CN111825745A - Immunoregulatory active heptapeptide and preparation method and application thereof - Google Patents
Immunoregulatory active heptapeptide and preparation method and application thereof Download PDFInfo
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- IBIDRSSEHFLGSD-UHFFFAOYSA-N valinyl-arginine Natural products CC(C)C(N)C(=O)NC(C(O)=O)CCCN=C(N)N IBIDRSSEHFLGSD-UHFFFAOYSA-N 0.000 description 1
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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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- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C12P21/00—Preparation of peptides or proteins
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Abstract
The invention discloses an immunoregulation active heptapeptide 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 with specific concentration, concentrating, purifying with Sephadex LH-20 chromatographic column, collecting ethanol solution eluate with specific concentration, concentrating, and drying to obtain target immunoregulatory active heptapeptide. The invention utilizes soy protein to prepare the immunomodulatory active heptapeptide, and the yield is 27-79 g/kg (the purity is 80-95%). The invention provides a new method for preparing immunoregulatory active heptapeptide, and has important significance for promoting deep processing and utilization of soybean protein, improving the added value of products and promoting 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 active heptapeptide, 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:
the first object of the present invention is to provide an immunomodulatory active heptapeptide isolated from soy protein.
The invention discovers and prepares an immunoregulatory active heptapeptide, and the structure of the immunoregulatory active heptapeptide is determined to be shown in a formula (I) by liquid chromatography-mass spectrometry:
Arg-Asp-Glu-Asn-Gly-Val-Arg
formula (I).
A second object of the present invention is to provide a method for preparing an immunomodulatory active heptapeptide, comprising the steps of:
dispersing soybean protein in water, adding protease for hydrolysis, inactivating enzyme at high temperature, centrifuging to obtain supernatant of protein hydrolysate, membrane separating, drying to obtain soybean protein hydrolysate, purifying active peptide with reversed phase chromatographic column, gradient eluting 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 immunoregulatory active heptapeptide.
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 a Sephadex LH-20 chromatographic column, collecting ethanol solution elution components with specific concentration, concentrating, and drying to obtain the target immunomodulatory active heptapeptide, namely purifying a soybean protein hydrolysate with a C18 reverse phase chromatographic column, gradually increasing the ethanol volume fraction from 0% to 20% according to the amplitude of 2% by using water-ethanol as an eluent, gradient elution with the volume fraction of 0% to 20%, collecting the target active peptide component eluted with the volume fraction of 8% ethanol concentration, concentrating, purifying with the Sephadex LH-20 chromatographic column, eluting with the volume fraction of 8% ethanol water solution, collecting the target active peptide component eluted with the volume fraction of 8% ethanol concentration, concentrating, and drying to obtain the target immunomodulatory active heptapeptide.
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, 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 neutral protease, 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 the supernatant by adopting an ultrafiltration membrane with the molecular weight cutoff of 3000 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 heptapeptide in preparing immunoregulation active medicines, foods or health products.
The fourth object of the present invention is to provide an immunomodulatory active pharmaceutical, food or health product comprising the immunomodulatory active heptapeptide as an active ingredient.
The fifth object of the present invention is to provide the use of soybean protein for preparing the above immunomodulatory active heptapeptide.
The yield of the immunomodulatory active heptapeptide prepared from the soybean protein is 27-79 g/kg (the purity is 80-95%), the immunomodulatory active heptapeptide has good immunomodulatory activity, and the immunomodulatory active heptapeptide can be applied to preparation of immunomodulatory active medicines, foods or health-care products. The invention provides a new method for preparing immunoregulatory active heptapeptide, and has important significance for promoting deep processing and utilization of soybean protein, improving the added value of products and promoting sustainable development of the industry.
Description of the drawings:
FIG. 1 is a secondary mass spectrum of an immunomodulatory active heptapeptide;
FIG. 2 is a graph showing the amount of NO production induced by macrophages by immunomodulatory active heptapeptides.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1: preparation, separation and structural identification of immunomodulatory active heptapeptides
Preparation and separation of immunoregulatory active heptapeptide
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 (Nuoweixin) according to the weight of the soybean protein isolate, carrying out enzymolysis for 3 hours at the pH of 8.5 and the temperature of 50 ℃, and then adding 0.5% neutral protease (Nuoweixin) for carrying out enzymolysis for 3 hours at the pH 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 3000 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 20% according to the amplitude of 2%, collecting target active peptide components eluted by the ethanol concentration with the volume fraction of 8%, purifying by using a Sephadex LH-20 chromatographic column (15 x 460mm) after concentrating, eluting by using ethanol water solution with the volume fraction of 8%, collecting target active peptide components eluted by the ethanol concentration with the volume fraction of 8%, concentrating, and drying to obtain the target immunomodulatory active heptapeptide.
The yield of the immunomodulatory active heptapeptide obtained by the method is 69g/kg, and the purity is 95%.
Structural identification of immunomodulatory active heptapeptides
The immunomodulatory active heptapeptide is soluble in water, and the immunomodulatory active heptapeptide is analyzed by high-resolution liquid chromatography-mass spectrometry, and the result is shown in fig. 1. According to the second-order mass spectrum information, the structure of the immunomodulatory activity heptapeptide is identified as shown in the formula (I) (amino acid sequence).
Arg-Asp-Glu-Asn-Gly-Val-Arg
Formula (I)
The molecular ion peak m/z of the immunomodulatory active heptapeptide in the cationic mode is 423.215, and z is 2. m/z689.321, 331.172, 274.048 and 175.119 are y ion signals of fragments Asp-Glu-Asn-Gly-Val-Arg, Val-Arg and Arg respectively; m/z 671.311, 515.257 and 272.135 are b-ion signals of fragments Arg-Asp-Glu-Asn-Gly-Val, Arg-Asp-Glu-Asn and Arg-Asp, respectively. The presence of these signals confirms that the structure of the immunomodulatory active heptapeptide is Arg-Asp-Glu-Asn-Gly-Val-Arg. 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.
Activity test of immunomodulatory active heptapeptides
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, Dulbecco's Modified Eagle's Medium (DMEM) containing different concentrations of the polypeptide was added, and the 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 a nitric oxide assay kit (Griess Reagent 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 results of the experiment are shown in FIG. 2, and FIG. 2 shows the immunomodulatory activity of the immunomodulatory heptapeptide, using the amount of NO produced by induced macrophages as an indicator. 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 0.1-1.2 mg/ml, the immunoregulation activity of the active peptide presents an obvious dose-effect relationship. When the highest dose of 1.2mg/ml in this experiment was used, the induced NO concentration reached 4.3. + -. 0.4. mu.M.
Example 2:
preparation and separation of immunoregulatory active heptapeptide
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 according to the weight of the soybean protein isolate, carrying out enzymolysis for 1 hour at the pH of 7.5 and the temperature of 30 ℃, and then adding 0.1% neutral protease, carrying out enzymolysis for 1 hour at the pH of 6.0 and the temperature of 40 ℃. 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 3000 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 20% according to the amplitude of 2%, collecting target active peptide components eluted by the ethanol concentration with the volume fraction of 8%, purifying by using a Sephadex LH-20 chromatographic column (15 x 460mm) after concentrating, eluting by using ethanol water solution with the volume fraction of 8%, collecting target active peptide components eluted by the ethanol concentration with the volume fraction of 8%, concentrating, and drying to obtain the target immunomodulatory active heptapeptide.
The yield of the immunomodulatory active heptapeptide obtained by the method is 27-41 g/kg, and the purity is 80-95%.
Example 3:
preparation and separation of immunoregulatory active heptapeptide
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% alkaline protease according to the weight of the soybean protein isolate, carrying out enzymolysis for 5 hours at the pH of 8.5 and the temperature of 50 ℃, and then adding 1.0% neutral protease, carrying out enzymolysis for 5 hours at the pH 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 3000 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 20% according to the amplitude of 2%, collecting target active peptide components eluted by the ethanol concentration with the volume fraction of 8%, purifying by using a Sephadex LH-20 chromatographic column (15 x 460mm) after concentrating, eluting by using ethanol water solution with the volume fraction of 8%, collecting target active peptide components eluted by the ethanol concentration with the volume fraction of 8%, concentrating, and drying to obtain the target immunomodulatory active heptapeptide.
The yield of the immunomodulatory active heptapeptide obtained by the method is 66-79 g/kg, and the purity is 80-95%.
Example 4:
preparation and separation of immunoregulatory active heptapeptide
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% alkaline protease according to the weight of the soybean protein isolate, carrying out enzymolysis for 3 hours at the pH of 9.5 and the temperature of 65 ℃, and then adding 0.5% neutral protease, carrying out enzymolysis for 3 hours at the pH 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 3000 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 20% according to the amplitude of 2%, collecting target active peptide components eluted by the ethanol concentration with the volume fraction of 8%, purifying by using a Sephadex LH-20 chromatographic column (15 x 460mm) after concentrating, eluting by using ethanol water solution with the volume fraction of 8%, collecting target active peptide components eluted by the ethanol concentration with the volume fraction of 8%, concentrating, and drying to obtain the target immunomodulatory active heptapeptide.
The yield of the immunomodulatory active heptapeptide obtained by the method is 36-52 g/kg, and the purity is 80-95%.
Claims (9)
1. An immunomodulatory active heptapeptide, having the amino acid sequence: Arg-Asp-Glu-Asn-Gly-Val-Arg.
2. A method for preparing the immunomodulatory active heptapeptide of claim 1, wherein the immunomodulatory active heptapeptide is isolated by performing biological enzymatic hydrolysis of soy protein.
3. The method as claimed in claim 2, wherein the target immunomodulatory active heptapeptide is obtained by dispersing soy protein in water, hydrolyzing with protease, inactivating the enzyme at high temperature, centrifuging to obtain supernatant of protein hydrolysate, membrane separating, drying to obtain soy protein hydrolysate, purifying the active peptide from the soy protein hydrolysate with reverse phase chromatography column, gradient eluting with water and ethanol water solution, collecting the ethanol solution eluate of specific concentration, concentrating, purifying with Sephadex LH-20 chromatography column, collecting the ethanol solution eluate of specific concentration, concentrating, and drying.
4. The method as claimed in claim 3, wherein the step of purifying the active peptide from the soy protein hydrolysate by using a reverse phase chromatographic column, gradient eluting with water and ethanol water, collecting the ethanol solution eluate with a specific concentration, concentrating, purifying by using a Sephadex LH-20 chromatographic column, collecting the ethanol solution eluate with a specific concentration, concentrating, and drying to obtain the target immunomodulatory active heptapeptide comprises the steps of purifying the soy protein hydrolysate by using a C18 reverse phase chromatographic column, gradient eluting from 0% to 20% according to the gradient of 2% of ethanol volume fraction, collecting the target active peptide component eluted with 8% of ethanol volume fraction, concentrating, purifying by using a Sephadex LH-20 chromatographic column, eluting with 8% of ethanol water, collecting the target active peptide component eluted with 8% of ethanol volume fraction, concentrating and drying to obtain the target immunoregulation activity heptapeptide.
5. The method according to claim 3, wherein the soybean protein is dispersed in water by adding the soybean protein to 5-20 times by mass of water and then stirring.
6. The method of claim 3, wherein the step of obtaining the soy protein hydrolysate by adding alkaline protease in an amount of 0.1-1.0% by mass, pH 7.5-9.5, temperature 30-65 ℃, and performing enzymolysis for 1-5 hours, then adding neutral protease in an amount of 0.1-1.0% by mass, pH 6.0-8.0, temperature 40-60 ℃, and performing enzymolysis for 1-5 hours, heating to 90 ℃ for 20 minutes to inactivate the enzyme, centrifuging to obtain the supernatant of the soy protein hydrolysate, filtering with an ultrafiltration membrane with a molecular weight cutoff of 3000 daltons, collecting the filtrate, and spray-drying the filtrate to obtain the soy protein hydrolysate.
7. Use of the immunomodulatory active heptapeptide of claim 1 in the preparation of an immunomodulatory active pharmaceutical, food or health product.
8. An immunomodulatory active pharmaceutical, food or health product comprising the immunomodulatory active heptapeptide of claim 1 as an active ingredient.
9. Use of soy protein for the preparation of an immunomodulatory active heptapeptide of claim 1.
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| CN113215212A (en) * | 2021-04-16 | 2021-08-06 | 国肽生物工程（常德）有限公司 | Soybean protein peptide with antioxidant and ACE (angiotensin converting enzyme) inhibiting functions and preparation method thereof |
Citations (3)
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| CN108396047A (en) * | 2018-01-25 | 2018-08-14 | 浙江海洋大学 | A kind of preparation method of small public fish immunomodulatory peptides |
| CN109400678A (en) * | 2018-10-18 | 2019-03-01 | 大连深蓝肽科技研发有限公司 | A kind of anti-oxidant and DPP-IV inhibitory activity peptide in stichopus japonicus source |
| CN110655556A (en) * | 2019-10-31 | 2020-01-07 | 福州大学 | Preparation and method of immunoregulatory peptide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108396047A (en) * | 2018-01-25 | 2018-08-14 | 浙江海洋大学 | A kind of preparation method of small public fish immunomodulatory peptides |
| CN109400678A (en) * | 2018-10-18 | 2019-03-01 | 大连深蓝肽科技研发有限公司 | A kind of anti-oxidant and DPP-IV inhibitory activity peptide in stichopus japonicus source |
| CN110655556A (en) * | 2019-10-31 | 2020-01-07 | 福州大学 | Preparation and method of immunoregulatory peptide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215212A (en) * | 2021-04-16 | 2021-08-06 | 国肽生物工程（常德）有限公司 | Soybean protein peptide with antioxidant and ACE (angiotensin converting enzyme) inhibiting functions and preparation method thereof |
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