CN107699601B - Cordyceps militaris protein peptide with ACE (angiotensin converting enzyme) inhibition function and DPP-IV inhibition function - Google Patents

Cordyceps militaris protein peptide with ACE (angiotensin converting enzyme) inhibition function and DPP-IV inhibition function Download PDF

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CN107699601B
CN107699601B CN201711085032.6A CN201711085032A CN107699601B CN 107699601 B CN107699601 B CN 107699601B CN 201711085032 A CN201711085032 A CN 201711085032A CN 107699601 B CN107699601 B CN 107699601B
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张恒
罗永康
刘怀高
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Anhui Guotai Biotechnology Co ltd
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Abstract

The invention provides a cordyceps militaris protein peptide with ACE (angiotensin converting enzyme) inhibition function and DPP-IV inhibition function, and a preparation method thereof comprises the following steps: (1) pulverizing Cordyceps militaris into powder, adding water, and treating at high temperature to obtain Cordyceps militaris solution; (2) carrying out ultrasonic treatment on cordyceps militaris liquid; (3) adding compound protease for enzymolysis, and synchronously performing ultrasonic treatment; preserving heat at high temperature, cooling, filtering and separating, and collecting separated liquid; (4) treating the separated liquid by a two-step ultrafiltration method; (5) separating the filtrate with column, and collecting the elution peak. The method has the advantages of simple operation, less enzyme dosage, no need of adding acid or alkali to adjust the pH value in the preparation process, no addition of any additive, better maintenance of the functional characteristics of the product, high yield and good flavor, and the obtained cordyceps militaris protein peptide has excellent ACE inhibition and DPP-IV inhibition functions and is widely applied to the manufacture of special foods and nutritional foods.

Description

Cordyceps militaris protein peptide with ACE (angiotensin converting enzyme) inhibition function and DPP-IV inhibition function
Technical Field
The invention relates to the technical field of food processing, in particular to a cordyceps militaris protein peptide with ACE (angiotensin converting enzyme) inhibition and DPP-IV (dipeptidyl peptidase-IV) inhibition functions and a preparation method thereof.
Background
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, reports about the development of ACE inhibitory peptides are mainly that the ACE inhibitory peptides are obtained from whey protein sources of aquatic animals or milk by an enzymolysis method, the pH value of a system is usually regulated by adding acid or alkali in the enzymolysis process, the functional characteristics of the obtained ACE inhibitory peptide products are undoubtedly influenced, the complexity of operation is increased, the operation steps and the production cost are increased by the methods to different degrees, and the production of industrial large-scale ACE inhibitory peptides and the yield of protein peptides are restricted. At present, no report of preparing cordyceps militaris protein peptide with DPP-IV (dipeptidyl peptidase IV) inhibition function and ACE (angiotensin converting enzyme) inhibition function by using cordyceps militaris as a raw material is found in domestic and foreign patents.
The blood sugar reducing functional factor refers to a bioactive component capable of reducing blood sugar concentration of diabetic patients and improving symptoms of the diabetic patients. At present, the research on more natural products such as blood sugar reducing factors, mineral blood sugar reducing factors and vitamin blood sugar reducing factors is different in action mechanism. The natural product hypoglycemic factors can be divided into flavonoids, active polysaccharides, alkaloids, saponins, terpenes, polypeptides and the like according to chemical structures. Since the artificial synthesis of insulin, various oral or injection medicaments such as sulfonylurea medicaments, biguanides, insulin sensitizers, sugar inhibitors and the like come out in succession, however, chemical medicaments often generate certain toxic and side effects, and natural hypoglycemic components have the advantages of mild and lasting action, stable property, almost no toxic reaction, coexistence of various hypoglycemic components, comprehensive action and the like, are favored by patients and the medical field, and also become the main research direction of hypoglycemic functional factors.
DPP IV dipeptidyl peptidase (an enzyme responsible for degradation of GLP 1): the function of the polypeptide is to split and degrade GLP-1, and DPPIV is an enzyme which can rapidly inactivate insulinotropic hormone GLP-1. Inhibiting DPP-IV can improve the hormone activity of GLP-1 and other bioactive peptides, thereby stimulating the release of insulin, reducing the secretion of glucagon and being beneficial to the regulation of high blood sugar level of type 2 diabetes patients.
Aiming at the current situation of the development of the cordyceps militaris protein product at present, the cordyceps militaris powder is used as a raw material, multiple proteases are used for compound enzymolysis under the condition of not adding any acid and alkali, and the cordyceps militaris protein peptide with specific functions of reducing blood pressure and blood sugar is developed through the membrane separation and gel separation technology, so that a simple and efficient multifunctional preparation method of the cordyceps militaris protein peptide is established.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide the cordyceps militaris protein peptide with ACE (angiotensin converting enzyme) inhibition and DPP-IV (dipeptidyl peptidase-IV) inhibition functions.
The invention also aims to provide a preparation method of the cordyceps militaris protein peptide.
The invention further aims to provide application of the cordyceps militaris protein peptide.
In order to realize the technical purpose, the invention provides cordyceps militaris protein peptide with ACE (angiotensin converting enzyme) inhibition and DPP-IV (dipeptidyl peptidase-IV) inhibition functions, which comprises the following steps:
(1) pulverizing clean Cordyceps militaris into powder, adding water, and treating at high temperature to obtain Cordyceps militaris solution;
(2) carrying out ultrasonic treatment on cordyceps militaris liquid;
(3) adding compound protease for enzymolysis, and performing ultrasonic treatment synchronously in the enzymolysis process; preserving heat at high temperature, cooling, filtering and separating, and collecting separated liquid;
(4) treating the separated liquid by a two-step ultrafiltration method;
(5) separating the filtrate with column, and collecting the elution peak.
The mass ratio of the water to the cordyceps militaris powder in the step (1) is 15-20:1, and the mixture is stirred for 1-4 hours at the high temperature of 100-121 ℃.
The ultrasonic treatment method in step (2) comprises adjusting the Cordyceps militaris liquid obtained in step (1) to 65-75 deg.C, and treating with ultrasonic frequency of 80-100kH for 15-25 min.
A large number of experiments show that the cordyceps militaris liquid is adjusted to 65-75 ℃ for 80-100kH ultrasonic treatment, the effect is best, the tissue structure of cordyceps militaris protein can be better changed, the enzymolysis time is shortened in the following enzymolysis steps, and high-quality protein peptide can be obtained by using less enzyme.
Adding compound protease according to the weight percentage of 0.2-0.8% of the total weight of the cordyceps militaris liquid, wherein the compound protease consists of alkaline protease, neutral protease and flavourzyme, and the mass ratio of the alkaline protease to the neutral protease to the flavourzyme is 1: (1-2):1.
The enzymolysis condition of the step (3) is enzymolysis for 1.5 to 3.5 hours at the temperature of between 50 and 65 ℃; the ultrasonic treatment in the step (3) is 25-40kH treatment; and (3) after enzymolysis, preserving the heat for 10-20min at the temperature of 90-95 ℃, cooling to 65-75 ℃, filtering and separating, and collecting separated liquid. In the examples of the present invention, the separation is performed by filtration through celite, and then the separated liquid is collected. The filtration separation can be carried out by any separation means having an equivalent effect by those skilled in the art.
The two-step ultrafiltration method in the step (4) is to firstly use a membrane with the aperture of 5000D for ultrafiltration and then use a membrane with the aperture of 2000D for ultrafiltration to obtain the protein peptide with the molecular weight of less than 2000D.
Specifically, the method utilizes a ceramic membrane with the aperture of 5000 daltons for ultrafiltration, firstly separates proteins and polypeptides with the molecular weight of less than 5000 daltons, and then separates protein peptides with the molecular weight of less than 1500 daltons by using a membrane with the aperture of 2000 daltons.
And (5) performing column separation on the filtrate, wherein the eluent is deionized water, detecting an elution peak at 280nm, collecting the 2 nd elution peak, concentrating, and performing freeze drying to obtain the cordyceps militaris protein peptide.
Specifically, taking protein peptide liquid with molecular weight less than 2000, 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; obtaining cordyceps militaris protein peptide by concentration and freeze drying; separating by RP-HPLC reversed-phase high performance liquid chromatography, and collecting the peptide liquid eluted at 9-10 min in RP-HPLC reversed-phase high performance liquid chromatography; and (5) concentrating and freeze-drying the peptide solution obtained in the step (5) to obtain cordyceps militaris protein peptide powder.
The main components of the cordyceps militaris protein peptide are determined by LC-MS/MS, the amino acid sequences of the cordyceps militaris protein peptide are NVEIDPE (asparagine-valine-glutamic acid-isoleucine-aspartic acid-proline-glutamic acid), SIIAEVK (serine-isoleucine-alanine-glutamic acid-valine-lysine), and RDLTDY (arginine-aspartic acid-leucine-threonine-aspartic acid-tyrosine), wherein the content of the peptide is 85-95%, and the amino acid sequences of the peptide are NVEIDPE (20-27%), SIIAEVK (35-45%) and RDLTDY (22-29%). The percentages are mass percentages.
The cordyceps militaris protein peptide prepared by the preparation method belongs to the protection range of the invention. Experiments show that the cordyceps militaris protein peptide prepared by the method has excellent DPP-IV inhibition and ACE inhibition functions.
Furthermore, the invention provides application of the cordyceps militaris protein peptide in preparing a medicine for reducing blood pressure or blood sugar. The food, health care product or medicine containing the cordyceps militaris protein peptide prepared by the method also belongs to the protection scope of the invention.
The invention discovers that the peptides with the amino acid sequences shown as SEQ ID NO.1-3 have ACE inhibitory function and DPP-IV inhibitory function.
Further, the invention provides application of the peptides with the amino acid sequences shown as SEQ ID NO.1-3 in preparing food, health products or medicines.
The invention has the advantages that:
(1) the whole preparation process of the cordyceps militaris protein peptide synchronously utilizes ultrasonic treatment with different frequencies, the preparation method 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.
(2) The developed cordyceps militaris protein peptide has good DPP-IV inhibition and ACE inhibition functions, the DPP-IV inhibition activity (IC50 value) of the prepared cordyceps militaris protein peptide is lower than 190 mu g/mL, the ACE inhibition activity (IC50 value) is lower than 305 mu g/mL, the DPP-IV inhibition activity (IC50 value) of the developed cordyceps militaris SIIAEVK peptide is 98 mu g/mL, and the ACE inhibition activity (IC50 value) of the NVEIDPE peptide is 208 mu g/mL.
(3) The cordyceps militaris protein liquid treated by the specific frequency ultrasonic technology can obviously improve the sensitivity of cordyceps militaris protein to enzyme and reduce the using amount of the enzyme.
(4) The developed product is safe, the invention adopts a plurality of food-grade compound proteases (alkaline protease, neutral protease and flavourzyme), obtains the cordyceps militaris protein peptide with specific molecular weight and peptide composition by moderate enzymolysis under mild conditions, and 100 percent of the cordyceps militaris protein peptide is not added with any additive.
(5) The protein peptide developed by the invention has good flavor and color and can be widely applied to special foods and nutritional foods.
(6) The protein peptide developed by the invention has a peptide composition which is very clear, and the proportion of peptides with molecular weight less than 2000 in the protein peptide is more than 95%.
Detailed Description
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specified, the reagents and materials used in the present invention are commercially available products or products obtained by a known method. The% in the following examples are all by mass. The Cordyceps militaris dregs are purchased from Shenyang Vandada biotechnology, and neutral protease, alkaline protease and flavourzyme are purchased from Novoxin (China) investment, Inc.
Example 1 preparation of Cordyceps militaris protein peptide with DPP-IV inhibition and ACE inhibition functions
(1) Selecting 100 g of dried cordyceps militaris, cleaning the cordyceps militaris by clean water meeting the drinking water sanitary standard, pulverizing the cordyceps militaris into powder by a pulverizer after the cordyceps militaris is cleaned, adding water 15 times of the total amount of cordyceps militaris powder, and treating the cordyceps militaris powder at 121 ℃ for 2 hours to obtain cordyceps militaris liquid.
(2) Adjusting the temperature of the cordyceps militaris liquid obtained in the step (1) to 75 ℃, and treating the cordyceps militaris liquid for 15 minutes by using an ultrasonic generator through ultrasonic waves (the frequency is 80kH) to change the tissue structure of the cordyceps militaris protein.
(3) Adding compound protease (composed of alkaline protease, neutral protease and flavourzyme in a mass ratio of 1: 1: 1) according to a weight percentage of 0.6% of the weight of the cordyceps militaris in the cordyceps militaris liquid for enzymolysis, carrying out enzymolysis reaction for 3.0 hours at the temperature of 55 ℃, and synchronously treating the enzymolysis process by using an ultrasonic generator (with the frequency of 40 kH); preserving the heat at 95 ℃ for 10 minutes, cooling to 75 ℃, filtering and separating through diatomite, and collecting a separation solution;
(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 daltons, and separating protein peptides with the molecular weight of less than 2000 daltons by using a membrane with the aperture of 2000 daltons.
(5) Taking protein peptide liquid with molecular weight less than 2000, and then carrying out Sephadex G-15 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; obtaining cordyceps militaris protein peptide by concentration and freeze drying; separating by RP-HPLC reversed-phase high performance liquid chromatography, and collecting the peptide liquid eluted at 9-10 min in RP-HPLC reversed-phase high performance liquid chromatography;
(6) and (5) concentrating and freeze-drying the peptide solution obtained in the step (5) to obtain cordyceps militaris protein peptide powder. The main components of the cordyceps militaris protein peptide are determined by LC-MS/MS, the amino acid sequences of the cordyceps militaris protein peptide are NVEIDPE (asparagine-valine-glutamic acid-isoleucine-aspartic acid-proline-glutamic acid), SIIAEVK (serine-isoleucine-alanine-glutamic acid-valine-lysine), and RDLTDY (arginine-aspartic acid-leucine-threonine-aspartic acid-tyrosine), wherein the content of the peptide is 88%, and the amino acid sequences of the cordyceps militaris protein peptide are NVEIDPE (23%), SIIAEVK (38%) and RDLTDY (27%).
Example 2 preparation of Cordyceps militaris protein peptide with DPP-IV inhibition and ACE inhibition functions
(1) Selecting 500 g of dried cordyceps militaris, cleaning the cordyceps militaris by clean water meeting the sanitary standard of drinking water, pulverizing the cordyceps militaris into powder by a pulverizer after the cordyceps militaris is cleaned, adding water which is 20.0 times of the total amount of cordyceps militaris powder, and treating the cordyceps militaris for 3 hours at 110 ℃ to obtain cordyceps militaris liquid.
(2) Adjusting the temperature of the cordyceps militaris liquid obtained in the step (1) to 70 ℃, and treating the cordyceps militaris liquid for 15 minutes by using an ultrasonic generator through ultrasonic waves (the frequency is 100kH) to change the tissue structure of the cordyceps militaris protein.
(3) Adding compound protease (composed of alkaline protease, neutral protease and flavourzyme in a mass ratio of 1: 2: 1) according to a weight percentage of 0.8% of the weight of the cordyceps militaris in the cordyceps militaris liquid for enzymolysis, carrying out enzymolysis reaction for 3.5 hours at the temperature of 60 ℃, and synchronously treating the enzymolysis process by using an ultrasonic generator (with the frequency of 25 kH); keeping the temperature at 90 ℃ for 15 minutes, cooling to 75 ℃, filtering and separating through diatomite, and collecting a separation solution;
(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 daltons, and separating protein peptides with the molecular weight of less than 2000 daltons by using a membrane with the aperture of 2000 daltons.
(5) Taking protein peptide liquid with molecular weight less than 2000, and then carrying out Sephadex G-15 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; obtaining cordyceps militaris protein peptide by concentration and freeze drying; separating by RP-HPLC reversed-phase high performance liquid chromatography, and collecting the peptide liquid eluted at 9-10 min in RP-HPLC reversed-phase high performance liquid chromatography;
(6) and (5) concentrating and freeze-drying the peptide solution obtained in the step (5) to obtain cordyceps militaris protein peptide powder. The main components of the cordyceps militaris protein peptide are determined by LC-MS/MS, the amino acid sequences of the cordyceps militaris protein peptide are NVEIDPE (asparagine-valine-glutamic acid-isoleucine-aspartic acid-proline-glutamic acid), SIIAEVK (serine-isoleucine-alanine-glutamic acid-valine-lysine) and RDLTDY (arginine-aspartic acid-leucine-threonine-aspartic acid-tyrosine), wherein the content of the peptide is 90%, and the amino acid sequences of the peptide are NVEIDPE (24%), SIIAEVK (39%) and RDLTDY (27%).
Example 3 preparation of Cordyceps militaris protein peptide with DPP-IV inhibition and ACE inhibition functions
(1) Selecting 1000 g of dried cordyceps militaris, cleaning the cordyceps militaris by clean water meeting the drinking water sanitary standard, pulverizing the cordyceps militaris into powder by a pulverizer after cleaning the cordyceps militaris, adding water with the amount being 18.0 times of the total amount of cordyceps militaris powder, and treating the cordyceps militaris for 4 hours at the temperature of 100 ℃ to obtain cordyceps militaris liquid.
(2) Adjusting the temperature of the cordyceps militaris liquid obtained in the step (1) to 65 ℃, and treating the cordyceps militaris liquid for 20 minutes by using an ultrasonic generator through ultrasonic waves (the frequency is 100kH) to change the tissue structure of the cordyceps militaris protein.
(3) Adding compound protease (composed of alkaline protease, neutral protease and flavourzyme in a mass ratio of 1: 2: 1) according to a weight percentage of 0.8% of the weight of the cordyceps militaris in the cordyceps militaris liquid for enzymolysis, carrying out enzymolysis reaction for 3.5 hours at the temperature of 60 ℃, and synchronously treating the enzymolysis process by using an ultrasonic generator (with the frequency of 30 kH); keeping the temperature at 95 ℃ for 12 minutes, cooling to 65 ℃, filtering and separating through diatomite, and collecting a separation solution;
(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 daltons, and separating protein peptides with the molecular weight of less than 2000 daltons by using a membrane with the aperture of 2000 daltons.
(5) Taking protein peptide liquid with molecular weight less than 2000, and then carrying out Sephadex G-15 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; obtaining cordyceps militaris protein peptide by concentration and freeze drying; separating by RP-HPLC reversed-phase high performance liquid chromatography, and collecting the peptide liquid eluted at 9-10 min in RP-HPLC reversed-phase high performance liquid chromatography;
(6) and (5) concentrating and freeze-drying the peptide solution obtained in the step (5) to obtain cordyceps militaris protein peptide powder. The main components of the cordyceps militaris protein peptide are determined by LC-MS/MS, the amino acid sequences of the cordyceps militaris protein peptide are NVEIDPE (asparagine-valine-glutamic acid-isoleucine-aspartic acid-proline-glutamic acid), SIIAEVK (serine-isoleucine-alanine-glutamic acid-valine-lysine), and RDLTDY (arginine-aspartic acid-leucine-threonine-aspartic acid-tyrosine), wherein the content of the peptide is 88%, and the amino acid sequences of the cordyceps militaris protein peptide are NVEIDPE (23%), SIIAEVK (37%) and RDLTDY (28%).
Example 4 determination test of DPP-IV inhibitory Activity of Cordyceps militaris protein peptide
1. Test samples: samples 1, 2 and 3 are the cordyceps militaris protein peptides prepared in example 1, example 2 and example 3, respectively, and sample 4 is the cordyceps militaris powder obtained by crushing in example 1.
2. The method comprises the following steps:
the sample was diluted to an appropriate concentration with 100mmol/L Tris-HCl (pH8.0) buffer, 25. mu.L of the sample dilution was aspirated, mixed with 25. mu.L of a substrate (concentration: 1.6mmol/L), and added to a 96-well plate. After incubation for 10min at 37 ℃, adding 50 mu L of DPP-IV enzyme liquid (the enzyme activity is 8U/L), mixing uniformly, then incubating for 60min at 37 ℃, immediately adding 100 mu L of 1mol/L acetic acid-sodium acetate (pH 4.0) buffer solution to terminate the reaction, measuring the absorbance A at 405nm, and calculating the DPP-IV inhibition rate of the sample according to the following formula.
DPP-IV inhibition = {1- (sample absorbance a-sample blank absorbance B))/(negative control absorbance C-negative blank absorbance D) } × 100
Sample absorbance a: the absorbance value of the sample reaction solution at 405nm is obtained;
sample blank control absorbance B: Tris-HCL buffer solution is used for replacing DPP-IV enzyme solution as the light absorption value of a sample blank control at 405 nm;
negative control absorbance C: the absorbance value at 405nm of a sample is replaced by a Tris-HCL buffer solution as a negative control;
negative blank control absorbance D: Tris-HCL buffer solution is used for replacing DPP-IV enzyme solution and sample as the light absorption value of negative blank control at 405nm
Determination of IC50 values for DPP-IV inhibition:
the DPP-IV inhibition rate of the sample at different concentrations is measured, and a regression curve is drawn by taking the logarithm value of the polypeptide concentration and the inhibition rate to obtain a regression equation, so that the IC50 is calculated, namely, 50 percent of the DPP-IV enzyme activity inhibition is the concentration of the peptide.
The results are shown in Table 1, and it can be seen that the Cordyceps militaris protein peptide of the invention has very good propertiesGood DPP-IV inhibitory ability, DPP-IV inhibitory activity (IC)50Value) is lower than 190 mug/mL and is obviously lower than IC of the same kind of compound protein peptide50The SIIAEVK peptide has good DPP-IV inhibition ability and DPP-IV inhibition activity (IC)50Value) was 98. mu.g/mL.
Example 5 determination of inhibitory Activity of Cordyceps militaris protein peptide Angiotensin Converting Enzyme (ACE)
Test samples: samples 1, 2 and 3 are the cordyceps militaris protein peptides prepared in example 1, example 2 and example 3, respectively, and sample 4 is the cordyceps militaris powder crushed in example 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) Method for drawing hippuric acid standard curve
Dissolving hippuric acid into standard concentrations of 1mmol/L, 0.5mmol/L, 0.25mmol/L, 0.1mmol/L, 0.05mmol/L, 0.01mmol/L and 0.005mmol/L by using double distilled water, filtering by using a 0.22ul filter membrane, performing sample injection analysis on an HPLC instrument to obtain the areas of hippuric acid with different concentrations, and then establishing a linear relation between the hippuric acid peak area and the concentration. The concentration and the peak area of hippuric acid can reflect the content of hippuric acid in a sample, and further reflect the inhibitory activity of the ACE inhibitor.
(5) 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 (see table 1) show that the cordyceps militaris protein peptide has better ACE inhibitory capacity and ACE inhibitory activity (IC)50Value) is lower than 305 mug/mL and is obviously lower than the IC of the same kind of compound protein peptide50Value, wherein NVEIDPE peptide ACE inhibitory Activity (IC)50Value) was 208. mu.g/mL, and had a good ACE inhibitory ability.
TABLE 1 DPP-IV inhibitory ability and ACE inhibitory activity of Cordyceps militaris protein peptide of the present invention
Figure BDA0001459881220000111
The above examples are only for describing the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Sequence listing
<110> Anhui peptide Biotechnology Ltd
<120> cordyceps militaris protein peptide with ACE (angiotensin converting enzyme) inhibition function and DPP-IV inhibition function
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>7
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>1
Asn Val Glu Ile Asp Pro Glu
1 5
<210>2
<211>7
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>2
Ser Ile Ile Ala Glu Val Lys
1 5
<210>3
<211>6
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>3
Arg Asp Leu Thr Asp Tyr
1 5

Claims (4)

1. A preparation method of cordyceps militaris protein peptide with ACE inhibition and DPP-IV inhibition functions is characterized by comprising the following steps:
(1) pulverizing clean Cordyceps militaris into powder, adding water, and treating at high temperature to obtain Cordyceps militaris solution;
(2) carrying out ultrasonic treatment on cordyceps militaris liquid, adjusting the cordyceps militaris liquid obtained in the step (1) to 65-75 ℃, and carrying out ultrasonic frequency treatment at 80-100kH for 15-25min to change the tissue structure of cordyceps militaris protein;
(3) adding compound protease for enzymolysis, wherein ultrasonic treatment of 25-40kH is synchronously performed by using an ultrasonic generator in the enzymolysis process without adding acid and alkali; preserving heat at high temperature, cooling, filtering and separating, and collecting a separation solution, wherein the compound protease consists of alkaline protease, neutral protease and flavourzyme;
(4) treating the separated liquid by two-step ultrafiltration, firstly performing ultrafiltration by using a membrane with the aperture of 5000D, and then performing ultrafiltration by using a membrane with the aperture of 2000D to obtain a protein peptide liquid with the molecular weight of less than 2000D;
(5) taking protein peptide liquid with molecular weight less than 2000D, 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, and freeze drying; separating by RP-HPLC reversed-phase high performance liquid chromatography, collecting protein peptide liquid eluted in 9-10 minutes in RP-HPLC reversed-phase high performance liquid chromatography, concentrating, and freeze-drying to obtain Cordyceps militaris protein peptide, wherein the Cordyceps militaris protein peptide comprises peptides with amino acid sequences of asparagine-valine-glutamic acid-isoleucine-aspartic acid-proline-glutamic acid (NVEIDPE), serine-isoleucine-alanine-glutamic acid-valine-lysine (SIIAEVK), arginine-aspartic acid-leucine-threonine-aspartic acid-tyrosine (RDLTDY) in the mass percentage of 85-95%;
wherein no acid or alkali is added to adjust the pH in the whole processing process of the cordyceps militaris protein peptide;
the mass ratio of the water to the cordyceps militaris powder in the step (1) is 15-20:1, and the mixture is stirred for 1-4 hours at the high temperature of 100-121 ℃;
adding compound protease according to the weight percentage of 0.2-0.8% of the total weight of the cordyceps militaris liquid, wherein the mass ratio of alkaline protease, neutral protease and flavourzyme in the compound protease is 1: (1-2) 1;
the enzymolysis condition of the step (3) is enzymolysis for 1.5 to 3.5 hours at the temperature of between 50 and 65 ℃; and (3) after enzymolysis, preserving the heat for 10-20min at the temperature of 90-95 ℃, cooling to 65-75 ℃, filtering and separating, and collecting separated liquid.
2. The cordyceps militaris protein peptide prepared by the preparation method of claim 1.
3. The application of the cordyceps militaris protein peptide disclosed in claim 2 in preparing a medicine for reducing blood sugar or blood pressure.
4. Food, health care product or medicine containing the cordyceps militaris protein peptide of claim 2.
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CN104403012A (en) * 2014-11-10 2015-03-11 苏州蔻美新材料有限公司 Extraction technology of cordyceps militaris polysaccharide and polypeptide
CN106520874A (en) * 2016-11-07 2017-03-22 中国农业大学 Swimming bladder protein peptide having functions of reducing blood sugar and resisting oxidation and preparation method of swimming bladder protein peptide

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CN104403012A (en) * 2014-11-10 2015-03-11 苏州蔻美新材料有限公司 Extraction technology of cordyceps militaris polysaccharide and polypeptide
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