阅读说明：本技术 一种地龙生物活性小肽及其制备方法和应用 (Earthworm bioactive small peptide and preparation method and application thereof ) 是由 侯焘 李泊远 何慧 马雪琪 李凯凯 于 2021-10-20 设计创作，主要内容包括：本发明公开了一种来源于地龙且具有抗氧化生物活性的小肽,所述小肽的氨基酸序列如SEQ ID No.1或SEQ ID No.2所示。申请人首先从中药地龙中提取分离生物活性肽,接着通过液质联用、质谱解析、序列比对、分子对接和活性验证等大量试验从中筛选出具有较强抗氧化活性的小肽。该小肽为首次从中药地龙中分离鉴定,为充分利用地龙药材提供了新途径,同时也为抗氧化剂的开发提供了新资源。(The invention discloses a small peptide which is derived from earthworm and has antioxidant bioactivity, and 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 biological active peptide from traditional Chinese medicine earthworm, and then screens out small peptide with stronger antioxidant activity from the biological active peptide through a large amount of tests such as liquid chromatography-mass spectrometry, 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 a new resource for developing an antioxidant.)

1. A small peptide, characterized by: 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 Pheretima, and separating target peptide fragment from the bioactive peptide, wherein the extraction of bioactive peptide comprises the following steps:

(1) taking traditional Chinese medicine earthworm as a raw material, extracting crude protein by an alkali extraction and acid precipitation method;

(2) adding protease into the crude protein for enzymolysis, and performing membrane separation to obtain polypeptide with molecular weight less than 5 kDa.

3. A method of preparing the small peptide of claim 2, wherein: the protease is alkaline protease.

4. A method of preparing the small peptide of claim 3, wherein: the enzymolysis method comprises dissolving crude protein with water to obtain 1-10% solution, adding protease 1-5% of the weight of crude protein into the solution, performing enzymolysis at 40-60 deg.C and pH of 7.5-10 for 1-5 hr, heating for inactivation, centrifuging, and collecting supernatant.

5. The method of preparing the small peptide of claim 4, wherein: dissolving crude protein into 3-4% solution with water, adding protease 2-3% of the weight of the crude protein into the solution, performing enzymolysis for 2-3 hours at pH of 8-9 and 50-55 ℃, heating for inactivation, centrifuging, and taking supernatant.

6. A method of preparing the small peptide of claim 2, wherein: the alkali extraction and acid precipitation method comprises grinding Lumbricus, adding sodium hydroxide solution with pH of 8-11, leaching at 50-70 deg.C for 1-5 hr, centrifuging, adjusting pH of supernatant to 3-6 with HCl solution, standing for precipitation, centrifuging again, and freeze drying the precipitate.

7. The use of the small peptide of claim 1, wherein: the small peptide plays a role in vivo or in vitro antioxidation by removing ABTS free radicals and chelating ferrous ions, and can be used for preparing antioxidant dietary supplements or antioxidant skin care products.

Technical Field

The invention relates to a small peptide, in particular to a small peptide which is derived from earthworm and has antioxidant bioactivity, and also relates to a preparation method and application of the small peptide.

Background

Human cells can generate a large amount of Reactive Oxygen Species (ROS) in the metabolic process, and an antioxidant defense system of an organism can effectively eliminate the ROS, namely under a normal condition, the generation and elimination of the ROS are in a delicate balance state. When the ROS production is excessive or the body's antioxidant defense system fails, this balance is broken and the body is put under oxidative stress. It has been shown 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 which is widely researched in recent years, is used as an antioxidant, has a relatively simple structure, is easy to absorb, has good stability and low antigen content, has stronger antioxidant activity, has health care effects of reducing blood pressure, resisting cancer and the like, and is more and more concerned by people in the fields of food and medical health care products.

Earthworm is a rare Chinese medicinal material, and researches show that the earthworm peptide has pharmacological actions in various aspects such as anticoagulation, blood pressure reduction, antibiosis, anti-inflammation, cerebral apoplexy resistance, neuroprotection and the like. However, there has been no report on the antioxidant activity of the small peptide of earthworm protein hydrolysate. Therefore, the earthworm protein is used as a raw material to develop the antioxidant peptide, and a new way for deeply utilizing earthworm protein resources can be provided.

Disclosure of Invention

The invention aims to provide a small peptide, a preparation method and application thereof.

The above purpose is realized by the following technical scheme:

the applicant firstly extracts and separates biological active peptide from traditional Chinese medicine earthworm, and then screens out small peptide with stronger antioxidant activity from the earthworm through a large amount of tests such as liquid chromatography-mass spectrometry, 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 a new resource for developing an antioxidant.

The small peptide has the advantages of small molecular weight and easy preparation, can be extracted and separated from the traditional Chinese medicine earthworm, can also be prepared by using an artificial synthesis method well known in the field, and has the advantages of simple process, low cost and the like when the small peptide is 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 two steps of extracting bioactive peptide from traditional Chinese medicine earthworm and then separating target peptide segment from the bioactive peptide, wherein the extraction of the bioactive peptide comprises the following steps:

(1) taking traditional Chinese medicine earthworm as a raw material, extracting crude protein by an alkali extraction and acid precipitation method;

(2) adding protease into the crude protein for enzymolysis, and performing membrane separation to obtain polypeptide with molecular weight less than 5 kDa.

The target peptide fragment can be isolated by chromatography and the like well known in the art, for example, using semi-preparative or preparative liquid chromatography, and the skilled person need not overcome technical hurdles in using these methods based on the well-established 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 1-5% of the weight of crude protein into the solution, performing enzymolysis at pH 7.5-10 and temperature 40-60 deg.C for 1-5 hr, heating for inactivation, centrifuging, and collecting supernatant.

Preferably, the enzymolysis method comprises dissolving crude protein with water to obtain 3-4% solution, adding protease 2-3% of the weight of crude protein into the solution, performing enzymolysis at 50-55 deg.C and pH of 8-9 for 2-3 hr, heating for inactivation, centrifuging, and collecting supernatant.

Preferably, the alkali extraction and acid precipitation method comprises grinding Lumbricus, adding sodium hydroxide solution with pH of 8-11, leaching at 50-70 deg.C for 1-5 hr, centrifuging, adjusting pH of supernatant to 3-6 with HCl solution, standing for precipitation, centrifuging again, and lyophilizing the precipitate.

Still further, the invention provides an application of the small peptide, the small peptide plays an in-vivo or in-vitro antioxidant role by removing ABTS free radicals and chelating ferrous ions, and can be used for preparing antioxidant dietary supplements or antioxidant skin care products.

The technical scheme is more thoroughly described by referring to specific examples.

Drawings

FIG. 1 shows the results of screening test of protease species.

FIG. 2 shows the results of a screening test for crude protein concentration.

FIG. 3 shows the results of screening tests for the amount of enzyme added.

FIG. 4 shows the results of the screening test for enzymatic hydrolysis time.

Fig. 5 is a total ion flow diagram of earthworm antioxidant peptide.

Detailed Description

For a better understanding of the present invention, the following embodiments are described in further detail, but it will be understood by those skilled in the art that the following embodiments are not intended to limit the scope of the present invention, and any changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention should be construed as being equivalent to the scope of the present invention.

In the following examples, the method for determining antioxidant activity in vitro is as follows:

(1) determination of ABTS free radical clearance

Dissolving 3mg ABTS in 0.735mL distilled water to obtain 7.4mmol/L ABTS stock solution, and collecting 1mg K₂S₂O₈Dissolved in 1.43mL of distilled water to give 2.6mmol/L of K₂S₂O₈Stock solution, taking the above two solutions respectivelyMixing 0.2mL, reacting at room temperature in dark for 12h, diluting with 95% ethanol solution 40-50 times to obtain ABTS⁺And (4) working fluid. The sample was dissolved in ultrapure water to give a 2mg/mL sample solution. Add 40. mu.L of sample solution and 160. mu.L of LABTS to 96-well plate in sequence⁺Shaking the working solution for 10s, standing for 6min, replacing sample solution with 95% ethanol solution as blank control, measuring light absorption value at 734nm, and making 3 replicates for each sample.

ABTS free radical clearance rate ═ A₀-A)/A₀*100％

(2) Determination of ferrous ion chelation rate

Taking 0.0056g of ferrous sulfate heptahydrate, and adding distilled water to a constant volume of 10mL to obtain a ferrous sulfate solution of 2mmol/L, and taking 0.0246g of felazine sodium salt, and adding distilled water to a constant volume of 10mL to obtain a felazine solution of 5 mmol/L. The sample was dissolved in ultrapure water to give a 2mg/mL sample solution. And sequentially adding 50 mu L of sample liquid, 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 phenazine solution, mixing uniformly, and standing at room temperature for 10 min. The absorbance was measured at 562nm using distilled water instead of the sample solution as a blank, and 3 replicates were run for each sample.

Chelating rate of ferrous ion ═ a₀-A)/A₀*100％

EXAMPLE 1 preparation of active Polypeptides

(1) Extracting crude earthworm protein: grinding dried Lumbricus into powder, placing 40g Lumbricus powder in a beaker, adding 1L deionized water, leaching at 60 deg.C and pH 10 for 2.5h, centrifuging at 3800r/min for 20min, adjusting supernatant pH to 4.5 with 1mol/L HCl solution, standing for a certain time, centrifuging again, and freeze drying the precipitate to obtain crude Lumbricus protein.

(2) Preparing and grading polypeptide: dissolving crude protein with water to obtain 3% solution, adding alkaline protease 3% of the crude protein, performing enzymolysis at 55 deg.C and pH of 8 for 2 hr, heating for inactivation, centrifuging, and collecting supernatant. And finally, separating the supernatant by an ultrafiltration membrane with the molecular weight cutoff of 5kDa, and freeze-drying to obtain the polypeptide.

The applicant used a one-way comparison test to optimize the protease species and the addition amount thereof, the concentration of the crude protein solution, and the enzymolysis time in step (2), respectively, and the results are shown in fig. 1-4.

The results show that:

as can be seen from FIG. 1, the degree of hydrolysis was the highest with alkaline protease (10.5%) and the lowest with trypsin (2.8%); in terms of ABTS free radical clearance, the inhibition rate of the enzymolysis product of the compound protease is the highest (87.2%), the inhibition rate of the enzymolysis product of the compound protease is the second (82.5%), and the two have no significant difference (p is more than 0.05); in terms of ferrous ion chelation rate, the chelation rate of the enzymolysis product of the alkaline protease is the highest (77.5%), the rate of the compound protease is the second (65.4%), and the two are significantly different (p is less than 0.05). As the hydrolysis degree and the product activity of the alkaline protease are both higher, the enrichment degree of 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 broken lines are substantially the same, and when the crude protein concentration 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, when the amount of the enzyme added was 2%, the ABTS radical clearance was the highest (86.0%); when the addition amount of the enzyme is 3%, the chelating rate of ferrous ions is the highest (84.6%). The enzymolysis effect is comprehensively considered, and the addition amount of the selected enzyme is more preferably 3%.

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 the enzymolysis time is preferably selected to be 2 hours.

Example 2: screening of active peptide fragments

(1) Earthworm antioxidant peptide structure identification

The earthworm peptide prepared in example 1 was structurally characterized by using liquid chromatography-ESI-MS/MS. UPLC conditions: a chromatographic column: accucore 100 x 2.1mm,2.6 μm; mobile phase A: 0.1% formic acid-acetonitrile; b: 0.1% aqueous formic acid. Mass spectrum conditions: ESI + MS ion source, spraying voltage 3800V, capillary temperature 275 deg.C, and total earthworm antioxidant peptide ion flow diagram shown in FIG. 5. Analyzing the liquid quality result by means of Xcalibur software, and predicting that the first 80 peptide fragments may have better antioxidant activity by comparing the liquid quality result with the earthworm protein sequence in the UniProt protein database and sequencing according to Abundans values.

(2) Molecular docking

By utilizing Autodock molecular docking software, 80 peptides are used as receptors, ABTS free radicals are used as ligands to carry out molecular docking, and 3 peptide segments with optimal antioxidant activity are screened out according to docking scores: PGAGAVY, VADGDVLL, KDLY.

(3) Activity verification of peptide fragments

The screened three peptide fragments PGAGAVY, VADGDVLL and KDLY are artificially synthesized, the antioxidant activity of the three peptide fragments is measured, the sample detection concentrations of ABTS free radical clearance rate and ferrous ion chelation rate are respectively 62.5 mu g/mL and 1mg/mL, and the results are shown in Table 1.

TABLE 1 antioxidant Activity of three peptides and Mixed Polypeptides

The result shows that PGAGAVY and KDLY have better ABTS free radical scavenging effect and certain ferrous ion chelating capacity. Under the same concentration, the clearance rate and ferrous ion chelation rate of the two peptides to ABTS free radicals are higher than those of the mixed polypeptide prepared in example 1, and the other peptide fragment VADGDVLL screened out has poor antioxidant activity. The PGAGAVY and KDLY obtained by the invention are peptide fragments with good antioxidant activity, and can be subsequently used for preparing antioxidant dietary supplements and antioxidant skin care essence.

<110> university of agriculture in Huazhong

<120> earthworm bioactive small peptide, preparation method and application thereof

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<170> SIPOSequenceListing 1.0

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<213> Pheretima (Geosaurus)

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Pro Gly Ala Gly Ala Val Tyr

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<212> PRT

<213> Pheretima (Geosaurus)

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Lys Asp Leu Tyr

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