CN107082807B - Yak bone protein peptide with ACE (angiotensin converting enzyme) inhibition function as well as preparation method and application thereof - Google Patents

Yak bone protein peptide with ACE (angiotensin converting enzyme) inhibition function as well as preparation method and application thereof Download PDF

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CN107082807B
CN107082807B CN201710345158.6A CN201710345158A CN107082807B CN 107082807 B CN107082807 B CN 107082807B CN 201710345158 A CN201710345158 A CN 201710345158A CN 107082807 B CN107082807 B CN 107082807B
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张恒
罗永康
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Anhui Guotai Biotechnology Co ltd
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Abstract

The invention provides a yak bone protein peptide with an ACE (angiotensin converting enzyme) inhibition function, and a preparation method and application thereof. The yak bone protein peptide is prepared from a zymolyte of yak bone protein subjected to stepwise enzymolysis by composite protease; the compound protease is alkaline protease, papain, trypsin and flavourzyme. The yak bone protein peptide has an excellent ACE inhibitory function, the ACE inhibitory activity (IC50 value) is less than 0.32mg/mL, and the yak bone protein peptide can be used as an ACE inhibitor to be applied to special foods and nutritional foods. The preparation method of the yak bone protein peptide is simple, no acid or alkali is added in the whole processing process for adjusting the pH value, the product keeps better functional characteristics, and the industrial production is easy to realize.

Description

Yak bone protein peptide with ACE (angiotensin converting enzyme) inhibition function as well as preparation method and application thereof
Technical Field
The invention relates to the field of animal food processing, in particular to a yak bone protein peptide with an ACE (angiotensin converting enzyme) inhibition function, a preparation method and application thereof.
Background
Hypertension is a disease with great harm to human health, and has long incubation period. Early stages of hypertension generally do not experience significant discomfort until death occurs with clinical signs of heart attack, and rupture of the cerebral vessels.
Angiotensin Converting Enzyme (ACE) is a key enzyme of the renin-Angiotensin system. Angiotensinogen is converted into angiotensin I under the action of renin, and angiotensin I is converted into angiotensin II under the action of ACE, which stimulates vasoconstriction and causes blood pressure rise. Inhibition of ACE activity has a positive effect on lowering blood pressure, and development of effective ACE inhibitors has attracted considerable attention. Although the ACE inhibitory peptide derived from food has weaker effect than that of an artificially synthesized ACE inhibitor, the ACE inhibitory peptide has the unique advantages of no excessive pressure reduction problem, high safety and no side effect, and has the functions of promoting immunity, being easy to digest and absorb and the like besides the pressure reduction function. Because of the side effects of pharmacotherapy, the development and utilization of the blood pressure lowering functional factors in natural foods become an important part of non-drug treatment of hypertension in the future.
In the prior art, fish resources or clams are used for preparing ACE inhibitory peptides, for example, Chinese patent CN106399435A discloses a method for preparing ACE inhibitory peptides by using fish scales, which comprises the steps of washing the fish scales with water to remove impurities, soaking the fish scales in NaCl solution for 1-2 days to remove impurity proteins and fish silver powder, fishing out the fish scales, and soaking the fish scales in HCl solution for 1-2 hours to remove inorganic salts such as calcium salt and the like, wherein the material-liquid ratio is 1:10-1: 20; soaking fish scales in distilled water for swelling, performing enzymolysis with alkaline protease, centrifuging to obtain a supernatant, and freeze-drying the enzymolysis solution to obtain ACE inhibitory peptide; the obtained product has higher ACE inhibitory rate, and compared with the ACE inhibitory peptide synthesized chemically, the prepared ACE inhibitory peptide is safe, non-toxic and easy to digest; for example, chinese patent CN104593466A discloses a method for preparing ACE inhibitory peptide from clam protein source by using endogenous enzyme, the method uses endogenous protease in the clam body for enzymolysis, natural ACE inhibitory peptide is obtained by autolysis, no exogenous protease is added in the preparation process, the whole preparation process comprises fresh clam pretreatment, homogenate, endogenous enzyme activation, enzyme competition, enzyme deactivation, fishy smell removal, centrifugation, and freeze drying; the method realizes the comprehensive utilization of tilapia wastes, particularly takes tilapia skin as a raw material, water extracts the skin protein in the tilapia skin, adopts protease to hydrolyze, obtains fish skin protease hydrolysate (TSP-1) through the processes of enzyme inactivation, debitterizing, decoloring, deodorizing, filtering, ion exchange, concentration and the like, collects high-activity components through a gel layer after the TSP-1 is subjected to ultrafiltration treatment, and prepares a high-activity ACE inhibitory peptide product through freeze drying; chinese patent CN104131055A discloses a preparation method of phycoerythrin ACE inhibitory peptide, which comprises the steps of phycoerythrin extraction, pepsin enzymolysis is added, and trypsin enzymolysis is added; dissolving the freeze-dried powder after enzymolysis in pure water, loading the solution to a SephadexG-15 gel column, and collecting the component with the highest activity peak as phycoerythrin ACE inhibition component. The components are continuously loaded on a high performance liquid chromatography ZORBAX300SB-C18 column for separation so as to detect the preparation of phycoerythrin ACE inhibitory peptide in the components.
However, in these methods, pH is usually adjusted by adding acid or alkali, which affects the quality of the product and makes the main composition of the protein peptide unclear. China has rich yak bone resources, and at present, no patent literature report for preparing angiotensin converting enzyme inhibitory peptide by using yak bone protein resources is found in domestic and foreign patents.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a yak bone protein peptide with an ACE (angiotensin converting enzyme) inhibition function, and a preparation method and application thereof.
In order to realize the purpose of the invention, on one hand, the invention provides the yak bone protein peptide with the ACE inhibitory function, and the yak bone protein peptide is prepared by performing stepwise enzymolysis on yak bone protein by using compound protease; wherein the compound protease is alkaline protease, papain, trypsin and flavourzyme.
The peptide obtained by carrying out distributed enzymolysis on the yak bone protein by using the specific compound protease has ACE inhibitory activity, and reasonably and effectively utilizes the yak bone protein.
In order to obtain relatively pure yak bone protein peptide, the distributed enzymolysis using the composite protease preferably comprises the following steps:
regulating the protein content in the yak bone protein liquid to 8-10 wt%, and adding compound protease according to the weight percentage of 0.4-1.0 wt% of the weight of the protein in the yak bone protein liquid for step-by-step enzymolysis.
In order to improve the inhibitory activity of the obtained yak protein peptide, in a preferred embodiment of the invention, the enzymolysis by using the composite protease distribution specifically comprises the following steps:
a first step of enzymolysis reaction: adding 0.2-0.5 wt% of first compound protease into yak bone protein liquid accounting for 8-10 wt% of protein weight, and performing enzymolysis reaction at 50-60 ℃ for 0.5-1.5 h, wherein the first compound protease is alkaline protease and papain;
the second step of enzymolysis reaction: and adding 0.2-0.5 wt% of second compound protease into the product of the first-step enzymolysis reaction, and carrying out enzymolysis reaction for 1.0-2.0 h at the temperature of 45-55 ℃, wherein the second compound protease is trypsin and flavourzyme.
In a preferred embodiment of the invention, the mass ratio of the alkaline protease to the papain in the first compound protease is 1-2: 1.
In a preferred embodiment of the invention, the mass ratio of the trypsin to the flavourzyme in the second compound protease is 1: 1-2.
In order to further improve the inhibitory activity of the obtained yak protein peptide, in a preferred embodiment of the invention, the preparation method of the yak bone protein peptide comprises the following steps:
1) preparing yak bone protein liquid;
2) carrying out enzymolysis on the yak bone protein liquid prepared in the step 1) by using compound protease step by step;
3) removing impurities from the product obtained after enzymolysis in the step 2), performing membrane separation, gel separation and reversed-phase HPLC separation, concentrating, and freeze-drying to obtain the product.
The preparation method does not add any acid or alkali.
In a preferred embodiment of the invention, the step 1) further comprises the step of subjecting the prepared yak bone protein liquid to ultrasonic treatment at the temperature of 75-85 ℃ and the frequency of 80-100kH for 15-25 minutes. Under the condition, the tissue structure of the yak bone collagen can be effectively changed, and the yak bone protein liquid processed by the specific frequency ultrasonic technology at the temperature can obviously improve the sensitivity of the yak bone protein to the enzyme and effectively reduce the using amount of the enzyme.
In step 3), a conventional method for removing impurities in the art can be used, and in order to obtain a polypeptide with better inhibitory activity, in a preferred embodiment of the present invention, the removing impurities in step 3) are specifically:
preserving the heat for 10-20 minutes at 90-95 ℃, cooling to room temperature, adding active carbon with the weight of 0.2-0.6 wt% of the enzymolysis liquid into the enzymolysis liquid, separating through diatomite, and collecting the separation liquid.
In the preferred embodiment, the protein peptide has good flavor and color by removing impurities by using activated carbon, and can be widely applied to special foods, medicaments and nutritional foods.
In order to make the inhibition effect of the obtained yak bone protein liquid better, the membrane separation in the step 3) can be specifically as follows: the membrane separation in the step 3) may specifically be:
and (3) treating the product after impurity removal by a two-step ultrafiltration method, and sequentially performing ultrafiltration by using a membrane with the aperture of 5000 daltons and a membrane with the aperture of 3000 daltons.
Further preferably, the membrane separation step is:
treating the obtained separated liquid by a two-step ultrafiltration method, performing ultrafiltration by using a ceramic membrane with the aperture of 5000 daltons, separating proteins and polypeptides with the molecular weight of less than 5000 daltons, and separating protein peptides with the molecular weight of less than 3000 daltons by using a membrane with the aperture of 3000 daltons.
In a preferred embodiment of the present invention, the gel separation and reverse phase HPLC in step 3) may be:
separating the product after membrane separation by Sephadex G-25 gel, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; concentrating, freezing and drying to obtain a yak bone protein peptide crude product; separating by RP-HPLC reversed-phase high performance liquid chromatography for 1 time, and collecting the peptide eluate in 16-18 minutes after RP-HPLC reversed-phase high performance liquid chromatography separation.
Concentrating the obtained peptide eluent, and freeze-drying to obtain the most preferable yak bone protein peptide.
The ACE inhibitory activity (IC50 value) of the yak bone protein peptide is less than 0.32g/mL, and when the concentration of the protein peptide is 1.0mg/mL, the ACE inhibitory rate can reach more than 80%, preferably more than 90%.
The proportion of peptides with the molecular weight lower than 1000 in the yak bone protein peptides extracted from yak bone protein is more than 90%.
In a preferred embodiment of the invention, the yak bone polypeptide prepared by the method comprises a polypeptide having the following amino acid sequence:
Glu-Ile-Arg-Met-Leu (GIAML, shown in SEQ ID NO. 1).
In a preferred embodiment of the invention, the yak bone polypeptide prepared by the method comprises a polypeptide having the following amino acid sequence:
Thr-Val-Ser-Leu-Pro-Arg (TVSLPA, shown in SEQ ID NO. 2)
In a preferred embodiment of the invention, the yak bone polypeptide prepared by the method comprises a polypeptide having the following amino acid sequence: Ser-Pro-Ile-Leu-Gly-Tyr-Trp (SPILGTT, shown in SEQ ID NO. 3).
The yak bone protein peptide containing any one of the 3 polypeptides has a good ACE inhibitory function, and the ACE inhibitory activity (IC50 value) is less than 0.32 g/mL.
In a preferred embodiment of the present invention, the active ingredient in the yak bone protein peptide comprises the above 3 polypeptides, more preferably, the active ingredient is a combination of the above 3 polypeptides, and more preferably, the content of the combination of the above 3 polypeptides is 50% or more.
The yak bone protein peptide can be in powder form.
In order to make the whole processing process simpler and not need to add acid or alkali to adjust the pH, the yak bone protein liquid in the step 1) is obtained by taking yak bones as raw materials and performing high-temperature cooking and degreasing.
Namely, the invention also provides a preparation method of the yak bone protein peptide, which comprises the following steps:
1) taking yak bones as raw materials, and obtaining yak bone protein liquid through high-temperature cooking and degreasing;
2) carrying out enzymolysis on the yak bone protein liquid prepared in the step 1) by using compound protease step by step;
3) removing impurities from the product obtained after enzymolysis in the step 2), performing membrane separation, gel separation and reversed-phase HPLC separation, concentrating, and freeze-drying to obtain the product.
Among them, the preferable embodiments of step 2) and step 3) refer to the above.
In a preferred embodiment of the present invention, the preparation of the yak bone protein liquid in the step 1) may specifically be:
selecting frozen fresh yak bones, cleaning the frozen fresh yak bones with clean water meeting the sanitary standard of drinking water, beating the yak bones into bone particles after cleaning the yak bones, adding water which is 1.0-3.0 times of the total amount of the yak bone particles, treating the bone particles at the temperature of 125-135 ℃ for 2-5 hours, then obtaining yak bone high-temperature cooking liquor without bone residues through centrifugal filtration, cooling the yak bone high-temperature cooking liquor to 15-30 ℃, and removing fat through a liquid separation tank to obtain yak bone protein liquor.
In order to maintain better functional characteristics and easily realize industrial production, in a preferred embodiment of the present invention, the preparation method of the yak bone protein peptide specifically comprises:
1) selecting frozen fresh yak bones, cleaning the yak bones with clean water meeting the sanitary standard of drinking water, crushing the yak bones into bone particles after cleaning the yak bones, adding water which is 1.0-3.0 times of the total amount of the yak bone particles, treating the yak bones at the temperature of 125-135 ℃ for 2-5 hours, then obtaining high-temperature yak bone cooking liquor without bone residues through centrifugal filtration, cooling the yak bone cooking liquor to 15-30 ℃, and removing fat through a liquid separation tank to obtain yak bone protein liquor;
adjusting the temperature of the yak bone protein liquid obtained in the step 1) to 75-85 ℃, and treating the yak bone protein liquid for 15-25 minutes by using an ultrasonic generator through ultrasonic waves (the frequency is 80-100kH), so as to change the tissue structure of yak bone collagen.
2) Adjusting the protein content in the yak bone protein liquid to 8-10 wt%, adding compound protease according to the weight percentage of 0.4-1.0 wt% of the weight of the protein in the yak bone protein liquid for step-by-step enzymolysis:
in the first step, the detergent consists of 0.2-0.5 wt% of compound protease I (alkaline protease and papain), and the mass ratio of the compound protease I to the compound protease I is 1-2: 1) carrying out enzymolysis reaction at 50-60 ℃ for 0.5-1.5 h;
then, performing enzymolysis reaction for 1.0-2.0 h at 45-55 ℃ by using 0.2-0.5% of a second compound protease (composed of trypsin and flavourzyme in a mass ratio of 1: 1-2);
3) removing impurities: preserving heat for 10-20 minutes at 90-95 ℃, cooling to room temperature, adding active carbon with the weight of 0.2-0.6 wt% of the enzymolysis liquid into the enzymolysis liquid, separating through diatomite, and collecting a separation liquid;
membrane separation: and (3) treating the separation liquid obtained in the step (3) by a two-step ultrafiltration method, carrying out ultrafiltration by using a ceramic membrane with the aperture of 5000 daltons, separating proteins and polypeptides with the molecular weight of less than 5000 daltons, and separating protein peptides with the molecular weight of less than 3000 daltons by using a membrane with the aperture of 3000 daltons.
Gel separation and reverse phase HPLC separation: taking protein peptide liquid with molecular weight less than 3000, and then carrying out SephadexG-25 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; concentrating, freezing and drying to obtain yak bone protein peptide; separating by RP-HPLC reversed-phase high performance liquid chromatography for 1 time, and collecting the peptide eluate in 16-18 minutes after RP-HPLC reversed-phase high performance liquid chromatography separation;
and concentrating and freeze-drying the obtained peptide solution to obtain the yak bone protein peptide powder.
The proportion of the peptides with the molecular weight of the yak bone protein peptides lower than 1000 obtained by the preparation method is more than 90%.
The invention also provides the application of the yak bone protein peptide and the method for preparing the yak bone protein peptide in preparing medicines or foods for preventing or treating diseases benefiting from ACE inhibition.
The yak protein peptide can be used as a medicine, or a dietary supplement, or can be used as a food base material to be added into common foods such as beverages, dairy products and the like, and the medicine or the food can be used for treating hypertension and other diseases which can be treated by an ACE inhibitor. The dosage used can be adjusted appropriately by one skilled in the art depending on the higher or lower IC50 value in combination with the actual condition of the subject being treated.
The yak bone protein peptide extracted from yak bone protein has an excellent ACE inhibitory function, and the ACE inhibitory activity (IC50 value) is less than 0.32 mg/mL; the preparation method of the yak bone protein peptide developed by the invention is simple, no acid or alkali is added in the whole processing process for adjusting the pH value, the product keeps better functional characteristics, and the industrial production is easy to realize; the product developed by the invention is safe, the invention adopts various food-grade specific compound proteases (alkaline protease, papain, trypsin and flavourzyme), and the yak bone protein peptide consisting of peptides with specific molecular weight is obtained by moderate enzymolysis under mild conditions, and the yak bone protein peptide is 100% yak bone protein peptide without any additive.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional technical means well known to those skilled in the art. The reagents used in the examples are commercially available unless otherwise specified.
Example 1 preparation method of Yak bone protein peptide having ACE inhibitory function
(1) Selecting 100 g of frozen fresh yak bones, cleaning the yak bones with clean water meeting the sanitary standard of drinking water, crushing the yak bones into bone particles by using a yak bone crusher after cleaning the yak bones, adding water which is 2 times of the total amount of the yak bone particles, treating the bone particles at 125 ℃ for 4 hours, obtaining yak bone cooking liquor without bone residues through centrifugal filtration, cooling the yak bone cooking liquor to 20 ℃, and removing fat through a liquid separation tank to obtain yak bone protein liquor.
(2) Adjusting the temperature of the yak bone protein liquid obtained in the step (1) to 80 ℃, and treating the yak bone protein liquid for 15 minutes by using an ultrasonic generator through ultrasonic waves (the frequency is 85 kH).
(3) Regulating the protein content in the yak bone protein liquid to be 8%, adding the compound protease according to the weight percentage of 0.8% of the weight of the protein in the yak bone protein liquid for step-by-step enzymolysis, and performing enzymolysis reaction for 1.5h at the temperature of 55 ℃ by using 0.4% of the compound protease I (the mass ratio of the alkaline protease to the papain is 1: 1); then, carrying out a second step of carrying out enzymolysis reaction for 1.0h at 50 ℃ by using 0.4% of a second compound protease (trypsin and flavourzyme in a mass ratio of 1: 2); preserving the heat at 95 ℃ for 10 minutes, cooling to room temperature, adding active carbon with the weight of 0.2 percent of that of the enzymolysis liquid into the enzymolysis liquid, separating through diatomite, and collecting a separation liquid;
(4) and (4) carrying out ultrafiltration on the separation liquid obtained in the step (3) by using a ceramic membrane with the aperture of 5000 daltons, firstly separating proteins and polypeptides with the molecular weight of less than 5000, and then separating protein peptides with the molecular weight of less than 3000 daltons by using a membrane with the aperture of 3000 daltons.
(5) Taking protein peptide liquid with molecular weight less than 3000, and then carrying out Sephadex G-25 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; concentrating, freezing and drying to obtain yak bone protein peptide separated by gel; separating by RP-HPLC reversed-phase high performance liquid chromatography for 1 time, and collecting 16-18 parts of peptide solution;
(6) and (5) concentrating and freeze-drying the peptide solution obtained in the step (5) to obtain the bone collagen peptide powder with the ACE inhibitory function. The main components of the collagen peptide are determined by LC-MS/MS, and the content of the peptide with the amino acid sequence of Glu-Ile-Arg-Met-Leu, Thr-Val-Ser-Leu-Pro-Arg and Ser-Pro-Ile-Leu-Gly-Tyr-Trp is 50.9 percent.
Example 2 preparation method of Yak bone protein peptide with ACE inhibitory function
(1) Selecting 500 g of frozen fresh yak bones, cleaning the yak bones with clean water meeting the sanitary standard of drinking water, crushing the yak bones into bone particles by using a yak bone crusher after cleaning the yak bones, adding water which is 1.5 times of the total amount of the yak bone particles, treating the bone particles for 3 hours at the temperature of 130 ℃, obtaining yak bone cooking liquor without bone residues through centrifugal filtration, cooling the yak bone cooking liquor to 25 ℃, and removing fat through a liquid separation tank to obtain yak bone protein liquor.
(2) Adjusting the temperature of the yak bone protein liquid obtained in the step (1) to 82 ℃, and treating the yak bone protein liquid for 20 minutes by using an ultrasonic generator through ultrasonic waves (the frequency is 90 kH).
(3) Regulating the protein content in the yak bone protein liquid to 9%, adding composite protease according to the weight percentage of 1.0% of the weight of the protein in the yak bone protein liquid for step-by-step enzymolysis, and performing enzymolysis reaction for 1.5h at 50 ℃ by using 0.4% of composite protease I (the mass ratio of alkaline protease to papain is 1: 1); then, carrying out a second step of carrying out enzymolysis reaction for 1.0h at 50 ℃ by using 0.6% of a second compound protease (trypsin and flavourzyme in a mass ratio of 1: 1); keeping the temperature at 95 ℃ for 10 minutes, cooling to room temperature, adding active carbon with the weight of 0.4 percent of that of the enzymolysis liquid into the enzymolysis liquid, separating through diatomite, and collecting a separation liquid;
(4) and (4) carrying out ultrafiltration on the separation liquid obtained in the step (3) by using a ceramic membrane with the aperture of 5000 daltons, firstly separating proteins and polypeptides with the molecular weight of less than 5000, and then separating protein peptides with the molecular weight of less than 3000 daltons by using a membrane with the aperture of 3000 daltons.
(5) Taking protein peptide liquid with molecular weight less than 3000, and then carrying out Sephadex G-25 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; concentrating, freezing and drying to obtain yak bone protein peptide separated by gel; separating by RP-HPLC reversed-phase high performance liquid chromatography for 1 time, and collecting 16-18 parts of peptide solution;
(6) and (5) concentrating and freeze-drying the peptide solution obtained in the step (5) to obtain the bone collagen peptide powder with the ACE inhibitory function. The main components of the collagen peptide are determined by LC-MS/MS, and the content of the peptide with the amino acid sequence of Glu-Ile-Arg-Met-Leu, Thr-Val-Ser-Leu-Pro-Arg and Ser-Pro-Ile-Leu-Gly-Tyr-Trp is 51.8 percent.
Example 3 preparation method of Yak bone protein peptide with ACE inhibitory function
(1) Selecting 1000 g of frozen fresh yak bones, cleaning the yak bones with clean water meeting the sanitary standard of drinking water, crushing the yak bones into bone particles by using a yak bone crusher after cleaning the yak bones, adding water which is 3.0 times of the total amount of the yak bone particles, treating the bone particles for 3 hours at 132 ℃, then obtaining yak bone high-temperature cooking liquor without bone residues through centrifugal filtration, cooling the yak bone high-temperature cooking liquor to 25 ℃, and removing fat through a liquid separation tank to obtain yak bone protein liquor.
(2) Adjusting the temperature of the yak bone protein liquid obtained in the step (1) to 85 ℃, and treating the yak bone protein liquid for 25 minutes by using an ultrasonic generator through ultrasonic waves (the frequency is 90 kH).
(3) Regulating the protein content in the yak bone protein liquid to 9%, adding composite protease according to the weight percentage of 0.6% of the weight of the protein in the yak bone protein liquid for step-by-step enzymolysis, and performing enzymolysis reaction for 1.0h at the temperature of 60 ℃ by using 0.3% of composite protease I (composed of alkaline protease and papain in a mass ratio of 2: 1); then, carrying out a second step of carrying out enzymolysis reaction for 2.0h at 50 ℃ by using 0.3% of a second compound protease (composed of trypsin and flavourzyme in a mass ratio of 1: 1); preserving the heat at 90 ℃ for 20 minutes, cooling to room temperature, adding active carbon with the weight of 0.6 percent of that of the enzymolysis liquid into the enzymolysis liquid, separating through diatomite, and collecting a separation liquid;
(4) and (3) treating the separation liquid obtained in the step (3) by a two-step ultrafiltration method, carrying out ultrafiltration by using a ceramic membrane with the aperture of 5000 daltons, separating proteins and polypeptides with the molecular weight of less than 5000, and separating protein peptides with the molecular weight of less than 3000 daltons by using a membrane with the aperture of 3000 daltons.
(5) Taking protein peptide liquid with molecular weight less than 3000, and then carrying out Sephadex G-25 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; concentrating, freezing and drying to obtain yak bone protein peptide separated by gel; separating by RP-HPLC reversed-phase high performance liquid chromatography for 1 time, and collecting 16-18 parts of peptide solution;
(6) and (5) concentrating and freeze-drying the peptide solution obtained in the step (5) to obtain the bone collagen peptide powder with the ACE inhibitory function. The main components of the collagen peptide are determined by LC-MS/MS, and the content of the peptide with the amino acid sequence of Glu-Ile-Arg-Met-Leu, Thr-Val-Ser-Leu-Pro-Arg and Ser-Pro-Ile-Leu-Gly-Tyr-Trp is 52.6 percent.
Example 4 measurement test of inhibitory Activity of Yak bone protein peptide Angiotensin Converting Enzyme (ACE)
Test samples: the yak bone protein peptide prepared in the embodiment 1, the embodiment 2 and the embodiment 3 and the sample 4 are yak bone protein powder obtained by freeze drying the yak bone protein liquid in the embodiment 1. ACE inhibitory capacity was performed as follows:
methods of ACE inhibition. The amount of the released Hip can be quantitatively detected at 228nm by using high performance liquid chromatography, so that the ACE inhibition rate of the polypeptide can be calculated.
1. Preparation of reagents
phosphate buffer solution at ph 8.3: preparing with ultrapure water, wherein the pH value is adjusted to 8.3, and the phosphate content is 50mmol/L and the NaCl content is 300 mmol/L;
ACE enzyme solution: 2mL of phosphate buffer was added to 1U of ACE so that the concentration became 0.5U/mL.
HHL solution: HHL was dissolved in phosphate buffer to a final concentration of 5 mmol/L.
Sample solution: appropriate amount of sample is weighed and used with the solution of the required concentration of phosphate buffer.
2. Chromatographic conditions for ACE inhibition assay
Detection wavelength: 228 nm; the flow rate is 1 mL/min; mobile phase A is ultrapure water (containing 0.1% of trifluoroacetic acid), and mobile phase B is methanol (containing 0.1% of trifluoroacetic acid); the sample introduction amount is 10 mu L, and manual sample introduction is carried out;
3. method for determining ACE inhibitory activity
Taking 120 mu L of HHL substrate solution, adding 20 mu L of sample, mixing uniformly, and preserving heat in a constant-temperature water bath at 37 ℃ for 10 min. Then 10 mul of ACE enzyme solution is added to react for 30min in a thermostatic water bath at 37 ℃, and 150 mul of 1mol/L HCl is added to stop the reaction, thus obtaining reaction solution. The reaction solution was analyzed by HPLC, and a blank control group was set. The ACE inhibitory activity was calculated as follows:
ACE inhibitory activity% ((M-N)/M × 100),
wherein M is the peak area of hippuric acid in the control group, and N is the peak area of hippuric acid in the added sample group.
4. Determination of the semi-inhibitory concentration
The inhibitory activity of the ACE inhibitory peptide is measured according to an in vitro detection method of the ACE inhibitory peptide, a smooth curve is drawn by taking the concentration as the abscissa and the ACE inhibitory rate as the ordinate, and the IC50 value is calculated from the curve. The results are shown in Table 1.
5. Measuring the molecular weight distribution in the yak bone protein peptide: the determination is carried out by referring to a GB/T22729-2008 method.
As can be seen from Table 1, the yak bone protein peptide prepared by the method has good ACE inhibitory activity, the IC50 value of the yak bone protein peptide is less than 0.32mg/mL, and the molecular weight of the yak bone protein peptide is less than 1000 and reaches more than 90%.
TABLE 1 ACE inhibitory Activity test results of Yak bone protein peptides
Figure BDA0001296280650000121
Finally, the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Anhui peptide Biotechnology Ltd
<120> Yak bone protein peptide with ACE inhibitory function, preparation method and application
<130>KHP171112765.1
<160>3
<170>PatentIn version 3.5
<210>1
<211>5
<212>PRT
<213> Yak bone
<400>1
Glu Ile Arg Met Leu
1 5
<210>2
<211>6
<212>PRT
<213> Yak bone
<400>2
Thr Val Ser Leu Pro Arg
1 5
<210>3
<211>7
<212>PRT
<213> Yak bone
<400>3
Ser Pro Ile Leu Gly Tyr Trp
1 5

Claims (3)

1. A yak bone protein peptide with ACE inhibitory function is characterized in that the yak bone protein peptide is prepared by the following method:
1) the method comprises the following steps of taking yak bones as raw materials, cooking at high temperature and removing fat to obtain yak bone protein liquid, and carrying out ultrasonic treatment on the prepared yak bone protein liquid at 75-85 ℃ for 15-25 minutes at the frequency of 80-100kH to change the tissue structure of the yak bone protein;
2) performing stepwise enzymolysis on the yak bone protein liquid prepared in the step 1) by using compound protease without adding acid and alkali, wherein the stepwise enzymolysis by using the compound protease comprises the following steps: a first step of enzymolysis reaction: adding 0.2-0.5 wt% of first compound protease into yak bone protein liquid accounting for 8-10 wt% of protein weight, and performing enzymolysis reaction at 50-60 ℃ for 0.5-1.5 h, wherein the first compound protease is alkaline protease and papain; the second step of enzymolysis reaction: adding 0.2-0.5 wt% of second compound protease into the product of the first-step enzymolysis reaction, and carrying out enzymolysis reaction for 1.0-2.0 h at the temperature of 45-55 ℃, wherein the second compound protease is trypsin and flavourzyme;
3) removing impurities from the product obtained after enzymolysis in the step 2), performing membrane separation, gel separation and reversed-phase HPLC separation, concentrating, and freeze-drying to obtain the product;
wherein, no acid or alkali is added in the whole processing process of the yak bone protein peptide for adjusting the pH value; the impurity removal in the step 3) is specifically as follows:
preserving heat for 10-20 minutes at 90-95 ℃, cooling to room temperature, adding active carbon with the weight of 0.2-0.6 wt% of the enzymolysis liquid into the enzymolysis liquid, separating through diatomite, and collecting a separation liquid;
the membrane separation in the step 3) is specifically as follows: treating the product after impurity removal by a two-step ultrafiltration method, and sequentially performing ultrafiltration by using a membrane with the aperture of 5000 daltons and a membrane with the aperture of 3000 daltons;
the gel separation and reversed phase HPLC in the step 3) are specifically as follows:
separating the product after membrane separation by Sephadex G-25 gel, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; concentrating, freezing and drying to obtain a yak bone protein peptide crude product; separating by RP-HPLC reversed-phase high performance liquid chromatography for 1 time, and collecting the peptide eluate after 16-18 min of RP-HPLC reversed-phase high performance liquid chromatography separation;
wherein the yak bone protein peptide comprises polypeptide with the following amino acid sequence content of more than 50 percent:
Glu-Ile-Arg-Met-Leu，
Thr-Val-Ser-Leu-Pro-Arg，
and Ser-Pro-Ile-Leu-Gly-Tyr-Trp.
2. A method for preparing the yak bone protein peptide of claim 1, comprising the steps of:
1) the method comprises the following steps of taking yak bones as raw materials, cooking at high temperature and removing fat to obtain yak bone protein liquid, and carrying out ultrasonic treatment on the prepared yak bone protein liquid at 75-85 ℃ for 15-25 minutes at the frequency of 80-100kH to change the tissue structure of the yak bone protein;
2) performing stepwise enzymolysis on the yak bone protein liquid prepared in the step 1) by using compound protease without adding acid and alkali, wherein the stepwise enzymolysis by using the compound protease comprises the following steps: a first step of enzymolysis reaction: adding 0.2-0.5 wt% of first compound protease into yak bone protein liquid accounting for 8-10 wt% of protein weight, and performing enzymolysis reaction at 50-60 ℃ for 0.5-1.5 h, wherein the first compound protease is alkaline protease and papain; the second step of enzymolysis reaction: adding 0.2-0.5 wt% of second compound protease into the product of the first-step enzymolysis reaction, and carrying out enzymolysis reaction for 1.0-2.0 h at the temperature of 45-55 ℃, wherein the second compound protease is trypsin and flavourzyme;
3) removing impurities from the product obtained after enzymolysis in the step 2), performing membrane separation, gel separation and reversed-phase HPLC separation, concentrating, and freeze-drying to obtain the product;
wherein, no acid or alkali is added in the whole processing process of the yak bone protein peptide for adjusting the pH value; the impurity removal in the step 3) is specifically as follows:
preserving heat for 10-20 minutes at 90-95 ℃, cooling to room temperature, adding active carbon with the weight of 0.2-0.6 wt% of the enzymolysis liquid into the enzymolysis liquid, separating through diatomite, and collecting a separation liquid;
the membrane separation in the step 3) is specifically as follows: treating the product after impurity removal by a two-step ultrafiltration method, and sequentially performing ultrafiltration by using a membrane with the aperture of 5000 daltons and a membrane with the aperture of 3000 daltons;
the gel separation and reversed phase HPLC in the step 3) are specifically as follows:
separating the product after membrane separation by Sephadex G-25 gel, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; concentrating, freezing and drying to obtain a yak bone protein peptide crude product; then RP-HPLC reversed-phase high performance liquid chromatography is used for 1 time of separation, and peptide eluent which is separated by RP-HPLC reversed-phase high performance liquid chromatography for 16-18 minutes is collected.
3. Use of a yak bone protein peptide of claim 1 in the manufacture of a medicament or food product for the prevention or treatment of a disease benefiting from ACE inhibition.
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