CN117482209A - Queen bee embryo active peptide composition with ACE inhibition effect, and preparation method and application thereof - Google Patents
Queen bee embryo active peptide composition with ACE inhibition effect, and preparation method and application thereof Download PDFInfo
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- CN117482209A CN117482209A CN202311510905.9A CN202311510905A CN117482209A CN 117482209 A CN117482209 A CN 117482209A CN 202311510905 A CN202311510905 A CN 202311510905A CN 117482209 A CN117482209 A CN 117482209A
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- queen bee
- peptide composition
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 53
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- 230000005764 inhibitory process Effects 0.000 title claims abstract description 36
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- 230000002401 inhibitory effect Effects 0.000 claims description 13
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- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a queen bee embryo active peptide composition with an ACE inhibition effect, and a preparation method and application thereof, and belongs to the technical field of food and medicine. The queen bee embryo active peptide composition provided by the invention comprises at least one of 5 small molecule peptides; the amino acid sequences of the 5 small molecule peptides are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5. The ACE inhibitory peptide composition prepared by the invention has high activity, high yield and strong operability, can be used for industrial production, and provides experimental basis for preparing the ACE inhibitory peptide with high activity from queen bee tyres.
Description
Technical Field
The invention relates to the technical field of food and medicine, in particular to a queen bee embryo active peptide composition with an ACE (angiotensin converting enzyme) inhibition effect, and a preparation method and application thereof.
Background
Hypertension is a clinical syndrome characterized by an increase in systemic arterial blood pressure (systolic and/or diastolic), and can cause myocardial infarction, stroke, coronary arteriosclerosis, and other diseases. Global hypertension patients are increasing year by year, and hypertension has become an important world public health problem. ACE is a Zn-containing compound widely present in the lungs, testes and kidneys 2+ Prosthetic dipeptidyl carboxypeptidase. ACE is part of the renin-angiotensin system (Renin angiotensin system, RAS) and can cleave His-Leu from the C-terminus to angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) which converts it to angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) which can constrict arterioles and accelerate heart beats leading to increased blood pressure. On the other hand, ACE is also an important component of the kallikrein system, which catalyzes bradykinin as an inactive fragment, thereby accelerating the increase in blood pressure in the body. Thus, inhibiting ACE activity may lower blood pressure. The general antihypertensive drugs used clinically achieve the aim of reducing blood pressure by inhibiting ACE activity, however, long-term administration of the chemical synthetic antihypertensive drugs can generate a series of side effects on human bodies, so that development of ACE inhibitory peptides of food-source proteins has become a hotspot for research of domestic and foreign students.
The queen bee embryo is larva body obtained by feeding fresh Lac Regis Apis to bee fertilized eggs, i.e. queen bee larva collected from queen bee larva. It was found that the moisture content in queen bee tires was about 80% by weight, the crude fat and crude protein were 18% and 56% by dry weight, the total content of 18 amino acids was 46g/100g by dry weight, the content of essential amino acids was 49% by weight, and the ratio of essential amino acids to non-essential amino acids was 0.98. And simultaneously contains rich vitamins and mineral elements. Therefore, the queen bee embryo can be used as a feeding protein resource with higher nutrition value, and has higher development and utilization values. China is a large country with long history of bee-keeping, the number of bee colonies and the yield of bee products are all the front of the world, and the annual yield of queen bee embryo can reach thousands of tons. However, as a byproduct of producing royal jelly, only about 50 tons of queen bee embryos are processed and utilized nationally each year, and 95% of queen bee embryos are discarded, so that resources are wasted greatly. Therefore, a scientific, reasonable and feasible production and processing technology is developed, the high-quality protein of the queen bee embryo is fully developed and utilized, and the method has important practical significance.
The research on queen bee embryo proteins is still in the starting stage, and is rarely reported. Relevant reports are: the research on the extraction process of queen bee larva protein is published by university of northeast agriculture scholars in 2006 of food industry technology. "A feeding queen bee embryo protein and its preparation process" are disclosed in Jiangxi Santong biosciences limited company. A patent 'bee pupa protein peptide with ACE inhibition and anti-fatigue functions' applied by the national peptide bioengineering (Changde) limited company and a preparation method thereof. A patent ' a bee pupa protein peptide with inhibiting and anti-fatigue functions ' applied by Qinghai national peptide biotechnology limited company ' and a preparation method thereof. The bee pupa is different from the queen bee embryo, the queen bee embryo protein needs to specially develop corresponding extraction and enzymolysis processes, and the patent for developing and utilizing the queen bee embryo protein source active peptide is not seen at present.
Disclosure of Invention
In order to overcome the defects in the prior art, effectively develop the queen bee embryo resources and improve the economic added value of the queen bee embryo, the invention provides a queen bee embryo active peptide composition with ACE inhibition effect and a preparation method and application thereof aiming at the characteristics of the queen bee embryo.
In order to achieve the above object, the present application provides the following technical solutions:
in one aspect, the present application provides a queen bee embryo active peptide composition with ACE inhibition, comprising at least one of 5 small molecule peptides;
the amino acid sequences of the 5 small molecule peptides are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
In a second aspect, the present application provides a preparation method of the above active peptide composition with ACE inhibition effect, comprising the following steps:
(1) Pretreatment: mixing queen bee embryo with water, pulping to obtain a prefabricated liquid A;
(2) Alkali dissolution: mixing the prefabricated liquid A with water for dilution, regulating the pH value to 7.0-9.0, stirring, centrifuging and taking supernatant fluid to obtain prefabricated liquid B;
(3) Enzymolysis: mixing the prefabricated liquid B with protease for enzymolysis, inactivating, centrifuging, and taking supernatant to obtain prefabricated liquid C;
(4) Purifying: purifying the prefabricated liquid C by gel filtration chromatography, anion exchange chromatography and reversed-phase high performance liquid chromatography in sequence to obtain the queen bee embryo active peptide composition with ACE inhibition effect.
Optionally, in the step (1), the mixing mass ratio of the queen bee larva to the water is 1:2-3, and the solid content in the prefabricated liquid A is 5-7%.
Optionally, in the step (2), the mixing mass ratio of the prefabricated liquid A to the water is 1:3-5;
the component for regulating the pH value is edible alkali;
the stirring speed is 100-300 rpm;
the stirring time is 1-3 h;
the centrifugal force of the centrifugation is 3000-10000 g;
the temperature of the centrifugation is 5-20 ℃;
the centrifugation time is 5-10 min.
Optionally, in step (3), the protease is an edible neutral protease;
the addition amount of the protease is 6000-9000U/g protein;
the enzymolysis temperature is 40-50 ℃;
the enzymolysis time is 2-4 hours;
the centrifugal force of the centrifugation is 3000-10000 g;
the temperature of the centrifugation is 5-20 ℃;
the centrifugation time is 5-10 min.
Optionally, in step (4), the conditions of the gel filtration chromatography are:
separating by Sephadex G-25 column chromatography, eluting with water at a flow rate of 0.4-0.6 mL/min, detecting absorbance at 250-300 nm, collecting the eluted component, collecting the component with highest ACE inhibition rate, and freeze drying.
Optionally, in step (4), the conditions of the anion exchange chromatography are:
purifying by HiTrap DEAE-FF anion exchange;
phosphate buffer solution with the mobile phase A liquid of 0.01-0.02 mol/L, pH-8;
the mobile phase B liquid is phosphate buffer solution containing 0.01-0.02 mol/L, pH-8 mol/L NaCl of 1.0-2.0 mol/L;
the sample loading liquid is the component obtained after gel filtration chromatography;
the elution condition is that the solution A with the concentration of 100 percent is eluted for 15 to 25 minutes, then the solution A is gradually changed to the solution B with the concentration of 100 percent within 55 to 65 minutes, and finally the solution B with the concentration of 100 percent is eluted for 15 to 25 minutes, and the elution flow rate is 0.5 to 1.5mL/min;
detecting absorbance at 250-300 nm to collect the eluted component, collecting the component with highest ACE inhibition rate, freeze drying.
Optionally, in step (4), the conditions of the reversed phase high performance liquid chromatography purification are:
adopting a C18 column;
the mobile phase A is ultrapure water containing 0.1-0.2 vt% of trifluoroacetic acid;
the mobile phase B is acetonitrile containing 0.1-0.2 vt% of trifluoroacetic acid;
the flow rate is 2-4 mL/min;
the sample loading liquid is a component obtained after anion exchange chromatography;
the gradient elution procedure was: 0 to 5min:100% of B, 5-25min:50% of A,50% of B, 25-40 min:100% A;
detecting absorbance at 250-300 nm to collect the eluted component, collecting the component with highest ACE inhibition rate, freeze drying.
In a third aspect, the application provides application of the queen bee embryo active peptide composition with ACE inhibition effect in preparation of ACE inhibition drugs.
In a fourth aspect, the application provides application of the queen bee embryo active peptide composition with ACE inhibition effect in preparation of health-care food for assisting in blood pressure reduction.
Compared with the prior art, the application has the following beneficial effects:
1. the invention is characterized in that queen bee embryo is pretreated and diluted, then alkali is directly added to promote protein dissolution, and then centrifugation is carried out to remove impurities, and supernatant is collected for enzymolysis, thus simplifying the complex process of heating and inactivating enzyme of diluent, adding acid to precipitate protein, centrifuging to collect precipitate, adding water to adjust to alkaline for redissolution, and then carrying out enzymolysis. Simplifying the process and improving the product yield.
2. The final step of separation and purification of the invention is completed by adopting a semi-preparative chromatographic column, and the pure peptide is not obtained, but the yield is larger, the operability is strong, the invention is suitable for industrial production, and the amino acid sequence composition of the main active peptide in the obtained ACE inhibitory peptide composition is IDFDF, VPIFD, FDYDFG, KNYPF and KPYPDWS respectively.
3. The prepared queen bee embryo protein source ACE inhibitory peptide composition is safe, free of toxic and side effects, scientific and reasonable in process, and can be developed as health-care food, food additives, drug synergists and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 shows ACE inhibition rates after different enzymes hydrolyze queen bee embryo proteins;
FIG. 2 is a Sephadex G-25 gel chromatographic profile of the enzyme substrate with maximum ACE inhibition of the present application;
FIG. 3 shows ACE inhibition ratios of components of Sephadex G-25 gel chromatography of the present application;
FIG. 4 is an anion exchange chromatogram of the F3 component;
FIG. 5 shows the ACE inhibition of the components after anion exchange chromatography of the F3 component;
FIG. 6 is a reversed phase HPLC purification chromatogram of the F3-4 fraction;
FIG. 7 shows ACE inhibition ratios of the F3-4 components after reversed-phase high performance liquid chromatography separation;
FIG. 8 is a total ion flow chromatogram of the F3-4-3 component;
FIG. 9 is a one-dimensional mass spectrum analysis of the first peptide content in the F3-4-3 fraction;
FIG. 10 is a two-dimensional mass spectrum analysis of the first peptide content in the F3-4-3 fraction;
FIG. 11 is a one-dimensional mass spectrum analysis of the second peptide content in the F3-4-3 fraction;
FIG. 12 is a two-dimensional mass spectrum analysis of the second peptide content in the F3-4-3 fraction;
FIG. 13 is a one-dimensional mass spectrum analysis of a peptide of the third content in the F3-4-3 component;
FIG. 14 is a two-dimensional mass spectrum analysis of the peptide of the third content in the F3-4-3 component;
FIG. 15 is a one-dimensional mass spectrum analysis of the fourth peptide in the F3-4-3 fraction;
FIG. 16 is a two-dimensional mass spectrum analysis of the fourth peptide in the F3-4-3 fraction;
FIG. 17 is a one-dimensional mass spectrum analysis of the peptide having the fifth content in the F3-4-3 component;
FIG. 18 is a two-dimensional mass spectrum analysis of the peptide having the fifth content in the F3-4-3 fraction.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Unless otherwise indicated, all starting materials in the examples of the present application were purchased commercially and used without any particular treatment.
Unless otherwise indicated, the analytical methods in the examples all employed conventional arrangements of instruments or equipment and conventional analytical methods.
Example 1
Enzymolysis of queen bee embryo proteins:
mixing fresh queen bee embryo with water according to a mass ratio of 1:2, and pulping to obtain a prefabricated liquid A, wherein the solid content in the prefabricated liquid A is about 7%; mixing the prefabricated liquid A with water according to the mass ratio of 1:4, diluting, adopting edible alkali to adjust the pH value to 8.0, stirring, centrifuging, and taking supernatant to obtain prefabricated liquid B; and (3) adding enzyme into the enzymolysis prefabricated solution B according to the amount of 8000U/g protein, and carrying out enzymolysis for 4 hours at 45 ℃ to obtain an enzymolysis solution. After the enzymolysis is finished, the enzymolysis liquid is subjected to enzyme deactivation at 90 ℃ for 15min, cooled to room temperature, centrifuged for 7min at 20 ℃ and 8000g, the supernatant is collected, and the ACE inhibition rate is determined by freeze-drying, and the result is shown in figure 1. The results prove that the obtained zymolyte has the highest ACE inhibition rate by adopting edible neutral protease for enzymolysis.
The protein content in the prefabricated liquid B is determined according to the Kjeldahl method.
Measurement of ACE inhibition ratio:
a100 mg/mL sample of the substrate was prepared (note: 70mg/mL concentration of aqueous solution was prepared for the chromatographic sample in example 2), 0.08mol/L of 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution (HEPES), 0.005mg/mL of (N- [3- (2-furyl) acryloyl ] -L-phenylalanyl-glycyl-glycine solution) (FAPGG), and 0.1. Mu.L of Angiotensin Converting Enzyme (ACE) solution. ACE 10. Mu.L, FAPGG 50. Mu.L, HEPES 40. Mu.L were added in blank wells. The sample wells were added with 10. Mu.L of ACE, 50. Mu.L of FAPGG, and 40. Mu.L of sample solution. Absorbance at 340nm before reaction was measured using a microplate reader. After the mixture on the ELISA plate was allowed to react at 37℃for 30 minutes, the absorbance value of the mixture was again measured.
And (3) calculating a formula: [ (A1-A2) - (B1-B2) ]/(A1-A2) x100%
A1: initial absorbance of blank wells; a2: absorbance for 30min in blank wells; b1: initial absorbance of the sample wells; b2: absorbance after 30min of reaction in the sample wells.
Example 2
Separation and purification of queen bee embryo active peptide composition with ACE inhibition effect:
the freeze-dried powder of the enzymolysis product prepared by the enzymolysis process with the highest ACE inhibition rate in the embodiment 1 is used as a raw material to purify ACE inhibitory peptide, and the method comprises the following steps:
(1) Gel chromatographic column separation
Dissolving the freeze-dried powder with pure water to prepare a solution with the concentration of 2g/mL, and filtering the solution with a filter membrane with the concentration of 0.22 mu m for standby. The Sephadex G-25 gel chromatographic column is selected, the specification phi of the chromatographic column is 1.5 multiplied by 60cm, the loading amount is 0.5mL, water is taken as a mobile phase, the flow rate is 0.5mL/min, effluent is detected at 280nm, a chromatographic peak F1, a chromatographic peak F2 and a chromatographic peak F3 are known from a spectrogram (shown in figure 2), the corresponding fractions of each chromatographic peak are collected, the ACE inhibitory activity of each fraction is detected (shown in figure 3), the inhibitory activity of the corresponding fraction of the chromatographic peak F3 is optimal, the steps are repeated continuously, the corresponding fraction of the chromatographic peak F3 is collected, and the concentration and freeze drying are carried out.
(2) Ion exchange chromatography column separation
Separating the corresponding fractions of the chromatographic peak F3 by using a HiTrap DEAE-FF anion exchange chromatographic column (25 mm multiplied by 150 mm), taking a phosphate buffer solution with pH of 7.0 and 0.01mol/L as a mobile phase A, taking a phosphate buffer solution with pH of 1mol/L as a mobile phase B and 0.1mol/L as a mobile phase B, eluting for 20min at the elution condition of 100% A, gradually changing to 80% A within 40min, eluting for 20min with 100% B, detecting at 280nm, obtaining the corresponding fractions of the chromatographic peak F3-1, the chromatographic peak F3-2, the chromatographic peak F3-3 and the chromatographic peak F3-4 by a spectrogram (shown in figure 4), collecting the corresponding fractions of each chromatographic peak, detecting ACE inhibition activity of each fraction (shown in figure 5), continuously repeating the steps, collecting the corresponding fractions of the chromatographic peak F3-4, concentrating, and drying.
(3) Semi-preparative reversed phase column chromatographic separation
The fractions corresponding to the chromatographic peak F3-4 were further separated using a semi-preparative Shim-pack prep-ods C18 column (20X 250mm,15 μm) at a loading of 100. Mu.L with ultrapure water (0.1% trifluoroacetic acid) as mobile phase A and acetonitrile (0.1% trifluoroacetic acid) as mobile phase B at a flow rate of 3mL/min, and the gradient elution procedure was: (1) 0-5min:100% B, (2)5-25 min:50% A,50% B, (3) 25-32min:100% A. Detection at 280nm, the chromatographic peak F3-4-1, the chromatographic peak F3-4-2 and the chromatographic peak F3-4-3 are known from the spectrogram (shown in figure 6), the corresponding fractions of each chromatographic peak are collected, ACE inhibitory activity of each fraction is detected (shown in figure 7), the ACE inhibitory activity of the corresponding fraction of the chromatographic peak F3-4-3 is optimal, the steps are repeated continuously, the corresponding fraction of the chromatographic peak F3-4-3 is collected, concentrated and freeze-dried.
Example 3
Mass spectrometry identification of queen bee embryo active peptide composition with ACE inhibition effect:
(1) Sample pretreatment
With 50mM NH 4 HCO 3 A sample of F3-4-3 powder was dissolved, and then DTT solution was added to a final concentration of 10mmol/L, followed by reduction in a 56℃water bath for 1 hour. IAM solution was added to give a final concentration of 55mmol/L and reacted in the dark for 40min. Desalting was performed using a self-packed desalting column, and the solvent was evaporated in a vacuum centrifugal concentrator at 45 ℃.
(2) Capillary liquid chromatography conditions
Pre-column: 300 μm i.d. times. 5mm,Packed with Acclaim PepMap RPLC C18,5 μm,10nm. Analytical column: 150 μm i.d.× 150mm,Packed with Acclaim PepMap RPLC C18,1.9 μm,10nm. Mobile phase a:0.1% formic acid; mobile phase B:0.1% formic acid, 80% acn; flow rate: 600nL/min; liquid chromatography gradient: 66min.
(3) Mass spectrometry conditions
Primary mass spectrometry parameters: resolution:70000; AGCtarget:3e6; maximumIT:100ms; scanrange:100-1500m/z. Secondary mass spectrometry parameters: resolution:75000; AGCtarget:1e5; maximumIT:50ms; topN:20, a step of; NCE/steppence: 28.
collecting raw data for mass spectrometry results may generate a total ion flow chromatogram (as shown in fig. 8). Mass spectrometry results showed that the most abundant peptides were: IDFDF, VPIFD, FDYDFG, KNYPF and KPYPDWS, etc. (as shown in Table 1), it can be seen that the major components of ACE inhibitory peptide compositions are small peptides of less than 1000Da, and the ratio of these five peptides is more than 40% of the total peptides. The results of primary and secondary mass spectrometry experiments on these small peptides are shown in FIGS. 9-18.
Table 1 mass spectrum data of five small peptides
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A queen bee embryo active peptide composition with an ACE inhibition effect, which is characterized by comprising at least one of 5 small molecule peptides;
the amino acid sequences of the 5 small molecule peptides are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
2. The method for preparing a queen bee embryo active peptide composition with ACE inhibition effect as claimed in claim 1, which is characterized by comprising the following steps:
(1) Pretreatment: mixing queen bee embryo with water, pulping to obtain a prefabricated liquid A;
(2) Alkali dissolution: mixing the prefabricated liquid A with water for dilution, regulating the pH value to 7.0-9.0, stirring, centrifuging and taking supernatant fluid to obtain prefabricated liquid B;
(3) Enzymolysis: mixing the prefabricated liquid B with protease for enzymolysis, inactivating, centrifuging, and taking supernatant to obtain prefabricated liquid C;
(4) Purifying: purifying the prefabricated liquid C by gel filtration chromatography, anion exchange chromatography and reversed-phase high performance liquid chromatography in sequence to obtain the queen bee embryo active peptide composition with ACE inhibition effect.
3. The method for preparing an ACE inhibitory active peptide composition of queen bee embryo according to claim 2, wherein in the step (1), the mixing mass ratio of the queen bee embryo to water is 1:2-3, and the solid content in the pre-preparation solution a is 5-7%.
4. The method for preparing a queen bee embryo active peptide composition with an ACE inhibitory effect according to claim 2, wherein in the step (2), the mixing mass ratio of the prefabricated liquid A to water is 1:3-5;
the component for regulating the pH value is edible alkali;
the stirring speed is 100-300 rpm;
the stirring time is 1-3 h;
the centrifugal force of the centrifugation is 3000-10000 g;
the temperature of the centrifugation is 5-20 ℃;
the centrifugation time is 5-10 min.
5. The method for producing an ACE inhibitory active peptide composition for queen bee of claim 2, wherein in step (3), the protease is an edible neutral protease;
the addition amount of the protease is 6000-9000U/g protein;
the enzymolysis temperature is 40-50 ℃;
the enzymolysis time is 2-4 hours;
the centrifugal force of the centrifugation is 3000-10000 g;
the temperature of the centrifugation is 5-20 ℃;
the centrifugation time is 5-10 min.
6. The method for preparing an ACE inhibitory active peptide composition of queen bee embryo according to claim 2, wherein in the step (4), the condition of the gel filtration chromatography is:
separating by Sephadex G-25 column chromatography, eluting with water at a flow rate of 0.4-0.6 mL/min, detecting absorbance at 250-300 nm, collecting the eluted component, collecting the component with highest ACE inhibition rate, and freeze drying.
7. The method for preparing an ACE inhibitory active peptide composition of queen bee embryo according to claim 2, wherein in the step (4), the conditions of the anion exchange chromatography are as follows:
purifying by HiTrap DEAE-FF anion exchange;
phosphate buffer solution with the mobile phase A liquid of 0.01-0.02 mol/L, pH-8;
the mobile phase B liquid is phosphate buffer solution containing 0.01-0.02 mol/L, pH-8 mol/L NaCl of 1.0-2.0 mol/L;
the loading solution is the component obtained in claim 7;
the elution condition is that the solution A with the concentration of 100 percent is eluted for 15 to 25 minutes, then the solution A is gradually changed to the solution B with the concentration of 100 percent within 55 to 65 minutes, and finally the solution B with the concentration of 100 percent is eluted for 15 to 25 minutes, and the elution flow rate is 0.5 to 1.5mL/min;
detecting absorbance at 250-300 nm to collect the eluted component, collecting the component with highest ACE inhibition rate, freeze drying.
8. The method for preparing an ACE inhibitory active peptide composition of queen bee embryo according to claim 2, wherein in the step (4), the conditions for the reversed-phase high performance liquid chromatography purification are as follows:
adopting a C18 column;
the mobile phase A is ultrapure water containing 0.1-0.2 vt% of trifluoroacetic acid;
the mobile phase B is acetonitrile containing 0.1-0.2 vt% of trifluoroacetic acid;
the flow rate is 2-4 mL/min;
the loading solution is the component obtained in claim 8;
the gradient elution procedure was: 0 to 5min:100% of B, 5-25min:50% of A,50% of B, 25-40 min:100% A;
detecting absorbance at 250-300 nm to collect the eluted component, collecting the component with highest ACE inhibition rate, freeze drying.
9. The use of the active peptide composition of queen bee embryo with ACE inhibition effect as defined in claim 1 in the preparation of ACE inhibition drugs.
10. The use of the active peptide composition of queen bee embryo with ACE inhibition effect as claimed in claim 1 for preparing health food for assisting blood pressure reduction.
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