CN117568432A - Preparation and application of novel cooking wine dreg ACE (angiotensin converting enzyme) inhibitory peptide - Google Patents
Preparation and application of novel cooking wine dreg ACE (angiotensin converting enzyme) inhibitory peptide Download PDFInfo
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- CN117568432A CN117568432A CN202410037192.7A CN202410037192A CN117568432A CN 117568432 A CN117568432 A CN 117568432A CN 202410037192 A CN202410037192 A CN 202410037192A CN 117568432 A CN117568432 A CN 117568432A
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- inhibitory peptide
- ace inhibitory
- enzymolysis
- cooking wine
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Classifications
-
- 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
- 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
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1019—Tetrapeptides with the first amino acid being basic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/485—Exopeptidases (3.4.11-3.4.19)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/17—Metallocarboxypeptidases (3.4.17)
- C12Y304/17023—Angiotensin-converting enzyme 2 (3.4.17.23)
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Public Health (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Biophysics (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Mycology (AREA)
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Abstract
The invention provides a novel cooking wine lees ACE inhibitory peptide, which belongs to the field of biological peptide, wherein cooking wine lees are diluted by water and subjected to enzymolysis by complex enzyme to obtain enzymolysis liquid; heating the enzymolysis liquid in a water bath to inactivate the compound enzyme, centrifuging and taking enzymolysis supernatant; mixing the enzymolysis supernatant with ethanol for alcohol precipitation, centrifuging, and taking an alcohol precipitation supernatant; separating and purifying the alcohol precipitation supernatant to obtain ACE inhibitory peptide; the amino acid sequence of the ACE inhibitory peptide is Arg-Glu-Glu-Phe. The invention can be applied to preparing antihypertensive drugs or health care products. The invention provides preparation and application of novel cooking wine lees ACE inhibitory peptide, solves the technical problem that the cooking wine lees are difficult to extract ACE inhibitory peptide, and increases the added value of the cooking wine lees.
Description
Technical Field
The invention relates to the technical field of biological peptides, in particular to preparation and application of novel cooking wine lees ACE inhibitory peptide.
Background
Hypertension is a clinical syndrome characterized by elevated arterial systolic or diastolic blood pressure. The causes of hypertension are numerous, and among them, the regulation of Angiotensin Converting Enzyme (ACE) in the body is considered to be one of the most important causes. Studies have shown that blood pressure is normal or not, and is closely related to the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS), whereas Angiotensin Converting Enzyme (ACE) plays an important role in both systems. ACE is a Zn-containing material 2+ The carboxypeptidase has a relative molecular mass of 120000-150000, is widely existing in human tissues and plasma, and mainly exists in vascular endothelial cells. In human physiological activities, renin can interpret the raw angiotensin water in plasma to release decapeptide angiotensin I, and ACE can cleave off a dipeptide group at the C-terminal end of inactive angiotensin I to form effective vasoconstrictor octapeptide-angiotensin II; at the same time, the effective vasodilator nine peptide-bradykinin can be deactivated, thereby achieving the effect of raising blood pressure.
ACE inhibitory peptides are polypeptide substances with ACE inhibitory activity, and achieve the effect of reducing blood pressure by inhibiting ACE activity. They have greater affinity for the ACE active region than angiotensin i and bradykinin and are difficult to release once bound to the ACE active region, thus impeding the two biochemical processes of ACE catalyzing the hydrolysis of angiotensin i to angiotensin ii and bradykinin to inactive fragments, which act to reduce blood pressure.
ACE inhibitors with antihypertensive effect are currently in wide clinical use. Typical chemically synthesized ACE inhibitors are captopril, perindopril, lisinopril, enalapril and the like, but clinical findings of such drugs cause adverse reactions such as elevated blood potassium levels, dysgeusia, cough, rash and the like. Compared with synthetic ACE inhibitors, the natural ACE inhibitory peptide has better food-borne property, better safety and stability and smaller side effect, and is therefore widely paid attention to.
The cooking wine lees are main byproducts obtained by fermentation and distillation in the process of brewing the cooking wine, and theoretically, due to abundant proteins in the brewing raw materials and enzymatic hydrolysis and microbial degradation in the brewing process, more difficult volatile polypeptides remain in the cooking wine lees. If the cooking wine lees are improperly treated, putrefactive fermentation and mildew are easily caused, and various toxic substances such as free acid, fusel oil, aldehyde and the like are generated. The research shows that the main way of comprehensively utilizing the wine and the vinegar residues is as feed and fertilizer at present, but the additional value is extremely low and the nutrition components are not analyzed and utilized, so that a new utilization mode with high additional value is necessary to be searched. Because the polypeptide content in the cooking wine lees is more, the ACE inhibitory peptide extracted from the polypeptide in the cooking wine lees can not only improve the added value of the cooking wine lees, but also provide a novel ACE inhibitory peptide.
The protein content in the wine lees can reach 10% -40%, but the separation, purification and utilization become difficult because a large number of metabolic inhibitors of microorganisms remain in the wine lees and the fiber structures therein are difficult to decompose. Therefore, the problem of difficult extraction of ACE inhibitory peptides in cooking wine lees needs to be solved.
Disclosure of Invention
In view of the above, the invention provides a preparation and application of novel cooking wine lees ACE inhibitory peptide, which can solve the technical problem that the cooking wine lees is difficult to extract ACE inhibitory peptide.
In order to achieve the above purpose, the invention provides a preparation method of novel cooking wine lees ACE inhibitory peptide, which comprises the steps of diluting cooking wine lees with water, and carrying out enzymolysis by complex enzyme to obtain enzymolysis liquid; heating the enzymolysis liquid in a water bath to inactivate the compound enzyme, centrifuging and taking enzymolysis supernatant; mixing the enzymolysis supernatant with ethanol for alcohol precipitation, centrifuging, and taking an alcohol precipitation supernatant; separating and purifying the alcohol precipitation supernatant to obtain ACE inhibitory peptide; the amino acid sequence of the ACE inhibitory peptide is Arg-Glu-Glu-Phe.
According to the preparation method of the novel cooking wine lees ACE inhibitory peptide, the polypeptide with ACE inhibitory activity can be obtained more by carrying out enzymolysis on the cooking wine lees with the complex enzyme, and the polypeptide with ACE inhibitory activity is purified by combining with a separation and purification technology after alcohol precipitation. The novel ACE inhibitory peptide has an amino acid sequence of Arg-Glu-Glu-Phe (REEF), has an ACE inhibition rate as high as 80.52 percent, and has better safety and stability and less side effect compared with the synthesized ACE inhibitor in the prior art because of being a natural ACE inhibitory peptide.
Optionally, diluting the cooking wine lees with water, adding 0.4% -0.6% of the compound enzyme for enzymolysis for 1.5-2.5 hours, wherein the enzymolysis condition is that the temperature is 45-55 ℃ and the PH is 6.0-7.0; mixing the enzymolysis supernatant with ethanol, precipitating with ethanol for 8-10 h, and keeping the alcohol precipitation temperature at 4-10 ℃; the separation and purification steps comprise organic roll type membrane separation and purification, ultrafiltration membrane separation and purification and nanofiltration membrane separation and purification to obtain ACE inhibitory peptide, wherein the ACE inhibitory peptide is preserved by spray drying and can also be preserved by other modes.
Optionally, the weight ratio of the complex enzyme to the papain to the bromelain is 2:1.
optionally, the wine lees is diluted to 10% of the raw material concentration.
Optionally, the volume ratio of ethanol to the supernatant of the enzymolysis liquid during the alcohol precipitation is 1:5, the ethanol is ethanol with the concentration of 50-80%.
Optionally, the organic roll type membrane separation and purification is to sequentially filter the supernatant liquid obtained by alcohol precipitation by a 20kDa membrane and a 5000Da membrane, wherein the pressure during the 20kDa membrane filtration is 0.3 MPa, and the pressure during the 5000Da membrane filtration is 0.5 MPa.
Optionally, the ultrafiltration membrane separation and purification is carried out by filtering by using a 3000Da ultrafiltration membrane, introducing nitrogen after sealing, and the pressure of an ultrafiltration system is 0.20 MPa when the 3000Da ultrafiltration membrane is used for filtering.
Optionally, the nanofiltration membrane separation and purification is carried out by using a 150Da nanofiltration roll membrane and matching with conventional membrane filtration equipment to separate and purify, and small molecules and water are removed, so as to obtain nanofiltration membrane trapped fluid.
Optionally, the spray drying is to dry the nanofiltration membrane trapped fluid by using a spray dryer, wherein the air inlet temperature is 130 ℃, the sample injection speed is 10.0 mL/min, and the fan frequency is 40 Hz.
The invention aims to improve the added value of the cooking wine lees, and in order to achieve the purpose, the invention provides the following technical scheme that the novel material lees ACE inhibitory peptide as described in claim 1 is applied to preparation of antihypertensive drugs or health care products.
The novel cooking wine lees ACE inhibitory peptide REEF provided by the invention can be applied to preparation of antihypertensive drugs or health care products, has important significance in inhibiting hypertension, has great development value and wide application prospect, and greatly increases the added value of cooking wine lees.
The technical scheme of the invention at least comprises the following beneficial effects:
the ACE inhibitory peptide prepared by the preparation method of the novel cooking wine lees ACE inhibitory peptide has better ACE inhibitory rate, has better safety and stability and smaller side effect compared with the synthesized ACE inhibitory peptide in the prior art, and is suitable for industrialized amplified production.
Drawings
FIG. 1 shows ACE inhibition rates of different enzymolysis raw material samples in the invention;
FIG. 2 shows ACE inhibition rates of a compound enzyme and an organic roll type membrane two-stage separation and purification sample in the invention;
FIG. 3 shows ACE inhibition of organic roll-type membrane separation and purification, ultrafiltration, nanofiltration, spray drying and captopril samples in the present invention;
FIG. 4 is a HPLC chart of the nanofiltration retentate fraction of the present invention;
FIG. 5 is a mass spectrum of the monomer H1 of the present invention;
FIG. 6 is a flow chart of a preparation process according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to fig. 1 to 6 of the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which are obtained by a person skilled in the art based on the described embodiments of the invention, fall within the scope of protection of the invention.
The invention provides a preparation method of novel cooking wine lees ACE inhibitory peptide, which comprises the steps of diluting cooking wine lees with water, adding 0.4% -0.6% of compound enzyme for enzymolysis for 1.5-2.5 h, wherein the enzymolysis condition is that the temperature is 45-55 ℃ and the PH is 6.0-7.0; heating in water bath to 80deg.C for inactivating for 30min, and collecting supernatant; mixing the supernatant of the enzymolysis liquid with ethanol, precipitating with ethanol for 8-10 h, keeping the temperature of the ethanol precipitation at 4-10 ℃, and taking the supernatant of the ethanol precipitation; and (3) sequentially carrying out organic roll type membrane separation and purification, ultrafiltration membrane separation and purification and nanofiltration membrane separation and purification on the alcohol precipitation supernatant to obtain ACE inhibitory peptide.
Example 1
Diluting with distilled water to 10% of the raw material concentration, and adding 0.5% of complex enzyme (the complex enzyme is composed of papain and bromelain in a weight ratio of 2:1) for enzymolysis for 2h, wherein the enzymolysis condition is that the temperature is 50 ℃, and the pH is 6.5; heating in water bath to 80deg.C for inactivating 30min, centrifuging at 4000 rpm for 10min, and collecting supernatant; mixing the supernatant of the enzymolysis liquid with ethanol, and precipitating for 9h, wherein the volume ratio of the ethanol to the supernatant of the enzymolysis liquid is 1:5, the concentration of ethanol is 50-80%, the temperature of ethanol precipitation is kept at 7 ℃, and centrifugation is carried out at 4000 rpm for 10min to obtain supernatant of ethanol precipitation;
the ethanol precipitation supernatant is subjected to two-stage separation and purification by using novel solid-liquid separation membrane equipment (patent number CN 215517229U) in the biological industry and an organic roll membrane which is matched with a roll membrane to be sequentially filtered by using a 20kDa membrane under the pressure of 0.3 MPa and filtered by using a 5000Da membrane under the pressure of 0.5 MPa, so as to obtain corresponding filtrate.
Filtering the filtrate at 7deg.C with 3000Da ultrafiltration membrane, sealing, introducing nitrogen, and regulating the pressure of ultrafiltration system to 0.20 MPa to obtain corresponding ultrafiltration membrane filtrate.
And separating and purifying the filtrate by using a 150Da nanofiltration roll type membrane at normal temperature and matching with conventional membrane filtration equipment to remove small molecules and water so as to obtain nanofiltration membrane trapped fluid.
According to the invention, the composite enzyme is adopted to carry out enzymolysis on the cooking wine lees, so that the fiber structure which is difficult to decompose in the lees can be decomposed, and after alcohol precipitation, the problem that ACE inhibitory peptide is difficult to separate and purify is solved by combining with multistage membrane filtration, and the purity and yield of the ACE inhibitory peptide are improved.
Example 2
Diluting the cooking wine lees with distilled water to 10% of the concentration of the raw materials, and adding 0.4% of compound enzyme (the compound enzyme is composed of papain and bromelain in a weight ratio of 2:1) for enzymolysis for 1.5h, wherein the enzymolysis condition is that the temperature is 45 ℃ and the PH is 6.0; heating in water bath to 80deg.C for inactivating 30min, centrifuging at 4000 rpm for 10min, and collecting supernatant; mixing the supernatant of the enzymolysis liquid with ethanol, and precipitating for 8 hours, wherein the volume ratio of the ethanol to the supernatant of the enzymolysis liquid is 1:5, the concentration of ethanol is 50-80%, the temperature of ethanol precipitation is kept at 4 ℃, and centrifugation is carried out at 4000 rpm for 10min to obtain supernatant of ethanol precipitation;
the ethanol precipitation supernatant is subjected to two-stage separation and purification by using novel solid-liquid separation membrane equipment (patent number CN 215517229U) in the biological industry and an organic roll membrane which is matched with a roll membrane to be sequentially filtered by using a 20kDa membrane under the pressure of 0.3 MPa and filtered by using a 5000Da membrane under the pressure of 0.5 MPa, so as to obtain corresponding filtrate.
Filtering the filtrate at 4deg.C with 3000Da ultrafiltration membrane, sealing, introducing nitrogen, and regulating the pressure of ultrafiltration system to 0.20 MPa to obtain corresponding ultrafiltration membrane filtrate.
And separating and purifying the filtrate by using a 150Da nanofiltration roll type membrane at normal temperature and matching with conventional membrane filtration equipment to remove small molecules and water so as to obtain nanofiltration membrane trapped fluid.
And using a spray dryer to obtain a spray-dried sample, namely ACE inhibitory peptide, at the air inlet temperature of 130 ℃, the sample injection speed of 10.0 mL/min and the fan frequency of 40 Hz.
Example 3
Diluting with distilled water to 10% of the raw material concentration, and adding 0.6% of complex enzyme (the complex enzyme is composed of papain and bromelain in a weight ratio of 2:1) for enzymolysis for 2.5h, wherein the enzymolysis condition is that the temperature is 55deg.C and the pH is 7.0; heating in water bath to 80deg.C for inactivating 30min, centrifuging at 4000 rpm for 10min, and collecting supernatant; mixing the supernatant of the enzymolysis liquid with ethanol, and precipitating for 10 hours, wherein the volume ratio of the ethanol to the supernatant of the enzymolysis liquid is 1:5, the concentration of ethanol is 50-80%, the temperature of ethanol precipitation is kept at 10 ℃, and centrifugation is carried out at 4000 rpm for 10min to obtain supernatant of ethanol precipitation;
the ethanol precipitation supernatant is subjected to two-stage separation and purification by using novel solid-liquid separation membrane equipment (patent number CN 215517229U) in the biological industry and an organic roll membrane which is matched with a roll membrane to be sequentially filtered by using a 20kDa membrane under the pressure of 0.3 MPa and filtered by using a 5000Da membrane under the pressure of 0.5 MPa, so as to obtain corresponding filtrate.
Filtering the filtrate at 10deg.C with 3000Da ultrafiltration membrane, sealing, introducing nitrogen, and regulating the pressure of ultrafiltration system to 0.20 MPa to obtain corresponding ultrafiltration membrane filtrate.
And separating and purifying the filtrate by using a 150Da nanofiltration roll type membrane at normal temperature and matching with conventional membrane filtration equipment to remove small molecules and water so as to obtain nanofiltration membrane trapped fluid.
And using a spray dryer to obtain a spray-dried sample, namely ACE inhibitory peptide, at the air inlet temperature of 130 ℃, the sample injection speed of 10.0 mL/min and the fan frequency of 40 Hz.
Example 4
Diluting with distilled water to 10% of the raw material concentration, and adding 0.5% of complex enzyme (the complex enzyme is composed of papain and bromelain in a weight ratio of 2:1) for enzymolysis for 2h, wherein the enzymolysis condition is that the temperature is 50 ℃, and the pH is 6.5; heating in water bath to 80deg.C for inactivating 30min, centrifuging at 4000 rpm for 10min, and collecting supernatant; mixing the supernatant of the enzymolysis liquid with ethanol, and precipitating for 9h, wherein the volume ratio of the ethanol to the supernatant of the enzymolysis liquid is 1:5, the concentration of ethanol is 50-80%, the temperature of ethanol precipitation is kept at 7 ℃, and centrifugation is carried out at 4000 rpm for 10min to obtain supernatant of ethanol precipitation;
the ethanol precipitation supernatant is subjected to two-stage separation and purification by using novel solid-liquid separation membrane equipment (patent number CN 215517229U) in the biological industry and an organic roll membrane which is matched with a roll membrane to be sequentially filtered by using a 20kDa membrane under the pressure of 0.3 MPa and filtered by using a 5000Da membrane under the pressure of 0.5 MPa, so as to obtain corresponding filtrate.
Filtering the filtrate at 7deg.C with 3000Da ultrafiltration membrane, sealing, introducing nitrogen, and regulating the pressure of ultrafiltration system to 0.20 MPa to obtain corresponding ultrafiltration membrane filtrate.
And separating and purifying the filtrate by using a 150Da nanofiltration roll type membrane at normal temperature and matching with conventional membrane filtration equipment to remove small molecules and water so as to obtain nanofiltration membrane trapped fluid.
And using a spray dryer to obtain a spray-dried sample, namely ACE inhibitory peptide, at the air inlet temperature of 130 ℃, the sample injection speed of 10.0 mL/min and the fan frequency of 40 Hz.
Comparative example 1
Diluting with distilled water to 10% of the raw material concentration, adding bromelain 0.5%, and performing enzymolysis for 2 hr at 50deg.C and pH of 6.5; heating in water bath to 80deg.C for inactivating 30min, centrifuging at 4000 rpm for 10min, and collecting supernatant; mixing the supernatant of the enzymolysis liquid with ethanol, and precipitating for 9h, wherein the volume ratio of the ethanol to the supernatant of the enzymolysis liquid is 1:5, the concentration of ethanol is 50-80%, the temperature of ethanol precipitation is kept at 7 ℃, and the ethanol precipitation supernatant is obtained by centrifugation at 4000 rpm for 10 min.
Comparative example 2
Diluting with distilled water to 10% of the raw material concentration, adding 0.5% papain, and performing enzymolysis for 2 hr at 50deg.C and pH of 6.5; heating in water bath to 80deg.C for inactivating 30min, centrifuging at 4000 rpm for 10min, and collecting supernatant; mixing the supernatant of the enzymolysis liquid with ethanol, and precipitating for 9h, wherein the volume ratio of the ethanol to the supernatant of the enzymolysis liquid is 1:5, the concentration of ethanol is 50-80%, the temperature of ethanol precipitation is kept at 7 ℃, and the ethanol precipitation supernatant is obtained by centrifugation at 4000 rpm for 10 min.
Comparative example 3
Diluting with distilled water to 10% of the raw material concentration, and adding 0.5% of complex enzyme (the complex enzyme is composed of papain and bromelain in a weight ratio of 2:1) for enzymolysis for 2h, wherein the enzymolysis condition is that the temperature is 50 ℃, and the pH is 6.5; heating in water bath to 80deg.C for inactivating 30min, centrifuging at 4000 rpm for 10min, and collecting supernatant; mixing the supernatant of the enzymolysis liquid with ethanol, and precipitating for 9h, wherein the volume ratio of the ethanol to the supernatant of the enzymolysis liquid is 1:5, the concentration of ethanol is 50-80%, the temperature of ethanol precipitation is kept at 7 ℃, and the ethanol precipitation supernatant is obtained by centrifugation at 4000 rpm for 10 min.
Comparative example 4
Diluting with distilled water to 10% of the raw material concentration, and adding 0.5% of complex enzyme (the complex enzyme is composed of papain and bromelain in a weight ratio of 2:1) for enzymolysis for 2h, wherein the enzymolysis condition is that the temperature is 50 ℃, and the pH is 6.5; heating in water bath to 80deg.C for inactivating 30min, centrifuging at 4000 rpm for 10min, and collecting supernatant; mixing the supernatant of the enzymolysis liquid with ethanol, and precipitating for 9h, wherein the volume ratio of the ethanol to the supernatant of the enzymolysis liquid is 1:5, the concentration of ethanol is 50-80%, the temperature of ethanol precipitation is kept at 7 ℃, and centrifugation is carried out at 4000 rpm for 10min to obtain supernatant of ethanol precipitation;
the supernatant obtained by alcohol precipitation is filtered by a novel solid-liquid separation membrane device (patent number CN 215517229U) in the biological industry and a 20kDa membrane under the pressure of 0.3 MPa in sequence by matching with a roll membrane to obtain 20kDa filtrate.
Comparative example 5
Diluting with distilled water to 10% of the raw material concentration, and adding 0.5% of complex enzyme (the complex enzyme is composed of papain and bromelain in a weight ratio of 2:1) for enzymolysis for 2h, wherein the enzymolysis condition is that the temperature is 50 ℃, and the pH is 6.5; heating in water bath to 80deg.C for inactivating 30min, centrifuging at 4000 rpm for 10min, and collecting supernatant; mixing the supernatant of the enzymolysis liquid with ethanol, and precipitating for 9h, wherein the volume ratio of the ethanol to the supernatant of the enzymolysis liquid is 1:5, the concentration of ethanol is 50-80%, the temperature of ethanol precipitation is kept at 7 ℃, and centrifugation is carried out at 4000 rpm for 10min to obtain supernatant of ethanol precipitation;
the ethanol precipitation supernatant is subjected to two-stage separation and purification by using novel solid-liquid separation membrane equipment (patent number CN 215517229U) in the biological industry and an organic roll membrane which is matched with a roll membrane to be sequentially filtered by using a 20kDa membrane under the pressure of 0.3 MPa and a 5000Da membrane under the pressure of 0.5 MPa, so as to obtain 5kDa filtrate.
Comparative example 6
Diluting with distilled water to 10% of the raw material concentration, and adding 0.5% of complex enzyme (the complex enzyme is composed of papain and bromelain in a weight ratio of 2:1) for enzymolysis for 2h, wherein the enzymolysis condition is that the temperature is 50 ℃, and the pH is 6.5; heating in water bath to 80deg.C for inactivating 30min, centrifuging at 4000 rpm for 10min, and collecting supernatant; mixing the supernatant of the enzymolysis liquid with ethanol, and precipitating for 9h, wherein the volume ratio of the ethanol to the supernatant of the enzymolysis liquid is 1:5, the concentration of ethanol is 50-80%, the temperature of ethanol precipitation is kept at 7 ℃, and centrifugation is carried out at 4000 rpm for 10min to obtain supernatant of ethanol precipitation;
the ethanol precipitation supernatant is subjected to two-stage separation and purification by using novel solid-liquid separation membrane equipment (patent number CN 215517229U) in the biological industry and an organic roll membrane which is matched with a roll membrane to be sequentially filtered by using a 20kDa membrane under the pressure of 0.3 MPa and a 5000Da membrane under the pressure of 0.5 MPa, so as to obtain 5kDa filtrate.
Filtering the filtrate at 7deg.C with 3000Da ultrafiltration membrane, sealing, introducing nitrogen, and regulating the pressure of ultrafiltration system to 0.20 MPa to obtain ultrafiltration membrane filtrate.
In vitro ACE Activity assay
(1) Preparation of the reaction solution
10. Mu.L of ACE solution with the concentration of 0.2U/mL is added to a sample tube and a blank tube respectively, 10. Mu.L of sample is added to the sample tube, 10. Mu.L of buffer solution and 80. Mu.L of 1M HCl are added to the blank tube, the solution is pre-incubated for 5 min at 37 ℃, 30. Mu.L of HHT solution with the concentration of 6.5 mmol/mL is added, the reaction is carried out at 37 ℃ for 1 h, and finally 80. Mu.L of 1M HCl is added to stop the reaction, so that a reaction solution is obtained. 0.22 μm was filtered and used for HPLC analysis.
The method comprises the steps of carrying out sample tests in groups, wherein the first group tests the influence of different enzymolysis raw materials on ACE inhibitory activity, and the samples are respectively cooking wine lees raw materials, alcohol precipitation supernatant obtained by enzymolysis of bromelain, alcohol precipitation supernatant obtained by enzymolysis of papain and alcohol precipitation supernatant obtained by enzymolysis of complex enzyme, namely cooking wine lees, comparative example 1, comparative example 2 and comparative example 3, so as to obtain a graph 1; the second group tests the influence of two-stage differentiation of an organic roll film on ACE inhibition activity, wherein samples are alcohol precipitation supernatant, 20kDa filtrate and 5kDa filtrate obtained by enzymolysis of complex enzyme, namely comparative example 3, comparative example 4 and comparative example 5, and FIG. 2 is obtained; the third group tested the effect of ultrafiltration, nanofiltration and drying on ACE inhibitory activity and comparison with existing drugs, samples were 5KDa filtrate, ultrafiltration membrane filtrate, nanofiltration membrane retentate, spray dried ACE inhibitory peptide samples and chemically synthesized ACE inhibitors, i.e. comparative example 5, comparative example 6, example 1, example 4 and captopril, respectively, giving fig. 3. The above sample concentrations were all diluted to 1mg/mL.
(2) Chromatographic conditions
Chromatographic column: ECOSIL C18 (260 mm X4.6 mm,5 μm);
mobile phase and chromatographic conditions: acetonitrile: ultrapure water=25: 75 (containing 0.1% (v/v) TFA), flow rate 1 mL/min; detection wavelength: 228 nm; column temperature: 30 ℃; sample injection amount: 20. and [ mu ] L.
(3) Result calculation
Hippuryl-His-Leu (HIL) is used as a substrate to produce Hippuric Acid (HA) upon enzymatic hydrolysis, which is maximally absorbed at 228 and nm. Therefore, the inhibition ratio of the sample to ACE activity was evaluated by measuring the amount of HA produced by reverse-phase high performance liquid chromatography.
The calculation formula is as follows:
ACE inhibition ratio (%) = (a-B-A0)/(a-A0) ×100%
Wherein: a: peak area of hippuric acid in control group;
b: peak area of hippuric acid in the sample set;
a0: peak area of hippuric acid in blank tube.
(4) Analysis of results
As can be seen from fig. 1, the raw material of the cooking wine lees has low ACE inhibition rate, but the activity is improved after the enzymolysis treatment. Wherein the enzymolysis effect of the compound enzyme is optimal, and the inhibition rate of the enzymolysis product ACE is 29.73 percent. The inhibition rate of the papain and bromelain zymolytes ACE is about 13%, and the result shows that the protein contained in the distiller's grains has no good biological activity, and the active polypeptide in the distiller's grains can be exposed and exert the effect through the hydrolysis of enzymes.
As can be seen from fig. 2, the ACE inhibition rate of the polypeptide increases with two organic roll membrane separation and purification steps. It is known that after macromolecules are removed, the activity of the polypeptide in the complex enzyme enzymolysis liquid sample is improved to a certain extent, the 5000Da filtrate activity is 47.68%, and is improved by about 17% compared with the complex enzymolysis liquid.
As shown in FIG. 3, after separation and purification by an ultrafiltration cup, the ACE inhibitory activity of the polypeptide in the sample reaches 66.74%, and the activity of the 5000Da filtrate in the previous step is improved by about 10%. After the nano-filtration membrane removes small molecules, the ACE inhibitory activity of the polypeptide in the sample reaches 80.52 percent, and the ACE inhibitory activity is greatly improved. The ACE inhibition rate of the polypeptide in the sample is increased along with the ultrafiltration and nanofiltration steps. For the convenience of preservation, ACE inhibition peptides can be selected to be spray dried, but after spray drying, the ACE inhibition rate of polypeptides in a sample is reduced to 75.28 percent to some extent, and presumably, due to the influence of temperature, some active peptide structures can be damaged, so that the ACE inhibition rate is reduced, and the balance of drying efficiency and activity is comprehensively considered. Compared with captopril for ACE inhibition rate, the ACE inhibition peptide provided by the invention has a slightly lower inhibition rate than captopril ACE inhibition rate, but is food-borne ACE inhibition peptide, and has better safety and smaller side effect.
(5) ACE inhibitory peptide structural analysis
To further define the functional monomers of ACE antihypertensive peptides and their monomer structures, we selected the most active sample, i.e. nanofiltration membrane retentate, whose composition was analyzed using HPLC, and the major components thereof were sampled. Qualitative analysis was then performed using a UPLC-LTQ-Orbitrap-Velos Pro mass spectrometer, combined with mass spectrometry software Compound Discovery (CD) analytical identification.
HPLC conditions were as follows: detection wavelength a280, mobile phase a:0.1% (v/v) aqueous trifluoroacetic acid, mobile phase B:0.1% (v/v) acetonitrile trifluoroacetic acid solution; 0-20min, and the concentration of mobile phase B is 5% -30%;20-40min, the concentration of mobile phase B is 45% -70%;40-50min, mobile phase B concentration 90%.
The mass spectrometry conditions were as follows: mobile phase a:0.1% (v/v) aqueous formic acid, mobile phase B:0.1% (v/v) acetonitrile formate solution; separation conditions: 0-8min, mobile phase B concentration 5% -50%;8-10min, and the concentration of mobile phase B is 50-90%;10-12min, mobile phase B concentration 90%; the flow rate is 300 mu L/min; the sample volume was 10. Mu.L. Negative ion mode is selected, and the ion source is set: capillary temperature 380 ℃, source heater temperature 350 ℃, sheath gas flow rate 50arb, auxiliary gas flow rate 20arb, collision mode: CID, scan range 50-1000m/z.
The condition parameters involved in mass spectrometry are as follows: the parameter settings refer to default parameters in the CD3.1 Metabolic Chinese tutorial, search the database, and all metaolika metabolic pathways.
FIG. 4 is obtained according to the experiment.
As can be seen from FIG. 4, one of the main peaks in ACE inhibitory peptides was found to be the main substance, designated as H1. This peak was sampled and its structure was identified by mass spectrometry. After mass spectrum data are obtained, comparison is carried out through CD software, and good matching results are found in the data, so that FIG. 5 is obtained. As can be seen from FIG. 5, the structure of the H1 monomer was identified as Arg-Glu-Glu-Phe.
In conclusion, the high-activity ACE inhibitory peptide sample is obtained from the raw material of the cooking wine lees through enzymolysis and solvent extraction and combining the separation and purification steps of the coiled film and the ultrafiltration cup, the ACE inhibitory rate of the sample is 75% -80%, and the main monomer structure is analyzed to obtain the brand new ACE inhibitory peptide structure Arg-Glu-Glu-Phe, so that the ACE inhibitory peptide provided by the invention can greatly increase the added value of the raw material of the cooking wine lees when being used for preparing the antihypertensive drugs or health care products.
The foregoing is 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 and are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A preparation method of novel cooking wine lees ACE inhibitory peptide is characterized in that cooking wine lees are diluted by water and subjected to enzymolysis by complex enzyme to obtain enzymolysis liquid; heating the enzymolysis liquid in a water bath to inactivate the compound enzyme, centrifuging and taking enzymolysis supernatant; mixing the enzymolysis supernatant with ethanol for alcohol precipitation, centrifuging, and taking an alcohol precipitation supernatant; separating and purifying the alcohol precipitation supernatant to obtain ACE inhibitory peptide; the amino acid sequence of the ACE inhibitory peptide is Arg-Glu-Glu-Phe.
2. The method for preparing ACE inhibitory peptide of novel material vinasse according to claim 1, wherein the material vinasse is diluted by water, 0.4% -0.6% of the compound enzyme is added for enzymolysis for 1.5-2.5 hours, and the enzymolysis condition is that the temperature is 45-55 ℃ and the PH is 6.0-7.0; mixing the enzymolysis supernatant with ethanol, precipitating with ethanol for 8-10 h, and keeping the alcohol precipitation temperature at 4-10 ℃; the separation and purification steps comprise organic roll type membrane separation and purification, ultrafiltration membrane separation and purification and nanofiltration membrane separation and purification to obtain ACE inhibitory peptide, and the ACE inhibitory peptide is preserved by spray drying.
3. The method for preparing novel cooking wine lees ACE inhibiting peptide according to claim 2, wherein the complex enzymes are papain and bromelain, and the weight ratio of papain to bromelain is 2:1.
4. the method for producing a novel cooking wine lees ACE inhibiting peptide as claimed in claim 2, wherein the cooking wine lees are diluted 10 times with water.
5. The method for preparing novel cooking wine lees ACE inhibitory peptide according to claim 2, wherein the volume ratio of ethanol to the supernatant of the enzymolysis liquid during alcohol precipitation is 1:5, the concentration of the ethanol is 50% -80%.
6. The method for preparing the novel cooking wine lees ACE inhibitory peptide according to claim 2, wherein the organic roll type membrane separation and purification is characterized in that the alcohol precipitation supernatant is sequentially filtered by a 20kDa membrane and a 5000Da membrane, the pressure is 0.3 MPa during the filtration of the 20kDa membrane, and the pressure is 0.5 MPa during the filtration of the 5000Da membrane.
7. The method for preparing the novel cooking wine lees ACE inhibitory peptide, according to claim 2, wherein the ultrafiltration membrane separation and purification is carried out by using a 3000Da ultrafiltration membrane, nitrogen is introduced after sealing, and the ultrafiltration system pressure is 0.20 MPa during the 3000Da ultrafiltration membrane filtration.
8. The method for preparing the novel cooking wine lees ACE inhibitory peptide, according to claim 2, wherein the nanofiltration membrane separation and purification is carried out by using a 150Da nanofiltration roll membrane to remove small molecules and water, so as to obtain nanofiltration membrane trapped fluid.
9. The method for producing ACE inhibitory peptides of distillers' grains according to claim 8, wherein the spray drying is to dry the nanofiltration membrane retentate with a spray dryer, the inlet air temperature is 130 ℃, the sample injection speed is 10.0 mL/min, and the fan frequency is 40 Hz.
10. A novel distillers grains ACE inhibitory peptide according to claim 1 for use in preparation of a medicament or health product for lowering hypertension.
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| MINGZHE MA ET AL.: "Revealing the Sequence Characteristics and Molecular Mechanisms of ACE Inhibitory Peptides by Comprehensive Characterization of 160, 000 Tetrapeptides", FOODS, vol. 12, no. 8, 7 April 2023 (2023-04-07), pages 1573 * |
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