A polypeptide composition for enhancing immunity and its preparation method and application

Abstract

The present invention discloses a polypeptide composition for enhancing immunity, a preparation method and application thereof, and belongs to the technical field of food and health care. The polypeptide composition for enhancing immunity provided by the present invention comprises the following components by weight: 15-25 parts of pine pollen peptide, 60-70 parts of sea cucumber oligopeptide powder, 2-6 parts of broccoli extract, 8-12 parts of sugar apple extract, and 0.2-1.8 parts of antimicrobial peptide LL-37. The preparation method of the polypeptide composition for enhancing immunity provided comprises the following steps: mixing pine pollen peptide, sea cucumber oligopeptide powder, broccoli extract and sugar apple extract to obtain a premix, and mixing the premix with antimicrobial peptide LL-37 to obtain a polypeptide composition. The present invention achieves the effect of enhancing immunity of the polypeptide composition by adjusting the type and amount of polypeptides used in combination, and adding plant extracts at the same time, and by affecting the metabolism of energy substances.

Description

Technical Field

[0001] The invention belongs to the technical field of food health care, and in particular relates to an immunity enhancing polypeptide composition and a preparation method and application thereof. Background Art

[0002] Sub-health refers to a state in which the human body is between disease and health. Sub-health is characterized by fatigue, decreased vitality, and reduced immunity as the main clinical manifestations, and has become one of the hidden dangers that endanger health. Therefore, it is very important to prevent sub-health by improving immunity. Immunity refers to the sum of biological effects produced by the body in the process of identifying and responding to "antigenic foreign bodies" or "foreign components". Under normal circumstances, it is a specific physiological reaction to maintain the stability of the internal environment, and immunity is a defense mechanism of the body itself. The immune system is composed of immune organs, immune cells, and immune molecules. According to the results of immunology research in medicine, the immune system has the immune defense function of anti-infection immunity, the immune self-stabilization function that can timely identify and remove damaged or aging cells in the body, and the immune surveillance function that can timely identify cell replication errors or cell mutations and remove them.

[0003] Immunology is one of the fastest-growing disciplines today. Due to the increasing number of sub-healthy people, people are more and more eager to improve the demand for drugs for immune function. With the progress of time, the research on the immune enhancement effect has been deepened, and some immune-related diseases have brought new breakthroughs. For example, the peptide-Zn complex formed by the combination of Zn and protein peptides belongs to the category of organic zinc, which not only has the characteristics of fast digestion and absorption, but also has certain biological activity. In addition, there are many reports on amino acid and zinc chelates, as well as chelates prepared by small peptides and iron, manganese, chromium, etc. as ligands, in improving animal growth and reproduction, enhancing disease resistance, regulating physiological metabolism, etc. The above-mentioned prior art shows that it has the function of improving immunity to a certain extent, but it mainly plays a single effect and bioavailability, which still needs to be improved. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a polypeptide composition for enhancing immunity, a preparation method and application thereof, by adjusting the types and amounts of polypeptides to be used in combination, and adding plant extracts, the metabolism of energy substances is affected, so as to achieve the effect of enhancing immunity of the polypeptide composition.

[0005] To achieve the above object, the present invention provides a polypeptide composition for enhancing immunity, comprising the following components in parts by weight:

[0006] 15-25 parts of pine pollen peptide, 60-70 parts of sea cucumber oligopeptide powder, 2-6 parts of broccoli extract, 8-12 parts of sugar apple extract, and 0.2-1.8 parts of antibacterial peptide LL-37.

[0007] Pine pollen peptide: rich in various amino acids and contains a variety of nutrients to maintain human health; it has antioxidant and anti-aging effects, can inhibit the peroxidation reaction of lipids and proteins in the body, accelerate the removal of free radicals, inhibit the aging of tissue cells, and delay aging. At the same time, it has the effects of significantly lowering blood lipids and cholesterol, strengthening the brain, and improving sleep quality.

[0008] Sea cucumber oligopeptide powder: rich in protein, vitamins, and mineral nutrients. Due to its small molecules, it is easier to be absorbed and utilized by the intestines. It has good antioxidant, anti-fatigue, anti-inflammatory, anti-tumor, immune regulation and blood pressure lowering effects.

[0009] Broccoli extract: contains a variety of beneficial ingredients, including glucosinolates, isothiocyanates, glucosinolates, and glucosinolate enzymes. These ingredients have strong antioxidant and antibacterial effects, especially glucosinolates and isothiocyanates, which are the most important antioxidant ingredients in broccoli extract and have a significant effect on preventing oxidative stress. Broccoli extract also contains compounds similar to metformin, which can play an auxiliary role in controlling blood sugar.

[0010] Sugar apple extract: has anti-tumor, antibacterial and antioxidant activities, and can significantly inhibit the activity of α-glucosidase and α-amylase, thereby improving normal glucose tolerance.

[0011] Antimicrobial peptide LL-37: It has antibacterial and anti-endotoxin effects, can promote wound healing, has immunomodulatory effects, and also has a food preservative effect.

[0012] Preferably, the Annona custard apple extract is Annona custard apple fruit extract.

[0013] The present invention also provides a method for preparing the polypeptide composition, comprising the following steps:

[0014] Pine pollen peptide, sea cucumber oligopeptide powder, broccoli extract and custard apple extract are mixed to obtain a premix, and the premix is ​​mixed with antimicrobial peptide LL-37 to obtain a polypeptide composition.

[0015] Preferably, the method for preparing the custard apple extract comprises the following steps:

[0016] The sugar apple fruit is crushed and sieved to obtain sugar apple powder; the sugar apple powder is mixed with anhydrous ethanol, refluxed and extracted for 5 times, the extracts are combined, and vacuum freeze-dried to obtain the sugar apple extract.

[0017] Preferably, the sieved particle size is required to be 120-500 mesh, and anhydrous ethanol is used as the extractant to limit the particle size of the custard apple to the range of 25-120 μm, thereby increasing the surface area and the contact area with the solvent, thereby enhancing the leaching rate, promoting the dissolution and diffusion of the active ingredients without losing the active ingredients, and improving the extraction efficiency.

[0018] Preferably, the mixing ratio of the sugar apple powder and anhydrous ethanol is 100g:300-400mL, and the dosage ratio of the anhydrous ethanol as the extraction solvent to the sugar apple powder substrate is adjusted. A low material-liquid ratio will result in a low content of active ingredients in the extract, and a high material-liquid ratio will decompose and lose the effective ingredients. By adjusting the dosage ratio of the anhydrous ethanol extraction solvent to the sugar apple powder substrate, the content of active ingredients in the sugar apple extract is guaranteed, and its efficacy is guaranteed.

[0019] Preferably, the temperature of the reflux extraction is 100-180° C., the time of each reflux extraction is 20-30 min, and the appropriate number of extractions is adjusted to ensure the highest extraction rate of the effective active ingredients of the custard apple without precipitation of by-products.

[0020] Preferably, the vacuum degree of the vacuum freeze-drying is 4 Pa, the temperature of the vacuum freeze-drying is -55 to -65°C, and the time of the vacuum freeze-drying is 12 to 36 hours.

[0021] The present invention also provides the use of the polypeptide composition or the polypeptide composition prepared by the preparation method in preparing products for enhancing immunity.

[0022] Preferably, the immunity enhancing product also includes auxiliary materials.

[0023] Compared with the prior art, the present invention has the following advantages and technical effects:

[0024] The polypeptide composition of the present invention improves the body's energy utilization rate by combining broccoli extract and custard apple extract to regulate the body's energy metabolism. At the same time, the pine pollen peptide can inhibit the peroxidation reaction of lipids and proteins in the body and accelerate the removal of free radicals. The small molecule activity and rapid absorption characteristics of sea cucumber oligopeptide powder successfully improve the sub-health of the human body and achieve the effect of enhancing the human body's immunity. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Unless otherwise specified, the "parts" described in the present invention are all based on mass parts.

[0031] The sources of materials used in the present invention are as follows: pine pollen peptide was purchased from Xi'an Shilin Biotechnology Co., Ltd., with an effective ingredient content of 98%; sea cucumber oligopeptide powder was purchased from Liaoning Tai'ai Peptide Bioengineering Technology Co., Ltd.; antibacterial peptide LL-37 was purchased from Nanjing Taopu Biotechnology Co., Ltd.; broccoli extract was purchased from Shanyang Lianfeng Biotechnology Co., Ltd.; chia seed polypeptide was purchased from Ningxia Vanilla Biotechnology Co., Ltd.

[0032] Example 1

[0033] The sugar apple fruit was crushed and passed through a 270-mesh sieve to obtain sugar apple powder. The sugar apple powder was mixed with anhydrous ethanol in a ratio of 100 g: 350 mL, and refluxed at 120° C. for 5 times, each time for 30 min. The extracts were combined and vacuum freeze-dried for 24 h at a vacuum degree of 4 Pa ​​and a temperature of -60° C. to obtain the sugar apple extract.

[0034] 20 parts of pine pollen peptide, 65 parts of sea cucumber oligopeptide powder, 4 parts of broccoli extract and 10 parts of custard apple extract are mixed to obtain a premix, and the premix is ​​mixed with 1 part of antimicrobial peptide LL-37 to obtain a polypeptide composition.

[0035] Example 2

[0036] The sugar apple fruit was crushed and passed through a 120-mesh sieve to obtain sugar apple powder. The sugar apple powder was mixed with anhydrous ethanol in a ratio of 100 g:400 mL, and refluxed at 100 ° C for 5 times, each time for 40 min. The extracts were combined and vacuum freeze-dried for 36 hours at a vacuum degree of 4 Pa ​​and a temperature of -55 ° C to obtain the sugar apple extract.

[0037] 15 parts of pine pollen peptide, 70 parts of sea cucumber oligopeptide powder, 2 parts of broccoli extract and 12 parts of custard apple extract were mixed to obtain a premix, and the premix was mixed with 0.2 parts of antimicrobial peptide LL-37 to obtain a polypeptide composition.

[0038] Example 3

[0039] The sugar apple fruit was crushed and passed through a 500-mesh sieve to obtain sugar apple powder. The sugar apple powder was mixed with anhydrous ethanol in a ratio of 100 g:300 mL, and refluxed at 180° C. for 5 times, each time for 20 min. The extracts were combined and vacuum freeze-dried for 12 h at a vacuum degree of 4 Pa ​​and a temperature of -65° C. to obtain the sugar apple extract.

[0040] 25 parts of pine pollen peptide, 60 parts of sea cucumber oligopeptide powder, 6 parts of broccoli extract and 8 parts of sugar apple extract were mixed to obtain a premix, and the premix was mixed with 1.8 parts of antimicrobial peptide LL-37 to obtain a polypeptide composition.

[0041] Example 4

[0042] The sugar apple fruit was crushed and passed through a 200-mesh sieve to obtain sugar apple powder. The sugar apple powder was mixed with anhydrous ethanol in a ratio of 100 g: 380 mL, and refluxed at 150 ° C for 5 times, each time for 35 min. The extracts were combined and vacuum freeze-dried for 24 h at a vacuum degree of 4 Pa ​​and a temperature of -60 ° C to obtain the sugar apple extract.

[0043] 18 parts of pine pollen peptide, 66 parts of sea cucumber oligopeptide powder, 3 parts of broccoli extract and 11 parts of custard apple extract were mixed to obtain a premix, and the premix was mixed with 0.7 parts of antimicrobial peptide LL-37 to obtain a polypeptide composition.

[0044] Example 5

[0045] The sugar apple fruit was crushed and passed through a 300-mesh sieve to obtain sugar apple powder. The sugar apple powder was mixed with anhydrous ethanol in a ratio of 100 g:320 mL, and refluxed at 160° C. for 5 times, each time for 25 min. The extracts were combined and vacuum freeze-dried for 24 h at a vacuum degree of 4 Pa ​​and a temperature of -60° C. to obtain the sugar apple extract.

[0046] 23 parts of pine pollen peptide, 62 parts of sea cucumber oligopeptide powder, 5 parts of broccoli extract and 9 parts of custard apple extract were mixed to obtain a premix, and the premix was mixed with 1.5 parts of antimicrobial peptide LL-37 to obtain a polypeptide composition.

[0047] Comparative Example 1

[0048] The sugar apple fruit was crushed and passed through a 270-mesh sieve to obtain sugar apple powder. The sugar apple powder was mixed with anhydrous ethanol in a ratio of 100 g: 350 mL, and refluxed at 120° C. for 5 times, each time for 30 min. The extracts were combined and vacuum freeze-dried for 24 h at a vacuum degree of 4 Pa ​​and a temperature of -60° C. to obtain the sugar apple extract.

[0049] 20 parts of chia seed polypeptide, 65 parts of sea cucumber oligopeptide powder, 4 parts of broccoli extract and 10 parts of custard apple extract were mixed to obtain a premix, and the premix was mixed with 1 part of antimicrobial peptide LL-37 to obtain a polypeptide composition.

[0050] Chia seed polypeptide has the function of regulating insulin secretion and maintaining blood sugar balance; it contains a variety of vitamins, which can eliminate free radicals, and also contains a variety of trace elements, which has the function of supplementing human nutrients.

[0051] Comparative Example 2

[0052] The sugar apple fruit was crushed and passed through a 50-mesh sieve to obtain sugar apple powder. The sugar apple powder was mixed with anhydrous ethanol in a ratio of 100 g:150 mL, and refluxed at 120°C for 3 times, each time for 30 min. The extracts were combined and vacuum freeze-dried for 24 h at a vacuum degree of 4 Pa ​​and a temperature of -60°C to obtain the sugar apple extract.

[0053] 20 parts of pine pollen peptide, 65 parts of sea cucumber oligopeptide powder, 4 parts of broccoli extract and 10 parts of custard apple extract are mixed to obtain a premix, and the premix is ​​mixed with 1 part of antimicrobial peptide LL-37 to obtain a polypeptide composition.

[0054] Experimental example

[0055] 400 female healthy clean-grade ICR mice weighing 18.0-22.0 g were selected. The mice were adaptively fed for one week before the experiment and given sterilized mouse food and sterilized water, with free access to food and water. The mice were randomly divided into 8 groups, each with 50 mice, a normal control group, which was fed with normal mouse food and water; an experimental group 1, which was fed with normal mouse food and water, and fed with the polypeptide composition prepared in Example 1 at 5 mg / g (body weight) per day; an experimental group 2, which was fed with normal mouse food and water, and fed with the polypeptide composition prepared in Example 2 at 5 mg / g (body weight) per day; an experimental group 3, which was fed with normal mouse food and water, and fed with the polypeptide composition prepared in Example 3 at 5 mg / g (body weight) per day; an experimental group 4, which was fed with normal mouse food and water, and fed with the polypeptide composition prepared in Example 4 at 5 mg / g (body weight) per day; an experimental group 5, which was fed with normal mouse food and water, and fed with the polypeptide composition prepared in Example 5 at 5 mg / g (body weight) per day; an experimental group 6, which was fed with normal mouse food and water, and fed with the polypeptide composition prepared in Comparative Example 1 at 5 mg / g (body weight) per day; and an experimental group 7, which was fed with normal mouse food and water, and fed with the polypeptide composition prepared in Comparative Example 2 at 5 mg / g (body weight) per day. The experimental period was 30 days, and the immune indicators were measured after 30 days.

[0056] Immune index determination experiments: Dinitrofluorobenzene (DNFB) induced delayed-type hypersensitivity (DTH) experiment in mice; Concanavalin A (ConA) induced mouse spleen lymphocyte transformation experiment (MTT method); NK cell activity determination; agarose plate method to detect antibody-producing cells; serum hemolysin by half-hemolysis value determination method; mouse carbon clearance experiment; mouse peritoneal macrophage phagocytosis of chicken red blood cells experiment (drop plate method); the above experiments were carried out in accordance with the 2003 version of the Ministry of Health's "Technical Specifications for Inspection and Evaluation of Health Foods", and the mouse immune organ index, ear swelling, optical density difference, hemolytic plaque number, serum half-hemolysis value (HC 50 ), phagocytic index and NK cell activity.

[0057] SPSS statistical software and one-way analysis of variance were used to process the data, and Dunnett-t test was used for pairwise comparison of data with P < 0.05.

[0058] Table 1 Statistical results of the effects of the control group and different experimental groups on the transformation of mouse spleen lymphocytes

[0059] ;

[0060] As shown in Table 1, the results of the ConA-induced mouse spleen lymphocyte transformation experiment show that the optical density difference of the experimental groups 1, 2, 3, 4 and 5 prepared by the present invention is significantly higher than that of the control group. After the experimental group 6 replaced the pine pollen peptide with the chia seed peptide, the optical density difference decreased significantly, destroying the regulatory pathway of the combined polypeptide composition for the body's energy metabolism, indicating that the combined use of the components in the composition of the present invention can enhance the effect of improving immunity. The change in the preparation method of the sugar apple extract in the experimental group 7 affected the efficacy of the sugar apple extract, and further affected the efficacy of the polypeptide composition.

[0061] Table 2 Statistics of the effects of the control group and different experimental groups on carbon clearance in mice

[0062] ;

[0063] As shown in Table 2, which is the result of the carbon clearance experiment in mice, it can be seen that the phagocytic index a of the mice in Experimental Groups 1, 2, 3, 4 and 5 prepared by the present invention was increased compared with the control group. After the pine pollen peptide in Experimental Group 6 was replaced with chia seed peptide, and after the preparation method of the sugar apple extract in Experimental Group 7 was changed, the phagocytic index a of the mice was not improved compared with the control group, indicating that the adjustment of the composition of the polypeptide composition in Experimental Groups 6 and 7 affected its effect of enhancing immunity.

[0064] In addition, according to statistics, the swelling degree of the ear of the mice in the control group was (13.75±5.53) mg, the number of hemolytic plaques in the mice was 408.9±88.2, the half hemolytic value of the mouse serum was 161.23±3.35, the percentage of macrophage phagocytosis was about 13.8%, and the activity of NK cells was (41.3±5.2)%; the swelling degree of the ear of the mice in the experimental group was (25.04±3.74) mg, the number of hemolytic plaques in the mice was 502.5±151.4, the half hemolytic value of the mouse serum was 165.10±1.12, the percentage of macrophage phagocytosis was about 16.5%, and the activity of NK cells was (53.8±9. 6)%; the ear swelling degree of mice in the second experimental group was (24.52±2.41) mg, the number of hemolytic plaques was 491.7±155.7, the half hemolytic value of mouse serum was 162.39±1.06, the percentage of macrophage phagocytosis was about 15.1%, and the activity of NK cells was (51.7±10.1)%; the ear swelling degree of mice in the third experimental group was (25.01±3.21) mg, the number of hemolytic plaques was 495.2±154.9, the half hemolytic value of mouse serum was 163.14±1.15, the percentage of macrophage phagocytosis was about 15.5%, and the activity of NK cells was (51.5±9.1 )%; the swelling degree of the auricle of the mice in the fourth group was (23.78±3.75) mg, the number of hemolytic plaques was 488.4±150.1, the half hemolytic value of the serum was 163.83±1.20, the percentage of macrophage phagocytosis was about 15.8%, and the activity of NK cells was (50.0±9.5)%; the swelling degree of the auricle of the mice in the fifth group was (23.01±2.97) mg, the number of hemolytic plaques was 496.3±153.7, the half hemolytic value of the serum was 163.95±1.54, the percentage of macrophage phagocytosis was about 15.4%, and the activity of NK cells was (52.4±10.5 )%; the swelling degree of the auricle of the mice in the sixth group of experiments was (18.84±6.01) mg, the number of hemolytic plaques was 426.1±95.8, the half hemolytic value of mouse serum was 161.98±3.01, the percentage of macrophage phagocytosis was about 14.7%, and the activity of NK cells was (45.7±6.6)%; the swelling degree of the auricle of the mice in the seventh group of experiments was (15.99±4.46) mg, the number of hemolytic plaques was 450.9±102.1, the half hemolytic value of mouse serum was 161.77±4.01, the percentage of macrophage phagocytosis was about 14.9%, and the activity of NK cells was (47.7±5.6)%.

[0065] In summary, it can be seen that the polypeptide compositions of Experiment 1 to Experiment 5 prepared in Examples 1 to 5 of the present invention significantly increased the swelling degree of the mouse auricle and the activity of NK cells compared with the control group, with significant differences; it is verified that the polypeptide composition prepared by the present invention has a good effect of enhancing immunity. In addition, the polypeptide compositions of Experiment 6 and Experiment 7 prepared in Comparative Examples 1 to 2, when the polypeptide components are replaced or the preparation method is changed, affect the efficacy of the polypeptide composition, and reduce the efficacy of the polypeptide composition in enhancing immunity.

[0066] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A polypeptide composition for enhancing immunity, characterized in that: It is composed of the following components in parts by weight: 15-25 parts of pine pollen peptide, 60-70 parts of sea cucumber oligopeptide powder, 2-6 parts of broccoli extract, 8-12 parts of sugar apple extract, 0.2-1.8 parts of antimicrobial peptide LL-37; The preparation method of the sugar apple extract comprises the following steps: crushing sugar apple fruit, sieving to obtain sugar apple powder; mixing the sugar apple powder with anhydrous ethanol, refluxing and extracting for 5 times, combining the extracts, and vacuum freeze-drying to obtain the sugar apple extract; The particle size of the sieved particles is required to be 120-500 meshes; The ratio of the sugar apple powder to anhydrous ethanol is 100 g: 300-400 mL; The temperature of the reflux extraction is 100-180° C., and the time of each reflux extraction is 20-30 minutes.

2. The method for preparing the polypeptide composition according to claim 1, characterized in that: The following steps are involved: Pine pollen peptide, sea cucumber oligopeptide powder, broccoli extract and custard apple extract are mixed to obtain a premix, and the premix is ​​mixed with antimicrobial peptide LL-37 to obtain a polypeptide composition.

3. The polypeptide composition according to claim 1, characterized in that: The vacuum degree of the vacuum freeze drying is 4 Pa, the temperature of the vacuum freeze drying is -55 to -65°C, and the time of the vacuum freeze drying is 12 to 36 hours.

4. Use of the polypeptide composition according to claim 1 or 3 or the polypeptide composition prepared by the preparation method according to claim 2 in preparing products that enhance immunity.

5. The application according to claim 4, characterized in that: The immunity enhancing product also includes auxiliary materials.