CN111848735B - Immunoregulation active peptide and preparation method and application thereof - Google Patents
Immunoregulation active peptide and preparation method and application thereof Download PDFInfo
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 78
- 230000007365 immunoregulation Effects 0.000 title claims description 8
- 238000002360 preparation method Methods 0.000 title abstract description 11
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- 230000002519 immonomodulatory effect Effects 0.000 claims abstract description 44
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- HSQGMTRYSIHDAC-BQBZGAKWSA-N Leu-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(O)=O HSQGMTRYSIHDAC-BQBZGAKWSA-N 0.000 description 1
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Classifications
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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
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- 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
Abstract
The invention discloses an immunomodulatory bioactive peptide, and a preparation method and application thereof. Dispersing soybean protein in water, hydrolyzing with protease, inactivating enzyme at high temperature, centrifuging to obtain supernatant of protein hydrolysate, drying after membrane separation, purifying active peptide with reversed phase chromatographic column, gradient eluting with water and ethanol solution, collecting ethanol solution eluate of specific concentration, concentrating, purifying with Sephadex LH-20 chromatographic column, collecting ethanol solution eluate of specific concentration, concentrating, and drying to obtain target immunoregulatory active peptide. The invention utilizes soy protein to prepare the immunomodulatory bioactive peptide, and the yield of the immunomodulatory bioactive peptide is 30-92 g/kg (the purity is 80-95%). The invention provides a new method for preparing the immunomodulatory active peptide, and has important significance for promoting the deep processing and utilization of the soybean protein, improving the added value of the product and promoting the sustainable development of the industry.
Description
The technical field is as follows:
the invention belongs to the field of agricultural product processing, and particularly relates to an immunomodulatory bioactive peptide, and a preparation method and application thereof.
Background art:
the active peptide belongs to an important food functional factor, has the biological activities of regulating immunity, resisting oxidation, preventing osteoporosis and the like, is widely applied to the production of products such as health-care food and the like, and is deeply welcomed by consumers. The soybean protein is a bulk protein raw material, has low price, is easy to obtain, and is very suitable for processing high-value products such as active peptide and the like. The biological activity of an active peptide is highly correlated with its chemical structure, in particular the amino acid sequence. Although there are many reports on soy-active peptides, there are still few active peptide products with higher activity. Therefore, it is necessary to develop a preparation process, structure identification and activity evaluation research of soybean active peptides to reveal the chemical structure of the main active peptides, which is of great significance for high-value utilization of soybean proteins.
The invention content is as follows:
it is a first object of the present invention to provide an immunomodulatory active peptide isolated from soy protein.
The invention discovers and prepares an immunomodulatory active peptide, and the structure of the immunomodulatory active peptide is determined to be shown in formula (I) through chromatography and mass spectrometry:
Leu-Ala-Gly-Asn-Pro-Asp-Ile-Glu-His-Pro-Glu
formula (I).
A second object of the present invention is to provide a method for preparing immunomodulatory active peptides, comprising the steps of:
adding water to soybean protein for dispersion, adding protease for hydrolysis, inactivating enzyme at high temperature, centrifuging to obtain supernatant of protein hydrolysate, drying after membrane separation to obtain soybean protein hydrolysate, purifying the soybean protein hydrolysate by using a reverse phase chromatographic column for active peptide, performing gradient elution by using water and ethanol water solutions, collecting ethanol solution elution components with specific concentration, concentrating, purifying by using a Sephadex LH-20 chromatographic column, collecting ethanol solution elution components with specific concentration, concentrating, and drying to obtain the target immunomodulatory active peptide.
Preferably, the soy protein hydrolysate is purified by reverse phase chromatography of the active peptide, water, gradient elution with ethanol water solution, collecting ethanol solution elution components with specific concentration, concentrating, purifying with SephadexLH-20 chromatographic column, collecting ethanol solution elution components with specific concentration, concentrating, and drying to obtain the target immunomodulatory bioactive peptide, namely purifying soybean protein hydrolysate with C18 reversed phase chromatographic column, gradient elution with water-ethanol as eluent according to the gradient that ethanol volume fraction gradually rises from 0% to 100% according to the amplitude of 5%, collecting target bioactive peptide component eluted with 10% ethanol concentration by volume fraction, concentrating, purifying with SephadexLH-20 chromatographic column, eluting with 10% ethanol water solution by volume fraction, collecting target bioactive peptide component eluted with 10% ethanol concentration by volume fraction, concentrating, and drying to obtain the target immunomodulatory bioactive peptide.
Preferably, the soybean protein is dispersed by adding water, namely adding the soybean protein into water with the mass of 5-20 times of that of the soybean protein, and then stirring and dispersing.
Preferably, the step of adding protease for hydrolysis, inactivating enzyme at high temperature, centrifuging to obtain supernatant of protein hydrolysate, and drying after membrane separation to obtain soybean protein hydrolysate is to add 0.1-1.0% of alkaline protease according to the mass of soybean protein, perform enzymolysis at the pH of 7.5-9.5 and the temperature of 30-65 ℃ for 1-5 hours, add 0.1-1.0% of protease SmPA, perform enzymolysis at the pH of 6.0-8.0 and the temperature of 40-60 ℃ for 1-5 hours, heat up to 90 ℃ for 20 minutes to inactivate enzyme, centrifuge to obtain supernatant of protein hydrolysate, filter the supernatant by adopting an ultrafiltration membrane with the molecular weight cutoff of 5000 daltons, collect permeate and spray-dry the permeate to obtain the soybean protein hydrolysate.
The third purpose of the invention is to provide the application of the immunoregulation active peptide in preparing the immunoregulation active medicine, food or health care products.
It is a fourth object of the present invention to provide an immunomodulatory active peptide, a food or a health product comprising the immunomodulatory active peptide as an active ingredient.
The fifth object of the present invention is to provide the use of soy protein for preparing the above immunomodulatory active peptides.
The method prepares the immunomodulatory bioactive peptide from the soybean protein, the yield of the immunomodulatory bioactive peptide is 30-92 g/kg (the purity is 80-95%), the immunomodulatory bioactive peptide has good immunomodulatory activity, and the immunomodulatory bioactive peptide can be applied to preparation of immunomodulatory bioactive medicaments, foods or health-care products. The invention provides a new method for preparing the immunomodulatory active peptide, and has important significance for promoting the deep processing and utilization of the soybean protein, improving the added value of the product and promoting the sustainable development of the industry.
Description of the drawings:
FIG. 1 is a secondary mass spectrum of an immunomodulatory active peptide;
FIG. 2 is a graph showing the amount of NO production induced by macrophages by immunomodulatory active peptides.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The amino acid sequence of the protease SmPA used in the examples below is as follows:
MSKTFSMPISGAGKRRSAIAAGTVVAAAALLVSGLTTGTAGAAPSAKSGAQPTALSASARAELLREANATKVDTASSLGLGAKEKLVVKDVIKDADGTTHTRYDRTYDGLPVLGGDLIVHRAKGGDVKGVTKATKATVKVASTTAGIAPTTAAKAAVKLAKADDTTQAAADQAPRKVIWAADGKPVLAYETVVGGVQKDGTPNELHVITDAATGKKLFERQGIETGKGESEYSGSVELGTSKEGSGYTLTDADRGGHKTTNLENGESGEGKAFTDDDDNWGTGKPDDPQTAAVDAHYGAAVTWDYYKNVHGRNGIADDGKGAYSRVHYGDSYVNAFWDDSCFCMTYGDGEGNKAPLTAIDVAAHEMSHGVTSATAGLEYSGESGGLNEATSDIFGTSVEFSADNSTDVGDYLIGEEIDINGDGSPLRYMDKPSKDGQSADEWSDGVGDMDVHYSSGVANHFFYLLSEGSGAKEINGVKYDSPTSDGSKVEGIGRDKAEKIWYKALTTYMTSNTDYHAAREATQKAATDLFGADSAEAKGVDAAWAGVNVK。
example 1: preparation, separation and structure identification of immune-modulating active peptide
Preparation and separation of immune-regulating active peptide
1) Selecting materials: soy protein isolate is selected.
2) Preparing: adding water with 10 times of the mass of the soybean protein isolate, and then stirring and dispersing.
3) And (3) enzymatic hydrolysis: adding 0.5% alkaline protease, pH8.5, and 50 deg.C according to the weight of soybean protein isolate, performing enzymolysis for 3 hr, adding 0.5% protease SmPA, pH7.0, and performing enzymolysis for 3 hr at 50 deg.C. Heating to 90 deg.C, maintaining for 20 min to inactivate enzyme, centrifuging to obtain supernatant of protein hydrolysate, filtering with ultrafiltration membrane with cut-off molecular weight of 5000 Dalton, collecting filtrate, and spray drying to obtain soybean protein hydrolysate.
4) Column purification: purifying the soybean protein hydrolysate by using a C18 reversed phase chromatographic column (15 x 460mm), performing gradient elution by using water and ethanol water solution, specifically, gradually increasing the volume fraction of ethanol from 0% to 100% according to the amplitude of 5%, collecting target active peptide components eluted by the ethanol concentration with the volume fraction of 10%, purifying by using a SephadexLH-20 chromatographic column (15 x 460mm) after concentrating, eluting by using the ethanol water solution with the volume fraction of 10%, collecting target active peptide components eluted by the ethanol concentration with the volume fraction of 10%, concentrating, and drying to obtain the target immunomodulatory active peptide.
The yield of the immunomodulatory bioactive peptide obtained by the method is 72-85 g/kg, and the purity is 80-95%.
Structural identification of immunoregulatory active peptide
The immunomodulatory bioactive peptide is soluble in water, and the result of the immunomodulatory bioactive peptide is shown in figure 1 after high-resolution liquid chromatography and mass spectrometry. According to the second-order mass spectrum information, the structure of the immunomodulatory active peptide is identified as shown in the formula (I) (amino acid sequence).
Leu-Ala-Gly-Asn-Pro-Asp-Ile-Glu-His-Pro-Glu
Formula (I)
The molecular ion peak m/z of the immune-modulating active peptide in a cation mode is 596.285, and z is 2. m/z836.380, 739.328, 624.300, 511.216, 382.173 and 245.114 are y ion signals of fragments Pro-Asp-Ile-Glu-His-Pro-Glu, His-Pro-Glu and Pro-Glu respectively; m/z947.461, 810.402, 356.194 and 185.129 are b ion signals of fragments Leu-Ala-Gly-Asn-Pro-Asp-Ile-Glu-His, Leu-Ala-Gly-Asn-Pro-Asp-Ile-Glu, Leu-Ala-Gly-Asn and Leu-Ala, respectively. The presence of these signals confirms that the structure of the immunomodulatory active peptide is Leu-Ala-Gly-Asn-Pro-Asp-Ile-Glu-His-Pro-Glu. After the identification structure is artificially synthesized, the identification structure is detected by liquid chromatography-mass spectrometry, and the peak retention time of the identification structure is consistent with that of the identified substance and the mass spectrum fragments are consistent. Therefore, the structure was determined to be the above structure.
Third, activity test of immunomodulatory active peptides
RAW264.7 cells in the logarithmic growth phase are inoculated in a 96-well plate and cultured in a carbon dioxide incubator at 37 ℃ for 24 hours. After discarding the medium, medium containing different concentrations of polypeptide (Dulbecco's microbial eagle's medium (DMEM)) was added, and control wells were added with medium without sample. After further incubation for 24h in the incubator, the medium was collected and the amount of Nitric Oxide (NO) released was determined using the nitric oxide detection kit (GriessReagent assay) with three replicates per test. The concentration of NO in the medium was calculated from the standard curve, with cells treated without the sample as blank.
The experimental results are shown in FIG. 2, and FIG. 2 shows the immunomodulatory activity of the immunomodulatory peptide, which is indicated by the amount of NO produced by induced macrophages. Compared with a blank control, the active peptide can generate a remarkable effect of promoting NO generation under the condition of the lowest dosage (0.1mg/ml), and the remarkable immunoregulation activity is proved. Within the dosage range of 0.1-0.4 mg/ml, the immunoregulation activity of the active peptide presents an obvious dose-effect relationship. Higher amounts, such as 0.4 and 0.6mg/ml, did not significantly differ in the NO production inducing effect.
Example 2:
preparation and separation of immune-regulating active peptide
1) Selecting materials: soy protein isolate is selected.
2) Preparing: adding water 5 times of the weight of the soybean protein isolate, and stirring and dispersing.
3) And (3) enzymatic hydrolysis: adding 0.1% alkaline protease, pH7.5, temperature 30 deg.C, and performing enzymolysis for 1 hr, and adding 0.1% protease SmPA, pH6.0, temperature 40 deg.C, and performing enzymolysis for 1 hr. Heating to 90 deg.C, maintaining for 20 min to inactivate enzyme, centrifuging to obtain supernatant of protein hydrolysate, filtering with ultrafiltration membrane with cut-off molecular weight of 5000 Dalton, collecting filtrate, and spray drying.
4) Column purification: purifying the soybean protein hydrolysate by using a C18 reversed phase chromatographic column (15 x 460mm), performing gradient elution by using water and ethanol water solution, specifically, gradually increasing the volume fraction of ethanol from 0% to 100% according to the amplitude of 5%, collecting target active peptide components eluted by the ethanol concentration with the volume fraction of 10%, purifying by using a SephadexLH-20 chromatographic column (15 x 460mm) after concentrating, eluting by using the ethanol water solution with the volume fraction of 10%, collecting target active peptide components eluted by the ethanol concentration with the volume fraction of 10%, concentrating, and drying to obtain the target immunomodulatory active peptide.
The yield of the immunomodulatory bioactive peptide obtained by the method is 30-43 g/kg, and the purity is 80-95%.
Example 3:
preparation and separation of immune-regulating active peptide
1) Selecting materials: selecting soy protein isolate;
2) preparing: adding water 20 times of the mass of the soybean protein isolate, and then stirring and dispersing.
3) And (3) enzymatic hydrolysis: adding 1.0% of alkaline protease according to the mass of the soybean protein isolate, carrying out enzymolysis for 5 hours at the pH value of 8.5 and the temperature of 50 ℃, and then adding 1.0% of self-made protease SmPA, carrying out enzymolysis for 5 hours at the pH value of 7.0 and the temperature of 50 ℃. Heating to 90 deg.C, maintaining for 20 min to inactivate enzyme, centrifuging to obtain supernatant of protein hydrolysate, filtering with ultrafiltration membrane with cut-off molecular weight of 5000 Dalton, collecting filtrate, and spray drying to obtain soybean protein hydrolysate.
4) Column purification: purifying the soybean protein hydrolysate by using a C18 reversed phase chromatographic column (15 x 460mm), performing gradient elution by using water and ethanol water solution, specifically, gradually increasing the volume fraction of ethanol from 0% to 100% according to the amplitude of 5%, collecting target active peptide components eluted by the ethanol concentration with the volume fraction of 10%, purifying by using a SephadexLH-20 chromatographic column (15 x 460mm) after concentrating, eluting by using the ethanol water solution with the volume fraction of 10%, collecting target active peptide components eluted by the ethanol concentration with the volume fraction of 10%, concentrating, and drying to obtain the target immunomodulatory active peptide.
The yield of the immunomodulatory bioactive peptide obtained by the method is 76-92 g/kg, and the purity is 80-95%.
Example 4:
preparation and separation of immune-regulating active peptide
1) Selecting materials: selecting soy protein isolate;
2) leaching: adding water with the mass 10 times of that of the soybean protein isolate, and then stirring and dispersing;
3) and (3) enzymatic hydrolysis: adding 0.5% of alkaline protease according to the mass of the soybean protein isolate, carrying out enzymolysis for 3 hours at the pH value of 9.5 and the temperature of 65 ℃, and then adding 0.5% of self-made protease SmPA, carrying out enzymolysis for 3 hours at the pH value of 8.0 and the temperature of 60 ℃. Heating to 90 deg.C, maintaining for 20 min to inactivate enzyme, centrifuging to obtain supernatant of protein hydrolysate, filtering with ultrafiltration membrane with cut-off molecular weight of 5000 Dalton, collecting filtrate, and spray drying to obtain soybean protein hydrolysate.
4) Column purification: purifying the soybean protein hydrolysate by using a C18 reversed phase chromatographic column (15 x 460mm), performing gradient elution by using water and ethanol water solution, specifically, gradually increasing the volume fraction of ethanol from 0% to 100% according to the amplitude of 5%, collecting target active peptide components eluted by the ethanol concentration with the volume fraction of 10%, purifying by using a SephadexLH-20 chromatographic column (15 x 460mm) after concentrating, eluting by using the ethanol water solution with the volume fraction of 10%, collecting target active peptide components eluted by the ethanol concentration with the volume fraction of 10%, concentrating, and drying to obtain the target immunomodulatory active peptide.
The yield of the immunomodulatory bioactive peptide obtained by the method is 45-57 g/kg, and the purity is 80-95%.
Claims (2)
1. A process for preparing the immunoregulation active peptide includes such steps as adding water to soybean protein, dispersing, adding protease, hydrolyzing,
inactivating enzyme at high temperature, centrifuging to obtain supernatant of protein hydrolysate, performing membrane separation, drying to obtain soybean protein hydrolysate, purifying the soybean protein hydrolysate with reversed phase chromatographic column to obtain active peptide, performing gradient elution with water and ethanol water solution, collecting ethanol solution eluate with specific concentration, concentrating, purifying with Sephadex LH-20 chromatographic column, collecting ethanol solution eluate with specific concentration, concentrating, and drying to obtain target immunomodulatory active peptide; the 'adding protease for hydrolysis, inactivating enzyme at high temperature, centrifuging to obtain supernatant of protein hydrolysate, and drying after membrane separation to obtain soybean protein hydrolysate' is to add 0.1-1.0% of alkaline protease according to the mass of soybean protein, carry out enzymolysis at the pH of 7.5-9.5 and the temperature of 30-65 ℃ for 1-5 hours, then add 0.1-1.0% of protease SmPA, carry out enzymolysis at the pH of 6.0-8.0 and the temperature of 40-60 ℃ for 1-5 hours, heat up to 90 ℃ for 20 minutes to inactivate enzyme, centrifuge to obtain supernatant of protein hydrolysate, filter by adopting an ultrafiltration membrane with the molecular weight cutoff of 5000 daltons, collect permeate and spray-dry to obtain soybean protein hydrolysate;
the amino acid sequence of the immunoregulation active peptide is as follows: Leu-Ala-Gly-Asn-Pro-Asp-Ile-Glu-His-Pro-Glu;
the soybean protein hydrolysate is purified by reversed phase chromatographic column, and is purified by water, gradient elution with ethanol water solution, collecting ethanol solution elution components with specific concentration, concentrating, purifying with Sephadex LH-20 chromatographic column, collecting ethanol solution elution components with specific concentration, concentrating, and drying to obtain the target immunomodulatory bioactive peptide, namely purifying soybean protein hydrolysate with C18 reversed phase chromatographic column, gradient elution with water-ethanol as eluent according to the gradient that ethanol volume fraction gradually rises from 0% to 100% according to the amplitude of 5%, collecting target bioactive peptide component eluted with 10% ethanol concentration by volume fraction, concentrating, purifying with Sephadex LH-20 chromatographic column, eluting with 10% ethanol water solution by volume fraction, collecting target bioactive peptide component eluted with 10% ethanol concentration by volume fraction, concentrating, and drying to obtain the target immunomodulatory bioactive peptide.
2. The method according to claim 1, wherein the soybean protein is dispersed in water by adding the soybean protein to 5-20 times by mass of water and then stirring.
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