CN114621993B - Soybean peptide and soybean oligopeptide with blood lipid reducing effect, and preparation method and application thereof - Google Patents
Soybean peptide and soybean oligopeptide with blood lipid reducing effect, and preparation method and application thereof Download PDFInfo
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- CN114621993B CN114621993B CN202210475808.XA CN202210475808A CN114621993B CN 114621993 B CN114621993 B CN 114621993B CN 202210475808 A CN202210475808 A CN 202210475808A CN 114621993 B CN114621993 B CN 114621993B
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Classifications
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- 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
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- 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
-
- 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
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- 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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- 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
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- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- Obesity (AREA)
- General Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biophysics (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides soybean peptide and soybean oligopeptide with a blood lipid reducing effect, and a preparation method and application thereof, and relates to the technical field of active peptides. The invention takes the isolated soy protein as the raw material, and the sensitized protein and the anti-nutritional factors in the soy protein can be removed by processing through a special process, thereby obviously improving the utilization rate and the action effect of the soy protein. The invention also separates, purifies and identifies two oligopeptides with hyperlipidemia activity from soybean peptide, the amino acid sequences are shown as SEQ ID NO.1 and SEQ ID NO.2, and the invention has obvious blood lipid reducing effect through the verification of examples.
Description
Technical Field
The invention belongs to the technical field of active peptides, and particularly relates to a soybean peptide and a soybean oligopeptide with a blood lipid reducing effect, and a preparation method and application thereof.
Background
Hyperlipidemia is a disease in which one or more lipids in serum or plasma are higher than normal due to abnormal fat metabolism or operation, and is generally represented by hypercholesterolemia, hypertriglyceridemia, or a mixed type of both (mixed hyperlipidemia). A large number of clinical data researches show that the hyperlipidemia is a primary risk factor of atherosclerosis, has close correlation with the incidence rate of coronary heart disease, cardiovascular and cerebrovascular diseases and other diseases, seriously endangers the life and health of human beings, however, along with the change of dietary structures of residents, the incidence of the hyperlipidemia shows rising and younger trend, and the prevention and treatment research of the hyperlipidemia is also receiving more and more attention.
At present, drugs such as fibrates, statins, nicotinic acids and the like are often used for preventing and treating the hyperlipidemia clinically, so that the medicine has good clinical effects; however, these drugs also have some side effects, such as long-term administration of statin drugs, which can cause liver damage, and blocked muscle synthesis; the fibrate can cause gastrointestinal tract function injury, kidney function injury and other problems; the hydrochloric acid medicine can cause allergic reaction of patients taking the medicine and aggravate the problems of rising of blood sugar and the like caused by insulin resistance. Thus, there is an urgent need for a new drug that is safer and more effective and does not cause side effects.
Disclosure of Invention
In view of the above, the present invention aims at a soybean peptide and a soybean oligopeptide with blood lipid reducing effect, and a preparation method and application thereof, wherein the soybean peptide and the soybean oligopeptide have blood lipid reducing effect and are non-sensitized.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of soybean peptide with blood lipid reducing effect, which comprises the following steps: (1) Sequentially carrying out colloid mill and homogenization treatment on an alkaline solution of the soybean protein isolate, and carrying out heat preservation on the obtained homogenate at 55 ℃ to obtain heat preservation liquid;
(2) Regulating the pH value of the heat preservation solution to a proper pH value of alkaline protease, sequentially carrying out enzymolysis by using the alkaline protease, neutral protease and flavourzyme, and collecting components with the molecular weight less than or equal to 1kDa after enzyme deactivation of the enzymolysis solution to obtain the soybean peptide.
Preferably, the preparation method of the isolated soy protein in the step (1) comprises the following steps: extracting soybean meal with alkali solution under stirring to obtain alkali extract, centrifuging, and collecting supernatant; and (3) carrying out acid precipitation on the supernatant, centrifuging, and collecting precipitate to obtain the soybean protein isolate.
Preferably, the preparation method of the alkaline solution of the isolated soy protein in the step (1) comprises the following steps: deionized water is heated to 50 ℃, and is mixed with the soy protein isolate after the pH value is adjusted to 10.0 by using alkaline solution.
Preferably, the treatment time of the colloid mill in the step (1) is 1.5h;
the pressure of the homogenizing treatment is 25MPa, and the time is 2h.
Preferably, in the step (2), after the pH value of the heat preservation solution is adjusted to 10.0, the heat preservation solution is mixed with alkaline protease for first enzymolysis to obtain a first enzymolysis solution;
when the pH value of the first enzymolysis liquid is 7.0-7.5, mixing the first enzymolysis liquid with neutral protease for second enzymolysis to obtain a second enzymolysis liquid; the second enzymolysis time is 4 hours;
and mixing the second enzymolysis liquid with flavourzyme for third enzymolysis, wherein the time of the third enzymolysis is 2 hours.
Preferably, in the step (2), the enzyme-deactivated enzymolysis solution is cooled to room temperature, and then passes through a 30000Da ceramic membrane, collects the membrane-mounted solution, passes through a 1kDa organic membrane, and collects the membrane-mounted solution.
The invention also provides the soybean peptide obtained by the preparation method.
The invention also provides a soybean oligopeptide separated and identified from the soybean peptide, wherein the soybean oligopeptide comprises P6-1 and P6-2, the amino acid of the P6-1 is shown as SEQ ID NO.1, and the amino acid of the P6-2 is shown as SEQ ID NO. 2.
The invention also provides application of the soybean peptide or the soybean oligopeptide in preparing a medicine for reducing blood fat or a health-care product for assisting in treating the blood fat.
The invention also provides a medicine for reducing blood fat or a health care product for assisting in treating the blood fat, which takes the soybean peptide or the soybean oligopeptide as an active ingredient.
The beneficial effects are that: the invention provides a preparation method of soybean peptide with blood lipid reducing effect, which takes soybean protein isolate as raw material, and the soybean peptide is treated by a special process, so that sensitization protein and anti-nutritional factors in the soybean protein can be removed, thereby remarkably improving the utilization rate and the action effect of the soybean protein. The invention also separates, purifies and identifies two oligopeptides with hyperlipidemia activity from soybean peptide, the amino acid sequences are shown as SEQ ID NO.1 and SEQ ID NO.2, and the invention has obvious blood lipid reducing effect through the verification of examples.
Drawings
FIG. 1 is a graph showing the efficacy of soybean peptide samples in reducing blood lipid;
FIG. 2 is a soybean peptide gel chromatogram;
FIG. 3 is a liquid chromatogram of soybean peptide fraction 3;
FIG. 4 is a secondary mass spectrum of soybean peptide P6-1;
FIG. 5 is a second-order mass spectrum of soybean peptide P6-2;
FIG. 6 is a graph showing typical intensity of vascular staining of the tail of zebra fish after soybean peptide treatment of example 1;
FIG. 7 is a typical graph of blood vessel staining intensity of the tail of zebra fish after sample treatment of soybean peptide after gel chromatography separation and purification;
FIG. 8 is a typical graph of blood vessel staining intensity of the tail of zebra fish after sample treatment of soybean peptide by high performance liquid phase separation and purification;
FIG. 9 is a graph showing typical blood vessel staining intensity of the tail of zebra fish after treatment with soybean peptide P6-1 and soybean peptide P6-2.
Detailed Description
The invention provides a preparation method of soybean peptide with blood lipid reducing effect, which comprises the following steps: (1) Sequentially carrying out colloid mill and homogenization treatment on an alkaline solution of the soybean protein isolate, and carrying out heat preservation on the obtained homogenate at 55 ℃ to obtain heat preservation liquid;
(2) Regulating the pH value of the heat preservation solution to a proper pH value of alkaline protease, sequentially carrying out enzymolysis by using the alkaline protease, neutral protease and flavourzyme, and collecting components with the molecular weight less than or equal to 1kDa after enzyme deactivation of the enzymolysis solution to obtain the soybean peptide.
The invention sequentially carries out colloid mill and homogenization treatment on alkaline solution of soybean protein isolate, and keeps the temperature of the obtained homogenate at 55 ℃ to obtain heat preservation liquid. The preparation method of the isolated soy protein is not particularly limited, and is preferably prepared by alkali extraction and acid precipitation, and more preferably comprises: extracting soybean meal with alkali solution under stirring to obtain alkali extract, centrifuging, and collecting supernatant; and (3) carrying out acid precipitation on the supernatant, centrifuging, and collecting precipitate to obtain the soybean protein isolate. The alkali solution is preferably NaOH solution, the mass and volume percentage of the NaOH solution is preferably 30%, and the pH value is adjusted to 10.0 by using the NaOH solution with the concentration. The stirring extraction is preferably carried out at 50 ℃ with stirring, and the time of the stirring extraction is preferably 90min; the rotational speed of the stirring is preferably 30rpm.
The preparation method of the soybean protein isolate firstly prepares the soybean protein isolate into an alkaline solution, and preferably comprises the following steps: deionized water is heated to 50 ℃, and is mixed with the soy protein isolate after the pH value is adjusted to 10.0 by using alkaline solution. In the embodiment of the invention, the isolated soy protein is preferably diluted into an alkaline solution of isolated soy protein with a mass percent of 5%.
The soybean protein isolate alkaline solution is sequentially subjected to colloid mill treatment and homogenization treatment to obtain homogenate, wherein the treatment time of the colloid mill is preferably 1.5h; the pressure of the homogenization treatment is preferably 25MPa and the time is preferably 2 hours. The particle size of the protein particles in the homogenate obtained by the invention can reach 50-150 mu m. The invention heats the homogenate, preferably to 55 ℃, keeps the temperature and stirs for 1h, and the heating and stirring help the isolated protein to be dissolved quickly and ensure the uniformity.
After the heat preservation liquid is obtained, the pH value of the heat preservation liquid is regulated to be a proper pH value of alkaline protease, the alkaline protease, neutral protease and flavourzyme are sequentially utilized for enzymolysis, and components with the molecular weight less than or equal to 1kDa are collected after the enzyme is inactivated to obtain the soybean peptide. In the embodiment of the invention, the pH value is preferably adjusted at a constant temperature of 55 ℃, and in the embodiment of the invention, the pH value of the heat preservation solution is preferably adjusted to 10.0, and then the heat preservation solution is mixed with alkaline protease for first enzymolysis to obtain a first enzymolysis solution; when the pH value of the first enzymolysis liquid is 7.0-7.5, mixing the first enzymolysis liquid with neutral protease for second enzymolysis to obtain a second enzymolysis liquid; the second enzymolysis time is 4 hours; and mixing the second enzymolysis liquid with flavourzyme for third enzymolysis, wherein the time of the third enzymolysis is 2 hours.
The mass of the alkaline protease, neutral protease and flavourzyme according to the present invention is preferably 1%, 0.8% and 0.2% of the mass of the isolated soy protein at a time, based on the mass of the isolated soy protein.
The invention kills the enzyme of the third enzymolysis liquid, wherein the enzyme killing preferably comprises the step of keeping the temperature of 110 ℃ for 5min and inactivating the protease. The invention preferably reduces the temperature of the enzyme-deactivated enzymolysis liquid to room temperature, passes through a 30000Da ceramic membrane, collects the membrane-down liquid, passes through a 1kDa organic membrane, and collects the membrane-down liquid. The invention preferably dries the last collected membrane dope to obtain soy peptide. The method of drying is not particularly limited in the present invention, and preferably includes spray drying.
The invention also provides the soybean peptide obtained by the preparation method.
In the embodiment of the invention, the soybean peptide prepared by the method has the protein content of 94.2%, the water content of 3.6% and the ash content of 2.2%; the soybean peptide has efficacy trend of reducing blood fat under different administration concentration conditions, and has statistical difference.
The invention also provides a soybean oligopeptide separated and identified from the soybean peptide, wherein the soybean oligopeptide comprises P6-1 and P6-2, the amino acid of the P6-1 is shown as SEQ ID NO.1, and the amino acid of the P6-2 is shown as SEQ ID NO. 2.
The soybean peptide is preferably subjected to gel chromatography separation and purification, the obtained fraction 3 is subjected to high performance liquid phase separation and purification, the soybean peptide component P6 in the fraction is subjected to structural identification, and finally two soybean oligopeptides are obtained, namely 1 strip of 9 peptide SEQ ID NO.1: LVIPPGVPY (Leu-Val-Ile-Pro-Pro-Gly-Val-Pro-Tyr) and 110 peptide SEQ ID NO.2: AIPVNKPGRY (Ala-Ile-Pro-Val-Lys-Pro-Gly-Arg-Tyr).
The invention also provides application of the soybean peptide or the soybean oligopeptide in preparing a medicine for reducing blood fat or a health-care product for assisting in treating the blood fat.
In the embodiment of the invention, soybean peptides with different dosages are orally administrated to rats, so that the TC and TG content levels of the blood of the rats can be obviously reduced, the HDLC content level is improved, but the HDLC content level has no statistical significance, and the result indicates that the soybean peptides have the function of reducing blood fat and can be applied to the preparation of corresponding medicaments or health-care products.
The invention also provides a medicine for reducing blood fat or a health care product for assisting in treating the blood fat, which takes the soybean peptide or the soybean oligopeptide as an active ingredient.
The invention does not limit the dosage form of the medicine or health care product, and the corresponding dosage form and auxiliary materials are prepared by using the conventional preparation method in the field.
The soybean peptide, soybean oligopeptide with hypolipidemic effect, the preparation method and application thereof provided by the invention are described in detail below with reference to examples, but they are not to be construed as limiting the scope of the invention; in the examples, the animal models and the methods for evaluating the efficacy of the samples were the same, unless otherwise specified.
EXAMPLE 1 preparation of Soybean peptide
Step 1, preparation of isolated soy protein
(1) Adding 100L of deionized water into an extraction tank, regulating the pH to 10.0 by using 30% NaOH, heating to 50 ℃ by using steam, weighing 10kg of soybean meal, adding the soybean meal, stirring and extracting at a speed of 30rpm for 90min to obtain an alkali extract A;
(2) Transporting the alkali extract A into a horizontal centrifuge by using a pipeline pump, centrifuging at the rotating speed of 2800rpm, and collecting supernatant to obtain centrifugate B;
(3) And transporting the centrifugate B into an extraction tank by using a pipeline pump again, regulating the pH value to 4.0 by using 30% HCl, and carrying out acid precipitation for 1h at the speed of 30rpm to obtain an acid precipitation solution C.
(5) And transporting the acid precipitation liquid C into a centrifugal machine by using a pipeline pump again, centrifuging at the rotating speed of 2800rpm, collecting precipitate, and drying a filter cake by using a tube bundle dryer to obtain the soybean protein isolate.
(6) And detecting by using a GB5009.5-2010 method, and finally obtaining the soybean protein isolate with the protein content of 88.6%.
Step 2, preparation of soybean peptide
Step one, preparing deionized water by 100L, heating to 50 ℃, regulating the pH to 10.0 by using 30% NaOH, and taking 50kg of soy protein isolate for pulping to obtain a soy protein solution with the concentration of 5%.
And secondly, uniformly stirring the soy protein solution, treating for 1.5 hours by using a colloid mill, and then treating for 2 hours by using a high-pressure homogenizer under the pressure of 25MPa, wherein the particle size of protein particles in the homogenate is 50-150 mu m.
And thirdly, heating the homogenate after high-pressure homogenization to 55 ℃, and preserving heat and stirring for 1h.
And fourthly, the heat-preserving soybean protein solution is subjected to pH value adjustment again to 10.0 by using 0.30 percent NaOH solution, and the temperature is kept constant.
And fifthly, adding 1% alkaline protease (0.5 Kg) into the soybean protein solution in the step four, continuously detecting the pH during enzymolysis, adding 0.8% neutral protease (0.4 Kg) when the pH is reduced to 7.0-7.5, continuously carrying out enzymolysis for 4 hours, adding 0.2% flavor protease (0.1 Kg), and continuously carrying out enzymolysis for 2 hours.
And step six, heating the enzymolysis liquid obtained in the step five to 110 ℃ and keeping for 5min, and inactivating protease.
And seventh, cooling the enzyme-deactivated enzymolysis liquid to room temperature, then passing through a 30000Da ceramic membrane, collecting the membrane-lower liquid, passing through a 1kDa organic membrane, collecting the membrane-lower liquid, performing spray drying to obtain soybean peptide, and detecting the molecular weight distribution of the soybean peptide by referring to GB22492, as shown in figure 2.
The basic physicochemical properties of the soybean peptide mixture were measured as follows: protein content measurement (GB 5009.9), moisture content measurement (GB 5009.3), ash content measurement (GB 5009.4). The results are shown in Table 1.
TABLE 1 basic physicochemical Properties of Soybean peptide mixtures
Example 2 evaluation of hypolipidemic efficacy
1.1. Sample formulation information
The soybean peptides prepared in example 1 were prepared as mother liquor of 2.00mg/mL with standard dilution water and used as it is.
Positive control: pioglitazone hydrochloride, white tablet, lot number 2108102104, jiangsu De Yuan pharmaceutical Co., ltd, were prepared into 10.0mg/mL mother liquor with DMSO, ground with a grinder, centrifuged to obtain supernatant, and stored at 4 ℃.
1.2. Experimental animal
Zebra fish are all bred in water for breeding fish at 28 ℃ (water quality: 200mg of instant sea salt is added into 1L of reverse osmosis water, conductivity is 450-550 mu S/cm, pH is 6.5-8.5, hardness is 50-100 mg/LCaCO 3), and the water is bred by a fish breeding center of the company, and the use license number of experimental animals is: SYXK (Zhe) 2012-0171, the feeding management meets the requirements of International AAALAC authentication (authentication number: 001458).
The melanin allele mutant Albino strain zebra fish is carried out in a natural pairing mating propagation mode. Zebra fish aged 5 days after fertilization (5 dpf) were used for evaluation of blood lipid lowering efficacy of the samples.
1.3. Instrument, consumable and reagent
Dissecting microscope (SZX 7, OLYMPUS, japan); CCD camera (VertA 1, shanghai Tusen Vision technologies Co., ltd.); precision electronic balances (CP 214, OHAUS, USA); 6-well plates (Nest Biotech, china); high-speed cryocentrifuge (Heraeus freesco 17, thermo fisher, germany); high speed tissue grinder (OSE-Y30, tiangen Biochemical technology (Beijing) Co., ltd., china).
Dimethyl sulfoxide (DMSO, lot 20171016, chinese chemical agents limited, china); methylcellulose (lot number C2004046, china, gladine biochemical technologies inc.); oil Red O (Oil Red O, lot number SHBN4926, sigma, USA); 1.2-propanediol (lot 20210817, national pharmaceutical group chemical company, china); egg yolk powder (lot 20200809, china, angry biotechnology, inc., zhejiang); d- (+) -glucose (lot 20201105, national pharmaceutical group chemical company, china).
Detection method
2.1. Evaluation of blood lipid lowering efficacy of sample
The melanin allele mutant Albino strain zebra fish were randomly selected in beakers with 15 tails per cup (experimental group). Samples (concentrations are shown in Table 2) were given in water, positive control pioglitazone hydrochloride 10.0 μg/mL, and normal control and model control were set simultaneously with a capacity of 25mL per cup. Except for the normal control group, all the other experimental groups are water-soluble and are given with high-sugar and high-fat feed, and a zebra fish high-sugar and high-fat model is built. Samples were co-treated with high fat diet for 15h (7.5 h daily), treated at 28 ℃ for 48h, and given oil red O for bulk fat staining. After the dyeing is finished, 8 zebra fish are randomly selected from each experimental group, photographed under an dissecting microscope, analyzed by Image-Pro Plus software and collected data advanced Image processing software to analyze the blood vessel dyeing intensity of the zebra fish tail, and the blood lipid reducing efficacy of the sample is evaluated by the statistical analysis result of the index. Statistical treatment results are expressed in mean+ -SE. Statistical analysis was performed with SPSS26.0 software, p <0.05 indicated that the differences were statistically significant.
Auxiliary hypolipidemic efficacy (%) = (model group tail blood vessel staining intensity-sample group tail blood vessel staining intensity)/model group tail blood vessel staining intensity 100%
3. Detection result
Under the experimental conditions, the soybean peptide powder has the efficacy trend of reducing blood fat under the condition of different administration concentrations, and has statistical differences, and the specific blood fat reducing effect is shown in figure 6.
Table 2 experimental results of blood lipid lowering efficacy evaluation of samples (n=8)
P <0.05, p <0.01, p <0.001 compared to model control.
The experiment is shown in figure 1: the tail vessel staining intensity of the model control group is 8684 pixels, and p <0.001 is compared with the normal control group (37.1 pixels), which indicates that the model establishment is successful. The blood vessel staining intensity of the tail part of the zebra fish in the positive control group is 61.6 pixels, and compared with the positive control group (8684 pixels), the p is less than 0.001, and the auxiliary blood lipid reducing effect is 99.3%, which shows that the pioglitazone hydrochloride has obvious auxiliary triglyceride reducing effect on the high-sugar and high-fat zebra fish under the experimental concentration condition.
Example 1 soybean peptide has tail blood vessel staining intensity of 7647 pixels, 7077 pixels and 6531 pixels respectively under the concentration conditions of 500, 1000 and 2000 mug/mL, and has auxiliary triglyceride reducing effects of 23.2%, 28.9% and 34.4% respectively compared with a model control group (11080 pixels), so that the soybean peptide of example 1 has obvious auxiliary blood lipid reducing effects on high-sugar and high-fat zebra fish under the concentration conditions of the experiment.
Example 3 isolation and purification of the Soybean peptide prepared in example 1
(1) Gel chromatography separation and purification
Swelling of the gel: weighing a proper amount of Sephadex G-50 gel dry powder, adding excessive deionized water, placing at room temperature for expansion for 2 days, vacuumizing to remove air in gel gaps, fully swelling for 2 hours under the condition of boiling water bath, removing small particles which are not easy to sink and float on the surface and air and bacteria in the gel after fully swelling, and repeatedly washing with deionized water for 2-3 times for standby.
Column loading and balancing: a clean chromatographic column with the length of 3cm multiplied by 100cm is vertically fixed on an iron stand, a water outlet is closed, distilled water (about 1/3 part) is added into the chromatographic column, the swollen gel is poured into the chromatographic column while being stirred, and the gel is completely settled under the action of gravity, so that the column loading is completed. Note that: the gel in the column cannot be delaminated, if delamination occurs, a constant flow pump is connected, the column is equilibrated with 2 times of column volume of eluent (the eluent is tris HCl of pH7.10.05 mmol/L) to stabilize the column bed.
Loading: the soybean peptide produced in example 1 was dissolved in water, 15G of the soybean peptide was weighed, a 500mg/mL solution was prepared with deionized water, and separation and purification were carried out using a Sephadex G-50 column (3 cm. Times.100 cm), the mobile phase was a 10% aqueous methanol solution, the flow rate was 1.0mL/min, absorbance was measured at 214nm of the eluent, fractions were collected according to the chromatographic peak and each fraction was concentrated and freeze-dried to powder using a vacuum dryer.
10 fractions were collected, and the peak time of the 10 fractions was as follows:
12-14 min of flow 1; 14-16 min of fraction 2; 16-18 min of fraction 3; 18-20 min of fraction 4; 20-22 min of flow 5; 22-24 min of flow 6; 24-26 min of flow 7. The blood lipid lowering effect was evaluated by freeze-drying the 7 fractions (fig. 7), and the fractions having a good blood lipid lowering effect were further separated and purified.
Table 3 experimental results of sample hypolipidemic efficacy evaluation (n=8)
P <0.05, p <0.01, p <0.001 compared to model control.
The soybean peptide is separated and purified by gel chromatography to obtain 7 fractions, the 7 fractions are freeze-dried, and under the concentration condition of 1000 mug/mL, the blood lipid reducing effect of the soybean peptide is verified by using a hyperlipoidemia zebra fish model; wherein the blood vessel staining intensity of the tail part of the zebra fish treated by the soybean peptide fraction 3 is the lowest, and the auxiliary blood fat reducing effect can reach 58.6%; the result shows that under the condition of the experimental concentration, the soybean peptide fraction 3 has the best efficacy of assisting in reducing blood fat for the high-sugar and high-fat zebra fish, and the component is separated and purified in the next step.
(2) High performance liquid phase separation and purification
HPLC apparatus: aglient 1260-6470 liquid chromatograph (Aglient company of America)
Chromatographic column: kromasil 100-5C18 (4.6 mm. Times.250 mm,5 m. Acron)
Detection wavelength: 214nm; column temperature: 30 ℃; flow rate: 1ml/min;
mobile phase a: water +0.1% trifluoroacetic acid; mobile phase B: acetonitrile +0.1% trifluoroacetic acid
The elution gradient was as follows: 0-10 min, 95-85% mobile phase B (linear gradient); 10-50 min, 85-5% mobile phase B (linear gradient); mobile phase B (linear gradient) 50-55 min; 55 min-60 min mobile phase B linear gradient).
The liquid chromatogram of soybean peptide fraction 3 is shown in fig. 3, and the blood vessel staining intensity of the tail part of the zebra fish after the sample treatment of the soybean peptide after high performance liquid phase separation and purification is shown in table 5 and fig. 8.
Table 4 Peak time cases of 10 Components
| Component (A) | Peak time |
| P1 | 0～3min |
| P2 | 3～6min |
| P3 | 6～9min |
| P4 | 9～12min |
| P5 | 12～15min |
| P6 | 15～18min |
| P7 | 18～21min |
| P8 | 21～24min |
| P9 | 24～27min |
| P10 | 27～30min |
Table 5 experimental results of sample hypolipidemic efficacy evaluation (n=8)
P <0.05, p <0.01, p <0.001 compared to model control group
The soybean peptide is separated and purified by reverse high performance liquid chromatography to obtain 10 fractions, the 10 fractions are freeze-dried, and under the concentration condition of 1000 mug/mL, the hyperlipidemia zebra fish model is utilized to verify the blood lipid reducing effect; wherein the blood vessel dyeing intensity of the tail part of the zebra fish after the soybean peptide component P6 is treated is the lowest, and the auxiliary blood fat reducing effect can reach 67.3 percent; the soybean peptide component P6 has the best auxiliary blood lipid reducing effect on the high-sugar and high-fat zebra fish under the experimental concentration condition, and the next structural identification is carried out on the component.
(3) Structural identification
And further carrying out purity and structure identification on the obtained soybean peptides P5 and P6 by using a nano-upgrading liquid chromatography-Q EXACTIVE mass spectrometry system.
1. Detection conditions:
(1) Mobile phase: phase A, 100% pure water and 0.1% formic acid; phase B, 100% acetonitrile +0.1% formic acid;
(2) Mobile phase flow rate: 300nl/min;
(3) Sample injection amount: 1 (L supernatant;
(4) The mobile phase gradient procedure is shown in table 6.
TABLE 6 Mobile phase gradient procedure
| Time (min) | 0 | 2.0 | 36.0 | 38.0 | 41.0 | 42.0 | 45.0 |
| A(％) | 97 | 97 | 63 | 10 | 10 | 97 | 97 |
| B(％) | 3 | 3 | 37 | 90 | 90 | 3 | 3 |
The structures of P6-1 and P6-2 were identified using a combination of Napromoting liquid chromatography-Q EXACTIVE mass spectrometry, and as a result, 19 peptides LVIPPGVPY (Leu-Val-Ile-Pro-Pro-Gly-Val-Pro-Tyr) and 110 peptides AIPVNKPGRY (Ala-Ile-Pro-Val-Lys-Pro-Gly-Arg-Tyr) were identified together. The soybean peptide ratios of the detected P6-1 and P6-2 in example 1 were 0.12% and 0.09%, respectively, and the secondary structures of the polypeptides for P6-1 and P6-2 are shown in FIGS. 4 and 5. The marchantia polymorpha medical Co., ltd is entrusted to synthesize the two single-chain polypeptides, and an in vitro digestion test is carried out, so that the results show that the two single-chain polypeptides are not degraded by digestive proteases.
The same model experiments as described above were conducted on P6-1 and P6-2, and the hypolipidemic effects were evaluated, and the results are shown in Table 7 and FIG. 9.
Table 7 experimental results of blood lipid lowering efficacy evaluation of samples (n=8)
P <0.05, p <0.01, p <0.001 compared to model control group
The experimental results show that: the tail vessel staining intensity of the model control group is 8631 pixels, and p <0.001 is compared with that of the normal control group (69.1 pixels), so that the model establishment is successful. The blood vessel staining intensity of the tail part of the zebra fish in the positive control group is 59.3 pixels, and compared with the p <0.001 in the model control group (8631 pixels), the auxiliary blood lipid reducing effect is 99.3%, which shows that the pioglitazone hydrochloride has obvious auxiliary blood lipid reducing effect on the high-sugar and high-fat zebra fish under the experimental concentration condition.
The blood vessel dyeing intensity of the tail part of the hyperlipoidemia zebra fish treated by the soybean peptide P6-1 is 183 pixels, the auxiliary blood lipid reducing effect is 97.9%, and compared with a model control group, the P is less than 0.001; the blood vessel dyeing intensity of the tail part of the hyperlipoidemia zebra fish treated by the soybean peptide P6-2 is 195 pixels, the auxiliary blood lipid reducing effect is 98.9%, and compared with a model control group, the P is less than 0.001; the soybean peptide P6-1 and the soybean peptide P6-2 obtained after separation and purification have remarkable blood lipid reducing effect, and can be further developed into blood lipid reducing medicines or health-care foods. Example 4 evaluation of efficacy of soybean peptide in reducing blood lipid
1 materials and methods
1.1 sample: the recommended intake of soybean peptide (prepared in example 1) by human is 5.0g per day.
1.2 experimental animals: healthy SPF-grade Wistar male rats are selected, the experimental environment temperature is 22+/-2 ℃, the relative humidity is 50+/-10%, and the weight is 150-200g.
1.3 Gao Xie feed: 78.8% of basic feed, 1% of cholesterol, 10% of yolk powder, 10% of lard and 0.2% of bile salt.
1.4 experimental method: rats were fed with basal feed and after 7 days of observation, tail blood was taken, serum Total Cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) levels were determined, and animals were randomly divided into 4 groups of high fat control group, low, medium, and high dose group, 10 rats per group, and 40 total animals based on serum total cholesterol levels. After the initiation of the official experiments, animals of each group were replaced with high-fat feed.
The recommended intake of the sample into the human body is 5g per person per day, and each person is equivalent to 83mg/kg.bw per day according to 60kg body weight. The low, medium and high doses of rats are determined according to the recommended amount of the human body which is 5 times, 10 times and 30 times, namely, the daily eating amount of each group of rats is 415 mg/kg/bw, 830 mg/kg/bw and 2490 mg/kg/bw respectively. The test substance is prepared by deionized water, and the test substance solution is administrated to rats through gastric lavage once daily, wherein the gastric lavage volume is 1mL/100 g.bw. The high fat control group was gavaged with a considerable volume of deionized water. And continuously administering for 35 days, and taking tail blood to measure various blood lipid indexes.
1.5 major instrumentation and reagents: animal balance, electronic balance, low-speed centrifuge, 722 type spectrophotometer, constant temperature water bath, microscale sampler, and surgical instrument. The kit for measuring TC, TG, HDL-C is provided by North control Biotech Co., ltd.
1.6 statistics of experimental data: the experimental data are all numerical variable data. All data were analyzed by SPSS statistical software line variance. Variance is analyzed by variance analysis, variance is verified by rank sum.
2. Results
2.1 effects on rat body weight: as can be seen from table 8, throughout the experiment, animals of each group had normal growth activity, and the body weight of animals of each dose group of the test substance was not significantly different from that of the high-fat control group (P > 0.05).
TABLE 8 influence on rat body weight during the experiment (x soil s)
2.2 effects on lipid levels in rats
2.2.1 levels of blood Fu in rats prior to the experiment: the difference in TC, TG, HDL-C content of the blood of rats in each group before the experiment has no statistical significance (P > 0.05) through analysis of variance. See table 9.
Table 9 comparison of the blood TC, TG, HDLC content (x.+ -. S) of rats in each group prior to the experiment
(note: in comparison to the model control group, * P<0.05， ** P<0.01)
2.2.2 post-experiment rat blood fetal content level: the TC and TG levels were significantly reduced (P <0.05, P < 0.01) in both the medium and high dose groups compared to the high fat control group, as shown in table 10.
Table 10 comparison of blood TC, TG, HDLC content (x.+ -. S) of rats in each group after the experiment
(note: in comparison to the model control group, * P<0.05， ** P<0.01)
in summary, it is known that different doses of soybean peptide powder are orally administered to rats, wherein the high dose of soybean peptide can significantly reduce the blood TC and TG content levels of rats, (p < 0.01); HDLC content level increased, (p less than 0.01); but has no statistical significance, and the result indicates that the soybean peptide powder has the function of reducing blood fat.
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.
Sequence listing
<110> Jiangsu pray pharmaceutical technology Co., ltd
<120> soybean peptide and soybean oligopeptide with hypolipidemic effect, and preparation method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Leu Val Ile Pro Pro Gly Val Pro Tyr
1 5
<210> 2
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Ala Ile Pro Val Asn Lys Pro Gly Arg Tyr
1 5 10
Claims (3)
1. The identified soybean oligopeptide is separated from soybean peptide, wherein the soybean oligopeptide is P6-1 and P6-2, the amino acid of the P6-1 is shown as SEQ ID NO.1, and the amino acid of the P6-2 is shown as SEQ ID NO. 2.
2. The use of the soybean oligopeptide of claim 1 in the preparation of a medicament for reducing blood lipid.
3. A hypolipidemic agent comprising the soybean oligopeptide of claim 1 as an active ingredient.
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| CN103014112A (en) * | 2012-12-20 | 2013-04-03 | 广州合诚实业有限公司 | Soybean peptide and industrial production method and application of soybean peptide |
| CN104719611A (en) * | 2013-12-18 | 2015-06-24 | 中粮营养健康研究院有限公司 | Method for preparing soybean peptides through enzymolysis of soy protein |
| CN113215212A (en) * | 2021-04-16 | 2021-08-06 | 国肽生物工程（常德）有限公司 | Soybean protein peptide with antioxidant and ACE (angiotensin converting enzyme) inhibiting functions and preparation method thereof |
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| CN103014112A (en) * | 2012-12-20 | 2013-04-03 | 广州合诚实业有限公司 | Soybean peptide and industrial production method and application of soybean peptide |
| CN104719611A (en) * | 2013-12-18 | 2015-06-24 | 中粮营养健康研究院有限公司 | Method for preparing soybean peptides through enzymolysis of soy protein |
| CN113215212A (en) * | 2021-04-16 | 2021-08-06 | 国肽生物工程（常德）有限公司 | Soybean protein peptide with antioxidant and ACE (angiotensin converting enzyme) inhibiting functions and preparation method thereof |
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| 多酶复合水解法生产高水解度大豆寡肽的工艺优化;梁秋丽等;食品工业;第33卷(第09期);第82-86页 * |
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