CN114133431B - Mare milk-derived small molecule peptide and application thereof - Google Patents

Abstract

The invention discloses a small molecular peptide, the amino acid sequence of which is Phe-Pro-Gly-Gly-Pro, the molecular weight of which is 545.6Da, the small molecular peptide has the inhibitory activity of dipeptidyl peptidase IV and the half Inhibitory Concentration (IC) of the dipeptidyl peptidase IV ₅₀ ) 0.664 mg/mL; the high-sugar-induced (human liver cancer) Hepg2 cell insulin resistance can be improved, the activity of the Hepg2 cell is obviously improved, and the glucose consumption and glucose uptake of the Hepg2 cell are improved; the small molecular peptide can be applied to the preparation of special medical foods, health care products and medicines for treating or assisting in treating type2 diabetes, is simple to prepare, and is suitable for industrial production and market popularization and application.

Description

Mare milk-derived small molecule peptide and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a small molecular peptide derived from fermented mare milk and application thereof.

Background

Diabetes has become a global public health problem. The international diabetes consortium (IDF) predicts that the total number of people with diabetes worldwide will break through 7.0 million people by 2045 years. In recent years, with the change of population and life style in China, the incidence rate of diabetics is increasing year by year, and the age of diabetics is becoming lower. In addition, diabetics are in a hyperglycemia state for a long time, various complications such as peripheral neuropathy, vascular disease, visual system abnormality, endocrine metabolism disorder and the like are extremely easy to be induced, and the physical health and the quality of life of the patients are seriously affected.

Diabetes is a metabolic disease in which hyperglycemia occurs due to in vivo insulin deficiency or a decrease in its biological effect, or both, and thus causes metabolic disorders. Common diabetes mellitus is classified into Type1 diabetes (Type 1 Diabetes Mellitus, T1 DM), type2 diabetes (Type 2 Diabetes Mellitus, T2 DM), gestational sugars (Gestational Diabetes Mellitus, GDM) and other special diabetes mellitus, wherein the onset population of Type2 diabetes mellitus is more common and accounts for 90% of the total number of diabetics. In the treatment of type2 diabetes mellitus, DPP-IV inhibitor is one of newer target drugs, and has better curative effect in clinic. Most of the DPP-IV inhibitors commonly used at present are chemically synthesized, and side effects such as anaphylactic reaction, heart rhythm disorder, upper respiratory tract infection, gastrointestinal discomfort and the like are easily caused after long-term administration. In contrast, the food-borne DPP-IV inhibitory polypeptide has become a research hotspot in the field of foods at home and abroad due to the advantages of simple and convenient preparation method, low cost, small side effect and the like. Since mare milk is rich in high-quality milk proteins (including casein, beta-lactoglobulin and alpha-lactalbumin) and has been paid attention to by more and more researchers, mare milk is reported to have a certain curative effect on diabetes, and mare milk is prepared from mare milk by co-fermentation of lactic acid bacteria and saccharomycetes, so that mare milk is an ideal protein source for separating and purifying DPP-IV inhibitor and has wide application prospect.

Disclosure of Invention

The invention aims to provide a small molecular peptide which is derived from fermented mare milk, has an amino acid sequence of Phe-Pro-Gly-Gly-Pro, has a molecular weight of 545.6Da and has dipeptidyl peptidase IV inhibition activity.

The invention aims at realizing the following scheme:

1. inoculating lactobacillus and saccharomycetes into the fresh mare milk, wherein the inoculum size is 0.1-10%, the fermentation temperature is 20-37 ℃, the rotation speed of a shaking table is 50-300 rpm, and the fermentation time is 16-90 h; the lactobacillus is one or more of Lactobacillus bulgaricus, lactobacillus helveticus, lactobacillus plantarum, lactobacillus casei, lactococcus lactis and Streptococcus thermophilus; the microzyme is one or more of Saccharomyces cerevisiae, pichia pastoris, yarrowia lipolytica and Hansenula polymorpha;

2. after the fermented mare milk is regulated to pH 4.8-4.0, centrifuging at the temperature of 4 ℃ and at the temperature of 3000-12000 g for 5-20 min, regulating the pH of the supernatant to 7.0-8.0 by NaOH, centrifuging at the temperature of 3000-12000 g for 5-20 min, freeze-drying the supernatant by a freeze dryer, and collecting freeze-dried powder;

3. dissolving the freeze-dried powder in double distilled water to prepare 0.05-0.5 g/mL solution, filtering the solution by ultrafiltration interception films with different interception molecular weights to obtain liquids with different components, determining the inhibition activity of the dipeptidyl peptidase IV of each liquid, selecting the liquid with the best inhibition activity for sephadex column chromatography and trans-high performance liquid chromatography (RP-HPLC) separation and purification, selecting the peak with the inhibition activity of the dipeptidyl peptidase IV after RP-HPLC purification for LC-MS identification, and performing the prediction of the inhibition activity of the dipeptidyl peptidase IV on Biopep by the peptide sequence obtained after identification, and selecting the solid phase synthesis verification with the best prediction activity to finally obtain the small molecular peptide of the invention.

The fermented mare milk small molecular peptide Phe-Pro-Gly-Gly-Pro (FPGGP) disclosed by the invention can obviously inhibit the activity of dipeptidyl peptidase IV, improve the high-sugar-induced insulin resistance of Hepg2 cells, obviously improve the activity of the Hepg2 cells and improve the glucose consumption and glucose uptake of the Hepg2 cells; the small molecular peptide can be applied to the preparation of special medical foods, health care products and medicines for treating or assisting in treating type2 diabetes, is simple to prepare, and is suitable for industrial production and market popularization and application.

Drawings

FIG. 1 shows the result of inhibition of dipeptidyl peptidase IV by solutions containing substances of different molecular weights after ultrafiltration interception;

FIG. 2 is a schematic diagram showing the peak separation result of the sephadex chromatography;

FIG. 3 shows the result of detection of dipeptidyl peptidase IV inhibitory activity of a liquid obtained after sephadex chromatographic separation;

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

FIG. 5 shows the result of detection of dipeptidyl peptidase IV inhibitory activity of the liquid corresponding to the separation peak after RP-HPLC separation;

FIG. 6 shows the results of liquid dipeptidyl peptidase IV inhibition activity assays corresponding to the 9 separation peaks over a period of 20-25 min;

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

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

FIG. 9 shows the results of enzyme inhibition kinetics experiments for the small molecule peptide FPGGP;

FIG. 10 is a schematic diagram showing the result of molecular docking of the small peptide FPGGP and DPP-IV;

FIG. 11 is the effect of the small molecule peptide FPGGP on the activity of Hepg2 cells, wherein Metformin is Metformin and NT is a negative control;

FIG. 12 is the effect of the small molecule peptide FPGGP on glucose consumption by high glucose-induced insulin resistant Hepg2 cells;

FIG. 13 is the effect of the small molecule peptide FPGGP on glucose uptake by high glucose-induced insulin resistant Hepg2 cells.

Detailed Description

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

the method for detecting the dipeptidyl peptidase IV inhibition activity in the following examples comprises the following steps:

DPP-IV enzyme and glycylproline para-nitroaniline (GPP) were formulated as solutions of 0.025U/mL and 2 mmol/L, respectively, with 100 mmol/L Tris-HCl buffer pH 8.0. Transferring 25 μL GPP and 25 μL sample polypeptide solution into 96-well plate, mixing thoroughly, incubating at 37deg.C for 10min, then adding 50 μL DPP-IV enzyme solution to start reaction, reacting at 37deg.C for 60 min; adding 100 mu L of acetic acid-sodium acetate solution with the pH of 4.0 and the concentration of 1mmoL/L to terminate the reaction, detecting and recording the absorbance value at the wavelength of 405 and nm by an enzyme-labeling instrument, and carrying out three parallel reactions on each sample;

since the reaction system was slightly turbid, the experiment set up an enzyme activity group (buffer+enzyme+substrate), an enzyme blank group (buffer+substrate), a sample group (enzyme+sample+substrate) and a sample control group (buffer+sample+substrate). The enzyme activity group only reacts with GPP and DPP-IV enzyme, and the equivalent buffer solution replaces the polypeptide sample solution to obtain the highest content of PNP which can be generated under the same reaction condition, so that whether the polypeptide sample has an inhibition effect is compared; the enzyme blank group is only added with GPP, and the rest is replaced by equivalent buffer solution, so that interference of the GPP in naturally decomposing and generating display substance PNP in the incubation process is eliminated; and (3) simultaneously adding a buffer solution, a sample polypeptide and a substrate into the sample control group, and eliminating interference of the sample polypeptide on absorbance in the incubation process, and calculating the DPP-IV enzyme inhibition activity:

inhibition (%) =1- (a) _{Sample group} −A _{Sample control group} ）/（B _{Enzyme active group} −B _{Enzyme blank} ）×100%

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

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

B _{enzyme active group} (buffer+dpp-iv+gpp): PNP absorbance measured without sample addition;

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

Example 1: acquisition of small molecule peptides

1. Fermenting mare milk: sterilizing 400 mL fresh Mare milk at 60deg.C for 30 min, and washing activated lactococcus lactis with physiological saline water for two generationsLactococcus lactis) Yarrowia lipolyticaYarrowia lipolytica) Y7, each inoculated with 2mL into sterilized horse milk, then fermented 68 h at 32 ℃, 225 rpm; wherein yarrowia lipolytica isYarrowia lipolytica) Y7 is disclosed in Tang Rong, etc. research on ACE inhibitory peptide produced by fermenting mare's milk with saccharomycetes and lactobacillus, food science, 2021, lactococcus lactis @, andLactococcus lactis) Purchased from kohansen;

2. pretreatment of fermentation liquor: regulating pH of fermented mare milk to 4.3 with 0.1mol/L HCL, centrifuging at 10000 g and 4deg.C for 10min, and collecting supernatant; regulating pH to 7.0 with 0.1mol/L NaOH, centrifuging at 10000 g and 4deg.C for 10min, and lyophilizing;

3. preparation of small molecule peptides: dissolving the freeze-dried powder in double distilled water to prepare 0.1 g/mL solution, filtering the liquid by using an ultrafiltration interception membrane with interception quantity of 10 kDa and 3kDa to obtain a solution containing components with molecular weight of more than 10 kDa, 3-10 kDa and 3kDa, freeze-drying each level of solution, preparing 50 mg/mL solution by using double distilled water, and determining the inhibition activity of the solution on dipeptidyl peptidase IV; as a result, as shown in FIG. 1, it was revealed from FIG. 1 that the most excellent inhibitory activity was obtained by subjecting a solution containing a component having a molecular weight of < 3kDa to Sephadex xG-10 chromatography at 0.1/g/mL, eluting with double distilled water at 0.5 mL/min, monitoring the change in absorbance value of the eluate at 280 nm, observing the separation of the components, collecting the liquids F1, F2, F3 and F4 corresponding to the separation peaks (FIG. 2), freeze-drying the liquids, preparing a solution having a concentration of 50 mg/mL with water, measuring the inhibitory activity of each solution on dipeptidyl peptidase IV (FIG. 3), selecting the F1 liquid having the best activity, further separating by semi-preparative RP-HPLC (FIG. 4), collecting separated liquids in different time periods, spin-evaporating, freeze-drying, preparing into solutions with the concentration of 10 mg/mL by using water, measuring the inhibitory activity of different solutions on the dipeptidyl peptidase IV, selecting the liquid with the highest activity from the solutions with the concentration of 20-25min for the next separation by using RP-HPLC, collecting 9 liquids corresponding to the separation peaks, spin-evaporating, freeze-drying, preparing into solutions with the concentration of 5 mg/mL by using water, measuring the inhibitory activity of different solutions on the dipeptidyl peptidase IV, measuring the inhibitory activity of different solutions, and measuring the liquid activity of the peak No. 2 in the figure, wherein the high-efficiency liquid chromatogram of the peak No. 2 liquid is shown in the figure 7, and the liquid phase purification is completed.

Peak No. 2 liquid was mass analyzed with a Q exact mass spectrometer (Thermo Fisher) for the duration of the analysis: 35 And (5) min. The detection mode is as follows: positive ions. The mass-to-charge ratio of the polypeptide and fragments of the polypeptide was collected as follows: 10 fragment patterns (MS 2 scan) were acquired after each full scan (full scan);

data analysis (beijing Baitai park): searching a corresponding database (Eulus bacillus) by using software Mascot2.2 to obtain 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 FPGGP by comparing the parent sequences of the database, and the amino acid sequence is shown as SEQ ID NO. 1;

the small molecular peptide FPGGP with the purity of more than or equal to 98 percent is synthesized by Shanghai engineering, and is dissolved into small molecular peptide solutions with different concentrations in a gradient way, so that the dipeptide of the small molecular peptide with different concentrations is detectedInhibitory activity of dipeptidyl peptidase IV (DPP-IV) and half Inhibitory Concentration (IC) of small molecule peptide FPGGP obtained by calculation ₅₀ ) Is 0.664 mg/mL.

Chromatographic conditions for semi-preparative RP-HPLC in the above-described method: sample injection amount 1mL, flow rate 2 mL/min and detection wavelength 215 nm; mobile phase a contained 0.1% (v/v) trifluoroacetic acid (TFA) in ultrapure water and mobile phase B was acetonitrile containing 0.1% (v/v) TFA, elution conditions: gradient elution with mobile phases 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).

Experimental example 2: enzyme inhibition kinetics experiment of small molecule peptide FPGGP on dipeptidyl peptidase IV (DPP-IV)

The following solutions were prepared using 0.1mol/L Tris-HCl buffer (pH 8.0) as a solvent: DPP-IV solution with enzyme activity of 80U/L; gly-Pro-pNA substrate solutions at concentrations of 0.4, 0.6, 0.8, 1.2, 1.6 and 2 mmol/L; the small molecular peptide FPGGP solution with the concentration range of 0-2000 mu m is adopted to obtain the change of absorbance values (namely initial reaction speed) of the small molecular peptide with different concentrations on substrates with different concentrations by adopting the dipeptidyl peptidase IV inhibition activity detection experimental method, and the inhibition type of the small molecular peptide FPGGP on DPP-IV is determined by adopting the Lineweaver-Burk double reciprocal mapping; the results are shown in FIG. 9, from which it can be seen that the small molecule peptide FPGGP acts on dipeptidyl peptidase IV through anti-competitive inhibition.

Experimental example 3: molecular docking of small molecule peptide FPGGP and dipeptidyl peptidase IV

The corresponding DPP-IV (1N 1M) model is downloaded in the RCBS PDB protein database (https:// www.rcsb.org /). The small molecule peptide model was drawn using chemdraw 19.0 and the NM2 force field was added to minimize its energy. Flexible molecular docking was performed using Gold 5.3.0, ligand extraction was performed on the receptor, water molecules were removed, polar hydrogen was added, and docking model screening was performed using 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. As can be seen from fig. 10, two hydrogen bonds are formed between FPGGP and dipeptidyl peptidase iv, and the binding site is close to the charge pocket of dipeptidyl peptidase iv S' 2.

Experimental example 4: small molecule peptide FPGGP functional evaluation

1. High sugar induced Hepg2 cell modeling

After resuscitating the Hepg2 cells, they were cultured in high-sugar DMEM medium containing 10% fbs and 1% penicillin-streptomycin; at 37℃with 5% CO ₂ Culturing in a carbon dioxide incubator until the fusion degree is 90%, digesting with 0.25% pancreatin, and carrying out passage according to 1:3, wherein the culture medium is replaced every 2 d; after the Hepg2 cells are plated, the supernatant of the original culture medium is discarded, and a DMEM culture medium containing 36mmol/L glucose is used for inducing an insulin resistance model;

negative control (NT) Hepg2 cells were cultured in DMEM medium containing 5.5 mmol/L glucose;

the small molecule peptide groups were incubated with different concentrations of the polypeptide (200. Mu.g/mL, 100. Mu.g/mL, 50. Mu.g/mL, 10. Mu.g/mL) and DMEM containing 36mmol/L glucose;

the positive control group (Metformin) was incubated with 36mmol/L glucose and 50. Mu. Mol/L Metformin in DMEM medium;

model group (IR) Hepg2 cells were cultured in DMEM medium containing 36mmol/L glucose;

all groups were subjected to subsequent experiments after 24h of culture to harvest cells;

2. CCK8 cell Activity assay

After the cultured Hepg2 cells were digested with trypsin, a cell suspension was prepared in DMEM medium containing 10% FBS according to 1.5X10 ⁴ Adding into 96-well plate, modeling in group according to step 1, and heating to 37deg.C and 5% CO ₂ After that, 10 mu L of CCK8 reagent is added into each hole of each group, the mixture is cultured for 4 hours in the cell culture box, the absorbance is detected at OD 450, 4 auxiliary holes are arranged in each group and used as a control, the result is shown in figure 11, and as can be seen from figure 11, the small molecular peptide FPGGP has no toxic or side effect on Hepg2 cells, can improve the activity of the cells and has dose dependency;

3. glucose consumption

After the cultured Hepg2 cells were digested with trypsin, a cell suspension was prepared in DMEM medium containing 10% FBS according to 1.5X10 ⁴ Adding into 96-well plate, modeling in group according to step 1, and heating at 37deg.C and 5% CO ₂ Is cultured in a cell culture box for 24 hours; the original medium was discarded, and 100. Mu.L of DMEM containing 100nmol/L insulin was added per well based on 5% CO at 37 ℃ ₂ The culture medium is discarded after 10min of culture in a cell culture box, and 100 mu L of DMEM serum-free medium containing 11mmol/L glucose is added into each group of holes for culture for 24h; detecting the glucose concentration in the culture medium by adopting a GOD-POD glucose detection kit, and detecting according to the operation instructions of the kit; mu.L of the sample to be tested and 300. Mu.L of working reagent (phenol reagent and enzyme reagent are mixed uniformly according to the ratio of 1:1) are added into each well of a 96-well plate, incubated for 15min at 37 ℃, and the mixture is subjected to OD (optical density) by using an enzyme-labeled instrument ₅₀₅ Measuring at nm; as a result of setting 4 sub-wells per group as a control, see fig. 12, it is clear from fig. 12 that the small molecule peptide FPGGP can significantly improve glucose consumption by high glucose-induced Hepg2 insulin resistant cells.

4. Glucose uptake assay

After the cultured Hepg2 cells were digested with trypsin, a cell suspension was prepared in DMEM medium containing 10% FBS according to 2X 10 ⁵ Adding into 24-well plate, modeling in group according to step 1, and heating at 37deg.C and 5% CO ₂ Is cultured in a cell culture box for 24 hours; the original medium was discarded, and 1mL of DMEM containing 100nmol/L insulin was added per well based on 5% CO at 7deg.C ₂ The culture medium is discarded, 1mL of serum-free DMEM medium containing 100 nmol/L2-NBDG is added to each well to culture for 1 h in the cell culture medium at 37 ℃,1 XPBS is washed twice to stop the reaction, a fluorescent enzyme-labeled instrument is used for detecting glucose uptake, the excitation wavelength is 475 nm, the emission wavelength is 535 nm, and 4 auxiliary wells are arranged in each group to serve as a control; as a result, as shown in fig. 13, it is clear from fig. 13 that FPGGP significantly improved glucose uptake by high glucose-induced Hepg2 insulin resistant cells.

Sequence listing

<110> university of Kunming engineering

<120> a mare milk-derived small molecule peptide and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> Mare milk (Horse milk)

<400> 1

Phe Pro Gly Gly Pro

1 5

Claims (2)

1. A small molecule peptide has an amino acid sequence of Phe-Pro-Gly-Gly-Pro.

2. The use of the small molecule peptide of claim 1 in the preparation of a medicament for the treatment of type2 diabetes.