CN111662361B - Oligopeptide, weight-losing composition, preparation method and application - Google Patents
Oligopeptide, weight-losing composition, preparation method and application Download PDFInfo
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- CN111662361B CN111662361B CN202010571947.3A CN202010571947A CN111662361B CN 111662361 B CN111662361 B CN 111662361B CN 202010571947 A CN202010571947 A CN 202010571947A CN 111662361 B CN111662361 B CN 111662361B
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- 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
- 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
- A61P3/04—Anorexiants; Antiobesity agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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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- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Public Health (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- Obesity (AREA)
- General Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
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- Biotechnology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides an oligopeptide, a weight-losing composition, a preparation method and an application. Wherein the oligopeptide has the following amino acid sequence: Glu-Glu-Ala-Ala-Ser-Leu-Arg. The oligopeptide has effect of inhibiting pancreatic lipase activity, is natural, safe, has no adverse side effects, and can be taken for a long time.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to an oligopeptide, a weight-losing composition, a preparation method and an application.
Background
In recent years, obesity is the primary health problem of human beings, and not only can cause cardiovascular diseases such as diabetes, hypertension, dyslipidemia and coronary heart disease, but also can cause some chronic diseases and even psychological diseases, so that the quality of life is reduced. Heredity, disease and excess energy all cause obesity, and especially the intake of food fat is more likely to increase the risk of obesity. Therefore, the control of the intake or absorption of dietary fat can effectively control obesity.
The main reason why dietary triglyceride is degraded into diglyceride, monoglyceride, glycerol and fatty acid only by the action of lipase in esophagus is absorbed by human body. Among these lipases, Pancreatic Lipase (PL) is an important enzyme for hydrolyzing fat, and thus inhibition of Pancreatic lipase activity and prevention of fat hydrolysis and absorption are one of the most effective methods for treating obesity.
At present, most of the drugs for losing weight by inhibiting the lipase activity in the market are synthetic drugs, such as orlistat, dihydroberberine and the like. Although having the efficacy of losing weight, all have side effects of different degrees, such as: rebound, anorexia, insomnia, palpitation, etc. Therefore, the search for pancreatic lipase inhibitors which are natural, safe, free of toxic and side effects and can be taken for a long time becomes a hot point of research.
Fructus Hippophae is also called as Hippophae Rhamnoides, Hippophae Sprensis, Hippophae rhamnoides, and Hippophae rhamnoides of Elaeagnaceae. The sea-buckthorn is wild, is widely planted for soil conservation and sand fixation in recent years, and at present, reports on preparation of enzymatic hydrolysis products rich in pancreatic lipase inhibitory activity by controlled enzymatic hydrolysis of sea-buckthorn protein are not seen.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the defect that most of the drugs for losing weight by inhibiting the activity of lipase have side effects in the prior art, so that an oligopeptide with pancreatic lipase inhibition activity is prepared by enzymolysis of sea buckthorn seeds, and specifically, an oligopeptide, a weight-losing composition, a preparation method and an application are provided.
Therefore, the invention provides the following technical scheme:
an oligopeptide having the amino acid sequence: Glu-Glu-Ala-Ala-Ser-Leu-Arg.
The invention also provides a weight-losing composition comprising the oligopeptide.
Furthermore, the content of the oligopeptides in the weight-losing composition is 0.5-1.5g/100 g.
Further, the weight loss composition is a dietary supplement.
The invention also provides a preparation method of the weight-losing composition, which comprises the following steps:
pulverizing semen Hippophae, adding water, adjusting pH to 8.0-10.0, performing enzymolysis, inactivating enzyme, and collecting supernatant to obtain the composition for reducing weight.
Further, the mass ratio of the enzyme to the crushed sea buckthorn seeds is (0.2-2.0): 100.
further, the mass ratio of the enzyme to the crushed sea buckthorn seeds is (0.2-0.5): 100.
further, alkaline protease and/or compound protease are/is adopted in enzymolysis.
Further, the flavourzyme may be subjected to enzymolysis in combination with the above-mentioned enzyme.
Further, the enzymolysis temperature is 45-60 deg.C, and the time is 6-24 h.
Further, the mass ratio of the crushed sea buckthorn seeds to water is 1: (10-15).
Further, a centrifugation step before supernatant taking is also included; the temperature of the centrifugation is 3-5 ℃, the time is 10-20min, and the rotating speed is 8000-12000 rpm.
Further, a freeze drying step after taking the supernatant is also included.
The invention also provides a method for purifying the oligopeptide, which comprises the following steps:
sequentially carrying out ultrafiltration, neutral resin and chromatographic column separation on the weight-reducing composition or the weight-reducing composition prepared by the preparation method of the weight-reducing composition to obtain the oligopeptide.
Further, collecting the filtrate with the molecular weight less than 3kDa during ultrafiltration.
Further, the weight-reducing composition is prepared into 0.1-0.2mg/mL aqueous solution, and the aqueous solution is filtered by an ultrafiltration membrane at the speed of 0.1-1mL/min, and the filtrate with the molecular weight of less than 3kDa is collected.
Further, drying the filtrate after ultrafiltration, preparing into 5-10mg/mL aqueous solution, mixing with neutral resin, and eluting with 30-60% ethanol solution at 0.1-0.2 mL/min.
Further, drying the eluent, preparing into 5-10mg/mL aqueous solution, and separating by using a chromatographic column under the following elution conditions: eluting with 20-40% acetonitrile or methanol, and collecting eluate to obtain the oligopeptide.
The elution rate and the eluent can be adjusted according to actual conditions.
The invention also provides the application of the oligopeptide or the oligopeptide extracted by the oligopeptide purification method or the weight-reducing composition prepared by the preparation method of the weight-reducing composition in inhibiting the activity of pancreatic lipase.
The invention also provides the application of the oligopeptide or the oligopeptide extracted by the oligopeptide purification method or the weight-losing composition prepared by the preparation method of the weight-losing composition in weight-losing foods, medicines or health-care products.
The weight-losing composition provided by the invention can obviously inhibit the activity of pancreatic lipase under the dosage of 2.0-10.0 mg/kg.d.
The technical scheme of the invention has the following advantages:
1. the oligopeptide provided by the invention has the following amino acid sequence: Glu-Glu-Ala-Ala-Ser-Leu-Arg. The oligopeptide has effect of inhibiting pancreatic lipase activity, is natural, safe, has no adverse side effects, and can be taken for a long time.
2. The invention provides a weight-losing composition, which comprises the following oligopeptides in amino acid sequence: Glu-Glu-Ala-Ala-Ser-Leu-Arg, the weight-reducing composition has the effect of inhibiting the activity of pancreatic lipase, is natural and safe, has no toxic or side effect, and can be taken for a long time.
3. The weight-losing composition provided by the invention has the oligopeptide content of 0.5-1.5g/100 g. By limiting the content of the oligopeptide, the effect of inhibiting the activity of pancreatic lipase can be further improved.
4. The weight-losing composition provided by the invention can be used as a dietary supplement. The dietary supplement has the effect of inhibiting the activity of pancreatic lipase, is natural and safe, has no toxic or side effect, and can be taken for a long time.
5. The preparation method of the weight-losing composition provided by the invention comprises the following steps: pulverizing semen Hippophae, adding water, adjusting pH to 8.0-10.0, performing enzymolysis, inactivating enzyme, and collecting supernatant. By adopting the method and controlling the pH value to be 8.0-10.0, the weight-losing composition containing the oligopeptide of the amino acid sequence Glu-Glu-Ala-Ala-Ser-Leu-Arg can be obtained by enzymolysis, has the effect of inhibiting the activity of pancreatic lipase, is natural, safe, free from toxic and side effects and can be taken for a long time.
6. According to the preparation method of the weight-reducing composition provided by the invention, the enzymolysis effect and the content of oligopeptides in the weight-reducing composition can be improved by limiting the mass ratio of the enzyme to the crushed sea buckthorn seeds, so that the effect of inhibiting the activity of pancreatic lipase is improved.
7. The preparation method of the weight-reducing composition provided by the invention can improve the content of oligopeptides in the weight-reducing composition by limiting the type of enzyme and the temperature and time of enzymolysis.
8. The oligopeptide is obtained by sequentially subjecting the weight-reducing composition to ultrafiltration, neutral resin and chromatographic column separation. If the order is changed or other resins such as acidic or basic resins are used, the oligopeptide cannot be separated.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a second mass spectrum of oligopeptide in the slimming composition prepared in example 1 of the present invention;
FIG. 2 is a graph showing the pancreatic lipase inhibition ratios measured in three different addition sequences of inhibitor (I), pancreatic lipase solution (E) and white olive oil substrate emulsion (S) in Experimental example 2 of the present invention;
fig. 3 retention of pancreatic lipase inhibitory activity at different temperatures for the slimming compositions prepared in example 1 of the present invention.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
The structural identification of the oligopeptides adopts HPLC-MS/MS.
The content of oligopeptide in the weight-reducing composition is determined by High Performance Liquid Chromatography (HPLC). The specific detection conditions of the high performance liquid chromatography are as follows:
flow rate: 0.2 mL/min; ultraviolet detection wavelength: 220 nm; a positive ion mode, and the molecular weight is selected from 50 to 3000; a C18 chromatography column;
example 1
This example provides a composition for weight loss containing Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR) oligopeptide, which is prepared by the following steps:
pulverizing sea buckthorn seeds to 100 meshes, and mixing the ground sea buckthorn seeds with water according to a mass ratio of 1: 10 to obtain an aqueous solution of seabuckthorn seeds, adjusting the pH value to 10.0, adding alkaline protease (the mass ratio of the alkaline protease to the crushed seabuckthorn seeds is 0.25: 100), stirring and hydrolyzing at 50 ℃ for 8 hours, and inactivating enzyme at 90 ℃ for 20min after the hydrolysis is finished to obtain a seabuckthorn seed protein hydrolysate;
centrifuging the above protein hydrolysate at 4 deg.C and 8000rpm for 20min, collecting supernatant, and lyophilizing to obtain weight reducing composition containing EEAASLR oligopeptide. Through detection, the content of EEAASLR oligopeptide in the weight-losing composition is 1.35g/100 g.
This example also provides a method for purifying Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR) oligopeptide, comprising the steps of:
preparing the weight-reducing composition into 0.1mg/mL aqueous solution, filtering with PLCGC ultrafiltration membrane at the rate of 0.6mL/min, and collecting filtrate with molecular weight less than 3 kDa; then freeze-drying to obtain powder;
preparing the powder into 8mg/mL aqueous solution, mixing the aqueous solution with DA201-C macroporous resin, eluting by adopting 50% ethanol solution at the rate of 0.2mL/min, and collecting eluent;
freeze-drying the eluate to obtain 5mg/mL aqueous solution, separating with SEP-C18 chromatography column under the following conditions: eluting with 40% ethanol, collecting eluate, freeze drying, and identifying the dried material by HPLC-MS/MS as oligopeptide with amino acid sequence Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR). As shown in fig. 1, specific values are shown in table 1 below.
TABLE 1
Example 2
This example provides a composition for weight loss containing Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR) oligopeptide, which is prepared by the following steps:
pulverizing sea buckthorn seeds to 100 meshes, and mixing the ground sea buckthorn seeds with water according to a mass ratio of 1: 10 mixing to obtain aqueous solution of semen Hippophae, adjusting pH to 9.0, adding compound protease (the mass ratio of the compound protease to pulverized semen Hippophae is 0.5: 100), stirring at 50 deg.C for hydrolysis for 24 hr, and inactivating enzyme at 90 deg.C for 20min to obtain semen Hippophae protein hydrolysate;
centrifuging the above protein hydrolysate at 4 deg.C and 8000rpm for 10min, collecting supernatant, and lyophilizing to obtain weight reducing composition containing EEAASLR oligopeptide. Through detection, the content of EEAASLR oligopeptide in the weight-losing composition is 0.68g/100 g.
This example also provides a method for purifying Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR) oligopeptide, comprising the steps of:
preparing the weight-reducing composition into 0.2mg/mL aqueous solution, filtering with PLCGC ultrafiltration membrane at the rate of 0.1mL/min, and collecting filtrate with molecular weight less than 3 kDa; then freeze-drying to obtain powder;
preparing the powder into 5mg/mL aqueous solution, mixing the aqueous solution with DA201-C macroporous resin, eluting by adopting 30% ethanol solution at the rate of 0.1mL/min, and collecting eluent;
freeze-drying the eluent to prepare 10mg/mL aqueous solution, and separating by using a SEP-C18 chromatographic small column under the following elution conditions: eluting with 20% ethanol, collecting eluate, freeze drying, and identifying the dried substance by HPLC-MS/MS to obtain oligopeptide with the following amino acid sequence Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR).
Example 3
This example provides a composition for weight loss containing Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR) oligopeptide, which is prepared by the following steps:
pulverizing sea buckthorn seeds to 100 meshes, and mixing the ground sea buckthorn seeds with water according to a mass ratio of 1: 10 mixing to obtain an aqueous solution of seabuckthorn seeds, adjusting the pH value to 9.0, adding alkaline protease and compound protease (the mass ratio of the alkaline protease to the compound protease to the crushed seabuckthorn seeds is 0.25: 025: 100), stirring and hydrolyzing at 60 ℃ for 6h, and inactivating the enzyme at 90 ℃ for 20min after the hydrolysis is finished to obtain a seabuckthorn seed protein hydrolysate;
centrifuging the above protein hydrolysate at 4 deg.C and 8000rpm for 10min, collecting supernatant, and lyophilizing to obtain weight reducing composition containing EEAASLR oligopeptide. Through detection, the content of EEAASLR oligopeptide in the weight-losing composition is 0.82g/100 g.
This example also provides a method for purifying Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR) oligopeptide, comprising the steps of:
preparing the weight-reducing composition into 0.15mg/mL aqueous solution, filtering with PLCGC ultrafiltration membrane at a rate of 1mL/min, and collecting filtrate with molecular weight less than 3 kDa; freeze drying to obtain powder 1;
preparing the powder 1 into 10mg/mL aqueous solution, mixing the aqueous solution with DA201-C macroporous resin, then eluting by adopting 60% ethanol solution at the rate of 0.1mL/min, and collecting eluent;
freeze-drying the eluent to prepare 8mg/mL aqueous solution, and separating by using a SEP-C18 chromatographic small column under the following elution conditions: eluting with 30% ethanol, collecting eluate, freeze drying, and identifying the dried substance by HPLC-MS/MS to obtain oligopeptide with the following amino acid sequence Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR).
Example 4
This example provides a composition for weight loss containing Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR) oligopeptide, which is prepared by the following steps:
pulverizing sea buckthorn seeds to 100 meshes, and mixing the ground sea buckthorn seeds with water according to a mass ratio of 1: 15 mixing to obtain aqueous solution of semen Hippophae, adjusting pH to 8.0, adding alkaline protease, compound protease, and flavourzyme (the mass ratio of alkaline protease, compound protease, flavourzyme and pulverized semen Hippophae is 0.4:0.4:0.2:100), stirring at 45 deg.C for hydrolysis for 18h, inactivating enzyme at 85 deg.C for 25min to obtain semen Hippophae protein hydrolysate;
centrifuging the above protein hydrolysate of semen Hippophae at 4 deg.C and 10000rpm for 15min, collecting supernatant, and lyophilizing to obtain weight reducing composition containing EEAASLR oligopeptide. Through detection, the content of EEAASLR oligopeptide in the weight-losing composition is 1.24g/100 g.
This example also provides a method for purifying Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR) oligopeptide, comprising the steps of:
preparing the weight-reducing composition into 0.15mg/mL aqueous solution, filtering with PLCGC ultrafiltration membrane at a rate of 1mL/min, and collecting filtrate with molecular weight less than 3 kDa; then freeze-drying to obtain powder 1;
preparing the powder 1 into a 10mg/mL aqueous solution, mixing the aqueous solution with HPD826 resin, eluting the solution at the rate of 0.1mL/min by adopting an ethanol solution with the volume content of 60%, and collecting eluent;
freeze-drying the eluate to obtain 8mg/mL aqueous solution, separating with SEP-C18 chromatography column under the following conditions: eluting with 30% ethanol, collecting eluate, freeze drying, and identifying the dried substance by HPLC-MS/MS to obtain oligopeptide with the following amino acid sequence Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR).
Example 5
This example provides a composition for weight loss containing Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR) oligopeptide, which is prepared by the following steps:
pulverizing sea buckthorn seeds to 100 meshes, and mixing the ground sea buckthorn seeds with water according to a mass ratio of 1: 12 to obtain aqueous solution of seabuckthorn seeds, adjusting the pH value to 8.0, adding alkaline protease and flavourzyme (the mass ratio of the alkaline protease to the flavourzyme to the crushed seabuckthorn seeds is 1.3: 0.7: 100), stirring and hydrolyzing at 50 ℃ for 12h, and inactivating the enzyme at 95 ℃ for 15min after the hydrolysis is finished to obtain seabuckthorn seed protein hydrolysate;
centrifuging the above protein hydrolysate at 4 deg.C and 9000rpm for 12min, collecting supernatant, and lyophilizing to obtain weight reducing composition containing EEAASLR oligopeptide. Through detection, the content of EEAASLR oligopeptide in the weight-losing composition is 1.13g/100 g.
This example also provides a method for purifying Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR) oligopeptide, comprising the steps of:
preparing the weight-reducing composition into 0.15mg/mL aqueous solution, filtering with PLCGC ultrafiltration membrane at a rate of 1mL/min, and collecting filtrate with molecular weight less than 3 kDa; then freeze-drying to obtain powder 1;
preparing the powder 1 into a 10mg/mL aqueous solution, mixing the aqueous solution with HPD826 resin, eluting the solution at the rate of 0.1mL/min by adopting an ethanol solution with the volume content of 60%, and collecting eluent;
freeze-drying the eluate to obtain 8mg/mL aqueous solution, separating with SEP-C18 chromatography column under the following conditions: eluting with 30% ethanol, collecting eluate, freeze drying, and identifying the dried substance by HPLC-MS/MS to obtain oligopeptide with the following amino acid sequence Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR).
Example 6
This example provides a composition for weight loss containing Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR) oligopeptide, which is prepared by the following steps:
pulverizing sea buckthorn seeds to 100 meshes, and mixing the ground sea buckthorn seeds with water according to a mass ratio of 1: 18 to obtain aqueous solution of seabuckthorn seeds, adjusting the pH value to 10.0, adding alkaline protease (the mass ratio of the alkaline protease to the crushed seabuckthorn seeds is 0.25: 100), stirring and hydrolyzing at 50 ℃ for 8h, and inactivating enzyme at 90 ℃ for 20min after the hydrolysis is finished to obtain protein hydrolysate of seabuckthorn seeds;
centrifuging the above protein hydrolysate at 4 deg.C and 8000rpm for 20min, collecting supernatant, and lyophilizing to obtain weight reducing composition containing EEAASLR oligopeptide. Through detection, the content of EEAASLR oligopeptide in the weight-losing composition is 0.57g/100 g.
This example also provides a method for purifying Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR) oligopeptide, comprising the steps of:
preparing the weight-reducing composition into 0.15mg/mL aqueous solution, filtering with PLCGC ultrafiltration membrane at a rate of 1mL/min, and collecting filtrate with molecular weight less than 3 kDa; then freeze-drying to obtain powder 1;
preparing the powder 1 into a 10mg/mL aqueous solution, mixing the aqueous solution with HPD826 resin, eluting the solution at the rate of 0.1mL/min by adopting an ethanol solution with the volume content of 60%, and collecting eluent;
freeze-drying the eluate to obtain 8mg/mL aqueous solution, separating with SEP-C18 chromatography column under the following conditions: eluting with 30% ethanol, collecting eluate, freeze drying, and identifying the dried substance by HPLC-MS/MS to obtain oligopeptide with the following amino acid sequence Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR).
Example 7
This example provides a composition for weight loss containing Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR) oligopeptide, which is prepared by the following steps:
pulverizing sea buckthorn seeds to 100 meshes, and mixing the ground sea buckthorn seeds with water according to a mass ratio of 1: 10 to obtain aqueous solution of seabuckthorn seeds, adjusting the pH value to 10.0, adding alkaline protease (the mass ratio of the alkaline protease to the crushed seabuckthorn seeds is 0.25: 100), stirring and hydrolyzing at 40 ℃ for 8h, and inactivating enzyme at 90 ℃ for 20min after the hydrolysis is finished to obtain protein hydrolysate of seabuckthorn seeds;
centrifuging the above protein hydrolysate at 4 deg.C and 8000rpm for 20min, collecting supernatant, and lyophilizing to obtain weight reducing composition containing EEAASLR oligopeptide. Through detection, the content of EEAASLR oligopeptide in the weight-losing composition is 0.52g/100 g.
This example also provides a method for purifying Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR) oligopeptide, comprising the steps of:
preparing the weight-reducing composition into 0.15mg/mL aqueous solution, filtering with PLCGC ultrafiltration membrane at a rate of 1mL/min, and collecting filtrate with molecular weight less than 3 kDa; then freeze-drying to obtain powder 1;
preparing the powder 1 into a 10mg/mL aqueous solution, mixing the aqueous solution with HPD826 resin, eluting the solution at the rate of 0.1mL/min by adopting an ethanol solution with the volume content of 60%, and collecting eluent;
freeze-drying the eluate to obtain 8mg/mL aqueous solution, separating with SEP-C18 chromatography column under the following conditions: eluting with 30% ethanol, collecting eluate, freeze drying, and identifying the dried substance by HPLC-MS/MS to obtain oligopeptide with the following amino acid sequence Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR).
Example 8
This example provides a composition for weight loss containing Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR) oligopeptide, which is prepared by the following steps:
pulverizing sea buckthorn seeds to 100 meshes, and mixing the ground sea buckthorn seeds with water according to a mass ratio of 1: 10 to obtain aqueous solution of seabuckthorn seeds, adjusting the pH value to 10.0, adding alkaline protease (the mass ratio of the alkaline protease to the crushed seabuckthorn seeds is 0.25: 100), stirring and hydrolyzing at 65 ℃ for 8h, and inactivating enzyme at 90 ℃ for 20min after the hydrolysis is finished to obtain protein hydrolysate of seabuckthorn seeds;
centrifuging the above protein hydrolysate at 4 deg.C and 8000rpm for 20min, collecting supernatant, and lyophilizing to obtain weight reducing composition containing EEAASLR oligopeptide. Through detection, the content of EEAASLR oligopeptide in the weight-losing composition is 0.51g/100 g.
This example also provides a method for purifying Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR) oligopeptide, comprising the steps of:
preparing the weight-reducing composition into 0.15mg/mL aqueous solution, filtering with PLCGC ultrafiltration membrane at a rate of 1mL/min, and collecting filtrate with molecular weight less than 3 kDa; then freeze-drying to obtain powder 1;
preparing the powder 1 into a 10mg/mL aqueous solution, mixing the aqueous solution with HPD826 resin, eluting the solution at the rate of 0.1mL/min by adopting an ethanol solution with the volume content of 60%, and collecting eluent;
freeze-drying the eluate to obtain 8mg/mL aqueous solution, separating with SEP-C18 chromatography column under the following conditions: eluting with 30% ethanol, collecting eluate, freeze drying, and identifying the dried substance by HPLC-MS/MS to obtain oligopeptide with the following amino acid sequence Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR).
Comparative example 1
The present comparative example provides a weight-loss composition, the method of preparation comprising:
pulverizing sea buckthorn seeds to 100 meshes, and mixing the ground sea buckthorn seeds with water according to a mass ratio of 1: 10 mixing to obtain aqueous solution of semen Hippophae, adjusting pH to 11.0, adding alkaline protease (the mass ratio of the alkaline protease to pulverized semen Hippophae is 0.25: 100), stirring at 50 deg.C for hydrolysis for 8 hr, and inactivating enzyme at 90 deg.C for 20min to obtain semen Hippophae protein hydrolysate;
centrifuging the above protein hydrolysate at 4 deg.C and 8000rpm for 20min, collecting supernatant, and lyophilizing to obtain weight reducing composition containing EEAASLR oligopeptide. Through detection, the weight-reducing composition does not contain oligopeptide with the amino acid sequence of Glu-Glu-Ala-Ala-Ser-Leu-Arg.
Preparing the weight-reducing composition into 0.1mg/mL aqueous solution, filtering with PLCGC ultrafiltration membrane at the rate of 0.6mL/min, and collecting filtrate with molecular weight less than 3 kDa; then freeze-drying to obtain powder;
preparing the powder into 8mg/mL aqueous solution, mixing the aqueous solution with DA201-C macroporous resin, eluting by adopting 50% ethanol solution at the rate of 0.2mL/min, and collecting eluent;
freeze-drying the eluate to obtain 5mg/mL aqueous solution, separating with SEP-C18 chromatography column under the following conditions: eluting with 40% ethanol, collecting eluate, freeze drying, and identifying the dried substance by HPLC-MS/MS to determine oligopeptide with amino acid sequence of Glu-Glu-Ala-Ala-Ser-Leu-Arg (EEAASLR).
Experimental example 1
The weight-reducing composition prepared in each example is tested for pancreatic lipase inhibition rate, and the specific test method is as follows:
preparation of white olive oil substrate emulsion: heating polyvinyl alcohol 20g and water 800g to dissolve completely, filtering warp cloth, mixing 150ml of filtrate with 50ml of olive oil, and homogenizing for 10 min.
Respectively adding 2ml of the white olive oil substrate emulsion and 2.5ml of phosphate buffer solution PBS (0.005M, pH 7.5) into a 25ml colorimetric tube, uniformly mixing by vortex oscillation, preheating in a constant-temperature water bath (40 ℃) for 5min, respectively adding 1ml of weight-reducing composition (1mg/ml) and 0.5ml of pancreatic lipase solution (3mg/ml), fully mixing, accurately reacting in a constant-temperature water bath (40 ℃) for 20min (shaking for 10 times at intervals), rapidly adding 6ml of 95% ethanol and 1ml of 6M hydrochloric acid for stopping reaction, then adding 3ml of isooctane for vortex oscillation for 90s, standing and layering in a 60 ℃ water bath, cooling at room temperature, taking 1ml of supernatant in a 10ml centrifuge tube, adding 4ml of isooctane and 1ml of copper acetate for vortex oscillation for 90s, standing and layering, taking an upper organic phase, and measuring absorbance at the wavelength of 714 nm. Blank experiments were performed in parallel, except that no slimming composition was added, and the other operations were identical. The calculation formula of the pancreatic lipase inhibition rate is as follows:
pancreatic lipase inhibition (%) ═ 1-ab/Aa)×100％
Wherein A isbAbsorbance of blank experiment; a. theaIs the absorbance measured after addition of the weight-loss composition. Specific test results are shown in the following table.
TABLE 2 pancreatic lipase inhibition of the slimming compositions prepared in the examples and comparative examples: (
n＝3)
Experimental example 2
Preparing the weight-losing composition prepared in the example 1 into a solution with the oligopeptide content of which the amino acid sequence is Glu-Glu-Ala-Ala-Ser-Leu-Arg being 1mg/ml and serving as an inhibitor (I); preparing a pancreatic lipase solution (E) with the concentration of 0.1 mg/ml; preparing white olive oil substrate emulsion (S) with the concentration of 9.1 mg/ml; then adding inhibitor (I), pancrelipase solution (E) and white olive oil substrate emulsion (S) in three sequences:
(1) adding 2ml of white olive oil substrate emulsion (S) and 1ml of inhibitor (I) into a 25ml colorimetric tube respectively, preheating for 15min, and adding 0.5ml of pancreatic lipase solution (E);
(2) adding 1ml of inhibitor (I) and 0.5ml of pancrelipase solution (E) into a 25ml colorimetric tube respectively, preheating for 15min, and adding 2ml of white olive oil substrate emulsion (S);
(3) respectively adding 2ml of white olive oil substrate emulsion (S) and 0.5ml of pancreatic lipase solution (E) into a 25ml colorimetric tube, reacting for 15min, and adding 1ml of inhibitor (I);
fully mixing the components according to the adding sequence, then respectively placing the components in a constant temperature water bath (40 ℃) to accurately react for 20min (shaking at intervals for 10 times), quickly adding 6ml of 95% ethanol and 1ml of 6M hydrochloric acid to stop the reaction, then adding 3ml of isooctane to perform vortex oscillation for 90s, then placing the mixture in a water bath kettle at 60 ℃ for layering, cooling the mixture at room temperature, taking 1ml of supernatant to a 10ml centrifuge tube, then adding 4ml of isooctane and 1ml of copper acetate developer to perform vortex oscillation for 90s, placing the mixture to perform layering, taking an upper organic phase, and measuring the absorbance at the 714nm wavelength. Blank experiments were performed in parallel, except that no slimming composition was added, and the other operations were identical.
The pancreatic lipase inhibition rates were calculated according to the calculation formula in the above experimental example 1, and the results are shown in fig. 2, where the "i" in fig. 2 represents the standard deviation, and as can be seen from fig. 2, the pancreatic lipase inhibition rate in the (1) th feeding sequence was the highest, indicating that the inhibitor mainly interacts with the substrate emulsion, thereby inhibiting the action of the substrate and the enzyme and achieving the inhibition effect. Specific data are shown in the following table.
TABLE 3 pancreatic lipase inhibition: (
n＝3)
Experimental example 3
The weight-reducing composition prepared in example 1 was prepared into a solution having an oligopeptide content of 1mg/ml and an amino acid sequence of Glu-Ala-Ser-Leu-Arg, and the solution was divided into 5 parts on average, and then the solution was incubated at 25 ℃, 45 ℃, 65 ℃, 85 ℃ and 100 ℃ for 60min, and then rapidly cooled to room temperature, and finally the pancreatic lipase inhibitory activity was measured according to the test method of example 1.
The experimental group at 25 ℃ served as a control group, and the inhibitory activity was 100%, and the inhibitory activity measured in each of the other groups was compared with the control group to obtain the activity retention rate. The calculation formula is as follows:
pancreatic lipase inhibitory Activity Retention%t/Ic×100％；
Wherein, IcIs the inhibition ratio of pancreatic lipase at 25 ℃, ItPancreatic lipase inhibition at other temperatures.
The specific experimental results are shown in FIG. 3. The letter b in the figure represents a significant difference compared to the control group (p < 0.05); the "I" in FIG. 3 represents the standard deviation. Specific data are shown in the following table. As shown in FIG. 3, the oligopeptide weight-reducing composition containing Glu-Glu-Ala-Ala-Ser-Leu-Arg has a reduced inhibitory activity with increasing temperature. After the inhibitory activity is substantially higher than 85 ℃ at a temperature lower than 65 ℃, the pancreatic lipase inhibitory activity decreases with an increase in temperature. The results show that although high temperature has certain influence on the activity of the weight-reducing composition, the pancreatic lipase inhibition activity retention rate is still above 84% even if the weight-reducing composition is treated at 100 ℃ for 1h, which indicates that the weight-reducing composition has good stability during heating.
TABLE 4 pancreatic lipase inhibition Retention at different temperatures: (
n＝3)
Experimental example 4
The oligopeptide of Glu-Glu-Ala-Ala-Ser-Leu-Arg is prepared by the following chemical synthesis method:
(1) 1g of polystyrene resin was weighed into a reaction column, DCM (dichloromethane) was added to swell for 30min, then DCM was taken out, 2g of the first amino acid (Glu) in the sequence, 2g of DIEA (diisopropylethylamine), 5ml of DMF (dimethylformamide) and 5ml of DCM were added, and a reaction was carried out for 60min from the start of the system to the timing of bubbling with nitrogen gas (0.025 m/min) to the reaction system. Then 5 equivalents of methanol were added, reaction was carried out for 30min, the reaction solution was drained so that carboxyl groups in Glu were supported on the resin, and then the resin was washed with DMF (5ml) and MeOH (5ml), respectively;
(2) adding 2g of the second amino acid (Glu of which the amino group is protected by Fmoc) in the sequence, 2g of HBTU (benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate) and 5ml of DIEA into a reaction column, blowing nitrogen (0.025 cubic meters per minute) into the reaction system, reacting for 30min from the beginning of the system to the timing of bubbling, washing off liquid, adding 5ml of decapping liquid to remove the Fmoc (9-fluorenylmethoxycarbonyl) protecting group, washing the resin with DMF (5ml) and MeOH (5ml) respectively, and carrying out the next step after detecting that the amino group is deprotected by ninhydrin;
(3) sequentially adding different amino acids (amino groups are protected by Fmoc) in the sequence according to the method in the step 2 and carrying out various modifications;
(4) blowing the resin to dry by using nitrogen, taking the resin out of the reaction column, pouring the resin into a flask, adding cutting fluid (the cutting fluid consists of 95 wt% of trichloroacetic acid, 2 wt% of ethanedithiol, 2 wt% of triisopropylsilane and 1 wt% of water) into the flask, oscillating, and filtering the resin to obtain filtrate; wherein, the proportion of the cutting fluid to the resin is 10 ml: 1g of a compound;
(5) then adding 10ml of ether (generated by precipitate) into the filtrate, centrifuging, and washing the precipitate with ether to obtain a crude product of Glu-Glu-Ala-Ala-Ser-Leu-Arg oligopeptide;
(6) separating the crude oligopeptide product by high performance liquid chromatography to a purity of not less than 98%;
(7) and (3) putting the oligopeptide solution with the purity not less than 98% separated by the high performance liquid chromatography into a freeze dryer for concentration, and freeze-drying to obtain white powdery Glu-Glu-Ala-Ala-Ser-Leu-Arg oligopeptide.
Respectively preparing the oligopeptide of Glu-Glu-Ala-Ala-Ser-Leu-Arg obtained by purification into aqueous solutions with different concentrations, and then determining pancreatic fat inhibition rates of the oligopeptides at different concentrations according to the method in the experimental example 1; the concentration is used as the abscissa and the inhibition rate of pancreatic lipase is used as the ordinate, a curve is prepared, and the half-inhibited inhibition concentration IC is calculated from the curve50. The results are shown in the table below.
TABLE 5 results of the experiment
As can be seen from the data in the above table, the pancreatic lipase inhibition rate increases with the increase of the oligopeptide concentration, and the concentration at which the half inhibition rate, that is, the inhibition rate is 50%, is 510.55. + -. 0.12. mu.g/ml.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
SEQUENCE LISTING
<110> Perfect (Guangdong) daily necessities Co., Ltd
<120> oligopeptide, weight-losing composition, preparation method and application
<130> HA202001475
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 7
<212> PRT
<213> Artificial sequence
<400> 1
Glu Glu Ala Ala Ser Leu Arg
1 5
Claims (8)
1. An oligopeptide, characterized in that its amino acid sequence is: Glu-Glu-Ala-Ala-Ser-Leu-Arg.
2. A weight loss composition comprising the oligopeptide of claim 1.
3. The slimming composition of claim 2, wherein the oligopeptide content of the slimming composition is 0.5 to 1.5g/100 g.
4. The weight loss composition of claim 2 or 3, wherein the weight loss composition is a dietary supplement.
5. The method for purifying the oligopeptide according to claim 1, which comprises the steps of:
sequentially subjecting the composition of any one of claims 2-4 to ultrafiltration, neutral resin, and chromatographic column separation to obtain the oligopeptide.
6. The method for purifying oligopeptide according to claim 5, wherein the filtrate having a molecular weight of less than 3kDa is collected during ultrafiltration.
7. Use of the oligopeptide according to claim 1 or the oligopeptide extracted by the method for purifying the oligopeptide according to claim 5 or 6 or the slimming composition according to any one of claims 2 to 4 for inhibiting pancreatic lipase activity for non-therapeutic purposes.
8. Use of the oligopeptide according to claim 1 or the oligopeptide extracted by the method for purifying the oligopeptide according to claim 5 or 6 or the weight-reducing composition according to any one of claims 2 to 4 in the preparation of weight-reducing food, medicine or health-care product.
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