CN114805479A

CN114805479A - Bioactive peptide with dipeptidyl peptidase IV inhibitory activity

Info

Publication number: CN114805479A
Application number: CN202210385683.1A
Authority: CN
Inventors: 郭元晟; 朱建军; 刘彦敏; 陈子衡; 伊日布斯; 郭梁; 雅梅
Original assignee: XILINGOL VOCATIONAL COLLEGE; Kunming University of Science and Technology
Current assignee: XILINGOL VOCATIONAL COLLEGE; Kunming University of Science and Technology
Priority date: 2022-04-13
Filing date: 2022-04-13
Publication date: 2022-07-29
Anticipated expiration: 2042-04-13
Also published as: CN114805479B

Abstract

The invention discloses a biological active peptide, which is derived from fermented horse milk, the amino acid sequence of the biological active peptide is Ile-Ala-Phe-Pro, the molecular weight of the biological active peptide is 500.6Da, the biological active peptide has dipeptidyl peptidase IV inhibiting activity, and the semi-Inhibiting Concentration (IC) of the biological active peptide on dipeptidyl peptidase IV ₅₀ ) Is 0.001 mol/L; the glucose consumption capability of the high-glucose-induced Hepg2 cell model can be improved, the effect of inhibiting the glucose uptake of the Hepg2 cell model is achieved, the application prospect in the aspect of preparing health care products and medicines for treating or assisting in treating type 2 diabetes mellitus is achieved, the preparation is simple, and the method is suitable for industrial production and market popularization and application.

Description

Bioactive peptide with dipeptidyl peptidase IV inhibitory activity

Technical Field

The invention relates to a bioactive peptide with DPP-IV inhibitory activity in fermented horse milk, and belongs to the technical field of bioactive peptide biology.

Background

With the development of social economy and the change of life style of people, the global incidence situation of diabetes is more and more severe, and the incidence population is younger and younger, and simultaneously, the incidence population can cause a plurality of chronic complications. Diabetes is an endocrine metabolic disease (WHO, 2006) in which hyperglycemia occurs due to insulin deficiency or a decrease in its biological effect or both in vivo and which causes disorders in fat and protein metabolism. Of all types of diabetes, type 2 diabetes is the most prevalent, accounting for 90-95% of all diabetes. In the pathogenesis of the diabetes mellitus, DPP-IV can quickly inactivate various hormones such as incretin glucagon-like peptide-1 and glucose-dependent insulin release peptide, so that the blood sugar is increased, and the DPP-IV becomes a new target for treating the type 2 diabetes mellitus. DPP-IV inhibitors have become an important direction for the development of diabetes drugs, and the commonly used drugs include: vildagliptin (Vitagliptin), Saxagliptin (Saxagliptin), alogliptin (aliliptin), and linagliptin (lilatidine). Prolonged administration of these chemically synthesized DPP-IV inhibitor drugs may result in side effects such as allergic reactions, heart rate disorders, upper respiratory tract infections, gastrointestinal distress, etc.

In the research related to diabetes reduction of milk-derived bioactive peptides, cow's milk and related products have been widely studied. Compared with cow milk, the cow milk has casein in 1.05% and soluble protein as high as 1.03%, is albumin milk, is easy to digest and absorb, can be used in the fields of functional food and health care products, and has wide market application prospect.

Disclosure of Invention

The invention provides a biological active peptide derived from fermented horse milk, the amino acid sequence of the biological active peptide is Ile-Ala-Phe-Pro, and the biological active peptide has dipeptidyl peptidase IV inhibiting activity.

The method takes fresh horse milk as a raw material, and prepares fermented horse milk by using lactic acid bacteria and milk-derived yeast for co-fermentation; separating and purifying by using a cut-off membrane, sephadex and a reverse high-performance liquid phase to obtain the bioactive peptide with DPP-IV inhibitory activity, wherein the amino acid sequence of the bioactive peptide is as follows: isoleucine-alanine-phenylalanine-proline (IAFP), with a molecular weight of 500.6Da, has a half-inhibitory concentration of 0.001mol/L to DPP-IV, and has the functions of remarkably improving the activity of a high-glucose-induced insulin resistance Hepg2 cell model and remarkably improving the glucose consumption and glucose uptake capacity of a high-glucose-induced insulin resistance cell Hepg 2.

The invention obtains the small molecular peptide IAFP which can obviously inhibit the DPP-IV activity by separating and purifying the horse milk co-fermented by lactic acid bacteria and saccharomycetes.

The preparation process of the DPP-IV inhibitory peptide comprises the following steps:

(1) activating strains: inoculating lactobacillus into an MRS liquid culture medium according to the inoculation amount of 0.5-2% by volume, and standing and culturing at 25-42 ℃ for 8-20 h, wherein the viable count of the activated bacterium liquid is not less than 10 ⁹ CFU/mL, inoculating yeast 0.5-3% by volume into YPD liquid culture medium, culturing at 20-35 deg.C and 100-300 rpm for 8-48 hr, and activating to obtain bacterial liquid with viable count of not less than 10 ⁸ CFU/mL; continuously proliferating the two bacteria for 2-4 generations for later use;

(2) filtering or centrifuging fresh horse milk to purify the milk, and sterilizing at 60-150 ℃ for 2 s-30 min;

(3) inoculating activated lactobacillus and yeast when horse milk is cooled to 20-40 ℃, wherein the inoculation amount is 0.1-5%, and the viable count in the horse milk after inoculation is 10 ⁶ ～ ⁹ CFU/mL；

(4) Fermenting the horse milk under the following conditions: fermenting for 4-60 h at 20-40 ℃, stirring speed of 20-200 rpm and ventilation of 10-200 vvm;

(5) centrifuging to obtain a supernatant after the fermentation of the horse milk is finished, adjusting the pH of the supernatant to 6-9, and centrifuging again to remove protein; the centrifugation conditions were: centrifuging for 10-30 min at 9000-12000 g at 2-10 ℃;

(6) carrying out vacuum freeze drying or rotary evaporation concentration on the obtained supernatant;

(7) dissolving the supernatant concentrate with water, separating the solution by using ultrafiltration cut-off membranes with different cut-off molecular weights to obtain liquids containing different components, and measuring the DPP-IV inhibitory activity of the different liquids;

(8) selecting a liquid with the best inhibitory activity, carrying out chromatography by using sephadex to obtain chromatographic liquids at different time intervals, and determining the DPP-IV inhibitory activity of different chromatographic liquids;

(9) selecting the chromatographic solution with the best inhibitory activity, and separating and purifying by using reverse phase high performance liquid chromatography (RP-HPLC); an Agilent chromatographic column is used for separation and purification, and the specification is as follows: c18, 5 μm, 250X 4.6id, 100A; the separation and purification conditions are as follows: the sample volume is 1mL, the flow rate is 2 mL/min, and the detection wavelength is 215 nm; mobile phase a was ultrapure water containing 0.1% (v/v) trifluoroacetic acid (TFA), mobile phase B was acetonitrile containing 0.1% (v/v) TFA, elution conditions: gradient eluting with mobile phase A and B (0-5 min, 10% B, 5-40 min, 10-50% B, 40-50 min, 50-80% B, 50-60 min, 80-10% B, 60-70 min, 10% B);

(10) selecting a peptide with DPP-IV inhibitory activity after RP-HPLC purification for LC-MS identification, predicting the DPP-IV inhibitory activity of a peptide sequence obtained after identification in a Biopep database, selecting the peptide with the best predicted activity for solid phase synthesis verification, and finally obtaining the small molecular peptide;

in the above steps, the DPP-IV inhibitory activity of the sample is measured by using an enzyme labeling method.

The invention has the following advantages and positive effects:

1. the invention proves that the horse milk co-fermented by lactic acid bacteria and saccharomycetes can be used for preparing the micromolecular peptide with DPP-IV inhibitory activity;

2. the preparation method disclosed by the invention can be used for preparing bioactive peptides with DPP-IV inhibitory activity from fermented horse milk, and the sequences are as follows: isoleucine-alanine-phenylalanine-proline (IAFP), molecular weight: 500.6Da, can remarkably inhibit DPP-IV activity (IC) ₅₀ =0.001 M）；

3. The bioactive peptide IAFP disclosed by the invention can obviously improve the glucose consumption and glucose uptake capacity of a cell model Hepg2 of high-glucose-induced insulin resistance, thereby improving the insulin resistance of Hepg 2;

4. the bioactive peptide with DPP-IV inhibitory activity in the fermented horse milk disclosed by the invention is simple in preparation method and stable in activity, can be applied to the fields of functional foods and health-care products, and has wide market application prospect.

Drawings

FIG. 1 shows the result of DPP-IV inhibitory activity of solutions containing substances of different molecular weights after ultrafiltration interception;

FIG. 2 is a schematic diagram showing the result of chromatographic peak separation by Sephadex chromatography;

FIG. 3 shows the result of DPP-IV inhibitory activity detection of a chromatography liquid obtained after separation by Sephadex chromatography;

FIG. 4 is a schematic representation of the separation peaks after RP-HPLC separation;

FIG. 5 shows the DPP-IV inhibitory activity detection results of components corresponding to the separation peaks after RP-HPLC separation;

FIG. 6 shows the DPP-IV inhibitory activity detection results of components corresponding to 9 separation peaks within 20-25 min;

FIG. 7 is a high performance liquid chromatogram of peak 2 sample;

FIG. 8 is a mass spectrometric identification of active peptides;

FIG. 9 shows the results of enzyme inhibition kinetics experiment of bioactive peptide IAFP;

FIG. 10 is a schematic diagram of the docking result of bioactive peptide IAFP and DPP-IV molecules;

FIG. 11 shows the effect of the bioactive peptide IAFP on Hepg2 cell activity, where Metformin is Metformin, NT is negative control, IR is model group, and the rest is bioactive peptide group;

FIG. 12 is the effect of the bioactive peptide IAFP on glucose consumption by high glucose-induced insulin resistance Hepg2 cells, where Metformin is Metformin, NT is negative control, and the rest is bioactive peptide group;

FIG. 13 is the effect of the bioactive peptide IAFP on glucose uptake in high-glucose-induced insulin resistance Hepg2 cells, where Metformin is Metformin, NT is negative control, IR is model group, and the rest is bioactive peptide group.

Detailed Description

The technical scheme of the invention is further described in detail by the following examples, but the content of the invention is not limited to the examples, the methods in the examples are all conventional methods unless otherwise specified, and the materials, reagents and the like used in the examples are obtained from commercial approaches unless otherwise specified;

the species used in the following examples are as follows:

yarrowia lipolytica (Yarrowia lipolytica) Y7 is disclosed in "Tangrong et al, saccharomycetes and lactic acid bacteria fermented Mare milk to produce ACE inhibitory peptide, food science, 2021", lactococcus lactis (Lactococcus lactis) Purchased from cohansen;

the DPP-IV inhibitory activity of the samples is determined by the enzyme assay in the examples as follows:

taking 25 mu L of 2mmol/L GPP (glycylproline p-nitroaniline) and 25 mu L of samples to be put in a 96-well plate, fully and uniformly mixing, incubating for 10min at 37 ℃, then adding 50 mu L of 0.025U/mL DPP-IV, uniformly mixing, fully reacting for 60min at 37 ℃, then adding 100 mu L of 1mmol/L acetic acid-sodium acetate solution with pH4.0 to terminate the reaction, using a microplate reader to measure the light absorption value at 405nm, and enabling three samples to be parallel; DPP-IV and glycylproline p-nitroaniline (GPP) were formulated at corresponding concentrations using 100mmol/L Tris-HCl buffer, pH 8.0.

The calculation formula of DPP-IV inhibitory activity is as follows:

）×100%

A _{sample set} (DPP-IV + sample + GPP): measuring the PNP absorbance of the polypeptide sample after adding the polypeptide sample;

A _{sample control group} (buffer + sample + GPP): absorbance of the polypeptide sample;

A _{enzyme activity group} (buffer + DPP-IV + GPP): PNP absorbance measured without adding a sample;

A _{enzyme blank} (buffer + GPP): PNP absorbance measured with buffer instead of enzyme.

Example 1: preparation of bioactive peptides having DPP-IV inhibitory Activity

(1) Inoculating lactococcus lactis into an MRS liquid culture medium according to the inoculation amount of 2% of the volume percentage, and standing and culturing for 12 hours at the temperature of 35 ℃; inoculating yarrowia lipolytica into YPD liquid culture medium at 2 vol%, and culturing at 29 deg.C and 200rpm for 12 hr; the two bacteria are continuously proliferated for 2 generations, and the viable count of activated lactobacillus and yeast is not less than 10 ⁹ CFU/mL and 10 ⁸ CFU/mL for standby;

(2) centrifuging fresh horse milk, and sterilizing at 95 deg.C for 10 min;

(3) inoculating activated yarrowia lipolytica and lactococcus lactis when horse milk is cooled to 30 ℃, wherein the viable count in the inoculated horse milk is 10 ⁶ ～ ⁸ CFU/mL；

(4) Fermenting the horse milk under the following conditions: fermenting for 16h at 30 ℃ under the conditions of stirring speed of 150rpm and ventilation capacity of 40 vvm;

(5) centrifuging 9000g of fermented mare milk for 30min to obtain supernatant, adjusting pH of the supernatant to 8.0 with sodium hydroxide, and centrifuging again to obtain supernatant;

(6) the supernatant is balanced for 0.5h at room temperature, prefreezed for 0.5h at 4 ℃, prefreezed for 1h at-20 ℃, prefreezed for 1h at-80 ℃, and then freeze-dried for 12h under the conditions of 4Pa and-52 ℃ to prepare freeze-dried powder;

(7) dissolving the lyophilized powder with double distilled water to 50mg/mL solution, filtering with ultrafiltration cut-off membranes with cut-off amounts of 10kDa and 3kDa to obtain solution containing components with molecular weights of >10kDa, 3-10kDa and <3kDa, and respectively measuring DPP-IV inhibitory activity of each component, wherein the result is shown in figure 1, selecting the solution with the best inhibitory activity and containing components with molecular weights of <3kDa, performing Sephadex G10 Sephadex gel chromatography, eluting with 0.5mL/min double distilled water, monitoring change of absorbance value of eluent at 280nm, collecting chromatographic liquids 1, 2, 3 and 4 corresponding to separation peaks, respectively freeze-drying, preparing the lyophilized powder with distilled water into solution with concentration of 10mg/mL, measuring DPP-IV inhibitory activity of each component (figure 3), selecting the chromatographic liquid 1 with the best activity, further separating with RP-HPLC (figure 4), collecting separated liquid in different time periods, performing rotary evaporation, performing freeze drying to obtain freeze-dried powder, preparing a solution with the concentration of 10mg/mL by using water, determining the DPP-IV inhibitory activity of different solutions, obtaining a result shown in figure 5, selecting liquid corresponding to the time period of 20-25min with the highest activity from the solutions, performing next-step separation by using RP-HPLC, collecting 9 liquids corresponding to separation peaks, performing rotary evaporation, performing freeze drying to obtain freeze-dried powder, preparing a solution with the concentration of 5mg/mL by using water, determining the DPP-IV inhibitory activity of different solutions, obtaining a result shown in figure 6, showing that the liquid activity of the No. 2 peak is highest in the figure, showing a high performance liquid chromatogram of the No. 2 peak liquid in the figure 7, and obtaining a pure peak with higher activity after purification;

wherein the RP-HPLC conditions are as follows: c18, 5 μm, 250 × 4.6id, 100A, sample volume of 1mL, flow rate of 2 mL/min, and detection wavelength of 215 nm; mobile phase a was ultrapure water containing 0.1% (v/v) trifluoroacetic acid (TFA), mobile phase B was acetonitrile containing 0.1% (v/v) TFA, elution conditions: gradient eluting with mobile phase A and B (0-5 min, 10% B, 5-40 min, 10-50% B, 40-50 min, 50-80% B, 50-60 min, 80-10% B, 60-70 min, 10% B);

(7) and (3) performing LC-MS identification on the peak component No. 2, wherein the analysis time is as follows: and (5) 35 min. The detection mode is as follows: a positive ion. The mass-to-charge ratio of the small molecule peptide and the fragment of the small molecule peptide was collected as follows: 10 fragment patterns (MS 2 scan) were acquired after each full scan (full scan); a mass spectrometry test original File (Raw File) searches a corresponding database (Equus caballus) by using Mascot2.2 software, and finally obtains a protein identification result, wherein the result is shown in figure 8, the corresponding intensity and charge-mass ratio of corresponding fragments can be obtained from the figure, and the small molecular peptide sequence is confirmed to be IAFP by database parent sequence comparison, and is shown in SEQ ID NO: 1;

synthesizing small molecular peptide IAFP with purity not less than 98% by Shanghai, dissolving into small molecular peptide solution with different concentrations in gradient, detecting DPP-IV inhibitory activity of small molecular peptide with different concentrations, and calculating to obtain half Inhibitory Concentration (IC) of small molecular peptide IAFP ₅₀ ) Is 0.001 mol/L.

Example 2: enzyme inhibition kinetics research of DPP-IV of bioactive peptide IAFP

(1) Preparing a DPP-IV solution with the enzyme activity of 80U/L by using 0.1mol/L Tris-HCl buffer solution (pH8.0), wherein the solution concentration is 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.2mmol/L, 1.6mmol/L and 2mmol/L Gly-Pro-Pna substrate solution (GPP);

(2) preparing a bioactive peptide IAFP solution with the concentration range of 0-2000 mmol/L;

(3) the DPP-IV inhibitory activity of a sample is determined by adopting an enzyme labeling method, the DPP-IV inhibitory activity of the bioactive peptide IAFP with different concentrations under different concentrations GPP is determined, and the initial reaction speed is obtained;

(4) determining the inhibition action type of the bioactive peptide IAFP on the DPP-IV by adopting a Lineweaver-Burk double reciprocal mapping; the results are shown in FIG. 9, from which it can be seen that the bioactive peptide IAFP acts on DPP-IV by means of anti-competitive inhibition.

Example 3: docking of small molecule peptide IAFP and dipeptidyl peptidase IV molecule

The corresponding DPP-IV (1N 1M) model was downloaded in the RCBS PDB protein database (https:// www.rcsb.org /). Small molecule peptide was modeled using Chem Draw 19.0, and NM2 force field was added to minimize its energy. Performing flexible molecular docking by using Gold 5.3.0, performing ligand extraction on a receptor, removing water molecules, adding polar hydrogen, and performing docking model screening by using a Gold score scoring system. Visual analysis of the docking molecule complex was performed using Moe2019.10 software, and the results are shown in FIG. 10. from FIG. 10, IAFP and DPP-IV form three hydrogen bonds, and the binding site is close to the DPP-IV S' 2 charge pocket.

Experimental example 4: functional evaluation of bioactive peptide IAFP

1. Construction of high-glucose-induced insulin resistance cell model Hepg2

(1) Culturing Hepg2 cells in high-glucose DMEM medium containing 10% FBS and 1% penicillin-streptomycin; the culture conditions were: 37 ℃ and 5% CO ₂ The incubator of (1);

(2) culturing the above cells until the fusion degree is 90%, digesting with 0.25% pancreatin, subculturing according to a ratio of 1:3, and replacing the culture medium every 2 d;

(3) hepg2 cells were packed at 1.5X 10 ⁴ Inoculating each cell/well into a 96-well plate, after the cells are plated, removing supernatant, and inducing an insulin resistance model by using a DMEM medium containing 36mmol/L glucose;

(4) treating cells according to the following different experimental groups, and performing subsequent experiments after the experimental groups are treated for 24 hours;

the experimental groups were as follows:

negative control group (NT): culturing Hepg2 cells in DMEM medium containing 5.5mmol/L glucose;

sample group: DMEM cultured cells containing different concentrations of bioactive peptide IAFP (200. mu.g/mL, 100. mu.g/mL, 50. mu.g/mL, 10. mu.g/mL) and 36mmol/L glucose;

positive control group (Metformin): culturing the cells in a DMEM medium containing 50 mu mol/L of metformin and 36mmol/L of glucose;

model set (IR): culturing the cells in a DMEM medium containing 36mmol/L glucose;

2. CCK8 cell activity detection

After each experimental group in the step 1 is treated, adding 10 mu L of CCK8 reagent into each hole of each group, continuing culturing for 4h, then measuring absorbance at 450nm, setting 4 auxiliary holes in each group as a control, and obtaining a result shown in figure 11, wherein the result is shown in figure 11, and the bioactive peptide IAFP has no toxic or side effect on Hepg2 cells, can improve cell activity and is dose-dependent;

3. glucose consumption

(1) After treatment of each experimental group as in 1 above, the original medium was discarded, and 100. mu.L of DMEM containing 100nmol/L of insulin was added to each well and cultured at 37 ℃ with 5% CO ₂ Culturing for 10min in the cell culture box;

(2) culturing for 10min, discarding supernatant, adding 100 μ L DMEM serum-free medium containing 11mmol/L glucose, and culturing for 24 hr;

(3) detecting the glucose concentration in the culture medium by adopting a GOD-POD glucose detection kit;

the detection method comprises the following steps:

adding 3 μ L of sample to be detected and 300 μ L of working reagent (phenol reagent and enzyme reagent are mixed uniformly according to a ratio of 1: 1) into each hole of a 96-hole plate, incubating for 15min at 37 ℃, and measuring absorbance at 505nm by using an enzyme-labeling instrument; the results are shown in fig. 12, and it can be seen from fig. 12 that the bioactive peptide IAFP significantly improves the glucose consumption ability of the Hepg2 cell model and has a significant improvement effect on the Hepg2 cell model insulin resistance.

4. Glucose uptake assay

(1) After treatment of each experimental group as in 1 above, the medium was discarded, and 1mL of DMEM containing 100nmol/L of insulin was added to each well and cultured at 37 ℃ with 5% CO ₂ Culturing for 10min in the cell culture box;

(2) culturing for 10min, discarding supernatant, adding 1mL of 100 nmol/L2-NBDG serum-free DMEM medium into each well, and culturing in a cell culture box at 37 ℃ for 1 h;

(3) after the culture is finished, washing the cells twice by using 1 XPBS to stop reaction, detecting glucose uptake by using a fluorescence microplate reader, wherein the excitation wavelength is 475nm, the emission wavelength is 535nm, and each group is provided with 4 auxiliary holes as a reference; the results are shown in FIG. 13, from which FIG. 13, the bioactive peptide IAFP is shown.

Sequence listing

<110> Stannum Guo professional college, Kunming university

<120> a biologically active peptide having dipeptidyl peptidase IV inhibitory activity

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4

<212> PRT

<213> horse milk (horse milk)

<400> 1

Ile Ala Phe Pro

1

Claims

1. The application of the bioactive peptide in preparing the dipeptidyl peptidase IV inhibitor is characterized in that the bioactive peptide is derived from fermented horse milk, and the amino acid sequence of the bioactive peptide is Ile-Ala-Phe-Pro.