Soybean protein hydrolysate containing anti-inflammatory peptide sequence

Abstract

The present invention relates to a soy protein hydrolyzate containing an anti-inflammatory peptide sequence. The present invention provides a soy protein hydrolyzate containing an anti-inflammatory peptide sequence that can be used for developing anti-inflammatory related pharmaceutical products.

Description

Technical Field

[0001] The present invention relates to a soybean protein hydrolyzate containing an anti-inflammatory peptide sequence and an application method of the soybean protein hydrolyzate as an anti-inflammatory functional material. Background Art

[0002] Inflammation is a local reaction that occurs when cells and tissues of an organism are subjected to adverse stimuli. The adverse stimuli to which the organism is subjected include biological stresses such as microbial infections, physical stresses such as temperature changes and attacks, and chemical stresses such as acids and bases. Inflammation is caused by the response of the tissues that are subjected to these stimuli. Inflammation causes immune cells and T cells in the innate immune system to aggregate in tissues and causes blood vessel dilation and increased vascular permeability, thereby inducing tumors, pain, and causing dysfunction of various tissues. There are many diseases caused by inflammation, including various diseases such as cancer, lifestyle diseases, autoimmune diseases, neurological diseases, obesity, inflammatory bowel disease, and arthritic diseases. Research and development of functional materials that inhibit inflammation are also actively underway.

[0003] It has been reported that soybeans have various physiological functions, and there are also many reports on the anti-inflammatory function. Regarding soy protein, it has also been reported that the hydrolyzate has antioxidant activity, and furthermore, 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 determined, and it is not clear whether they are sequences having anti-inflammatory functions in soybean protease hydrolyzates.

[0004] In addition, regarding the functional material described in Patent Document 2, the anti-inflammatory function of the soybean protease decomposition product is disclosed, but it is a functional material based on a conjugate with phytic acid, and peptide sequences having anti-inflammatory functions generated by enzymatic hydrolysis of soy protein are not disclosed.

[0005] Prior Art Documents

[0006] Patent Documents

[0007] Patent Document 1: Japanese Patent No. 5729297

[0008] Patent Document 2: Japanese Patent No. 6933246 Summary of the Invention

[0009] Problems to be Solved by the Invention

[0010] The technical problem of the present invention is to provide an anti-inflammatory functional material that can be used in medicines and shows anti-inflammatory activity.

[0011] Solutions to the Problems

[0012] The present inventors conducted in-depth research on this technical problem, and as a result, by enzymatically hydrolyzing soy protein with an appropriate protease, they successfully cleaved out peptide sequences that exhibit anti-inflammatory functions in the soy protein hydrolysate sequence.

[0013] That is, the present invention provides the following (1) to (11).

[0014] (1) A soy protein hydrolysate, characterized in that it contains the peptide sequence PFPRPPHQ that exhibits anti-inflammatory function.

[0015] (2) A soy protein hydrolysate, characterized in that it contains the peptide sequence PFPRPPH that exhibits anti-inflammatory function.

[0016] (3) A soy protein hydrolysate, characterized in that it contains the peptide sequence GEIPRPRPRPQHPE that exhibits anti-inflammatory function.

[0017] (4) A soy protein hydrolysate, characterized in that it contains the peptide sequence FPFPRPPHQKEE that exhibits anti-inflammatory function.

[0018] (5) A soy protein hydrolysate, characterized in that it contains the peptide sequence FPFPRPPHQKE that exhibits anti-inflammatory function.

[0019] (6) A soy protein hydrolysate, characterized in that it contains the peptide sequence FPFPRPPHQK that exhibits anti-inflammatory function.

[0020] (7) A soy protein hydrolysate, characterized in that it contains the peptide sequence FPFPRPPHQ that exhibits anti-inflammatory function.

[0021] (8) A soy protein hydrolysate, characterized in that it contains the peptide sequence FPFPRPPH that exhibits anti-inflammatory function.

[0022] (9) A soy protein hydrolysate, characterized in that it contains the peptide sequence ERQFPFPRPPHQKE that exhibits anti-inflammatory function.

[0023] (10) A soy protein hydrolysate, characterized in that it contains two selected from the peptide sequences PFPRPPHWQ, PFPRPPPH, GEIPRPRPRPQHPE, FPFPRPPHQKEE, FPFPRPPHQKE, FPFPRPPHQK, FPFPRPPHQ, FPFPRPPH, ERQFPFPRPPHQKE that exhibit anti-inflammatory functions.

[0024] (11) A soy protein hydrolyzate, characterized by comprising three selected from the peptide sequences PFPRPPHWQ, PFPRPPPH, GEIPRPRPRPQHPE, FPFPRPPHQKEE, FPFPRPPHQKE, FPFPRPPHQK, FPFPRPPHQ, FPFPRPPH, ERQFPFPRPPHQKE that exhibit anti-inflammatory functions.

[0025] Effects of the Invention

[0026] According to the present invention, an anti-inflammatory functional material that exhibits anti-inflammatory functions can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Results of anti-inflammatory tests on cell culture performed according to the peptide sequences synthesized in Test Example 1.

[0028] Figure 2 Measurement of the concentration change of inflammatory cytokine IL-6 based on ELISA according to the peptide sequences synthesized in Test Example 1. (Partial measurement results).

[0029] Figure 3 Results of evaluation of the anti-inflammatory effects of the synthetic peptide sequences selected in Test Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0030] The anti-inflammatory functional material of the present invention is characterized by comprising a peptide sequence that exhibits anti-inflammatory activity. The present invention will be specifically described below.

[0031] (Preparation of Synthetic Peptides)

[0032] Download the primary amino acid sequences of proteins from GENBANK (https: / / www.ncbi.nlm.nih.gov / genbank / ), and use a peptide synthesizer Respep (manufactured by Intavis) to perform Fmoc solid-phase synthesis on all amino acid sequences in a manner of 10 residues per sequence and staggering by 2 residues. Measure the weight of the purified synthetic peptide and dissolve it in Tris buffer calculated according to the molecular weight of each synthesized peptide sequence to prepare a 10 mmol / l synthetic peptide solution.

[0033] (Soybean Protein Raw Material)

[0034] The soy protein hydrolyzate involved in the present invention is made by hydrolyzing soy protein raw materials with proteases. Here, as an example of the soy protein raw material, whole soy milk or 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 the soy protein raw material, soy protein isolate obtained by concentrating protein from these soy milks by treatment with an ultrafiltration membrane or isoelectric precipitation using an acid, etc. can be cited. As still another example of the soy protein raw material, protein concentrates containing fibers, decomposition products of soybean flour can be cited. These soy protein raw materials can be sterilized and dried. It should be noted that the finally obtained soy protein raw material preferably contains 80% by weight or more of protein based on the dry weight, and is preferably soy protein isolate, etc.

[0035] Soy protein isolate is usually prepared as follows. That is, water is added to defatted soybeans, extraction is carried out near neutral pH, soybean meal is separated, and soy milk is obtained. Then, the pH value of the soy milk is adjusted to about 4.5, and the isoelectric precipitate is recovered. Water and an alkaline agent are added to the precipitate to obtain an aqueous solution having a solid component concentration of 5 to 15% by weight, a pH of 5.7 to 8.0, preferably about pH 6.8 to 7.5. The solution of soy protein isolate thus obtained can be directly used in the following processes, or the non-sterilized material or the sterilized material can be dried and then dissolved again for use. However, the production method of soy protein isolate is not limited to the above method, and of course, various changes can be made to this production method.

[0036] (Soy protein hydrolyzate)

[0037] The soy protein hydrolyzate involved in the present invention is a peptide mixture obtained by subjecting the above soy protein raw materials to protease treatment. This protein hydrolyzate preferably has a relatively high degree of decomposition, and 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 hydrolyzate is high.

[0038] (Protease)

[0039] The protease used to obtain the soy protein hydrolyzate involved in the present invention can be derived from animals, plants, or microorganisms, and can be appropriately selected from proteases classified as "metalloprotease", "acid protease", "thiol protease", "serine protease" in protease classification, and is preferably appropriately selected from proteases classified as "metalloprotease", "thiol protease", "serine protease". 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 peptides with relatively small molecular weights such as dipeptides and tripeptides, and is therefore preferred.

[0040] The classification of such proteases is a classification method commonly used in the field of enzymology based on the types of amino acids in the active center. As representative examples of each, "metalloproteases" include neutral proteases from Bacillus, neutral proteases from Streptomyces, neutral proteases from Aspergillus, "TERMOASE", etc.; "acid proteases" include pepsin, acid proteases from Aspergillus, "Sumizyme FP", etc.; "thiol proteases" include bromelain, papain, etc.; "serine proteases" include trypsin, chymotrypsin, subtilisin, alkaline protease from Streptomyces, "Alcalase", "Bioprase", etc. Enzymes other than these can also have their classification confirmed based on the pH at which they act and their reactivity with inhibitors. Since the sites of action on substrates differ greatly among enzymes with different active centers, "cutting residues" are reduced, and enzyme decomposition products can be obtained with high efficiency. In addition, enzyme decomposition products can be produced more effectively by using enzymes from different sources (source organisms) in combination. Even within the same classification, if the sources are different, the sites of action on the protein serving as the substrate are different, and as a result, the proportion of dipeptides and tripeptides can be increased. These proteases preferably have low exopeptidase activity.

[0041] The reaction pH and reaction temperature for protease treatment can be set according to the properties of the protease used. Generally, the reaction pH is carried out near the optimum pH, and the reaction temperature is carried out near the optimum temperature. Generally, the reaction temperature is 20 to 80°C, preferably 40 to 60°C. After the reaction, it is heated to a temperature sufficient to inactivate the enzyme (about 60 to 170°C) to inactivate the remaining enzyme.

[0042] The reaction solution after protease treatment can be used directly or after concentration, and is usually sterilized, spray-dried, freeze-dried, etc. and used in the form of a dry powder. Sterilization is preferably heat sterilization, and the heating temperature is preferably 110 to 170°C, more preferably 130 to 170°C. The heating time is preferably 3 to 20 seconds. In addition, the reaction solution can be adjusted to any pH. Insoluble substances (precipitates, suspensions) generated during protease treatment and pH adjustment can be removed by centrifugation, filtration, etc. Removing insoluble substances can improve the antioxidant activity of the soy protein hydrolyzate, so it is preferred. It can be further purified by activated carbon and adsorption resin.

[0043] (Anti-inflammatory evaluation using macrophages)

[0044] To evaluate the anti-inflammatory effect on macrophages, cell culture was performed by the following method. As mouse macrophages, "J774.1 cells" (provided by the RIKEN) 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 a final concentration of 100 μg / ml were added as antibiotics to prepare a cell culture medium. In the cell culture medium, J774.1 cells were cultured under the conditions of 37°C and 5% CO2 and cultured in a 10 cm culture dish until confluence was reached. The confluent cells were recovered and diluted to 2.5×10 5 cells / ml, and 1 ml was added to each well of a 24-well cell culture plate. The cells were cultured under the conditions of 37°C and 5% CO2 for 24 hours, and the medium was removed with a suction device. After removal, 350 μl of a synthetic peptide and soy protein hydrolyzate adjusted to a final concentration of 5 μg / ml were added, and 350 μl of "Pam3CSK4" (manufactured by InvivoGen) as an inflammation-inducing substance was added to a final concentration of 10 ng / ml, and the cells were cultured under the conditions of 37°C and 5% CO2 for 24 hours. Then, 200 μl of the culture supernatant was recovered.

[0045] (Measurement of IL-6 concentration based on ELISA)

[0046] The concentration of the 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 0.5 μg / ml with 0.1 M Na2HPO4 buffer (pH 9.0) and added to a 96-well microplate at 50 μl / well, and left standing at room temperature for 2 hours. Then, it was washed 3 times with PBS-Tween20 (PBS-T) and thoroughly drained. Next, 3% BSA in PBS-T was added at 100 μl / well, left standing at room temperature for 1 hour, washed 3 times with PBS-T, and thoroughly drained. The culture supernatant sample appropriately diluted with PBS-T was added at 50 μl / well, left standing at 4°C for 24 hours, washed 3 times with PBS-T, and thoroughly drained. The secondary antibody adjusted to 0.5 μg / ml with PBS-T was added at 50 μl / well, left standing at room temperature for 1 hour with aluminum foil shielding from light, washed 3 times with PBS-T, and thoroughly drained. "Streptavidin-Alkalin Phosphate in PBS-T" was added at 50 μl / well, left standing at room temperature for 1 hour with aluminum foil shielding from light, washed 3 times with PBS-T, and thoroughly drained. 1 M diethanolamine buffer containing 0.2 mg / ml of disodium 4-nitrophenyl phosphate was added at 50 μl / well, left standing for 20 minutes with aluminum foil shielding from light, and the absorbance at 405 nm (reference wavelength 492 nm) was measured twice with an enzyme-linked immunosorbent assay reader, and the average value was calculated.

[0047] <Peptide sequence analysis>

[0048] The prepared soy protein hydrolysate was diluted with ultrapure water and adjusted to 0.1% by weight, 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 carried out 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 the MS conditions, scanning analysis was performed under the conditions of ESI+, spray voltage 4 kV, capillary temperature 300°C, and mass range 100 - 2000.

[0049] (Application in drugs)

[0050] The soy protein hydrolyzate containing an anti-inflammatory peptide sequence involved in the present invention can also be used as a drug. Examples of diseases that cause inflammation in organisms include arteriosclerosis, myocardial infarction, cancer, diabetes, Alzheimer's disease, sarcopenia, etc., and it can be used as a drug for preventing and treating these diseases. When provided in the form of a drug, it can be used in various forms such as liquid, powder, tablet, capsule, etc.

[0051] Examples

[0052] The present invention will be described more specifically below by way of examples.

[0053] (Production Example 1) Preparation of synthetic peptides

[0054] Download the amino acid primary sequences of the main storage proteins, β-conglycinin (α, α', β subunits), glycinin (A1aB1b, A2B1a, A1bB2, A3B4, A5A4B3 subunits), LP (Basic 7S, Cytochrome C, Gly m Bd 28k, Bly m Bd 30k, Lipoxygenase, Oleosin Isoform A, Oleosin Isoform B) that make up soy protein from GENBANK, and use a peptide synthesizer Respep (manufactured by Intavis) to perform solid-phase synthesis on all amino acid sequences in the manner of 10 residues per sequence and staggering by 2 residues. Measure the weight of the purified synthetic peptide and dissolve it in Tris buffer calculated based on the molecular weight of each synthesized peptide sequence to prepare a 10 mmol / l synthetic peptide solution.

[0055] (Production Example 2) Preparation of isolated soy protein

[0056] Isolated soy protein was prepared from low-temperature-deflavored defatted soybeans as follows.

[0057] (1) Extract the protein part from 1 kg of low-temperature-deflavored defatted soybeans with water, remove the soybean residue components by a centrifuge, and prepare defatted soy milk.

[0058] (2) Adjust the pH of the obtained defatted soy milk to 4.5 and perform isoelectric precipitation, obtain acid-precipitated curd by a centrifuge, and neutralize it.

[0059] (3) Neutralize each component obtained, and heat-sterilize it at 120 °C for 10 seconds.

[0060] (Production Example 3) Preparation of soy protein hydrolyzate A

[0061] Prepare a 3% solution of the isolated soy protein obtained in Production Example 2, and add 1% or 2% of the metalloprotease "TERMOASE" (derived from Bacillus thermoproteolyticus Rokko, Daiwa Kasei Co., Ltd.) relative to the protein, and carry out an enzymatic hydrolysis reaction at pH 9.0 and 58 °C for 60 minutes.

[0062] Next, add 0.5% or 1% of the serine protease "Bioprase" (derived from Bacillus sp., Nagase ChemteX Corporation) relative to the protein, and carry out an enzymatic hydrolysis reaction at pH 7.5 and 58 °C for 60 minutes.

[0063] Next, add 0.5% or 1% of the metalloprotease "Sumizyme FP" (derived from Aspergillus oryzae, Shin Nippon Chemical Industry Co., Ltd.) relative to the protein, and carry out an enzymatic hydrolysis reaction at pH 7.5 and 58 °C for 60 minutes.

[0064] After the above enzymatic reaction, the reaction was stopped by heating the enzymatic hydrolysis reaction solution at 90 °C for 20 minutes, and then freeze-dried to obtain a soy protein hydrolyzate A.

[0065] (Test Example 1)

[0066] For all the synthetic peptides obtained in Production Example 1, anti-inflammatory evaluation using cultured cells was carried out. The addition amount of the synthetic peptide was uniformly 10 μmol. The results showed that the synthetic peptides tested in this test did not play a good role in the effect of inhibiting cell proliferation. ( Figure 1 ) However, without further confirming the anti-inflammatory effect of the synthetic peptide, the measurement of the concentration of the inflammatory cytokine IL-6 based on ELISA was carried out. Figure 2 Part of them is shown. All the synthetic peptides were evaluated, and they were summarized in order from the synthetic peptides with high anti-inflammatory effects (Table 1). As a result, PRP (phenylalanine-arginine-phenylalanine) was detected at a high frequency as a sequence common to the peptide sequences showing anti-inflammatory functions.

[0067] (Test Example 2)

[0068] The peptide sequence analysis of the soy protein hydrolyzate A obtained in Production Example 3 was carried out, and the sequences containing the PRP sequence were summarized (Table 2). For these sequences, synthetic peptides were prepared and anti-inflammatory evaluation using cultured cells was carried out ( Figure 3). The added amount of the synthetic peptide was uniformly 1 μmol. As a result, among the synthesized peptides, PFPRPPHQ, PFPRPPH, GEIPRPRPRPQHPE, FPFPRPPHQKEE, FPFPRPPHQKE, FPFPRPPHQK, FPFPRPPHQ, FPFPRPPH, ERQFPFPRPPHQKE exhibited anti-inflammatory functions.

[0069]

[0070]

Claims

1. A soy protein hydrolyzate, characterized in that, Contains the peptide PFPRPPH that exhibits anti-inflammatory function.

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