CN113845565B - Lumbricus bioactive small peptide, and preparation method and application thereof - Google Patents
Lumbricus bioactive small peptide, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a small peptide derived from earthworm and having antioxidant biological activity, wherein the amino acid sequence of the small peptide is shown as SEQ ID No.1 or SEQ ID No. 2. The applicant firstly extracts and separates bioactive peptide from traditional Chinese medicine earthworm, and then screens out small peptide with stronger antioxidant activity through mass experiments such as liquid chromatography-mass spectrometry, mass spectrometry analysis, sequence comparison, molecular docking, activity verification and the like. The small peptide is separated and identified from the traditional Chinese medicine earthworm for the first time, provides a new way for fully utilizing earthworm medicinal materials, and also provides new resources for developing antioxidants.
Description
Technical Field
The invention relates to a small peptide, in particular to a small peptide which is derived from earthworm and has antioxidant biological activity, and also relates to a preparation method and application of the small peptide.
Background
The human cells produce a large amount of Reactive Oxygen Species (ROS) during metabolism, and the body's antioxidant defense system can effectively scavenge these ROS, i.e., the ROS production and elimination are normally in a subtle equilibrium state. When ROS production is excessive or the body's antioxidant defense system fails, this balance is broken and the body will be in an oxidative stress state. It has been demonstrated that oxidative stress damage can cause a variety of chronic diseases such as cancer, diabetes, inflammation, cardiovascular disease, asthma, and Alzheimer's disease.
The antioxidant peptide is a biological active peptide widely studied in recent years, is relatively simple in structure, easy to absorb, good in stability and low in antigen, has stronger antioxidant activity, has health care effects of reducing blood pressure, resisting cancer and the like, and is more and more concerned in the fields of foods and medical care products.
Lumbricus is a rare Chinese medicinal material in China, and has been studied to show that the earthworm peptide has various pharmacological effects of anticoagulation, blood pressure reduction, antibiosis, anti-inflammatory, cerebral apoplexy resistance, neuroprotection and the like. However, no antioxidant effect of small peptides of the earthworm protein hydrolysate has been reported. Therefore, the earthworm protein is used as a raw material to develop the antioxidant peptide, and a new way can be provided for the deep utilization of earthworm protein resources.
Disclosure of Invention
The invention aims to provide a small peptide, a preparation method and application thereof, wherein the small peptide is derived from traditional Chinese medicine earthworm and has good antioxidation effect.
The above purpose is achieved by the following technical scheme:
the applicant firstly extracts and separates bioactive peptide from traditional Chinese medicine earthworm, and then screens out small peptide with stronger antioxidant activity from the bioactive peptide through mass experiments such as liquid chromatography-mass spectrometry, mass spectrometry analysis, sequence comparison, molecular docking, activity verification and the like, wherein the amino acid sequence of the small peptide is PGAGAVY (SEQ ID No. 1) or KDLY (SEQ ID No. 2). The small peptide is separated and identified from the traditional Chinese medicine earthworm for the first time, provides a new way for fully utilizing earthworm medicinal materials, and also provides new resources for developing antioxidants.
The small peptide has the advantages of small molecular weight and easy preparation, can be extracted and separated from traditional Chinese medicine earthworm, can be prepared by using an artificial synthesis method well known in the art, and has the advantages of simple process, low cost and the like when being prepared by using the synthesis method due to the small molecular weight.
Further, the invention provides a method for preparing the small peptide, which comprises the steps of extracting bioactive peptide from traditional Chinese medicine earthworm and then separating target peptide fragments from the bioactive peptide, wherein the extraction of the bioactive peptide comprises the following steps:
(1) Extracting crude protein from traditional Chinese medicine earthworm by an alkali extraction and acid precipitation method;
(2) Adding protease into the crude protein for enzymolysis, and obtaining the polypeptide with molecular weight less than 5kDa after membrane separation.
The separation of the target peptide fragment can be performed by chromatography methods well known in the art, for example, using semi-preparative or preparative liquid chromatography, and the skilled person need not overcome technical hurdles when using these methods on the basis of the well-defined molecular structure of the target peptide fragment.
Preferably, the protease is an alkaline protease.
Preferably, the enzymolysis method comprises dissolving crude protein with water to obtain 1-10% solution, adding protease into the solution according to 1-5% of the weight of crude protein, performing enzymolysis at pH of 7.5-10 and temperature of 40-60deg.C for 1-5 hr, heating for inactivating, centrifuging, and collecting supernatant.
Preferably, the enzymolysis method is to dissolve crude protein into a solution with the concentration of 3-4%, then adding protease into the solution according to the weight of 2-3% of the crude protein, carrying out enzymolysis for 2-3 hours at the pH of 8-9 and the temperature of 50-55 ℃, heating, inactivating, centrifuging, and taking supernatant.
Preferably, the alkali extraction and acid precipitation method comprises pulverizing Lumbricus, adding sodium hydroxide solution with pH of 8-11, leaching at 50-70deg.C for 1-5 hr, centrifuging, adjusting pH of supernatant to 3-6 with HCl solution, standing for precipitation, centrifuging again, and lyophilizing.
Still further, the present invention provides an application of the small peptide, which exerts an antioxidant effect in vivo or in vitro by scavenging ABTS free radicals and chelating ferrous ions, and can be used for preparing antioxidant dietary supplements or antioxidant skin care products.
See the specific examples for further details.
Drawings
FIG. 1 shows the results of a screening test for protease species.
FIG. 2 shows the results of a screening test for crude protein concentration.
FIG. 3 shows the results of a screening test for enzyme addition.
FIG. 4 shows the results of the screening test for the enzymatic hydrolysis time.
FIG. 5 is a total ion flow chart of the earthworm antioxidant peptide.
Detailed Description
For a better understanding of the present invention, reference will now be made in detail to the present embodiments, but it should be understood by those skilled in the art that the following examples are not intended to limit the scope of the present invention, and any changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present invention should be construed as equivalent arrangements.
In the following examples, the in vitro antioxidant activity was measured as follows:
(1) Determination of ABTS radical clearance
Dissolving 3mg of ABTS in 0.735mL of distilled water to obtain 7.4mmol/L of ABTS stock solution, and taking 1mg of K 2 S 2 O 8 Dissolved in 1.43mL of distilled water to obtain 2.6mmol/L K 2 S 2 O 8 Mixing the stock solution with 0.2mL of each of the two solutions, performing light-shielding reaction at room temperature for 12h, and diluting with 95% ethanol solution for 40-50 times to obtain ABTS + And (5) working fluid. The sample was dissolved in ultrapure water to obtain a sample solution of 2 mg/mL. 40. Mu.L of sample solution and 160. Mu.LABSS were sequentially added to a 96-well plate + Working solution is oscillated for 10s, and is kept stand for 6min, 95% ethanol solution is used for replacing sample solution as blank control, the absorbance value is measured at 734nm, and each sample is in parallel with 3 samples.
ABTS radical scavenging = (a 0 -A)/A 0 *100％
(2) Determination of ferrous ion chelation Rate
Taking 0.0056g of ferrous sulfate heptahydrate to reach 10mL by distilled water to obtain 2mmol/L ferrous sulfate solution, taking 0.0246g of phenanthroline sodium salt to reach 10mL by distilled water to obtain 5mmol/L phenanthroline solution. The sample was dissolved in ultrapure water to obtain a sample solution of 2 mg/mL. Sequentially adding 50 mu L of sample solution, 180 mu L of distilled water and 4 mu L of ferrous sulfate solution into a 96-well plate, fully mixing uniformly, standing at room temperature for 5min, adding 8 mu L of phenanthroline solution, mixing uniformly, and standing at room temperature for 10min. Distilled water was used as a blank instead of the sample solution, and its absorbance was measured at 562nm, and 3 replicates were made for each sample.
Ferrous ion chelation rate= (a 0 -A)/A 0 *100％
EXAMPLE 1 preparation of active Polypeptides
(1) Extracting crude earthworm protein: grinding dry Lumbricus into powder, collecting 40g Lumbricus powder in beaker, adding 1L deionized water, leaching at 60deg.C and pH of 10 for 2.5 hr, centrifuging at 3800r/min for 20min, adjusting pH of supernatant to 4.5 with 1mol/L HCl solution, standing for a certain time, centrifuging again, and lyophilizing precipitate to obtain Lumbricus crude protein.
(2) Preparation and fractionation of the polypeptide: dissolving crude protein with water to obtain solution with concentration of 3%, adding alkaline protease into the solution according to 3% of crude protein weight, performing enzymolysis at pH of 8 and temperature of 55deg.C for 2 hr, heating for inactivating, centrifuging, and collecting supernatant. Finally separating the supernatant by an ultrafiltration membrane with the molecular weight cutoff of 5kDa, and freeze-drying to obtain the polypeptide.
The applicant uses a single factor comparison test to optimize the protease type and the addition amount thereof, the concentration of the crude protein solution and the enzymolysis time in the step (2), and the results are shown in figures 1-4.
The results show that:
as can be seen from FIG. 1, the alkaline protease has the highest degree of hydrolysis (10.5%) and trypsin has the lowest degree of hydrolysis (2.8%); in terms of ABTS free radical clearance, the inhibition rate of the enzymolysis product of the composite protease is the highest (87.2%), the inhibition rate of the alkaline protease is the next (82.5%), and the inhibition rate are not obviously different (p > 0.05); in terms of ferrous ion chelation rate, the chelation rate of alkaline protease enzymatic hydrolysis products is highest (77.5%), the complex protease is inferior (65.4%), and the chelation rates are significantly different (p < 0.05). Because the hydrolysis degree of the alkaline protease and the activity of the product are higher, the enrichment degree of the active peptide in the product is higher, and the yield is higher, so that the alkaline protease is finally selected.
As can be seen from FIG. 2, the trends of the two folding lines are basically the same, and when the concentration of crude protein reaches 3%, the ABTS free radical clearance rate and the ferrous ion chelation rate of the enzymolysis product are higher.
As can be seen from fig. 3, ABTS radical scavenging was highest (86.0%) when the enzyme addition was 2%; when the enzyme addition amount was 3%, the ferrous ion chelation rate was the highest (84.6%). Considering the enzymolysis effect comprehensively, the addition amount of the selected enzyme is 3 percent better.
As can be seen from FIG. 4, when the enzymolysis time is 2 hours, the ABTS free radical clearance and the ferrous ion chelation rate reach the highest simultaneously, and are respectively 85.8% and 86.1%, so that the enzymolysis time is better than 2 hours.
Example 2: screening of active peptide fragments
(1) Structural identification of earthworm antioxidant peptide
The structure of the earthworm peptide prepared in example 1 was identified by liquid chromatography-mass spectrometry (UPLC-ESI-MS/MS). UPLC condition: chromatographic column: accucore 100 x 2.1mm,2.6 μm; mobile phase a:0.1% formic acid-acetonitrile; b:0.1% formic acid in water. Mass spectrometry conditions: the ion source ESI+MS, spray voltage 3800V, capillary temperature 275 deg.C, and total ion flow diagram of Lumbricus antioxidant peptide as shown in figure 5. Analysis of the results by Xcalibur software, comparison with the Lumbricus protein sequences in the UniProt protein database, and sequencing according to Abundance values, predicting that the first 80 peptide fragments may have good antioxidant activity.
(2) Molecular docking
Using Autodock molecular docking software, using 80 peptides as receptors, using ABTS free radical as ligand to make molecular docking, screening out 3 peptide segments with optimal antioxidant activity according to docking scoring: PGAGAVY, VADGDVLL, KDLY.
(3) Verification of Activity of peptide fragments
The three peptide fragments PGAGAVY, VADGDVLL, KDLY were synthesized artificially and their antioxidant activity was measured, and the concentrations of the sample detection of ABTS radical scavenging rate and ferrous ion chelating rate were 62.5. Mu.g/mL and 1mg/mL, respectively, and the results are shown in Table 1.
Table 1 antioxidant Activity of three peptide fragments and Mixed Polypeptides
The result shows that PGAGAVY and KDLY have better ABTS free radical scavenging effect and certain ferrous ion chelating ability. The two peptides had higher scavenging rates for ABTS free radicals and ferrous ion chelating rates than the mixed polypeptide prepared in example 1 at the same concentration, while the other peptide fragment screened was inferior in the anti-oxidant activity of vaggdvll. The PGAGAVY and KDLY obtained by the invention are peptide fragments with good antioxidant activity, and can be used for preparing antioxidant dietary supplements and antioxidant skin care essence subsequently.
<110> university of agriculture in China
<120> an earthworm bioactive small peptide, its preparation method and application
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<170> SIPOSequenceListing 1.0
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<213> Lumbricus (Geosaurus)
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Pro Gly Ala Gly Ala Val Tyr
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<213> Lumbricus (Geosaurus)
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Lys Asp Leu Tyr
1
Claims (6)
1. A small peptide, characterized in that: the amino acid sequence of the small peptide is shown as SEQ ID No.1 or SEQ ID No. 2.
2. A method for preparing the small peptide of claim 1, comprising two steps of extracting bioactive peptide from traditional Chinese medicine earthworm and then separating target peptide fragment from bioactive peptide, wherein the extraction of bioactive peptide comprises the following steps:
(1) Extracting crude protein from traditional Chinese medicine earthworm by an alkali extraction and acid precipitation method;
(2) Adding alkaline protease into crude protein for enzymolysis, and membrane separating to obtain polypeptide with molecular weight less than 5 kDa.
3. The method for producing a small peptide according to claim 2, wherein: the enzymolysis method comprises dissolving crude protein with water to obtain 1-10% solution, adding alkaline protease into the solution according to 1-5% of the weight of crude protein, performing enzymolysis at pH of 7.5-10 and temperature of 40-60deg.C for 1-5 hr, heating for inactivating, centrifuging, and collecting supernatant.
4. A method of producing a small peptide according to claim 3, wherein: the enzymolysis method comprises dissolving crude protein with water to obtain solution with concentration of 3-4%, adding alkaline protease into the solution according to 2-3% of the weight of crude protein, performing enzymolysis at pH of 8-9 and temperature of 50-55deg.C for 2-3 hr, heating for inactivating, centrifuging, and collecting supernatant.
5. The method for producing a small peptide according to claim 2, wherein: the alkaline extraction and acid precipitation method comprises pulverizing Lumbricus, adding sodium hydroxide solution with pH of 8-11, leaching at 50-70deg.C for 1-5 hr, centrifuging, regulating pH of supernatant to 3-6 with HCl solution, standing for precipitation, centrifuging again, and lyophilizing.
6. Use of a small peptide according to claim 1, characterized in that: the small peptide plays an in-vivo or in-vitro antioxidation role by removing ABTS free radicals and chelating ferrous ions, and can be used for preparing an antioxidation dietary supplement or an antioxidation skin care product.
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| CN103845273B (en) * | 2014-03-14 | 2017-06-30 | 江苏隆力奇生物科技股份有限公司 | A kind of earthworm peptide of resisting age of skin and its preparation method and application |
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