CN118530340B - A bioactive peptide with antioxidant and anti-allergic effects, composition and application - Google Patents

Abstract

The invention discloses a bioactive peptide with antioxidant and antiallergic effects, a composition and application thereof, and belongs to the technical field of bioactive peptides. The amino acid sequence of the bioactive peptide with the antioxidant and antiallergic effects is at least one of FPYTTQ, ADSQGRFL, GGTGPMDEY. The invention separates and purifies phycocyanin to obtain a plurality of bioactive peptides with antioxidant and antiallergic effects, chemically synthesizes the obtained peptide sequences, detects the antioxidant activity and antiallergic activity of single peptide and compound peptide of the synthesized peptide, the bioactive peptide and the compound peptide section thereof have good free radical removal and hyaluronidase inhibition activities, have good application prospects in the preparation of products with antioxidant and antiallergic effects, and lay a foundation for the high-added-value utilization of phycocyanin and the promotion of the research, development and application of functional active peptides.

Description

A bioactive peptide with antioxidant and anti-allergic effects, composition and application

Technical Field

The present invention belongs to the technical field of bioactive peptides, and in particular relates to a bioactive peptide with antioxidant and anti-allergic effects, a composition and an application thereof.

Background Art

Antioxidants are substances that can effectively inhibit free radical oxidation reactions. At present, most of the antioxidants used are chemically synthesized, such as L-ascorbic acid and its sodium salt, isoascorbic acid and its sodium salt. Anti-allergy mainly relies on drug treatment, such as oral antihistamines: loratadine, cetirizine, chlorpheniramine maleate, etc. However, the safety of these chemical synthetic reagents and drugs is controversial, and the presence of chemical residues and toxic side effects cannot meet people's demand for the safety and sustainable development of health food.

Although some natural functional proteins have antioxidant, anti-allergic and anti-tumor effects, as biological macromolecules, these natural proteins are mostly unstable, easily inactivated, and difficult to digest and absorb by the human body. These characteristics limit their further application in the fields of food, health care and medicine. Bioactive peptides are specific protein fragments. Compared with complete proteins, small molecule peptide fragments have high activity and small molecular weight and are easier to be absorbed and utilized by the human body.

Summary of the invention

In view of the problems existing in the prior art, the object of the present invention is to provide a bioactive peptide, composition and application with antioxidant and anti-allergic effects.

In order to achieve the above object, the present invention adopts the following technical solution:

A bioactive peptide with antioxidant and anti-allergic effects, wherein the amino acid sequence of the bioactive peptide is at least one of FPYTTQ, ADSQGRFL, and GGTGPMDEY.

A bioactive peptide composition with antioxidant and anti-allergic effects, the composition comprising at least one of the above-mentioned bioactive peptides with antioxidant and anti-allergic effects; preferably, the bioactive peptide composition comprises any two of FPYTTQ, ADSQGRFL, and GGTGPMDEY; further preferably, the bioactive peptide composition comprises FPYTTQ and GGTGPMDEY, and the mass ratio of FPYTTQ to GGTGPMDEY is 1:1.

Based on the above scheme, the antioxidant has the ability to scavenge free radicals.

Based on the above scheme, the free radical is at least one of DPPH, -OH, and ABTS+.

On the basis of the above scheme, the anti-allergic agent has the effect of inhibiting hyaluronidase.

Application of the above bioactive peptides with antioxidant and anti-allergic effects and the above bioactive peptide composition in the preparation of products with antioxidant and anti-allergic effects.

On the basis of the above scheme, the product includes food, medicine or health care product, and the product also contains excipients acceptable in pharmacy, food or health care product.

The above-mentioned bioactive peptide with antioxidant and anti-allergic effects is prepared by the following method:

(1) dissolving phycocyanin in distilled water to prepare a phycocyanin solution;

(2) adding protease to the phycocyanin solution for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, centrifuging to obtain a supernatant;

(3) ultrafiltration of the supernatant of step (2) to obtain a protease hydrolysate with a molecular weight range of <3 kDa, 3-10 kDa, and >10 kDa;

(4) taking the protease hydrolysate with a molecular weight of <3 kDa and further purifying it by gel permeation chromatography to obtain multiple polypeptide components;

(5) determining the antioxidant activity and anti-allergic activity of the polypeptide components obtained in step (4), selecting the components with the highest antioxidant and anti-allergic activities and further purifying them by reverse phase HPLC to obtain multiple further purified polypeptide components;

(6) Determine the antioxidant activity and anti-allergic activity of the multiple polypeptide components further purified in step (5), select the component with the highest antioxidant and anti-allergic activity, and determine the amino acid sequence of the obtained peptide fragment by liquid chromatography-mass spectrometry.

Based on the above scheme, the phycocyanin in step (1) is Spirulina platensis protein.

On the basis of the above scheme, the protease in step (2) is alkaline protease, the addition amount of the alkaline protease is 2000 U/g, the enzymatic hydrolysis conditions are 50°C, pH 10.5, and the enzymatic hydrolysis is 6 h.

Advantages of the technical solution of the present invention

The present invention separates and purifies a plurality of bioactive peptides with antioxidant and anti-allergic effects from phycocyanin by enzymatic hydrolysis, ultrafiltration, gel chromatography, and liquid chromatography separation techniques, and preliminarily identifies its components and sequences by liquid chromatography-mass spectrometry. Chemical synthesis is performed based on the identified peptide sequence, and the antioxidant and anti-allergic activities of the single peptide and compound peptide of the synthetic peptide are tested. The bioactive peptide and its compound peptide have good free radical scavenging and hyaluronidase inhibitory activities, and have good application prospects in the preparation of products with antioxidant and anti-allergic effects, laying a foundation for the high added value utilization of phycocyanin and promoting the research and development and application of functional active peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 Degree of hydrolysis of different phycocyanin hydrolysates;

FIG2 is a measurement result of the free radical scavenging ability of different phycocyanin hydrolysates;

FIG3 is a result of measuring the hyaluronidase inhibition rate of different phycocyanin hydrolysates;

Fig. 4 is an electrophoretic analysis of the products obtained by enzymatic hydrolysis of phycocyanin by alkaline protease (wherein the molecular weight of the protein marker A is 11-180 KDa; the molecular weight of the protein marker B is 3.3-20.1 KDa);

FIG5 is a result of measuring the free radical scavenging ability of products of different molecular weights obtained by enzymatic hydrolysis of phycocyanin with alkaline protease;

FIG6 is a result of measuring the hyaluronidase inhibition rate of products of different molecular weights obtained by enzymatic hydrolysis of phycocyanin with alkaline protease;

Figure 7 Superdex™ Increase 10/300 GL gel chromatogram of the molecular weight <3 KDa fraction;

FIG8 is a result of determining the free radical scavenging ability of each fraction collected from Superdex™ Increase 10/300 GL filtration;

FIG9 is a result of measuring the hyaluronidase inhibition rate of each fraction collected from Superdex™ Increase 10/300 GL filtration;

Figure 10 is the RP-HPLC chromatogram of the active fraction of Superdex™ Increase 10/300 GL loaded on a _C18 column;

FIG11 is the result of determination of free radical scavenging ability of each component collected from RP-HPLC;

FIG12 is the result of determination of hyaluronidase inhibition rate of each component collected from RP-HPLC;

FIG13 is the mass spectrometry analysis result of phycocyanin polypeptide FPYTTQ;

FIG14 is a mass spectrometry analysis result of the phycocyanin polypeptide ADSQGRFL;

FIG15 is the mass spectrometry analysis result of phycocyanin polypeptide GGTGPMDEY;

Figure 16 shows the molecular weight of the mass spectrum of the peptide FPYTTQ;

Figure 17 Molecular weight diagram of the mass spectrum of peptide ADSQGRFL;

Figure 18 shows the molecular weight of the mass spectrum of peptide GGTGPMDEY;

FIG19 is a result of measuring the free radical scavenging ability of chemically synthesized FPYTTQ, ADSQGRFL, and GGTGPMDEY peptides;

FIG20 is the measurement result of hyaluronidase inhibition rate of chemically synthesized FPYTTQ, ADSQGRFL, and GGTGPMDEY peptide segments;

Figure 21 Results of the free radical scavenging abilities of the composite peptides FPYTTQ+ADSQGRFL, FPYTTQ+GGTGPMDEY, and GGTGPMDEY+ADSQG RFL;

Figure 22 Results of hyaluronidase inhibition rates of the complex peptides FPYTTQ+ADSQGRFL, FPYTTQ+GGTGPMDEY, and GGTGPMDEY+ADSQG RFL.

DETAILED DESCRIPTION

The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below in conjunction with specific examples and with reference to data. The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

In the following embodiments,

The determination of phycocyanin hydrolysis was carried out using the o-phthalaldehyde (OPA) method, and the specific method is as follows:

80 mg of OPA was dissolved in 2 mL of β-mercaptoethanol, 5 mL of 10% SDS (w/v) and 92.8 mL of 0.1 M sodium tetraborate to prepare 100 mL of OPA reagent solution. 4 mL of phycocyanin hydrolysate hydrolyzed by different proteases was mixed with 4 mL of OPA reagent, incubated at room temperature for 2 min, and the absorbance at 340 nm was measured.

Phycocyanin was placed in 6M HCl and reacted at 4°C for 24 hours. The amount of free amino acids was determined using a serine standard curve as the amount of free amino acids produced by the complete hydrolysis of phycocyanin.

The degree of hydrolysis of phycocyanin was calculated according to the following formula:

Degree of hydrolysis (%) = [(NH ₂ ) _t - (NH ₂ ) _t0 )] / (NH ₂ ) _T

Wherein: (NH ₂ ) _t represents the amount of free amino acids in the enzymatic hydrolysis product at time t; (NH ₂ ) _t0 represents the amount of free amino acids that have not been hydrolyzed by enzyme; (NH ₂ ) _T represents the amount of free amino acids after complete hydrolysis.

Determination of DPPH free radical scavenging ability

The phycocyanin peptide solution was mixed with an equal volume of 0.2 mmol/L DPPH ethanol solution and reacted at room temperature in the dark for 30 min. The absorbance was measured at a wavelength of 517 nm (A sample). The corresponding absorbance was measured by replacing the DPPH ethanol solution with an equal amount of ethanol (A blank). The corresponding absorbance was measured by replacing the phycocyanin peptide solution with an equal amount of distilled water (A control). The DPPH free radical scavenging rate was calculated as follows:

Clearance rate (%) = [1 – (A sample – A blank) / A control] × 100.

Determination of -OH radical scavenging ability

0.5 mL 9 mmol/L ferrous sulfate solution, 1 mL 9 mmol/L salicylic acid ethanol solution, 1 mL 4.4 mmol/L hydrogen peroxide and 2 mL distilled water were added to 1 mL phycocyanin peptide solution in sequence. After mixing, the mixture was reacted in a 37°C water bath for 30 min. The absorbance was measured at a wavelength of 510 nm (B sample). The corresponding absorbance was measured by replacing the salicylic acid ethanol solution with an equal amount of ethanol (B blank). The corresponding absorbance was measured by replacing the phycocyanin peptide solution with an equal amount of distilled water (B control). The hydroxyl radical scavenging rate was calculated using the following formula:

Clearance rate (%) = (B control - B sample + B blank) / B control × 100.

Determination of free radical scavenging ability of ABTS+

Preparation of ABTS+ working solution: Mix equal volumes of 7.0 mM ABTS+ aqueous solution and 4.9 mM K ₃ S ₂ O ₈ and react at room temperature in the dark for 24 h to obtain ABTS+ solution. Use ethanol to dilute the ABTS+ solution to an absorbance of 0.70±0.02 at 734 nm to obtain ABTS+ working solution. Mix 150 μL of sample (C sample) or ethanol (C blank) with 4 mL of ABTS+ working solution, replace the ABTS+ working solution with an equal amount of ethanol and mix it with the sample (C control), incubate at room temperature in the dark for 10 minutes, and use a visible light spectrophotometer to detect the absorbance of the reaction solution at 734 nm. Calculate the ABTS+ clearance rate using the following formula:

Clearance rate (%) = [C blank - (C sample - C control)] / C control × 100.

Determination of hyaluronidase inhibitory activity

Antiallergic activity was determined using a modified version of the Morgan-Elson method as follows:

The four EP tubes were labeled A, B, C, and D. 100 μL of 0.2 M sodium acetate buffer (pH 4.6, containing 0.15 M NaCl) was added to tubes A and B, 100 μL of sample solution was added to tubes C and D, 50 μL of hyaluronidase (500 U/mL, prepared in sodium acetate buffer) was added to tubes A and C, and 50 μL of sodium acetate buffer was added to tubes B and D. After incubation at 37°C for 20 min, 20 μL of 2.5 mol/L CaCl ₂ was added to the reaction solution and incubated at 37°C for 20 min. Then 50 μL of hyaluronic acid (3 mg/mL, prepared in sodium acetate buffer), 100 μL of sodium acetate buffer, and 250 μL of deionized water were added to each tube and incubated at 37°C for 40 min. The enzymatic reaction was then stopped by adding 110 μL of alkaline borate solution and then heating in a boiling water bath for 5 min. The test tubes were then placed in ice water for 20 min, and then 1.5 mL of p-dimethylaminobenzaldehyde solution was added. To produce maximum coloration of the reaction mixture, the test tubes were incubated at 37°C for 20 min. The absorbance of the colored product was measured at 585 nm. The test samples were replaced with buffer solution instead of control, while the enzyme solution was replaced with blank buffer solution.

The antiallergic activity was calculated using the following formula:

Inhibition(%)=[(A-B)-(C-D)]/(A-B)×100

Where A is the control absorbance; B is the control blank absorbance; C is the sample absorbance; and D is the sample blank absorbance.

The phycocyanin used in the following examples was purchased from Zhejiang Binmei Biotechnology Co., Ltd.

Example 1

A bioactive peptide with antioxidant and anti-allergic effects, the bioactive peptide comprising a peptide having at least one amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 3:

SEQ ID NO: 1: FPYTTQ;

SEQ ID NO:2: ADSQGRFL;

SEQ ID NO:3: GGTGPMDEY.

Example 2

The preparation method of the bioactive peptide with antioxidant and anti-allergic effects comprises the following steps:

(1) Phycocyanin was dissolved in distilled water at a ratio of 1:20 (w/v). Then, alkaline protease at a concentration of 2000 U/g was added to the phycocyanin solution according to the amount of phycocyanin added. The solution was incubated at 50°C, pH 10.5, and enzymatic hydrolysis was performed for 6 h.

(2) After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is boiled at 100°C for 10 min to terminate the reaction. The enzymatic hydrolyzate is then centrifuged at 6000 r/min and 4°C for 15 min, and the supernatant is retained.

Effects of different proteases on the hydrolysis degree, free radical scavenging ability related to antioxidant activity and inhibition of hyaluronidase activity related to antiallergic activity of phycocyanin hydrolysates

Phycocyanin was hydrolyzed by papain, trypsin, pepsin, neutral protease and acid protease, respectively. The temperature and pH of the hydrolysis were carried out at the optimal temperature and pH of each protease (Table 1), and the other conditions were the same as those of alkaline protease. After the hydrolysis was completed, the effects of different proteases on the degree of hydrolysis (DH,%), the free radical scavenging ability related to antioxidant activity, and the inhibitory effect of hyaluronidase activity related to anti-allergic activity of the phycocyanin hydrolyzate were determined. The results are shown in Figure 1. The results show that alkaline protease has a good hydrolysis ability (Figure 1), and the antioxidant and anti-allergic effects of the hydrolyzate are significant (Figures 2 and 3).

Table 1 Optimal temperature and pH of different proteases

(3) The alkaline protease was used to hydrolyze phycocyanin to obtain the enzymatic product supernatant, which was fractionated into different molecular weights using an ultrafiltration centrifuge tube, with a molecular weight cutoff of 10 and 3 kDa. The fractions were collected as follows: >10 kDa, the peptide was retained but did not pass through the 10 kDa membrane; 3–10 kDa, the peptide permeated the 10 kDa membrane but not the 3 kDa membrane; <3 kDa, the peptide permeated the 3 kDa membrane. Thus, the protease hydrolysate with a molecular weight range of <3 kDa, 3-10 kDa, and >10 kDa was obtained (Figure 4).

The free radical scavenging ability related to antioxidant activity and the inhibitory effect of hyaluronidase activity related to anti-allergic activity of components with different molecular weights were measured. The results showed that protein peptides with a smaller molecular weight of < 3KDa showed better effects in scavenging free radicals and inhibiting hyaluronic acid (Figures 5 and 6).

(4) The digest of <3 kDa was further purified by filtration on a Superdex™ Increase 10/300 GL gel permeation column (10×300 mm). The column was eluted with deionized water (pH 10) and fractions were collected at a flow rate of 1 mL/min. The fractions were detected at 280 nm.

According to the time sequence of the peaks, six peptide components were collected and named F1, F2, F3, F4, F5 and F6 (Figure 7). The free radical scavenging ability related to antioxidant activity and the inhibitory effect of hyaluronidase activity related to anti-allergic activity of these six peptide components were determined. The results showed that all six components had the ability to scavenge free radicals (Figure 8), among which component F3 had the most significant effect. Similarly, the hyaluronic acid inhibition rate of component F3 was also the highest (Figure 9).

(5) Fraction F3 obtained from gel permeation chromatography was further purified using reverse phase HPLC on a C18 column. A linear gradient of acetonitrile (0–40%) containing 0.1% trifluoroacetic acid (TFA) was used at a flow rate of 1 mL/min. The elution peak was detected at 215 nm.

According to the time sequence of the peaks, five peptide components were collected and named F3-Ⅰ, F3-Ⅱ, F3-Ⅲ, F3-Ⅳ and F3-Ⅴ (Figure 10). The active peak was concentrated using a rotary evaporator. The free radical scavenging ability and hyaluronic acid inhibition rate were determined. The results showed that F3-Ⅲ showed good effects on free radical scavenging ability and hyaluronic acid inhibition (Figures 11 and 12).

(6) After centrifugation and drying, the F3-Ⅲ peptide sample was redissolved in Nano-LC mobile phase A (0.1% formic acid/water) and bottled for loading. The amino acid sequence was determined by liquid chromatography-mass spectrometry (LC-MS). The liquid phase was Easy nLC 1200 nanoliter liquid phase system. The sample was desalted and retained on the pre-column and then separated by the analytical column. The analytical column specification was C18 reverse phase chromatography (Acclaim PepMap RSLC, 75 μm×25 cm C18-2 μm 100 A). The gradient used in the experiment was mobile phase B (80% acetonitrile, 0.1% formic acid) increased from 5% to 38% within 30 min. The mass spectrometer used a ThermoFisher Q Exactive system (ThermoFisher, USA) combined with a nanospray Nano Flex ion source (ThermoFisher, USA). The spray voltage was 1.9 kV and the ion transfer tube heating temperature was 275°C. The mass spectrometer scanning mode was in the information-dependent acquisition mode (DDA, Data Dependent Analysis), with a primary mass spectrometer scanning resolution of 70000, a scanning range of 350-2000 m/z, and a maximum injection time of 100 ms. A maximum of 20 secondary spectra with charges of 2+ to 5+ were collected in each DDA cycle, and the maximum injection time of secondary mass spectrometer ions was 50 ms. The collision cell energy (high-energy collision-induced dissociation, HCD) was set to 28 eV, applicable to all precursor ions, and the dynamic exclusion was set to 25 s.

As a result, three peptides were obtained (Table 2). The secondary mass spectrum analysis showed that the peptide had a molecular weight of 755.349 and a sequence of FPYTTQ (Figure 13), a peptide had a molecular weight of 892.4402 and a sequence of ADSQGRFL (Figure 14), and a peptide had a molecular weight of 925.3488 and a sequence of GGTGPMDEY (Figure 15).

Table 2 List of amino acid sequences of component F3-Ⅲ determined by LC-MS

The sequences FPYTTQ, ADSQGRFL, and GGTGPMDEY in Table 2 above were chemically synthesized (Figures 16, 17, and 18). The free radical scavenging ability and hyaluronic acid inhibition rate of the synthesized peptides were measured for a single peptide (0.5 mg/mL). The results showed that the effects of free radical scavenging ability and hyaluronic acid inhibition were: FPYTTQ>GGTGPMDEY>ADSQGRFL (Figures 19 and 20).

Example 3

A bioactive peptide composition with antioxidant and anti-allergic effects contains FPYTTQ and ADSQGRFL, wherein the mass ratio of FPYTTQ to ADSQGRFL is 1:1.

Example 4

A bioactive peptide composition with antioxidant and anti-allergic effects contains FPYTTQ and GGTGPMDEY, wherein the mass ratio of FPYTTQ to GGTGPMDEY is 1:1.

Example 5

A bioactive peptide composition with antioxidant and anti-allergic effects contains ADSQGRFL and GGTGPMDEY, wherein the mass ratio of ADSQGRFL to GGTGPMDEY is 1:1.

The FPYTTQ, ADSQGRFL, and GGTGPMDEY peptides were compounded in pairs to obtain three compositions of FPYTTQ+ADSQGRFL, FPYTTQ+GGTGPMDEY, and ADSQGRFL+GGTGPMDEY. The mass ratio of the two peptides in the composition was 1:1; the concentration of each peptide was 0.5 mg/mL.

The free radical scavenging ability and hyaluronic acid inhibition rate of the composite peptide were measured. The results showed that the free radical scavenging ability and hyaluronic acid inhibition rate of the composite peptide were the best, which were significantly better than the effects of FPYTTQ or GGTGPMDEY alone (Figures 21 and 22).

The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims (10)

1. A bioactive peptide with antioxidant and antiallergic effects, which is characterized in that the amino acid sequence of the bioactive peptide is FPYTTQ, ADSQGRFL, GGTGPMDEY.

2. A bioactive peptide composition having antioxidant and antiallergic effects, characterized in that the composition comprises at least one bioactive peptide having antioxidant and antiallergic effects as claimed in claim 1.

3. The bioactive peptide composition having antioxidant and antiallergic effects of claim 2, wherein said composition comprises any two of FPYTTQ, ADSQGRFL, GGTGPMDEY.

4. The bioactive peptide composition having antioxidant and antiallergic effect of claim 3, wherein said composition comprises FPYTTQ and GGTGPMDEY.

5. The bioactive peptide composition with antioxidant and antiallergic effect of claim 4, wherein said mass ratio of FPYTTQ to GGTGPMDEY is 1:1.

6. The bioactive peptide composition having antioxidant and antiallergic effects of claim 2, wherein said antioxidant is capable of scavenging free radicals.

7. The bioactive peptide composition with antioxidant and antiallergic effect of claim 6, wherein said free radical is at least one of DPPH, -OH, abts+.

8. The bioactive peptide composition having an antioxidant and antiallergic effect of claim 2, wherein said antiallergic effect is an effect of inhibiting hyaluronidase.

9. Use of a bioactive peptide having antioxidant and antiallergic effects according to claim 1 and a bioactive peptide composition according to any of claims 2-8 for the preparation of a product having antioxidant and antiallergic effects, characterized in that said product is a pharmaceutical.

10. The use according to claim 9, wherein the medicament further comprises pharmaceutically acceptable excipients.