An eggshell membrane antioxidant peptide, and a preparation method and application thereof

Abstract

An eggshell membrane antioxidant peptide and its preparation method and application, belonging to the field of biotechnology, identifies endophytes of Forsythia suspensa by LC-MS / MS LQ1 Active peptides produced by pepsin-assisted eggshell membrane hydrolysis were initially screened using ToxinPred, admetSAR, and BIOPEP-UWM analysis programs. Molecular docking technology was then used to identify peptide sequences with the greatest antioxidant potential due to their non-toxicity, high water solubility, and good intestinal absorption. These peptides were then synthesized for in vitro antioxidant validation. The amino acid sequence of this antioxidant peptide, STDVPRDPWVWG, is known to possess high antioxidant capacity and is suitable for use in food, beauty products, and health supplements. This provides a theoretical basis for the subsequent screening of natural antioxidant peptides from eggshell membranes.

Description

[0001] This case is a divisional application of the patent with application number "2023104348569".

[0002] The application date of the original application is April 21, 2023;

[0003] The application number is "2023104348569";

[0004] The invention name is "Eggshell membrane antioxidant peptide based on combined preparation of bacteria and enzymes and its preparation method and application". TECHNICAL FIELD

[0005] The present application belongs to the field of biotechnology, and relates to eggshell membrane antioxidant peptides, in particular to eggshell membrane antioxidant peptides based on combined preparation of bacteria and enzymes and their screening method and application. BACKGROUND

[0006] Chicken eggshells are a source of pollution that is difficult to manage in the environment and is a waste product of life and industry. Eggshell membranes are by-products of avian eggshells, also known as phoenix clothes, and their main components are various proteins, lipids, sugars, and sodium. By analyzing the main components, it is found that egg membranes are a potential source of polypeptides and amino acids with high utilization value. Currently, both at home and abroad, the hydrolysis of eggshell membranes is mainly by acid or alkali method. Protease is the best choice for hydrolyzing eggshell membranes due to its trace amount, high efficiency, safety, and harmlessness. Currently, there are also researchers who use acid-enzyme combination, alkali-enzyme combination, or enzyme method to hydrolyze eggshell membranes, and then study the antibacterial or antioxidant activity of the hydrolyzate. However, there are few reports on the use of bacteria and enzymes to hydrolyze eggshell membranes and the antibacterial and antioxidant activity of the hydrolyzate. Currently, the antibacterial preservatives used in the food and medical and cosmetic industries are mostly chemical reagents, and antioxidant is also a topic that has been discussed for a long time in the medical and cosmetic industries. If eggshell membranes can be utilized to obtain products with antibacterial and antioxidant activity, it will be of great benefit to the environment, food, medical, and cosmetic industries. SUMMARY

[0007] In view of the above deficiencies in the prior art, the present application provides an eggshell membrane antioxidant peptide based on combined preparation of bacteria and enzymes and a screening method thereof. The present application can effectively solve the problem of poor preparation and screening efficiency of existing eggshell membrane antioxidant peptides.

[0008] In order to achieve the above-mentioned purpose, the specific scheme adopted by the present application is as follows:

[0009] An eggshell membrane antioxidant peptide, whose amino acid sequence is: STDVPRDPWVWG.

[0010] The preparation method of the eggshell membrane antioxidant peptide uses eggshell membrane as raw material and adopts bacteria-enzyme combined preparation, the bacteria is Forsythia endophyte LQ1, and the enzyme is pepsin; the preservation number of the Forsythia endophyte LQ1 is CGMCC NO.8728.

[0011] As further optimization of the above technical solution, the preparation method specifically comprises the following steps:

[0012] (1) The eggshell membrane is dried at 60 DEG C, crushed, sieved, and eggshell membrane powder is obtained;

[0013] (2) The eggshell membrane powder is mixed with water in a ratio of 1:10-50, dissolved at 70-100 DEG C for 10-60 min, and cooled to room temperature; the pH of the solution is adjusted to 6.0, the Forsythia endophyte LQ1 bacterial liquid is added, fermented at 37 DEG C for 30-36 h, the bacteria are removed, the pepsin is added, and enzymolysis is carried out at 40 DEG C for 2-8 h, and then the solution is subjected to ultrasonic treatment; then the solution is subjected to enzyme inactivation treatment, centrifuged, and the pH of the solution is adjusted to neutral; the eggshell membrane hydrolysate is obtained, and the solution is filtered and concentrated;

[0014] (3) The concentrated solution is subjected to LC-MS / MS identification to obtain an enzymolysis peptide sequence, then the peptide sequence is simulated and screened by using a computer, the antioxidant peptide is preliminarily screened by using ToxinPred program, admetSAR program and BIOPEP-UWM program analysis, and after re-screening by using molecular docking technology, a peptide sequence with no toxicity, high water solubility, good intestinal absorption and the most antioxidant activity potential is obtained, and in-vitro antioxidant verification is carried out.

[0015] As further optimization of the above preparation method, in step (1), the particle size of the eggshell membrane powder is 120-140 mesh.

[0016] As further optimization of the above preparation method, in step (2), the pepsin enzymolysis conditions are as follows: the enzyme addition amount is 40 u / mg, the enzymolysis time is 4 h, the enzymolysis temperature is 40 DEG C, and the enzymolysis pH value is 4.

[0017] As further optimization of the above preparation method, in step (2), the bacterial liquid addition amount of the Forsythia endophyte LQ1 is 5-6.1% of the volume of the mixed liquid, and the bacterial liquid concentration is 1.5 x 10 8 cfu / mL.

[0018] As further optimization of the above preparation method, in step (2), the ultrasonic treatment temperature is 30-60 DEG C, the ultrasonic extraction time is 30-90 min, the ultrasonic extraction power is 60-90 W, and the ultrasonic frequency is 30-50 KHz.

[0019] As a further optimization of the above preparation method, in step (2), the centrifugation temperature is 3-4°C, the centrifugation speed is 10000-12000r, and the centrifugation time is 10-15min.

[0020] An antioxidant comprising the eggshell membrane antioxidant peptide.

[0021] Application of the eggshell membrane antioxidant peptide or antioxidant in food additives, cosmetics and health products.

[0022] The beneficial effects produced by the present invention are:

[0023] The eggshell membrane antioxidant peptide STDVPRDPWVWG described in this application exhibits high DPPH free radical scavenging activity, hydroxyl free radical scavenging rate, and high total antioxidant capacity. The antioxidant peptides obtained by extracting and screening the eggshell membrane antioxidant peptides from the formulation described in this application also exhibit certain antibacterial properties. The bacterial-enzyme synergistic technology combines microbial fermentation and enzymatic hydrolysis, offering two major advantages. The endophytic bacteria LQ1 from Forsythia suspensa can secrete cellulases and proteases during fermentation, which regulate animal intestinal health. Protease hydrolysis primarily involves enzymatically breaking disulfide bonds in proteins to produce soluble polypeptides and bioactive peptides. Therefore, screening for bacterial species capable of degrading eggshell membranes in natural plants, synergizing with pepsin, a proteinase that degrades eggshell membranes, and increasing the yield of bioactive eggshell membrane peptides is of great research significance. The process of validating the activity of a large number of bioactive peptides is time-consuming and labor-intensive. In contrast, the present invention overcomes the shortcomings of traditional methods by using computer-assisted bioinformatics screening of peptide activity, reducing the number of validation steps required and accurately screening the desired bioactive peptides for construction and biovalidation.

[0024] By adopting the extraction method of the present application, the antioxidant peptides in the eggshell membrane can be fully extracted and screened, thereby improving the efficiency of extracting the antioxidant peptides from the eggshell membrane. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Figure 1 is the electrophoresis result diagram; among them, 1 is the sample after 20% ammonium sulfate precipitation, and 2 is the sample after 80% ammonium sulfate precipitation;

[0026] Figure 2 It is a protein chromatogram;

[0027] Figure 3 This is a schematic diagram of the docking structure of the peptide IRDGWPH and the protein KEAP1; DETAILED DESCRIPTION

[0028] An eggshell membrane antioxidant peptide prepared by bacterial enzyme combination and a screening method thereof, comprising the following steps: drying and crushing the eggshell membrane at 60°C to obtain eggshell membrane powder; mixing the eggshell membrane powder with water at a ratio of 1:10-50, dissolving it at 70-100°C for 10-60 minutes, and then cooling it to room temperature to obtain a mixed solution; adjusting the pH of the solution to 6; adding 1.5×10 8 cfu / mL of a Forsythia suspensa endophyte LQ1 liquid was added at a rate of 5-6.1% by volume, and the mixture was fermented at 37°C for 30-36 hours to remove the bacteria. Pepsin was then added at a rate of 40 u / mg, and enzymatic hydrolysis was performed for 4 hours at a temperature of 40°C and a pH of 4. After completion, the enzyme was inactivated and centrifuged, and the supernatant was collected and concentrated under reduced pressure to obtain a concentrate. The concentrate was subjected to LC-MS / MS (liquid chromatography coupled with tandem mass spectrometry, LC-MS / MS) analysis to identify enzymatic peptide sequences. Peptide simulation screening was then performed using computers. Preliminary screening of antioxidant peptides was performed using the ToxinPred, admetSAR, and BIOPEP-UWM programs to analyze their absorption, distribution, metabolism, excretion, and toxicity characteristics. Molecular docking technology was then used to further screen the peptide sequences. The peptide sequences with the greatest antioxidant potential, high water solubility, and good intestinal absorption were synthesized and tested for in vitro antioxidant activity.

[0029] The antioxidant peptide sequences screened in this application are: IRDGWPH; STDVPRDPWVWG (SEQ ID NO: 1); EKIWHHTF.

[0030] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0031] Example

[0032] The preparation method of eggshell membrane antioxidant peptide comprises the following steps:

[0033] (1) Fresh eggshells were collected, the mucus attached to the eggshell membranes was washed off, and the eggshell membranes were peeled off. The eggshell membranes were washed with distilled water and then dried in an oven at 60°C. The dried eggshell membranes were crushed with a crusher, passed through a 120-mesh sieve, and stored in the dark for later use to obtain eggshell membrane powder.

[0034] (2) 20 g of ESM powder was mixed with 1000 mL of sterile water, dissolved at 85 °C for 40 min, and then cooled to room temperature; the pH of the solution was adjusted to 6, and 1.5 × 10 850 mL of the LQ1 endophyte of Forsythia suspensa with a CFU / mL of 108 was inoculated into the mixed solution, and after 36 h of fermentation at 37℃, the pH value of the fermentation liquid mixture was adjusted to 4, pepsin was added at an amount of 40 u / mg, and after enzymolysis at 40℃ for 4 h, the enzyme was inactivated by high-temperature, and the supernatant was obtained by centrifugation at 12000 r / min for 20 min, the solution pH was adjusted to neutral, and the ESM crude enzyme solution was obtained by filtration with a 0.45 μm filter membrane.

[0035] (3) The enzyme solution in (2) was subjected to ammonium sulfate fractionation precipitation

[0036] 1000 mL of the enzyme solution was evenly divided into 10 conical flasks, and pre-crushed ammonium sulfate powder was slowly added in an ice bath, and the protein precipitates with ammonium sulfate saturation degrees of 20%, 40%, 60%, and 80% were collected, respectively.

[0037] (4) Dialysis desalting and activity determination

[0038] The ammonium sulfate in the above precipitate was removed by dialysis. An appropriate amount of PBS buffer was added to resuspend the above precipitate, which was added into a treated dialysis bag (molecular weight cutoff 500D), sealed, and placed in a container filled with dialysis solution, and placed on a magnetic stirrer for dialysis. The dialysis solution was replaced every 3 h to speed up the dialysis process, and after 4-6 times of dialysis solution replacement, a small amount of sample solution in the dialysis bag was taken, and whether there was residual ammonium sulfate in the sample solution was detected by the principle that barium chloride and ammonium sulfate can produce white precipitate. After the ammonium sulfate was completely dialyzed and removed, the dialysis was completed and the sample solution in the dialysis bag was collected. The dialysis bag should not be directly contacted by hand during the whole process; the dialysis bag should be leak-tested before use; and the dialysis bag should be boiled in 50% ethanol and pure water for 20-30 min before and after use. After dialysis, the sample solution was freeze-dried, a certain amount of sample was resuspended with PBS buffer, and SDS-PAGE gel electrophoresis analysis and antioxidant activity determination were performed, respectively. According to the determination results, the sample for further purification was determined.

[0039] 0.1 g of sample was dissolved in 4 mL of water, and the DPPH clearance rate, hydroxyl radical clearance effect, and total antioxidant capacity were determined. The blank control solution was PBS buffer, and the positive control was VC solution. The results are shown in Table 1. The total antioxidant capacity, DPPH clearance rate, and hydroxyl radical clearance effect of the 80% ammonium sulfate precipitated sample were the best, reaching 16039.3 U / ml, 96.3%, and 84.28%, respectively.

[0040] Table 1 Comparison of antioxidant capacity of precipitated samples at different ammonium sulfate saturation degrees.

[0041]

[0042] The electrophoresis results are shown in Figure 1As can be seen from the figure, the sample after 20% ammonium sulfate precipitation, the molecular weight is larger, about 98KD, 80% ammonium sulfate precipitated sample, the molecular weight is relatively small, mainly concentrated in 14-22KD. It can be seen that the enzyme combined with eggshell membrane degradation solution of the material mainly for small molecule peptides, which will be further purified.

[0043] (6) Sephadex-G25 wet method column filtration chromatography

[0044] G-25 dextran can make different molecular weight of substances from the chromatographic column, so as to separate different molecular weight proteins. The above-80% ammonium sulfate treated ESM enzyme solution precipitate was dissolved with 0.9% NaCl, and further purified by chromatography. According to the fluctuation peak in the chromatographic data workstation, the samples collected in the same peak were combined.

[0045] The chromatographic data workstation spectrum parameters: full screen mileage (mV) 500, starting peak width level 3, minimum peak area 100. The results are shown in Figure 2 As shown in the figure, there are three main peaks, indicating that there are three different molecular weight polypeptides in the 80% ammonium sulfate precipitated sample. After freeze-drying, the 1 peak sample was collected 0.98g, the 2 peak sample was collected 1.69g, and the 3 peak sample was collected 0.87g.

[0046] Take 0.1g of each peak sample and add 4mL distilled water to dissolve. The blank solution for this determination is 0.9% NaCl solution, and the positive control is VC solution. The results are shown in Table 2. The 2 peak and 3 peak samples have better effect on hydroxyl radical scavenging than the 1 peak sample, and the 3 peak sample has the best effect. The 3 peak sample is mostly small molecule peptides (5-8KD). The 3 peak enzyme solution was freeze-dried for testing.

[0047] Table 2 Comparison of antioxidant capacity of each peak.

[0048]

[0049] (7) ESM antioxidant peptide extraction and analysis

[0050] ESM enzyme solution was taken out to centrifuge tube in frozen state; add appropriate amount of 0.1% TFA to dissolve; use Oasis HLB desalination column to desalt; use freeze centrifugal concentration dryer to dry the polypeptide; add appropriate amount of 50% ACN+0.5% TFA to dissolve; use Oasis MCX desalination column to desalt; use freeze centrifugal concentration dryer to dry the polypeptide; take 1 / 10 sample to dissolve polypeptide with 50 μL distilled water; use Pierce Quantitative Colorimetric Peptide Assay quantitative kit to determine the concentration and perform liquid chromatography tandem mass spectrometry analysis.

[0051] Table 3 EASY-nLC liquid phase gradient.

[0052]

[0053] Chromatographic separation time: 60 min; A phase: 2% acetonitrile 0.1% formic acid; B phase: 80% acetonitrile 0.1% formic acid; flow rate: 300 nL / min;

[0054] MS scan range (m / z): 300-1500, acquisition mode: DDA; Top 20 (select the 20 strongest signals in the parent ion for secondary fragmentation); primary mass resolution: 60000, AGC target: 3e6, maximum injection time: 20 ms, fragmentation mode HCD; secondary resolution: 15000, AGC target: 1e5, maximum injection time: 50 ms, fixed first mass: 100 m / z; minimum AGC target: 8e3, Intensity threshold: 1.6e5, dynamic exclusion time: 18 s.

[0055] Through liquid chromatography tandem mass spectrometry analysis, it was found that the number of amino acids in the ESM small molecule peptide after enzymolysis was within 2-10 oligopeptide segments, accounting for 95%. The proportion of oligopeptides obtained by this method was higher, and such peptides were more easily digested and absorbed, and had some physicochemical properties that large molecule proteins did not have. The structure of the smaller molecular weight peptide segment was easier to modify and modify, and the cost of artificial synthesis was lower. These characteristics provided a broad prospect for the development and utilization of ESM active polypeptide drugs.

[0056] Table 4 search result list.

[0057]

[0058] (8) Screening and physicochemical property evaluation of ESM enzyme solution antioxidant activity peptides

[0059] The potential toxicity of the resulting peptide sequences was predicted by the ToxinPred program (http: / / crdd.osdd.net / raghava / / toxinpred / ). The absorption, distribution, metabolism, excretion, and toxicity properties of the resulting peptide sequences were predicted by the admetSAR program (http: / / lmmd.ecust.edu.cn / admetsar2 / ), and the potential biological activity of the resulting peptide sequences was predicted by the BIOPEP-UWM program (http: / / www.uwm.edu.pl / biochemistry / index.php / pl / biopep).

[0060] ADMET analysis and activity prediction of the antioxidant peptides screened in Table 5.

[0061]

[0062] (9) Molecular docking screening

[0063] The polypeptide structure was drawn using ChemBioDraw Ultra 14.0, the structure was imported into ChemBio3D Ultra 14.0 for energy minimization, the Minimum RMS Gradient was set to 0.001, and the small molecule was saved as a mol2 format. The protein structure of Keap1 (PDB ID: 1ZGK) was downloaded from the PDB database, and Pymol 2.3.0 was used to remove protein crystal water, original ligand, etc. The polypeptide and protein structure were uploaded to HPEPDOCK for docking, and Pymol 2.3.0 was used for mapping.

[0064] As shown in Figure 3 , the binding energy of IRDGWPH with KEAP1 was -226.255 kcal / mol, indicating good binding. H7 of IRDGWPH formed hydrogen bonds with R380 and N382 of the protein, and the lengths of the hydrogen bonds were 2.01 and 2.02 Å, respectively. P6 of IRDGWPH formed hydrogen bonds with R380, N382, and R415 of the protein, and the lengths of the hydrogen bonds were 2.01, 2.02, and 2.03 Å, respectively. R2 of IRDGWPH formed hydrogen bonds with R415, Y525, and G527 of the protein, and the lengths of the hydrogen bonds were 2.02, 2.03, and 2.04 Å, respectively. I1 of IRDGWPH formed a hydrogen bond with Y572 of the protein, and the length of the hydrogen bond was 2.02 Å. The above sites may be the key sites for the action of the polypeptide.

[0065] (10) In vitro antioxidant verification of the synthesized eggshell membrane antioxidant peptides.

[0066] Synthesis of STDVPRDPWVWG, IRDGWPH, EKIWHHTF, with the same concentration of vitamin C as a positive control, and PBS buffer as a negative control.

[0067] Table 6 in vitro antioxidant verification.

[0068]

[0069] Comparative example

[0070] A common antioxidant vitamin C

[0071] The examples and comparative examples of the present application were prepared into 0.1 mg / ml solutions. The test results showed that the antioxidant capacity of the two was equivalent at the same concentration.

[0072] It should be noted that the above-mentioned embodiments should be understood as illustrative rather than limiting the scope of protection of the present application, and the scope of protection of the present application is subject to the claims. For those skilled in the art, some non-essential improvements and adjustments of the present application without departing from the spirit and scope of the present application still belong to the protection scope of the present application.

Claims

1. An eggshell membrane antioxidant peptide, whose amino acid sequence is: STDVPRDPWVWG.

2. The method for preparing the eggshell membrane antioxidant peptide according to claim 1, wherein: The invention uses eggshell membrane as raw material and adopts bacterial enzyme combination preparation, wherein the bacterial is an endophytic bacterium of Forsythia suspensa with a preservation number of CGMCC NO.8728. LQ1 , the enzyme is pepsin; the method comprises the following steps: (1) Dry the eggshell membranes at 60°C, crush them, and sieve them to obtain eggshell membrane powder; (2) Mix eggshell membrane powder and water in a ratio of 1:10-50, dissolve at 70-100°C for 10-60 min, and then cool to room temperature to obtain a mixed solution; adjust the pH of the solution to 6.0, and add endophytes of Forsythia suspensa. LQ1 The bacterial solution was fermented at 37°C for 30-36 hours, after which the bacterial cells were removed and pepsin was added. The solution was enzymatically hydrolyzed at 40°C for 2-8 hours and ultrasonically treated. The solution was then inactivated and centrifuged. The pH of the solution was adjusted to neutral to obtain the eggshell membrane hydrolyzate, which was filtered and concentrated under reduced pressure. (3) The concentrate was identified by LC-MS / MS to obtain the enzymatic peptide sequence, and then the peptide simulation screening was performed by computer. The antioxidant peptides were preliminarily screened by the ToxinPred program, admetSAR program and BIOPEP-UWM program analysis. The molecular docking technology was then used to screen again. After the peptide sequence with non-toxicity, high water solubility, good intestinal absorption and the greatest antioxidant activity potential was obtained, it was synthesized for in vitro antioxidant verification.

3. The preparation method according to claim 2, wherein: In step (1), the particle size of the eggshell membrane powder is 120-140 mesh.

4. The preparation method according to claim 2, wherein: In step (2), the pepsin hydrolysis conditions are as follows: enzyme addition amount is 40u / mg, hydrolysis time is 4h, hydrolysis temperature is 40°C, and hydrolysis pH value is 4.

5. The preparation method according to claim 2, wherein: In step (2), the amount of the bacterial solution of the Forsythia suspensa endophyte LQ1 is 5%-6.1% of the volume of the mixed solution, and the concentration of the bacterial solution is 1.5×10 8 cfu / mL.

6. The preparation method according to claim 2, wherein: In step (2), the temperature of the ultrasonic treatment is 30-60° C., the ultrasonic extraction is performed for 30-90 min, the ultrasonic extraction power is 60-90 W, and the ultrasonic frequency is 30-50 KHz.

7. The preparation method according to claim 2, characterized in that: In step (2), the centrifugation temperature is 3-4°C, the centrifugation speed is 10,000-12,000 r, and the centrifugation time is 10-15 min. An antioxidant comprising the eggshell membrane antioxidant peptide according to claim 1 .

9. Use of the eggshell membrane antioxidant peptide according to claim 1 or the antioxidant according to claim 8 in food additives, cosmetics, and health products.

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