CN113087773A

CN113087773A - Yak bone peptide with blood sugar reducing and antioxidant functions and preparation method thereof

Info

Publication number: CN113087773A
Application number: CN202110489295.3A
Authority: CN
Inventors: 党亚丽; 卜凡; 刘怀高; 衣大龙; 张恒
Original assignee: Anhui Guotai Biotechnology Co ltd
Current assignee: Anhui Guotai Biotechnology Co ltd
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2021-07-09
Anticipated expiration: 2041-04-29
Also published as: CN113087773B

Abstract

The invention relates to the technical field of animal food processing, in particular to yak bone peptide with functions of reducing blood sugar and resisting oxidation and a preparation method thereof. The preparation method of the yak bone peptide comprises the following steps: yak bone pretreatment, two-step enzymolysis, enzymolysis product ultrafiltration, polypeptide crude liquid gel chromatography, and reversed-phase high performance liquid chromatography separation and purification. The content of small molecular peptides of the yak bone peptide prepared by the method is high; good water solubility, easy absorption by human body, no bad smell and safe eating; has strong DPP-IV inhibitory activity, hypoglycemic activity and strong antioxidant capacity, and can be applied to food industry, pharmaceutical industry, feed industry and the like.

Description

Yak bone peptide with blood sugar reducing and antioxidant functions and preparation method thereof

Technical Field

The invention relates to the technical field of animal food processing, in particular to yak bone peptide with functions of reducing blood sugar and resisting oxidation and a preparation method thereof.

Background

The yak bone peptide contains 8 kinds of amino acids essential to human body, which cannot be synthesized by human body, and must depend on exogenous supplement, otherwise, the life cannot be continued healthily. The yak bone peptide contains just the essential amino acids. Through the carrier effect of the peptide and the special physiological function of participating in the whole body metabolism, the yak bone peptide has good nutrition and food therapy effects on improving the immunity, assisting special patients to strengthen the body, strengthening the bones and aiming at collagen pathological changes.

In recent years, many researchers have isolated and purified a variety of active peptides from different sources. The active peptides have great potential in long-term administration therapy due to their high specificity and potency, and low toxicity. The hypoglycemic and antioxidant peptide is one of active peptides, can reduce the blood sugar of a patient with hyperglycemia, has no influence on normal blood sugar, and has good application prospect in the fields of food and cosmetics due to strong antioxidant capacity. In the existing research, CN105624251B, a peony seed blood sugar lowering peptide, a purification method and an application thereof, adopts ultrasonic agitation to extract protein, and uses trypsin for ultrasonic-assisted enzymolysis to obtain the peony seed blood sugar lowering peptide. CN102428832A fungal medicinal mycoplasm with blood sugar reducing effect and preparation method thereof select medicinal substrates with blood sugar reducing effect and fungal strains as two-way fermentation to obtain medicinal mycoplasm with obvious blood sugar reducing effect without increasing insulin concentration. CN109336953A Tartary buckwheat Albumin enzyme hydrolysis peptide is obtained by preparing Tartary buckwheat albumin powder from Tartary buckwheat powder as raw material, and separating and purifying Tartary buckwheat albumin enzyme hydrolysis peptide to obtain Tartary buckwheat antioxidative peptide. CN105748426A intestinal tract location releasing tablet of pine nut source antioxidant peptide and its preparation method, pine nut source antioxidant peptide lyophilized powder with molecular weight less than 1000Da is used as raw material to be processed by high voltage pulse electric field to improve its antioxidant activity, and is made into pine nut source antioxidant peptide lyophilized powder, and the pine nut source antioxidant peptide tablet is prepared by shaping, granulating, mixing, tabletting and other operations. However, the peptide separated and purified from the yak bone has the functions of reducing blood sugar and resisting oxidation, and is a bifunctional peptide. The yak bone waste rate can be reduced, the additional value of the yak bones is increased, and the economic value of the yak bones is increased.

The small molecular peptide generated by the livestock and poultry bone after enzymolysis is beneficial to the digestion and absorption of human body and has functional characteristics. Therefore, the development of bone protein products and functional foods can not only generate high economic benefit, but also solve the problems of resource waste and environmental pollution, thereby forming a healthy and continuously-developed industry.

Polypeptide products of proteins in yak bones after enzymolysis are quite complex in composition, and comprise a large number of polypeptides with unknown sequences and unknown functions, and active peptides with specific functions may comprise a plurality of peptides with different amino acid compositions and different molecular weights, so that the peptides are difficult to distinguish through certain common characteristics. These all pose great difficulties in the development of bovine bone peptide with specific functions. Many yak bone peptides in the prior art do not have specific functions, and the yak bone peptides with better functions of reducing blood sugar and resisting oxidation are fewer.

Disclosure of Invention

The invention aims to provide yak bone peptide which has the functions of reducing blood sugar and resisting oxidation. The invention also aims to provide a preparation method of the yak bone peptide.

The invention aims to develop a yak bone peptide with the functions of reducing blood sugar and resisting oxidation, and develops a preparation process for producing the yak bone peptide with the functions of reducing blood sugar and resisting oxidation by taking yak bones as raw materials according to the characteristics of the raw materials and the composition characteristics of the yak bones.

Specifically, the invention provides the following technical scheme:

in a first aspect, the invention provides a yak bone protein peptide which comprises an amino acid sequence shown as SEQ ID NO.1 and has DPP-IV inhibitory activity and an antioxidant function.

Specifically, the invention provides a yak bone peptide with DPP-IV inhibitory activity and an antioxidant function, and the amino acid sequence of the yak bone peptide is PYPYEPYEPYPY.

In a second aspect, the invention provides a preparation method of yak bone peptide with DPP-IV inhibitory activity and antioxidant function, which is prepared by the following steps: taking yak bones as raw materials, crushing, and cooking at the temperature of 100-: performing neutral protease enzymolysis in the first step, performing compound protease enzymolysis in the second step to obtain antihypertensive peptide enzymatic hydrolysate, performing high-temperature inactivation, centrifuging and ultrafiltering the antihypertensive peptide enzymatic hydrolysate to obtain a crude product of yak bone protein peptide; wherein the compound protease is trypsin and flavourzyme, and the mass ratio of the trypsin to the flavourzyme is (2-4): 1.

The enzyme activity of the neutral protease is as follows: 300,000-550,000U/g; the enzyme activity of the trypsin is as follows: 300,000-500,000U/g; the enzyme activity of the flavourzyme is as follows: 150,000-200,000U/g.

In the preparation method provided by the invention, the raw materials are crushed and then cooked for 3-4 hours at the temperature of 100-125 ℃.

In the preparation method provided by the invention, the pH value is not adjusted, the temperature of the first step of enzymolysis reaction is 50-65 ℃ during two steps of enzymolysis, the addition amount of neutral protease is 0.2-0.5 wt%, and the reaction time is 1.75-2.25 h; the temperature of the second step of enzymolysis reaction is 55-65 ℃, the adding amount of compound protease (the dosage ratio of trypsin to flavourzyme is 2-4:1) is 0.2-0.5 wt%, and the reaction time is 1.0-2.0 h; after the two steps of enzymolysis are finished, the temperature of high-temperature inactivation is 95-105 ℃, and the time is 15-20 min.

In the preparation method provided by the invention, the inactivated DPP-IV inhibitory activity and antioxidant function peptidase hydrolyzed solution is centrifuged for 10-20 min at the rotating speed of 8000-10000 rpm, and then filtered by an ultrafiltration membrane below 3000 Da.

In the preparation method provided by the invention, when the SephadexG-25 gel column is used for separation, the absorbance is 220nm, and the 2 nd elution peak is collected; and when the reversed-phase high performance liquid chromatography is used for separation and purification, the detection wavelength is 220nm, and the peptide eluent which is separated by RP-HPLC reversed-phase high performance liquid chromatography for 9-12 min is collected.

As a preferred scheme of the invention, the preparation method of the yak bone peptide comprises the following steps:

(1) pretreating, namely crushing yak bones, and then cooking for 3-4 hours at the temperature of 100-125 ℃ to obtain yak bone protein liquid;

(2) adding neutral protease accounting for 0.3 wt% of the weight of the protein, performing enzymolysis at 55 ℃ for 2h, adding 0.3 wt% of compound protease into the product of the first step of enzymolysis reaction, performing enzymolysis at 55 ℃ for 2h, and inactivating enzyme at 100 ℃ for 15 min;

(3) performing ultrafiltration, namely centrifuging the inactivated peptidase hydrolyzed solution (8000r,4 ℃,15min), wherein the ultrafiltration membrane is an ultrafiltration membrane with the molecular weight cutoff below 3000Da to obtain a polypeptide crude solution, and concentrating and freeze-drying the polypeptide crude solution;

(4) gel chromatography, preparing the freeze-dried powder into a solution, and separating by using a Sephadex G-25 gel column under the separation conditions that: a glass column (1.5cm × 100cm), the sample loading concentration is 200mg/mL, the sample loading amount is 400 μ L, the flow rate is 60mL/h, the sensitivity is 1.0, and the absorbance is 220 nm;

(5) separating by reverse phase high performance liquid chromatography, and separating by gel chromatography to obtain IC₅₀Dissolving the most functional components in water to prepare a solution with the protein concentration of 1mg/mL, and separating and purifying by using reverse phase high performance liquid chromatography (RP-HPLC), wherein the separation conditions are as follows: sample introduction volume, 100 μ L; flow rate, 1 mL/min; eluent, solution A is 0.1% TFA (trifluoroacetic acid) water solution; solution B is 0.1% TFA acetonitrile solution; linear elution gradient, 0-10 min, 0-5% B; 10E up to ten20min, 5-19% of B; 20-29 min, 19% -90% B; 29-40 min, 90% -0% B; column temperature: 35 ℃; detection wavelength, 220 nm; collecting the most functional ICs₅₀Preparing components;

(6) and (3) mass spectrum sequencing, and identifying the structure and the sequence of the component with the highest activity by using ESI-MS-MS and MALDI-TOF-MS after the separation and purification are finished.

The invention also provides a functional polypeptide, which has an amino acid sequence shown as SEQ ID NO. 1.

Experiments prove that the functional polypeptide shown in any one of SEQ ID NO.1-3 has better functions of reducing blood sugar and resisting oxidation.

The invention also provides application of the yak bone peptide or the functional polypeptide in preparation of medicines, foods or food additives.

Preferably, the pharmaceutical product or the food product has the functions of inhibiting DPP-IV activity and antioxidant function.

The invention also provides a medicine, food or food additive containing the yak bone peptide or the functional polypeptide.

The medicine, food or food additive can contain auxiliary materials allowed in the medicine or food field besides the yak bone peptide or the functional polypeptide.

The invention has the beneficial effects that:

(1) the raw materials used by the preparation method are aggregate waste yak bones, and the yield is high in plateau areas.

(2) The preparation method of the invention adopts a double-enzyme method to carry out enzymolysis on yak bones, has good enzymolysis effect, and can effectively carry out enzymolysis on yak bone proteins into peptides which inhibit DPP-IV activity and antioxidant function.

(3) The yak bone DPP-IV active and antioxidant functional peptide has high edible safety, has strong DPP-IV inhibiting activity in vitro, also has strong in-vivo blood sugar reducing activity, and has no side effect on a self-C57 mouse.

(4) In the protection experiment of the oxidative damage of the HepG2 cell, the protection effect of the peptide PYPYEPYEPYPY on the oxidative damage model of the HepG2 cell is firstly determined.

(5) The yak bone peptide can be applied to the food industry, the pharmaceutical industry, the feed industry and the like; the preparation method is simple to operate and convenient for industrial production.

Drawings

FIG. 1 is a chromatogram of primary column chromatography purification of hypoglycemic and antioxidant peptide fragments.

FIG. 2 is a chromatogram of reversed phase liquid phase purification of hypoglycemic and antioxidant peptide fragments.

FIG. 3 is a mass spectrum of pure peptide fragment for lowering blood sugar and resisting oxidation.

FIG. 4 shows the effect of hypoglycemic and antioxidant peptides on lowering blood glucose from mice. Sitagliptin (Sitagliptin) and Metformin (Metformin) are used as positive Control groups, Control is used as a blank Control group, and PY-12 is used as a peptide sample group.

FIG. 5 shows the antioxidant effect of hypoglycemic and antioxidant peptides in HePG2 cells.

A, cytotoxicity of peptides on cells at a concentration of 10-600. mu.M. B: protection of damaged cells by the polypeptide. Effect of peptide on intracellular ROS content. And D, HepG-2 cell fluorescence image. Different letters indicate significant differences in peptide activity (p < 0.05).

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The composite protease used in the embodiment of the invention is a combination of trypsin (with the enzyme activity of 300,000-.

Example 1 preparation method of Yak bone peptide having hypoglycemic and antioxidant effects (1)

The embodiment provides a preparation method of yak bone peptide with the functions of reducing blood sugar and resisting oxidation, which comprises the following steps:

(1) pretreating, crushing 200g of yak bones, and cooking for 4 hours at 110 ℃;

(2) adding neutral protease accounting for 0.3 wt% of the weight of the yak bones, performing enzymolysis for 2h at 55 ℃, adding compound protease accounting for 0.3 wt% of the yak bones (trypsin accounts for 0.2 wt% of the yak bones and flavourzyme accounts for 0.1 wt% of the yak bones) into a product obtained in the first step of enzymolysis reaction, performing enzymolysis for 2h at 55 ℃, and inactivating enzymes for 15min at 100 ℃;

(4) gel chromatography, preparing the freeze-dried powder into a solution, and separating by using a Sephadex G-25 gel column under the separation conditions that: glass column (1.5 cm. times.100 cm), loading concentration of 200mg/mL, loading amount of 400. mu.L, flow rate of 60mL/h, sensitivity of 1.0, absorbance of 220nm, and collection of the 2 nd elution peak. The mass spectrum of column chromatography is shown in figure 1;

(5) separating by reverse phase high performance liquid chromatography, and separating by gel chromatography to obtain IC₅₀Dissolving the most functional components in water to prepare a solution with the protein concentration of 1mg/mL, and separating and purifying by using reverse phase high performance liquid chromatography (RP-HPLC), wherein the separation conditions are as follows: sample introduction volume, 100 μ L; flow rate, 1 mL/min; eluent, solution A is 0.1% TFA (trifluoroacetic acid) water solution; solution B is 0.1% TFA acetonitrile solution; linear elution gradient, 0-10 min, 0-5% B; 10-20 min, 5% -19% of B; 20-29 min, 19% -90% B; 29-40 min, 90% -0% B; column temperature: 35 ℃; detecting the wavelength at 220nm, and harvesting the peptide liquid collected for 9-12 min. The chromatogram of reversed phase liquid phase purification is shown in FIG. 2;

(6) mass spectrum sequencing, ESI-MS-MS and MALDI-TOF-MS for active IC after separation and purification₅₀Identifying the structure and sequence of the highest component; the mass spectrogram is shown in figure 3, and after the mass spectrogram is identified, the sequence of the pure peptide is Pro-Tyr-Pro-Tyr-Glu-Pro-Tyr-Glu-Pro-Tyr-Pro-Tyr which is an undiscovered novel antihypertensive peptide segment. The sample injection concentration of the gel chromatographic column is 200mg/mL, and the sample injection amount is 1 mL. Based on the ratio of the gel chromatogram peak G2 area to the total peak area and the ratio of the RP-HPLC peak F5 area to the total peak area, the peptide was estimated approximately at PYPYEPYEPYPY content of 0.16%.

Example 2 preparation method of Yak bone peptide having hypoglycemic and antioxidant effects (2)

(1) pretreating, crushing 600g of yak bones, and cooking for 3.5 hours at 120 ℃;

(2) adding neutral protease accounting for 0.4 wt% of the weight of the yak bones, performing enzymolysis at 60 ℃ for 2.2h, adding composite protease accounting for 0.4 wt% of the yak bones (trypsin is 0.3 wt% of the yak bones and flavourzyme is 0.1 wt% of the yak bones) into a product obtained in the first-step enzymolysis reaction, performing enzymolysis at 60 ℃ for 2h, and performing enzyme deactivation at 100 ℃ for 15 min;

(3) performing ultrafiltration, namely centrifuging the inactivated peptidase hydrolyzed solution (9000r,4 ℃ and 15min), wherein the ultrafiltration membrane is an ultrafiltration membrane with the molecular weight cutoff below 2000Da to obtain a polypeptide crude solution, and concentrating and freeze-drying the polypeptide crude solution;

(4) gel chromatography, preparing the freeze-dried powder into a solution, and separating by using a Sephadex G-25 gel column under the separation conditions that: a glass column (1.5cm multiplied by 100cm), the sample loading concentration is 200mg/mL, the sample loading amount is 400 mu L, the flow rate is 60mL/h, the sensitivity is 1.0, the absorbance is 220nm, and the 2 nd elution peak is collected;

(5) separating with reverse phase high performance liquid chromatography, dissolving the most functional components obtained by gel chromatography in water, preparing into solution with protein concentration of 1mg/mL, and separating and purifying with reverse phase high performance liquid chromatography (RP-HPLC), wherein the separation conditions are as follows: sample introduction volume, 100 μ L; flow rate, 1 mL/min; eluent, solution A is 0.1% TFA (trifluoroacetic acid) water solution; solution B is 0.1% TFA acetonitrile solution; linear elution gradient, 0-10 min, 0-5% B; 10-20 min, 5% -19% of B; 20-29 min, 19% -90% B; 29-40 min, 90% -0% B; column temperature: 35 ℃; detecting the wavelength at 220nm, and harvesting the peptide liquid collected for 9-12 min; the chromatogram is similar to that of FIG. 2.

(6) Mass spectrum sequencing, ESI-MS-MS and MALDI-TOF-MS for active IC after separation and purification₅₀Identifying the structure and sequence of the highest component; after the identification of mass spectrum, the sequence of the pure peptide is Pro-Tyr-Pro-Tyr-Glu-Pro-Tyr-Glu-Pro-Tyr-Pro-Tyr. Based on the ratio of the gel chromatogram peak G2 area to the total peak area and the ratio of the RP-HPLC peak F5 area to the total peak area, the peptide was estimated approximately at PYPYEPYEPYPY content of 0.17%.

Example 3 preparation method of Yak bone peptide having hypoglycemic and antioxidant effects (3)

(1) pretreating, crushing 1000g of yak bones, and cooking for 3 hours at 125 ℃;

(2) adding neutral protease accounting for 0.5 wt% of the weight of the yak bones, performing enzymolysis at 65 ℃ for 2.2h, adding composite protease accounting for 0.5 wt% of the yak bones (trypsin is 0.4 wt% of the yak bones and flavourzyme is 0.1 wt% of the yak bones) into a product obtained in the first-step enzymolysis reaction, performing enzymolysis at 60 ℃ for 2h, and performing enzyme deactivation at 100 ℃ for 15 min;

(3) performing ultrafiltration, namely centrifuging the inactivated peptidase hydrolyzed solution (10000r,4 ℃ and 20min), wherein the ultrafiltration membrane is an ultrafiltration membrane with the cut-off molecular weight of less than 2000Da to obtain a polypeptide crude solution, and concentrating and freeze-drying the polypeptide crude solution;

(6) Mass spectrum sequencing, namely identifying the structure and the sequence of the component with the highest activity by using ESI-MS-MS and MALDI-TOF-MS after the separation and purification are finished; after the identification of mass spectrum, the sequence of the pure peptide product is Pro-Tyr-Pro-Tyr-Glu-Pro-Tyr-Glu-Pro-Tyr-Pro-Tyr. Based on the ratio of the gel chromatogram peak G2 area to the total peak area and the ratio of the RP-HPLC peak F5 area to the total peak area, the peptide was roughly estimated to have a PYPYEPYEPYPY content of 0.18%.

Experimental example 1 detection of dipeptidyl peptidase-IV (DPP-IV) inhibitory Activity of Yak bone peptide

Test samples: yak bone peptide prepared in example 1, example 2 and example 3. DPP-IV inhibitory ability is performed as follows:

1. yak bone peptide PYPYEPYEPYPY

The detection was performed with DPP-IV inhibitor screening kit (MAK203, sigma) according to the manufacturer's instructions, sitagliptin (sitagliptin) as positive control.

(1) Sample preparation: a4 Xaqueous peptide solution was prepared, the sample diluted 4-fold with DPP-IV assay buffer to the final assay concentration, and 25. mu.L pipetted into a 96-well plate. Sample is derived from yak bone peptide (dissolved in double distilled water) with amino acid sequence of PYPYEPYEPYPY obtained in examples 1, 2 and 3

(2) Reaction liquid inhibition: the DPP-IV enzyme and DPP-IV analysis buffer are diluted according to the volume ratio of 1: 49. Adding 50 mu L of the solution into each hole of the reaction system, blowing, mixing uniformly, and incubating at 37 ℃ for 10 min. The blank was added with 50. mu.L of DPP-IV assay buffer.

(3) Enzyme reaction solution: DPP-IV substrate DPP-IV assay buffer is diluted 2:23 by volume. And after the incubation is finished, adding 25 mu L of the reagent into each hole of the reaction system, and detecting the fluorescence value after uniformly mixing the reagent by blowing. Fluorescence intensity was recorded at an excitation wavelength of 360nm and an emission wavelength of 460nm of Bioeksynergy2(BioTek, USA), measured every minute for 30 min.

(4) And (4) calculating a result: two time points (T1 and T2) were selected within the linear range of the resulting plot, fluorescence values (FLU1 and FLU2) were determined, and the slope of the plot (Δ FLU/minute) was determined using this.

Slope＝(FLU2–FLU1)/(T2–T1)＝ΔFLU/minute

The inhibition rate is (SlopeEC-SlopeSM)/SlopeEC × 100%

Wherein the slope of the SlopeSM is the slope of the sample; slope of SlopeEC as control

And (4) analyzing results:

yak bone peptide with the sequence of PYPYEPYEPYPY obtained in example 1 has higher activity on DPP-IV inhibition and IC thereof₅₀189.4 mu M is achieved; practice ofExample 2 the yak bone peptide with the sequence of PYPYEPYEPYPY has higher activity on DPP-IV inhibition and IC₅₀188.6 mu M is reached; the yak bone peptide with the sequence of PYPYEPYEPYPY obtained in example 3 has higher activity on DPP-IV inhibition and IC₅₀Reaches 187.6 μ M; the yak bone peptide with the DPP-IV inhibition effect has wide application prospect.

2. DPP-IV inhibition effect of polypeptide crude liquid freeze-dried powder

(1) Sample preparation: a4 Xaqueous peptide solution was prepared, the sample diluted 4-fold with DPP-IV assay buffer to the final assay concentration, and 25. mu.L pipetted into a 96-well plate. The sample solution is derived from the freeze-dried powder (dissolved in double distilled water) of the polypeptide crude liquid obtained by ultrafiltration in the step (3) of the examples 1, 2 and 3

Slope＝(FLU2–FLU1)/(T2–T1)＝ΔFLU/minute

Inhibition rate (SlopeEC-SlopeSM)/SlopeEC 100%

And (4) analyzing results:

crude peptide solution obtained in step (3) of example 1 has higher activity on DPP-IV inhibitionC₅₀The crude peptide solution obtained in step (3) of example 2 had a high activity on DPP-IV inhibition and IC thereof reached 5.33mg/mL₅₀The crude peptide solution obtained in step (3) of example 3 had a high activity on DPP-IV inhibition and IC thereof reached 5.13mg/mL₅₀5.06mg/mL was reached.

(Note: the molecular weight of the crude peptide solution is unknown and can only be expressed in mg/mL, whereas the molecular weight of the peptide PYPYEPYEPYPY is known and can be expressed in. mu.M.)

Experimental example 2 hypoglycemic and antioxidant peptide animal hypoglycemic experiment

Test samples: yak bone peptide prepared in example 1, example 2 and example 3.

20 female mice (18-20 g) were purchased from Beijing Wittiulihua laboratory animal technology, Inc. (China). Each C57 mouse received regular feed and purified water at a temperature of 25 ± 4 ℃ and a humidity of 40 ± 6%. After one week of acclimation, C57 mice were randomly divided into four groups, and 6 mice each group were subjected to the experiment. The blood glucose changes of C57 mice were measured by Oral Glucose Tolerance Test (OGTT) to determine the hypoglycemic effect of peptide PYPYEPYEPYPY in vivo. Mice were fasted for 14h prior to OGTT. Peptide PYPYEPYEPYPY, the positive drugs sitagliptin and metformin were dissolved in distilled water to prepare a sample group (100mg/kg BW) and a positive control group (100mg/kg BW), respectively. 0.9% saline was used as a negative control. The administration was performed 1 time per day, fasting without water deprivation was performed for 14 hours before the administration, blood was collected by cutting mouse tails at 0min, 15min, 30min, 60min, and 120min after the administration, and the effect of the DPP-IV inhibitor on C57 mice was measured at 37 ℃ using a Glutest glucometer (Sanwa Kagaku corporation, japan ancient house). Before measurement, the mice are kept at 38 ℃, the measurement is repeated for 5 times, and the average value is the blood sugar value of the C57 mice.

And (4) analyzing results: to determine the hypoglycemic effect of peptide PYPYEPYEPYPY in vivo, the hypoglycemic effect was determined by the change in blood glucose in C57 mice within 2h after oral administration. The blood glucose of the negative control group (0.9% saline solution) did not change significantly after 2h of oral administration (fig. 4). The final blood glucose of C57 mice was 6.5. + -. 0.26 mmol/L. However, the sample group (peptide, 100mg/kg) and the positive control group (sitagliptin and metformin, 100mg/kg) significantly reduced the blood glucose in the C57 mice. The peptide still has obvious hypoglycemic effect within 1 hour after oral administration. After oral administration, the maximum hypoglycemic activity appears at 1h, and the blood sugar is 10.28 +/-0.47 mmol/L. Notably, the maximum hypoglycemic rate of 100mg/kg peptide was faster than 100mg/kg (1h) of sitagliptin, indicating that PYPYEPYEPYPY may be superior to sitagliptin in vivo effect. Furthermore, the maximum blood glucose reduction was 10.27 and 9.33mmol/L for the sample group and the positive control group, respectively, at an oral dose of 100mg/kg, indicating that the blood glucose lowering ability of the peptide and sitagliptin was functional both in vivo and in vitro and not completely proportional in vivo. Interestingly, the hypoglycemic effect of the 100mg/kg peptide was the same as the overall trend for the positive control (metformin, 100mg/kg), indicating PYPYEPYEPYPY as a potential drug for the prevention and treatment of hypoglycemic events.

Experimental example 3 antioxidant cell experiment

Test samples: yak bone peptide 1.HepG2 cell culture and cytotoxicity test prepared in example 1, example 2 and example 3

DMEM complete medium was prepared and the cells were cultured in DMEM medium (10% bovine serum FBS; 1% penicillin streptomycin P/S) and maintained in a 37% incubator containing 5% carbon dioxide.

In DMEM medium at 2X 10⁴Cell/well density cells were cultured. Cells were cultured with different concentrations of peptide PYPYEPYEPYPY (10, 30, 100, 200, 300 and 600 μ M) for 24h, with five replicates for each concentration. After 24h incubation, 10. mu.L of CCK-8(Cell Counting Kit-8 Kit from Boster, Wuhan) was added to each well, which was then returned to the incubator and incubated at 37 ℃ for 2 h. In addition, the half-Lethality (LD) of the cells within 4h was also determined₅₀) H of (A) to (B)₂O₂And (4) concentration.

2. Protection effect of yak bone peptide on oxidative stress injury of HepG2 cells

HepG2 cells were cultured for 24h with different concentrations of peptides (20, 40, 80, 160. mu.M) which proved to be non-toxic to the cells. Subsequently, the old medium was removed and added to the medium containing 800. mu. M H₂O₂(LD₅₀Concentration) of the culture medium, and culturing for 4 hours. 40 μ M GSH solution was used as a positive control.

3. Effect of Yak bone peptide on intracellular ROS levels

Cell antioxidant activity was studied using fluorescence reduction of Dichlorofluorescein (DCF). Cells were seeded at a density of 2X 10⁴Cells/well in 96-well plates and cultured for 6 h. Then, the old medium was removed, a new medium containing the polypeptide was added, and the culture was carried out under the culture conditions for 24 hours. Then in a solution containing 1% H₂O₂The DMEM medium (1) was incubated for 4 h. And finally adding 10 mu L of DCFH-DA, then continuing incubation for 30min, after that, washing the cells twice by using PBS, and determining by using an enzyme-linked immunosorbent assay at 37 ℃, wherein the excitation wavelength is 488nm, and the generation wavelength is 525 nm. Positive control group (cells treated with GSH only) and negative control group (cells containing culture medium only).

4. And (4) analyzing results:

(1) effect of Yak bone peptide on HepG2 cytotoxicity

The effect of a sample on cell viability plays an important role in cytotoxicity experiments, which generally verify whether a sample promotes cell growth. Fig. 5 a shows the cytotoxic effect of different concentrations of peptide on HepG2 cells. This proliferation-promoting effect increased from 95.9% to 106.2% when added at concentrations ranging from 10 to 200. mu.M, indicating that peptide PYPYEPYEPYPY can promote the growth of HepG2 cells. At a concentration of 600. mu.M, slight cytostatic was shown, and the proliferation rate was 92.9%. This may be due to the cells exceeding the optimal growth concentration and causing slight toxicity.

(2) Protection effect of yak bone peptide on oxidative stress injury of HepG2 cells

Shown in B of FIG. 5, peptide PYPYEPYEPYPY vs H₂O₂The induced HepG2 cell oxidative damage has a protection effect and the range is 20-160 mu M. GSH is a common antioxidant in cells, often acting as a positive control. The results show that the protection of 40 μ M peptide is 1.5 times that of 40 μ M GSH. In addition, the peptide has protective effect on cells under the concentration of 20-160 mu M (p)<0.05)。

(3) Effect of Yak bone peptide on intracellular ROS levels

Of ROS in cellsThe content is one of the important indexes for determining the antioxidant of the sample. The change in ROS content is usually due to the digestion of DCFH-DA into DCFH by esterases on the cell membrane. DCFH can freely pass through the cell membrane and specifically bind cellular ROS, which produces fluorescence. The change in ROS content is determined by measuring the change in fluorescence intensity. The ROS content produced by the peptide in the cell is shown in fig. 5C and fig. 5D. ROS levels are continuously reduced at peptide concentrations of 20-40. mu.M, while the highest ROS levels are achieved at 160. mu.M, probably because high concentrations of peptide lead to reduced receptor binding. However, the ROS content at all concentrations was lower than H₂O₂And (4) a damage group. The experimental result shows that the peptide can effectively protect HepG2 cells from being damaged by free radicals.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Anhui peptide Biotechnology Ltd

<120> yak bone peptide with functions of reducing blood sugar and resisting oxidation and preparation method thereof

<130> KHP211112405.7

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Pro Tyr Pro Tyr Glu Pro Tyr Glu Pro Tyr Pro Tyr

1 5 10

Claims

1. A yak bone protein peptide with DPP-IV inhibitory activity and an antioxidant function is characterized by comprising a polypeptide with an amino acid sequence of PYPYEPYEPYPY.

2. A yak bone protein peptide with DPP-IV inhibitory activity and antioxidant function is characterized by being prepared by the following method: taking yak bones as a raw material, crushing, and cooking at the temperature of 100-125 ℃ for 3-4 hours to obtain yak bone protein liquid, wherein the protein liquid is subjected to two steps of enzymolysis: performing neutral protease enzymolysis in the first step, performing compound protease enzymolysis in the second step to obtain antihypertensive peptide enzymatic hydrolysate, performing high-temperature inactivation, centrifuging and ultrafiltering the antihypertensive peptide enzymatic hydrolysate to obtain a crude product of yak bone protein peptide; the composite protease is trypsin and flavourzyme, wherein the mass ratio of the trypsin to the flavourzyme is 2-4: 1.

3. The yak bone protein peptide as claimed in claim 2, wherein the enzyme activity of the neutral protease is: 300,000-550,000U/g; the enzyme activity of the trypsin is as follows: 300,000-500,000U/g; the enzyme activity of the flavourzyme is as follows: 150,000-200,000U/g.

4. The yak bone protein peptide as claimed in claim 2, wherein in the two-step enzymolysis, the first step enzymolysis reaction: adding neutral protease with the weight percent of 0.2-0.5% of raw bone into yak bone protein liquid, and carrying out enzymolysis reaction for 1.75-2.25 h at the temperature of 50-65 ℃.

5. The yak bone protein peptide as claimed in claim 2, wherein in the two-step enzymolysis, the second step enzymolysis reaction: and adding 0.2-0.5 wt% of compound protease of the raw material bone into the product of the first-step enzymolysis reaction, and carrying out enzymolysis reaction for 1.0-2.0 h at the temperature of 55-65 ℃.

6. The yak bone protein peptide according to any one of claims 2 to 5, wherein after the two steps of enzymolysis, the temperature for high-temperature inactivation is 95-105 ℃ for 15-20 min.

7. The yak bone protein peptide as claimed in claim 6, wherein the inactivated antihypertensive peptide enzymatic hydrolysate is centrifuged at 8000-10000 rpm for 10-20 min, and then filtered with an ultrafiltration membrane of 3000Da or less.

8. The yak bone protein peptide as claimed in claim 7, wherein when the filtrate is separated by Sephadex G-25 gel column, the absorbance is 220nm, and the 2 nd elution peak is collected; and when the reversed-phase high performance liquid chromatography is used for separation and purification, the detection wavelength is 220nm, and the peptide eluent which is separated by RP-HPLC reversed-phase high performance liquid chromatography for 9-12 min is collected.

9. Use of the yak bone protein peptide of any one of claims 1-8 in the preparation of a medicament, food or health product for relieving hypertension.

10. A product having DPP-IV inhibitory activity and antioxidant function, comprising the yak bone peptide according to any one of claims 1 to 8, wherein said product is a pharmaceutical, food or health product.