon. The π-hydrogen bond, π-cation, and hydrogen bond interactions are represented by green dashed lines. As can be seen from Figure 7 this, a suitable spatial complementarity was formed at the binding site of FLGPF and the human PTL protein. The nitrogen atom on the backbone of Phe1 of FLGPF formed a hydrogen bond with the oxygen atom on the side chain of Arg79 of the human PTL protein as a hydrogen bond donor. The nitrogen atom on the backbone of Phe1 of FLGPF formed a π-cation interaction with the benzene ring of Trp252 of the human PTL protein. The nitrogen atom on the backbone of Phe5 of FLGPF formed a π-hydrogen bond with the benzene ring of Tyr114 of the human PTL protein. There were also van der Waals forces between FLGPF and the human PTL protein. In summary, the short peptide FLGPF could interact with 3 amino acid residues on the human PTL protein, mainly forming π-hydrogen bonds, π-cation, and hydrogen bond interactions, and inhibiting the activity of pancreatic lipase by occupying the catalytic or substrate binding site.

[0096] Experimental Example 3 Verification of the Performance of the Polypeptide Extract of Chlorella pyrenoidosa

[0097] The different fractions (>10 kDa, 5 - 10 kDa, and <5 kDa) prepared in Example 1, Comparative Example 1, and Comparative Example 2 were tested for the inhibition rate of pancreatic lipase activity. The inhibitory effects of the Chlorella pyrenoidosa protease hydrolysate and each fraction on pancreatic lipase activity were observed at a concentration of 8 mg / mL. The method was the same as in Experimental Example 2, and the results are shown in Figure 8 shown, and the results showed that the <5 kDa fraction provided by the present invention had the best inhibitory effect on pancreatic lipase.

[0098] Respectively take the main peak F2 collected in Example 1 and the main peak F1 collected in Comparative Example 3, and observe the inhibitory effect of the two samples on pancreatic lipase activity at a concentration of 8 mg / mL. The method is the same as that in Experimental Example 2, and the results are as follows: Figure 9 shown, and the results show that the main peak F2 provided by the present invention has a better inhibitory effect on pancreatic lipase.

[0099] Determine the inhibitory rate of the Chlorella pyrenoidosa polypeptide extract obtained in Example 1 on pancreatic lipase activity at concentrations of 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 mg / mL according to the method in Experimental Example 2. Use orlistat (8 μg / mL) as a positive control, and the results show that when the concentration of the Chlorella pyrenoidosa polypeptide extract is 8 mg / mL, the inhibitory rate of pancreatic lipase activity reaches 42.33%, as shown in Figure 10 shown.

[0100] This experimental example also tested the inhibitory mechanism of the Chlorella pyrenoidosa polypeptide extract on pancreatic lipase, and the results are as shown in Figures 11 - 12 shown, Figures 11 - 12 indicating that the inhibitory type of the Chlorella pyrenoidosa polypeptide extract provided by the present invention on pancreatic lipase is reversible inhibition and non-competitive inhibition.

[0101] The specific measurement steps are as follows:

[0102] 4-Nitrophenol (pNP) standard curve: Prepare a series of pNP solutions with different concentrations (0.01, 0.02, 0.03, 0.04, 0.05 mmol / L), and measure the absorbance values of pNP with different concentrations at 405 nm. Take the pNP concentration as the abscissa and the absorbance value as the ordinate to obtain the 4-nitrophenol (pNP) standard curve.

[0103] Prepare solutions of the Chlorella pyrenoidosa polypeptide extract prepared in Example 1 with concentrations of 0 mg / mL and 1 mg / mL, fix the concentration of p-nitrophenyl palmitate (pNPP) at 0.8 mmol / L, and measure the initial velocity of the enzymatic reaction when the mass concentration of pancreatic lipase is 0, 5, 10, 15, and 20 mg / mL respectively. The reaction system is the same as the method for testing the inhibitory rate of pancreatic lipase activity in Experimental Example 2. According to the 4-nitrophenol (pNP) standard curve, obtain the content (C: mmol / L) of the corresponding reaction product (pNP), and then calculate the reaction rate of each reaction. The reaction rate V = C / t, where C refers to the content of 4-nitrophenol (pNP) after the reaction, and t refers to the reaction time (min) after the Chlorella pyrenoidosa polypeptide extract solution is added to p-nitrophenyl palmitate (pNPP). Plot the initial velocity of the enzymatic reaction against the enzyme mass concentration, as shown in Figure 11 shown. From Figure 11It can be seen that the curve of the addition of Chlorella pyrenoidosa polypeptide extract passes through the origin and has a lower slope than the curve without the lipid-lowering peptide, indicating that the inhibition type of Chlorella pyrenoidosa polypeptide extract on pancreatic lipase is reversible inhibition.

[0104] The Chlorella pyrenoidosa polypeptide extract prepared in Example 1 was formulated into solutions of 0 mg / mL, 1 mg / mL, and 2 mg / mL. The concentration of pancreatic lipase was fixed at 10 mg / mL, and the initial velocity of the enzymatic reaction was measured when the concentration of p-nitrophenyl palmitate (pNPP) was 8, 4, 2, and 1 mmol / L. The reaction system was the same as the test method for the inhibition rate of pancreatic lipase activity in Experimental Example 2. The reciprocal of the reaction rate (1 / v) was plotted against the reciprocal of the substrate concentration (1 / [S]) to obtain a Lineweaver-Burk double-reciprocal curve, as Figure 12 shown. As can be seen from 12, this inhibition type is non-competitive inhibition.

[0105] Experimental Example 4 Determination of the lipid-lowering effect of Chlorella pyrenoidosa polypeptide extract

[0106] The Chlorella pyrenoidosa polypeptide extract was obtained through Example 1.

[0107] The wild-type N2 of Caenorhabditis elegans was used as the model organism and fed OP50 (uracil-deficient Escherichia coli), both of which were purchased from Fujian Shangyuan Biotechnology Co., Ltd.

[0108] The triglyceride (TG) kit, total cholesterol (TC) kit, and total protein (TP) kit were purchased from Nanjing Jiancheng Bioengineering Institute.

[0109] Establish a high-fat model: The wild-type N2 was induced with a high-fat diet by culturing C. elegans in NGM medium containing 10 mM glucose to construct a C. elegans high-fat model.

[0110] The experiment was set up with 6 groups (3 culture medium plates in each group, 1000 worms / plate), including a normal control group, a high-fat model group, a positive control group, a low-dose group of Chlorella pyrenoidosa polypeptide extract, a medium-dose group of Chlorella pyrenoidosa polypeptide extract, and a high-dose group of Chlorella pyrenoidosa polypeptide extract. The specific experimental grouping and dosing are shown in Table 1 below.

[0111] Table 1 Experimental grouping and dosing

[0112]

[0113] C. elegans culture and synchronization: C. elegans were cultured using nematode growth medium (NGM) with OP50 bacterial solution coated on the surface as food for C. elegans, and cultured in a constant temperature incubator at 20°C. All C. elegans used in this experimental example were C. elegans that had been synchronized and grown to the L4 stage, and the lysis egg storage method was used to synchronize C. elegans.

[0114] Drug administration method: A stock solution of Chlorella pyrenoidosa polypeptide extract at 5 mg / mL was prepared. The stock solution was diluted 0, 2, and 4 times respectively, and after filtration sterilization, it was mixed evenly with OP50 bacterial solution at a mass ratio of 1:4. The drug concentrations after mixing were 1, 0.5, and 0.25 mg / mL in sequence. Orlistat was used as a positive control, and after filtration sterilization, the concentration after mixing with OP50 bacterial solution was 6 μg / mL. 400 μL of OP50 bacterial solution was aspirated onto the NGM culture medium plate to feed C. elegans every day, and the NGM culture medium was changed every day. After 72 h, the worms were collected. Under this condition, the drug entered the C. elegans body through ingestion.

[0115] Index determination: After culturing C. elegans for 72 h, the C. elegans were collected in a 1.5 mL centrifuge tube, washed 3 times with M9 buffer, centrifuged, and the supernatant was discarded. 1 mL of M9 buffer was added to resuspend, and the centrifuge tube was quickly frozen in liquid nitrogen for 10 min and then thawed at room temperature. After repeating three times, the worms were ground and broken to prepare a C. elegans homogenate. The contents of triglyceride (TG) and total cholesterol (TC) in C. elegans were determined according to the instructions of the kit from Nanjing Jiancheng Bioengineering Institute, and the results were standardized by protein concentration.

[0116] Experimental results: The contents of triglyceride (TG) and total cholesterol (TC) in C. elegans in different experimental groups are shown in Table 2 below.

[0117] Table 2 Contents of triglyceride (TG) and total cholesterol (TC) in C. elegans in different experimental groups

[0118] Group TG (mmol / g prot) TC (mmol / g prot) Normal control group <![CDATA[0.94±0.11 c > <![CDATA[0.42±0.04 c > High - fat model group <![CDATA[1.57±0.10 a > <![CDATA[0.68±0.08 a > Positive control group <![CDATA[1.16±0.09 b > <![CDATA[0.56±0.04 b > Low - dose group of Chlorella pyrenoidosa polypeptide extract <![CDATA[1.26±0.05 b > <![CDATA[0.53±0.07 b > Medium - dose group of Chlorella pyrenoidosa polypeptide extract <![CDATA[1.17±0.07 b > <![CDATA[0.52±0.05 bc > High - dose group of Chlorella pyrenoidosa polypeptide extract <![CDATA[1.14±0.06 b > <![CDATA[0.48±0.07 bc >

[0119] Note: The values are expressed in the form of mean ± standard deviation, and different letters represent significant differences, p < 0.05.

[0120] The results showed that the contents of TG and TC in the nematodes of the high-fat model group were significantly higher than those of the normal control group (p<0.05), indicating that culturing C.elegans with NGM medium containing 10 mM glucose could successfully establish a high-fat model of C.elegans. The TG of nematodes in each dose group of the polypeptide extract of Chlorella pyrenoidosa was significantly lower than that of the high-fat model group (p<0.05), and there was no significant difference from the positive control group (p>0.05), and there was no significant difference among each dose group (p>0.05). The TC of nematodes in each dose group of the polypeptide extract of Chlorella pyrenoidosa was significantly lower than that of the high-fat model group (p<0.05), and there was no significant difference from the positive control group (p>0.05), and there was no significant difference between the medium-dose group, high-dose group and the normal control group (p>0.05).

[0121] Experimental conclusion:

[0122] By establishing a high-fat model of Caenorhabditis elegans for in vivo lipid-lowering activity testing, within the range of the administration concentration of 0.25 - 1 mg / mL, the polypeptide extract of Chlorella pyrenoidosa of the present invention showed a certain lipid-lowering effect on the high-fat model nematodes, and could significantly reduce the contents of triglyceride and cholesterol in the nematodes. It shows that the polypeptide extract of Chlorella pyrenoidosa described in the present invention has good application prospects in the development of weight loss functional foods or health products.

[0123] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A short peptide, characterized in that, The amino acid sequence of the short peptide is shown in SEQ ID NO.1.

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