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

Abstract

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 the production of an opioid peptide. The present invention addresses the problem of providing a method for efficiently producing an opioid peptide. The problem can be solved by a method for producing an opioid peptide according to the present invention, the method comprising a step for producing a peptide containing an amino acid sequence represented by SEQ ID NO: 2 by bringing an Aspergillus alkaline protease, which recognizes the amino acid sequence of SLXX and is cleaved between two XXs, into contact with A2-type casein.

Description

Technical Field

[0001] This invention relates to a method for manufacturing opioid peptides, a method for improving the production efficiency of opioid peptides, and an alkaline protease composition for manufacturing opioid peptides. According to this invention, opioid peptides can be manufactured efficiently. Background Technology

[0002] In recent years, various stress-related illnesses have been reported, and opioid receptor agonists with morphine-like activity are expected to be developed as analgesics or neurotropic drugs. On the other hand, the effects of enzymatically hydrolyzed peptides of food proteins on opioid receptors have been reported (Non-Patent Literature 1), which show promise as food additives that can alleviate stress.

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: Japanese Patent Application Publication No. 2022-135015

[0006] Non-patent literature

[0007] Non-patent literature 1: Peptides, 1999 (Netherlands), Vol. 20, pp. 957-962 Summary of the Invention

[0008] [The problem the invention aims to solve]

[0009] The inventors of this case discovered that opioid peptides with high opioid receptor agonist activity can be obtained by decomposing casein with a protease from Aspergillus (Patent Document 1). However, obtaining the opioid peptides of Patent Document 1 efficiently is not an easy task.

[0010] Therefore, the purpose of this invention is to provide an efficient method for manufacturing opioid peptides.

[0011] [Technical means to solve the problem]

[0012] The inventors have conducted in-depth research on a method for producing highly efficient opioid peptides, and surprisingly discovered that novel opioid peptides can be obtained efficiently by using alkaline protease from Aspergillus and A2 type casein.

[0013] This invention is based on the aforementioned insights.

[0014] Therefore, this invention relates to:

[0015] [1] A method for manufacturing an opioid peptide, comprising the following steps: contacting an Aspergillus alkaline protease that recognizes the amino acid sequence of SLXX and is cleaved between the two XXs with A2 type casein to generate a peptide containing the amino acid sequence represented by sequence number 2.

[0016] [2] According to the method for manufacturing opioid peptides according to [1], the Aspergillus alkaline protease is (a) an Aspergillus alkaline protease having the amino acid sequence represented by sequence number 1; or (b) an Aspergillus alkaline protease containing an amino acid sequence that is 90% or more identical to the amino acid sequence represented by sequence number 1, and capable of generating an opioid peptide containing the amino acid sequence represented by sequence number 2 from A2 casein;

[0017] [3] A method for improving the production efficiency of opioid peptides, comprising the following steps: contacting an Aspergillus alkaline protease that recognizes the amino acid sequence of SLXX and is cleaved between 2 XX with A2 type casein to generate an opioid peptide containing the amino acid sequence represented by sequence number 2, and in order to separate the Aspergillus alkaline protease from contaminating proteases, the Aspergillus alkaline protease is recovered in the form of a low-adsorption component of anion exchange resin or in the form of an adsorption component of a cation exchange resin;

[0018] [4] The method for improving the production efficiency of opioid peptides according to [3], wherein the Aspergillus alkaline protease is (a) an Aspergillus alkaline protease having the amino acid sequence represented by sequence number 1; or (b) an Aspergillus alkaline protease containing an amino acid sequence having more than 90% identity with the amino acid sequence represented by sequence number 1, and capable of generating an opioid peptide containing the amino acid sequence represented by sequence number 2 from A2 casein;

[0019] [5] An alkaline protease composition comprising an Aspergillus alkaline protease that recognizes an amino acid sequence of SLXX and is cleaved between two XXs, for the production of an opioid peptide comprising an amino acid sequence represented by sequence number 2 from A2 type casein.

[0020] [6] The alkaline protease composition according to [5], wherein the Aspergillus alkaline protease is (a) an Aspergillus alkaline protease having the amino acid sequence represented by sequence number 1; or (b) an Aspergillus alkaline protease containing an amino acid sequence having more than 90% identity with the amino acid sequence represented by sequence number 1, and capable of generating an opioid peptide containing the amino acid sequence represented by sequence number 2 from A2 casein;

[0021] [7] An opioid peptide having an amino acid sequence represented by sequence number 2;

[0022] [8] An opioid receptor agonist comprising, as an active ingredient, the opioid peptide according to [7];

[0023] [9] A food composition for opioid receptor agonism comprising, as an active ingredient, the opioid peptide according to [7];

[0024]

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

[0025]

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

[0026]

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

[0027] [Invention Effects]

[0028] The method for manufacturing opioid peptides according to the present invention can efficiently produce opioid peptides exhibiting excellent effects. Attached Figure Description

[0029] Figure 1 The images (A) and (B) are obtained by analyzing the components of crude purified enzyme from Aspergillus oryzae using DEAE-agarose gel (Sepharose) via SDS-PAGE of 10, 50, 100, 250, and 500 mM NaCl.

[0030] Figure 2 The images (A) and (B) show the CM-12 peptide obtained by analyzing A1 or A2 type milk using purified alkaline protease.

[0031] Figure 3 This is a chart showing the generation of CM-12 peptide from A2 casein using purified alkaline protease and crude purified enzyme.

[0032] Figure 4 This is a chart showing the optimal pH (A) and optimal temperature (B) for purifying alkaline protease using CM-30.

[0033] Figure 5 This is a diagram showing the cleavage site of the alkaline protease of the present invention in A2 type casein. Detailed Implementation

[0034] [1] Methods for manufacturing opioid peptides

[0035] The method for manufacturing the opioid peptide of the present invention includes the following steps: contacting an Aspergillus alkaline protease that recognizes the amino acid sequence SLXX and is cleaved between two XXs with A2 type casein, thereby generating a peptide comprising the amino acid sequence represented by sequence number 2. In the amino acid sequence SLXX, S is serine, L is leucine, and X refers to any amino acid (any one of 20 amino acids).

[0036] The method for producing the opioid peptide of the present invention may include a step of purifying the generated opioid peptide. For example, the opioid peptide may be purified by adsorption using a hydrophobic resin and elution using solvents such as ethanol or acetonitrile. To concentrate the peptide to a higher purity, elution with acetonitrile can be performed by HPLC. In addition, insoluble peptides may be removed by adsorption with activated carbon.

[0037] Alkaline protease

[0038] The alkaline protease used in this invention is an alkaline protease derived from Aspergillus, which recognizes the amino acid sequence of SLXX and cleaves it between the two XXs.

[0039] The alkaline protease is not limited as long as it recognizes the amino acid sequence of SLXX and cleaves between the two XXs. For example, it can be (a) an Aspergillus alkaline protease having the amino acid sequence represented by sequence number 1. Alternatively, it can be an Aspergillus alkaline protease containing the amino acid sequence represented by sequence number 1. Furthermore, it can also be (b) an alkaline protease containing an amino acid sequence with more than 90% identity to the amino acid sequence represented by sequence number 1, and capable of generating an opioid peptide containing the amino acid sequence represented by sequence number 2 from A2 casein. The alkaline protease in (b) is not limited as long as it has the same function as the protease in (a) (e.g., 50% or more of the activity relative to the alkaline protease in (a), such as 60% or more, 70% or more, 80% or more, 90% or more, or equivalent activity). The identity of the amino acid sequence represented by sequence number 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 sequence number 1 is as follows.

[0040] MQSIKRTLLLLGAILPAVLGAPVQETRRAAEKLPGKYIVTFKPGIDEAKIQEHTTWATNIHQRSLERRGATGGDLPVGIERNYKINKFAAYAGSFDDATI EEIRKNEDVAYVEEDQIYYLDGLTTQKSAPWGLGSISHKGQQSTDYIYDTSAGEGTYAYVVDSGVNVDHEEFEGRASKAYNAAGGQHVDSIGHGTHVSGTI AGKTYGIAKKASILSVKVFQGESSSTSVILDGFNWAANDIVSKKRTSKAAINMSLGGGYSKAFNDAVENAFEQGVLSVVAAGNENSDAGQTSPASAPDAITVAAIQKSNNRASFSNFGKVVDVFAPGQDILSAWIGSSSATNTISGTSMATPHIVGLSLYLAALENLDGPAAVTKRIKELATKDVVKDVKGSPNLLAYNGNA

[0041] The amino acid sequence with an identity of 90% or more may be, for example, an amino acid sequence with one or more amino acids deleted, substituted, inserted, and / or added relative to its respective amino acid sequence. The polypeptide of the alkaline protease may be a polypeptide containing an amino acid sequence with one or more amino acids deleted, substituted, inserted, and / or added, provided that the amino acid sequence of sequence number 1 has an identity of 90% or more. In this specification, "amino acid sequence with one or more amino acids deleted, substituted, inserted, and / or added" refers to an amino acid sequence modified by amino acid substitution or the alkaline protease of closely related microorganisms. Regarding the number of amino acid modifications, there is no particular limitation as long as the amino acid identity is 90% or more; for example, it may be 1-40, 1-30, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, or 1-2. Examples of modified amino acid sequences of alkaline proteases that can be used in this invention are preferably amino acid sequences in which the amino acids have one or more (preferably one, two, three or four) conserved substitutions.

[0042] Even if multiple amino acids at the N-terminus and / or C-terminus are missing in the alkaline protease, it is still included in the alkaline protease of the present invention as long as the effects of the present invention can be obtained. In the amino acid sequence of Sequence Number 1, "an amino acid sequence with one or more amino acids missing, substituted, inserted, and / or added" or "an amino acid sequence with more than 90% identity" is formed by substitution of the amino acid sequence of Sequence Number 1, but the substitution of this amino acid sequence is a conservative substitution that maintains the function of the alkaline protease. In other words, "conservative substitution" refers to a substitution that does not cause the loss of the superior effects of the alkaline protease. That is, a substitution such that even in the case of insertion, substitution, deletion, or addition, the amino acid sequence of SLXX can be recognized and cleaved between the two XXs. Specifically, it refers to replacing an amino acid residue with another chemically similar amino acid residue. Examples include: replacing a hydrophobic residue with another hydrophobic residue, replacing a polar residue with another polar residue with the same charge, etc. For each amino acid, functionally similar amino acids formed by such substitution are known in the art. Examples of nonpolar (hydrophobic) amino acids include: alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include: glycine, serine, threonine, tyrosine, glutamic acid, asparagine, and cysteine. Examples of positively charged (basic) amino acids include: arginine, histidine, and lysine. Additionally, examples of negatively charged (acidic) amino acids include: aspartic acid and glutamic acid.

[0043] The alkaline protease is not limited to any particular type and is derived from Aspergillus. Specifically, examples include: Aspergillus flavus, Aspergillus clavatus, Aspergillus nidulans, Aspergillus funigatus, Aspergillus fischeri, and Aspergillus oryzae, but Aspergillus oryzae is preferred.

[0044] The alkaline protease used in this invention recognizes SLXX and cleaves the peptide (protein) between XX. Figure 5 The diagram shows the cleavage site when A2 casein is cleaved. That is, as shown... Figure 5As shown, it can recognize "SLSS" and cleave between SS, recognize "SLVY" and cleave between VY, recognize "SLPQ" and cleave between PQ, recognize "SLTL" and cleave between TL, and recognize "SLSQ" and cleave between SQ. Since it can recognize SLXX and cleave peptides (proteins) between XX, the opioid peptides of the present invention can be generated from A2 type casein as described below.

[0045] A2 casein

[0046] A2 casein is the A2 type β-casein found in bovine milk. β-casein is a protein containing approximately 30% of bovine milk, comprising 224 amino acids. β-casein includes both A1 casein (sequence number 4) and A2 casein (sequence number 3). The difference between A1 and A2 casein lies in whether the 67th amino acid is histidine or proline. Therefore, the opioid peptide represented by sequence number 2 is generated from A2 casein via the alkaline protease, but not from A1 casein.

[0047] A1 casein is commonly found in Holstein and other breeds, while A2 casein is commonly found in Guernsey and other breeds. Therefore, milk containing only A1 casein, milk containing only A2 casein, or milk containing both A1 and A2 casein are available on the market.

[0048] Opioid peptides

[0049] Regarding the opioid peptide obtained by the manufacturing method of the present invention, there are no particular limitations as long as the effects of the present invention can be obtained, but it is preferred to be a peptide containing the amino acid sequence represented by sequence number 2, that is, a peptide containing the amino acid sequence "YPFPGPIPNSLP (hereinafter, sometimes referred to as CM-12)".

[0050] Opioid peptides are peptides that bind to opioid receptors. Opioid receptors are cell surface receptor proteins expressed in response to morphine-like substances and are Gamma-ray receptors. i / G o Coupled 7-transmembrane receptors. δ receptors, κ receptors, and μ receptors are reported. The opioid receptors bound to the opioid peptides of this invention are not limited, but δ receptors or μ receptors are preferred.

[0051] Delta receptors have a strong affinity for leucine-enkephalin and are widely distributed in the central nervous system. Delta receptors also participate in antidepressant effects, physical / psychological dependence, and analgesia, and there are two types of receptors: δ1 and δ2. The opioid peptide of this invention has binding activity to opioid receptors, particularly μ and δ receptors. Through receptor binding, it is expected to exert anti-anxiety, analgesic, antidepressant, or anti-stress effects.

[0052] Peptide generation process

[0053] In the opioid peptide generation step of the manufacturing method of the present invention, Aspergillus alkaline protease is contacted with A2 type casein to generate an opioid peptide containing the amino acid sequence represented by sequence number 2.

[0054] Regarding the conditions for contacting the Aspergillus alkaline protease with A2 casein, there are no particular limitations as long as opioid peptides are generated. However, the pH value of the buffer solution is, for example, pH 4 to 10, preferably 5 to 9.5, more preferably pH 6 to 9, further preferably pH 6.5 to 8.5, and most preferably pH 7 to 8. Specific buffer solutions include acetate buffer, citrate buffer, or tartaric acid buffer. The reaction temperature of the Aspergillus alkaline protease is also not particularly limited as long as opioid peptides are generated, for example, 5 to 60°C, preferably 10 to 50°C, more preferably 20 to 48°C, more preferably 30 to 46°C, and further preferably 35 to 42°C. The reaction time is also not particularly limited, for example, the lower limit is 10 minutes or more, in one form 30 minutes or more, and in another form 1 hour or more. The upper limit of the reaction time is, for example, 72 hours or less, in one form 48 hours or less, in another form 24 hours or less, and in another form 12 hours or less.

[0055] The pH value, reaction temperature, and reaction time of the reaction can be appropriately set according to the various Aspergillus alkaline proteases used by the manufacturer.

[0056] Methods to Improve the Production Efficiency of Opioid Peptides

[0057] The method for improving the production efficiency of opioid peptides according to the present invention includes the following steps: contacting an Aspergillus alkaline protease that recognizes the amino acid sequence of SLXX and is cleaved between the two XXs with A2 type casein, thereby generating an opioid peptide containing the amino acid sequence represented by sequence number 2. In the method for improving the production efficiency of opioid peptides according to the present invention, although not limited, in order to separate the Aspergillus alkaline protease from contaminating proteases, the Aspergillus alkaline protease is recovered as a low-adsorption component of anion exchange resin or as an adsorption component of a cation exchange resin.

[0058] Purification (Preparation) Methods for Alkaline Proteases

[0059] The Aspergillus alkaline protease is not limited and can be purified (manufactured) from a mixture of Aspergillus proteases. The isoelectric point of the Aspergillus alkaline protease of the present invention is alkaline. On the other hand, many Aspergillus proteases are acidic enzymes and can be separated from these acidic proteases using anion exchange resins or cation exchange resins.

[0060] For example, when using anion exchange resins, adsorption becomes difficult if the pH is low. Specifically, at pH 8.5, adsorption occurs on the anion exchange resin, but elution can be performed using 10 mM NaCl. If the pH is further lowered, the low-adsorption component will be eluted. For example, when recovering the low-adsorption component from the anion exchange resin, a buffer solution with a pH of 7 or higher, preferably 8 or higher, is sufficient. Furthermore, when using cation exchange resins, the alkaline protease of the present invention can adsorb onto the cation exchange resin. On the other hand, the acidic protease of Aspergillus cannot adsorb onto the cation exchange resin, thus allowing the alkaline protease to be separated from the acidic protease. For example, when using the adsorbed component in the cation exchange resin, a buffer solution with a pH of 7 or lower, preferably 6 or lower, is sufficient.

[0061] [2] Alkaline protease composition for the production of opioid peptides from A2 casein.

[0062] The alkaline protease composition of the present invention comprises an Aspergillus alkaline protease that recognizes the amino acid sequence of SLXX and is cleaved between the two XXs, for the production of an opioid peptide comprising the amino acid sequence represented by sequence number 2 from A2 type casein.

[0063] The “A2 type casein” and “opioid peptide” are the A2 type casein and opioid peptide described in the technical solution “[1] Method for manufacturing opioid peptide”.

[0064] Alkaline protease

[0065] The alkaline protease contained in the composition of the present invention is an Aspergillus alkaline protease that recognizes the amino acid sequence of SLXX and cleaves between the two XXs, and the alkaline protease described in the above-mentioned technical solution "[1] Method for manufacturing opioid peptides" can be used without limitation.

[0066] The alkaline protease is not limited and can be obtained from crude purified enzymes derived from Aspergillus. Aspergillus is preferably a species of Aspergillus, and particularly preferably Aspergillus oryzae.

[0067] For example, the alkaline protease can be efficiently purified by using DEAE-agarose gel chromatography with 10 mM NaCl from the crude purified enzyme from Aspergillus oryzae. At a lower pH, elution into a low-adsorption fraction, by removing other contaminating proteases, can improve the productivity of the opioid peptides of the present invention.

[0068] In addition to the alkaline protease, the composition of the present invention may also contain a carrier or other components. Other components may include, for example, water-soluble solvents such as physiological saline or Ringer's solution, non-water-soluble solvents such as vegetable oil or fatty acid esters, and solubilizers, solubilizers, stabilizers, preservatives, suspending agents, or emulsifiers such as glucose or sodium chloride.

[0069] The content of alkaline phosphatase in the composition of the present invention is not particularly limited, and may contain more than 90% by weight, more than 50% by weight, more than 10% by weight, or more than 1% by weight of alkaline protease, with an upper limit of less than 100% by weight or less than 99% by weight.

[0070] [3] Opioid peptides

[0071] The opioid peptide of the present invention is an opioid peptide having the amino acid sequence represented by sequence number 2. The opioid peptide is not limited to the opioid peptide described in the above-described technical solution "[1] Method for Manufacturing Opioid Peptide". The opioid peptide can also be synthesized, but it is preferred to be manufactured by the manufacturing method of the present invention.

[0072] [4] Opioid receptor agonists

[0073] 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 sequence number 2 as an active ingredient. The opioid receptor agonist of the present invention is an agonist that, by binding to opioid receptors, can exhibit analgesic, antidepressant, or anti-stress effects.

[0074] Examples of opioid receptors include delta receptors, κ receptors, and μ receptors. The opioid receptors to which the peptides of the present invention act are not limited, but delta receptors or μ receptors are preferred.

[0075] [5] Food composition

[0076] Food compositions for opioid receptor agonists or for anxiolytic purposes may contain the opioid peptides of the present invention as active ingredients.

[0077] Specifically, food products include: fresh foods such as salads; heated foods such as steaks, pizzas, and hamburgers; stir-fried vegetables; vegetables such as tomatoes, bell peppers, celery, bitter melon, carrots, potatoes, and asparagus, and processed foods made from these vegetables; pasta products such as cookies, bread, biscuits, compressed biscuits, cakes, pancakes, yokan (sweet bean jelly), pudding, jelly, ice cream, chewing gum, crispy biscuits, potato chips, chocolate, and candy; noodles such as udon noodles, pasta, and soba noodles; surimi products such as fish cakes, ham, and fish sausages; dairy products such as cheese, cream, and butter; condiments such as miso, soy sauce, sauces, ketchup, salad dressing, soup stock, noodle seasoning, curry powder, mirin, fried flour, and spices; soy products such as tofu; konjac; and supplements.

[0078] Examples of beverages include: coffee beverages; cocoa beverages; vegetable juices obtained from the aforementioned vegetables; fruit juices such as grapefruit juice, orange juice, grape juice, and lemon juice; tea beverages such as green tea, black tea, sencha, and oolong tea; alcoholic beverages such as beer, wine (red wine, white wine, or sparkling wine, etc.), sake, plum wine, sparkling wine, whiskey, brandy, shochu, molasses, gin, and liqueurs; dairy beverages; soy milk beverages; liquid foods; and sports drinks.

[0079] Food or beverages include feed for animals. Examples of animals that are the subject of such feed include primates such as humans, cattle, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, or mice.

[0080] In these foods or beverages, two or more food additives and food ingredients may be formulated individually or in combination as needed, such as antioxidants, flavorings, acidulants, food 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, legumes, vegetables, meats, fish, shellfish, etc. The formulation amounts of these food ingredients and food additives may be appropriately determined within the scope of this invention without prejudice to its purpose.

[0081] Food and beverages include functional foods (beverages) and health foods (beverages). In this specification, "health foods (beverages)" refers to foods or beverages that impart a certain health benefit or anticipated health benefit, while "functional foods (beverages)" refers to foods or beverages that are designed and processed to fully exert their physiological regulatory functions (i.e., opioid receptor agonist effects). Functional foods and health foods may be in granular, solid, liquid, capsule, gel, or tablet form.

[0082] [6] Pharmaceutical Compositions

[0083] The opioid receptor agonist pharmaceutical compositions or anxiolytic pharmaceutical compositions of the present invention contain the opioid peptides of the present invention as active ingredients. The opioid receptor agonist pharmaceutical compositions of the present invention are not limited and can be used for anxiolytic, analgesic, antidepressant, or anti-stress purposes.

[0084] The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and oral and non-oral dosage forms may be listed, but oral dosage forms are preferred. Examples of oral dosage forms include: solid or powdered preparations such as granules, tablets, capsules, and pills, as well as liquid preparations such as suspensions, emulsions, syrups, and extracts. Examples of non-oral dosage forms include injectable preparations.

[0085] The pharmaceutical compositions of the present invention may be composed of, or may contain, the opioid peptides. When the pharmaceutical compositions of the present invention contain the opioid peptides, other additives may be included.

[0086] When the pharmaceutical composition of the present invention is an oral dosage form, other additives may include: excipients, binders, disintegrants, emulsifiers, lubricants, flow promoters, diluents, preservatives, colorants, flavorings, taste agents, stabilizers, humectants, preservatives, antioxidants, or suspending agents. Specifically, these may include, for example, gelatin, sodium alginate, starch, corn starch, white sugar, lactose, glucose, mannitol, carboxymethyl cellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soybean lecithin, sucrose, fatty acid esters, talc, magnesium stearate, polyethylene glycol, magnesium silicate, silicic anhydride, or synthetic aluminum silicate, etc.

[0087] When the pharmaceutical composition of the present invention is a non-oral preparation, other additives may include: water-soluble solvents such as physiological saline or Ringer's solution, non-water-soluble solvents such as vegetable oil or fatty acid esters, and solubilizers, solubilizers, stabilizers, preservatives, suspending agents, or emulsifiers such as glucose or sodium chloride.

[0088] The pharmaceutical composition of the present invention may contain 90% or more, 50% or more, 10% or more, or 1% or more of the peptide or peptide mixture.

[0089] The dosage or intake of the pharmaceutical composition of the present invention can be appropriately adjusted according to the formulation form and the age, sex, weight, and severity of disease symptoms of the user. However, it is preferably an amount that can exhibit analgesic, antidepressant, or anti-stress effects when administered or ingested. Specifically, the amount of opioid peptide added can be 0.01 to 1000 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, further preferably 5 to 400 mg / kg body weight / day, further preferably 10 to 300 mg / kg body weight / day, further preferably 15 to 200 mg / kg body weight / day, or most preferably 20 to 150 mg / kg body weight / day.

[0090] Of course, the method of administration described is one example, and other methods of administration may also be used. Ideally, the method of administration, dosage, administration time, and administration interval for administering a pharmaceutical composition to humans should be determined based on the managed clinical efficacy.

[0091] The pharmaceutical composition of the present invention can be administered to humans, but can also be administered to animals other than humans, such as: pets such as dogs, cats, rabbits, hamsters, guinea pigs and squirrels; livestock such as cattle and pigs; laboratory animals such as mice and rats; and animals kept in zoos, etc.

[0092] The pharmaceutical composition includes pharmaceuticals and quasi-pharmaceuticals. Examples of pharmaceuticals include preparations of raw medicinal herbs and traditional Chinese medicine preparations. Examples of quasi-pharmaceuticals include nutritional beverages and health supplements containing raw medicinal herbs.

[0093] [Example]

[0094] The present invention will be specifically described below through embodiments, but the scope of the present invention is not limited to these embodiments.

[0095] Example 1

[0096] In this embodiment, the alkaline protease used in this invention was purified from a crude purified enzyme derived from Aspergillus oryzae.

[0097] 50 mg of the crude purified enzyme was dissolved in 2 mL of 100 mM Tris-HCl buffer (pH 8.5, 20 °C). This crude purified enzyme solution was loaded onto a 1 mL DEAE-agarose gel Sepharose Fast Flow anion exchange chromatography column pre-equilibrated with 10 mL of 100 mM Tris-HCl buffer. The protein was eluted from the column in stages 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.

[0098] The obtained components were analyzed using SDS-PAGE. The results were as follows: Figure 1 The gel photograph represented by (A) in the image.

[0099] Each component was brought into contact with the amino acid sequence AQTQSLVYPFPGPIPNSLPQNIPPLTQTPV(CM-30) (sequence number 5) prepared from the A2 type β-casein sequence, and the ability of the present invention to generate opioid peptides was studied.

[0100] like Figure 1 As shown in (B), the activity of generating CM-12 (YPFPGPIPNSLP) from CM-30 was confirmed in the fraction with 10 mM NaCl. This fraction was used as the alkaline protease in the following examples.

[0101] The 34 kDa protein band from the 10 mM NaCl fraction was isolated from an SDS-10% PAGE gel. TOF-MS analysis was used to resolve the peptides obtained from the protein via trypsin digestion. Mass data of the trypsin peptides were analyzed using the MASCOT database. The 34 kDa protein was identified as sequence number 1, an alkaline protease. Specifically, it was considered a mature protease of 283 aa, excluding the 21 aa signal sequence and the 100 aa pre-sequence from the N-terminus.

[0102] Example 2

[0103] In this embodiment, the alkaline protease obtained in Example 1 was used to study the cleavage of the opioid peptides of the present invention using either A1-type casein or A2-type casein. A1-type casein was used as a comparative example.

[0104] 10 mL of A1 or A2 type milk was centrifuged at 7,500 x g at 4°C for 20 minutes to remove the cream layer. The supernatant was heated at 55°C for 5 minutes and then cooled to below 20°C. Subsequently, 1 M hydrochloric acid was added to the supernatant to adjust the pH to 4.6, and the precipitate was recovered by centrifugation at 1,300 x g at 20°C for 5 minutes. To dissolve the particles, the precipitate was dissolved in 10 mL of distilled water, and 1 M NaOH was added to adjust the pH to 7.0. The obtained casein protein was analyzed by SDS-12% PAGE. Figure 2 (A) Bands corresponding to A1 and A2 casein were observed (arrows). A1 and A2 casein were separated, and the CM-12 release capacity of purified Alp was evaluated. The results showed that a specific peak corresponding to CM-12 was detected in the A2 casein hydrolysate (…). Figure 2 B was detected in the A1 casein hydrolysate, but not in the A1 casein hydrolysate. Figure 2 (B in the middle).

[0105] Example 3

[0106] In this embodiment, purified alkaline protease and crude purified enzyme were used to study the generation of CM-12 peptide from A2 casein.

[0107] 3 μL of A2 casein (2 mg / mL) was hydrolyzed with 10 μL of purified alkaline protease (0.3 mg / mL) or 10 μL of crude purified enzyme (30 mg / mL), and the pH of the solution was adjusted to 8 using 0.2 M Na2HPO4 buffer. The reaction mixture was incubated at 45 °C for 1, 2, 4, 6, 8, 10, and 12 hours, and opioid peptides were measured.

[0108] With crude purified enzyme, the yield of CM-12 is very low, but by using purified alkaline protease, CM-12 can be produced and produced for a long time. Purified alkaline protease can release 18.6 times more CM-12 from A2 casein with 100% purity. Figure 3 ).

[0109] Example 4

[0110] In this embodiment, the reaction pH and reaction temperature of the alkaline protease obtained in Example 1 were investigated. Specifically, CM-30 was used as the substrate to study the activity of the purified alkaline protease. A 0.2 M citrate-Na₂HPO₄ buffer was used at pH 3.0-5.0, a 0.2 M Na₂HPO₄-NaH₂PO₄ buffer was used at pH 6.0-8.0, and a glycine-sodium hydroxide buffer was used at pH 9.0 and 10.0. The purified alkaline protease and substrate at a ratio of 10:1 (S / E, w / w) were incubated in the reaction buffer at 45°C for 1 hour. Figure 4 As shown in A, the optimal pH value is approximately 8.

[0111] Additionally, regarding reaction temperature, activity was tested at 30, 37, 40°C, 45, 50, 60, and 70°C using 0.2 M pH 8.0 Na₂HPO₄-NaH₂PO₄ buffer. The alkaline protease was incubated at a 10:1 (S / E, w / w) ratio for 1 hour. Figure 4 As shown in B, the optimal temperature is approximately 45℃.

[0112] [Industrial Applicability]

[0113] The manufacturing method of the present invention is effective for manufacturing opioid peptides in food or medicine for purposes such as anti-anxiety, analgesia, anti-depression, or anti-stress.

Claims

1. A method for manufacturing an opioid peptide, comprising the steps of: contacting an Aspergillus alkaline protease that recognizes the amino acid sequence of SLXX and is cleaved between the two XXs with A2 type casein, thereby generating a peptide containing the amino acid sequence represented by sequence number 2.

2. The method for manufacturing opioid peptides according to claim 1, wherein the Aspergillus alkaline protease is (a) an Aspergillus alkaline protease having the amino acid sequence represented by sequence number 1; or (b) an Aspergillus alkaline protease containing an amino acid sequence having more than 90% identity with the amino acid sequence represented by sequence number 1, and capable of generating an opioid peptide containing the amino acid sequence represented by sequence number 2 from A2 casein.

3. A method for improving the production efficiency of opioid peptides, comprising the following steps: contacting an Aspergillus alkaline protease that recognizes the amino acid sequence of SLXX and cleaves it between two XXs with A2 type casein, thereby generating an opioid peptide containing the amino acid sequence represented by sequence number 2; and To separate the Aspergillus alkaline protease from mixed proteases, the Aspergillus alkaline protease is recovered as a low-adsorption component of anion exchange resin or as an adsorption component of a cation exchange resin.

4. The method for improving the production efficiency of opioid peptides according to claim 3, wherein the Aspergillus alkaline protease is (a) an Aspergillus alkaline protease having the amino acid sequence represented by sequence number 1; or (b) an Aspergillus alkaline protease containing an amino acid sequence having more than 90% identity with the amino acid sequence represented by sequence number 1, and capable of generating an opioid peptide containing the amino acid sequence represented by sequence number 2 from A2 casein.

5. An alkaline protease composition comprising an Aspergillus alkaline protease that recognizes an amino acid sequence of SLXX and is cleaved between the two XXs, for the production of an opioid peptide comprising an amino acid sequence represented by sequence number 2 from A2 type casein.

6. The alkaline protease composition according to claim 5, wherein the Aspergillus alkaline protease is (a) an Aspergillus alkaline protease having the amino acid sequence represented by sequence number 1; or (b) an Aspergillus alkaline protease comprising an amino acid sequence having more than 90% identity with the amino acid sequence represented by sequence number 1, and capable of generating an opioid peptide comprising the amino acid sequence represented by sequence number 2 from A2 casein.

7. An opioid peptide having an amino acid sequence represented by sequence number 2.

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

9. A food composition for opioid receptor agonism 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 opioid receptor agonism 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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