CN111471086B

CN111471086B - Sesame polypeptide and preparation method and application thereof

Info

Publication number: CN111471086B
Application number: CN202010299270.2A
Authority: CN
Inventors: 芦鑫; 贾聪; 高锦鸿; 王瑞丹; 张丽霞; 宋国辉; 孙强; 黄纪念
Original assignee: Henan Academy of Agricultural Sciences
Current assignee: Henan Academy of Agricultural Sciences
Priority date: 2020-04-16
Filing date: 2020-04-16
Publication date: 2023-01-24
Anticipated expiration: 2040-04-16
Also published as: CN111471086A

Abstract

The invention belongs to the technical field of deep processing of food-derived protein, and particularly relates to sesame polypeptide, which consists of 8 amino acids and has the sequence: glu-Ala-Cys-Ile-Gln-Ala-Cys-Lys (EACIQACK). The sesame polypeptide can be obtained by separating and purifying enzymolysis sesame protein, and can also be artificially synthesized by adopting a chemical solid-phase synthesis method. The invention finds out by measuring DPPH clearance capacity, ABTS clearance capacity, total oxidation resistance (FRAP) and ACE inhibition capacity: the polypeptide has strong antioxidation and strong blood pressure lowering activity, can be used as a functional active component for preparing the fields of medicaments, health products, foods, nutrition enhancers, feeds, cosmetics, daily chemical products and the like, and has wide market prospect.

Description

Sesame polypeptide and preparation method and application thereof

Technical Field

The invention belongs to the field of deep processing of vegetable protein, and particularly relates to an active polypeptide EACIQACK from sesame 7S protein, a preparation method and application thereof in the aspects of antioxidant activity and blood pressure reduction activity.

Background

At present, research on functional peptides is one of hot spots in food research, and common functional peptides include antioxidant peptides, antihypertensive peptides, antibacterial peptides, flavor peptides and the like. Previous studies have often focused on the study of a single activity of a polypeptide, while studies of active peptides with multiple activities have been ignored. Tanzadehpanah et al structurally modify afkdedteeevpfr to have hypotensive activity and antioxidant activity. Polypeptide with blood pressure lowering activity and antioxidant activity is found by enzymolysis of hyacinth bean protein by Garcia-Mora et al, which shows that the multifunctional active peptide exists in the nature. Compared with the active peptide with single function, the multifunctional active peptide has wider application range, obvious advantages and higher research and application values.

With the change of life style, non-infectious chronic diseases (hypertension, cardiovascular and cerebrovascular diseases, diabetes and the like) seriously threaten the health of people all over the world. Research shows that the occurrence and development of most non-infectious chronic diseases are related to oxidative stress reaction caused by free radicals in vivo, inhibit free radical reaction and resist oxidation, and have positive significance for preventing and relieving the diseases. Meanwhile, the regulation and control of the activity of in vivo vascular tensinase have important significance for controlling blood pressure. Therefore, if a functional peptide with antioxidant and antihypertensive activities can be developed, the functional peptide has active research value and bright market value for preventing and adjunctively treating non-infectious chronic diseases.

China is a big country for sesame production and consumption, the sesame yield is about 60 ten thousand tons every year, and the sesame processing amount is about 150 ten thousand tons. As the main component of sesame, namely sesame protein (accounting for about 20 percent of the total weight of the sesame), the sesame protein is processed to a lower degree due to the lagging processing technology, and is mostly processed into feed or fertilizer for use, so that the sesame protein resource is not fully utilized. In order to utilize sesame protein, the former can prepare antioxidant peptide, antihypertensive peptide and chelated metal peptide by enzymolysis of sesame protein. On the basis, if the bifunctional sesame active peptide with the functions of resisting oxidation and reducing blood pressure can be prepared, the method has stronger market competitiveness and wider application range, and makes a contribution to improving the health of residents while improving the processing additional value of sesame protein.

Disclosure of Invention

In order to meet the requirements of healthy life of people and replace toxic side effects for artificially synthesizing antioxidants and antihypertensive drugs, the invention aims to provide sesame polypeptide which is derived from sesame protein and has antioxidant and antihypertensive activities.

The invention also provides a preparation method of the sesame polypeptide and application of the sesame polypeptide in preparing medicines with antioxidation and/or blood pressure reduction.

In order to realize the purpose, the invention adopts the following technical scheme:

the invention provides a sesame polypeptide with antioxidant and/or antihypertensive activity, which consists of eight amino acids, and has the sequence: glu-Ala-Cys-Ile-Gln-Ala-Cys-Lys (EACIQACK), the sesame polypeptide is derived from sesame 7S protein (Q9 AUD0_ SESIN).

The sesame polypeptide can be prepared by artificial chemical synthesis, such as solid phase synthesis according to the conventional process in the field.

The invention also provides another method for extracting the sesame polypeptide from the sesame protein, which is prepared by the steps of proteolysis, separation and purification and the like; the method specifically comprises the following steps:

1) Extracting sesame protein:

crushing and degreasing the sesame after impurity removal, adding distilled water, adjusting the pH to 8-10, centrifuging, collecting supernatant, adjusting the pH to 4-5, centrifuging, collecting precipitate, washing with water, and freeze-drying to obtain sesame protein;

2) And (3) proteolysis:

mixing the sesame protein obtained in the step 1) according to a feed-liquid ratio of 1g: adding distilled water into 6-15ml to obtain sesame protein solution, adjusting pH value to 1.5-2.5, adding 0.1-1.5% (w/v, i.e. adding 0.1-1.5g pepsin into 100ml sesame protein solution), stirring and performing enzymolysis for 1-5 h at 30-50 ℃, then adjusting pH to 7.0-8.5, adding 0.1-2.5% (w/v, i.e. adding 0.1-2.5g compound protease into 100ml sesame protein solution), performing enzymolysis for 3-6 h at 30-50 ℃, adjusting pH to 4.0-4.5, centrifuging to obtain supernatant, and keeping at low temperature of 4-10 ℃ for later use;

3) Separation and purification:

adjusting the pH of the supernatant obtained in the step 2) to 7.0-8.0, removing impurity polypeptide by strong cation exchange resin adsorption, performing ultrafiltration by an ultrafiltration membrane with the molecular weight cutoff of 1000-5000 Da, desalting and concentrating the permeate by nanofiltration, separating and purifying by preparative liquid chromatography, collecting the components with the retention time of 22-23 min, and freeze-drying.

Analyzing and identifying the polypeptide sample obtained by freeze drying through Nano-LC-ESI-MS/MS, and identifying the amino acid sequence of the polypeptide with the activities of resisting oxidation and reducing blood pressure as follows: glu-Ala-Cys-Ile-Gln-Ala-Cys-Lys (EACIQACK). EACIQACK is derived from sesame 7S protein 49-56, namely SKEQKEACIQACKEYIRQ 49-56。

Specifically, in the step 1), a solvent extraction method or a subcritical extraction method is adopted for sesame degreasing. In the solvent extraction, petroleum ether, n-hexane or ether is adopted for degreasing; and in subcritical extraction, butane and propane are adopted for degreasing. When protein is extracted, the ratio of material to liquid is 1g: distilled water is added into the mixture of 6ml to 24 ml. Adjusting pH to 8-10 with sodium hydroxide, and stirring at room temperature for 30-90 min. The centrifugation conditions were: centrifuging at 3500-5000 r/min for 20-40 min; the pH of the supernatant was adjusted to 4-5 with hydrochloric acid.

Specifically, the compound protease in the step 2) consists of trypsin and chymotrypsin in a mass ratio of 6-12.

Further, the strong cation exchange resin used in the step 3) is ANX Sephrose FF, after sample loading, the elution is carried out for 60-180 min by adopting the distilled water flow rate of 2-8 mL/min, and the effluent is collected for ultrafiltration. The molecular weight cut-off of the nanofiltration membrane selected during nanofiltration is 100-300 Da.

Further preferably, the preparative liquid chromatography conditions in step 3) are: chromatographic column Ultimate AQ-C18 (250X 20mm, 5 μm) with a mobile phase of 55% aqueous methanol containing 0.1% trifluoroacetic acid, i.e. 55% (v/v) methanol solution (containing 0.1% (v/v) trifluoroacetic acid); the flow rate is 8 mL/min, the sample loading is 4mL, and the detection wavelength is 220 nm.

The invention also provides application of the sesame polypeptide serving as a main functional component in the fields of preparation of anti-oxidation and/or blood pressure reduction medicines, health-care products, foods, nutrition enhancers, animal feeds, cosmetics, daily chemical products and the like.

Compared with the prior art, the invention has the following advantages:

1) The raw materials used in the invention are sesame or low-temperature sesame cake meal, have wide sources and price advantages, and do not influence the utilization of other sesame components, so the invention has the advantages of controllable cost, high utilization rate of the raw materials, environmental friendliness and the like;

2) The sesame active polypeptide provided by the invention is high in safety (shown in table 2), and has the effects of resisting oxidation peptide and/or reducing blood pressure activity;

3) The sesame polypeptide provided by the invention has obvious activity, can be used as a food additive, and can also be used as a functional component to be applied to medicines, health-care products, foods, nutrition enhancers, animal feeds, cosmetics and daily chemical products.

Drawings

FIG. 1 is a chromatogram of a liquid-phase separation ultrafiltration permeate prepared in example 1;

FIG. 2 is a secondary mass spectrum of the sesame polypeptide sample extracted by the method in Nano-LC-ESI-MS/MS;

FIG. 3 is an HPLC chart of the sesame polypeptide of the present invention;

FIG. 4 is a mass spectrum of the sesame polypeptide of the present invention;

FIG. 5 is a graph showing the prediction of the binding of the sesame polypeptide EACIQACK of the present invention to human angiotensin enzyme.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to the following embodiments and the accompanying drawings, but the scope of the present invention is not limited thereto. Other embodiments obtained by persons skilled in the art without making any creative effort based on the embodiments in the present invention belong to the protection scope of the present invention.

Example 1

A method for extracting sesame polypeptide from sesame protein specifically comprises the following steps:

1) Extracting sesame protein:

0.5 kg of sesame without impurities and mildew grains is taken, crushed to 60 meshes and degreased by a Soxhlet extractor, wherein the degreasing conditions are as follows: petroleum ether (boiling range 30-60 ℃) is used as an extracting agent, the temperature is 65 ℃, and degreasing is carried out for 8 hours. Adding distilled water into defatted sesame according to a material-liquid ratio (w/v) of 12ml, adjusting the pH value to 10 by using sodium hydroxide, stirring at room temperature for 90 min, then centrifuging at 4000 r/min for 30 min, taking a supernatant, adjusting the pH value to 4.5, centrifuging at 4500 r/min for 30 min, collecting a precipitate, washing with distilled water for 3 times, freeze-drying to obtain sesame protein, and sealing for later use.

2) And (3) proteolysis:

adding the sesame protein obtained in the step 1) into distilled water according to a feed-liquid ratio of 1g to 10ml (w/v) to obtain a sesame protein solution, adjusting the pH value to 2.0, adding 0.5 percent (w/v, namely adding 0.5g of pepsin into each 100ml of sesame protein solution), and stirring and performing enzymolysis for 4 hours at 37 ℃; then adjusting the pH to 8.0, adding 0.5% composite protease (w/v, namely adding 0.5g composite protease in each 100ml of sesame protein solution, wherein the composite protease is composed of trypsin and chymotrypsin according to the mass ratio of 6.

3) Separating and purifying

Adjusting the pH value of the supernatant obtained in the step 2) to 7.5, sampling 50 mL of the supernatant to an ANX Sepharose FF resin column (500 x 40 mm), eluting at the flow rate of 4 mL/min for 160 min, collecting effluent, performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cut-off of 3000 Da, performing desalination and concentration on permeate by using a nanofiltration membrane (with the molecular weight cut-off of 200 Da), and then performing separation and purification by using a preparative liquid chromatography under the conditions of: the mobile phase was 55% aqueous methanol containing 0.1% trifluoroacetic acid, i.e., 55% (v/v) methanol solution (containing 0.1% (v/v) trifluoroacetic acid), the flow rate was 8 mL/min, the loading was 4mL, the detection wavelength was 220nm, and the column was Ultimate AQ-C18 (250X 2 mm i.d., 5 μm); collecting the fraction with retention time of 22min-23 min (see figure 1), and freeze drying at 60 deg.C for 24 h to obtain sesame polypeptide sample.

FIG. 1 shows a chromatogram of a liquid-phase separation ultrafiltration permeate prepared in example 1; after the separation and purification, other polypeptide components still exist in the system, and the chromatographic peaks with the numbers of P1-P14 existing in the chromatogram map are reflected. And respectively collecting the components corresponding to the chromatographic peaks, and determining the activity. The result shows that the activity of the component corresponding to the chromatographic peak P8 is strongest, and the component is identified to find that the sequence is EACIQACK.

The sesame polypeptide sample obtained by freeze drying is analyzed and identified by Nano-LC-ESI-MS/MS (see figure 2), and the amino acid sequence of the polypeptide is identified as follows: glu-Ala-Cys-Ile-Gln-Ala-Cys-Lys (EACIQACK). The synthesized polypeptide was subjected to liquid phase mass spectrometry (see FIGS. 3 and 4), and its molecular weight was identical to that of Nano-LC-ESI-MS/MS.

Example 2

An artificial synthesis method of sesame polypeptide adopts a solid phase synthesis method. The basic flow is as follows: firstly, connecting an amino acid with an amino group protected by Fmoc group on an insoluble solid phase carrier Wang resin, and then removing the protecting group of the amino group, wherein the first amino acid is connected to the solid phase carrier; secondly, activating carboxyl of a second amino acid of which the amino group is protected by Fmoc group by using a condensing agent, and reacting the activated amino acid with the amino group of the first amino acid which is connected with the solid phase carrier to form a peptide bond, thereby generating the dipeptide with the protecting group on the solid phase carrier. Repeating the above peptide bond formation reaction to make the peptide chain grow from C terminal to N terminal until reaching the required peptide chain length, and finally cutting to obtain the target polypeptide Glu-Ala-Cys-Ile-Gln-Ala-Cys-Lys (EACIQACK). The liquid phase mass spectrum analysis chart of the synthesized sesame polypeptide sequence is shown in figure 3 and figure 4, the main ion peak mass-to-charge ratio of the high-purity synthetic peptide is 865.03, which accords with the molecular weight of the sequence to be synthesized, and the success of solid phase synthesis is shown.

The specific process for synthesizing sesame polypeptide by solid phase synthesis method is as follows: (1) huang Weide, chen Chang Qing, peptide Synthesis, scientific Press, 1985. (2) N. hound, h.d. gaku, liuclen et al, peptide: chemistry and biology, science press, 2005. ) The sesame polypeptide can also be synthesized by corresponding polypeptide biological companies, and the chemical synthesis process is not the key point of the application, so the chemical synthesis process is not described in detail herein.

Application test of antioxidant and blood pressure lowering activity

In the following application tests, DPPH, ABTS scavenging ability, total antioxidant ability, FRAP, and ACE inhibitory ability were measured as follows. The polypeptide solution is prepared by diluting lyophilized or artificially synthesized sesame polypeptide with distilled water to obtain aqueous solution with certain concentration.

1) DPPH clearance assay: adding 100 μ L polypeptide solution into 96-well enzyme-linked immunosorbent assay plate, adding 100 μ L0.2 mmol/L DPPH ethanol solution, oscillating for 30s, reacting at room temperature in dark place for 30 min, and measuring absorbance A at wavelength of 517 nm _i The absorbance A of a mixture of 100. Mu.L of the polypeptide solution and 100. Mu.L of ethanol was measured in the same manner _j And absorbance A of 100. Mu.L of a 0.2 mmol/L DPPH ethanol solution mixed with 100. Mu.L of ethanol _o . DPPH radical scavenging Rate (D) _I ) Calculating the formula:

adjusting the concentration of the polypeptide solution to 0.01-10 mg/mL, measuring the DPPH free radical clearance rate, and calculating the IC by adopting the probit regression of SPSS24 ₅₀ I.e. the concentration at which DPPH clearance reaches 50%.

2) ABTS clearance assay: adding 25 μ L polypeptide solution into 96-well enzyme-linked immunosorbent assay plate, adding 200 μ L3.7 mmol/L ABTS solution, standing at room temperature in dark place for 6 min, and measuring absorbance A at wavelength of 734 nm _i ', the absorbance A of each of the polypeptide solutions was measured after mixing 25. Mu.L of the polypeptide solution with 200. Mu.L of methanol _j ', absorbance A after mixing 25. Mu.L of methanol with 200. Mu.L of 3.7 mmol/L ABTS solution _o '. ABTS free radical clearance (A) _I ) The calculation formula is as follows:

determining ABTS free radical clearance of polypeptide with different concentrations, and calculating IC by adopting IBM SPSS24 probabilistic regression algorithm ₅₀ 。

3) FRAP determination: the determination can be made by reference to the literature (A. Aleman, restriction of Leu and hydroxy principles to inhibitory and ACE-inhibition activities from liquid gel hydrolyase. Food Chemistry 125 (2011) 334-341.) as follows:

the FRAP working solution is composed of 300 mmol/L pH3.6 acetic acid buffer solution, 10 mmol/L2, 4, 6-tri (2-pyridyl) -1,3, 5-triazine (TPTZ) and 20 mmol/L FeCl at the volume ratio of 10 ₃ And (4) forming. Adding 150 mu L of FRAP working solution into the holes of a 96-well plate, then adding 25 mu L of 3 mg/mL polypeptide solution, mixing uniformly, reacting for 30 min at 37 ℃ in the dark, and measuring the absorbance at 593 nm by using an enzyme-labeling instrument. The standard solution adopts 0-2 mmol/L FeSO ₄ The absorbance of (b) was used as a standard, and the result was expressed as μmol FeEq/g _Protein The higher the value, the stronger the oxidation resistance.

4) Determination of ACE inhibitory capacity: adding malonyl-histidyl-leucine (HHL) into 0.05 mol/L boric acid buffer solution (containing 0.3 mol/L sodium chloride) with pH8.2 to prepare the solution with the concentration of 5 mmol/L, adding 0.125 mL into a centrifuge tube, adding 0.025 mL of 4.66mmol/L ACE solution and 0.025 mL of polypeptide solution, reacting at 37 ℃ for 1h, and adding 0.2 mL of 1 mol/L hydrochloric acid to stop the reaction. Adding 0.4 mL pyridine and 0.2 mL benzene sulfonyl chloride into the solution, mixing for 1 min, cooling, and measuring absorbance at 410 nm (A) _S ). Respectively measuring the absorbance A of the solution without adding ACE, the polypeptide solution and the polypeptide solution _b And A _c Calculating the ACE inhibition ratio by adopting the following formula:

IC is calculated by adopting probability regression algorithm of IBM SPSS24 ₅₀ 。

Active application of sesame polypeptide, IC with DPPH and ABTS free radical scavenging ability ₅₀ FRAP value as antioxidant index, ACE inhibiting IC ₅₀ As an index for lowering blood pressure, the sesame polypeptide extracted from the sesame protein in example 1 and the artificially synthesized sesame polypeptide EACIQACK in example 2 are tested and analyzed to have antioxidant and blood pressure lowering activities which are basically consistent, and both show stronger antioxidant and blood pressure lowering capabilities (see Table 1).

Results of the activity of the sesame polypeptide EACIQACK obtained in Table 1, examples 1 and 2

Table 1 shows DPPH scavenging, ABTS scavenging, total antioxidant capacity (FRAP value), IC of ACE inhibition of the sesame polypeptides EACIQACK prepared in examples 1 and 2 ₅₀ . As can be seen from Table 1: the sesame polypeptide EACIQACK has strong antioxidant capacity and is suitable for being used as an antioxidant in a polar aqueous solution system. Meanwhile, the sesame polypeptide also has certain ACE inhibition capability, and can assist the antihypertensive drug to control the blood pressure of patients.

TABLE 2 prediction of security of the sesame polypeptide EACIQACK of the present invention

Note: the numbers in parentheses are the prediction probabilities, ^a prediction used is AlgPred (https:// webs. Iiitd. Edu. In/raghava/AlgPred/sub-transmission. Html), ^b ToxinPred (https:// webs. Iiitd. Edu. In/raghava/ToxinPred/design. Php) was used for prediction, ^c admeasar (http:// lmmd. House. Edu. Cn/admeasar 2 /).

Table 2 shows the security prediction of the sesame polypeptide EACIQACK of the present invention. As can be seen from Table 2: EACIQACK has no sensitization, no toxicity and no carcinogenicity, and simultaneously, does not interfere the function of normal cytochrome 450, and has high safety to human body.

FIG. 5 shows the prediction graph of the binding of the sesame polypeptide EACIQACK of the present invention to human angiotensin enzyme. As shown in FIG. 5, EACIQACK can react with amino acid residues Glu162, thr166, gln281, ala356, lys511 and Tyr523 in Angiotensin Converting Enzyme (ACE) through hydrogen bonds, thereby affecting the space structure of ACE, increasing the difficulty of combining with a substrate and playing a role in inhibiting.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims

1. A sesame polypeptide, which consists of eight amino acids, and has the sequence: glu-Ala-Cys-Ile-Gln-Ala-Cys-Lys.

2. A method for producing the sesame polypeptide according to claim 1, wherein the sesame polypeptide is produced by a solid phase synthesis method.

3. A method for preparing the sesame polypeptide of claim 1 from sesame protein, comprising the steps of:

1) Extracting sesame protein:

crushing the sesame after impurity removal, degreasing, adding distilled water, adjusting the pH to 8-10, centrifuging, collecting supernatant, adjusting the pH to 4-5, centrifuging, collecting precipitate, washing with water, and freeze-drying to obtain sesame protein;

2) And (3) proteolysis:

mixing the sesame protein obtained in the step 1) according to a feed-liquid ratio of 1g: adding distilled water into 6-15ml to obtain sesame protein solution, adjusting pH value to 1.5-2.5, adding 0.1-1.5% pepsin, stirring and performing enzymolysis at 30-50 ℃ for 1-5 h, then adjusting pH value to 7.0-8.5, adding 0.1-2.5% compound protease, performing enzymolysis at 30-50 ℃ for 3-6 h, adjusting pH value to 4.0-4.5, centrifuging to obtain supernatant, and keeping at 4-10 ℃ for later use;

3) Separation and purification:

adjusting the pH of the supernatant obtained in the step 2) to 7.0-8.0, removing impurities by using strong cation exchange resin, performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 1000-5000 Da, desalting and concentrating the permeate by using nanofiltration, separating and purifying by using preparative liquid chromatography, collecting the components with the retention time of 22-23 min, and freeze-drying.

4. The method for preparing sesame polypeptide from sesame protein according to claim 3, wherein the complex protease in step 2) consists of trypsin and chymotrypsin in a mass ratio of 6-12.

5. The method for preparing sesame polypeptide from sesame protein as claimed in claim 3, wherein the strong cation exchange resin used in step 3) is ANX sepharose FF and the nanofiltration membrane selected for nanofiltration has a molecular weight cut-off of 100-300 Da.

6. The method for preparing sesame polypeptide from sesame protein according to claim 3, wherein the conditions of the preparative liquid chromatography in the step 3) are: the chromatographic column Ultimate AQ-C18, mobile phase is 55% methanol aqueous solution containing 0.1% trifluoroacetic acid, flow rate is 8 mL/min, and detection wavelength is 220 nm.

7. The use of the sesame polypeptide of claim 1 in the preparation of a health product, a food product, a cosmetic product, an antioxidant and/or a hypotensive drug.