CN107226860B

CN107226860B - Bioactive polypeptide SKHSSLDCVL, and preparation method and application thereof

Info

Publication number: CN107226860B
Application number: CN201710546493.2A
Authority: CN
Inventors: 张少辉; 李阜烁; 汪超; 袁芳豪; 范梦珠; 陈静; 李婉茹
Original assignee: Zhejiang Huitai Life Health Technology Co ltd
Current assignee: Zhejiang Huitai Life Health Technology Co ltd
Priority date: 2017-07-06
Filing date: 2017-07-06
Publication date: 2020-04-10
Anticipated expiration: 2037-07-06
Also published as: CN107226860A

Abstract

The invention relates to the field of protein, and in particular relates to a bioactive polypeptide SKHSSLDCVL, a preparation method and application thereof, wherein the amino acid sequence of the bioactive polypeptide SKHSSLDCVL is Ser-Lys-His-Ser-Ser-Leu-Asp-Cys-Val-Leu. In-vitro antioxidant experiments and in-vitro immune function promotion experiments prove that the polypeptide SKHSSLDCVL has better antioxidant biological activity and immune regulation function, on one hand, the polypeptide has better antioxidant activity, can remove free radicals in a body and improve the quality of life; on the other hand, the bioactive polypeptide SKHSSLDCVL can enhance the in vitro proliferation capacity of lymphocytes and macrophages, promote the phagocytosis of neutral red by the macrophages, improve the resistance of the organism to infection by external pathogens, reduce the morbidity of the organism and have very important significance for developing foods, health-care products and medicines with immunoregulation function and antioxidant function.

Description

Bioactive polypeptide SKHSSLDCVL, and preparation method and application thereof

Technical Field

The invention relates to the field of proteins, in particular to a bioactive polypeptide SKHSSLDCVL, and a preparation method and application thereof.

Background

In the process of fermenting the cow milk by the lactic acid bacteria, a part of protein in the cow milk is metabolized and utilized by the lactic acid bacteria, and a series of physiological and biochemical reactions occur, so that the protein is changed into polypeptide or free amino acid which is digested and absorbed by a human body or directly enters the blood circulation of the human body through the absorption and transportation of small intestinal epithelial cells. Among these polypeptides, some have a specific physiological function and are called "bioactive peptides".

Oxidation reactions and oxidative metabolism are vital to food and the human body, and free radicals and active oxygen cause a series of oxidation reactions. When excessive free radicals are formed, they exceed the protective effects of protective enzymes such as superoxide dismutase, catalase, resulting in a series of side effects such as lipid oxidation, apoptosis, etc. The oxidation reaction not only affects the shelf life of the fat-containing food, but also causes certain harm to the health of human bodies, such as rheumatoid arthritis, diabetes, arteriosclerosis and the like. In addition, Collins et al, 2005 discovered that cancer development was also associated with oxidative damage to DNA.

Early synthetic antioxidants such as Butylated Hydroxyanisole (BHA), 2, 6-di-tert-butyl-4-methylphenol (BHT) were used in food as lipid antioxidants, but these synthetic additives all have potential risks to humans. Therefore, it is particularly important to find safe antioxidants in natural food sources. In recent years, it has been found that some food-derived polypeptides, such as short peptides of corn, soybean peptides, milk polypeptides, etc., have a good antioxidant effect. The polypeptides can be obtained through various ways such as microbial fermentation, digestion and enzymolysis and the like, and most of the polypeptides with antioxidant activity consist of 2-20 amino acid residues, have the molecular weight of less than 6000Da and contain a certain amount of hydrophobic amino acids and aromatic amino acids.

Immunoactive peptides are a class of bioactive polypeptides that are first obtained from milk following opioid peptide discovery and demonstrate their physiological activity. Jolles et al found in 1981 for the first time that a hexapeptide with an amino acid sequence Val-Glu-Pro-Ile-Pro-Tyr can be obtained by hydrolyzing human milk protein with trypsin, and in vitro experiments prove that the hexapeptide can enhance the phagocytosis of mouse abdominal cavity macrophages to sheep erythrocytes. Migliore-Samour et al found that the casein-derived hexapeptide Thr-Thr-Met-Pro-Leu-Trp was able to stimulate phagocytosis of murine peritoneal macrophages by sheep red blood cells and to enhance resistance to Klebsiella pneumoniae. Lemna hexandra et al, fed rats with synthetic milk-derived immunoregulatory peptide (PGPIPN), found that phagocytosis of macrophages in the abdominal cavity of rats and immunoregulatory function related to erythrocytes were significantly enhanced.

Researches show that the immune active peptide can not only enhance the immunity of the organism, stimulate the proliferation of lymphocytes of the organism, enhance the phagocytic function of macrophages, promote the release of cell factors, improve the capability of the organism for resisting the infection of external pathogens, reduce the morbidity of the organism, but also can not cause the immune rejection reaction of the organism.

At present, there are many researches on bioactive polypeptides, for example, chinese patent CN105254738A discloses a milk-derived bioactive polypeptide DELQDKIH derived from β -casein, chinese patent CN105254739A discloses a milk-derived bioactive polypeptide GTQYTD derived from α s 1-casein, and chinese patent CN105254740A discloses a milk-derived bioactive polypeptide NQFYQKF derived from α s 2-casein.

Disclosure of Invention

The invention aims to provide a bioactive polypeptide SKHSSLDCVL, and a preparation method and application thereof.

The purpose of the invention can be realized by the following technical scheme:

in the first aspect of the invention, a bioactive polypeptide SKHSSLDCVL is provided, the amino acid sequence of which is Ser-Lys-His-Ser-Ser-Leu-Asp-Cys-Val-Leu, as shown in SEQ ID NO: 1 is shown.

Preferably, the biologically active polypeptide is milk-derived. Specifically, the lactoferrin-containing composition is derived from lactoferrin and is an amino acid residue at the 418-427 th position of the lactoferrin. The lactoferrin amino acid sequence is shown as SEQ ID NO: 3, respectively.

The amino acid sequence and the corresponding nucleotide sequence of the lactoferrin are the prior art, and the nucleotide fragment for coding the 418 th to 427 th amino acid residues of the lactoferrin can code mature bioactive polypeptide SKHSSLDCVL.

Preferably, the bioactive polypeptide has antioxidant and immunoregulatory functions.

In a second aspect of the present invention, there is provided a nucleotide fragment encoding the biologically active polypeptide SKHSSLDCVL, the sequence of which is: 5'-tcc aaa cac agt agc cta gat tgt gtg ctg-3', as shown in SEQ ID NO: 2, respectively.

In the third aspect of the present invention, a preparation method of the bioactive polypeptide SKHSSLDCVL is provided, which can be artificially synthesized by a genetic engineering method, can be directly obtained from a dairy product by a separation and purification method, and can be directly prepared by chemical synthesis.

In the fourth aspect of the invention, the application of the bioactive polypeptide SKHSSLDCVL in preparing food, health products, medicines or cosmetics with antioxidant function is provided.

In the fifth aspect of the invention, the application of the bioactive polypeptide SKHSSLDCVL in preparing food, health-care products or medicines with immunoregulation function is provided.

In a sixth aspect, the invention provides an application of the bioactive polypeptide SKHSSLDCVL in preparing food, health care products or medicines with antioxidant function and immunoregulation function.

Specifically, the bioactive polypeptide SKHSSLDCVL can be used for preparing cosmetics for reducing free radical damage to skin, and medicines for resisting oxidation and/or regulating immunity of organism; and because the product of the bioactive polypeptide SKHSSLDCVL degraded by gastrointestinal tract still has bioactivity, the bioactive polypeptide SKHSSLDCVL can be used for preparing foods such as yoghourt and health-care products for regulating immunity, and can be orally taken for preparing medicines for resisting oxidation and/or regulating organism immunity.

In a seventh aspect of the invention, there is provided an antioxidant product comprising said biologically active polypeptide SKHSSLDCVL or a derivative of said biologically active polypeptide SKHSSLDCVL; the antioxidant product comprises antioxidant food, antioxidant health product, antioxidant medicine or antioxidant cosmetic; the derivative of the biologically active polypeptide SKHSSLDCVL refers to a polypeptide derivative obtained by performing modifications such as hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation on an amino acid side chain group, an amino terminal or a carboxyl terminal of the biologically active polypeptide SKHSSLDCVL.

In an eighth aspect of the invention, there is provided an immunomodulatory product comprising said biologically active polypeptide SKHSSLDCVL or a derivative of said biologically active polypeptide SKHSSLDCVL; the immunoregulation product comprises immunoregulation food, immunoregulation health-care products or immunoregulation medicines; the derivative of the biologically active polypeptide SKHSSLDCVL refers to a polypeptide derivative obtained by performing modifications such as hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation on an amino acid side chain group, an amino terminal or a carboxyl terminal of the biologically active polypeptide SKHSSLDCVL.

In the ninth aspect of the present invention, a product having both antioxidant function and immunoregulatory function is provided, which comprises the bioactive polypeptide SKHSSLDCVL or a derivative of the bioactive polypeptide SKHSSLDCVL; products with antioxidant and immunoregulatory effects include foods, health products or pharmaceuticals; the derivative of the biologically active polypeptide SKHSSLDCVL refers to a polypeptide derivative obtained by performing modifications such as hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation on an amino acid side chain group, an amino terminal or a carboxyl terminal of the biologically active polypeptide SKHSSLDCVL.

The bioactive polypeptide SKHSSLDCVL has the following beneficial effects: the milk-derived bioactive polypeptide SKHSSLDCVL has good antioxidant activity and activity for regulating the immunity of organisms; on one hand, the free radicals in the organism can be removed, and the harm of the free radicals to the human body is reduced; on the other hand, the bioactive polypeptide SKHSSLDCVL can also regulate the immunity of the organism, enhance the proliferation capacity of lymphocytes, improve the capability of the organism for resisting the infection of external pathogens, reduce the morbidity of the organism, and cannot cause the immune rejection reaction of the organism, thereby having very important significance for developing dairy products and health care products with the functions of resisting oxidation and regulating immunity.

Drawings

FIG. 1: mass chromatogram extraction (m/z 573.28);

FIG. 2: a primary mass spectrum of a fragment with a mass to charge ratio of 573.28;

FIG. 3: fragmentation of polypeptide az and by with mass-to-charge ratio of 573.28;

FIG. 4: [ DPPH. ] methanol Standard Curve;

FIG. 5: FeSO₄A standard curve.

Detailed Description

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS Inmolecular BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATINSTRUCUTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) Methods Inenzymolygy, Vol.304, Chromatin (P.M. Wassarman and A.P.Wolffe, eds.), academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

The invention is described in detail below with reference to the figures and specific embodiments.

Example 1 Artificial Synthesis of active peptide SKHSSLDCVL

Synthesis of bioactive peptide

1. 3g of RINK resin (degree of substitution 0.3mmol/g) was weighed into a 150ml reactor and soaked with 50ml of Dichloromethane (DCM).

After 2.2 hours, the resin was washed with 3 resin volumes of N-Dimethylformamide (DMF) and then drained, and this was repeated four times and the resin was drained until use.

3. The Fmoc protecting group on the resin was removed by adding a quantity of 20% piperidine (piperidine/DMF: 1:4, v: v) to the reactor and shaking on a decolourising shaker for 20 min. After deprotection, the resin was washed four times with 3 resin volumes of DMF and then drained.

4. And (3) detecting a small amount of resin by a ninhydrin (ninhydrin) method (detecting A and B, respectively, and reacting at 100 ℃ for 1min), wherein the resin is colored, which indicates that the deprotection is successful.

5. Weighing a proper amount of amino acid Ser and a proper amount of 1-hydroxy-benzotriazole (HOBT) into a 50ml centrifuge tube, adding 20ml of DMF to dissolve the amino acid Ser and the 1-hydroxy-benzotriazole (HOBT), then adding 3ml of N, N diisopropyl carbodiimide (DIC) to shake and shake for 1min, adding the solution into a reactor after the solution is clarified, and then placing the reactor into a30 ℃ shaking table to react.

After 6.2 hours, the column was capped with a suitable amount of acetic anhydride (acetic anhydride: DIEA: DCM ═ 1:1:2, v: v: v) for half an hour, then washed four times with 3 resin volumes of DMF and drained until needed.

7. The Fmoc protecting group on the resin was removed by adding a quantity of 20% piperidine (piperidine/DMF: 1:4, v: v) to the reactor and shaking on a decolourising shaker for 20 min. After deprotection was washed four times with DMF and then drained.

8. And (3) detecting a small amount of resin by a ninhydrin (ninhydrin) method (detecting A and B, respectively, and reacting at 100 ℃ for 1min), wherein the resin is colored, which indicates that the deprotection is successful.

9. Weighing a second proper amount of amino acid and a proper amount of HOBT in a 50ml centrifuge tube, adding 25ml of DMF to dissolve the amino acid and the HOBT, adding 2.5ml of DIC to shake and shake for 1min, adding the solution into a reactor after the solution is clarified, and then placing the reactor in a shaking table at 30 ℃ to react.

After 10.1 hours, a small amount of resin is taken for detection, and the detection is carried out by an indanthrone method (two drops are respectively detected A and B, and the reaction is carried out for 1min at 100 ℃), if the resin is colorless, the reaction is complete; if the resin is colored, the condensation is not complete and the reaction is continued.

11. After the reaction was completed, the resin was washed four times with DMF and then drained, and a certain amount of 20% piperidine (piperidine/DMF ═ 1:4, v: v) was added to the reactor, and the mixture was shaken on a decolorizing shaker for 20min to remove the Fmoc-protecting group from the resin. After the protection is removed, washing with DMF for four times, and then draining to detect whether the protection is removed.

12. And sequentially grafting amino acids Lys, His, Ser, Leu, Asp, Cys, Val and Leu according to the steps 9-11.

13. After the last amino acid had been grafted, the protection was removed, washed four times with DMF and the resin was drained with methanol. The polypeptide was then cleaved from the resin with 95 cleavage medium (trifluoroacetic acid: 1,2 ethanedithiol: 3, isopropylsilane: water: 95:2:2:1, v: v: v) (10 ml of cleavage medium per gram of resin) and centrifuged four times with glacial ethyl ether (cleavage medium: ethyl ether: 1:9, v: v).

To this end, bioactive peptide SKHSSLDCVL was synthesized.

The proper amount in the above steps 5 and 9 refers to the actual amount obtained by calculating a theoretical amount according to the target synthesis amount and yield, multiplying the theoretical amount by a coefficient (1.1 in this embodiment) on the basis of the theoretical amount.

Since the synthesis amount of the target peptide varies from one time to another and the yield varies, the amount of amino acid to be weighed varies from time to time.

For example, when the amount of the target peptide to be synthesized is 10 g and the yield (recovery rate) of the synthesized peptide is 90%, the theoretical amount of the amino acid is calculated by the following formula:

the theoretical weighed amount of amino acids was 10 grams amino acid molecular weight/target peptide molecular weight/90%.

The result calculated in this way is the theoretical weighing amount of amino acid, and in order to ensure that 10 g of polypeptide is obtained in the actual synthesis process, the amino acid weighing amount is a little more, and the amino acid weighing amount is generally 1.1 times of the theoretical weighing amount in the actual operation.

Similarly, 1-hydroxy-benzotriazole (HOBT) is used as a mediator in the synthesis of polypeptides, and is 1.1 times the theoretical nominal amount.

Since the theoretical amounts of amino acids and 1-hydroxy-benzotriazole (HOBT) used in the synthesis of bioactive peptides can be calculated by those skilled in the art according to the amount required for the target synthetic peptide, in actual operation, the target peptide can be synthesized by multiplying a coefficient on the basis of the theoretical amounts, and the bioactive peptides in this embodiment can be synthesized according to the conventional knowledge of those skilled in the art based on the synthesis steps and synthesis process conditions of this embodiment.

Confirmation of biologically active peptides

1) UPLC analysis

UPLC conditions were as follows:

the instrument comprises the following steps: waters ACQUITY UPLC ultra-high performance liquid-electrospray-quadrupole-time-of-flight mass spectrometer

Specification of chromatographic column: BEH C18 chromatographic column

Flow rate: 0.4mL/min

Temperature: 50 deg.C

Ultraviolet detection wavelength: 210nm

Sample introduction amount: 2 μ L

Gradient conditions: solution A: water containing 0.1% formic acid (v/v), liquid B: acetonitrile containing 0.1% formic acid (v/v)

2) Mass spectrometric analysis

The mass spectrometry conditions were as follows:

ion mode: ES +

Mass range (m/z): 100-1000

Capillary voltage (Capillary) (kV): 3.0

Sampling cone (V): 35.0

Ion source temperature (. degree. C.): 115

Desolvation temperature (. degree. C.): 350

Desolventizing gas stream (L/hr): 700.0

Collision energy (eV): 4.0

Scan time (sec): 0.25

Inner scan time (sec): 0.02

According to the above analysis method, ultra high performance liquid chromatography-electrospray-quadrupole-time-of-flight mass spectrometry is used to perform chromatographic analysis and mass spectrometry on bioactive peptide SKHSSLDCVL, the mass chromatogram extraction diagram is shown in FIG. 1, the primary and secondary mass spectrograms of the peak are shown in FIGS. 2 and 3, the polypeptide mass-to-charge ratio of the peak is 573.28Da, and the retention time is 17.74 min.

3) Results

As can be seen from fig. 3, the fragment sequence with the mass-to-charge ratio of 573.28Da was obtained by analysis and calculation with Progenesis QI software according to the cases of az and by fragmentation, and was Ser-Lys-His-Ser-Leu-Asp-Cys-Val-Leu (skhssldcvl) and was identified as SEQ ID NO: 1. the fragment corresponds to a residue sequence of 418-427 th positions of lactoferrin, the GenBank number of an amino acid sequence of the lactoferrin is AAA30617.1, and the sequence is shown in SEQ ID NO: 3.

example 2 antioxidant Activity assay of bioactive peptides

The bioactive polypeptide SKHSSLDCVL obtained in example 1 was tested for antioxidant activity by free radical scavenging (DPPH & method) and total antioxidant activity (Ferric Reducing AbilityPower FRAP method).

1. [ DPPH ] method for determining in vitro antioxidant activity of bioactive peptide SKHSSLDCVL

1) Experimental reagent and instrument

Reagent: 1, 1-Diphenyl-2-trinitrophenylhydrazine (1, 1-Diphenyl-2-piperidinylhydrazyl [ DPPH. ]), manufactured by Wako corporation of Japan; methanol, available from Shanghai national drug company; milk-derived bioactive polypeptide SKHSSLDCVL obtained in example 1.

The main apparatus is as follows: sunrise microplate reader, available from Tecan, austria; 96-well cell culture plates, manufactured by Millipore, usa; analytical balance, product of Meitelei-tolido.

2) Experimental methods

(1)1mmol/L of [ DPPH. ] methanol solution

0.349mg of [ DPPH ] is weighed by an analytical balance and dissolved in 1mL of methanol solution to prepare 1mmol/L of [ DPPH ] methanol solution, and the tinfoil is stored away from light and ready to use.

(2) Determination of [ DPPH. ] methanol Standard Curve

Add 100 μ L [ DPPH. cndot. ] methanol standard curve sample into 96-well plate according to table 1, let stand for 90min at room temperature, and detect the absorbance at 517nm with enzyme-linked immunosorbent assay.

TABLE 1[ DPPH. methanol Standard Curve solution preparation

From the experimental results, a curve was fitted using Excel and a regression equation was calculated, and the results are shown in fig. 4 (regression equation: y ═ 0.192x +0.2271, R²＝0.9991)。[DPPH·]The linear relation of the methanol standard curve is good, the correlation coefficient is 0.999, and the result shows that [ DPPH ]]The precision and accuracy of the methanol standard curve meet the detection requirements. From the results, the absorbance value was compared with [ DPPH ]]The contents are in inverse proportion, [ DPPH ]]The lower the content, the higher the absorbance, i.e.the greater the ability of the sample to scavenge free radicals.

(3) Method for measuring antioxidant activity of bioactive peptide SKHSSLDCVL by [ DPPH ]

1) Sample group: adding 80 μ L of 1mmol/L [ DPPH. cndot. ] methanol solution into a 96-well plate, and adding 20 μ L of samples to be tested (SKHSSLDCVL), positive control 1 (Trolox of 2.5 mg/mL), positive control 2 (Trolox of 0.025 mg/mL), and negative control (phytic acid) at different concentrations according to Table 2;

2) blank group: a blank was made on the same 96-well plate by adding 80. mu.L of a 1mmol/L [ DPPH. ] methanol solution and 20. mu.L of deionized water.

And (3) standing the sample to be detected for 90min at room temperature after the sample loading is finished, and detecting the light absorption value at 517nm by using an enzyme-labeling instrument. The radical scavenging rate was calculated according to the following formula and the experimental results are shown in table 2.

The formula:

TABLE 2 determination of antioxidant Activity of bioactive Polypeptides by the DPPH method

As can be seen from Table 2, 2.5mg/mL of Trolox as a positive control had the strongest ability to scavenge free radicals under the same conditions, almost all free radicals in solution were scavenged, followed by 0.025mg/mL of Trolox, phytic acid, active polypeptide. The rate of scavenging [ DPPH. ] free radicals by polypeptide SKHSSLDCVL was 28.39%, and with decreasing concentration of SKHSSLDCVL, the ability to scavenge free radicals decreased.

2. FARP method for measuring in-vitro antioxidant capacity of bioactive peptide SKHSSLDCVL

1) Experimental reagents and instruments

A total antioxidant capacity detection kit (a pharmaceutical Reducing activity of Plasma FRAP method) purchased from Shanghai Biyuntian Biotech company; FeSO₄Solution (10mmol/L), water-soluble vitamin E (Trolox solution) (10mmol/L), milk-derived bioactive polypeptide SKHSSLDCVL obtained in example 1.

The main apparatus is as follows: sunrise microplate reader, available from Tecan, austria; 96-well cell culture plates, manufactured by Millipore, usa; analytical balance, product of Meitelei-tolido; HWS26 model electric heating constant temperature water bath, manufactured by Shanghai-constant technology, Inc.

2) Experimental methods

(1) Preparation of FRAP working solution

According to the total antioxidant capacity detection kit, 7.5mL of TPTZ diluent, 750 mu L of TPTZ solution and 750 mu L of detection buffer solution are uniformly mixed, incubated in a water bath at 37 ℃ and used up within 2 hours.

(2)FeSO₄Preparation and determination of Standard Curve

Adding 180 mu LFRAP working solution into a 96-well plate, adding 5 mu L FeSO according to the table 3₄And (3) lightly mixing the standard curve solution, incubating for 3-5min at 37 ℃, and measuring the light absorption value at 593nm by using an enzyme-labeling instrument.

TABLE 3 FeSO₄Solution formulation for standard curve determination

FeSO₄The concentration and the light absorption value are in a good proportional relation, FeSO₄The higher the concentration, the higher the absorbance. FeSO of the invention₄The results of the standard curve are shown in FIG. 5, the linear relation of the standard curve is good, the correlation coefficient is 0.998, and the FeSO₄The precision and accuracy of the standard curve both meet the detection requirements and can be used for subsequent calculation.

(3) FRAP method for determining antioxidant capacity of bioactive polypeptide SKHSSLDCVL

Adding 180 mu L of FRAP working solution into a 96-well plate, and adding 5 mu L of ddH into a blank control well₂And O, adding 5 mu L of a sample to be detected into the sample detection hole and 5 mu L of phytic acid into the positive control, slightly mixing uniformly, incubating at 37 ℃ for 3-5min, and measuring the light absorption value at 593nm by using an enzyme-linked immunosorbent assay. Total antioxidant capacity is expressed as FeSO₄The concentration of the standard solution. The radical clearance was calculated according to the following formula and the results are shown in Table 4.

TABLE 4 FARP method for determining the Total antioxidant Capacity results of the bioactive polypeptide SKHSSLDCVL

The in vitro total antioxidant activity of the polypeptide SKHSSLDCVL is measured by a total antioxidant activity method (a Ferric Reducing activity Power FRAP method), and the bioactive polypeptide SKHSSLDCVL is found to have better capability of Reducing an oxidation substance; under the condition of concentration of 4mg/mL, the total antioxidant level of the polypeptide SKHSSLDCVL reaches 0.0200 mmol/g; the total antioxidant capacity of the bioactive polypeptide SKHSSLDCVL is proved to be higher than that of phytic acid with weak antioxidant activity under the same concentration, and the difference is significant (p is more than 0.05). Thus, the biologically active polypeptide SKHSSLDCVL of the invention was identified as having significant antioxidant capacity.

Example 3 Activity experiment of bioactive peptide for improving immunity

First, MTT method for testing in vitro lymphocyte proliferation capacity experiment of bioactive polypeptide SKHSSLDCVL

1) Experimental materials and instruments:

reagents and materials: experimental animals balb/c mice (male 6-8 weeks old, animal experiment center of Shanghai university of transportation, college of agriculture and biology); milk-derived bioactive polypeptide SKHSSLDCVL obtained in example 1; mouse lymphocyte extract (ex solibao); RPMI1640 medium (purchased from GIBCO); 3- (4, 5-Dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide salt (MTT, available from Amresco, Inc.); concanavalin (ConA, available from Sigma); bovine serum albumin (BSA, available from Genebase); pepsin (available from Sigma); pancreatin (Corolase PP, from AB).

The instrument comprises the following steps: LRH-250F Biochemical incubator, Shanghai Hengshi Co., Ltd; GL-22M high speed refrigerated centrifuge, shanghai luxiang instrument centrifuge instruments ltd; hera cell 150CO2 incubator, Heraeus; dragon wellscan mk3 microplate reader, Labsystems corporation; ALPHA 1-2-LD vacuum freeze drier, Christ company; ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometer, waters corporation.

2) The experimental method comprises the following steps:

taking mouse spleen under aseptic condition, extracting mouse lymphocyte with lymphocyte extract, and performing primary culture. The cell density was adjusted to 2.5X 10 with complete RPMI1640 medium⁶one/mL. To a 96-well cell culture plate were added in sequence: 100 μ L mouse lymphocyte suspension, 100 μ L RPMI1640 complete medium, 20 μ L concanavalin, 100 μ L sample. In addition, a blank control group (PBS with pH7.2-7.4 and 3 mol/L) and a negative control group (500 mu g/mL BSA) are arranged, and the research shows that the blank control group has no influence on the in vitro lymphocyte proliferation. Each set of 3 replicates. At 5% CO₂Culturing at 37 deg.C for 68h, adding 20 μ L MTT into each well under aseptic condition, culturing for 4h, carefully removing supernatant, adding 100 μ L dimethyl sulfoxide into each well, incubating at 37 deg.C for 10min, shaking, and measuring absorbance at 570nm with microplate reader.

The in vitro lymphocyte proliferation capacity is expressed by a stimulation index and is calculated as follows:

in the formula: a. the₁Absorbance at 570nm for the blank; a. the₂Absorbance at 570nm for the negative control, A₃The absorbance at 570nm for the experimental group.

3) Results and analysis of the experiments

The results are shown in Table 5. As can be seen from Table 5, under the condition that the mass concentration of the bioactive peptide SKHSSLDCVL is 100 μ g/mL, the stimulation index of the milk-derived bioactive peptide SKHSSLDCVL is greater than that of BSA, which indicates that SKHSSLDCVL can stimulate the proliferation of mouse lymphocytes in vitro to some extent. And SKHSSLDCVL reached a stimulation index of 1.184, which was significantly different from that of the negative control group (P < 0.05). Therefore, the active polypeptide SKHSSLDCVL can be considered to have the capacity of remarkably promoting the mouse lymphocyte proliferation, can be eaten as a health product or an additive, and can improve the immunity of animals and human bodies.

TABLE 5 Effect of biologically active polypeptide SKHSSLDCVL on in vitro lymphocyte proliferation

Experiment grouping	Stimulation index SI
		Negative control group	1
SKHSSLDCVL	1.184±0.072*

Note: the number marked as significant difference (P <0.05) compared to the negative control.

Second, MTT method for measuring in vitro macrophage proliferation ability experiment of bioactive polypeptide SKHSSLDCVL

1) Experimental reagent and instrument

Reagent: experimental animals balb/c mice (male 6-8 weeks old) were collected at the animal Experimental center of the college of agriculture and biology of Shanghai university of transportation; milk-derived bioactive polypeptide SKHSSLDCVL obtained in example 1; 3- (4, 5-Dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide (MTT) Amresco; LPS (lipopolysaccharide) Sigma company; bovine Serum Albumin (BSA) Genebase; triple solutions, aqueous solutions containing 10% SDS, 5% isobutanol and 0.012mol/L HCl.

The instrument equipment comprises: LRH-250F Biochemical incubator Shanghai Hengshi Co., Ltd; GL-22M high speed refrigerated centrifuge Shanghai Luxiang apparatus centrifuge Instrument Co., Ltd; hera cell 150CO₂Incubator Heraeus; dragon WellscanMK3 microplate reader Labsystems.

2) The test method comprises the following steps:

balb/c mice were injected intraperitoneally with 2ml of 2% (w/w) sterile starch solution for three consecutive days, and sacrificed by cervical dislocation 24 hours after the last injection. Peeling off the abdominal skin, sucking 4 ℃ Phosphate Buffer Solution (PBS) by using a syringe to repeatedly wash the abdominal cavity, centrifuging the washed solution by using a centrifuge tube for 10 minutes after collecting the washed solution, discarding the supernatant after centrifuging the washed solution (1000rpm and 4 ℃), washing the washed solution twice by using 4 ℃ RPMI1640 complete culture solution (containing 10% FBS), staining the washed solution by using 0.2% trypan blue solution to detect the vitality of the cells, and confirming that the collected viable macrophages account for more than 95%. After reading the cell counting plate, the cell concentration was adjusted to the appropriate concentration.

The cell suspension that had been blown to complete suspension was added to a 96-well cell culture plate at 37 ℃ with 5% CO in an appropriate volume₂After culturing for 4 hours under the environment, removing liquid in the holes, carefully cleaning the bottom of the holes of the cell culture plate by using a complete culture solution RPMI1640 at 37 ℃, and washing the cells and cell fragments which are not attached to the walls to obtain the purified attached abdominal cavity macrophages. 0.2ml of RPMI1640 complete medium was added to each well, and the small peptide sample for experiment and LPS were dissolved in the medium in advance and then added to start cell culture.

After obtaining purified adherent abdominal cavity macrophages, adding 200 mul/hole RPMI1640 complete culture solution (10% FBS) dissolved with bioactive polypeptide LPLP (1mg/ml) into each hole of the experimental group, and continuously culturing for 48 h; negative control group added BSA (500. mu.g/mL) dissolved in RPMI1640 complete medium (10% FBS) 200. mu.l/well; the blank group was continuously cultured for 48 hours with the addition of 200. mu.l/well of RPMI1640 complete medium (10% FBS). In addition, the experimental group, the negative control group and the blank group are respectively provided with a normal group and an inflammation group; LPS is added into the inflammation group when the inflammation group is cultured for 24 hours until the final concentration is 100 ng/ml; LPS is not added in a normal group; and the normal group and the inflammatory group were added with 5% MTT 20. mu.l/well at 44 h; after the cell culture reached 48h, 100. mu.l/well of the triple lysis buffer was added to terminate the culture, and after overnight lysis, the absorbance value (OD570) of each well was measured by a microplate reader at a wavelength of 570nm, and the Growth index (Growth Indices) was calculated as follows:

wherein the blank culture solution is RPMI1640 complete culture solution containing 10% FBS.

3) Results and analysis of the experiments

The results are shown in Table 6, and it is understood from Table 6 that macrophages were proliferated in both the normal group and the inflammatory group in the presence of 1mg/ml of the biologically active polypeptide SKHSSLDCVL. And compared with a negative control group, the two groups have significant difference (P is less than 0.01). The result shows that the bioactive polypeptide SKHSSLDCVL has obvious proliferation effect on in vitro macrophage.

TABLE 6 Effect of biologically active polypeptide SKHSSLDCVL on macrophage proliferation in vitro

Experiment grouping	Normal group GI	GI inflammation group
			Negative control group	1	1
SKHSSLDCVL(1mg/ml)	1.0883±0.0607**	1.126±0.0574**

Note: indicates a significant difference (P <0.05) compared to the negative control; indicates that there is a significant difference (P < 0.01) compared with the negative control group

Third, experiment of macrophage phagocytosis neutral red promoting ability of bioactive polypeptide SKHSSLDCVL

1) Experimental reagent and instrument

Reagent: experimental animals balb/c mice (male 6-8 weeks old) were collected at the animal Experimental center of the college of agriculture and biology of Shanghai university of transportation; milk-derived bioactive polypeptide SKHSSLDCVL obtained in example 1; LPS, purchased from Sigma; neutral red staining solution, produced by Biyuntian biotechnological research institute.

The instrument equipment comprises: LRH-250F Biochemical incubator Shanghai Hengshi Co., Ltd; GL-22M high speed refrigerated centrifuge Shanghai Luxiang apparatus centrifuge Instrument Co., Ltd; hera cell 150CO2 incubator Heraeus; dragon WellscanMK3 microplate reader Labsystems.

2) The test method comprises the following steps:

the number of the added cells was 2X 10⁶100 mul/well of cell suspension per ml, adding 200 mul/well of RPMI1640 complete culture solution (10% FBS) containing active peptide SKHSSLDCVL (1mg/ml) after adherent purification as experimental group, adding 200 mul/well of RPMI1640 complete culture solution (10% FBS) containing no active peptide for culture as blank group; and LPS is added into the experimental group and the blank group when the culture time reaches 24h to reach the final concentration of 10 mug/ml; after further culturing for 48h, the cell culture solution was aspirated. After washing the bottom of the well with PBS, 80. mu.l/well of neutral red dye solution at 37 ℃ was added, and after 10 minutes the dye solution was aspirated and washed twice with PBS, 150. mu.l of cell lysate (glacial acetic acid: absolute ethanol ═ 1:1, v/v) was added to each well. After overnight dissolution at 4 ℃ the absorbance value (OD540) was determined at a wavelength of 540 nm.

3) Experimental results and analysis:

TABLE 7 determination of the ability of the biologically active polypeptide SKHSSLDCVL to promote phagocytosis of neutral Red by macrophages

Note: significant difference compared to negative control (P <0.05)

Significant difference compared with negative control group (P < 0.01)

The experimental results are shown in Table 7, compared with the blank cell group, the macrophage phagocytosis ability of the inflammatory group macrophage added with 1mg/ml bioactive polypeptide SKHSSLDCVL is obviously increased, and compared with the blank cell group, the macrophage phagocytosis ability of the inflammatory group macrophage has significant difference (P is less than 0.01). The result shows that the bioactive polypeptide SKHSSLDCVL has obvious promotion effect on the ability of phagocytizing neutral red by macrophages in vitro under the condition of inflammation.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

<110> Zhejiang ghui peptide Life health science and technology Limited

<120> a bioactive polypeptide SKHSSLDCVL, and its preparation method and application

<160>3

<170>PatentIn version 3.3

<210>1

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> biologically active polypeptide

<400>1

Ser Lys His Ser Ser Leu Asp Cys Val Leu

5 10

<210>2

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> bioactive polypeptide coding sequence

<400>2

tccaaacaca gtagcctaga ttgtgtgctg 30

<210>3

<211>689

<212>PRT

<213> Artificial sequence

<220>

<223> Lactoferrin amino acid sequence

<400>3

Ala Pro Arg Lys Asn Val Arg Trp Cys Thr Ile Ser Gln Pro Glu

5 10 15

Trp Phe Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly

20 25 30

Ala Pro Ser Ile Thr Cys Val Arg Arg Ala Phe Ala Leu Glu Cys

35 40 45

Ile Arg Ala Ile Ala Glu Lys Lys Ala Asp Ala Val Thr Leu Asp

50 55 60

Gly Gly Met Val Phe Glu Ala Gly Arg Asp Pro Tyr Lys Leu Pro

65 70 75

Pro Val Ala Ala Glu Ile Tyr Gly Thr Lys Glu Ser Pro Gln Thr

80 85 90

His Tyr Tyr Ala Val Ala Val Val Lys Lys Gly Ser Asn Phe Gln

95 100 105

Leu Asp Gln Leu Gln Gly Arg Lys Ser Cys His Thr Gly Leu Gly

110 115 120

Arg Ser Ala Gly Trp Ile Ile Pro Met Gly Ile Leu Arg Pro Tyr

125 130 135

Leu Ser Trp Thr Glu Ser Leu Glu Pro Leu Gln Gly Ala Val Ala

140 145 150

Lys Phe Phe Ser Ala Ser Cys Val Pro Cys Ile Asp Arg Gln Ala

155 160 165

Tyr Pro Asn Leu Cys Gln Leu Cys Lys Gly Glu Gly Glu Asn Gln

170 175 180

Cys Ala Cys Ser Ser Arg Glu Pro Tyr Phe Gly Tyr Ser Gly Ala

185 190 195

Phe Lys Cys Leu Gln Asp Gly Ala Gly Asp Val Ala Phe Val Lys

200 205 210

Glu Thr Thr Val Phe Glu Asn Leu Pro Glu Lys Ala Asp Arg Asp

215 220 225

Gln Tyr Glu Leu Leu Cys Leu Asn Asn Ser Arg Ala Pro Val Asp

230 235 240

Ala Phe Lys Glu Cys His Leu Ala Gln Val Pro Ser His Ala Val

245 250 255

Val Ala Arg Ser Val Asp Gly Lys Glu Asp Leu Ile Trp Lys Leu

260 265 270

Leu Ser Lys Ala Gln Glu Lys Phe Gly Lys Asn Lys Ser Arg Ser

275 280 285

Phe Gln Leu Phe Gly Ser Pro Pro Gly Gln Arg Asp Leu Leu Phe

290 295 300

Lys Asp Ser Ala Leu Gly Phe Leu Arg Ile Pro Ser Lys Val Asp

305 310 315

Ser Ala Leu Tyr Leu Gly Ser Arg Tyr Leu Thr Thr Leu Lys Asn

320 325 330

Leu Arg Glu Thr Ala Glu Glu Val Lys Ala Arg Tyr Thr Arg Val

335 340 345

Val Trp Cys Ala Val Gly Pro Glu Glu Gln Lys Lys Cys Gln Gln

350 355 360

Trp Ser Gln Gln Ser Gly Gln Asn Val Thr Cys Ala Thr Ala Ser

365 370 375

Thr Thr Asp Asp Cys Ile Val Leu Val Leu Lys Gly Glu Ala Asp

380 385 390

Ala Leu Asn Leu Asp Gly Gly Tyr Ile Tyr Thr Ala Gly Lys Cys

395 400 405

Gly Leu Val Pro Val Leu Ala Glu Asn Arg Lys Ser Ser Lys His

410 415 420

Ser Ser Leu Asp Cys Val Leu Arg Pro Thr Glu Gly Tyr Leu Ala

425 430 435

Val Ala Val Val Lys Lys Ala Asn Glu Gly Leu Thr Trp Asn Ser

440 445 450

Leu Lys Asp Lys Lys Ser Cys His Thr Ala Val Asp Arg Thr Ala

455 460 465

Gly Trp Asn Ile Pro Met Gly Leu Ile Val Asn Gln Thr Gly Ser

470 475 480

Cys Ala Phe Asp Glu Phe Phe Ser Gln Ser Cys Ala Pro Gly Ala

485 490 495

Asp Pro Lys Ser Arg Leu Cys Ala Leu Cys Ala Gly Asp Asp Gln

500 505 510

Gly Leu Asp Lys Cys Val Pro Asn Ser Lys Glu Lys Tyr Tyr Gly

515 520 525

Tyr Thr Gly Ala Phe Arg Cys Leu Ala Glu Asp Val Gly Asp Val

530 535 540

Ala Phe Val Lys Asn Asp Thr Val Trp Glu Asn Thr Asn Gly Glu

545 550 555

Ser Thr Ala Asp Trp Ala Lys Asn Leu Asn Arg Glu Asp Phe Arg

560 565 570

Leu Leu Cys Leu Asp Gly Thr Arg Lys Pro Val Thr Glu Ala Gln

575 580 585

Ser Cys His Leu Ala Val Ala Pro Asn His Ala Val Val Ser Arg

590 595 600

Ser Asp Arg Ala Ala His Val Lys Gln Val Leu Leu His Gln Gln

605 610 615

Ala Leu Phe Gly Lys Asn Gly Lys Asn Cys Pro Asp Lys Phe Cys

620 625 630

Leu Phe Lys Ser Glu Thr Lys Asn Leu Leu Phe Asn Asp Asn Thr

635 640 645

Glu Cys Leu Ala Lys Leu Gly Gly Arg Pro Thr Tyr Glu Glu Tyr

650 655 660

Leu Gly Thr Glu Tyr Val Thr Ala Ile Ala Asn Leu Lys Lys Cys

665 670 675

Ser Thr Ser Pro Leu Leu Glu Ala Cys Ala Phe Leu Thr Arg

680 685

Claims

1. A bioactive polypeptide SKHSSLDCVL, characterized in that its amino acid sequence is Ser-Lys-His-Ser-Ser-Leu-Asp-Cys-Val-Leu.

2. A nucleotide fragment encoding the biologically active polypeptide SKHSSLDCVL of claim 1, wherein the nucleotide fragment has the sequence set forth in SEQ ID NO: 2, respectively.

3. The method of claim 1, wherein the biologically active polypeptide SKHSSLDCVL is synthesized by genetic engineering methods or is prepared directly by chemical synthesis.

4. The use of the biologically active polypeptide SKHSSLDCVL of claim 1, wherein the biologically active polypeptide SKHSSLDCVL is used in the preparation of a food, a health product, a pharmaceutical or a cosmetic product with antioxidant activity.

5. The use of the biologically active polypeptide SKHSSLDCVL of claim 1, wherein the biologically active polypeptide SKHSSLDCVL is used in the preparation of a food, a health product or a pharmaceutical product with immunomodulatory activity.

6. The use of the biologically active polypeptide SKHSSLDCVL of claim 1, wherein the biologically active polypeptide SKHSSLDCVL is used in the preparation of a food, a health product or a pharmaceutical product with antioxidant and immunomodulatory effects.

7. An antioxidant product comprising the biologically active polypeptide SKHSSLDCVL of claim 1; the antioxidant product comprises antioxidant food, antioxidant health product, antioxidant medicine or antioxidant cosmetic.

8. An immunomodulatory product comprising the biologically active polypeptide SKHSSLDCVL of claim 1; the immunoregulation product comprises immunoregulation food, immunoregulation health-care products or immunoregulation medicines.

9. A product having antioxidant and immunomodulating properties, comprising the biologically active polypeptide SKHSSLDCVL of claim 1; the product with antioxidant and immunoregulatory effects comprises food, health product or medicine.