Method for producing opioid peptide, method for improving production efficiency of opioid peptide, and alkaline protease composition for producing opioid peptide

Abstract

The problem addressed by the present invention is to provide an efficient method for producing an opioid peptide.　The problem can be solved by a method for producing an opioid peptide, the method comprising a step for bringing a koji mold alkaline protease of the present invention that recognizes the amino acid sequence of SLXX and cleaves between the two XX into contact with A2-type casein to produce a peptide comprising the amino acid sequence represented by SEQ ID NO: 2.

Description

Method for producing opioid peptides, method for improving production efficiency of opioid peptides, and alkaline protease composition for use in producing opioid peptides

[0001] The present invention relates to a method for producing an opioid peptide, a method for improving the production efficiency of an opioid peptide, and an alkaline protease composition for use in producing an opioid peptide. According to the present invention, opioid peptides can be produced efficiently.

[0002] In recent years, various stress-induced disorders have been reported, and opioid receptor agonists with morphine-like activity have been developed as analgesics or neurotropic drugs. Meanwhile, enzymatically hydrolyzed peptides derived from food proteins have been reported to act on opioid receptors (Non-Patent Document 1), and they are expected to be used as food additives to reduce stress.

[0003] Japanese Patent Application Laid-Open No. 2022-135015

[0004] Peptides, 1999 (Netherlands), Vol. 20, pp. 957-962

[0005] The present inventors have discovered that opioid peptides with high opioid receptor agonist activity can be obtained by hydrolyzing casein with a protease derived from Aspergillus oryzae (Patent Document 1). However, it has not been easy to efficiently obtain the opioid peptides described in Patent Document 1. Therefore, an object of the present invention is to provide an efficient method for producing opioid peptides.

[0006] The present inventors have conducted extensive research into efficient methods for producing opioid peptides, and as a result have surprisingly found that novel opioid peptides can be efficiently obtained by using an alkaline protease from Aspergillus oryzae and A2 type casein. The present invention is based on this finding. Accordingly, the present invention provides: [1] a method for producing an opioid peptide, comprising the step of contacting an Aspergillus oryzae alkaline protease that recognizes the amino acid sequence SLXX and cleaves between the two XXs with A2 type casein to produce a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2; [2] a method for producing an opioid peptide according to [1], wherein the Aspergillus oryzae alkaline protease is (a) an Aspergillus oryzae alkaline protease having the amino acid sequence represented by SEQ ID NO: 1, or (b) an Aspergillus oryzae alkaline protease that contains an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 1 and is capable of producing an opioid peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 from A2 type casein; [3] A method for improving the production efficiency of opioid peptides, comprising the step of contacting A2 type casein with a koji mold alkaline protease that recognizes the amino acid sequence SLXX and cleaves between the two XXs to produce an opioid peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, wherein the koji mold alkaline protease is recovered as a low-adsorption fraction on an anion exchange resin or as an adsorption fraction on a cation exchange resin to separate it from contaminating proteases; [4] The method for improving the production efficiency of opioid peptides according to [3], wherein the koji mold alkaline protease is (a) a koji mold alkaline protease having the amino acid sequence represented by SEQ ID NO: 1, or (b) a koji mold alkaline protease that contains an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 1 and is capable of producing an opioid peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 from A2 type casein; [5] An alkaline protease composition for producing an opioid peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 from A2 type casein, comprising an Aspergillus alkaline protease that recognizes the amino acid sequence SLXX and cleaves between the two XXs;[6] The alkaline protease composition according to [5], wherein the koji mold alkaline protease is (a) a koji mold alkaline protease having the amino acid sequence represented by SEQ ID NO: 1, or (b) a koji mold alkaline protease comprising an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 1 and that can produce an opioid peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 from A2 type casein; [7] an opioid peptide having the amino acid sequence represented by SEQ ID NO: 2; [8] an opioid receptor agonist comprising the opioid peptide according to [7] as an active ingredient; [9] a food composition for agonizing opioid receptors comprising the opioid peptide according to [7] as an active ingredient;

[10] an anti-anxiety food composition comprising the opioid peptide according to [7] as an active ingredient;

[11] a pharmaceutical composition for agonizing opioid receptors comprising the opioid peptide according to [7] as an active ingredient; and

[12] an anti-anxiety pharmaceutical composition comprising the opioid peptide according to [7] as an active ingredient.

[0007] According to the method for producing an opioid peptide of the present invention, an opioid peptide that exhibits excellent effects can be produced efficiently.

[0008] 1A is a photograph of SDS-PAGE analysis of 10, 50, 100, 250, and 500 mM NaCl fractions from a crude enzyme derived from Aspergillus oryzae using DEAE-Sepharose, and a graph (B) showing the ability of each fraction to produce opioid peptides. 1B is a photograph of SDS-PAGE analysis of A1 type milk or A2 type milk hydrolyzed with purified alkaline protease and a chart (B) showing the resulting CM-12 peptide. 1C is a graph showing the production of CM-12 peptide from A2 type casein using purified alkaline protease and crude enzyme. 1D is a graph showing the optimum pH (A) and optimum temperature (B) of purified alkaline protease using CM-30. 1E is a diagram showing the cleavage site of the alkaline protease of the present invention in A2 type casein.

[0009] [1] Method for Producing Opioid Peptides The method for producing an opioid peptide of the present invention comprises the step of contacting A2 type casein with Aspergillus oryzae alkaline protease, which recognizes the amino acid sequence SLXX and cleaves between the two XX residues, to produce a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2. In the amino acid sequence SLXX, S represents serine, L represents leucine, and X represents any amino acid (any of 20 amino acids). The method for producing an opioid peptide of the present invention may also comprise the step of purifying the produced opioid peptide. The opioid peptide can be purified, for example, by adsorption onto a hydrophobic resin and elution with a solvent such as ethanol or acetonitrile. To concentrate a more highly pure peptide, elution with acetonitrile can be performed by HPLC. Insoluble peptides can also be removed by adsorption through activated carbon.

[0010] <<Alkaline Protease>> The alkaline protease used in the present invention is an alkaline protease derived from Aspergillus oryzae that recognizes the amino acid sequence SLXX and cleaves between the two XXs. The alkaline protease is not limited as long as it recognizes the amino acid sequence SLXX and cleaves between the two XXs. For example, it may be (a) an Aspergillus oryzae alkaline protease having the amino acid sequence represented by SEQ ID NO: 1. It may also be an Aspergillus oryzae alkaline protease consisting of the amino acid sequence represented by SEQ ID NO: 1. It may also be (b) an alkaline protease that contains an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 1 and is capable of producing an opioid peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 from A2 type casein. The alkaline protease (b) is not limited as long as it has a function equivalent to that of the protease (a) (e.g., 50% or more activity of the alkaline protease (a), for example, 60% or more activity, for example, 70% or more activity, for example, 80% or more activity, for example, 90% or more activity, for example, equivalent activity), but the identity of the amino acid sequence represented by SEQ ID NO: 1 is preferably 92% or more, more preferably 94% or more, more preferably 96% or more, more preferably 98% or more, and most preferably 99% or more. The amino acid sequence of SEQ ID NO: 1 is as follows:MQSIKRTLLLLGAILPAVLGAPVQETRRAAEKLPGKYIVTFKPGIDEAKIQEHTTWATNIHQRSLERRGATGGDLPVGIERNYKINKFAAYAGSFDDATIE EIRKNEDVAYVEEDQIYYLDGLTTQKSAPWGLGSISHKGQQSTDYIYDTSAGEGTYAYVVDSGVNVDHEEFEGRASKAYNAAGGQHVDSIGHGTHVSGTIA GKTYGIAKKASILSVKVFQGESSSTSVILDGFNWAANDIVSKKRTSKAAINMSLGGGYSKAFNDAVENAFEQGVLSVVAAGNENSDAGQTSPASAPDAITV AAIQKSNNRASFSNFGKVVDVFAPGQDILSAWIGSSSATNTISGTSMATPHIVGLSLYLAALENLDGPAAVTKRIKELATKDVVKDVKGSPNLLAYNGNA.

[0011] The amino acid sequence having 90% or more identity may be, for example, an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added relative to the respective amino acid sequences. The alkaline protease polypeptide may be a polypeptide consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added relative to the amino acid sequence of SEQ ID NO: 1, as long as the identity is 90% or more. As used herein, "an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added" means that the amino acid sequence has been modified by amino acid substitution or the like, or that the alkaline protease is an alkaline protease of a closely related microorganism. The number of amino acid modifications is not particularly limited, as long as the amino acid identity is 90% or more, and may be, for example, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. An example of a modified amino acid sequence of an alkaline protease that can be used in the present invention is preferably an amino acid sequence having one or several (preferably 1, 2, 3, or 4) conservative substitutions in the amino acids.

[0012] The alkaline protease of the present invention includes the deletion of multiple amino acids at the N-terminus and / or multiple amino acids at the C-terminus as long as the effects of the present invention are still achieved. The "amino acid sequence with one or several amino acid deletions, substitutions, insertions, and / or additions" or "amino acid sequences with 90% or greater identity" to the amino acid sequence of SEQ ID NO: 1 is a substitution of the amino acid sequence of SEQ ID NO: 1, but this amino acid sequence substitution is a conservative substitution that maintains the function of the alkaline protease. In other words, a "conservative substitution" refers to a substitution that does not impair the excellent effects of the alkaline protease. That is, even after the insertion, substitution, deletion, or addition, the enzyme is able to recognize the SLXX amino acid sequence and cleave between the two XXs. Specifically, this refers to the replacement of an amino acid residue with another chemically similar amino acid residue. Examples include the replacement of a hydrophobic residue with another hydrophobic residue, or the replacement of a polar residue with another polar residue having the same charge. Functionally similar amino acids that can be obtained by such substitutions are known in the art for each amino acid. Nonpolar (hydrophobic) amino acids include, for example, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include, for example, glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged (basic) amino acids include, for example, arginine, histidine, and lysine. Negatively charged (acidic) amino acids include, for example, aspartic acid and glutamic acid.

[0013] The alkaline protease is derived from, but is not limited to, Aspergillus flavus, Aspergillus clavatus, Aspergillus nidulans, Aspergillus funigatus, Aspergillus fischeri, and Aspergillus oryzae, with Aspergillus oryzae being preferred.

[0014] The alkaline protease used in the present invention recognizes SLXX and cleaves peptides (proteins) between XX. Figure 5 shows the cleavage sites when A2 type casein is cleaved. That is, as shown in Figure 5, it can recognize "SLSS" and cleave between SS, "SLVY" and cleave between VY, "SLPQ" and cleave between PQ, "SLTL" and cleave between TL, and "SLSQ" and cleave between SQ. The ability to recognize SLXX and cleave peptides (proteins) between XX allows the production of the opioid peptides of the present invention from A2 type casein, as described below.

[0015] <A2 Type Casein> A2 type casein is the A2 type β-casein found in milk. β-casein is a protein consisting of 224 amino acids and accounts for approximately 30% of milk. β-casein exists as A1 type casein (SEQ ID NO: 4) and A2 type casein (SEQ ID NO: 3). A1 type casein and A2 type casein differ in whether the 67th amino acid is histidine or proline. Therefore, the opioid peptide represented by SEQ ID NO: 2 is produced from A2 type casein by the alkaline protease, but the opioid peptide is not produced from A1 type casein. A1 type casein is commonly found in Holstein breeds, while A2 type casein is commonly found in Guernsey breeds. Therefore, milk containing only A1 type casein, milk containing only A2 type casein, or milk containing both A1 and A2 type caseins is commercially available.

[0016] <Opioid Peptide> The opioid peptide obtained by the production method of the present invention is not particularly limited as long as the effects of the present invention can be obtained, but is preferably a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, i.e., a peptide consisting of the amino acid sequence "YPFPGPIPNSLP (hereinafter, sometimes referred to as CM-12)." The opioid peptide is a peptide that has binding activity to opioid receptors. Opioid receptors are cell surface receptor proteins involved in the expression of the effects of morphine-like substances, and ｉ / G ｏ It is a seven-transmembrane receptor of the opioid receptor type. δ receptor, κ receptor, and μ receptor have been reported. The opioid receptor to which the opioid peptide of the present invention binds is not limited, but is preferably the δ receptor or μ receptor. The δ receptor has a strong affinity for leucine-enkephalin and is widely distributed in the central nervous system. The δ receptor is also involved in antidepressant action, physical and mental dependence, and analgesia, and the δ receptor is also involved in antidepressant action, physical and mental dependence, and analgesia. １ and δ ２The opioid peptides of the present invention have binding activity to opioid receptors, particularly μ and δ receptors, and are expected to exert anxiolytic, analgesic, antidepressant, or anti-stress effects upon binding to the receptors.

[0017] <<Peptide Production Step>> In the opioid peptide production step of the production method of the present invention, a koji mold alkaline protease is contacted with A2 type casein to produce an opioid peptide consisting of the amino acid sequence represented by SEQ ID NO: 2. The conditions for contacting the koji mold alkaline protease with A2 type casein are not particularly limited, as long as an opioid peptide is produced. However, the pH of the buffer solution is, for example, 4 to 10, preferably 5 to 9.5, more preferably 6 to 9, even more preferably 6.5 to 8.5, and most preferably 7 to 8. Specific examples of buffer solutions include acetate buffer, citrate buffer, and tartrate buffer. The reaction temperature of the koji mold alkaline protease is also not particularly limited, as long as an opioid peptide is produced, but is, for example, 5 to 60°C, preferably 10 to 50°C, more preferably 20 to 48°C, more preferably 30 to 46°C, and even more preferably 35 to 42°C. The reaction time is not particularly limited, and for example, the lower limit is 10 minutes or more, in one embodiment 30 minutes or more, and in one embodiment 1 hour or more. The upper limit of the reaction time is, for example, 72 hours or less, in one embodiment 48 hours or less, in one embodiment 24 hours or less, and in one embodiment 12 hours or less. The reaction pH, reaction temperature, and reaction time can be appropriately set by one skilled in the art depending on the koji mold alkaline protease used.

[0018] <<Method for Improving Production Efficiency of Opioid Peptides>> The method for improving production efficiency of opioid peptides of the present invention comprises the step of contacting A2 type casein with a koji mold alkaline protease that recognizes the amino acid sequence SLXX and cleaves between the two XX residues, to produce an opioid peptide consisting of the amino acid sequence represented by SEQ ID NO: 2. In the method for improving production efficiency of opioid peptides of the present invention, the koji mold alkaline protease is recovered as a low-adsorption fraction on an anion exchange resin or as an adsorption fraction on a cation exchange resin to separate the koji mold alkaline protease from contaminating proteases, although this is not a limitation.

[0019] <<Method for Purifying (Manufacturing) Alkaline Protease>> The koji mold alkaline protease can be purified (manufactured) from a koji mold protease mixture, but is not limited thereto. The koji mold alkaline protease of the present invention has an alkaline isoelectric point. On the other hand, many koji mold proteases are acidic enzymes and can be separated from these acidic proteases using an anion exchange resin or a cation exchange resin. For example, when using an anion exchange resin, adsorption becomes difficult at low pH. Specifically, it is adsorbed to an anion exchange resin at pH 8.5 but eluted with 10 mM NaCl. Further lowering the pH results in elution as a low-adsorption fraction. For example, when recovering the alkaline protease as a low-adsorption component on an anion adsorption resin, a buffer solution with a pH of 7 or higher, preferably 8 or higher, can be used. Furthermore, when a cation exchange resin is used, the alkaline protease of the present invention can be adsorbed to the cation exchange resin. On the other hand, since the acid protease of koji mold cannot be adsorbed to a cation exchange resin, alkaline protease and acid protease can be separated. For example, when the acid protease is used as an adsorbed fraction on a cation exchange resin, a buffer solution of pH 7 or less, preferably pH 6 or less, may be used.

[0020] [2] Alkaline protease composition for producing opioid peptide from A2 type casein The alkaline protease composition of the present invention contains an Aspergillus alkaline protease that recognizes the amino acid sequence SLXX and cleaves between the two XXs, and is used for producing an opioid peptide from A2 type casein consisting of the amino acid sequence represented by SEQ ID NO: 2. The "A2 type casein" and "opioid peptide" are the A2 type casein and opioid peptide described in the section "[1] Method for producing opioid peptide" above.

[0021] <<Alkaline Protease>> The alkaline protease contained in the composition of the present invention is an Aspergillus oryzae alkaline protease that recognizes the amino acid sequence SLXX and cleaves between the two XXs. However, any alkaline protease described in the above section “[1] Method for producing opioid peptide” can be used without limitation.

[0022] The alkaline protease can be obtained from a crude enzyme derived from, but is not limited to, Aspergillus. As the Aspergillus, Aspergillus oryzae is preferred. For example, the alkaline protease can be efficiently purified from a crude enzyme derived from Aspergillus oryzae by DEAE-Sepharose chromatography and elution with 10 mM NaCl. At lower pH, the alkaline protease is eluted in a low-adsorption fraction, and by removing other contaminating proteases, productivity of the opioid peptide of the present invention can be increased.

[0023] The composition of the present invention may contain, in addition to the alkaline protease, a carrier or other components. Examples of other components that may be used include water-soluble solvents such as physiological saline or Ringer's solution, water-insoluble solvents such as vegetable oils or fatty acid esters, isotonic agents such as glucose or sodium chloride, solubilizers, stabilizers, preservatives, suspending agents, or emulsifiers. The content of the alkaline phosphatase in the composition of the present invention is not particularly limited, and may be 90% by weight or more, 50% by weight or more, 10% by weight or more, or 1% by weight or more, with the upper limit being less than 100% by weight or less than 99% by weight.

[0024] [3] Opioid Peptide The opioid peptide of the present invention is an opioid peptide having the amino acid sequence represented by SEQ ID NO: 2. As the opioid peptide, any of the opioid peptides described in the above section "[1] Method for producing an opioid peptide" can be used without limitation. The opioid peptide can be synthesized, but is preferably produced by the production method of the present invention.

[0025] [4] Opioid Receptor Agonist The opioid receptor agonist of the present invention comprises the opioid peptide of the present invention as an active ingredient. That is, the opioid receptor agonist of the present invention comprises an opioid peptide having the amino acid sequence represented by SEQ ID NO: 2 as an active ingredient. The opioid receptor agonist of the present invention is an agonist, and can exhibit analgesic, antidepressant, or anti-stress effects by binding to an opioid receptor. Examples of opioid receptors include the δ receptor, the κ receptor, and the μ receptor. The opioid receptor on which the peptide of the present invention acts is not limited, but is preferably the δ receptor or the μ receptor.

[0026] [5] Food Composition A food composition for activating opioid receptors or an anti-anxiety food composition contains the opioid peptide of the present invention as an active ingredient.

[0027] Specific examples of foods include fresh prepared foods such as salads; cooked foods such as steak, pizza, and hamburger steak; stir-fried foods such as stir-fried vegetables; vegetables such as tomatoes, bell peppers, celery, bitter melon, carrots, potatoes, and asparagus, and cooked foods made from these vegetables; sweets such as cookies, bread, biscuits, hardtack, cakes, rice crackers, yokan, puddings, jellies, ice cream, chewing gum, crackers, chips, chocolate, and candy; noodles such as udon, pasta, and soba; fish paste products such as kamaboko, ham, and fish sausage; dairy products such as cheese, cream, and butter; condiments such as miso, soy sauce, dressing, ketchup, mayonnaise, soup base, noodle soup, curry powder, mirin, roux, and seasoning spices; soy foods such as tofu; konjac; and supplements.

[0028] Examples of beverages include coffee beverages; cocoa beverages; vegetable juices obtained from the above-mentioned vegetables; fruit juice beverages such as grapefruit juice, orange juice, grape juice, and lemon juice; tea beverages such as green tea, black tea, green tea, and oolong tea; alcoholic beverages such as beer, wine (red wine, white wine, sparkling wine, etc.), sake, plum wine, happoshu, whiskey, brandy, shochu, rum, gin, and liqueurs; dairy beverages; soy milk beverages; liquid diets; and sports drinks.

[0029] The food or beverage includes feed for animals, such as primates including humans, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, and mice.

[0030] These foods or beverages may contain, as desired, food additives and food ingredients, such as antioxidants, flavorings, acidulants, colorings, emulsifiers, preservatives, seasonings, sweeteners, spices, pH adjusters, stabilizers, antioxidants, vegetable oils, animal oils, sugars and sugar alcohols, vitamins, organic acids, fruit juice extracts, vegetable extracts, grains, beans, vegetables, meat, seafood, etc. The amounts of these food ingredients and food additives to be added can be determined appropriately within the range that does not impair the object of the present invention.

[0031] Foods and beverages include functional foods (drinks) and health foods (drinks). As used herein, "health foods (drinks)" refers to foods or beverages that have or are expected to have some effect on health, and "functional foods (drinks)" refers to foods or beverages among the "health foods (drinks)" that are designed and processed to fully exhibit bioregulatory functions (i.e., opioid receptor agonism). Functional foods and health foods can be in granular, solid, liquid, capsule, gel, or tablet form.

[0032] [6] Pharmaceutical Composition The opioid receptor agonist pharmaceutical composition or anti-anxiety pharmaceutical composition of the present invention contains the opioid peptide of the present invention as an active ingredient. The opioid receptor agonist pharmaceutical composition of the present invention can be used for, but is not limited to, anti-anxiety, analgesia, antidepressant, or anti-stress purposes.

[0033] The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and includes oral and parenteral preparations, with oral preparations being preferred. Examples of the oral preparations include solid or powder preparations such as fine granules, granules, tablets, capsules, and pills, as well as liquid preparations such as suspensions, emulsions, syrups, and extracts. Examples of parenteral preparations include injections.

[0034] The pharmaceutical composition of the present invention may consist of the opioid peptide or may contain the opioid peptide. When the pharmaceutical composition of the present invention contains the opioid peptide, it may contain other additives.

[0035] When the pharmaceutical composition of the present invention is an oral dosage form, other additives may include excipients, binders, disintegrants, emulsifiers, lubricants, flow enhancers, diluents, preservatives, colorants, flavorings, corrigents, stabilizers, moisturizers, antiseptics, antioxidants, or suspending agents, and specific examples thereof include gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soybean lecithin, sucrose, fatty acid esters, talc, magnesium stearate, polyethylene glycol, magnesium silicate, anhydrous silicic acid, and synthetic aluminum silicate.

[0036] When the pharmaceutical composition of the present invention is a parenteral preparation, other additives may include water-soluble solvents such as physiological saline or Ringer's solution, water-insoluble solvents such as vegetable oils or fatty acid esters, isotonic agents such as glucose or sodium chloride, solubilizing agents, stabilizers, preservatives, suspending agents, or emulsifiers.

[0037] The pharmaceutical composition of the present invention may contain the peptide or peptide mixture in an amount of 90% by weight or more, 50% by weight or more, 10% by weight or more, or 1% by weight or more.

[0038] The dosage or intake of the pharmaceutical composition of the present invention can be adjusted as appropriate depending on the formulation, as well as the age, sex, body weight, and severity of disease symptoms of the subject, but is preferably an amount that can exert analgesic, antidepressant, anti-stress, or other effects upon administration or ingestion of the pharmaceutical composition. Specifically, the dosage, converted into the amount of opioid peptide added, can be 0.01 to 1,000 mg / kg body weight / day, preferably 0.1 to 750 mg / kg body weight / day, more preferably 1 to 500 mg / kg body weight / day, even more preferably 5 to 400 mg / kg body weight / day, even more preferably 10 to 300 mg / kg body weight / day, even more preferably 15 to 200 mg / kg body weight / day, and most preferably 20 to 150 mg / kg body weight / day.

[0039] Of course, the above administration method is merely an example, and other administration methods may be used. The administration method, dosage, administration period, administration interval, etc. of the pharmaceutical composition to humans are preferably determined through controlled clinical trials.

[0040] The pharmaceutical composition of the present invention can be administered to humans, but may also be administered to animals other than humans, including pets such as dogs, cats, rabbits, hamsters, guinea pigs, and squirrels; livestock such as cows and pigs; laboratory animals such as mice and rats; and animals kept in zoos, etc.

[0041] The pharmaceutical compositions include pharmaceuticals and quasi-drugs. Pharmaceuticals include, for example, herbal medicine preparations and herbal medicine preparations. Quasi-drugs include, for example, nutritional drinks and health medicines containing herbal medicines.

[0042] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0043] Example 1 In this example, the alkaline protease used in the present invention was purified from a crude enzyme derived from Aspergillus oryzae. 50 mg of the crude enzyme was dissolved in 2 mL of 100 mM Tris-HCl buffer (pH 8.5, 20°C). This crude enzyme solution was applied to a DEAE-Sepharose Fast Flow anion exchange chromatography column (1 mL) pre-equilibrated with 100 mM Tris-HCl buffer (10 mL). The protein was eluted from the column stepwise with 10, 50, 100, 250, and 500 mM NaCl in 100 mM Tris-HCl. The protein concentration was estimated by measuring the absorbance at 280 nm. The obtained fractions were analyzed by SDS-PAGE. The gel photograph shown in Figure 1(A) was obtained.

[0044] Each fraction was contacted with the amino acid sequence AQTQSLVYPFPGPIPNSLPQNIPPLTQTPV (CM-30) (SEQ ID NO: 5), prepared from the A2 type β-casein sequence, to examine its ability to produce the opioid peptide of the present invention. As shown in Figure 1(B), the 10 mM NaCl fraction was confirmed to have the activity of producing CM-12 (YPFPGPIPNSLP) from CM-30. In the following examples, this fraction was used as the alkaline protease.

[0045] The 34 kDa protein band in the 10 mM NaCl fraction was isolated from an SDS-10% PAGE gel. Peptides obtained from the protein by trypsin digestion were analyzed by TOF-MS analysis. The mass data of the tryptic peptides were used for analysis using the MASCOT database. The 34 kDa protein was considered to be the alkaline protease of SEQ ID NO: 1. That is, it was considered to be a 283 aa mature protease, excluding the 21 aa signal sequence and the 100 aa pro-sequence from the N-terminus.

[0046] Example 2 In this example, the cleavage of the opioid peptide of the present invention from A1-type casein or A2-type casein was investigated using the alkaline protease obtained in Example 1. A1-type casein was used as a comparative example. 10 mL of A1-type milk or A2-type milk was centrifuged at 7,500 × g and 4°C for 20 minutes, and the cream top layer was removed. Each supernatant was heated at 55°C for 5 minutes and cooled to below 20°C. The pH was then adjusted to 4.6 by adding 1 M hydrochloric acid, and the aggregates were recovered by centrifugation at 1,300 × g and 20°C for 5 minutes. To dissolve the pellet, the precipitate was dissolved in 10 mL of distilled water, and the pH was adjusted to 7.0 by adding 1 M NaOH. The resulting casein protein was analyzed by SDS-12% PAGE (Figure 2A). Bands corresponding to A1-type casein and A2-type casein (arrows) were observed. A1 and A2 caseins were separated, and the CM-12-releasing ability of purified Alp was evaluated. As a result, a specific peak corresponding to CM-12 was detected in the A2 casein hydrolysate (Fig. 2B), but not in the A1 casein hydrolysate (Fig. 2B).

[0047] Example 3 In this example, the production of CM-12 peptide from A2 type casein was investigated using purified alkaline protease and crude enzyme. 3 μL of A2 type casein (2 mg / mL) was hydrolyzed with 10 μL of purified alkaline protease (0.3 mg / mL) or 10 μL of crude enzyme (30 mg / mL), and the hydrolysis was performed in 0.2 M NaCl solution. ２ HPO ４ The pH of the solution was adjusted to 8 with buffer. The reaction mixture was incubated at 45°C for 1, 2, 4, 6, 8, 10, and 12 hours, and the opioid peptides were measured. While the crude enzyme produced very little CM-12, the use of purified alkaline protease resulted in prolonged CM-12 production. The purified alkaline protease was able to release 100% (18.6-fold) of CM-12 from A2-type casein (Figure 3).

[0048] Example 4 In this example, the reaction pH and reaction temperature of the alkaline protease obtained in Example 1 were investigated. Specifically, the activity of the purified alkaline protease was investigated using CM-30 as a substrate. ２ HPO ４ Buffer solution, pH 6.0-8.0 contains 0.2M Na ２ HPO ４ -NaH ２ P.O. ４ The buffers, pH 9.0 and 10.0, used were glycine-sodium hydroxide buffer. The purified alkaline protease was incubated with the substrate at a ratio of 10:1 (S / E, w / w) in the reaction buffer at 45°C for 1 hour. As shown in Figure 4A, the optimum pH was around 8. The reaction temperature was 0.2 M Na ２ HPO ４ -NaH ２ P.O. ４ The activity was tested using a buffer solution of pH 8.0 at 30, 37, 40, 45, 50, 60, and 70°C. The alkaline protease was incubated at a ratio of 10:1 (S / E, w / w) for 1 hour. As shown in Figure 4B, the optimum temperature was around 45°C.

[0049] The production method of the present invention is effective for producing opioid peptides to be used in foods or medicines for purposes such as anti-anxiety, analgesia, anti-depression, or anti-stress.

Claims

1. A method for producing an opioid peptide, comprising the step of contacting A2 type casein with Aspergillus alkaline protease, which recognizes the amino acid sequence SLXX and cleaves between the two XXs, to produce a peptide consisting of the amino acid sequence represented by SEQ ID NO:

2.

2. The method for producing an opioid peptide according to claim 1, wherein the koji mold alkaline protease is (a) a koji mold alkaline protease having the amino acid sequence represented by SEQ ID NO:1, or (b) a koji mold alkaline protease containing an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO:1 and capable of producing an opioid peptide consisting of the amino acid sequence represented by SEQ ID NO:2 from A2 type casein.

3. A method for improving the production efficiency of opioid peptides, comprising a step of contacting A2 type casein with an Aspergillus alkaline protease that recognizes the amino acid sequence SLXX and cleaves between two XXs to produce an opioid peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, wherein the Aspergillus alkaline protease is recovered as a low adsorption fraction on an anion exchange resin or as an adsorption fraction on a cation exchange resin in order to separate the Aspergillus alkaline protease from contaminating proteases.

4. The method for improving the production efficiency of opioid peptides according to claim 3, wherein the koji mold alkaline protease is (a) a koji mold alkaline protease having the amino acid sequence represented by SEQ ID NO:1, or (b) a koji mold alkaline protease containing an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO:1 and capable of producing an opioid peptide consisting of the amino acid sequence represented by SEQ ID NO:2 from A2 type casein.

5. An alkaline protease composition for producing an opioid peptide consisting of the amino acid sequence represented by SEQ ID NO:2 from A2 type casein, comprising an Aspergillus alkaline protease that recognizes the amino acid sequence SLXX and cleaves between the two XXs.

6. The alkaline protease composition according to claim 5, wherein the koji mold alkaline protease is (a) a koji mold alkaline protease having the amino acid sequence represented by SEQ ID NO:1, or (b) a koji mold alkaline protease which contains an amino acid sequence which is 90% or more identical to the amino acid sequence represented by SEQ ID NO:1 and which is capable of producing an opioid peptide consisting of the amino acid sequence represented by SEQ ID NO:2 from A2 type casein.

7. An opioid peptide having the amino acid sequence represented by SEQ ID NO:

2.

8. An opioid receptor agonist comprising the opioid peptide according to claim 7 as an active ingredient.

9. A food composition for activating opioid receptors, comprising the opioid peptide according to claim 7 as an active ingredient.

10. An anti-anxiety food composition comprising the opioid peptide according to claim 7 as an active ingredient.

11. A pharmaceutical composition for activating opioid receptors, comprising the opioid peptide according to claim 7 as an active ingredient.

12. An anti-anxiety pharmaceutical composition comprising the opioid peptide according to claim 7 as an active ingredient.

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