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

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

[Technical Field]

[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. [Background technology]

[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 that can reduce stress. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2022-135015 [Non-patent literature]

[0004] [Non-Patent Document 1] "Peptides" 1999 (Netherlands) Vol. 20, p957-962 Summary of the Invention [Problem to be solved by the invention]

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

[0006] The present inventors have conducted extensive research into efficient methods for producing opioid peptides and have surprisingly found that novel opioid peptides can be efficiently obtained by using alkaline protease from Aspergillus oryzae and A2 type casein. The present invention is based on this finding. Therefore, the present invention provides [1] A method for producing an opioid peptide, comprising the step of contacting A2 type casein with an Aspergillus alkaline protease that 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 [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 an opioid peptide, 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 to separate it from contaminating proteases. [4] The method for improving the production efficiency of an opioid peptide 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 that can produce 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 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. [7] An opioid peptide having an amino acid sequence represented by SEQ ID NO: 2. [8] An opioid receptor agonist containing the opioid peptide according to [7] as an active ingredient. [9] A food composition for activating opioid receptors, comprising the opioid peptide according to [7] as an active ingredient.

[10] An anti-anxiety food composition containing the opioid peptide according to [7] as an active ingredient.

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

[12] An anti-anxiety pharmaceutical composition containing the opioid peptide according to [7] as an active ingredient. Regarding. [Effects of the Invention]

[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. [Brief explanation of the drawings]

[0008] [Figure 1] A photograph shows the results of SDS-PAGE analysis of fractions of crude enzyme derived from Aspergillus oryzae at 10, 50, 100, 250, and 500 mM NaCl using DEAE-Sepharose (A), and a graph showing the ability of each fraction to produce opioid peptides (B). [Figure 2] A1 type milk or A2 type milk was digested with purified alkaline protease and analyzed by SDS-PAGE (A), and a chart showing the resulting CM-12 peptides (B). [Figure 3] This is a graph showing the results of an investigation into the production of CM-12 peptide from A2 type casein using purified alkaline protease and crude enzyme. [Figure 4] 1 is a graph showing the results of an investigation into the optimum pH (A) and optimum temperature (B) of purified alkaline protease using CM-30. [Figure 5] FIG. 1 shows the cleavage sites in A2 type casein by the alkaline protease of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[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 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. 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 include a 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 of SLXX and cleaves between the two XXs, but may be, for example, (a) an Aspergillus alkaline protease having the amino acid sequence shown in SEQ ID NO: 1. It may also be an Aspergillus alkaline protease consisting of the amino acid sequence shown in SEQ ID NO: 1. Furthermore, it may be (b) an alkaline protease that contains an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 1 and is capable of producing an opioid peptide consisting of the amino acid sequence shown in 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, or 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. MQSIKRTLLLLGAILPAVLGAPVQETRRAAEKLPGKYIVTFKPGIDEAKIQEHTTWATNIHQRSLERRGATGGDLPVGIERNYKINKFAAYAGSFDDATI EEIRKNEDVAYVEEDQIYYLDGLTTQKSAPWGLGSISHKGQQSTDYIYDTSAGEGTYAYVVDSGVNVDHEEFEGRASKAYNAAGGQHVDSIGHGTHVSGTI AGKTYGIAKKASILSVKVFQGESSSTSVILDGFNWAANDIVSKKRTSKAAINMSLGGGYSKAFNDAVENAFEQGVLSVVAAGNENSDAGQTSPASAPDAITVAAIQKSNNRASFSNFGKVVDVFAPGQDILSAWIGSSSATNTISGTSMATPHIVGLSLYLAALENLDGPAAVTKRIKELATKDVVKDVKGSPNLLAYNGNA

[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 it has 90% or more identity. 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 amino acid sequence 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 amino acid sequence SLXX 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 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 opioid peptides of the present invention to be produced 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 found in many breeds such as Holsteins, while A2 type casein is found in many breeds such as Guernseys. 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 peptides 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 are 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)." Opioid peptides are peptides that have binding activity to opioid receptors. Opioid receptors are cell surface receptor proteins involved in the expression of the effects of morphine-like substances, and G i / G o It is a seven-transmembrane coupled receptor. δ receptors, κ receptors, and μ receptors have been reported. The opioid receptor to which the opioid peptides of the present invention bind is not limited, but is preferably the δ receptor or μ receptor. δ receptors have a strong affinity for leucine-enkephalin and are widely distributed in the central nervous system. δ receptors are also involved in antidepressant effects, physical and mental dependence, and analgesia, and there are two types of receptors: δ1 and δ2. The 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 process In the opioid peptide production step of the production method of the present invention, the Aspergillus alkaline protease is brought into contact 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, so long as opioid peptides are 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, so long as opioid peptides are 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 also not particularly limited, but 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. Those skilled in the art can appropriately set the reaction pH, reaction temperature, and reaction time according to the type of koji mold alkaline protease used.

[0018] <Method for improving the production efficiency of opioid peptides> The method for improving the production efficiency of opioid peptides of the present invention comprises the step of contacting A2 type casein with an Aspergillus 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. In the method for improving the production efficiency of opioid peptides of the present invention, 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 to separate the Aspergillus alkaline protease from contaminating proteases, although this is not a limitation.

[0019] <Method for purifying (producing) alkaline protease> The koji mold alkaline protease can be purified (produced) 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 an anion exchange resin is used, adsorption becomes difficult at a low pH. Specifically, it adsorbs to the anion exchange resin at pH 8.5 but is eluted with 10 mM NaCl. Further lowering the pH results in elution as a low-adsorption fraction. For example, when recovering 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, the acid protease of Aspergillus oryzae cannot be adsorbed to the cation exchange resin, so the alkaline protease and the acid protease can be separated. For example, when using as an adsorption fraction on a cation exchange resin, a buffer solution with a pH of 7 or lower, preferably 6 or lower, can be used.

[0020] [2] Alkaline protease composition for producing opioid peptides 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 consisting of the amino acid sequence represented by SEQ ID NO: 2 from A2 type casein. 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."

[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 peptides" can be used without limitation.

[0022] The alkaline protease can be obtained from a crude enzyme derived from, but not limited to, koji mold, preferably the koji mold of the genus Aspergillus, and particularly preferably Aspergillus oryzae. For example, the alkaline protease can be efficiently purified from crude enzymes derived from Aspergillus oryzae by DEAE-Sepharose chromatography using 10 mM NaCl elution. At lower pH, it is eluted in the low-adsorption fraction, and the removal of other contaminating proteases can enhance the productivity of the opioid peptides of the present invention.

[0023] The composition of the present invention may contain, in addition to the alkaline protease, a carrier or other components, such as a water-soluble solvent such as physiological saline or Ringer's solution, a water-insoluble solvent such as vegetable oil or fatty acid ester, an isotonic agent such as glucose or sodium chloride, a solubilizer, a stabilizer, a preservative, a suspending agent, or an emulsifier. The content of the alkaline phosphatase in the composition of the present invention is not particularly limited, but may be 90% by weight or more, 50% by weight or more, 10% by weight or more, or 1% by weight or more of the alkaline protease, with the upper limit being less than 100% by weight or less than 99% by weight.

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

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

[0026] [5] Food composition The opioid receptor agonist food composition or 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 exert 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, and can be used for, but 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 when administered or ingested. 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. [Example]

[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 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 column (1 mL) pre-equilibrated with 100 mM Tris-HCl buffer (10 mL). Protein was eluted from the column stepwise with 10, 50, 100, 250, and 500 mM NaCl in 100 mM Tris-HCl. Protein concentration was estimated by measuring absorbance at 280 nm. The obtained fractions were analyzed by SDS-PAGE, and 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 activity of converting CM-30 to CM-12 (YPFPGPIPNSLP) was confirmed in the 10 mM NaCl fraction. In the following examples, this fraction was used as the alkaline protease.

[0045] A 34 kDa protein band in the 10 mM NaCl fraction was isolated from a 10% SDS-PAGE gel. Peptides isolated from the protein by trypsin digestion were analyzed by TOF-MS. The mass data of the tryptic peptides were analyzed using the MASCOT database. The 34 kDa protein was considered to be the alkaline protease of SEQ ID NO: 1. This suggests that it is 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, 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. Ten mL of A1 or A2 type milk was centrifuged at 7,500 x g for 20 minutes at 4°C, and the creamy top layer was removed. Each supernatant was heated at 55°C for 5 minutes and cooled to below 20°C. The pH of the supernatant was then adjusted to 4.6 with 1 M hydrochloric acid, and the aggregates were recovered by centrifugation at 1,300 x g for 5 minutes at 20°C. To dissolve the pellet, the precipitate was dissolved in 10 mL of distilled water and the pH was adjusted to 7.0 with 1 M NaOH. The resulting casein proteins were analyzed by SDS-PAGE (Figure 2A). Bands corresponding to A1 and A2 type caseins (arrows) were observed. A1 and A2 type caseins were separated, and the CM-12 release capacity of the 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. Three microliters 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 pH of the solution was adjusted to 8 with 0.2 M NaHPO 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 purified alkaline protease produced CM-12 for a long period of time. The purified alkaline protease was able to release 100% (18.6-fold) of CM-12 from A2 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 examined using CM-30 as a substrate. A 0.2 M citric acid-NaHPO buffer solution was used for pH 3.0-5.0, a 0.2 M NaHPO-NaHPO buffer solution for pH 6.0-8.0, and a glycine-sodium hydroxide buffer solution for pH 9.0 and 10.0. The purified alkaline protease was incubated with the substrate at a 10:1 (S / E, w / w) ratio in the reaction buffer at 45°C for 1 hour. As shown in Figure 4A, the optimum pH was around 8. The activity was tested using 0.2 M NaHPO-NaHPO buffer (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. [Industrial Applicability]

[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 sequence number 2.

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 that is 90% or more identical to 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 an opioid peptide, comprising the step of contacting A2 type casein with an Aspergillus 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, A method for improving the production efficiency of opioid peptides, wherein the koji mold alkaline protease is separated from contaminating proteases by recovering the koji mold alkaline protease as a low adsorption fraction on an anion exchange resin or as an adsorption fraction on a cation exchange resin.

4. 4. The method for improving opioid peptide production efficiency 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 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 oryzae alkaline protease that recognizes the amino acid sequence SLXX and cleaves between the two XXs.

6. 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 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.

7. The manufacturing method described in claim 1 or 2, wherein the opioid peptide is used for an opioid receptor agonist, an opioid receptor agonist food composition, an anti-anxiety food composition, an opioid receptor agonist pharmaceutical composition, or an anti-anxiety pharmaceutical composition.

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