CN117551167B - Oyster DPP-IV inhibitory peptide rich in branched chain amino acid, and preparation method and application thereof - Google Patents
Oyster DPP-IV inhibitory peptide rich in branched chain amino acid, and preparation method and application thereof Download PDFInfo
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- CN117551167B CN117551167B CN202311516908.3A CN202311516908A CN117551167B CN 117551167 B CN117551167 B CN 117551167B CN 202311516908 A CN202311516908 A CN 202311516908A CN 117551167 B CN117551167 B CN 117551167B
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- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- 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
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
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- 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
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- 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
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- 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
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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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Abstract
The invention discloses an oyster DPP-IV inhibitory peptide rich in branched chain amino acid, and a preparation method and application thereof, belonging to the technical fields of food nutrition and health care and biological medicine. The invention adopts alcohol precipitation desugarization and activated carbon adsorption to obtain oyster DPP-IV inhibitory peptide rich in branched chain amino acid, and in vitro experiments show that: after gastrointestinal digestion, the inhibition activity of the oyster peptide to DPP-IV is stable, which proves that the oyster DPP-IV inhibition peptide has good digestion stability; and the peptide spectrum sequence characteristics show that the branched chain amino acid sequence characteristics of the oyster peptide have close relation with the hypoglycemic activity and the digestion stability. Animal experiment results show that the oyster DPP-IV inhibitory peptide has good blood sugar reducing effect and better effect than single branched chain amino acid. The invention provides a new direction for reducing blood sugar and treating and preventing type II diabetes.
Description
Technical Field
The invention relates to the technical fields of food nutrition and health care and biological medicine, in particular to an oyster DPP-IV inhibitory peptide rich in branched chain amino acid, a preparation method and application thereof.
Background
Diabetes is a common chronic metabolic disease and is characterized by fasting hyperglycemia, impaired insulin signaling, disturbed glycolipid metabolism, etc., and in addition, diabetes can induce various chronic complications such as myocardial infarction, stroke, cardiovascular death, etc., and has become a third disease endangering human life health in the presence of cardiovascular and cerebrovascular diseases and cancers. Type II diabetes is the main onset type of diabetes, and prior studies indicate that alpha-amylase, alpha-glucosidase and dipeptidyl peptidase IV (DPP-IV) are important targets for treating type II diabetes. The international diabetes union (IDF) statistics indicate that the number of diabetics worldwide reaches 5.73 billion in 2021. Therefore, low cost and safe and effective hypoglycemic functional factors are sought to control blood glucose and inhibit the further development of hyperglycemic populations into diabetic patients. In recent years, low-cost and safe and effective alpha-amylase, alpha-glucosidase and DPP-IV inhibitors obtained from natural sources have become hot spots of interest in the fields of foods, biology and medicine.
A number of clinical studies have demonstrated that branched chain amino acids (Branchedchain amino acids, BCAAs), including leucine (Leu), isoleucine (Ile) and valine (Val), have been considered potential biomarkers of insulin resistance, pre-diabetes and mid-development. When the metabolism of branched chain amino acids is disturbed, the accumulation of branched chain amino acids in the body causes higher level, negative metabolic effect can occur, hyperlipidemia, obesity and insulin resistance are caused, and thus the risk of type II diabetes mellitus is increased. In daily diet, BCAAs mainly take proteins or peptides as carriers for ingestion into organisms, and the BCAAs not only endow the active peptides with nutritional functions, but also influence the digestion stability of the active peptides through the proportion and specific positions of the BCAAs, thereby deeply participating in the physiological functions of the active peptides, in particular to regulating the metabolism of the organisms. It has been reported that the active peptide rich in BCAAs has good digestion stability, is favorable for the exertion of physiological functions, and can increase insulin response, regulate glucose homeostasis and delay the development and transformation of hyperglycemia to diabetes by promoting the release of glucagon-like peptide-1 (GLP-1).
Oyster is commonly called oyster, is used as an important marine economic shellfish in coastal areas of China, has higher nutritive value, is called as marine milk, and is one of the first medicinal and edible seafood approved by the health department of China. Because of unique marine environment and unique oyster active peptide amino acid sequence, the oyster active peptide is an effective alpha-amylase, alpha-glucosidase and DPP-IV inhibitor, and has natural advantage in the aspect of reducing blood sugar. However, there are few reports on the research of the preparation method of oyster hypoglycemic peptide rich in BCAs and the research of the BCAs proportion and specific position of oyster bioactive peptide on the influence rule of the oyster hypoglycemic peptide on the digestion stability and hypoglycemic effect from the aspects of the composition and sequence of branched chain amino acids (BCAs).
Disclosure of Invention
The invention aims to provide an oyster DPP-IV inhibitory peptide rich in branched chain amino acid, and a preparation method and application thereof, so as to solve the problems in the prior art.
In order to achieve the above object, the present invention provides the following solutions:
The invention provides an oyster DPP-IV inhibitory peptide rich in branched chain amino acid, which comprises the following peptide fragments: LPA, LEI, VVD, ICIIM (SEQ ID NO: 1), LSEPSEVVGPITP (SEQ ID NO: 2) and SGEPGPEGPAGPI (SEQ ID NO: 3).
The invention also provides a preparation method of the oyster DPP-IV inhibitory peptide rich in branched-chain amino acid, which is characterized by comprising the following steps:
carrying out enzymolysis on oyster meat to obtain oyster peptide;
The oyster peptide is desugared by adopting an ethanol precipitation method to obtain ethanol precipitation desugared oyster peptide;
And (3) adsorbing and treating the alcohol precipitation desugared oyster peptide by using activated carbon to obtain the oyster DPP-IV inhibitory peptide rich in branched chain amino acids.
Preferably, the enzymolysis method comprises the following steps:
Homogenizing oyster meat according to a feed liquid ratio of 1g: adding 3mL of water, homogenizing, adding neutral protease for enzymolysis, centrifuging, collecting supernatant, and drying to obtain the oyster peptide.
Preferably, the homogenizing conditions are: regulating pH of the mixture of oyster pulp homogenate and water to 7.0, homogenizing at 8000r/min for 2min.
Preferably, the enzymolysis condition is water bath enzymolysis at 50 ℃ for 4 hours.
Preferably, the method for desugaring by the alcohol precipitation method comprises the following steps:
mixing and dissolving the oyster peptide and water according to the mass-volume ratio of 1:20, centrifuging, and collecting filtrate;
Mixing the filtrate with 95% ethanol according to a volume ratio of 1:3, standing overnight at low temperature, centrifuging, collecting supernatant, removing ethanol, and drying to obtain the ethanol precipitation desugared oyster peptide.
Preferably, the method for treating the desugared oyster peptide by the adsorption of the activated carbon comprises the following steps:
Dissolving the alcohol precipitation desugared oyster peptide, adding active carbon into oyster peptide solution according to the mass volume percentage of 5%, carrying out water bath for 3 hours at 20 ℃, stirring, centrifuging, collecting supernatant, filtering and drying to obtain the oyster DPP-IV inhibitory peptide rich in branched chain amino acid.
Preferably, the oyster peptide solution is adjusted to pH 7.0 before adding the activated carbon.
The invention also provides application of the oyster DPP-IV inhibitory peptide rich in branched-chain amino acid in preparation of hypoglycemic products.
The invention also provides a blood sugar reducing product, which takes the oyster DPP-IV inhibitory peptide rich in branched chain amino acid as an active ingredient.
The invention discloses the following technical effects:
The invention adopts alcohol precipitation desugarization and activated carbon adsorption to enrich the BCAs of oyster peptide, and utilizes an amino acid composition analyzer and UPLC-Q-TOF technology to analyze the amino acid composition and peptide spectrum sequence of oyster peptide; the hypoglycemic activity (alpha-amylase, alpha-glucosidase and dipeptidyl peptidase IV) and the peptide profile sequence characteristics of oyster peptides before and after gastrointestinal digestion were compared by an in vitro simulated digestion model. The results show that: the BCAs content (from 55.31mg/g to 101.66 mg/g) and the proportion (from 15.78% to 16.37%) of oyster peptide are obviously improved after the oyster peptide is desugared by alcohol precipitation; and the N-terminal position of the peptide chain of the activated carbon is rich in BCAs after adsorption, and proline is close to the C-terminal, thus being a typical structural mode of the hypoglycemic peptide. After gastrointestinal digestion, the inhibition activity of the oyster peptide to DPP-IV is relatively stable, which proves that the DPP-IV inhibition peptide rich in BCAs in the oyster peptide has good digestion stability; and the peptide spectrum sequence characteristics show that the BCAs sequence characteristics of the oyster peptide have close relation with the hypoglycemic activity and the digestion stability. The animal experiment result shows that: the oyster peptide rich in the BCAs has good blood sugar reducing effect and better effect than the BCAs alone. The invention provides a new direction for reducing blood sugar and treating and preventing type II diabetes.
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 needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the relative molecular mass distribution of oyster peptides after various treatments; the different letters represent a significant difference between the different molecular weights (P < 0.05);
FIG. 2 shows in vitro hypoglycemic activity of oyster peptides; a: inhibition of oyster peptide alpha-amylase; b: inhibition rate of oyster peptide alpha-glucosidase; c: DPP-IV inhibition rate of oyster peptide;
FIG. 3 shows the total amount of hydrolyzed amino acids and the proportions of various amino acids; y represents crude oyster peptide; t represents alcohol precipitation desugared oyster peptide; c represents activated carbon adsorption oyster peptide; YW represents crude oyster peptide after gastric digestion; TW represents the gastric digested alcohol-precipitated desugared oyster peptide; CW represents gastric digested activated carbon-adsorbed oyster peptide; YWC represents crude oyster peptide after gastrointestinal digestion; TWC means gastrointestinal digested alcohol precipitation desugared oyster peptide; CWC represents activated carbon-adsorbed oyster peptide after gastrointestinal digestion;
FIG. 4 shows in vitro hypoglycemic activity before and after oyster peptide digestion; a: hypoglycemic activity before and after digestion of oyster peptide crude products; b: hypoglycemic activity before and after digestion of the alcohol precipitation desugared oyster peptide; c: activity of reducing blood sugar before and after digestion of oyster peptide is adsorbed by activated carbon;
FIG. 5 shows the variation of the peptide sequence structure of crude oyster peptide before and after in vitro simulated digestion; a: digestion of crude oyster peptide with propeptide sequence features; b: crude gastric digestion peptide sequence characteristics of oyster peptide; c: crude oyster peptide gastrointestinal digestion peptide sequence characteristics;
FIG. 6 is a diagram showing the variation of the sequence structure of the in vitro simulated digestion of the desugared oyster peptide by alcohol precipitation; a: the sequence characteristics of the peptide digestion precursor of the alcohol precipitation desugared oyster peptide; b: the sequence characteristics of the gastric digestion peptide of the desugared oyster peptide by alcohol precipitation; c: the sequence characteristics of the gastrointestinal digestion peptide of the alcohol precipitation desugared oyster peptide;
FIG. 7 is a diagram showing the change of the sequence structure of activated carbon adsorption oyster peptide before and after in vitro simulated digestion; a: activated carbon adsorption oyster peptide digestion propeptide sequence characteristics; b: activated carbon adsorption oyster peptide gastric digestion peptide sequence characteristics; c: activated carbon adsorption oyster peptide gastrointestinal digestion peptide sequence characteristics;
FIG. 8 is a graph showing the effect of branched chain amino acid-rich oyster DPP-IV inhibitory peptides on food intake in type II diabetic mice; a: the influence of oyster DPP-IV inhibitory peptide rich in branched chain amino acid on the ingestion of type II diabetic mice; b: the influence of oyster DPP-IV inhibitory peptide rich in branched chain amino acid on water uptake of type II diabetes mice;
FIG. 9 is a graph showing the effect of a branched-chain amino acid-rich oyster DPP-IV inhibitor peptide on fasting blood glucose levels in type II diabetic mice;
FIG. 10 is a graph showing the effect of a branched-chain amino acid-rich oyster DPP-IV inhibitory peptide on oral glucose tolerance in type II diabetic mice; a: oral glucose tolerance (OGTT); b: area under OGTT curve AUC;
FIG. 11 is a graph showing the modulating effect of oyster DPP-IV inhibitory peptides rich in branched-chain amino acids on glucagon-like peptide-1 (GLP-1) and Glycated Serum Protein (GSP) content in type II diabetic mice; a: glucagon-like peptide-1 (GLP-1) content in mouse serum; b: glycosylated Serum Protein (GSP) content in mouse serum.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The following examples relate to the main experimental materials, reagents and equipment:
(1) The hong Kong oyster (Crassostrea hongkongenis) is purchased in the eastern wind market in Zhanjiang; neutral protease, alpha-glucosidase (70U/mL), trichloroacetic acid, biuret reagent, alpha-amylase (porcine pancreas, 5U/mg), soluble starch, p-nitrophenylpyranoside (p-NPG), absolute ethanol, activated carbon powder, pepsin, trypsin, and Phosphate Buffer (PBS) were purchased from shanghai-derived leaf biotechnology limited; dipeptidyl peptidase-IV (DPP-IV) inhibitor screening kit, purchased from Sigma Co., USA; the other reagents were all analytically pure.
(2) Varioskan Flash full-automatic microplate reader (Thermo company, usa); 5810R high-speed bench-top refrigerated centrifuge (Eppendorf Co., U.S.A.); semi-preparative liquid chromatograph (chromatography column: TSKgel2000SWXL300 mm. Times.7.8 mm) (Agilent Co., USA); FD-551 large vertical freeze dryer (Tokyo physical and chemical instruments Co.); spin-on concentration system N-4000 (Tokyo physical and chemical instruments Co.); full-automatic Kjeldahl apparatus VAPODEST 450 (Gehart analysis instruments, inc. of Germany); SHJ-6AB magnetic stirring water bath (Hexagon gold altar Liriod instruments Co., ltd.).
Example 1
1. Preparation of oyster peptide rich in BCAs
1.1 Preparation of oyster peptides
Washing fresh oyster meat with ultrapure water, pulping, adding ultrapure water according to a feed-liquid ratio of 1:3 (g: mL), adjusting pH to 7.0, homogenizing for 2min, adding neutral protease (3300U/g), performing enzymolysis in water bath at 50deg.C for 4h, inactivating enzyme in boiling water bath for 10min, cooling to room temperature, centrifuging for 15min, filtering, collecting supernatant, concentrating, freeze drying, and drying at 4deg.C.
1.2 Desugaring treatment of oyster peptides
Removing sugar from oyster peptide by ethanol precipitation, dissolving a certain amount of oyster peptide in 20 times of water under stirring, standing, centrifuging to remove insoluble substances, filtering with filter paper, slowly adding 3 times of 95% ethanol into the filtrate, standing overnight at 4deg.C, centrifuging, rotary evaporating supernatant to recover ethanol, lyophilizing, and storing at 4deg.C.
1.3 Enrichment of oyster peptide BCAs
2.5G of oyster peptide desugared with ethanol is weighed and dissolved in 50mL of water. Activated carbon (2.5 g) was added to oyster peptide solution (50 mL) at a concentration of 5% (w/v), and the solution was adjusted to pH 7.0 prior to addition of activated carbon powder. Subsequently, the mixture was water-bath at 20℃for 3 hours and stirred at a speed of 200 r/min. After centrifugation, the supernatant was collected, filtered through a 0.45 μm microfiltration membrane and freeze-dried to obtain BCAAs-enriched oyster peptides, which were stored at-20 ℃ for use.
2. Analysis of BCAAs-enriched oyster peptides
2.1 Protein content
Protein content determination is described in GB 5009.5-2016 "determination of proteins in food safety national Standard food".
2.2 Polysaccharide content
Polysaccharide determination is referred to GB/T9695.31-2008 determination of total sugar content of meat products.
2.3 Polypeptide content
2.5ML of the sample solution was taken, 2.5mL of 10% (w/v) aqueous trichloroacetic acid (TCA) was added, mixed uniformly on a vortex mixer, and after 10min of standing, centrifuged (4000 r/min,15 min), the supernatant was transferred to a 50mL volumetric flask in its entirety, and the volume was fixed to the scale with 5% TCA, and shaken uniformly. Then, 6.0mL of the solution is placed in another test tube, 4.0mL of biuret reagent (sample solution: biuret reagent=3:2, v/v) is added, the mixture is uniformly mixed on a vortex mixer, the mixture is kept stand for 10min, centrifugation is carried out for 10min at 2000r/min, the OD value of the supernatant is measured at 540nm, and the concentration c (mg/mL) of the polypeptide in the sample solution is calculated by comparing with a standard curve.
3. Structural characterization of BCAAs-enriched oyster peptides
3.1 Relative molecular Mass distribution
The molecular mass distribution of oyster peptides was analyzed by HPLC, reference GB/T22729-2008 "Marine fish oligopeptide powder".
3.2 Analysis of amino acid composition
Amino acid determination is described in GB 5009.124-2016, "determination of amino acids in food safety national Standard food".
3.3 Peptide Spectroscopy and peptide sequence analysis
And (3) carrying out peptide spectrum sequence identification on the oyster peptide by adopting UPLC-Q-TOF technology. C18 chromatographic column (150 mm. Times.2.0 mm) was selected, and the detection wavelength: 220nm, column temperature: 40 ℃, flow rate: sample injection amount of 0.3 mL/min: 10 mu L. Mobile phase a was pure acetonitrile (containing 0.1% formic acid) and mobile phase B was ultrapure water (containing 0.1% formic acid). The gradient elution mode is adopted: mobile phase B was from 5% to 80% in 50min and after completion the column was rinsed with 95% B.
4. In vitro hypoglycemic activity of oyster peptide rich in BCAs
4.1 Alpha-Amylase inhibitory Activity
Mu.L of amylase solution (1U/mL, 0.1mol/LPBS, pH 6.8) and 50. Mu.L of samples of different concentrations were added to a 2mL centrifuge tube and incubated at 37℃for 15min. Then 100. Mu.L of 1% soluble starch solution was added, incubated at 37℃for 20min, then 400. Mu.LDNS reagent was added, the mixture was cooled to room temperature after 10min in a boiling water bath, 1mL of distilled water was added for dilution, and absorbance at 540nm was measured using an enzyme-labeled instrument. The blank group replaced the sample solution with PBS. Acarbose was used as a positive control. The alpha-amylase inhibition rate was calculated as follows:
Wherein: a1 is the absorbance of the sample group; a2 is the absorbance of the blank group of the sample; a3 is the absorbance of the control group; a4 is the blank absorbance.
4.2 Alpha-glucosidase inhibitory Activity
Mu.L of glucosidase solution (1U/mL, 0.1mol/LPBS, pH 6.8) and 50. Mu.L of samples of different concentrations were added to a 2mL centrifuge tube and incubated at 37℃for 10min. Then, 100. Mu.L of a 6mol/LpNPG solution was added, and after incubation at 37℃for 50min, 1mL of a sodium carbonate solution (1 mol/L) was added, and absorbance at 400nm was measured using an enzyme-labeled instrument. The blank group replaced the sample solution with PBS. Acarbose was used as a positive control. The alpha-glucosidase inhibition rate was calculated as follows:
Wherein: a1 is the absorbance of the sample group; a2 is the absorbance of the blank group of the sample; a3 is the absorbance of the control group; a4 is the blank absorbance.
4.3 Dipeptidyl peptidase-IV (DPP-IV) inhibitory Activity
The operation is carried out according to the specification of DPP-IV kit: the buffer was first diluted 10-fold and kept at 4℃until use. 30. Mu.L of DPP-IV enzyme was added to 120. Mu.L of buffer, and the mixture was kept on ice for stabilization within 2 hours. mu.L of DPP-IV substrate is taken, 720 mu.L of buffer is added, and the mixture is put on ice for standby and stable within 6 hours. Then 10 mu L of enzyme solution, 10 mu L of sample and 50 mu L of substrate are sequentially added into a 96-well plate, the mixture is uniformly mixed and incubated for 30min at 37 ℃, and fluorescence values are measured at excitation wavelengths of 350-360nm and emission wavelengths of 450-465 nm. The DPP-IV enzyme inhibition rate has the following calculation formula:
wherein: a1 is the fluorescence value of a sample blank group; a2 is a blank fluorescence value; a3 is the sample group fluorescence value.
5. In vitro simulated digestion stability assay for BCAAs-enriched oyster peptides
5.1 In vitro simulation of gastric digestion
Preparation of simulated gastric digestion solution: 2.0g NaCl solid is weighed and dissolved in a beaker, the pH is adjusted to 2.0, 3.2g pepsin is added, and the volume is fixed to 1000mL by using a hydrochloric acid solution with the pH of 2.0. 2.0g oyster peptide is dissolved in 50mL gastric digestion liquid, and the gastric digestion liquid is put into a magnetic stirring water bath (37 ℃ C., 200 r/min) to simulate gastric digestion for 2h, and then the enzyme is inactivated by boiling water bath for 10min. And (3) cooling the digestive juice to room temperature, regulating the pH to 6.8 by using 1mol/LNaOH, fixing the volume to 100mL, centrifuging at the temperature of 4 ℃ for 10min under the condition of 8000r/min, collecting the supernatant, and storing at the temperature of 4 ℃ for later use.
5.2 In vitro simulated intestinal digestion
Preparation of simulated intestinal digestive juice: 6.8g KH 2PO4 solid was weighed, dissolved in 250mL water, adjusted to pH 6.8, 10g trypsin was added, and the volume was fixed to 1000mL with a solvent at pH 6.8. Taking 50mL of gastric digestion liquid and 50mL of simulated intestinal digestion liquid in 5.1, fully and uniformly mixing, and placing into a 37 ℃ magnetic stirring water bath kettle for simulated intestinal digestion for 4 hours, wherein the rotating speed is 200r/min. Inactivating enzyme in boiling water bath for 10min after intestinal digestion, cooling to room temperature, fixing volume to 100mL, centrifuging at 4deg.C and 8000r/min for 10min to obtain supernatant, and storing at 4deg.C.
5.3 Digestion stability analysis
Performing inhibition activity measurement of alpha-amylase, alpha-glucosidase and dipeptidyl peptidase-IV (DPP-IV) on the supernatant obtained after the 5.1 and 5.2 gastrointestinal digestion according to the 'in vitro hypoglycemic activity of 4 and BCAs-enriched oyster peptides'; and simultaneously, carrying out peptide sequence analysis on the obtained supernatant according to the 3.3 peptide spectrum characteristics and peptide sequence analysis, examining the influence of in vitro simulated digestion on the peptide sequence and the hypoglycemic activity of the oyster peptide rich in the BCAs, and evaluating the digestion stability.
6. In vivo hypoglycemic assay of BCAAs-enriched oyster peptides
6.1 Establishment of a mouse model for type II diabetes
After carrying out adaptive feeding on 84 SPF-class c57bl/6J male mice for 1 week, randomly selecting 72 mice to be fed with high-sugar and high-fat feed as an experimental group; another 12 mice were given normal diet as a normal control group, and both mice were given free water. After 4 weeks of feeding, mice of the experimental group were intraperitoneally injected with STZ (streptozotocin) injection at a dose of 90mg/kg twice within 3 days. The normal control group was injected with an equal amount of citric acid buffer in the same manner, and the fasting blood glucose value was measured after 3 days (blood was taken from the tail vein of the mouse, and the fasting blood glucose value was measured using a fish-strike glucometer). A mouse with fasting blood glucose higher than 11.1mmol/L is used as a successful model of type II diabetes.
6.2 Grouping
Mice successfully molded were selected and randomly divided into 6 groups: model group (DC), positive control group (positive control is acarbose, 50mg/kg, PC), branched chain amino acid group (0.09 g/kg, leucine Leu: isoleucine Ile: valine Val=1.4:1:1 in BCAs) and three oyster peptide groups (high dose group HD:1.2g/kg, medium dose group MD:0.9g/kg, low dose group LD:0.6 g/kg). After normal groups (NC) are fed with normal feed, the other 6 groups are continuously fed with high-sugar and high-fat feed, after 4 weeks of feeding, eyeballs of the mice are subjected to blood taking and centrifugation to obtain serum, then the mice are killed for dissection, and the taken liver and muscle tissues are cleaned by normal saline and stored in liquid nitrogen.
6.3 Analysis of diet and fasting blood glucose levels in mice
During the experiment, the ingestion and water intake of the mice are recorded every day; fasting blood glucose values (FBG) were recorded weekly: before measuring the fasting blood glucose level, taking blood from the tail vein of the mouse without water control overnight, standing at room temperature, centrifuging for 15min at 8000r/min, taking 250 mu L supernatant, measuring the fasting blood glucose level by using a glucose measuring kit instruction method, and evaluating the improvement effect of oyster peptide rich in BCAs on diet and fasting blood glucose level abnormality of the type II diabetic mouse.
6.4 Oral glucose tolerance (OGTT) analysis
OGTT analysis was performed 2 days prior to mice sacrifice. All experimental groups of mice were fasted for 12h and then filled with glucose solution (2.0 g/kg. W.). Blood glucose levels were then measured by tail bleeding at 0, 30, 60, 90, 120min after feeding, plotted on time as the abscissa and blood glucose levels as the ordinate, and the area under each curve (AUC, calculated by GRAPHPAD PRISM version 5.0 software) was compared to evaluate the improvement effect of BCAAs-enriched oyster peptides on glucose tolerance in type ii diabetic mice.
6.5 Determination of relevant indicators
6.5.1 Serum insulin related index determination
Analysis of insulin related index in serum: the blood glucose FBG (mmol L -1), insulin content FSI (mIU L -1), glucagon-like peptide-1 (GLP-1) and Glycosylated Serum Protein (GSP) content in the serum of each group of mice are measured by adopting a kit, and the following insulin related indexes are calculated by adopting a HOMA method, so that the regulation and control effect of oyster peptide rich in BCAs on the insulin of diabetic mice is examined.
Insulin resistance index HOMA-ir= (fbg×fsi)/22.5
Beta cell dysfunction index (HOMA-beta) = (20×fsi)/(FBG-3.5)
Saccharogenesis (glycogen) analysis in 6.5.2 liver and muscle
Hydrolyzing the extract of mouse liver and muscle in a water bath at 95deg.C for 20 min, centrifuging for 5min (at normal temperature and 8000 rpm) to obtain supernatant, measuring liver glycogen and muscle glycogen content in each group of mouse liver and muscle by using the kit, and analyzing gluconeogenesis.
7. Statistical analysis
The test results obtained are expressed as mean ± standard deviation, each set of tests being performed in parallel 3 times. The test data were analyzed using SPSS data analysis software, the significance of the differences between the samples was determined by single factor analysis of variance (One-wayANOVA), P <0.05 indicated the significance differences and was statistically significant, and data were plotted using the software of Origin 2023b, weblog, etc.
8. Results and analysis
8.1 Preparation and component analysis of oyster peptides enriched in BCAs
As shown in Table 1, after the oyster peptide prepared by the enzymolysis method is subjected to alcohol precipitation and desugarization, the polysaccharide content of the oyster peptide is obviously reduced from 20.23% to 14.68%, the protein content is increased from 53.84% to 67.27%, the polypeptide content is also increased from 34.42% to 43.56% (P is less than 0.05), and the short peptide enrichment effect is primarily achieved. The activated carbon adsorption is mainly aimed at removing free aromatic amino acid, and the method can prepare high F value (rich in BCAs) oligopeptide. The results of the invention show that after the treatment of alcohol precipitation and desugaring, the polysaccharide content of oyster peptide is obviously reduced, the protein and polypeptide content is also improved, and the oyster peptide basic component after the activated carbon adsorption has no obvious change (P is more than 0.05) compared with the oyster peptide basic component after the alcohol precipitation and desugaring.
TABLE 1 oyster peptide basic composition analysis
Note that: different letters represent significant differences (P < 0.05) in the composition (same column) of the different treated oyster peptides.
8.2 Structural characterization of oyster peptides enriched in BCAAs
8.2.1 Relative molecular Mass distribution
As shown in FIG. 1, the oyster peptides prepared by the neutral protease enzymolysis method have the relative molecular weight concentrated below 2000Da and account for 94.3% of the total proportion. After the alcohol precipitation desugarization treatment, the relative molecular mass of the oyster peptide is mainly concentrated below 2000Da and accounts for 90.7% of the total proportion, wherein the molecular weight is 180-1000Da and accounts for 62.21%, because macromolecular substances and partial polysaccharide in the oyster peptide are not dissolved by ethanol and separated out in the alcohol precipitation desugarization treatment process, and micromolecular peptide (< 1000 Da) is enriched in supernatant fluid. Then, after the free aromatic amino acid in the oyster peptide is further removed by activated carbon adsorption, the small molecular peptide (< 1000 Da) is increased to 64.12 percent.
8.2.2 Amino acid composition analysis
As shown in Table 2, after the alcohol precipitation desugarization treatment, macromolecular substances and partial polysaccharide in oyster peptide are removed, so that the micromolecular peptide is enriched, and the total amount of hydrolyzed amino acid (TAA) is increased from 350.44mg/g to 621.18mg/g. Wherein the ratio of Essential Amino Acid (EAA) to Hydrophobic Amino Acid (HAA) is improved, the content of the essential amino acid in the oyster peptide crude product is 33.69 percent, the content of the essential amino acid is improved to 37.14 percent after the oyster peptide crude product is subjected to alcohol precipitation and desugarization, and is improved to 38.75 percent after the oyster peptide crude product is subjected to activated carbon adsorption, which indicates that the alcohol precipitation and desugarization and the activated carbon adsorption treatment have a certain enrichment effect on the essential amino acid and branched amino acid of the oyster peptide.
In addition, compared with the oyster peptide crude product, the BCAs proportion after the ethanol precipitation and desugaring is improved from 15.78% to 16.37%; after the activated carbon adsorption treatment, the BCAAs ratio was further increased to 18.77%. It has been reported that polypeptides having high hypoglycemic activity generally contain a high proportion of hydrophobic amino acids, which inhibit the enzymatic activity by binding to their corresponding enzymes via different sequence structures. Branched-chain amino acids are essential amino acids that cannot be synthesized by the human body and can only be ingested by the diet, and thus oyster peptides are a good source of dietary supplementation of branched-chain amino acids.
TABLE 2 amino acid composition of oyster peptides after various treatments
Note that: different letters represent significant differences (P < 0.05) between oyster peptides (same row of data) of amino acids in different treatments; NEAA is a non-essential amino acid.
8.2.3 Peptide profiling and peptide sequence analysis
Identifying peptide spectrum sequences of oyster peptides by adopting UPLC-Q-TOF technology, comparing the oyster peptide crude products with a database, and detecting 261 peptide sequences by matching 41 proteins with the oyster peptide crude products; matching the oyster peptides after the desugared by alcohol precipitation to 57 proteins, and detecting 231 peptide sequences; the oyster peptides after activated carbon adsorption are matched with 21 proteins, 213 peptide sequences are detected, the reliability, the peptide chain length and the number of matching segments are comprehensively considered, and the main peptide sequences of the oyster peptide crude product and the oyster peptide subjected to desugaring and activated carbon adsorption treatment (6 peptide sequences are selected for each treatment) are shown in table 3. The specific structure of bioactive peptides, such as amino acid composition, sequence, chain length, hydrophobicity, electrostatic charge, and the like, is closely related to their bioactivity. The main peptide sequence of oyster peptide crude product has relative molecular mass mainly concentrated below 2000Da, and the composition content of hydrophobic amino acid in the sequence is relatively low.
After the alcohol precipitation and desugarization, the sequences with shorter peptide chains are enriched, the molecular weight is mainly concentrated at 180-500Da, and the main peptide sequences can be seen that the ratio of hydrophobic amino acids is improved, such as dipeptide IP and tripeptide LPA, the compositions are all hydrophobic amino acids, and the matching number is high. In addition, research shows that hydrophobic amino acids such as proline and alanine are present at the N1 position or the N2 position in the active peptide sequence, or aromatic amino acids such as isoleucine and phenylalanine are present at the N1 position, and the active peptide DPP-IV with the proline at the C1 position has remarkable blood glucose inhibition activity, so that the DPP-IV inhibition peptide of the oyster peptide after desugarization has remarkable structural characteristics. Compared with the oyster peptide desugared by alcohol precipitation, the oyster peptide has no obvious difference in relative molecular mass distribution of oyster peptide sequences and similar sequences after activated carbon adsorption treatment, but the branched chain amino acids (BCAs) at two ends of a peptide chain are more prominent in proportion, such as tripeptide LEI, and the BCAs are at two ends, and the result is consistent with the analysis result of the amino acid composition. Alpha-amylase inhibiting peptide sequences typically contain hydrophobic amino acids such as proline and leucine at the N-terminus, leucine at the C-terminus, and phenylalanine at both ends, while peptides with branched and cationic residues are more prone to binding to alpha-amylase. From the above three main peptide fragments of oyster peptide, it can be seen that oyster peptide after desugared by alcohol precipitation has 4 peptide chains, and leucine at N-terminal or C-terminal, which indicates that the alpha-amylase inhibitory activity is probably better.
Therefore, based on the sequence characteristic analysis, compared with the crude oyster peptide, the oyster peptide has the advantage that the hypoglycemic activity can be improved to different degrees after different treatments.
TABLE 3 oyster peptide Main peptide sequences of different treatments
8.3 In vitro hypoglycemic Activity of oyster peptides enriched with BCAs
As shown in FIG. 2, the IC 50 value of the oyster peptide crude product on alpha-amylase is 0.096mg/mL, the IC 50 value of the oyster peptide after alcohol precipitation desugarization on alpha-amylase is 0.025mg/mL, the oyster peptide crude product becomes gentle after the concentration is 0.0313mg/mL (the inhibition rate reaches 60%), the inhibition rate reaches more than 80%, and the inhibition rate is obviously higher than that of the oyster peptide crude product (P is less than 0.05). The inhibition rate of the oyster peptide adsorbed by the activated carbon to the alpha-amylase is greatly reduced, the trend of rising and then falling is presented, the highest inhibition rate is 21.9%, and the concentration is 0.188mg/mL. The intake ratio of branched-chain amino acid can influence intestinal flora and fat metabolism in human bodies, particularly leucine, the higher the leucine ratio in active peptide is, the lower the inhibition rate of alpha-amylase is, the synthesis of alpha-amylase and trypsin in pancreatic tissues of dairy cows can be enhanced in vivo, and the synthesis of alpha-amylase and trypsin can be stimulated as nutrition signals through an mTOR (mammalian target of rotation) pathway, so that the inhibition rate of the activated carbon adsorbed oyster peptide to the alpha-amylase can be reduced.
The IC 50 value of oyster peptide crude product to alpha-glucosidase is 0.273mg/mL. The IC 50 value of the desugared oyster peptide on the alpha-glucosidase is 0.2mg/mL, the inhibition rate of the desugared oyster peptide on the alpha-glucosidase is 0.5mg/mL, the inhibition rate of the desugared oyster peptide is up to 70%, and the inhibition effect of the whole alpha-glucosidase is better than that of the oyster peptide crude product (P is less than 0.05). The IC 50 value of the oyster peptide treated by the activated carbon for inhibiting the alpha-glucosidase is 0.2mg/mL, the inhibition rate reaches more than 76% along with the increase of the concentration, and the inhibition effect is better than that of the oyster peptide crude product (P is less than 0.05).
The IC 50 value of the oyster peptide crude product on DPP-IV enzyme is 1.1mg/mL. The IC 50 value of the desugared oyster peptide on DPP-IV enzyme inhibition is 0.8mg/mL, and the effect is better than that of the original product (P is less than 0.05). The IC 50 value of the oyster peptide after activated carbon adsorption on DPP-IV enzyme inhibition is 1.05mg/mL, and the effect is between the two.
In summary, after the oyster peptide is subjected to alcohol precipitation and desugarization treatment, the in vitro hypoglycemic activity (comprising the inhibition capability to alpha-amylase, alpha-glucosidase and DPP-IV) is improved, and after the oyster peptide is adsorbed by the activated carbon, the in vitro hypoglycemic activity is reduced, and particularly the inhibition activity to alpha-amylase is greatly reduced, which is possibly related to the adsorption of part of small molecular peptides by the activated carbon.
8.4 Effect of in vitro mock digestion on oyster peptide amino acid composition
As shown in FIG. 3, after desugaring and activated carbon adsorption treatment, the total content of leucine (L: leu), isoleucine (I: ile) and valine (V: val) (i.e., branched chain amino acid BCAs) in oyster peptide is increased in percentage of total amino acid content, which indicates that peptide fragments containing BCAs in oyster peptide are enriched after alcohol precipitation and activated carbon adsorption, and the oyster peptide has the amino acid composition characteristics of DPP-IV inhibitory peptide. Further analyzing the specific proportion of three kinds of BCAs in oyster peptide, the proportion of Leu, ile and Val in crude oyster peptide is found to be 2.1:1:1.2, the proportion of Leu, ile and Val in oyster peptide after alcohol precipitation desugarization is 1.4:1:1, the proportion of Leu, ile and Val in oyster peptide after activated carbon adsorption is 1.5:1:1, and meanwhile, the simulation of gastrointestinal digestion results shows that the content and proportion of Leu in oyster peptide after activated carbon adsorption are obviously higher than those of oyster peptide after alcohol precipitation desugarization, and research reports that rich BCAs, especially Leu possibly contributes to interaction of activated peptide and membrane lipid bilayer, promotes the interaction of activated peptide into cells, so that the activity of the oyster peptide for reducing sugar is improved, and the digestion stability is also better. The results in conclusion show that the oyster peptide rich in BCAs obtained after desugaring and activated carbon adsorption treatment has the potential of improving the hypoglycemic activity (especially DPP-IV inhibitory activity) and digestion stability.
8.5 In vitro simulation of changes in vitro hypoglycemic Activity of oyster peptides after digestion
As shown in FIG. 4, when the mass concentration of the oyster peptide crude product is 0.313mg/mL, the inhibition rate of alpha-amylase before digestion is 73.58%, and after gastric digestion, the inhibition rate is obviously reduced to 40.99% (P < 0.05); after gastrointestinal digestion, the inhibition rate of oyster peptide to alpha-amylase is reduced to 48.88%, which is 7.89% higher than that after gastric digestion. When the mass concentration of the oyster peptide crude product is 0.417mg/mL, the inhibition rate of alpha-glucosidase is 58.80% before digestion, and the inhibition rate is obviously reduced to 33.50% after gastric digestion; after gastrointestinal digestion, the inhibition rate of oyster peptide to alpha-glucosidase is reduced to 18.71%. When the mass concentration of the oyster peptide crude product is 1.0mg/mL, the inhibition rate of DPP-IV before digestion is 44.77%, and the inhibition rate of DPP-IV after gastric digestion is up to 48.78%; after gastrointestinal digestion, the inhibition rate of the oyster peptide on DPP-IV is increased to 53.78 percent, and the inhibition rate of the oyster peptide crude product on DPP-IV enzyme is relatively stable before and after digestion, which shows that the digestion stability is better, which is possibly related to the higher proline content in the peptide sequence and the positions at the two ends of the peptide chain, and is consistent with the analysis results of 8.2.2 and 8.2.3.
When the mass concentration of the oyster peptide subjected to the alcohol precipitation and desugarization is 0.313mg/mL, the inhibition rate of alpha-amylase before digestion is 83.12 percent (which is obviously higher than 73.58 percent of the crude oyster peptide), and the comparison of peptide sequence characteristics (Table 3) shows that the BCAs proportion in the oyster peptide chain after the oyster peptide is subjected to the alcohol precipitation and desugarization is increased (consistent with the analysis result of 8.2.2 amino acids), so that the inhibition rate of the oyster peptide subjected to the desugarization to the alpha-amylase is increased. The inhibition rate of the alcohol precipitation desugared oyster peptide is obviously reduced to 50.31 percent (P < 0.05) after the gastric digestion; after gastrointestinal digestion, the inhibition rate of oyster peptide to alpha-amylase is reduced to 59.59%, which is 9.28% higher than that after gastric digestion. When the mass concentration of the oyster peptide is 0.417mg/mL, the inhibition rate of alpha-glucosidase is 63.69% before digestion, and the inhibition rate is obviously reduced to 48.26% after gastric digestion; after gastrointestinal digestion, the inhibition rate of oyster peptide to alpha-glucosidase is reduced to 22.58%. When the mass concentration of oyster peptide after the desugared by alcohol precipitation is 1.0mg/mL, the inhibition rate of DPP-IV before digestion is 52.49 percent, and the inhibition rate is increased to 53.62 percent after gastric digestion; after gastrointestinal digestion, the inhibition rate of the oyster peptide to DPP-IV is obviously increased to 60.99%, the overall change is more gentle, and the digestion stability is better.
When the mass concentration of the activated carbon adsorbed oyster peptide is 0.313mg/mL, the inhibition rate of the activated carbon adsorbed oyster peptide to alpha-amylase before digestion is 8.65%, and the inhibition rate of the activated carbon adsorbed oyster peptide after gastric digestion is reduced to 5.97%; after gastrointestinal digestion, the inhibition rate of oyster peptide on alpha-amylase is reduced to 5.57%, which is possibly related to the increase of leucine content (8.2.2 amino acid analysis result), and researches report that the active peptide has a promotion effect on the activity of alpha-amylase when the leucine content is higher. When the mass concentration of the activated carbon adsorbed oyster peptide is 0.417mg/mL, the inhibition rate of alpha-glucosidase before digestion is 58.45%, and the inhibition rate is obviously reduced to 35.50% after gastric digestion; after gastrointestinal digestion, the inhibition rate of the oyster peptide on the alpha-glucosidase is reduced to 20.53%, which shows that the gastrointestinal digestion affects the structure on which the oyster peptide inhibits the alpha-glucosidase activity. When the mass concentration of the activated carbon adsorbed oyster peptide is 1.0mg/mL, the inhibition rate of DPP-IV before digestion is 48.46%, and after gastric digestion, the inhibition rate is increased by 51.58%; after gastrointestinal digestion, the inhibition rate of the oyster peptide to the DPP-IV is increased to 54.67%, which shows that the DPP-IV inhibition peptide in the oyster peptide has better digestion stability.
In conclusion, the oyster peptides subjected to alcohol precipitation and desugarization have the best hypoglycemic activity, but the digestion stability is not improved, the hypoglycemic activity of the three oyster peptides is obviously reduced after gastrointestinal digestion, and the DPP-IV inhibition peptide fragments in the oyster peptides subjected to alcohol precipitation and desugarization and activated carbon adsorption treatment are more stable.
8.6 In vitro simulation of structural changes in oyster peptide sequence after digestion
Literature studies have shown that the position of the amino acid at the C-terminus or N-terminus is a key feature of biologically active peptides, and that the amino acid sequence positions that currently have a greater influence on the activity of the active peptide are mainly at the terminal (C1 and N1), the penultimate (C2 and N2) and the penultimate (C3 and N3) positions, so that the amino acid sequence features at the C1-3 and N1-3 positions are key to the activity of the active peptide. In addition, the amino acid sequence structure of oyster peptides has a close relationship with their digestion stability, e.g., active peptides containing proline and hydroxyproline residues are generally resistant to degradation by digestive enzymes, whereas active peptides with lysine or arginine residues at the C-terminus are susceptible to degradation by trypsin. As shown in A in FIG. 5, A in FIG. 6 and A in FIG. 7, the C3-terminal proline characteristics of the oyster peptides in three different treatment modes are obvious, the C1-terminal lysine content of the oyster peptide crude product is lower than that of the oyster peptide crude product, and the C2-terminal lysine and arginine characteristic sequences are obvious, which indicates that the oyster peptides have poor digestion stability and are easily degraded by protease to influence the hypoglycemic activity.
As shown in A-C in FIG. 5, the proportion of peptide fragments with alanine (Ala) at the N1 end is increased from 3.3% to 6% before digestion of the crude oyster peptide, the proportion of peptide sequences with isoleucine (Ile) at the N2 end is gradually and obviously increased from 4% to 20% along with the progress of digestion, and the in vitro hypoglycemic activity result also shows that the DPP-IV inhibition activity of the oyster peptide has an increasing trend, which indicates that the structural characteristics of the oyster peptide sequences, particularly the structural characteristics of the oyster peptide sequences, including the amino acids, including the leucine and the isoleucine, are related to the DPP-IV inhibition activity of the oyster peptide sequences.
The presence or absence and the size of the alpha-glucosidase inhibitory activity are determined by the characteristic amino acids of the active peptide and the sequence positions thereof, including serine, threonine, tyrosine, lysine and arginine at the N-terminus, proline at the C2-terminus, and methionine or alanine at the C-terminus. As shown in FIG. 5, the proportion of leucine (Leu) at the N1 end of oyster peptide after desugared ethanol precipitation is reduced from 10% to 8% after gastric digestion, and the proportion is reduced to less than 1% after gastrointestinal digestion, which shows that the degradation of the alpha-glucosidase inhibitory activity is influenced by rapid change of gastrointestinal acid-base environment and is possibly caused by the degradation of gastrointestinal digestive enzymes to change the sequence characteristics of the active peptide. In addition, the literature reports that peptide chains containing hydrophobic amino acids, essential amino acids and branched amino acids can exhibit remarkable inhibitory activity on α -glucosidase by interaction forces such as hydrophobic interaction, especially peptide chains containing leucine and proline. As shown in the A of figure 7, the top three positions of the N1 end amino acid residue are all hydrophilic amino acids (Arg, lys, thr), so that the inhibition activity of the peptide to alpha-amylase is greatly influenced, as shown in the B-C of figure 7, the peptide characteristics of the peptide sequence N1 end and N2 end of the peptide sequence are not obvious (P is more than 0.05) after gastric digestion and gastrointestinal digestion of the peptide sequence N1 end and N2 end of the peptide sequence are branched amino acids, and the digestion stability of DPP-IV inhibition peptide in the peptide is better.
8.7 In vivo experiment results
(1) The mice with type II diabetes induced by high sugar and high fat feed combined with STZ have hyperglycemia and increased intake of food and water. As can be seen from fig. 8, the normal group mice showed less variation in food intake and water intake at 1-4 weeks of feeding, while the diabetic model group (DC) mice showed significantly increased food intake (P < 0.05). Compared with the DC group mice, the intake and water uptake of the mice fed with the oyster peptide rich in the BCAs are obviously reduced (P < 0.05), and especially the improvement effect of the oyster peptide (LD) group rich in the BCAs with low dosage is obvious, which shows that the oyster peptide rich in the BCAs with low dosage has better effect on improving the intake and water drinking of the diabetic mice, and the improvement effect on the water uptake is better than that of the BCAs standard group.
(2) As can be seen from fig. 9, in the fed 1-4 weeks, the fasting glycemia (FBG) values of the diabetic model group (DC) mice were all significantly higher than that of the normal group mice (P < 0.05), while the FBG of the oyster peptide fed group mice enriched with branched chain amino acids was significantly lower than that of the diabetic model group mice (P < 0.05), and especially the low dose group (LD) of oyster peptide enriched with BCAAs had a more pronounced trend of decreasing than that of the BCAAs standard group (BCAAs), the effect was better, i.e., the oyster peptide enriched with BCAAs had a more remarkable effect in improving FBG of the diabetic mice; as can be seen from fig. 10 (OGTT, AUC), the glucose tolerance of LD mice was close to that of Positive Control (PC), and the effect was significantly better than that of BCAAs and oyster peptides in medium and high dose groups (p < 0.01). Taken together, the results show that oyster peptides rich in BCAAs have remarkable effects in improving fasting glucose (FBG) and oral glucose tolerance (OGTT) of diabetic mice.
(3) The main clinical symptoms of diabetics include hyperglycemia, insulin resistance, beta cell dysfunction, etc. As can be seen from table 4, the Insulin (INS) content in serum of the oyster peptide group (LD, MD, HD) mice enriched with BCAAs was significantly reduced (P < 0.05) compared to the diabetes model group (DC) mice, and the oyster peptide group insulin content was significantly lower than that of the BCAAs standard group. In addition, data analysis of insulin resistance index (HOMA-IR) and beta cell dysfunction index (HOMA- β) showed that BCAAs-enriched oyster peptide groups (LD, MD, HD) significantly improved both beta cell dysfunction and insulin resistance in diabetic mice compared to DC group mice, wherein BCAAs-enriched oyster peptide effects were superior to BCAAs standard groups in improving insulin resistance.
TABLE 4 oyster peptides enriched in BCAs modulating effects on insulin resistance in type II diabetic mice
Note that: NC: normal group; DC: a diabetes model group; PC: positive control group (acarbose); BCAAs group: branched chain amino acid group (0.09 g/kg); LD: oyster peptide low dose group (0.6 g/kg); MD: dose group in oyster peptide (0.9 g/kg); HD: oyster peptide high dose group (1.2 g/kg). All data are expressed as mean ± standard deviation SD (n=12). Shoulder-annotated different letters in the same column of data indicate that P <0.05 was statistically significant by analysis of variance. All data are expressed as mean ± standard deviation SD (n=12).
(4) Glucagon-like peptide 1 (GLP-1), a polypeptide hormone secreted by intestinal L cells, is capable of promoting insulin secretion by islet beta cells in a glucose-dependent manner; reducing alpha cell reaction, inhibiting glucagon secretion and the like, namely GLP-1 can systematically regulate blood sugar level in a multi-layer way, is an advantageous target for treating type II diabetes, but GLP-1 is extremely easy to be degraded in vivo by dipeptidyl peptidase IV (DPP-IV) enzyme (about 95% of activity is lost), and has extremely short half-life. Glycated Serum Proteins (GSPs) reflect the more recent effects of diabetes treatment, knowing that diabetes controls blood glucose levels for 1-2 weeks. From fig. 11, the GLP-1 content of the model group is significantly lower than that of the normal group, while the GLP-1 content of the oyster peptide group rich in BCAAs is significantly higher than that of the model group (P < 0.05), which indicates that the oyster peptide rich in BCAAs has the effects of significantly inhibiting DPP-iv activity (in vitro hypoglycemic activity data also confirms the result) and prolonging the half-life of GLP-1. And the effect of the oyster peptide rich in BCAs for prolonging the half life of GLP-1 is better than that of a BCAs standard group. GSP content analysis shows that the effects of the BCAs group and the positive group are equivalent in terms of blood glucose level control effect, but the effects of the oyster peptide rich in BCAs on blood glucose level control are obviously superior to those of the BCAs group and the positive group (P < 0.05), and particularly, the oyster peptide rich in BCAs with low dosage has the best effect. Taken together, the results show that oyster peptides rich in BCAs have remarkable effect in regulating blood sugar of type II diabetes.
(5) Difficulty in glycogen synthesis in the liver and muscle is also one of the manifestations of type ii diabetes, and therefore liver glycogen content and muscle glycogen content are important indicators for evaluation of improvement of the condition in diabetic patients. As can be seen from table 2, the liver glycogen and myoglycogen in the diabetic model group mice were significantly reduced (P < 0.05) compared to the normal group mice, whereas the liver glycogen and myoglycogen content in the oyster peptide group enriched with BCAAs was significantly higher than that in the model group (P < 0.05), and particularly the myoglycogen and liver glycogen content in the low dose group was comparable to that in the positive group. In addition, BCAAs group also had significant liver glycogen and myoglycogen synthesis promoting effects. Taken together, the data demonstrate that BCAAs-enriched oyster peptides regulate glycometabolism by promoting liver glycogen and myoglycogen synthesis, alleviating symptoms of diabetes-induced glycometabolism disorders.
TABLE 5 Effect of oyster peptides on liver glycogen and myoglycogen in type II diabetic mice
Note that: NC: normal group; DC: a diabetes model group; PC: positive control group (acarbose); BCAAs group: branched chain amino acid group (0.09 g/kg); LD: oyster peptide low dose group (0.6 g/kg); MD: dose group in oyster peptide (0.9 g/kg); HD: oyster peptide high dose group (1.2 g/kg). All data are expressed as mean ± standard deviation SD (n=12). Shoulder-annotated different letters in the same column of data indicate that P <0.05 was statistically significant by analysis of variance.
The embodiment shows that the invention explores the enrichment effect of alcohol precipitation desugarization and activated carbon adsorption treatment on branched chain amino acids in oyster peptides, and researches the correlation between the branched chain amino acid sequence structure of oyster peptides and the in-vitro hypoglycemic activity and digestion stability. The results show that the hypoglycemic activity and the digestion stability of oyster peptide are related to the peptide fragment position of branched chain amino acid. The oyster peptide after desugaring has the branched chain amino acid enriched in the peptide segment of the front three positions of the C end and the N end, so that the activity of lowering blood sugar is improved; the oyster peptide branched-chain amino acid after the activated carbon adsorption treatment is further enriched, but the inhibition activity of starch digestive enzyme is not obviously improved, and particularly the inhibition activity of alpha-amylase is obviously reduced; however, the oyster peptide has more stable inhibitory activity on DPP-IV, namely the DPP-IV inhibitory peptide rich in branched chain amino acid in the oyster peptide has good digestion stability. The invention analyzes the in vitro hypoglycemic activity and digestion stability of oyster peptide from the angles of branched chain amino acid enrichment and sequence structure, and provides theoretical basis and scientific guidance for development and utilization of oyster hypoglycemic products.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (5)
1. An oyster DPP-iv inhibitory peptide mixture enriched in branched-chain amino acids, wherein said oyster DPP-iv inhibitory peptide comprises the following peptide fragments: LPA, LEI, VVD, ICIIM, LSEPSEVVGPITP and SGEPGPEGPAGPI;
The preparation method of the oyster DPP-IV inhibitory peptide comprises the following steps:
Homogenizing oyster meat according to a feed liquid ratio of 1g: adding 3mL of water, homogenizing, adding neutral protease for enzymolysis, centrifuging, collecting supernatant, and drying to obtain oyster peptide; the enzymolysis condition is water bath enzymolysis for 4 hours at 50 ℃;
The oyster peptide is desugared by adopting an ethanol precipitation method to obtain ethanol precipitation desugared oyster peptide;
Adsorbing and treating the alcohol precipitation desugared oyster peptide by using activated carbon to obtain oyster DPP-IV inhibitory peptide rich in branched chain amino acid;
the method for desugaring by the alcohol precipitation method comprises the following steps:
mixing and dissolving the oyster peptide and water according to the mass-volume ratio of 1:20, centrifuging, and collecting filtrate;
Mixing the filtrate with 95% ethanol according to a volume ratio of 1:3, standing overnight at low temperature, centrifuging, collecting supernatant, removing ethanol, and drying to obtain the ethanol precipitation desugared oyster peptide;
The method for adsorbing and treating the alcohol precipitation desugared oyster peptide by using the activated carbon comprises the following steps:
Dissolving the alcohol precipitation desugared oyster peptide, adding active carbon into oyster peptide solution according to the mass volume percentage of 5%, carrying out water bath for 3 hours at 20 ℃, stirring, centrifuging, collecting supernatant, filtering and drying to obtain the oyster DPP-IV inhibitory peptide rich in branched chain amino acid.
2. The oyster DPP-iv inhibitory peptide mixture rich in branched-chain amino acids according to claim 1, wherein the homogenization conditions are: regulating pH of the mixture of oyster pulp homogenate and water to 7.0, homogenizing at 8000r/min for 2min.
3. The oyster DPP-iv inhibitory peptide mixture rich in branched-chain amino acids according to claim 1, wherein the activated carbon is added after the pH of the oyster peptide solution is adjusted to 7.0.
4. Use of the oyster DPP-iv inhibitory peptide mixture enriched in branched-chain amino acids according to claim 1 for the preparation of a hypoglycemic product.
5. A hypoglycemic product, characterized in that the oyster DPP-IV inhibitory peptide mixture rich in branched-chain amino acids as defined in claim 1 is used as an active ingredient.
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