Soybean protein hydrolysate containing anti-inflammatory peptide sequence

Abstract

The invention relates to a soy protein hydrolysate containing an anti-inflammatory peptide sequence. The invention provides a soybean protein hydrolysate containing an anti-inflammatory peptide sequence, which can be used for developing an anti-inflammatory related medicine product or a food with a certain anti-inflammatory function.

Description

Technical Field

[0001] The invention relates to a soybean protein hydrolysate containing an anti-inflammatory peptide sequence and a method for using the soybean protein hydrolysate as an anti-inflammatory functional material. Background Art

[0002] Inflammation is a local reaction of cells and tissues of organisms when they are adversely stimulated. Adverse stimuli to organisms include biological stress such as microbial infection, physical stress such as temperature change and attack, and chemical stress such as acid-base. Inflammation is caused by the response of tissues to these stimuli. Inflammation causes immune cells and T cells in the natural immune system to gather in tissues and cause vasodilation and increased vascular permeability, which in turn induces tumors, pain, and causes dysfunction of various tissues. There are many diseases caused by inflammation, including cancer, lifestyle diseases, autoimmune diseases, neurological diseases, obesity, inflammatory bowel disease, arthritis and other diseases. Research and development of functional materials that inhibit inflammation are also being actively carried out.

[0003] It is reported that soybeans have a variety of physiological functions, and there are many reports on anti-inflammatory functions. Regarding soybean protein, it is also reported that hydrolyzates have antioxidant activity, and further, peptide sequences showing anti-inflammatory functions have been identified. Among them, as described in Patent Document 1, it is disclosed that peptides having FLV or VPY sequences have anti-inflammatory functions. However, only two sequences have been identified, and it is not clear whether they are sequences in soybean protease hydrolyzates that have anti-inflammatory functions.

[0004] Furthermore, the functional material described in Patent Document 2 discloses the anti-inflammatory function of a soybean protease decomposition product, but it is a functional material based on a conjugate with phytic acid, and does not disclose a peptide sequence having an anti-inflammatory function generated by enzymatic hydrolysis of soybean protein.

[0005] Prior art literature Patent Literature Patent Document 1: Japanese Patent No. 5729297 Patent Document 2: Japanese Patent No. 6933246 Summary of the invention

[0006] Problem that the invention aims to solve The technical problem of the present invention is to provide an anti-inflammatory functional material that can be used in food or medicine and exhibits anti-inflammatory activity.

[0007] Solutions for solving problems The present inventors have conducted in-depth research on this technical problem and have successfully cut out a peptide sequence showing anti-inflammatory function from the sequence of soybean protein hydrolysate by enzymatically hydrolyzing soybean protein with an appropriate proteolytic enzyme.

[0008] That is, the present invention provides the following (1) to (11).

[0009] (1) A soybean protein hydrolyzate, characterized by comprising a peptide sequence PFPRPPHWQ showing anti-inflammatory function.

[0010] (2) A soybean protein hydrolyzate, characterized in that it contains a peptide sequence PFPRPPPH showing anti-inflammatory function.

[0011] (3) A soybean protein hydrolyzate, characterized in that it contains a peptide sequence GEIPRPRPRPQHPE showing anti-inflammatory function.

[0012] (4) A soybean protein hydrolyzate, characterized in that it contains a peptide sequence FPFPRPPHQKEE showing anti-inflammatory function.

[0013] (5) A soybean protein hydrolyzate, characterized in that it contains a peptide sequence FPFPRPPHQKE showing anti-inflammatory function.

[0014] (6) A soybean protein hydrolyzate, characterized in that it contains a peptide sequence FPFPRPPHQK showing anti-inflammatory function.

[0015] (7) A soybean protein hydrolyzate, characterized in that it contains a peptide sequence FPFPRPPHQ showing anti-inflammatory function.

[0016] (8) A soybean protein hydrolyzate, characterized in that it contains a peptide sequence FPFPRPPH showing anti-inflammatory function.

[0017] (9) A soybean protein hydrolyzate, characterized in that it contains a peptide sequence ERQFPFPRPPHQKE showing anti-inflammatory function.

[0018] (10) A soybean protein hydrolyzate, characterized in that it contains two selected from the group consisting of peptide sequences PFPRPPHWQ, PFPRPPPH, GEIPRPRPRPQHPE, FPFPRPPHQKEE, FPFPRPPHQKE, FPFPRPPHQK, FPFPRPPHQ, FPFPRPPH, and ERQFPFPRPPHQKE showing anti-inflammatory function.

[0019] (11) A soybean protein hydrolyzate, characterized in that it contains three peptide sequences selected from the group consisting of PFPRPPHWQ, PFPRPPPH, GEIPRPRPRPQHPE, FPFPRPPHQKEE, FPFPRPPHQKE, FPFPRPPHQK, FPFPRPPHQ, FPFPRPPH, and ERQFPFPRPPHQKE that exhibit anti-inflammatory function.

[0020] Effects of the Invention According to the present invention, an anti-inflammatory functional material exhibiting an anti-inflammatory function can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The anti-inflammatory test results of cell culture were performed according to the peptide sequence synthesized in Experimental Example 1.

[0022] Figure 2 The concentration change of the inflammatory cytokine IL-6 was measured by ELISA according to the peptide sequence synthesized in Test Example 1. (Partial measurement results).

[0023] Figure 3 Evaluation results of the anti-inflammatory effects of the synthetic peptide sequences screened in Experimental Example 2. DETAILED DESCRIPTION

[0024] The anti-inflammatory functional material of the present invention is characterized by comprising a peptide sequence showing anti-inflammatory activity. The present invention is described in detail below.

[0025] (Preparation of synthetic peptides) The primary amino acid sequence constituting the protein was downloaded from GENBANK (https: / / www.ncbi.nlm.nih.gov / genbank / ), and all amino acid sequences were subjected to Fmoc solid phase synthesis using the peptide synthesizer Respep (manufactured by Intavis) in a manner of 10 residues per sequence and staggered by 2 residues. The weight of the purified synthetic peptide was measured and dissolved in a Tris buffer calculated based on the molecular weight of each synthesized peptide sequence to prepare a 10 mmol / l synthetic peptide solution.

[0026] (Soybean protein raw material) The soy protein hydrolysate to which the present invention relates is made by hydrolyzing a soy protein raw material with protease. Here, as an example of a soy protein raw material, full-fat soy milk and defatted soy milk obtained by extracting protein components from whole beans, defatted soybeans, etc. with water and removing soybean meal components can be cited. As another example of a soy protein raw material, isolated soy protein obtained by concentrating protein from these soy milks by treatment with an ultrafiltration membrane or by using isoelectric precipitation of an acid can be cited. As another example of a soy protein raw material, a protein concentrate containing fiber and a decomposition product of soy flour can be cited. These soy protein raw materials can be sterilized and dried. It should be noted that the soy protein raw material finally obtained preferably contains more than 80% by weight of protein by dry weight, preferably, for example, isolated soy protein, etc.

[0027] Isolated soy protein is usually prepared as follows. That is, water is added to defatted soybeans, extraction is performed near pH neutrality, and soybean meal is separated to obtain soy milk. The pH value of the soy milk is then adjusted to about 4.5, and the isoelectric point precipitate is recovered. Water and an alkali agent are added to the precipitate to obtain an aqueous solution with a solid content concentration of 5 to 15% by weight, a pH of 5.7 to 8.0, and preferably a pH of about 6.8 to 7.5. The solution of isolated soy protein thus obtained can be used directly in the following steps, or the unsterilized material or the sterilized material can be dried and then dissolved again for use. However, isolated soy protein is not limited to the above-mentioned production method, and of course various changes can be made to the production method.

[0028] (Soy protein hydrolysate) The soy protein hydrolysate of the present invention is a peptide mixture obtained by treating the above-mentioned soy protein raw material with protease. The protein hydrolysate preferably has a relatively high degree of decomposition, and is particularly preferably a peptide mixture in which the proportion of dipeptides and tripeptides (sometimes collectively referred to as "di / tripeptides") in the total amount of peptides and free amino acids in the hydrolysate is high.

[0029] (Protease) The protease used to obtain the soy protein hydrolyzate according to the present invention may be derived from animals, plants, or microorganisms, and may be appropriately selected from proteases classified as "metalloproteases", "acid proteases", "thiol proteases", and "serine proteases" in the protease classification, and preferably appropriately selected from proteases classified as "metalloproteases", "thiol proteases", and "serine proteases". In particular, a decomposition method in which two or more or three or more enzymes belonging to different classifications act sequentially or simultaneously can increase the proportion of relatively small molecular weight peptides such as dipeptides and tripeptides, and is therefore preferred.

[0030] Such classification of proteases is a classification method based on the type of amino acids in the active center commonly used in the field of enzymology. As representative examples of each, "metalloprotease" can include neutral proteases from Bacillus, neutral proteases from Streptomyces, neutral proteases from Aspergillus, "TERMOASE", etc., "acidic proteases" can include pepsin, acidic proteases from Aspergillus, "Sumizyme FP", etc., "thiol proteases" can include bromelain, papain, etc., and "serine proteases" can include trypsin, chymotrypsin, subtilisin, alkaline proteases from Streptomyces, "Alcalase", "Bioprase", etc. Other enzymes can also be classified according to their pH and reactivity with inhibitors. Since the sites of action on substrates are very different between enzymes with different active centers, "cleavage residues" are reduced, and enzyme decomposition products can be obtained efficiently. In addition, enzyme degradation products can be produced more efficiently by combining enzymes from different sources (source organisms). Even within the same classification, if the sources are different, the sites of action on the protein as a substrate are also different, resulting in an increase in the proportion of dipeptides and tripeptides. These proteases preferably have low exo-activity.

[0031] The reaction pH and reaction temperature of the protease treatment can be set according to the properties of the protease used. Usually, the reaction pH is near the optimal pH and the reaction temperature is near the optimal temperature. Generally speaking, the reaction temperature is 20-80°C, preferably 40-60°C. After the reaction, the reaction is heated to a temperature sufficient to inactivate the enzyme (about 60-170°C) to inactivate the remaining enzyme.

[0032] The reaction solution after protease treatment can be used directly or concentrated, and is usually sterilized, spray-dried, freeze-dried, etc. and used in the form of dry powder. Sterilization is preferably heat sterilization, and the heating temperature is preferably 110~170°C, more preferably 130~170°C. The heating time is preferably 3~20 seconds. In addition, the reaction solution can be adjusted to any pH. Insoluble matter (precipitate, suspended matter) produced during protease treatment and pH adjustment can be removed by centrifugation, filtration, etc. Removal of insoluble matter can improve the antioxidant activity of soy protein hydrolyzate, so it is preferred. It can be further purified by activated carbon and adsorption resin.

[0033] (Anti-inflammatory evaluation using macrophages) In order to evaluate the anti-inflammatory effect on macrophages, cell culture was performed by the following method. As mouse macrophages, "J774.1 cells" (provided by RIKEN, Japan) were used. In addition, bovine serum was added to the "RPMI-1640" medium (manufactured by Wako Pure Chemical Industries, Ltd.) at a final concentration of 10%, and penicillin at a final concentration of 100 IU / ml and streptomycin at 100 μg / ml were added as antibiotics to prepare a cell culture medium. J774.1 cells were cultured in a cell culture medium at 37°C and 5% CO2, and cultured in a 10 cm culture dish until confluence was reached. The cells that had reached confluence were recovered and diluted to 2.5×10 5 / ml, add 1ml to each 24-well cell culture plate. Culture for 24 hours at 37°C and 5% CO2, and remove the culture medium with an aspirator. After removal, add 350μl of synthetic peptides and soy protein hydrolysates adjusted to a final concentration of 5μg / ml, and add 350μl of "Pam3CSK4" (manufactured by InvivoGen) as an inflammation-inducing substance to a final concentration of 10ng / ml, and culture for 24 hours at 37°C and 5% CO2. Then, collect 200μl of the culture supernatant.

[0034] (Determination of IL-6 concentration based on ELISA) The concentration of inflammatory cytokine IL-6 in each culture medium after cell culture was measured by ELISA. "Purified rat anti-mouse IL-6" (primary antibody), "Biotinylated rat anti-mouse IL-6" (secondary antibody), "Recombinant mouse IL-6" (standard IL-6), and "Streptavidin-Alkalin Phosphate" were purchased from BD Biosciences and used. The primary antibody was adjusted to 0.5μg / ml with 0.1M Na2HPO4 buffer (pH9.0), and added to a 96-well microplate at 50μl / well and left to stand at room temperature for 2 hours. Then, it was washed three times with PBS-Tween20 (PBS-T) and the water was drained thoroughly. Then, 3% BSA in PBS-T was added at 100μl / well, left to stand at room temperature for 1 hour, washed three times with PBS-T, and the water was drained thoroughly. Add 50 μl / well of the culture supernatant sample diluted appropriately with PBS-T, leave it at 4°C for 24 hours, wash it three times with PBS-T, and drain it thoroughly. Add 50 μl / well of the secondary antibody adjusted to 0.5 μg / ml with PBS-T, leave it at room temperature for 1 hour with aluminum foil, wash it three times with PBS-T, and drain it thoroughly. Add "Streptavidin-Alkalin Phosphatein PBS-T" at 50 μl / well, leave it at room temperature for 1 hour with aluminum foil, wash it three times with PBS-T, and drain it thoroughly. Add 50 μl / well of 1M diethanolamine buffer containing 0.2 mg / ml of 4-dinitrophenylphosphate disodium, leave it at room temperature for 20 minutes with aluminum foil, measure the absorbance at 405 nm (reference wavelength 492 nm) twice with an enzyme reader, and calculate the average value.

[0035] <Peptide sequence analysis> The prepared soy protein hydrolysate was diluted with ultrapure water and adjusted to 0.1% by weight, and passed through a 0.22 μm filter to prepare a sample for peptide sequence analysis. Peptide sequence analysis was performed by LC-MS / MS analysis. LC-MS / MS analysis was performed using an Agilent 1200 series (manufactured by Agilent Technologies), LTQ Orbitrap XL (manufactured by Thermo Fisher Scientific), and an OSHO TSKgel ODS-100V column (4.6 mm×250 mm, 5 μm). As MS conditions, scanning analysis was performed under the conditions of ESI+, spray voltage 4 kV, capillary temperature 300°C, and mass range 100-2000.

[0036] (Application in food) The soy protein hydrolyzate containing the anti-inflammatory peptide sequence of the present invention can be used as an anti-inflammatory functional material with the soy protein hydrolyzate as an effective ingredient in various foods. For example, beverages, tablet products, bar foods, salad dressings, meat products, snacks, desserts, cakes, and nutritional supplements can be obtained that exert anti-inflammatory functions as physiological functions on the ingesting user.

[0037] (Application in medicines) The soy protein hydrolysate containing the anti-inflammatory peptide sequence of the present invention can also be used as a medicine. Diseases that cause inflammation in organisms include arteriosclerosis, myocardial infarction, cancer, diabetes, Alzheimer's disease, sarcopenia, etc., and can be used as a medicine for preventing and treating these diseases. When provided as a medicine, it can be used in various forms such as liquid, powder, tablet, capsule, etc.

[0038] Example

[0039] The present invention is described in more detail below by way of examples.

[0040] (Production Example 1) Preparation of synthetic peptide The primary amino acid sequences of β-conglycinin (α, α', β subunits), glycinin (A1aB1b, A2B1a, A1bB2, A3B4, A5A4B3 subunits), and LP (Basic 7S, Cytochrome C, Gly m Bd 28k, Bly m Bd 30k, Lipoxygenase, Oleosin Isoform A, and Oleosin Isoform B), which are the main storage proteins constituting soybean protein, were downloaded from GENBANK, and all amino acid sequences were solid-phase synthesized using a peptide synthesizer Respep (manufactured by Intavis) in a manner that each sequence had 10 residues and was staggered by 2 residues. The weight of the purified synthetic peptide was measured, and it was dissolved in a Tris buffer calculated based on the molecular weight of each synthesized peptide sequence to prepare a 10 mmol / l synthetic peptide solution.

[0041] (Production Example 2) Preparation of isolated soybean protein Isolated soy protein was prepared from low-denatured defatted soybeans as follows.

[0042] (1) The protein portion was extracted from 1 kg of low-denatured defatted soybeans using water, and the dregs were removed by centrifuge to prepare defatted soy milk.

[0043] (2) The pH of the defatted soy milk is adjusted to 4.5 and isoelectric precipitation is performed. The acid precipitated curd is obtained by centrifugation and then neutralized.

[0044] (3) The obtained components are neutralized and sterilized by heating at 120°C for 10 seconds.

[0045] (Production Example 3) Preparation of Soy Protein Hydrolyzate A A 3% solution of the isolated soybean protein obtained in Production Example 2 was prepared, and 1% or 2% of the metalloprotease "TERMOASE" (derived from Bacillus thermoproteolyticus Rokko, Yamato Chemical Industry Co., Ltd.) was added to the protein, and an enzymatic reaction was carried out at pH 9.0 and 58° C. for 60 minutes.

[0046] Next, 0.5% or 1% of serine protease "Bioprase" (derived from Bacillus sp., Nagase ChemteX Corporation) was added to the protein, and enzymatic hydrolysis was performed at pH 7.5 and 58°C for 60 minutes.

[0047] Next, 0.5% or 1% of metalloprotease "Sumizyme FP" (derived from Aspergillus oryzae, Shin Nippon Chemical Co., Ltd.) was added to the protein, and enzymatic hydrolysis was performed at pH 7.5 and 58° C. for 60 minutes.

[0048] After the enzyme reaction, the enzymatic reaction solution was heated at 90° C. for 20 minutes to stop the reaction, and then freeze-dried to obtain soybean protein hydrolyzate A.

[0049] (Test Example 1) For all synthetic peptides obtained in Preparation Example 1, anti-inflammatory evaluation using cultured cells was performed. The amount of synthetic peptide added was uniformly 10 μmol. The results showed that the synthetic peptides tested in this test did not play a good role in inhibiting cell proliferation. ( Figure 1 ). However, the anti-inflammatory effect of the synthetic peptide was not further confirmed, and the concentration of the inflammatory cytokine IL-6 was measured based on ELISA. Figure 2 A portion of them is shown in the figure. All synthetic peptides were evaluated and sorted in order of the most effective anti-inflammatory peptides (Table 1). As a result, PRP (phenylalanine-arginine-phenylalanine) was detected at a high frequency as a common sequence in peptide sequences showing anti-inflammatory function.

[0050] (Test Example 2) The soy protein hydrolyzate A obtained in Preparation Example 3 was subjected to peptide sequence analysis, and sequences containing the PRP sequence were summarized (Table 2). Synthetic peptides were prepared from these sequences, and anti-inflammatory evaluation using cultured cells was performed ( Figure 3 ). The amount of synthetic peptides added was uniformly 1 μmol. As a result, among the synthetic peptides, PFPRPPHWQ, PFPRPPPH, GEIPRPRPRPQHPE, FPFPRPPHQKEE, FPFPRPPHQKE, FPFPRPPHQK, FPFPRPPHQ, FPFPRPPH, and ERQFPFPRPPHQKE exerted anti-inflammatory function.

[0051]

[0052]

Claims

1. A soybean protein hydrolysate, characterized in that Contains the peptide sequence PFPRPPHWQ showing anti-inflammatory function.

2. A soybean protein hydrolysate, characterized in that Contains the peptide sequence PFPRPPPH which exhibits anti-inflammatory function.

3. A soybean protein hydrolysate, characterized in that It contains the peptide sequence GEIPRPRPRPQHPE which exhibits anti-inflammatory function.

4. A soybean protein hydrolysate, characterized in that Contains the peptide sequence FPFPRPPHQKEE which exhibits anti-inflammatory function.

5. A soybean protein hydrolysate, characterized in that Contains the peptide sequence FPFPRPPHQKE which exhibits anti-inflammatory function.

6. A soybean protein hydrolysate, characterized in that: Contains the peptide sequence FPFPRPPHQK which exhibits anti-inflammatory function.

7. A soybean protein hydrolysate, characterized in that: It contains the peptide sequence FPFPRPPHQ which exhibits anti-inflammatory function.

8. A soybean protein hydrolysate, characterized in that: Contains the peptide sequence FPFPRPPH showing anti-inflammatory function.

9. A soybean protein hydrolysate, characterized in that: It contains the peptide sequence ERQFPFPRPPHQKE which exhibits anti-inflammatory function.

10. A soybean protein hydrolysate, characterized in that: It contains two peptide sequences selected from the group consisting of PFPRPPHWQ, PFPRPPPH, GEIPRPRPRPQHPE, FPFPRPPHQKEE, FPFPRPPHQKE, FPFPRPPHQK, FPFPRPPHQ, FPFPRPPH, and ERQFPFPRPPHQKE that show anti-inflammatory function.

11. A soybean protein hydrolysate, characterized in that: It contains three peptide sequences selected from the group consisting of PFPRPPHWQ, PFPRPPPH, GEIPRPRPRPQHPE, FPFPRPPHQKEE, FPFPRPPHQKE, FPFPRPPHQK, FPFPRPPHQ, FPFPRPPH, and ERQFPFPRPPHQKE that show anti-inflammatory function.

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