CN115894619A - Short peptide, chlorella pyrenoidosa polypeptide extract as well as preparation method and application thereof - Google Patents

Abstract

The invention provides a short peptide, a chlorella pyrenoidosa polypeptide extract, a preparation method and application thereof, and belongs to the technical field of functional polypeptides. The invention can overcome the problem that the performance of the microalgae polypeptide lipid-lowering product needs to be improved in the prior art. The short peptide FLGPF provided by the invention has smaller molecular weight and higher inhibition rate on pancreatic lipase activity, and the content of the short peptide FLGPF in the chlorella pyrenoidosa polypeptide extract obtained by the preparation method can reach 7.1-8.6%, so that the chlorella pyrenoidosa polypeptide extract has good utilization prospect. The chlorella pyrenoidosa polypeptide extract provided by the invention can obviously reduce the content of triglyceride and cholesterol in caenorhabditis elegans, and has a good application prospect in the development of weight-losing functional foods or health-care products. The invention provides technical support for developing safe and effective pancreatic lipase inhibitors, and has important significance for promoting the development of chlorella industry.

Description

Short peptide, chlorella pyrenoidosa polypeptide extract, and preparation method and application thereof

Technical Field

The invention relates to the technical field of functional polypeptides, and particularly relates to a short peptide, a chlorella pyrenoidosa polypeptide extract, and a preparation method and application thereof.

Background

Chlorella (Chlorophyta), chlorophyceae (Chlorophyceae), chlorococcales (Chlorococcales), oocystoceae (Oocystaceae) and Chlorella (Chlorella) are a kind of common unicellular green algae, and have the advantages of wide ecological distribution, high growth speed, easy culture and high application value. The chlorella pyrenoidosa contains a large amount of protein and chlorophyll, the content of the protein and the chlorophyll respectively reaches 42-58% and 3-5% of the dry weight, the amino acid composition of the chlorella pyrenoidosa is higher than the protein standard for human nutrition issued by the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) of the United nations, and the chlorella pyrenoidosa is a high-quality protein resource with safe eating. Various biological activities of chlorella protein and hydrolyzed polypeptide thereof have been reported, and mainly focus on antioxidant activity, anti-tumor activity, blood sugar reduction, blood pressure reduction, mineral chelation and the like.

With the continuous improvement of the physical life of people, unhealthy high-fat diet and poor living habits induce the occurrence of various chronic diseases. The investigation shows that the proportion of the adult lipid metabolism disorder in China is as high as 30 percent, the conventional lipid-lowering medicament has high hepatotoxicity, and the curative effect on the secondary lipid metabolism disorder is not ideal. The search for natural biological resources with the function of improving lipid metabolism and the development of high-efficiency and low-toxicity nutritional health-care food and biological medicine for regulating blood fat become hot spots of current global attention and research.

In recent years, many studies show that bioactive peptides derived from natural proteins have a significant blood lipid reducing effect, long-lasting drug effect and small side effect, the bioactive peptides have small molecular weight and good cell permeability and are easy to digest and absorb by human bodies, and the bioactive peptides are concerned by broad scholars as a natural active substance capable of effectively improving blood lipid balance in vivo, show great potential in high-tech industries such as functional foods, health care products, medicines and the like, and the existing microalgae source polypeptides have large molecular weight and still need to be improved in the aspect of bioavailability.

Disclosure of Invention

The invention aims to provide a short peptide, a chlorella pyrenoidosa polypeptide extract, and a preparation method and application thereof, so as to solve the problem that the performance of polypeptide lipid-lowering products in the prior art needs to be improved.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a short peptide, and the amino acid sequence of the short peptide is shown in SEQ ID No. 1.

The invention also provides application of the short peptide in preparing a product for inhibiting the activity of pancreatic lipase.

The invention also provides a chlorella pyrenoidosa polypeptide extract which contains the short peptide.

The invention also provides a preparation method of the chlorella pyrenoidosa polypeptide extract, which comprises the following steps:

sequentially carrying out enzymolysis, enzyme deactivation and centrifugation on chlorella protein solution to obtain supernatant;

sequentially carrying out freeze drying and ultrafiltration on the supernatant to obtain an ultrafiltration product;

and sequentially carrying out vacuum concentration, freeze drying and sephadex chromatography purification on the ultrafiltration product to obtain the chlorella pyrenoidosa polypeptide extract.

Preferably, the chlorella protein solution takes water as a solvent, and the concentration of the chlorella protein solution is 10-20 g/kg;

papain is adopted for enzymolysis;

calculated according to the mass of chlorella protein, the dosage of the papain is 4000-8000U/g;

the temperature of the enzymolysis is 40-60 ℃;

the enzymolysis time is 3-6 h.

Preferably, the temperature of enzyme deactivation is 90-100 ℃;

the enzyme deactivation time is 5-15 min;

the rotating speed of the centrifugation is 6000 to 10000r/min;

the centrifugation time is 10-20 min.

Preferably, the temperature of a cold trap adopted by the freeze drying is-50 to-60 ℃, and the vacuum degree of the freeze drying is 60 to 100Pa;

the ultrafiltration product has a molecular weight <5kDa;

the temperature of the vacuum concentration is 50-60 ℃;

the pressure of vacuum concentration is 0.08-0.09 MPa.

Preferably, the main peak F2 is collected after the purification and separation of the sephadex chromatography, and the freeze drying is carried out to obtain the chlorella pyrenoidosa polypeptide extract.

Preferably, the specification of the Sephadex used for the Sephadex chromatographic purification is Sephadex G-25;

the sample injection amount of the sephadex chromatographic 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 220nm.

The invention also provides application of the chlorella pyrenoidosa polypeptide extract in preparation of lipid-lowering products.

The invention has the technical effects and advantages that:

when the concentration of the short peptide FLGPF provided by the invention is 8mg/mL, the inhibition rate on the pancreatic lipase activity reaches 50.12%, the inhibition type on the pancreatic lipase activity is reversible inhibition and non-competitive inhibition, the short peptide FLGPF can interact with 3 amino acid residues on human PTL protein, mainly forms pi-hydrogen bond, pi-cation and hydrogen bond action, and inhibits the pancreatic lipase activity by occupying catalytic or substrate binding sites. The content of short peptide FLGPF in the chlorella pyrenoidosa polypeptide extract obtained by the preparation method can reach 7.1-8.6%, and the chlorella pyrenoidosa polypeptide extract can obtain better inhibition effect on pancreatic lipase compared with other main peaks obtained by ultrafiltration fractionation and purification. When the concentration of the chlorella pyrenoidosa polypeptide extract provided by the invention is 8mg/mL, the inhibition rate on pancreatic lipase activity reaches 42.33%, and the inhibition effect type is reversible inhibition and noncompetitive inhibition. The chlorella pyrenoidosa polypeptide extract has a certain lipid-lowering effect on high-fat model caenorhabditis elegans, and can remarkably reduce the content of triglyceride and cholesterol in the nematode body. The chlorella pyrenoidosa polypeptide extract has a good application prospect in the development of weight-losing functional foods or health-care products. The invention provides technical support for developing safe and effective pancreatic lipase inhibitors, and has important significance for promoting the development of chlorella industry.

Drawings

FIG. 1 is a Sephadex G-25 gel chromatogram;

FIG. 2 is a secondary mass spectrum of the FLGPF (579.3057 Da) short peptide identified by LC-MS/MS;

FIG. 3 is a chromatogram of FLGPF synthetic peptide;

FIG. 4 shows the inhibitory effect of FLGPF synthetic peptide on pancreatic lipase at different concentrations;

FIG. 5 shows the type of inhibition of pancreatic lipase by FLGPF synthetic peptides;

FIG. 6 is a double reciprocal curve of reversible inhibition of pancreatic lipase by FLGPF synthetic peptide;

FIG. 7 is a diagram of the two-dimensional (A), surface (B) and three-dimensional (C) binding patterns of FLGPF and human pancreatic lipase (PTL);

FIG. 8 is a comparison of the inhibitory effect of different ultrafiltration fractions on pancreatic lipase;

FIG. 9 shows the inhibitory effect of Sephadex G-25 gel chromatography purified peptide fragment on pancreatic lipase;

FIG. 10 shows the pancrelipase inhibitory effect of Chlorella pyrenoidosa polypeptide extracts at different concentrations;

FIG. 11 shows the type of pancreatic lipase inhibition by Chlorella pyrenoidosa polypeptide extract;

FIG. 12 is a double reciprocal curve of the reversible inhibition of pancreatic lipase by Chlorella pyrenoidosa polypeptide extracts.

Detailed Description

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

The invention also provides application of the short peptide in preparing products for inhibiting the activity of pancreatic lipase, wherein the products are preferably health-care products, functional foods and medicines; the product preferably takes the short peptide as the only active component; the product preferably also comprises auxiliary materials, and the types of the auxiliary materials can be fillers, sweeteners or other auxiliary materials for adjusting taste, disintegrants, lubricants, binders, coatings, colorants, preservatives and the like; the medicament dosage form of the invention is preferably powder, tablets, granules, capsules, solutions, emulsions, suspensions or injections, and the functional food is preferably functional beverage, jelly products, candy products, flour products, and the like.

The invention also provides a chlorella pyrenoidosa polypeptide extract, which contains the short peptide, and the content of the short peptide in the chlorella pyrenoidosa polypeptide extract is more than 6%.

The invention also provides a preparation method of the chlorella pyrenoidosa polypeptide extract, which comprises the following steps:

sequentially carrying out enzymolysis, enzyme deactivation and centrifugation on chlorella protein solution to obtain supernatant;

sequentially carrying out freeze drying and ultrafiltration on the supernatant to obtain an ultrafiltration product;

and sequentially carrying out vacuum concentration, freeze drying and purification on the ultrafiltration product to obtain the chlorella pyrenoidosa polypeptide extract.

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 to 20g/kg, and more preferably 12 to 18g/kg, and the chlorella protein in the chlorella protein solution is preferably extracted by the following steps: the chlorella pyrenoidosa is taken as a raw material, pure water is added according to the mass-volume ratio of 1 to 48-52, the chlorella pyrenoidosa is soaked for 1 to 3 hours at room temperature, the mixture is fully stirred and swelled for repeated freeze thawing, the pH value of the mixture is adjusted to 11.5 to 12.5 by using 0.5 to 1.5mol/L NaOH solution after the repeated freeze thawing is preferably carried out for 4 to 6 times at the temperature of minus 20 to 37 ℃, the mixture is leached for 1 to 3 hours in a constant-temperature water bath at the temperature of 60 to 80 ℃, the mixture is centrifuged for 10 to 20 minutes at the speed of 5000 to 7000r/min, the supernatant is taken, the supernatant is filtered, the pH value of the mixture is adjusted to 3 to 4 by using 0.5 to 1.5mol/L HCL solution, the mixture is stood for 1 to 3 hours at the room temperature of 7000 to 9000r/min, the supernatant is discarded, the precipitate is washed to be neutral by the pure water, the cold trap temperature of minus 50 ℃ and the vacuum degree of 60 to 100Pa, and the chlorella pyrenoidosa protein is obtained after the freeze drying.

In the invention, the chlorella protein solution is subjected to enzymolysis, and papain is preferably adopted as the enzymolysis; the dosage of the papain is preferably 4000-8000U/g, the dosage refers to an enzyme-substrate ratio [ E ]/[ S ], and the dosage is further preferably 5000-7000U/g; the temperature of the enzymolysis is preferably 40-60 ℃, and more preferably 45-55 ℃; the enzymolysis time is preferably 3 to 6 hours, and more preferably 4.5 to 5.5 hours; the pH value of the enzymolysis is preferably 5.5-6.5; the enzyme is inactivated after enzymolysis, and the temperature of the enzyme inactivation is preferably 90-100 ℃, and is further preferably 95-100 ℃; the enzyme deactivation time is preferably 5-15 min, and more preferably 8-12 min; preferably cooling to room temperature after enzyme deactivation, wherein the room temperature is preferably 24-30 ℃, centrifuging after cooling, and the rotating speed of centrifuging is preferably 6000-10000 r/min, and more preferably 7000-9000 r/min; the time for centrifugation is preferably 10 to 20min, and more preferably 13 to 17min; centrifuging to obtain a supernatant, and freeze-drying the supernatant, wherein the temperature of a cold trap adopted for freeze-drying is preferably-50 to-60 ℃, more preferably-54 to-58 ℃, and the vacuum degree of freeze-drying is preferably 60 to 100Pa, more preferably 70 to 90Pa; performing ultrafiltration on the freeze-dried product to obtain an ultrafiltration product, wherein the molecular weight of the ultrafiltration product is preferably less than 5kDa, and performing vacuum concentration on the collected ultrafiltration product, wherein the temperature of the vacuum concentration is preferably 50-60 ℃, and further preferably 53-57 ℃; the pressure of the vacuum concentration is preferably 0.08-0.09 MPa; after vacuum concentration, freeze drying is carried out again, the temperature of a cold trap adopted by freeze drying is preferably-50 to-60 ℃, more preferably-54 to-58 ℃, and the vacuum degree of freeze drying is preferably 60 to 100Pa, more preferably 70 to 90Pa; and (2) performing purification after freeze drying, wherein the purification preferably adopts Sephadex chromatography, the main peak F2 is collected after separation, and the freeze dried chlorella pyrenoidosa polypeptide extract is obtained, the specification of the Sephadex G-25 is preferably adopted in the process, the adopted sample volume is preferably 1-5%, more preferably 2-4%, the sample 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 220nm, the temperature of a cold trap adopted by freeze drying is preferably-50-60 ℃, more preferably-54-58 ℃, and the vacuum degree of freeze drying is preferably 60-100 Pa, and more preferably 70-90 Pa.

The invention also provides application of the chlorella pyrenoidosa polypeptide extract in preparation of lipid-lowering products, wherein the products are preferably health products, functional foods and medicines; the product preferably takes the chlorella pyrenoidosa polypeptide extract as the only active component; the product preferably also comprises auxiliary materials, and the types of the auxiliary materials can be fillers, sweeteners or other auxiliary materials for adjusting taste, disintegrants, lubricants, binders, coatings, colorants, preservatives and the like; the medicament dosage form of the invention is preferably powder, tablets, granules, capsules, solutions, emulsions, suspensions or injections and the like, and the functional food is preferably functional beverage, jelly products, candy products, flour products and the like.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

(1) The chlorella pyrenoidosa is taken as a raw material, pure water is added according to the mass-volume ratio of 1.

(2) Weighing a proper amount of chlorella protein, adding pure water to prepare 15g/kg of chlorella protein solution, adjusting the pH to 6.0, adding papain, wherein the enzyme-substrate ratio [ E ]/[ S ] is 6000U/g, the enzymolysis temperature is 50 ℃, the enzymolysis time is 5 hours, after the enzymolysis is finished, heating to 100 ℃ to inactivate enzyme for 10 minutes, cooling to room temperature, centrifuging at 8000r/min for 15 minutes, taking supernatant to obtain chlorella protease hydrolysate, and freeze-drying at the cold trap temperature of-50 ℃ and the vacuum degree of 90Pa to obtain chlorella protein hydrolysate.

(3) And (3) carrying out ultrafiltration on the chlorella protein enzymolysis liquid collected in the step (2) by using ultrafiltration membranes with molecular weight cut-off of 10kDa and 5kDa to obtain different fractions (> 10kDa, 5-10 kDa and <5 kDa). The <5kDa fraction was collected, concentrated in vacuo and freeze dried, with the vacuum concentration parameters: the temperature is 55 ℃, the pressure is 0.09MPa, and the freeze-drying parameters are as follows: the cold trap temperature was-50 ℃ and vacuum 90Pa, resulting in a <5kDa fraction.

(4) And (3) further purifying the fraction of <5kDa by using sephadex G-25, eluting with ultrapure water as eluent, wherein the sample injection amount is 3% of the column volume, the sample injection concentration is 20mg/mL, the flow rate is 1mL/min, and the detection wavelength is 220nm. Collecting main peak F2 (Sephadex G-25 gel chromatogram shown in figure 1), and freeze-drying with freeze-drying parameters: the cold trap temperature is-50 ℃, and the vacuum degree is 90Pa, so as to obtain the chlorella pyrenoidosa polypeptide extract.

Example 2

(1) The chlorella pyrenoidosa is taken as a raw material, pure water is added according to the mass-volume ratio of 1 to 30, the chlorella pyrenoidosa is soaked for 2 hours at room temperature, fully stirred and swollen, then repeatedly frozen and thawed, the repeated freezing and thawing is carried out for 4 times between minus 20 ℃ and 37 ℃, the pH value is adjusted to 10.0 by using 1mol/L NaOH solution, the centrifugation is carried out for 15 minutes in 50 ℃ constant-temperature water bath for leaching for 4 hours and 6000r/min, the supernatant is taken, filtered, the pH value is adjusted to 3.0 by using 1mol/L HCL solution, the supernatant is removed after standing for 2 hours at room temperature, the centrifugation is carried out for 15 minutes at 8000r/min, the supernatant is discarded, the precipitate is washed to be neutral by pure water, the temperature of a cold trap is minus 56 ℃, and the freeze drying is carried out under the vacuum degree of 80Pa, and the chlorella protein is obtained.

(2) Weighing a proper amount of chlorella protein, adding pure water to prepare 20g/kg of chlorella protein solution, adjusting the pH to 5.5, adding papain, wherein the enzyme-substrate ratio [ E ]/[ S ] is 5000U/g, the enzymolysis temperature is 55 ℃, the enzymolysis time is 4h, after the enzymolysis is finished, heating to 100 ℃ to inactivate enzyme for 10min, cooling to room temperature, centrifuging at 7000r/min for 15min, taking supernatant to obtain chlorella protease hydrolysate, and freeze-drying at the cold trap temperature of-56 ℃ and the vacuum degree of 80Pa to obtain chlorella protein hydrolysate.

(3) And (3) performing ultrafiltration on the chlorella protein enzymolysis liquid collected in the step (2) by using ultrafiltration membranes with molecular weight cut-off of 10kDa and 5kDa to obtain different fractions (> 10kDa, 5-10 kDa and <5 kDa). The <5kDa fraction was collected, concentrated in vacuo and freeze-dried, with the vacuum concentration parameters: the temperature is 60 ℃, the pressure is 0.09MPa, and the freeze-drying parameters are as follows: the cold trap temperature was-56 ℃ and vacuum 80Pa, resulting in a <5kDa fraction.

(4) And (3) further purifying the fraction of <5kDa by using sephadex chromatography, wherein the specification of the sephadex is SephadexG-25, the eluent is ultrapure water, the sample injection amount is 2% of the column volume, the sample injection concentration is 25mg/mL, the flow rate is 1mL/min, and the detection wavelength is 220nm. Collecting main peak F2 (Sephadex G-25 gel chromatogram shown in figure 1), and freeze-drying with freeze-drying parameters: the temperature of the cold trap is minus 56 ℃, and the vacuum degree is 80Pa, so as to obtain the chlorella pyrenoidosa polypeptide extract.

Example 3

(1) The chlorella pyrenoidosa is taken as a raw material, pure water is added according to the mass-volume ratio of 1.

(2) Weighing a proper amount of chlorella protein, adding pure water to prepare 10g/kg of chlorella protein solution, adjusting the pH to 6.5, adding papain, wherein the enzyme-substrate ratio [ E ]/[ S ] is 4000U/g, the enzymolysis temperature is 60 ℃, the enzymolysis time is 3h, after the enzymolysis is finished, heating to 100 ℃ to inactivate enzyme for 10min, cooling to room temperature, centrifuging at 6000r/min for 20min, taking supernatant to obtain chlorella protease hydrolysate, and freeze-drying at the cold trap temperature of-58 ℃ and the vacuum degree of 70Pa to obtain chlorella protein hydrolysate.

(3) And (3) carrying out ultrafiltration on the chlorella protein enzymolysis liquid collected in the step (2) by using ultrafiltration membranes with molecular weight cut-off of 10kDa and 5kDa to obtain different fractions (> 10kDa, 5-10 kDa and <5 kDa). The <5kDa fraction was collected, concentrated in vacuo and freeze dried, with the vacuum concentration parameters: the temperature is 50 ℃, the pressure is 0.09MPa, and the freeze-drying parameters are as follows: the cold trap temperature was-58 ℃ and vacuum 70Pa, resulting in a <5kDa fraction.

(4) And (3) further purifying the fraction of <5kDa by using sephadex chromatography, wherein the specification of the sephadex is SephadexG-25, the eluent is ultrapure water, the sample injection amount is 1% of the column volume, the sample injection concentration is 30mg/mL, the flow rate is 1mL/min, and the detection wavelength is 220nm. Collecting main peak F2 (Sephadex G-25 gel chromatogram shown in figure 1), and freeze drying with freeze drying parameters: the cold trap temperature is-58 ℃, and the vacuum degree is 70Pa, so as to obtain the chlorella pyrenoidosa polypeptide extract.

Example 4

FLGPF synthetic peptide (FLGPF, shown as SEQ ID NO. 1) is prepared by Fmoc solid phase synthesis method, and is synthesized by Competition Biotechnology engineering (Shanghai) GmbH.

Comparative example 1

The only difference from example 1 is that the chlorella protein hydrolysate collected in step (2) was subjected to ultrafiltration with ultrafiltration membranes with molecular weight cut-off of 10kDa and 5kDa to obtain different fractions (> 10kDa, 5-10 kDa and <5 kDa). The >10kDa fraction was collected.

Comparative example 2

The only difference from example 1 is that the chlorella protein hydrolysate collected in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cut-off of 10kDa and 5kDa to obtain different fractions (> 10kDa, 5-10 kDa and <5 kDa). Fractions of 5-10 kDa were collected.

Comparative example 3

Only different from example 1, the main peak F1 was collected after further purification by sephadex chromatography.

Experimental example 1

The content of FLGPF short peptides in the chlorella pyrenoidosa polypeptide extracts prepared in examples 1-3 was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

The desalted polypeptide sample was dried by centrifugation, re-dissolved in 100uL Nano-LC mobile phase A (0.1% formic acid/water), bottled and analyzed by on-line LC-MS/MS. The solubilized sample was applied to a NanoViper C18 pre-column (3 μm, 100A) in a volume of 2 μ L, followed by desalting by a volume wash of 20 μ L. The liquid phase was Easy nLC 1200 nanoliter liquid phase system (ThermoFisher, USA), the sample was desalted and retained on the pre-column and then separated by analytical column, which was a C18 reverse phase chromatographic column (Acclaim PepMap RSLC,75 μm × 25cm C18-2 μm 100A) with a gradient of mobile phase B (80% acetonitrile, 0.1% formic acid) increasing from 5% to 38% over 30 min. Mass Spectrometry A ThermoFisher Q active system (ThermoFisher, USA) was used in combination with a nanoliter nebulizing Nano Flex ion source (ThermoFisher, USA), the nebulizing voltage was 1.9kV, and the ion transfer tube heating temperature was 275 ℃. The raw profile file collected by mass spectrometry was processed and analyzed by PEAKS Studio 8.5 (Bioinformatics Solutions Inc. Waterloo, canada) software, and the database was a Uniprot downloaded database of Chlorella pyritinoidosa species proteins with the following search parameters: the mass tolerance of the primary mass spectrum is 10ppm, and the mass tolerance of the secondary mass spectrum is 0.05Da.

LC-MS/MS identified 999 polypeptides, of which the secondary mass spectrum of the identified FLGPF (579.3057 Da) short peptide is shown in FIG. 2. The identification results are as follows: the proportion of the FLGPF short peptide in the total amount of the polypeptide extract of the chlorella pyrenoidosa polypeptide extract in example 1 is 7.9%, the proportion of the FLGPF short peptide in the total amount of the polypeptide extract of the chlorella pyrenoidosa polypeptide extract in example 2 is 8.6%, and the proportion of the FLGPF short peptide in the total amount of the polypeptide extract of the chlorella pyrenoidosa polypeptide extract in example 3 is 7.1%.

Experimental example 2 Performance verification of short peptide FLGPF

The FLGPF synthetic peptide in example 4 is taken, and the purity of the FLGPF synthetic peptide is determined to be more than 99% by high performance liquid chromatography and mass spectrometry, as shown in figure 3.

The inhibition rates of pancreatic lipase at concentrations of 0.25, 0.5, 1.0, 2.0, 4.0, 8.0mg/ml for FLGPF synthetic peptide were determined as follows:

80 mu L of Tris-HCl buffer solution with pH8.0, 40 mu L of sample solution and 120 mu L of pancrelipase solution with 10mg/mL are mixed uniformly, incubated at 37 ℃ for 10min, then 160 mu L of 0.8mmol/L p-nitrophenylpalmitate (pNPP) is added immediately as a substrate, and placed at 37 ℃ for reaction again for 20min. Immediately after the reaction was completed, the reaction was terminated by water bath at 100 ℃ for 5 min. Centrifuging the reaction solution after termination at 10000r/min for 10min, removing precipitate, taking 200 mu L of supernatant liquid to a 96-hole enzyme label plate, and recording the light absorption value at 405nm by an enzyme label instrument. Orlistat (8 μ g/mL) was used as a positive control and calculated according to the following formula:

in the formula: a: comparing the light absorption values of the test groups; b: the light absorption value of the sample test group;

a: comparing the light absorption value of the blank group; b: absorbance of sample blank.

The result shows that the inhibition rate of the FLGPF synthetic peptide on the activity of the pancreatic lipase reaches 50.12% when the concentration of the FLGPF synthetic peptide is 8mg/mL, as shown in a figure 4.

The inhibition mechanism of FLGPF synthetic peptide for inhibiting the activity of pancreatic lipase is determined by the following method:

4-nitrophenol (pNP) standard curve: a series of pNP solutions (0.01, 0.02, 0.03, 0.04 and 0.05 mmol/L) with different concentrations are prepared, and the absorbance values of pNP with different concentrations at 405nm are measured. Taking pNP concentration as an abscissa and a light absorption value as an ordinate to obtain a 4-nitrophenol (pNP) standard curve.

Preparing FLGPF synthetic peptide into 0mg/mL and 1mg/mL solutions, fixing the concentration of para-nitrophenylpalmitate (pNPP) to be 0.8mmol/L, respectively measuring the initial speed of enzymatic reaction when the mass concentration of pancreatic lipase is 0, 5, 10, 15 and 20mg/mL, and using the reaction system as the pancreatic lipase activity inhibition rate test method. The content (C: mmol/L) of the corresponding reaction product (pNP) was obtained from the 4-nitrophenol (pNP) standard curve, and the respective reaction rates were determined. Reaction speed 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 FLGPF synthetic peptide solution was added to p-nitrophenylpalmitate (pNPP). The initial rate of the enzymatic reaction was plotted against the enzyme mass concentration, as shown in FIG. 5. FLGPF synthetic peptide is prepared into solutions of 0mg/mL, 1mg/mL and 2mg/mL, the concentration of the immobilized pancreatic lipase is 10mg/mL, the initial speeds of enzymatic reactions when the concentration of p-nitrophenylpalmitate (pNPP) is 8, 4, 2 and 1mmol/L are respectively determined, and the reaction system is the same as the pancreatic lipase activity inhibition rate test method. The reciprocal of the reaction rate (1/v) was plotted against the reciprocal of the substrate concentration (1/[ S ]), yielding a Lineweaver-Burk double reciprocal curve, as shown in FIG. 6.

The results show that: the inhibition type of FLGPF short peptide on the activity of pancreatic lipase is reversible inhibition, and the inhibition is noncompetitive inhibition.

The method comprises the steps of using a crystal structure of human pancreatic lipase (PTL) as a receptor (PDB: 1 LPB), using MOE software to carry out flexible docking on the short peptide FLGPF and the receptor, determining key amino acid residues and interaction force of the FLGPF and the receptor, and drawing a binding mode diagram by adopting PyMOL, wherein as shown in figure 7, the FLGPF is represented as a yellow stick model, residues around a binding pocket are represented as a blue stick model, a main chain of the receptor is represented as blue cartoon, and pi-hydrogen bond, pi-cation and hydrogen bond actions are represented by green dotted lines. As can be seen in FIG. 7, FLGPF forms a suitable spatial complement to the binding site of the human PTL protein. The nitrogen atom on the Phe1 main chain of FLGPF acts as a hydrogen bond donor, and forms a hydrogen bond with the oxygen atom of the Arg79 side chain of the human-derived PTL protein. The nitrogen atom of the Phe1 backbone of FLGPF forms a pi-cation with the benzene ring of Trp252 of the human PTL protein. The nitrogen atom on Phe5 backbone of FLGPF forms pi-hydrogen bond with the benzene ring of Tyr114 of the human PTL protein. FLGPF also presents van der waals forces with the human-derived PTL protein. In conclusion, the short peptide FLGPF can interact with 3 amino acid residues on the human PTL protein, mainly forms pi-hydrogen bond, pi-cation and hydrogen bond action, and inhibits the activity of pancreatic lipase by occupying catalytic or substrate binding sites.

Experimental example 3 Performance verification of Chlorella pyrenoidosa polypeptide extract

The different fractions (> 10kDa, 5-10 kDa and <5 kDa) prepared in example 1, comparative example 1 and comparative example 2 were tested for pancreatic lipase activity inhibition rate, chlorella proteolysis and inhibition of pancreatic lipase activity by each fraction were observed at a concentration of 8mg/mL, the same procedure as in experimental example 2, and the results are shown in fig. 8, which shows that the <5kDa fraction provided by the present invention has the best pancreatic lipase inhibition effect.

The main peak F2 collected in example 1 and the main peak F1 collected in comparative example 3 are respectively taken to observe the inhibition effect of 2 samples on the activity of pancreatic lipase under the concentration of 8mg/mL, the method is the same as the experimental example 2, the result is shown in figure 9, and the result shows that the main peak F2 provided by the invention has better inhibition effect on the pancreatic lipase.

Pancreatic lipase activity inhibition rates of the chlorella pyrenoidosa polypeptide extract obtained in example 1 were measured at concentrations of 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0mg/mL according to the method in experimental example 2, and orlistat (8 μ g/mL) was used as a positive control, and the results are shown. When the concentration of the chlorella pyrenoidosa polypeptide extract is 8mg/mL, the inhibition rate of the activity of pancreatic lipase reaches 42.33%, as shown in figure 10.

The experimental example also tests the inhibition mechanism of the chlorella pyrenoidosa polypeptide extract on pancreatic lipase, the results are shown in fig. 11-12, and fig. 11-12 show that the inhibition type of the chlorella pyrenoidosa polypeptide extract on pancreatic lipase provided by the invention is reversible inhibition and noncompetitive inhibition.

The specific determination steps are as follows:

4-nitrophenol (pNP) standard curve: a series of pNP solutions (0.01, 0.02, 0.03, 0.04, 0.05 mmol/L) with different concentrations were prepared, and the absorbance at 405nm of pNP with different concentrations was measured. Taking pNP concentration as an abscissa and a light absorption value as an ordinate to obtain a 4-nitrophenol (pNP) standard curve.

The chlorella pyrenoidosa polypeptide extract prepared in example 1 is prepared into 0mg/mL and 1mg/mL solutions, the concentration of the immobilized p-nitrophenylpalmitate (pNPP) is 0.8mmol/L, the initial speed of enzymatic reaction is respectively measured when the mass concentration of pancreatic lipase is 0, 5, 10, 15 and 20mg/mL, and the reaction system is the same as the method for testing the pancreatic lipase activity inhibition rate in experimental example 2. The content (C: mmol/L) of the corresponding reaction product (pNP) was obtained from the 4-nitrophenol (pNP) standard curve, and the respective reaction rates were determined. The reaction speed V = C/t, wherein 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 with p-nitrophenylpalmitate (pNPP). The initial rate of the enzymatic reaction was plotted against the mass concentration of enzyme as shown in FIG. 11. As can be seen from fig. 11, the curve with the chlorella pyrenoidosa polypeptide extract added passes through the origin and has a lower slope than the curve without the addition of the lipid-lowering peptide, indicating that the type of inhibition of pancreatic lipase by the chlorella pyrenoidosa polypeptide extract is reversible.

The chlorella pyrenoidosa polypeptide extract prepared in example 1 is prepared into solutions of 0mg/mL, 1mg/mL and 2mg/mL, the concentration of the immobilized pancreatic lipase is 10mg/mL, the initial speed of enzymatic reaction is respectively measured when the concentration of p-nitrophenylpalmitate (pNPP) is 8, 4, 2 and 1mmol/L, and the reaction system is the same as the method for testing the pancreatic lipase activity inhibition rate in experiment example 2. The reciprocal of the reaction rate (1/v) was plotted against the reciprocal of the substrate concentration (1/[ S ]), resulting in a Lineweaver-Burk double reciprocal curve, as shown in FIG. 12. From 12, this type of inhibition is a non-competitive inhibition.

Experimental example 4 measurement of lipid-lowering Effect of Chlorella pyrenoidosa polypeptide extract

Chlorella pyrenoidosa polypeptide extract was obtained by example 1.

Caenorhabditis elegans (C. Elegans) wild type N2 is taken as a model organism to feed OP50 (uracil-deficient escherichia coli), and the Caenorhabditis elegans is purchased from Fujian Shangyuan bioscience technology, inc.

Triglyceride (TG) kit, total Cholesterol (TC) kit and protein quantification (TP) kit, which are purchased from Nanjing institute of bioengineering.

Establishing a high fat model: high fat diet induction of wild type N2 was performed by culturing c.elegans using NGM medium containing 10mM glucose to construct a c.elegans high fat model.

Experiment set 6 groups (each group comprises 3 medium plates and 1000 medium plates) including a normal control group, a high-fat model group, a positive control group, a chlorella pyrenoidosa polypeptide extract low-dose group, a chlorella pyrenoidosa polypeptide extract medium-dose group and a chlorella pyrenoidosa polypeptide extract high-dose group. The specific experimental groups and dosages administered are shown in table 1 below.

TABLE 1 Experimental groups and dosages administered

Nematode culture and synchronization: the nematode is cultured in a constant temperature incubator at 20 ℃ by using a Nematode Growth Medium (NGM) coated with OP50 bacteria solution as the nematode food. All nematodes used in the experimental example are nematodes which grow to the L4 stage through synchronization treatment, and the nematodes are subjected to synchronization treatment by adopting a cracking egg storage method.

The administration mode comprises the following steps: 5mg/mL of chlorella pyrenoidosa polypeptide extract is prepared as mother liquor. Respectively diluting the mother liquor by 0, 2 and 4 times, filtering and sterilizing, and then uniformly mixing with OP50 bacterial liquid according to the mass ratio of 1:4, wherein the drug concentration after mixing is 1, 0.5 and 0.25mg/mL in sequence. Orlistat was used as a positive control, and the concentration was 6 μ g/mL after mixing with OP50 bacterial suspension after filter sterilization. Sucking 400 mu L OP50 bacterial liquid every day to feed nematodes on a nematode growth medium plate, replacing the NGM medium every day, and collecting the larvae after 72 hours. Under these conditions, the drug enters the nematode through nematode feeding.

Index measurement: and (3) after the nematodes are cultured for 72h, collecting the nematodes in a 1.5mL centrifuge tube, washing the nematodes 3 times by using M9 buffer solution, centrifuging, and discarding supernatant. Adding 1mL M9 buffer solution for resuspension, placing the centrifuge tube into liquid nitrogen for quick freezing for 10min, thawing at room temperature, repeating for three times, grinding and crushing the worm body, and making into nematode homogenate. The contents of Triglyceride (TG) and Total Cholesterol (TC) in the nematode are measured according to the specification of the kit of Nanjing institute of biological engineering, and the result is standardized by protein concentration.

The experimental results are as follows: triglyceride (TG) and Total Cholesterol (TC) levels in the nematode bodies of the different experimental groups are shown in Table 2 below.

TABLE 2 Triglyceride (TG) and Total Cholesterol (TC) levels in nematodes of different experimental groups

Group of	TG(mmol/gprot)	TC(mmol/gprot)
			Normal control group	0.94±0.11 c	0.42±0.04 c
High fat model group	1.57±0.10 a	0.68±0.08 a
			Positive control group	1.16±0.09 b	0.56±0.04 b
Chlorella pyrenoidosa polypeptide extract low dose group	1.26±0.05 b	0.53±0.07 b
			Dosage group in chlorella pyrenoidosa polypeptide extract	1.17±0.07 b	0.52±0.05 bc
Chlorella pyrenoidosa polypeptide extract high-dose composition	1.14±0.06 b	0.48±0.07 bc

Note: values are expressed as mean ± standard deviation, different letters represent significant differences, p <0.05.

The results show that the contents of TG and TC in the nematodes in the high-fat model group are remarkably higher than those in the normal control group (p is less than 0.05), which indicates that C.elegans can be successfully constructed by culturing C.elegans in NGM medium containing 10mM glucose. Nematode TG (nematode TG) of each dosage group of the chlorella pyrenoidosa polypeptide extract is remarkably lower than that of a high-fat model group (p < 0.05), the difference with a positive control group is not remarkable (p > 0.05), and the difference between each dosage group is not remarkable (p > 0.05). Nematode TC (T & ltp & gt 0.05) of each dosage group of the chlorella pyrenoidosa polypeptide extract is remarkably lower than that of a high-fat model group (p & lt 0.05), the nematode TC is not remarkably different from that of a positive control group (p & gt 0.05), and the nematode TC is not remarkably different from that of a medium-dosage group, a high-dosage group and a normal control group (p & gt 0.05).

The experimental conclusion is that:

by constructing a high-fat model of caenorhabditis elegans to carry out in-vivo lipid-lowering activity test, the chlorella pyrenoidosa polypeptide extract has a certain lipid-lowering effect on the high-fat model nematodes within the administration concentration range of 0.25-1 mg/mL, and can remarkably reduce the content of triglyceride and cholesterol in the nematodes. The chlorella pyrenoidosa polypeptide extract has a good application prospect in the development of weight-losing functional foods or health-care products.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims (10)

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

2. Use of the short peptide according to claim 1 for the preparation of a product for inhibiting pancreatic lipase activity.

3. A Chlorella pyrenoidosa polypeptide extract, which contains the short peptide according to claim 1.

4. The method of preparing a chlorella pyrenoidosa polypeptide extract as set forth in claim 3, comprising the steps of:

sequentially carrying out enzymolysis, enzyme deactivation and centrifugation on chlorella protein solution to obtain supernatant;

sequentially carrying out freeze drying and ultrafiltration on the supernatant to obtain an ultrafiltration product;

and sequentially carrying out vacuum concentration, freeze drying and sephadex chromatography purification on the ultrafiltration product to obtain the chlorella pyrenoidosa polypeptide extract.

5. The method for producing a chlorella pyrenoidosa polypeptide extract as claimed in claim 4, wherein the chlorella pyrenoidosa protein solution is water as a solvent, and the concentration of the chlorella pyrenoidosa protein solution is 10 to 20g/kg;

papain is adopted for enzymolysis;

calculated according to the mass of chlorella protein, the dosage of the papain is 4000-8000U/g;

the temperature of the enzymolysis is 40-60 ℃;

the enzymolysis time is 3-6 h.

6. The method for preparing a Chlorella pyrenoidosa polypeptide extract as claimed in claim 4, wherein the temperature for inactivating the enzyme is 90 to 100 ℃;

the enzyme deactivation time is 5-15 min;

the rotating speed of the centrifugation is 6000 to 10000r/min;

the centrifugation time is 10-20 min.

7. The method for preparing a chlorella pyrenoidosa polypeptide extract as claimed in claim 4, wherein the temperature of a cold trap used for freeze drying is-50 to-60 ℃, and the vacuum degree of the freeze drying is 60 to 100Pa;

the ultrafiltration product has a molecular weight <5kDa;

the temperature of the vacuum concentration is 50-60 ℃;

the pressure of vacuum concentration is 0.08-0.09 MPa.

8. The method for preparing a chlorella pyrenoidosa polypeptide extract as claimed in claim 4, wherein the chlorella pyrenoidosa polypeptide extract is obtained by collecting the main peak after purification by sephadex chromatography and freeze-drying.

9. The method for preparing a chlorella pyrenoidosa polypeptide extract as claimed in claim 8, wherein the Sephadex specification used for Sephadex purification;

the sample injection amount of the sephadex chromatographic 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 220nm.

10. Use of a chlorella pyrenoidosa polypeptide extract as defined in claim 3 or a chlorella pyrenoidosa polypeptide extract obtained by the method of preparation as defined in any one of claims 4 to 9 for the preparation of a lipid lowering product.

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