WO2024071404A1 - Novel peptide, and antioxidant composition - Google Patents

Novel peptide, and antioxidant composition Download PDF

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Publication number
WO2024071404A1
WO2024071404A1 PCT/JP2023/035715 JP2023035715W WO2024071404A1 WO 2024071404 A1 WO2024071404 A1 WO 2024071404A1 JP 2023035715 W JP2023035715 W JP 2023035715W WO 2024071404 A1 WO2024071404 A1 WO 2024071404A1
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Prior art keywords
peptide
present
composition
food
antioxidant
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PCT/JP2023/035715
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French (fr)
Japanese (ja)
Inventor
直樹 湯田
佐保 朝倉
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森永乳業株式会社
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Publication of WO2024071404A1 publication Critical patent/WO2024071404A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/20—Milk; Whey; Colostrum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

Definitions

  • the present invention relates to a novel peptide having antioxidant activity, and further to an antioxidant composition containing the peptide.
  • Oxidative stress occurs when reactive oxygen species overwhelm the body's antioxidant function, or when the antioxidant function is unable to cope due to aging or other factors, and can lead to serious damage to cellular components. Therefore, strengthening antioxidant function through diet is considered to be a rational and practical approach to lowering the level of oxidative stress.
  • peptides having specific amino acid sequences exhibit blood pressure lowering effects, antithrombotic effects, antibacterial effects, antioxidant effects, etc.
  • peptides to be used in foods are peptide compositions obtained by enzymatic hydrolysis or microbial fermentation of proteins, taking into consideration safety, production costs, etc. Therefore, in terms of industrial use, it is necessary that not only the effects of a specific peptide, but also the peptide composition containing it be effective.
  • approximately 80% of the protein derived from milk is casein, and the remaining approximately 20% is whey protein. Due to its high yield, effective use of casein has great industrial merit.
  • Patent Document 1 discloses peptides with ORAC activity consisting of amino acid sequences found in milk proteins, but because they use synthetic peptides that have been organically synthesized, they cannot be used in foods as is for safety reasons.
  • Non-Patent Document 1 reveals that ⁇ -lactoglobulin ( ⁇ -Lg), ⁇ -lactalbumin ( ⁇ -La) and specific peptides derived from these have ORAC activity, but does not show that specific peptides derived from casein have ORAC activity.
  • the present invention aims to obtain an antioxidant peptide that can be used in foods and an antioxidant composition containing the same.
  • the gist of the present invention relates to the following.
  • [1] A peptide comprising the amino acid sequence Gln-Pro-Glu-Val-Met.
  • [2] A casein hydrolysate containing the peptide according to [1].
  • [3] An antioxidant composition comprising the peptide described in [1] or the casein hydrolysate described in [2].
  • [4] A food or drink comprising the composition according to [3].
  • a pharmaceutical comprising the composition according to [3].
  • the present invention can also employ the following configuration.
  • An antioxidant method comprising administering to a subject a peptide comprising the amino acid sequence Gln-Pro-Glu-Val-Met.
  • a method for producing an antioxidant composition comprising formulating a peptide comprising the amino acid sequence Gln-Pro-Glu-Val-Met.
  • the present invention provides antioxidant peptides that can be used in foods and antioxidant compositions that contain the same.
  • One aspect of the present invention relates to a peptide comprising the amino acid sequence Gln-Pro-Glu-Val-Met (SEQ ID NO: 1) (hereinafter sometimes referred to as "the peptide of the present invention”).
  • the peptide of the present invention comprises the amino acid sequence represented by Gln-Pro-Glu-Val-Met (QPEVM) and has antioxidant activity.
  • QPEVM Gln-Pro-Glu-Val-Met
  • a preferred example of the peptide of the present invention is a peptide consisting of Gln-Pro-Glu-Val-Met.
  • Gln (Q) represents a glutamine residue
  • Pro (P) represents a proline residue
  • Glu (E) represents a glutamic acid residue
  • Val (V) represents a valine residue
  • Met (M) represents a methionine residue. It is preferable that each of the amino acids is an L-amino acid.
  • the peptide of the present invention may be a peptide in which one or more arbitrary amino acid residues have been added to the N-terminus or C-terminus of a specific amino acid sequence of the peptide, and more specifically, may be a peptide in which 1 to 10 residues, preferably 1 to 5 residues, and more preferably 1 to 3 residues have been added, and the sequence may have an antioxidant effect.
  • the peptide of the present invention may be in the form of a salt, and examples of salts include alkali metals such as potassium and sodium, and alkaline earth metals such as calcium magnesium.
  • the method for measuring the antioxidant activity of the peptide of the present invention is not particularly limited, and can be measured, for example, according to the method of Watanabe J et al. (Anal Sci. 2012;28(2):159-165.).
  • the antioxidant activity can be measured by the method described in the Examples below.
  • the antioxidant activity (oxygen radical absorbance capacity; ORAC value) measured in this manner may preferably be 1 ⁇ mol Trolox equivalent (TE)/mol or more, 1.1 ⁇ mol TE/mol or more, 1.2 ⁇ mol TE/mol or more, 1.3 ⁇ mol TE/mol or more, 1.4 ⁇ mol TE/mol or more, or 1.45 ⁇ mol TE/mol or more.
  • the antioxidant activity (ORAC value) of the peptide-containing casein hydrolysate or composition may preferably be 400 ⁇ mol TE/g or more, 450 ⁇ mol TE/g or more, 500 ⁇ mol TE/g or more, 510 ⁇ mol TE/g or more, 520 ⁇ mol TE/g or more, 530 ⁇ mol TE/g or more, or 540 ⁇ mol TE/g or more.
  • the peptides of the present invention can be obtained, for example, by (1) hydrolyzing a protein or peptide containing a specific amino acid sequence of the peptide of the present invention with an enzyme, acid, alkali, etc., and isolating and purifying the resulting hydrolyzate; (2) synthesizing the peptide of the present invention by a peptide synthesis method, and then isolating and purifying the desired peptide from the resulting synthetic product; or (3) extracting the peptide of the present invention or peptides containing the peptide from a plant, animal, or microorganism that produces the peptide of the present invention or peptides containing the peptide, and then isolating and purifying the resulting extract.
  • the outline of the method for producing the peptide of the present invention is that a milk-derived protein such as casein is dispersed, suspended or dissolved in water, and then hydrolyzed by adding a protease, acid or alkali, and the reaction is stopped when the decomposition has progressed appropriately, thereby producing a component containing the target peptide.
  • a milk-derived protein such as casein
  • it may be purified by methods such as ion exchange chromatography, adsorption chromatography, reverse phase chromatography, partition chromatography, gel filtration chromatography, solvent precipitation, and salting out.
  • the peptide of the present invention can be produced by hydrolyzing a natural protein.
  • natural proteins that can be used as raw materials include proteins derived from milk, soybeans, eggs, wheat, barley, rice, potatoes, sweet potatoes, peas, corn, livestock meat, fish meat, seafood, etc., and casein contained in the milk of mammals (cows, goats, sheep, pigs, humans, etc.) is preferred.
  • natural proteins that can be used as raw materials, so long as they contain the sequence Gln-Pro-Glu-Val-Met (QPEVM).
  • the peptides of the present invention do not necessarily have to be derived from a single naturally occurring protein, but may be derived from different naturally occurring proteins.
  • Casein includes ⁇ -casein, ⁇ -casein, ⁇ -casein, etc., and in this technology, a mixture of these can be used, but it is preferable to use ⁇ -casein.
  • a protein solution is prepared by dissolving or dispersing the raw natural protein in a solvent such as water.
  • the solvent is not particularly limited, but distilled water is preferably used.
  • the pH may be adjusted appropriately.
  • the concentration of the solution is not particularly limited, but it is usually 5 to 20% by mass, more preferably 6 to 18% by mass, and more preferably 7 to 15% by mass in terms of protein, from the viewpoints of efficiency and operability. By setting the solution concentration to 5% or more, the efficiency of production can be improved. In addition, by setting the solution concentration to 20% or less, it is possible to prevent a decrease in decomposition efficiency, burning during heat treatment, an increase in viscosity during cooling, and the like.
  • the pH of the solution is adjusted to about the optimum pH of the enzyme used, for example, preferably to pH 5 to 10, and more preferably to pH 7 to 8.
  • the alkaline agent used for adjusting the pH is not particularly limited, but examples thereof include sodium hydroxide, potassium hydroxide, potassium carbonate, and the like.
  • a pre-decomposition treatment step can also be performed as appropriate, such as a heat treatment or ion exchange treatment, either before or after the pH adjustment, or both.
  • protease is added to the substrate solution.
  • the protease is not particularly limited as long as it can generate QPEVM from natural proteins, and examples of the protease include those derived from plants, animals, and microorganisms, and one or more of these can be used in combination.
  • An endoprotease is preferable as the protease.
  • the endoprotease include serine proteases, metalloproteases, cysteine proteases, and aspartic acid proteases, and one or more of these can be selected and used. Of these, it is preferable to use serine proteases and/or metalloproteases.
  • Proteases are further classified into alkaline proteases, neutral proteases and acidic proteases, of which neutral proteases are preferably used.
  • the proteolytic enzyme is not particularly limited, but one or a combination of two or more known endoproteases can be used.
  • endoproteases include commercially available products such as Bioprase (manufactured by Nagase Biochemical Industries Co., Ltd.), Proleather (manufactured by Amano Enzyme Co., Ltd.), PTN6.0S (manufactured by Novozymes Co., Ltd.), Savinase (manufactured by Novozymes Co., Ltd.), GODOB.A.P (manufactured by Godo Shusei Co., Ltd.), Protease N Amano (manufactured by Amano Enzyme Co., Ltd.), Protease S (manufactured by Amano Enzyme Co., Ltd.), Sumiteam LP (Shin Nippon Chemical Industry Co., Ltd.), GODO B.N.P (manufactured by Godo Shusei Co., Ltd.),
  • the amount of endoprotease used relative to the protein is not particularly limited and may be adjusted appropriately depending on the substrate concentration, enzyme titer, reaction temperature, reaction time, etc., but it is generally preferable to add 100 to 30,000 activity units per gram of protein in the protein.
  • protease From the standpoint of efficiency and ease of use, it is preferable to disperse and dissolve the protease in cold water at 4 to 10°C before use.
  • the protease can also be added all at once or at appropriate intervals, and immobilized enzymes can also be used.
  • the temperature of the reaction system during the enzyme reaction can be appropriately determined within a practical range that includes the optimal temperature range for the enzyme action to be expressed.
  • the temperature of the reaction system is preferably 30 to 60°C, and more preferably 40 to 55°C.
  • the reaction duration varies depending on reaction conditions such as reaction temperature and initial pH. For example, if the reaction duration of the enzyme reaction is constant, there is a problem that decomposition products with different physicochemical properties may be generated for each production batch, and therefore it cannot be determined uniformly. Therefore, the reaction duration is determined by monitoring the enzyme reaction so that the peptide used in this technology can be obtained. In this technology, for example, the reaction duration is preferably determined between 1 and 48 hours, and more preferably between 4 and 18 hours.
  • the reaction duration of the protease may be determined by monitoring the decomposition rate of the enzyme reaction and continuing the reaction until a desired decomposition rate is reached. For example, when the casein protein is used as the raw material, the decomposition rate is preferably 10 to 50%, and more preferably 15 to 25%.
  • the decomposition rate of the raw material protein is calculated by measuring the total nitrogen content of the sample using the Kjeldahl method (Food Analysis Methods, edited by the Japan Society of Food Industry, p. 102, Korin Co., Ltd., 1984) and measuring the formol nitrogen content of the sample using the formol titration method (Mitsuda et al., eds., Food Engineering Experiment Book, Vol. 1, p. 547, Yokendo, 1970), and then calculating the decomposition rate from these measured values using the following formula.
  • Decomposition rate (%) (formol nitrogen amount/total nitrogen amount) x 100
  • the enzyme reaction is stopped.
  • the enzyme reaction is stopped by inactivating the enzyme in the hydrolysis solution.
  • the inactivation treatment can be carried out by a conventional method, for example, a heat inactivation treatment.
  • the conditions for the heat inactivation treatment can be appropriately set so as to sufficiently inactivate the enzyme, taking into account the thermal stability of the enzyme used.
  • the enzyme can be inactivated by holding the enzyme at a temperature range of 80 to 130°C for a holding time of 30 minutes to 2 seconds.
  • the resulting solution containing the peptide may be sterilized.
  • the sterilization method may be a conventional heat treatment method or the like.
  • the heating temperature and holding time during the heat treatment may be appropriately set so as to provide sufficient sterilization.
  • sterilization can be achieved by heat treatment at 70 to 140°C for 2 seconds to 30 minutes.
  • the heat sterilization method may be either a batch method or a continuous method, and in the continuous method, methods such as a plate heat exchange method, an infusion method, and an injection method may be used.
  • the resulting peptide-containing solution can be used as is, or, if necessary, can be used after being treated by known methods, such as homogenization, concentration, drying, granulation, sieving, etc. That is, the peptide of the present invention may be in the form of a concentrate, a dry product, a granulated product, etc. The peptide of the present invention may be in the form of a liquid, a solid, or a powder.
  • the peptide of the present invention can also be produced by chemical synthesis.
  • Examples of chemical synthesis of peptides include the liquid phase method or solid phase method commonly used for synthesizing oligopeptides.
  • the synthesized peptide can be deprotected as necessary, and unreacted reagents and by-products can be removed to isolate and purify the peptide of the present invention.
  • Such peptide synthesis can be performed using a commercially available peptide synthesizer.
  • the peptides of the present invention can also be produced by biosynthesis.
  • Peptide biosynthesis can be carried out by a conventional method, for example, by introducing a peptide expression vector into a host organism to produce and secrete the peptide.
  • the peptide of the present invention can exert its efficacy even when used in an unpurified state. That is, the unpurified peptide of the present invention may be ingested or administered in the form of an antioxidant composition, food or drink, or pharmaceutical product described below. Furthermore, the obtained peptide of the present invention may be subjected to appropriate separation and purification methods known in the art.
  • the casein hydrolysate containing the peptide of the present invention produced as described above also has antioxidant activity. That is, one aspect of the present invention relates to a casein hydrolysate containing the peptide of the present invention (hereinafter sometimes referred to as "the casein hydrolysate of the present invention” or “the casein hydrolysate of the present invention”).
  • the casein hydrolysate is not limited as long as it contains the peptide of the present invention, but is preferably a casein hydrolysate.
  • the casein hydrolysate is preferably a hydrolysate produced by a protease, and examples of the protease include the protease mentioned above in the section on Peptides.
  • the casein hydrolysate may be used after it has been processed into a liquid, concentrated or purified form, or into a solid, granular or powder form.
  • the content of peptide QPEVM in the casein hydrolysate of the present invention is not particularly limited, but the lower limit is preferably 0.001% by mass or more, more preferably 0.005% by mass or more, even more preferably 0.01% by mass or more, and even more preferably 0.1% by mass or more, from the viewpoint of better exerting the efficacy of the present invention.
  • the upper limit of the content of peptide QPEVM in the casein hydrolysate of the present invention is preferably 1% by mass or less, more preferably 0.5% by mass or less, even more preferably 0.4% by mass or less, and even more preferably 0.3% by mass or less, from the viewpoint of production efficiency of the casein hydrolysate of the present invention.
  • the content of peptide QPEVM in the casein hydrolysate of the present invention may be 0.001 to 1 mass%, 0.005 to 0.5 mass%, 0.01 to 0.4 mass%, or 0.1 to 0.3 mass%.
  • the peptide content in the casein hydrolysate of the present invention can be measured by the following method.
  • the sample powder is diluted and dissolved in 0.1% formic acid solution to a concentration of 1.0 mg/mL, and then ultrasonically crushed for 10 minutes.
  • the powder solution is then filtered through a 0.22 ⁇ m PVDF filter (Millipore) to prepare a powder solution, and LC/MS analysis is performed under the following measurement conditions. Meanwhile, a calibration curve is prepared using a chemically synthesized standard peptide (Peptide Institute) of the peptide to be measured.
  • peaks in the analysis of the powder solution those having the same molecular weight and retention time as the standard peptide are identified as having the same sequence as the standard peptide.
  • the content of the target peptide in the powder solution is determined by comparing the peak area of the standard peptide with the peak area of the sample powder.
  • Target peptide content [measured target peptide content in the obtained casein hydrolysate (mg)] / [mass of the obtained casein hydrolysate (g)] [Measured amount (mg) of target peptide in the obtained casein hydrolysate] is the measured amount of the target peptide in the sample by "LC/MS" described below.
  • the average molecular weight of the casein hydrolysate in the present invention is typically more than 190 daltons (hereinafter "Da"), 200 Da or more, 250 Da or more, 300 Da or more, or 350 Da or more, and may be less than 1000 Da, 800 Da or less, 600 Da or less, 500 Da or less, or 400 Da or less. Furthermore, the average molecular weight of the casein hydrolysate in the present invention may usually be more than 190 and less than 1000 Da, 200 to 800 Da, 250 to 600 Da, 300 to 500 Da, or 350 to 400 Da.
  • Da 190 daltons
  • the average molecular weight of the casein hydrolysate can be determined, for example, by the following procedure.
  • the molecular weight distribution is measured by HPLC to calculate the number average molecular weight of the protein hydrolysate.
  • a polyhydroxyethyl aspartamide column (Poly LC, diameter 4.6 mm, length 220 mm) is used, and elution is performed with 20 mM sodium chloride and 50 mM formic acid at an elution rate of 0.5 mL/min.
  • Detection is performed using a UV detector (Shimadzu Corporation), and data analysis is performed using a GPC analysis system (Shimadzu Corporation).
  • protein hydrolysates contain free amino acids during the production process.
  • the free amino acid ratio of the casein hydrolysate of the present invention is usually 5% by mass or more, 10% by mass or more, or 15% by mass or more, and may be 60% by mass or less, 40% by mass or less, 30% by mass or less, or 20% by mass or less.
  • the free amino acid ratio of the casein hydrolysate in the present invention may usually be 5 to 60% by mass, 5 to 40% by mass, 10 to 30% by mass, or 15 to 20% by mass.
  • the free amino acid ratio can be determined, for example, by the following procedure.
  • (a) Measurement of Amino Acid Composition For amino acids other than tryptophan, cysteine, and methionine, the sample is hydrolyzed with 6N hydrochloric acid at 110°C for 24 hours, tryptophan is alkaline decomposed with barium hydroxide at 110°C for 22 hours, and cysteine and methionine are hydrolyzed with 6N hydrochloric acid at 110°C for 18 hours after treatment with performic acid, and each is analyzed with an amino acid analyzer (e.g., Hitachi, Model 835) to measure the mass of the amino acid.
  • an amino acid analyzer e.g., Hitachi, Model 835
  • the amount of glutamine and glutamic acid in the sample is quantified as the glutamic acid analysis value, which is the total amount of both.
  • composition of the present invention containing the peptide of the present invention or the casein hydrolysate of the present invention.
  • the composition of the present invention preferably contains the peptide of the present invention or the casein hydrolysate of the present invention as an active ingredient.
  • composition of the present invention can be administered to a subject to be used for the prevention or treatment of fatigue, aging, lifestyle-related diseases, and the like, which are associated with oxidative stress.
  • Another aspect of the composition of the present invention is that it can be used as a food ingredient to prevent or improve deterioration in flavor, discoloration, and other physical property changes of foods caused by oxidation.
  • Non-therapeutic purposes is a concept that does not include medical procedures, i.e., treatments on the human body by treatment. For example, health promotion, beauty treatments, etc. can be mentioned.
  • “amelioration” refers to the improvement of a disease, symptom, or condition in a subject; the prevention or delay of the worsening of a disease, symptom, or condition; or the reversal, prevention, or delay of the progression of a disease or symptom.
  • prevention refers to preventing or delaying the onset of a disease or symptom or the occurrence of a condition in a subject; or reducing the risk of developing a disease or symptom in a subject.
  • composition of the present invention When the composition of the present invention is used for non-therapeutic purposes, it can be administered to a healthy subject.
  • a healthy subject may mean a subject who does not have a disease or symptom caused by oxidative stress.
  • the composition of the present invention When the composition of the present invention is used for non-therapeutic purposes, it is possible to prevent a disease or symptom caused by oxidative stress in a healthy subject.
  • diseases or symptoms caused by oxidative stress include high blood pressure, diabetes, aging symptoms, fatigue symptoms, etc.
  • aging refers to undesirable changes that occur with age. In this specification, “fatigue” means physical fatigue and mental fatigue.
  • the composition of the present invention When the composition of the present invention is used for non-therapeutic purposes, it reduces the level of oxidative stress in healthy individuals, thereby making it possible to prevent hypertension, diabetes, aging symptoms, fatigue symptoms, and the like in healthy individuals.
  • the composition of the present invention can be used as a general food ingredient.
  • the composition of the present invention as a food ingredient, it is possible to prevent phenomena caused by oxidation of other food ingredients combined with it, and of the food as a whole. Phenomena caused by oxidation include deterioration of flavor and discoloration.
  • composition of the present invention When the composition of the present invention is used for therapeutic purposes, it can be administered to a non-healthy subject.
  • a non-healthy subject may mean a subject having a disease or symptom caused by oxidative stress.
  • the composition of the present invention When the composition of the present invention is used for therapeutic purposes, it is possible to prevent or treat a disease or symptom caused by oxidative stress in a non-healthy subject. Examples of diseases or symptoms caused by oxidative stress include hypertension, diabetes, aging symptoms, fatigue symptoms, etc.
  • the composition of the present invention When the composition of the present invention is used for therapeutic purposes, the level of oxidative stress in unhealthy individuals is reduced, making it possible to prevent or treat hypertension, diabetes, aging symptoms, fatigue symptoms, and the like in unhealthy individuals.
  • the content of peptide QPEVM in the composition of the present invention is not particularly limited, but the lower limit is preferably 0.001% by mass or more, more preferably 0.005% by mass or more, even more preferably 0.01% by mass or more, and even more preferably 0.1% by mass or more, from the viewpoint of better exerting the efficacy of the present invention.
  • the upper limit of the content of peptide QPEVM in the composition of the present invention is preferably 1% by mass or less, more preferably 0.5% by mass or less, even more preferably 0.4% by mass or less, even more preferably 0.3% by mass or less, and even more preferably 0.2% by mass or less, from the viewpoint of production efficiency of the composition of the present invention.
  • the content of peptide QPEVM in the composition of the present invention may be 0.001-1% by weight, 0.005-0.5% by weight, 0.01-0.4% by weight, 0.01-0.3% by weight, or 0.1-0.2% by weight.
  • the content of peptide QPEVM in the composition of the present invention can be measured based on known methods, for example, the method described above in the section on casein hydrolysates.
  • the administration time of the composition of the present invention as an active ingredient is not particularly limited and can be appropriately selected depending on the condition of the subject to be administered.
  • the administration time of the composition of the present invention is not particularly limited and can be appropriately selected, for example, before meals, after meals, between meals, before going to bed, etc.
  • the dosage of the composition of the present invention as an active ingredient is not particularly limited, but when used in the antioxidant composition of the present invention, the amount of peptide is 0.001 to 3000 mg/day, preferably 0.01 to 30 mg/day, and is determined depending on age, sex, severity of symptoms, etc.
  • the daily dosage may be divided into one to three times a day.
  • Examples of the administration route include oral administration and parenteral administration (intraperitoneal administration, intravenous administration, intramuscular administration, transmucosal administration, intranasal administration, intrarectal administration, etc.).
  • the subjects of administration are usually humans, but also include mammals other than humans, such as pet animals such as dogs and cats, and livestock such as cows, sheep, and pigs.
  • administering a peptide to an animal may be synonymous with "allowing an animal to ingest a peptide.”
  • Ingestion may be voluntary (free intake) or forced (forced intake). That is, the administration step may specifically be, for example, a step of blending the peptide into food, drink, or feed and supplying it to the subject, thereby allowing the subject to ingest the peptide freely.
  • the composition of the present invention When the composition of the present invention is intended to be orally ingested, it is preferably in the form of a food or drink, although there are no limitations thereon. That is, one aspect of the present invention relates to a food or drink comprising the composition of the present invention (hereinafter, may be referred to as "the food or drink of the present invention").
  • the food and beverage of the present invention can be administered to any subject, including healthy and unhealthy individuals, but when the food and beverage is labeled with a specific use (particularly a health use) or function, it is used for the above-mentioned non-therapeutic purposes.
  • Food and drink products include any form of liquid, paste, gel, solid, powder, etc., such as tablet confectionery; liquid food (nutritional food for tube feeding); wheat flour products such as bread, macaroni, spaghetti, noodles, cake mix, fried chicken flour, breadcrumbs, etc.; instant noodles, cup noodles, retort/prepared foods, canned foods, microwave foods, instant soups/stews, instant miso soup/cleansing liquids, canned soups, freeze-dried foods, other instant foods, etc.; canned agricultural products, canned fruit.
  • liquid food nutritional food for tube feeding
  • wheat flour products such as bread, macaroni, spaghetti, noodles, cake mix, fried chicken flour, breadcrumbs, etc.
  • instant noodles, cup noodles, retort/prepared foods canned foods, microwave foods, instant soups/stews, instant miso soup/cleansing liquids, canned soups, freeze-dried foods, other instant foods, etc.
  • canned agricultural products canned fruit.
  • Agricultural processed products such as stuffing, jams and marmalades, pickles, boiled beans, dried agricultural goods, cereals (processed grain products), etc.; marine processed products such as canned seafood, fish ham and sausages, marine paste products, marine delicacies, and tsukudani (fried fish paste); livestock processed products such as canned livestock and pastes, livestock ham and sausages, etc.; dairy products such as processed milk, milk drinks, yogurt, lactic acid bacteria drinks, cheese, ice cream, modified milk powder, cream, other dairy products, etc.; fats and oils such as butter, margarines, vegetable oils, etc.; Basic seasonings such as soy sauce, miso, sauces, processed tomato seasonings, mirin, vinegar, etc.; complex seasonings and foods such as cooking mixes, curry bases, sauces, dressings, noodle soups, spices, and other complex seasonings; frozen foods such as frozen ingredients, semi-cooked frozen foods, and cooked frozen foods; sweets such as caramel, candy, chewing gum, chocolate, cookies,
  • the food and drink of the present invention is preferably prepared as a milk preparation, such as a powdered milk preparation or a liquid milk preparation.
  • milk preparation include infant formula, follow-up formula, low birth weight infant formula, pediatric formula, adult formula, elderly formula, allergy formula, lactose intolerance formula, and congenital metabolic disorder formula.
  • the food and drink of the present invention is preferably used as an infant formula, follow-up formula, low birth weight infant formula, and pediatric formula.
  • Infant formula refers to infant formula for infants aged 0-12 months, follow-up milk for infants aged 6-9 months or older and young children (up to 3 years), low birth weight infant formula for newborns weighing less than 2500g at birth (low birth weight infants), and various therapeutic milks used to treat children with pathological conditions such as cow's milk allergy and lactose intolerance.
  • the formula of the present invention may be administered to any subject, including healthy and unhealthy subjects.
  • the formula of the present invention When the formula of the present invention is administered to healthy individuals, it can be used as an infant formula, a follow-up formula, a formula for low birth weight infants, a pediatric formula, an adult formula, or a formula for the elderly, and can be used for the non-therapeutic purposes described above.
  • the formula of the present invention When the formula of the present invention is administered to non-healthy individuals, it can be used as a formula for allergies, lactose intolerance, or congenital metabolic disorders, and can be used for the above-mentioned therapeutic purposes.
  • the feed can be used.
  • the feed include pet food, livestock feed, and fish feed.
  • the form of the feed is not particularly limited, and may contain, in addition to the peptide QPEVM, for example, grains such as corn, wheat, barley, rye, and milo; vegetable oil cakes such as soybean oil cake, rapeseed oil cake, palm oil cake, and linseed oil cake; bran such as wheat bran, rice bran, and defatted rice bran; manufacturing residues such as corn gluten meal and corn jam meal; animal feeds such as fish meal, skimmed milk powder, whey, yellow grease, and tallow; yeasts such as torula yeast and brewer's yeast; mineral feeds such as calcium triphosphate and calcium carbonate; oils and fats; simple amino acids; and sugars.
  • grains such as corn, wheat, barley, rye, and milo
  • vegetable oil cakes such as soybean oil cake, rapeseed oil cake, palm oil cake, and linseed oil cake
  • composition of the present invention when the composition of the present invention is in the form of a food or beverage (including feed), it can be provided and sold as a food or beverage labeled with an antioxidant use, such as a use for preventing fatigue, aging, lifestyle-related diseases, etc., associated with oxidative stress.
  • an antioxidant use such as a use for preventing fatigue, aging, lifestyle-related diseases, etc., associated with oxidative stress.
  • the peptide of the present invention described herein can be used for the production of such food or beverage.
  • Such "indication” acts include all acts aimed at informing consumers of the above-mentioned uses, and any expression that can recall or infer the above-mentioned uses falls under the category of "indication” acts in this invention, regardless of the purpose of the indication, the content of the indication, the object or medium on which it is displayed, etc.
  • the "labeling" be done in an expression that allows consumers to directly recognize the above-mentioned uses.
  • Specific examples include the act of transferring, delivering, displaying for transfer or delivery, or importing food and beverage products or product packaging on which the above-mentioned uses are written, displaying or distributing advertisements, price lists, or transaction documents related to the products and writing the above-mentioned uses in information containing the above-mentioned uses and providing them by electromagnetic means (such as the Internet), etc.
  • the content of the labeling be approved by the government (for example, labeling approved based on various systems established by the government and made in a manner based on such approval). It is also preferable that such content of the labeling be affixed to packaging, containers, catalogs, pamphlets, POP and other sales promotion materials, other documents, etc.
  • labeling also includes labeling as health food, functional food, enteral nutrition food, special purpose food, health functional food, food for specified health use, nutrient functional food, functional food, functional labeling food, medical drug, etc.
  • labeling approved by the Consumer Affairs Agency for example, labeling approved under the system related to food for specified health use, nutrient functional food, or functional labeling food, or a similar system, etc., can be mentioned.
  • labeling as food for specified health use labeling as food for conditional specified health use, labeling that affects the structure or function of the body, labeling for reducing disease risk, labeling of functionality based on scientific evidence, etc.
  • labeling as food for specified health use especially labeling of health use
  • similar labeling as specified in the Cabinet Office Ordinance on the Permission of Labeling for Special Uses Prescribed in the Health Promotion Act are typical examples. Examples of such claims include "alleviates oxidative stress,””improves antioxidant power,””supports antioxidant function,” and the like.
  • the composition of the present invention may also be in the form of a pharmaceutical product. That is, one aspect of the present invention relates to a pharmaceutical product (hereinafter, sometimes referred to as "the pharmaceutical product of the present invention") that contains the composition of the present invention.
  • the pharmaceutical product of the present invention can be manufactured by a conventional method using raw materials that are generally used for pharmaceutical products, except that it contains the peptide QPEVM.
  • the pharmaceutical agent of the present invention can be administered to non-healthy subjects and used for the above-mentioned therapeutic purposes.
  • the route of administration of pharmaceuticals may be either oral or parenteral, with oral being preferred. Parenteral intake (administration) includes transdermal, intravenous, rectal, inhalation, etc.
  • the pharmaceutical form can be formulated into a desired dosage form depending on the administration method.
  • oral administration it can be formulated into solid preparations such as powders, granules, tablets, and capsules; liquid preparations such as solutions, syrups, suspensions, and emulsions.
  • parenteral administration it can be formulated into suppositories, ointments, injections, and the like.
  • components that are usually used in formulations such as a pH adjuster, a colorant, etc., can be used. It is also possible to use other medicinal ingredients, known or future components with antioxidant activity, and other medicines in combination.
  • formulation can be carried out by a known method according to the dosage form.
  • a carrier usually used in formulation may be appropriately blended.
  • Such carriers include excipients, binders, disintegrants, lubricants, stabilizers, flavorings, etc.
  • the excipients include, for example, sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, ⁇ -starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, and calcium carboxymethyl cellulose; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; and sulfate derivatives such as calcium sulfate.
  • sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol
  • starch derivatives such as corn starch, potato starch, ⁇ -starch, dextrin, and carb
  • the binder may be, for example, gelatin, polyvinylpyrrolidone, macrogol, etc., in addition to the above excipients.
  • the disintegrants include, for example, the above-mentioned excipients, as well as chemically modified starch or cellulose derivatives such as croscarmellose sodium, sodium carboxymethyl starch, and cross-linked polyvinylpyrrolidone.
  • the lubricants include, for example, talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as veegum and galangal; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylates such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; starch derivatives, etc.
  • the stabilizer examples include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; and sorbic acid.
  • flavoring agent examples include sweeteners, acidulants, and fragrances.
  • examples of carriers to be used include solvents such as water, flavoring agents, etc.
  • the timing of taking the pharmaceutical of the present invention is not particularly limited, and may be, for example, before or after meals, between meals, or before going to bed.
  • Preparation of Peptide Compositions 90g of water was added to 10g of sodium caseinate (manufactured by Tatua Dairy Company) derived from milk, and the mixture was thoroughly dispersed. Sodium hydroxide was added to adjust the pH of the solution to 7.0, and then casein was completely dissolved to prepare an aqueous casein solution with a concentration of about 10% by mass. The aqueous casein solution was sterilized by heating at 85°C for 10 minutes, the temperature was adjusted to 50°C, and 75,000 activity units of Sumiteam LP (manufactured by Shin Nippon Chemical Industry Co., Ltd.) were added and reacted for 7 hours.
  • Sumiteam LP manufactured by Shin Nippon Chemical Industry Co., Ltd.
  • peptide composition A with an average molecular weight of 350 Da and a free amino acid ratio of 15% by mass.
  • peptide compositions B average molecular weight 1000 Da, free amino acid ratio ⁇ 5% by mass
  • C average molecular weight 190 Da, free amino acid ratio >60% by mass
  • Peptide compositions A to C were dissolved in distilled water to a concentration of 10 mg/ml, and after ultrasonic treatment, the solution was passed through a Millex-GV 0.22 ⁇ m PVDF filter, and the permeate was used as a sample.
  • the ORAC value was measured using an H-ORAC measurement kit (FUJIFILM Wako Pure Chemical Corporation) according to the kit's protocol. Specifically, the measurement was performed as follows.
  • Trolox (6-hydroxy-2,5,7,8-tetoramethyl-chroman-2-carboxilic acid) was dissolved in potassium phosphate buffer to prepare a Trolox standard solution.
  • AAPH (2,2'-Azobis(2-amidinopropane)dihydrochloride) was dissolved in potassium phosphate buffer to prepare an active oxygen solution (prepared just before use).
  • Fluorescent reagent sodium fluorescein was dissolved in potassium phosphate buffer to prepare a fluorescent reagent solution.
  • peptide composition A showed a high ORAC value, so the antioxidant peptide contained in peptide composition A was identified.
  • Peptide composition A was subjected to reverse-phase HPLC using a CD-C18 column (Cadenza; 250 x 10 mm).
  • the peptide contained in this peak was analyzed using a Q Exactive Orbitrap (Thermo Fisher Scientific) and was confirmed to be Gln-Pro-Glu-Val-Met (QPEVM). It was confirmed that neither peptide composition B nor peptide composition C contained Gln-Pro-Glu-Val-Met (QPEVM).
  • Peptide QPEVM was obtained by solid-phase synthesis.
  • QPEVM was dissolved in distilled water to a concentration of 1 mg/ml and subjected to ultrasonic treatment.
  • the antioxidant activity of the peptide solution was measured using an H-ORAC measurement kit (FUJIFILM Wako Pure Chemical Corporation). As a result, the ORAC value of QPEVM was 1.457 ⁇ mol TE/mol.
  • peptide composition A prepared according to the method of the above Example is dissolved in 360 g of purified water, and the pH is adjusted to 3.6 with citric acid to obtain a peptide composition solution.
  • the peptide composition solution 110 g of sugar, 2 g of calcium lactate, 1 g of emulsifier, and 10 g of soybean polysaccharides are added, and purified water is added to make the total volume 0.8 L.
  • the mixture is heated to 80°C and then cooled to obtain an acidic beverage.
  • an antioxidant effect in the subject can be expected.
  • the beverage obtained can be expected to have an effect of preventing deterioration in flavor and color change due to oxidation.
  • Peptide composition A prepared according to the method of the above example and crystalline cellulose are placed in an agitator granulator and mixed. Purified water is then added to granulate, and the granules are dried to obtain a granule (pharmaceutical composition).
  • an antioxidant effect in the subject can be expected.
  • the obtained pharmaceutical product can be expected to have an effect of preventing deterioration and color change due to oxidation.
  • the present invention is useful in the fields of food and beverages, health foods, functional foods, supplements, pharmaceuticals, etc.

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Abstract

The present invention addresses the problem of providing a food-applicable antioxidant peptide and an antioxidant composition containing same. The present invention provides a peptide comprising the amino acid sequence of Gln-Pro-Glu-Val-Met and an antioxidant composition containing said peptide.

Description

新規ペプチド、及び抗酸化用組成物Novel peptide and antioxidant composition
　本発明は、抗酸化作用を有する新規ペプチドに関し、さらに該ペプチドを含有する抗酸化用組成物に関する。 The present invention relates to a novel peptide having antioxidant activity, and further to an antioxidant composition containing the peptide.
　酸化ストレスは、活性酸素が身体の抗酸化機能に過剰に作用した時、あるいは加齢等により抗酸化機能が対応できなかった時に生じ、細胞成分の重大な損傷につながる可能性がある。そのため、食事による抗酸化機能の強化は、酸化ストレスのレベルを下げるための合理的かつ実用的なアプローチであると考えられている。 Oxidative stress occurs when reactive oxygen species overwhelm the body's antioxidant function, or when the antioxidant function is unable to cope due to aging or other factors, and can lead to serious damage to cellular components. Therefore, strengthening antioxidant function through diet is considered to be a rational and practical approach to lowering the level of oxidative stress.
　ところで、特定のアミノ酸配列を有するペプチドが、血圧低下効果や抗血栓効果、抗菌効果、抗酸化効果等を示すことが知られている。一方、食品に供されるペプチドは安全性や製造コスト等を考慮して、タンパク質を酵素加水分解や微生物発酵して得られるペプチド組成物が用いられる。したがって、特定のペプチドの効果だけでなく、それを含むペプチド組成物にも効果があることが産業利用上、必要である。
　また、牛乳由来のタンパク質は約80%がカゼイン、残りの約20%が乳清タンパク質であり、収率の高さからカゼインを有効に利用することは産業上メリットが大きい。 It is known that peptides having specific amino acid sequences exhibit blood pressure lowering effects, antithrombotic effects, antibacterial effects, antioxidant effects, etc. On the other hand, peptides to be used in foods are peptide compositions obtained by enzymatic hydrolysis or microbial fermentation of proteins, taking into consideration safety, production costs, etc. Therefore, in terms of industrial use, it is necessary that not only the effects of a specific peptide, but also the peptide composition containing it be effective.
In addition, approximately 80% of the protein derived from milk is casein, and the remaining approximately 20% is whey protein. Due to its high yield, effective use of casein has great industrial merit.
　特許文献１では、乳タンパク質中に見いだされるアミノ酸配列からなるORAC活性を有するペプチドが開示されているが、有機合成された合成ペプチドを用いており、安全性の観点からそのまま食品に供することができない。非特許文献１では、β-lactoglobulin（β-Lg）、α-lactalbumin（α-La）およびこれらに由来する特定のペプチドがORAC活性を有することが明らかにされているが、カゼインに由来する特定のペプチドがORAC活性を有することは示されていない。 Patent Document 1 discloses peptides with ORAC activity consisting of amino acid sequences found in milk proteins, but because they use synthetic peptides that have been organically synthesized, they cannot be used in foods as is for safety reasons. Non-Patent Document 1 reveals that β-lactoglobulin (β-Lg), α-lactalbumin (α-La) and specific peptides derived from these have ORAC activity, but does not show that specific peptides derived from casein have ORAC activity.
特許第5714206号Patent No. 5714206
　このような状況を鑑み、本発明は、食品利用が可能な抗酸化ペプチドおよびそれを含む抗酸化用組成物を得ることを課題とする。 In view of this situation, the present invention aims to obtain an antioxidant peptide that can be used in foods and an antioxidant composition containing the same.
　本発明者らは、抗酸化ペプチドを開発すべく鋭意検討した結果、カゼイン由来の抗酸化効果を有するペプチドおよびそれを含む抗酸化用組成物を知見し、本発明の完成に至った。 As a result of intensive research into the development of antioxidant peptides, the inventors discovered a peptide derived from casein that has antioxidant effects and an antioxidant composition containing the same, leading to the completion of the present invention.
　すなわち、本発明の要旨は以下に関する。
［１］　アミノ酸配列Gln-Pro-Glu-Val-Metを含むペプチド。
［２］　［１］に記載のペプチドを含有する、カゼイン分解物。
［３］　［１］に記載のペプチド、または［２］に記載のカゼイン分解物を含有する、抗酸化用組成物。
［４］　［３］に記載の組成物を含む、飲食品。
［５］　［３］に記載の組成物を含む、医薬品。 That is, the gist of the present invention relates to the following.
[1] A peptide comprising the amino acid sequence Gln-Pro-Glu-Val-Met.
[2] A casein hydrolysate containing the peptide according to [1].
[3] An antioxidant composition comprising the peptide described in [1] or the casein hydrolysate described in [2].
[4] A food or drink comprising the composition according to [3].
[5] A pharmaceutical comprising the composition according to [3].
　なお、本発明は、以下の構成を採用することも可能である。
［６］　アミノ酸配列Gln-Pro-Glu-Val-Metを含むペプチドを対象に投与することを含む、抗酸化方法。
［７］　抗酸化のための、アミノ酸配列Gln-Pro-Glu-Val-Metを含むペプチドの使用。
［８］　抗酸化の使用のための、アミノ酸配列Gln-Pro-Glu-Val-Metを含むペプチド。
［９］　抗酸化用組成物の製造における、アミノ酸配列Gln-Pro-Glu-Val-Metを含むペプチドの使用。
［１０］　アミノ酸配列Gln-Pro-Glu-Val-Metを含むペプチドを製剤化することを含む、抗酸化用組成物の製造方法。 The present invention can also employ the following configuration.
[6] An antioxidant method, comprising administering to a subject a peptide comprising the amino acid sequence Gln-Pro-Glu-Val-Met.
[7] Use of a peptide comprising the amino acid sequence Gln-Pro-Glu-Val-Met for antioxidant purposes.
[8] A peptide comprising the amino acid sequence Gln-Pro-Glu-Val-Met for use as an antioxidant.
[9] Use of a peptide comprising the amino acid sequence Gln-Pro-Glu-Val-Met in the production of an antioxidant composition.
[10] A method for producing an antioxidant composition, comprising formulating a peptide comprising the amino acid sequence Gln-Pro-Glu-Val-Met.
　本発明により、食品利用が可能な抗酸化ペプチドおよびそれを含む抗酸化用組成物を提供することができる。 The present invention provides antioxidant peptides that can be used in foods and antioxidant compositions that contain the same.
　次に、本発明を詳細に説明する。ただし、本発明は以下の実施形態に限定されず、本発明の範囲内で自由に変更することができるものである。
　なお、本明細書において、数値範囲を「下限～上限」で表現するものに関しては、上限は「以下」であっても「未満」であってもよく、下限は「以上」であっても「超」であってもよい。 The present invention will now be described in detail. However, the present invention is not limited to the following embodiments and can be freely modified within the scope of the present invention.
In this specification, when a numerical range is expressed as "lower limit to upper limit," the upper limit may be "equal to or less than" or "less than," and the lower limit may be "equal to or greater than" or "exceeding."
＜ペプチド＞
　本発明の一態様は、アミノ酸配列Gln-Pro-Glu-Val-Met（配列番号１）を含むペプチド（以下、「本発明のペプチド」ということがある）に関する。
　本発明のペプチドは、Gln-Pro-Glu-Val-Met（QPEVM）で表されるアミノ酸配列を含み、抗酸化活性を有する。本発明のペプチドとして、好ましくはGln-Pro-Glu-Val-Metからなるペプチドが挙げられる。 <Peptide>
One aspect of the present invention relates to a peptide comprising the amino acid sequence Gln-Pro-Glu-Val-Met (SEQ ID NO: 1) (hereinafter sometimes referred to as "the peptide of the present invention").
The peptide of the present invention comprises the amino acid sequence represented by Gln-Pro-Glu-Val-Met (QPEVM) and has antioxidant activity. A preferred example of the peptide of the present invention is a peptide consisting of Gln-Pro-Glu-Val-Met.
　ここで、Gln(Q)はグルタミン残基を、Pro(P)はプロリン残基を、Glu(E)はグルタミン酸残基を、Val(V)はバリン残基を、Met(M)はメチオニン残基を、それぞれ示す。いずれのアミノ酸も、L-型アミノ酸であることが好ましい。 Here, Gln (Q) represents a glutamine residue, Pro (P) represents a proline residue, Glu (E) represents a glutamic acid residue, Val (V) represents a valine residue, and Met (M) represents a methionine residue. It is preferable that each of the amino acids is an L-amino acid.
　なお、本発明のペプチドは、該ペプチドの所定のアミノ酸配列のN末端またはC末端にペプチド残基が１または複数個の任意のアミノ酸が付加したものであってもよく、より具体的には1～10残基、望ましくは1～5残基、さらに望ましくは1～3残基付加したペプチドであって、かつ抗酸化作用を有する配列であってもよい。 The peptide of the present invention may be a peptide in which one or more arbitrary amino acid residues have been added to the N-terminus or C-terminus of a specific amino acid sequence of the peptide, and more specifically, may be a peptide in which 1 to 10 residues, preferably 1 to 5 residues, and more preferably 1 to 3 residues have been added, and the sequence may have an antioxidant effect.
　本発明のペプチドは塩類であってもよく、塩類として例えば、カリウム、ナトリウム等のアルカリ金属類、カルシウムマグネシウム等のアルカリ土類金属類等が挙げられる。 The peptide of the present invention may be in the form of a salt, and examples of salts include alkali metals such as potassium and sodium, and alkaline earth metals such as calcium magnesium.
　本発明のペプチドの抗酸化活性の測定方法は、特に限定されないが、例えば、Watanabe Jらの方法（Anal Sci. 2012;28(2):159-165.）に準じて測定することが可能である。また、抗酸化活性は、後記実施例に記載の方法等によって測定することが可能である。
　このようにして測定される抗酸化活性（Oxygen radical absorbance capacity;ORAC値）は、好ましくは、1 μmol トロロックス当量(Trolox Equivalent:TE)/mol以上、1.1 μmol TE/mol以上、1.2 μmol TE/mol以上、1.3 μmol TE/mol以上、1.4 μmol TE/mol以上、または1.45 μmol TE/mol以上であってよい。
　ペプチドを含有するカゼイン分解物または組成物の抗酸化活性（ORAC値）は、好ましくは、400 μmol TE/g以上、450 μmol TE/g以上、500 μmol TE/g以上、510 μmol TE/g以上、520 μmol TE/g以上、530 μmol TE/g以上、または540 μmol TE/g以上であってよい。 The method for measuring the antioxidant activity of the peptide of the present invention is not particularly limited, and can be measured, for example, according to the method of Watanabe J et al. (Anal Sci. 2012;28(2):159-165.). In addition, the antioxidant activity can be measured by the method described in the Examples below.
The antioxidant activity (oxygen radical absorbance capacity; ORAC value) measured in this manner may preferably be 1 μmol Trolox equivalent (TE)/mol or more, 1.1 μmol TE/mol or more, 1.2 μmol TE/mol or more, 1.3 μmol TE/mol or more, 1.4 μmol TE/mol or more, or 1.45 μmol TE/mol or more.
The antioxidant activity (ORAC value) of the peptide-containing casein hydrolysate or composition may preferably be 400 μmol TE/g or more, 450 μmol TE/g or more, 500 μmol TE/g or more, 510 μmol TE/g or more, 520 μmol TE/g or more, 530 μmol TE/g or more, or 540 μmol TE/g or more.
（ペプチドの製造方法）
　本発明のペプチドは、例えば、（1）本発明のペプチドの特定のアミノ酸配列を含むタンパク質やペプチドを酵素や酸、アルカリ等にて分解し、得られた分解物から分離精製して得る方法、（2）ペプチドの合成法によって本発明のペプチドを合成した後、得られた合成物から所望のペプチドを分離精製して得る方法、（3）本発明のペプチドおよびこれを含むペプチド等を生産する植物、動物や微生物から抽出し、得られた抽出物から分離精製する方法等により得ることができる。
　より具体的には、例えば、本発明のペプチドの製造方法の概略は、カゼイン等の乳由来のタンパク質を、水に分散、懸濁または溶解し、これにタンパク質分解酵素や酸、アルカリを添加して加水分解し、適宜分解が進んだところで反応を停止することによって、目的のペプチドを含む成分を製造することができる。
　なお、さらに本発明のペプチドを単離することを目的として、イオン交換クロマトグラフィー、吸着クロマトグラフィー、逆相クロマトグラフィー、分配クロマトグラフィー、ゲルろ過クロマトグラフィー、溶媒沈殿、塩析等の方法で精製してもよい。 (Method of Producing Peptides)
The peptides of the present invention can be obtained, for example, by (1) hydrolyzing a protein or peptide containing a specific amino acid sequence of the peptide of the present invention with an enzyme, acid, alkali, etc., and isolating and purifying the resulting hydrolyzate; (2) synthesizing the peptide of the present invention by a peptide synthesis method, and then isolating and purifying the desired peptide from the resulting synthetic product; or (3) extracting the peptide of the present invention or peptides containing the peptide from a plant, animal, or microorganism that produces the peptide of the present invention or peptides containing the peptide, and then isolating and purifying the resulting extract.
More specifically, for example, the outline of the method for producing the peptide of the present invention is that a milk-derived protein such as casein is dispersed, suspended or dissolved in water, and then hydrolyzed by adding a protease, acid or alkali, and the reaction is stopped when the decomposition has progressed appropriately, thereby producing a component containing the target peptide.
For the purpose of further isolating the peptide of the present invention, it may be purified by methods such as ion exchange chromatography, adsorption chromatography, reverse phase chromatography, partition chromatography, gel filtration chromatography, solvent precipitation, and salting out.
　以下、本発明のペプチドの製造方法の一例について具体的に説明するが、これに限定されるものではない。 Below, an example of a method for producing the peptide of the present invention is specifically described, but is not limited to this.
（1）タンパク質の加水分解により得る方法
　本技術においては、公知の天然タンパク質の加水分解で用いられる様々な工程を、自由に選択して採用することができる。 (1) Method Obtained by Hydrolysis of Proteins In the present technology, various steps used in the hydrolysis of known natural proteins can be freely selected and employed.
（原料）
　本発明のペプチドは、天然タンパク質を加水分解することによって製造することが可能である。原料となる天然タンパク質としては、例えば、乳、大豆、卵、小麦、大麦、米、じゃが芋、さつま芋、えんどう豆、トウモロコシ、畜肉、魚肉、魚介などに由来するタンパク質などが挙げられ、哺乳動物（ウシ、ヤギ、ヒツジ、ブタ、ヒト等）の乳に含まれるカゼインが好ましい。原料となる天然タンパク質はGln-Pro-Glu-Val-Met（QPEVM）の配列を含んでいるものであれば特に限定されない。
　また、本発明のペプチドは、必ずしも単一の天然タンパク質由来である必要はなく、異なる天然タンパク質由来であってもよい。 (material)
The peptide of the present invention can be produced by hydrolyzing a natural protein. Examples of natural proteins that can be used as raw materials include proteins derived from milk, soybeans, eggs, wheat, barley, rice, potatoes, sweet potatoes, peas, corn, livestock meat, fish meat, seafood, etc., and casein contained in the milk of mammals (cows, goats, sheep, pigs, humans, etc.) is preferred. There are no particular limitations on the natural proteins that can be used as raw materials, so long as they contain the sequence Gln-Pro-Glu-Val-Met (QPEVM).
Furthermore, the peptides of the present invention do not necessarily have to be derived from a single naturally occurring protein, but may be derived from different naturally occurring proteins.
　カゼインには、α-カゼイン、β-カゼイン、κ-カゼイン等が含まれており、本技術においては、それらの混合物を使用することが可能であるが、なかでもβ-カゼインを使用することが好ましい。 Casein includes α-casein, β-casein, κ-casein, etc., and in this technology, a mixture of these can be used, but it is preferable to use β-casein.
（基質溶液の調製）
　原料の天然タンパク質を水等の溶媒に溶解または分散させ、タンパク質溶液を調製する。溶媒は特に限定されないが、蒸留水を用いることが好ましい。タンパク質を可溶化させるために、適宜pH調整を行ってもよい。また、前記溶液の濃度は特に限定されないが、通常、タンパク質換算で5～20質量%、より好ましくは6～18質量%、より好ましくは7～15質量%とすることが効率および操作性の点から好ましい。溶液濃度を5%以上とすることで、製造上の効率を向上させることができる。また、溶液濃度を20%以下とすることで、分解効率の低下、加熱処理時の焦付き、冷却時の粘度上昇等を防止することができる。 (Preparation of substrate solution)
A protein solution is prepared by dissolving or dispersing the raw natural protein in a solvent such as water. The solvent is not particularly limited, but distilled water is preferably used. In order to solubilize the protein, the pH may be adjusted appropriately. The concentration of the solution is not particularly limited, but it is usually 5 to 20% by mass, more preferably 6 to 18% by mass, and more preferably 7 to 15% by mass in terms of protein, from the viewpoints of efficiency and operability. By setting the solution concentration to 5% or more, the efficiency of production can be improved. In addition, by setting the solution concentration to 20% or less, it is possible to prevent a decrease in decomposition efficiency, burning during heat treatment, an increase in viscosity during cooling, and the like.
　次に、前記溶液のpHを、使用する酵素の至適pH付近に調整する。例えば、pH5～10に調整することが好ましく、pH7～8に調整することがより好ましい。
　pH調整に用いるアルカリ剤は特に限定されないが、例えば、水酸化ナトリウム、水酸化カリウム、炭酸カリウム等が挙げられる。 Next, the pH of the solution is adjusted to about the optimum pH of the enzyme used, for example, preferably to pH 5 to 10, and more preferably to pH 7 to 8.
The alkaline agent used for adjusting the pH is not particularly limited, but examples thereof include sodium hydroxide, potassium hydroxide, potassium carbonate, and the like.
　本技術においては、さらに、分解前処理工程として、pH調整前若しくは後、またはその両方で加熱処理、イオン交換処理等を適宜実施することもできる。 In this technology, a pre-decomposition treatment step can also be performed as appropriate, such as a heat treatment or ion exchange treatment, either before or after the pH adjustment, or both.
　次に、前記基質溶液にタンパク質分解酵素を添加する。タンパク質分解酵素は、天然タンパク質からQPEVMを生成することができれば特に限定されず、例えば、植物由来、動物由来、微生物由来等が挙げられ、これらから１種または２種以上組み合わせて使用できる。当該タンパク質分解酵素としては、エンドプロテアーゼが好適である。前記エンドプロテアーゼとしては、例えば、セリンプロテアーゼ、メタロプロテアーゼ、システインプロテアーゼ、アスパラギン酸プロテアーゼが挙げられ、これらを１種または２種以上選択して用いることができる。このうち、セリンプロテアーゼおよび/またはメタロプロテアーゼを用いるのが好適である。
　また、プロテアーゼは、アルカリ性プロテアーゼ、中性プロテアーゼおよび酸性プロテアーゼに分類される。このうち中性プロテアーゼを用いるのが好適である。 Next, a protease is added to the substrate solution. The protease is not particularly limited as long as it can generate QPEVM from natural proteins, and examples of the protease include those derived from plants, animals, and microorganisms, and one or more of these can be used in combination. An endoprotease is preferable as the protease. Examples of the endoprotease include serine proteases, metalloproteases, cysteine proteases, and aspartic acid proteases, and one or more of these can be selected and used. Of these, it is preferable to use serine proteases and/or metalloproteases.
Proteases are further classified into alkaline proteases, neutral proteases and acidic proteases, of which neutral proteases are preferably used.
　タンパク質分解酵素は、特に限定されないが、公知のエンドプロテアーゼを１種または２種以上組み合わせて使用することができる。エンドプロテアーゼとしては、例えば、ビオプラーゼ（長瀬生化学工業社製）、プロレザー（天野エンザイム社製）、PTN6.0S（ノボザイムズ社製）、サビナーゼ（ノボザイムズ社製）、GODOB.A.P（合同酒精社製）、プロテアーゼNアマノ（天野エンザイム社製）、プロテアーゼS（天野エンザイム社製）、スミチームLP（新日本化学工業社製）、GODO B．N．P（合同酒精社製）、ニュートラーゼ（ノボザイムズ社製）、アルカラーゼ（ノボザイムズ社製）、トリプシン（ノボザイムズ社製）、キモトリプシン（ノボザイムズ社製）、パパイン（天野エンザイム社製）、ブロメライン（天野エンザイム社製）等の市販品が挙げられる。 The proteolytic enzyme is not particularly limited, but one or a combination of two or more known endoproteases can be used. Examples of endoproteases include commercially available products such as Bioprase (manufactured by Nagase Biochemical Industries Co., Ltd.), Proleather (manufactured by Amano Enzyme Co., Ltd.), PTN6.0S (manufactured by Novozymes Co., Ltd.), Savinase (manufactured by Novozymes Co., Ltd.), GODOB.A.P (manufactured by Godo Shusei Co., Ltd.), Protease N Amano (manufactured by Amano Enzyme Co., Ltd.), Protease S (manufactured by Amano Enzyme Co., Ltd.), Sumiteam LP (Shin Nippon Chemical Industry Co., Ltd.), GODO B.N.P (manufactured by Godo Shusei Co., Ltd.), Neutrase (manufactured by Novozymes Co., Ltd.), Alcalase (manufactured by Novozymes Co., Ltd.), trypsin (manufactured by Novozymes Co., Ltd.), chymotrypsin (manufactured by Novozymes Co., Ltd.), papain (manufactured by Amano Enzyme Co., Ltd.), and bromelain (manufactured by Amano Enzyme Co., Ltd.).
　前記タンパク質に対するエンドプロテアーゼの使用量は、特に限定されず、基質濃度、酵素力価、反応温度および反応時間等により適宜調整すればよいが、一般的には、タンパク質中のタンパク質1g当り100～30,000活性単位の割合で添加することが好ましい。 The amount of endoprotease used relative to the protein is not particularly limited and may be adjusted appropriately depending on the substrate concentration, enzyme titer, reaction temperature, reaction time, etc., but it is generally preferable to add 100 to 30,000 activity units per gram of protein in the protein.
　タンパク質分解酵素は、効率性および操作性の観点から、4～10℃の冷水に分散し、溶解してから使用することが好ましい。また、タンパク質分解酵素は、一括添加、または適宜の間隔で添加することもでき、さらに、固定化酵素を使用することもできる。 From the standpoint of efficiency and ease of use, it is preferable to disperse and dissolve the protease in cold water at 4 to 10°C before use. The protease can also be added all at once or at appropriate intervals, and immobilized enzymes can also be used.
　本技術において、酵素反応中の反応系の温度は、酵素作用の発現する最適温度範囲を含む実用に供され得る範囲内で、適宜決定することができる。例えば、反応系の温度を、30～60℃とすることが好ましく、40～55℃とすることがより好ましい。 In this technology, the temperature of the reaction system during the enzyme reaction can be appropriately determined within a practical range that includes the optimal temperature range for the enzyme action to be expressed. For example, the temperature of the reaction system is preferably 30 to 60°C, and more preferably 40 to 55°C.
　また、本技術において、反応継続時間は、反応温度、初発pH等の反応条件によって異なる。例えば、酵素反応の反応継続時間を一定とすると、製造バッチ毎に異なる理化学的性質を有する分解物が生じる可能性等の問題があるため、一該に決定することができない。したがって、酵素反応をモニターすることにより、本技術に用いられるペプチドが得られるように、反応継続時間を決定する。本技術においては、例えば、反応継続時間は、1～48時間の間で決定することが好ましく、4～18時間の間で決定することがより好ましい。前記タンパク質分解酵素の反応継続時間は、酵素反応の分解率をモニターしながら、好ましい分解率に達するまで反応を続けてもよく、例えば、原料に前記カゼインタンパク質を用いた場合の分解率は10～50%であることが好ましく、15～25%であることがより好ましい。 In addition, in this technology, the reaction duration varies depending on reaction conditions such as reaction temperature and initial pH. For example, if the reaction duration of the enzyme reaction is constant, there is a problem that decomposition products with different physicochemical properties may be generated for each production batch, and therefore it cannot be determined uniformly. Therefore, the reaction duration is determined by monitoring the enzyme reaction so that the peptide used in this technology can be obtained. In this technology, for example, the reaction duration is preferably determined between 1 and 48 hours, and more preferably between 4 and 18 hours. The reaction duration of the protease may be determined by monitoring the decomposition rate of the enzyme reaction and continuing the reaction until a desired decomposition rate is reached. For example, when the casein protein is used as the raw material, the decomposition rate is preferably 10 to 50%, and more preferably 15 to 25%.
　なお、原料タンパク質の分解率の算出方法は、ケルダール法（日本食品工業学会編、「食品分析法」、第102頁、株式会社光琳、昭和59年）により試料の全窒素量を測定し、ホルモール滴定法（満田他編、「食品工学実験書」、上巻、第547ページ、養賢堂、1970年）により試料のホルモール態窒素量を測定し、これらの測定値から分解率を次式により算出する。
　分解率（%）=（ホルモール態窒素量/全窒素量）×100 The decomposition rate of the raw material protein is calculated by measuring the total nitrogen content of the sample using the Kjeldahl method (Food Analysis Methods, edited by the Japan Society of Food Industry, p. 102, Korin Co., Ltd., 1984) and measuring the formol nitrogen content of the sample using the formol titration method (Mitsuda et al., eds., Food Engineering Experiment Book, Vol. 1, p. 547, Yokendo, 1970), and then calculating the decomposition rate from these measured values using the following formula.
Decomposition rate (%) = (formol nitrogen amount/total nitrogen amount) x 100
　次に、酵素反応を停止させる。酵素反応の停止は、加水分解液中の酵素を失活させることにより行われる。失活処理は、常法、例えば、加熱失活処理等により実施することができる。加熱失活処理の条件（加熱温度、加熱時間等）は、使用した酵素の熱安定性を考慮し、十分に失活できる条件を適宜設定することができる。本技術においては、例えば、80～130℃の温度範囲で30分間～2秒間の保持時間で、酵素を失活させることができる。 Next, the enzyme reaction is stopped. The enzyme reaction is stopped by inactivating the enzyme in the hydrolysis solution. The inactivation treatment can be carried out by a conventional method, for example, a heat inactivation treatment. The conditions for the heat inactivation treatment (heating temperature, heating time, etc.) can be appropriately set so as to sufficiently inactivate the enzyme, taking into account the thermal stability of the enzyme used. In this technology, for example, the enzyme can be inactivated by holding the enzyme at a temperature range of 80 to 130°C for a holding time of 30 minutes to 2 seconds.
（殺菌処理）
　得られたペプチドを含有する溶液を殺菌してもよい。殺菌方法は、常法による加熱処理方法等を用いることができる。加熱処理時の加熱温度と保持時間は、充分に殺菌できる条件を適宜設定すればよく、例えば、70～140℃で2秒間～30分間加熱処理することにより殺菌できる。加熱殺菌の方式は、バッチ方式、連続方式のいずれの方式も可能であり、連続方式においてもプレート熱交換方式、インフュージョン方式、インジェクション方式等の方式を用いることができる。 (Sterilization treatment)
The resulting solution containing the peptide may be sterilized. The sterilization method may be a conventional heat treatment method or the like. The heating temperature and holding time during the heat treatment may be appropriately set so as to provide sufficient sterilization. For example, sterilization can be achieved by heat treatment at 70 to 140°C for 2 seconds to 30 minutes. The heat sterilization method may be either a batch method or a continuous method, and in the continuous method, methods such as a plate heat exchange method, an infusion method, and an injection method may be used.
（濃縮・乾燥）
　さらに、得られたペプチドを含有する溶液は、そのまま使用することもでき、また、必要に応じて、該溶液を公知の方法により処理したものを使用することもできる。公知の方法としては、例えば、均質化、濃縮、乾燥、造粒、篩別等が挙げられる。
　すなわち、本発明のペプチドの態様は、濃縮物、乾燥物、造粒物等とすることができる。本発明のペプチドの態様は、液体、固体、粉末にすることができる。 (Concentration/Drying)
Furthermore, the resulting peptide-containing solution can be used as is, or, if necessary, can be used after being treated by known methods, such as homogenization, concentration, drying, granulation, sieving, etc.
That is, the peptide of the present invention may be in the form of a concentrate, a dry product, a granulated product, etc. The peptide of the present invention may be in the form of a liquid, a solid, or a powder.
（2）ペプチドを化学合成により得る方法
　本発明のペプチドは、化学合成によっても製造することができる。ペプチドの化学合成は、例えば、オリゴペプチドの合成に通常用いられている液相法または固相法が挙げられる。合成されたペプチドは必要に応じて脱保護され、未反応試薬や副生物等を除去して、本発明のペプチドを単離精製することが可能である。このようなペプチドの合成は、市販のペプチド合成装置を用いて行うことができる。 (2) Method for obtaining peptide by chemical synthesis The peptide of the present invention can also be produced by chemical synthesis. Examples of chemical synthesis of peptides include the liquid phase method or solid phase method commonly used for synthesizing oligopeptides. The synthesized peptide can be deprotected as necessary, and unreacted reagents and by-products can be removed to isolate and purify the peptide of the present invention. Such peptide synthesis can be performed using a commercially available peptide synthesizer.
（3）ペプチドを生合成により得る方法
　本発明のペプチドは、生合成によっても製造することができる。ペプチドの生合成は、例えば、宿主生物にペプチド発現ベクターを導入して生成・分泌させるといった常法により行うことができる。 (3) Method for obtaining peptides by biosynthesis The peptides of the present invention can also be produced by biosynthesis. Peptide biosynthesis can be carried out by a conventional method, for example, by introducing a peptide expression vector into a host organism to produce and secrete the peptide.
　本発明のペプチドは、未精製のままの状態で使用しても効能を発揮することが可能である。すなわち、未精製の本発明のペプチドを後述の抗酸化用組成物、飲食品や医薬品の態様として、摂取や投与に供してもよい。
　また、さらに、得られた本発明のペプチドに対して、適宜公知の分離精製を行ってもよい。 The peptide of the present invention can exert its efficacy even when used in an unpurified state. That is, the unpurified peptide of the present invention may be ingested or administered in the form of an antioxidant composition, food or drink, or pharmaceutical product described below.
Furthermore, the obtained peptide of the present invention may be subjected to appropriate separation and purification methods known in the art.
＜カゼイン分解物＞
　前記のように製造された本発明のペプチドを含むカゼイン分解物も、抗酸化活性を有する。
　すなわち、本発明の一態様は、本発明のペプチドを含有する、カゼイン分解物（以下、「本発明のカゼイン分解物」、「本発明のカゼイン加水分解物」ということがある）に関する。
　カゼイン分解物は、本発明のペプチドを含有するものであれば、限定されないが、好ましくはカゼイン加水分解物である。カゼイン加水分解物は、好ましくはタンパク質分解酵素による加水分解物であり、タンパク質分解酵素としては、例えば、前記＜ペプチド＞の項に前記したタンパク質分解酵素が挙げられる。 <Casein hydrolysate>
The casein hydrolysate containing the peptide of the present invention produced as described above also has antioxidant activity.
That is, one aspect of the present invention relates to a casein hydrolysate containing the peptide of the present invention (hereinafter sometimes referred to as "the casein hydrolysate of the present invention" or "the casein hydrolysate of the present invention").
The casein hydrolysate is not limited as long as it contains the peptide of the present invention, but is preferably a casein hydrolysate. The casein hydrolysate is preferably a hydrolysate produced by a protease, and examples of the protease include the protease mentioned above in the section on Peptides.
　カゼイン分解物は、カゼイン分解物を液状、濃縮または精製してから、あるいは固体状、顆粒状または粉末状に加工してから用いてもよい。 The casein hydrolysate may be used after it has been processed into a liquid, concentrated or purified form, or into a solid, granular or powder form.
（QPEVMの含有量）
　本発明のカゼイン分解物中のペプチドQPEVMの含有量は特に限定されないが、その下限値は、本発明の効能をより良好に発揮させる観点から、好ましくは0.001質量%以上であり、より好ましくは0.005質量%以上であり、さらに好ましくは0.01質量%以上であり、よりさらに好ましくは0.1質量%以上である。本発明のカゼイン分解物中のペプチドQPEVMの含有量の上限値は、本発明のカゼイン分解物の製造効率の観点から、好ましくは1質量%以下であり、より好ましくは0.5質量%以下であり、さらに好ましくは0.4質量%以下であり、よりさらに好ましくは0.3質量%以下である。
　また、本発明のカゼイン分解物中のペプチドQPEVMの含有量は、0.001～1質量%、0.005～0.5質量%、0.01～0.4質量%、または0.1～0.3質量%であってよい。 (QPEVM content)
The content of peptide QPEVM in the casein hydrolysate of the present invention is not particularly limited, but the lower limit is preferably 0.001% by mass or more, more preferably 0.005% by mass or more, even more preferably 0.01% by mass or more, and even more preferably 0.1% by mass or more, from the viewpoint of better exerting the efficacy of the present invention. The upper limit of the content of peptide QPEVM in the casein hydrolysate of the present invention is preferably 1% by mass or less, more preferably 0.5% by mass or less, even more preferably 0.4% by mass or less, and even more preferably 0.3% by mass or less, from the viewpoint of production efficiency of the casein hydrolysate of the present invention.
Furthermore, the content of peptide QPEVM in the casein hydrolysate of the present invention may be 0.001 to 1 mass%, 0.005 to 0.5 mass%, 0.01 to 0.4 mass%, or 0.1 to 0.3 mass%.
　本発明のカゼイン分解物に含まれるペプチド含有量は、下記の方法にて測定できる。
（a）試料粉末を、1.0mg/mLとなるように、0.1%ギ酸水溶液に希釈溶解し、10分間超音波破砕したのち、0.22μm口径のPVDFフィルター（Millipore社製）でろ過して粉末溶液を調製し、下記測定条件によるLC/MS分析を実施する。一方、測定対象のペプチドの化学合成標準ペプチド（ペプチド研究所社製）を用いて検量線を作成する。
　前記粉末溶液の分析におけるピークのうち、標準ペプチドと分子量およびリテンションタイムが一致するものを、標準ペプチドと同一の配列として同定する。標準ペプチドのピーク面積と試料粉末のピーク面積を対比することにより、前記粉末溶液中に対象ペプチドの含有量を求める。 The peptide content in the casein hydrolysate of the present invention can be measured by the following method.
(a) The sample powder is diluted and dissolved in 0.1% formic acid solution to a concentration of 1.0 mg/mL, and then ultrasonically crushed for 10 minutes. The powder solution is then filtered through a 0.22 μm PVDF filter (Millipore) to prepare a powder solution, and LC/MS analysis is performed under the following measurement conditions. Meanwhile, a calibration curve is prepared using a chemically synthesized standard peptide (Peptide Institute) of the peptide to be measured.
Among the peaks in the analysis of the powder solution, those having the same molecular weight and retention time as the standard peptide are identified as having the same sequence as the standard peptide. The content of the target peptide in the powder solution is determined by comparing the peak area of the standard peptide with the peak area of the sample powder.
（b）対象ペプチド含有量（mg/カゼイン加水分解物1g）
　対象ペプチド含有量（mg/カゼイン加水分解物1g）=〔得られたカゼイン加水分解物中の対象ペプチド測定値（mg）〕/〔得られたカゼイン加水分解物の質量（g）〕
　〔得られたカゼイン加水分解物中の対象ペプチド測定値（mg）〕は、下記「LC/MS」による、試料中の対象ペプチドの測定値である。 (b) Content of target peptide (mg/g casein hydrolysate)
Target peptide content (mg/g of casein hydrolysate) = [measured target peptide content in the obtained casein hydrolysate (mg)] / [mass of the obtained casein hydrolysate (g)]
[Measured amount (mg) of target peptide in the obtained casein hydrolysate] is the measured amount of the target peptide in the sample by "LC/MS" described below.
（c）LC/MS使用機器 
　質量分析計：Q Exactive Orbitrap（Thermo Fisher Scientific社）。
　高速液体クロマトグラフ：Vanquish （Thermo Fisher Scientific社）、カラム：Acquity　UPLCBEH C18 φ2.1 mm×150 mm，1.7 μm（Waters社製）。 (c) LC/MS equipment used
Mass spectrometer: Q Exactive Orbitrap (Thermo Fisher Scientific).
High performance liquid chromatograph: Vanquish (Thermo Fisher Scientific), column: Acquity UPLCBEH C18 φ2.1 mm × 150 mm, 1.7 μm (Waters).
（d）LC/MS測定条件
　A液：0.1重量%  ギ酸-水溶液
　B液：0.1重量%  ギ酸-アセトニトリル溶液
　タイムプログラム：B液2%（0.0分）-B液22%（20.0分）-B液85%（22.1分）-B液85%（28.0分）-B液2%（28.1分）-STOP（38.0分）。
　試料注入量：5μL、カラム温度：40℃、液体流量：200μL／min
　分析モード：SIM測定。
　設定質量数（m/z）：m/z＝603.28 (d) LC/MS measurement conditions. Solution A: 0.1 wt% formic acid-water solution. Solution B: 0.1 wt% formic acid-acetonitrile solution. Time program: Solution B 2% (0.0 min)-Solution B 22% (20.0 min)-Solution B 85% (22.1 min)-Solution B 85% (28.0 min)-Solution B 2% (28.1 min)-STOP (38.0 min).
Sample injection volume: 5 μL, column temperature: 40°C, liquid flow rate: 200 μL/min
Analysis mode: SIM measurement.
Set mass number (m/z): m/z = 603.28
（平均分子量）
　本発明におけるカゼイン分解物の平均分子量は、通常190ダルトン（以下、「Da」）超、200Da以上、250Da以上、300Da以上、または350Da以上であり、1000Da未満、800Da以下、600Da以下、500Da以下、または400Da以下であってよい。
　また、本発明におけるカゼイン分解物の平均分子量は、通常190超1000Da未満、200～800Da、250～600Da、300～500Da、または350～400Daであってよい。 (Average Molecular Weight)
The average molecular weight of the casein hydrolysate in the present invention is typically more than 190 daltons (hereinafter "Da"), 200 Da or more, 250 Da or more, 300 Da or more, or 350 Da or more, and may be less than 1000 Da, 800 Da or less, 600 Da or less, 500 Da or less, or 400 Da or less.
Furthermore, the average molecular weight of the casein hydrolysate in the present invention may usually be more than 190 and less than 1000 Da, 200 to 800 Da, 250 to 600 Da, 300 to 500 Da, or 350 to 400 Da.
（平均分子量の測定）
　本発明において、カゼイン分解物の平均分子量は、例えば、以下の手順で求めることができる。HPLCにより分子量分布を測定して、タンパク質加水分解物の数平均分子量を算出する。ポリハイドロキシエチル・アスパルタミド・カラム［Poly Hydroxyethyl Aspartamide Column:ポリ・エル・シー（Poly LC社製）、直径4.6ｍｍおよび長さ220mm］を用い、20mM塩化ナトリウム、50mMギ酸により溶出速度0.5mL/minで溶出する。検出はUV検出器（島津製作所社製）を用い、データ解析はGPC分析システム（島津製作所社製）を使用する。 (Measurement of average molecular weight)
In the present invention, the average molecular weight of the casein hydrolysate can be determined, for example, by the following procedure. The molecular weight distribution is measured by HPLC to calculate the number average molecular weight of the protein hydrolysate. A polyhydroxyethyl aspartamide column (Poly LC, diameter 4.6 mm, length 220 mm) is used, and elution is performed with 20 mM sodium chloride and 50 mM formic acid at an elution rate of 0.5 mL/min. Detection is performed using a UV detector (Shimadzu Corporation), and data analysis is performed using a GPC analysis system (Shimadzu Corporation).
（遊離アミノ酸比率）
　タンパク質分解物には、一般に製造過程において遊離アミノ酸が含まれる。本発明におけるカゼイン分解物のアミノ酸遊離比率は、通常5質量%以上、10質量%以上、または15質量%以上であり、60質量%以下、40質量%以下、30質量%以下、または20質量%以下であってよい。
　また、本発明におけるカゼイン分解物のアミノ酸遊離比率は、通常5～60質量%、5～40質量%、10～30質量%、または15～20質量%であってよい。 (Free amino acid ratio)
In general, protein hydrolysates contain free amino acids during the production process. The free amino acid ratio of the casein hydrolysate of the present invention is usually 5% by mass or more, 10% by mass or more, or 15% by mass or more, and may be 60% by mass or less, 40% by mass or less, 30% by mass or less, or 20% by mass or less.
Furthermore, the free amino acid ratio of the casein hydrolysate in the present invention may usually be 5 to 60% by mass, 5 to 40% by mass, 10 to 30% by mass, or 15 to 20% by mass.
（遊離アミノ酸比率の計算）
　本発明において、遊離アミノ酸比率は、例えば、以下の手順で求めることができる。
（a）アミノ酸組成の測定
　トリプトファン、システインおよびメチオニン以外のアミノ酸については、試料を6N塩酸で110℃、24時間加水分解し、トリプトファンについては、水酸化バリウムで110℃、22時間アルカリ分解し、システインおよびメチオニンについては、過ギ酸処理後、6N塩酸で110℃、18時間加水分解し、それぞれアミノ酸分析機（例えば、日立製作所製、835型）により分析し、アミノ酸の質量を測定する。なお、この方法では、試料のグルタミンとグルタミン酸の量は、両者を合わせた合計量であるグルタミン酸分析値として定量される。 (Calculation of free amino acid ratio)
In the present invention, the free amino acid ratio can be determined, for example, by the following procedure.
(a) Measurement of Amino Acid Composition For amino acids other than tryptophan, cysteine, and methionine, the sample is hydrolyzed with 6N hydrochloric acid at 110°C for 24 hours, tryptophan is alkaline decomposed with barium hydroxide at 110°C for 22 hours, and cysteine and methionine are hydrolyzed with 6N hydrochloric acid at 110°C for 18 hours after treatment with performic acid, and each is analyzed with an amino acid analyzer (e.g., Hitachi, Model 835) to measure the mass of the amino acid. In this method, the amount of glutamine and glutamic acid in the sample is quantified as the glutamic acid analysis value, which is the total amount of both.
（b）遊離アミノ酸比率の算定
　試料中の各アミノ酸組成を前記（a）アミノ酸組成の測定の方法により測定し、これを合計して試料中の全アミノ酸の質量を算出する。次いで、スルホサリチル酸で試料を除蛋白し、残留する各遊離アミノ酸の質量を前記（a）の方法により測定し、これを合計して試料中の全遊離アミノ酸の質量を算出する。これらの値から、試料中の遊離アミノ酸の比率を算出する。 (b) Calculation of free amino acid ratio Each amino acid composition in the sample is measured by the method of measuring amino acid composition in (a) above, and the mass of the total amino acids in the sample is calculated by adding them up. The sample is then deproteinized with sulfosalicylic acid, and the mass of each remaining free amino acid is measured by the method of (a) above, and the mass of the total free amino acids in the sample is calculated by adding them up. From these values, the ratio of free amino acids in the sample is calculated.
　なお、前記＜ペプチド＞の項において説明された事項は、本発明の上記カゼイン分解物の説明に全て適用され得る。 All of the matters explained in the section on Peptides above can be applied to the explanation of the casein hydrolysates of the present invention.
＜抗酸化用組成物＞
　本発明の一態様は、本発明のペプチド、または本発明のカゼイン分解物を含有する、抗酸化用組成物（以下、「本発明の組成物」ということがある）に関する。本発明の組成物は、本発明のペプチド、または本発明のカゼイン分解物を、好ましくは有効成分として含有する。 <Antioxidant Composition>
One aspect of the present invention relates to an antioxidant composition (hereinafter, sometimes referred to as the "composition of the present invention") containing the peptide of the present invention or the casein hydrolysate of the present invention. The composition of the present invention preferably contains the peptide of the present invention or the casein hydrolysate of the present invention as an active ingredient.
　本発明の組成物は、対象に投与することにより、酸化ストレスが関与する疲労、老化、生活習慣病等の予防または治療用途に利用することができる。
　本発明の組成物の別の側面としては、食品原料として用いることで、酸化による食品の風味劣化、退色等の物性変化の予防または改善用途に利用することができる。 The composition of the present invention can be administered to a subject to be used for the prevention or treatment of fatigue, aging, lifestyle-related diseases, and the like, which are associated with oxidative stress.
Another aspect of the composition of the present invention is that it can be used as a food ingredient to prevent or improve deterioration in flavor, discoloration, and other physical property changes of foods caused by oxidation.
　本実施形態の用途は、治療目的使用であってもよく、非治療目的使用であってもよい。「非治療目的」とは、医療行為、すなわち、治療による人体への処置行為を含まない概念である。例えば、健康増進、美容行為等が挙げられる。
　本明細書において、「改善」とは、適用対象における疾患、症状または状態の好転；疾患、症状または状態の悪化の防止、遅延；或いは、疾患または症状の進行の逆転、防止または遅延をいう。
　また、本明細書において、「予防」とは、適用対象における疾患または症状の発症または状態の発生の防止または遅延；或いは、適用対象の疾患または症状の発症の危険性を低下させることをいう。 The use of the present embodiment may be for therapeutic purposes or for non-therapeutic purposes. "Non-therapeutic purposes" is a concept that does not include medical procedures, i.e., treatments on the human body by treatment. For example, health promotion, beauty treatments, etc. can be mentioned.
As used herein, "amelioration" refers to the improvement of a disease, symptom, or condition in a subject; the prevention or delay of the worsening of a disease, symptom, or condition; or the reversal, prevention, or delay of the progression of a disease or symptom.
In addition, as used herein, "prevention" refers to preventing or delaying the onset of a disease or symptom or the occurrence of a condition in a subject; or reducing the risk of developing a disease or symptom in a subject.
　本発明の組成物が非治療目的で使用される場合は、健常者を対象に投与されうる。健常者とは、酸化ストレスに起因する疾患又は症状を有していない者を意味してよい。本発明の組成物が非治療目的で使用される場合は、健常者の酸化ストレスに起因する疾患又は症状の予防が可能になる。酸化ストレスに起因する疾患又は症状としては、高血圧、糖尿病、老化症状、疲労症状等が挙げられる。
　本明細書において「老化」とは、加齢に伴う好ましくない変化を意味する。
　本明細書において「疲労」とは、身体的疲労、精神的疲労を意味する。
　本発明の組成物が非治療目的で使用される場合は、健常者における酸化ストレスのレベルが下がることにより、健常者における高血圧、糖尿病、老化症状、疲労症状等の予防が可能になる。 When the composition of the present invention is used for non-therapeutic purposes, it can be administered to a healthy subject. A healthy subject may mean a subject who does not have a disease or symptom caused by oxidative stress. When the composition of the present invention is used for non-therapeutic purposes, it is possible to prevent a disease or symptom caused by oxidative stress in a healthy subject. Examples of diseases or symptoms caused by oxidative stress include high blood pressure, diabetes, aging symptoms, fatigue symptoms, etc.
As used herein, "aging" refers to undesirable changes that occur with age.
In this specification, "fatigue" means physical fatigue and mental fatigue.
When the composition of the present invention is used for non-therapeutic purposes, it reduces the level of oxidative stress in healthy individuals, thereby making it possible to prevent hypertension, diabetes, aging symptoms, fatigue symptoms, and the like in healthy individuals.
　本発明の組成物は一般の食品原料として使用されうる。本発明の組成物が食品原料として使用されることで、組み合わせた他の食品原料、及び食品全体の酸化に起因する現象を予防することが可能である。酸化に起因する現象としては、風味劣化、退色等が挙げられる。 The composition of the present invention can be used as a general food ingredient. By using the composition of the present invention as a food ingredient, it is possible to prevent phenomena caused by oxidation of other food ingredients combined with it, and of the food as a whole. Phenomena caused by oxidation include deterioration of flavor and discoloration.
　本発明の組成物が治療目的で使用される場合は、非健常者を対象に投与されうる。非健常者とは、酸化ストレスに起因する疾患又は症状を有している者を意味してよい。本発明の組成物が治療目的で使用される場合は、非健常者の酸化ストレスに起因する疾患又は症状の予防又は治療が可能になる。酸化ストレスに起因する疾患又は症状としては、高血圧、糖尿病、老化症状、疲労症状等が挙げられる。
　本発明の組成物が治療目的で使用される場合は、非健常者における酸化ストレスのレベルが下がることにより、非健常者における高血圧、糖尿病、老化症状、疲労症状等の予防又は治療が可能になる。 When the composition of the present invention is used for therapeutic purposes, it can be administered to a non-healthy subject. A non-healthy subject may mean a subject having a disease or symptom caused by oxidative stress. When the composition of the present invention is used for therapeutic purposes, it is possible to prevent or treat a disease or symptom caused by oxidative stress in a non-healthy subject. Examples of diseases or symptoms caused by oxidative stress include hypertension, diabetes, aging symptoms, fatigue symptoms, etc.
When the composition of the present invention is used for therapeutic purposes, the level of oxidative stress in unhealthy individuals is reduced, making it possible to prevent or treat hypertension, diabetes, aging symptoms, fatigue symptoms, and the like in unhealthy individuals.
（QPEVMの含有量）
　本発明の組成物中のペプチドQPEVMの含有量は特に限定されないが、その下限値は、本発明の効能をより良好に発揮させる観点から、好ましくは0.001質量%以上であり、より好ましくは0.005質量%以上であり、よりさらに好ましくは0.01質量%以上であり、よりさらに好ましくは0.1質量%以上である。本発明の組成物中のペプチドQPEVMの含有量の上限値は、本発明の組成物の製造効率の観点から、好ましくは1質量%以下であり、より好ましくは0.5質量%以下であり、さらに好ましくは0.4質量%以下であり、よりさらに好ましくは0. 3質量%以下であり、よりさらに好ましくは0.2質量%以下である。
　また、本発明の組成物中のペプチドQPEVMの含有量は、0.001～1質量%、0.005～0.5質量%、0.01～0.4質量%、0.01～0.3質量%、または0.1～0.2質量%であってよい。 (QPEVM content)
The content of peptide QPEVM in the composition of the present invention is not particularly limited, but the lower limit is preferably 0.001% by mass or more, more preferably 0.005% by mass or more, even more preferably 0.01% by mass or more, and even more preferably 0.1% by mass or more, from the viewpoint of better exerting the efficacy of the present invention. The upper limit of the content of peptide QPEVM in the composition of the present invention is preferably 1% by mass or less, more preferably 0.5% by mass or less, even more preferably 0.4% by mass or less, even more preferably 0.3% by mass or less, and even more preferably 0.2% by mass or less, from the viewpoint of production efficiency of the composition of the present invention.
The content of peptide QPEVM in the composition of the present invention may be 0.001-1% by weight, 0.005-0.5% by weight, 0.01-0.4% by weight, 0.01-0.3% by weight, or 0.1-0.2% by weight.
　本発明の組成物中のペプチドQPEVMの含有量は、公知の方法、例えば、前記＜カゼイン分解物＞の項における方法に基づいて測定することができる。 The content of peptide QPEVM in the composition of the present invention can be measured based on known methods, for example, the method described above in the section on casein hydrolysates.
　有効成分としての本発明の組成物の投与時期は、特に限定されず、投与対象の状態に応じて適宜選択することが可能である。本発明の組成物の投与のタイミングは特に限定されず、例えば食前、食後、食間、就寝前等を適宜選択することが可能である。
　有効成分としての本発明の組成物の投与量は、特に限定されないが、本発明の抗酸化用組成物に用いられる場合、ペプチド量として0.001～3000mg/日、好ましくは0.01～30mg/日であり、年齢、性別、症状の程度等に応じて決定される。また、１日の投与量を１日１回から３回に分けてもよい。投与経路は、例えば経口投与、非経口投与（腹腔内投与、静脈内投与、筋肉内投与、経粘膜投与、鼻腔内投与、直腸内投与等）が挙げられる。なお、投与対象は、通常、ヒトであるが、ヒト以外の哺乳動物、例えばイヌ、ネコ等のペット動物、ウシ、ヒツジ、ブタ等の家畜も含むものとする。 The administration time of the composition of the present invention as an active ingredient is not particularly limited and can be appropriately selected depending on the condition of the subject to be administered. The administration time of the composition of the present invention is not particularly limited and can be appropriately selected, for example, before meals, after meals, between meals, before going to bed, etc.
The dosage of the composition of the present invention as an active ingredient is not particularly limited, but when used in the antioxidant composition of the present invention, the amount of peptide is 0.001 to 3000 mg/day, preferably 0.01 to 30 mg/day, and is determined depending on age, sex, severity of symptoms, etc. The daily dosage may be divided into one to three times a day. Examples of the administration route include oral administration and parenteral administration (intraperitoneal administration, intravenous administration, intramuscular administration, transmucosal administration, intranasal administration, intrarectal administration, etc.). The subjects of administration are usually humans, but also include mammals other than humans, such as pet animals such as dogs and cats, and livestock such as cows, sheep, and pigs.
　なお、本明細書において「ペプチドを動物に投与すること」は、「ペプチドを動物に摂取させること」と同義であってよい。摂取は、自発的なもの（自由摂取）であってもよく、強制的なもの（強制摂取）であってもよい。すなわち、投与工程は、具体的には、例えば、ペプチドを飲食品や飼料に配合して対象に供給し、以て対象にペプチドを自由摂取させる工程であってもよい。 In this specification, "administering a peptide to an animal" may be synonymous with "allowing an animal to ingest a peptide." Ingestion may be voluntary (free intake) or forced (forced intake). That is, the administration step may specifically be, for example, a step of blending the peptide into food, drink, or feed and supplying it to the subject, thereby allowing the subject to ingest the peptide freely.
　なお、前記＜ペプチド＞、＜カゼイン分解物＞の項において説明された事項は、本発明の上記組成物の説明に全て適用され得る。 All of the matters explained in the sections "Peptides" and "Casein hydrolysates" above can be applied to the description of the above composition of the present invention.
＜飲食品＞
　本発明の組成物を経口摂取される組成物とする場合は、限定されないが、飲食品の態様とすることが好ましい。
　すなわち、本発明の一態様は、本発明の組成物を含む、飲食品（以下、「本発明の飲食品」ということがある）に関する。
　本発明の飲食品は、健常者、非健常者を含むあらゆる対象に投与され得るが、特定の用途（特に保健の用途）や機能が表示された飲食品とする場合は、上記の非治療目的として使用される。 <Food and drink>
When the composition of the present invention is intended to be orally ingested, it is preferably in the form of a food or drink, although there are no limitations thereon.
That is, one aspect of the present invention relates to a food or drink comprising the composition of the present invention (hereinafter, may be referred to as "the food or drink of the present invention").
The food and beverage of the present invention can be administered to any subject, including healthy and unhealthy individuals, but when the food and beverage is labeled with a specific use (particularly a health use) or function, it is used for the above-mentioned non-therapeutic purposes.
　飲食品としては、本発明の効果を損なわず、経口摂取できるものであれば形態や性状は特に制限されず、ペプチドQPEVMを含有させること以外は、通常飲食品に用いられる原料を用いて通常の方法によって製造することができる。 There are no particular limitations on the form or properties of the food or beverage product, so long as it does not impair the effects of the present invention and can be taken orally, and it can be produced by ordinary methods using raw materials normally used for food or beverage products, except that it contains the peptide QPEVM.
　飲食品としては、液状、ペースト状、ゲル状固体、粉末等の形態を問わず、例えば、錠菓；流動食（経管摂取用栄養食）；パン、マカロニ、スパゲッティ、めん類、ケーキミックス、から揚げ粉、パン粉等の小麦粉製品；即席めん、カップめん、レトルト・調理食品、調理缶詰め、電子レンジ食品、即席スープ・シチュー、即席みそ汁・吸い物、スープ缶詰め、フリーズ・ドライ食品、その他の即席食品等の即席食品類；農産缶詰め、果実缶詰め、ジャム・マーマレード類、漬物、煮豆類、農産乾物類、シリアル（穀物加工品）等の農産加工品；水産缶詰め、魚肉ハム・ソーセージ、水産練り製品、水産珍味類、つくだ煮類等の水産加工品；畜産缶詰め・ペースト類、畜肉ハム・ソーセージ等の畜産加工品；加工乳、乳飲料、ヨーグルト類、乳酸菌飲料類、チーズ、アイスクリーム類、調製粉乳類、クリーム、その他の乳製品等の乳・乳製品；バター、マーガリン類、植物油等の油脂類；しょうゆ、みそ、ソース類、トマト加工調味料、みりん類、食酢類等の基礎調味料；調理ミックス、カレーの素類、たれ類、ドレッシング類、めんつゆ類、スパイス類、その他の複合調味料等の複合調味料・食品類；素材冷凍食品、半調理冷凍食品、調理済冷凍食品等の冷凍食品；キャラメル、キャンディー、チューインガム、チョコレート、クッキー、ビスケット、ケーキ、パイ、スナック、クラッカー、和菓子、米菓子、豆菓子、デザート菓子、ゼリー、その他の菓子等の菓子類；炭酸飲料、天然果汁、果汁飲料、果汁入り清涼飲料、果肉飲料、果粒入り果実飲料、野菜系飲料、豆乳、豆乳飲料、コーヒー飲料、お茶飲料、粉末飲料、濃縮飲料、スポーツ飲料、栄養飲料、アルコール飲料、その他の嗜好飲料等の嗜好飲料類、ベビーフード、ふりかけ、お茶漬けのり等のその他の市販食品等；育児用調製粉乳；経腸栄養食；機能性食品（特定保健用食品、栄養機能食品）等が挙げられる。 Food and drink products include any form of liquid, paste, gel, solid, powder, etc., such as tablet confectionery; liquid food (nutritional food for tube feeding); wheat flour products such as bread, macaroni, spaghetti, noodles, cake mix, fried chicken flour, breadcrumbs, etc.; instant noodles, cup noodles, retort/prepared foods, canned foods, microwave foods, instant soups/stews, instant miso soup/cleansing liquids, canned soups, freeze-dried foods, other instant foods, etc.; canned agricultural products, canned fruit. Agricultural processed products such as stuffing, jams and marmalades, pickles, boiled beans, dried agricultural goods, cereals (processed grain products), etc.; marine processed products such as canned seafood, fish ham and sausages, marine paste products, marine delicacies, and tsukudani (fried fish paste); livestock processed products such as canned livestock and pastes, livestock ham and sausages, etc.; dairy products such as processed milk, milk drinks, yogurt, lactic acid bacteria drinks, cheese, ice cream, modified milk powder, cream, other dairy products, etc.; fats and oils such as butter, margarines, vegetable oils, etc.; Basic seasonings such as soy sauce, miso, sauces, processed tomato seasonings, mirin, vinegar, etc.; complex seasonings and foods such as cooking mixes, curry bases, sauces, dressings, noodle soups, spices, and other complex seasonings; frozen foods such as frozen ingredients, semi-cooked frozen foods, and cooked frozen foods; sweets such as caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice snacks, bean snacks, dessert sweets, jellies, and other sweets; beverages such as carbonated drinks, natural fruit juice, fruit juice drinks, soft drinks with fruit juice, fruit pulp drinks, fruit drinks with fruit particles, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks, and other beverages; other commercially available foods such as baby food, furikake, and ochazuke nori; infant formula; enteral nutrition; functional foods (foods for specified health uses, foods with nutritional functions), etc.
　本発明の飲食品は、特に調製粉乳、調製液状乳等の調製乳とすることが好ましい。調製乳としては、乳児用調製乳、フォローアップ用調製乳、低出生体重児用調製乳、小児用調製乳、成人用調製乳、高齢者用調製乳、アレルギー用調製乳、乳糖不耐症用調製乳、先天代謝異常用調製乳等が挙げられる。特に乳児用調製乳、フォローアップ用調製乳、低出生体重児用調製乳、小児用調製乳に用いられることが好ましい。乳幼児用調製乳とは、０～１２か月の乳児を対象とする乳児用調製乳、６～９か月以降の乳児および年少幼児（３歳まで）を対象とするフォローアップミルク、出生時の体重が２５００ｇ未満の新生児（低出生体重児）を対象とする低出生体重児用調製乳、牛乳アレルギーや乳糖不耐症等の病的状態を有する児の治療に用いられる各種治療用ミルク等を指す。 The food and drink of the present invention is preferably prepared as a milk preparation, such as a powdered milk preparation or a liquid milk preparation. Examples of the milk preparation include infant formula, follow-up formula, low birth weight infant formula, pediatric formula, adult formula, elderly formula, allergy formula, lactose intolerance formula, and congenital metabolic disorder formula. In particular, the food and drink of the present invention is preferably used as an infant formula, follow-up formula, low birth weight infant formula, and pediatric formula. Infant formula refers to infant formula for infants aged 0-12 months, follow-up milk for infants aged 6-9 months or older and young children (up to 3 years), low birth weight infant formula for newborns weighing less than 2500g at birth (low birth weight infants), and various therapeutic milks used to treat children with pathological conditions such as cow's milk allergy and lactose intolerance.
　本発明の調製乳は健常者、非健常者を含むあらゆる対象に投与され得る。
　本発明の調製乳が健常者に投与される場合は、乳児用調製乳、フォローアップ用調製乳、低出生体重児用調製乳、小児用調製乳、成人用調製乳、高齢者用調製乳に用いられ、上記の非治療目的として使用され得る。 The formula of the present invention may be administered to any subject, including healthy and unhealthy subjects.
When the formula of the present invention is administered to healthy individuals, it can be used as an infant formula, a follow-up formula, a formula for low birth weight infants, a pediatric formula, an adult formula, or a formula for the elderly, and can be used for the non-therapeutic purposes described above.
　本発明の調製乳が非健常者に投与される場合は、アレルギー用調製乳、乳糖不耐症用調製乳、先天代謝異常用調製乳に用いられ、上記の治療目的として使用され得る。 When the formula of the present invention is administered to non-healthy individuals, it can be used as a formula for allergies, lactose intolerance, or congenital metabolic disorders, and can be used for the above-mentioned therapeutic purposes.
　飲食品の一態様として飼料とすることもできる。飼料としては、ペットフード、家畜飼料、養魚飼料等が挙げられる。飼料の形態としては特に制限されず、ペプチドQPEVMの他に例えば、トウモロコシ、小麦、大麦、ライ麦、マイロ等の穀類；大豆油粕、ナタネ油粕、ヤシ油粕、アマニ油粕等の植物性油粕類；フスマ、麦糠、米糠、脱脂米糠等の糠類；コーングルテンミール、コーンジャムミール等の製造粕類；魚粉、脱脂粉乳、ホエイ、イエローグリース、タロー等の動物性飼料類；トルラ酵母、ビール酵母等の酵母類；第三リン酸カルシウム、炭酸カルシウム等の鉱物質飼料；油脂類；単体アミノ酸；糖類等を含有するものであってよい。 As an embodiment of the food and beverage, the feed can be used. Examples of the feed include pet food, livestock feed, and fish feed. The form of the feed is not particularly limited, and may contain, in addition to the peptide QPEVM, for example, grains such as corn, wheat, barley, rye, and milo; vegetable oil cakes such as soybean oil cake, rapeseed oil cake, palm oil cake, and linseed oil cake; bran such as wheat bran, rice bran, and defatted rice bran; manufacturing residues such as corn gluten meal and corn jam meal; animal feeds such as fish meal, skimmed milk powder, whey, yellow grease, and tallow; yeasts such as torula yeast and brewer's yeast; mineral feeds such as calcium triphosphate and calcium carbonate; oils and fats; simple amino acids; and sugars.
　本発明の組成物が飲食品（飼料を含む）の態様である場合、抗酸化に関する用途、例えば酸化ストレスが関与する疲労、老化、生活習慣病等の予防等の用途が表示された飲食品として提供・販売されることが可能である。また、本明細書に係る本発明のペプチドは、これら飲食品等の製造のために使用可能である。 When the composition of the present invention is in the form of a food or beverage (including feed), it can be provided and sold as a food or beverage labeled with an antioxidant use, such as a use for preventing fatigue, aging, lifestyle-related diseases, etc., associated with oxidative stress. In addition, the peptide of the present invention described herein can be used for the production of such food or beverage.
　かかる「表示」行為には、需要者に対して前記用途を知らしめるための全ての行為が含まれ、前記用途を想起・類推させうるような表現であれば、表示の目的、表示の内容、表示する対象物・媒体等の如何に拘わらず、全て本発明における「表示」行為に該当する。
　また、「表示」は、需要者が上記用途を直接的に認識できるような表現により行われることが好ましい。具体的には、飲食品に係る商品または商品の包装に前記用途を記載したものを譲渡し、引き渡し、譲渡若しくは引き渡しのために展示し、輸入する行為、商品に関する広告、価格表若しくは取引書類に上記用途を記載して展示し、若しくは頒布し、またはこれらを内容とする情報に上記用途を記載して電磁気的（インターネット等）方法により提供する行為等が挙げられる。 Such "indication" acts include all acts aimed at informing consumers of the above-mentioned uses, and any expression that can recall or infer the above-mentioned uses falls under the category of "indication" acts in this invention, regardless of the purpose of the indication, the content of the indication, the object or medium on which it is displayed, etc.
Furthermore, it is preferable that the "labeling" be done in an expression that allows consumers to directly recognize the above-mentioned uses.Specific examples include the act of transferring, delivering, displaying for transfer or delivery, or importing food and beverage products or product packaging on which the above-mentioned uses are written, displaying or distributing advertisements, price lists, or transaction documents related to the products and writing the above-mentioned uses in information containing the above-mentioned uses and providing them by electromagnetic means (such as the Internet), etc.
　一方、表示内容としては、行政等によって認可された表示（例えば、行政が定める各種制度に基づいて認可を受け、そのような認可に基づいた態様で行う表示等）であることが好ましい。また、そのような表示内容を、包装、容器、カタログ、パンフレット、ＰＯＰ等の販売現場における宣伝材、その他の書類等へ付することが好ましい。 On the other hand, it is preferable that the content of the labeling be approved by the government (for example, labeling approved based on various systems established by the government and made in a manner based on such approval). It is also preferable that such content of the labeling be affixed to packaging, containers, catalogs, pamphlets, POP and other sales promotion materials, other documents, etc.
　また、「表示」には、健康食品、機能性食品、経腸栄養食品、特別用途食品、保健機能食品、特定保健用食品、栄養機能食品、機能性表示食品、医薬用部外品等としての表示も挙げられる。この中でも特に、消費者庁によって認可される表示、例えば、特定保健用食品、栄養機能食品、若しくは機能性表示食品に係る制度、またはこれらに類似する制度にて認可される表示等が挙げられる。具体的には、特定保健用食品としての表示、条件付き特定保健用食品としての表示、身体の構造や機能に影響を与える旨の表示、疾病リスク減少表示、科学的根拠に基づいた機能性の表示等を挙げることができ、より具体的には、健康増進法に規定する特別用途表示の許可等に関する内閣府令（平成二十一年八月三十一年内閣府令第五十七号）に定められた特定保健用食品としての表示（特に保健の用途の表示）およびこれに類する表示が典型的な例である。
　かかる表示としては、例えば、「酸化ストレスを緩和する」、「抗酸化力を向上させる」、「抗酸化機能をサポートする」等と表示することが挙げられる。 In addition, "labeling" also includes labeling as health food, functional food, enteral nutrition food, special purpose food, health functional food, food for specified health use, nutrient functional food, functional food, functional labeling food, medical drug, etc. Among these, in particular, labeling approved by the Consumer Affairs Agency, for example, labeling approved under the system related to food for specified health use, nutrient functional food, or functional labeling food, or a similar system, etc., can be mentioned. Specifically, labeling as food for specified health use, labeling as food for conditional specified health use, labeling that affects the structure or function of the body, labeling for reducing disease risk, labeling of functionality based on scientific evidence, etc., can be mentioned, and more specifically, labeling as food for specified health use (especially labeling of health use) and similar labeling as specified in the Cabinet Office Ordinance on the Permission of Labeling for Special Uses Prescribed in the Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009) are typical examples.
Examples of such claims include "alleviates oxidative stress,""improves antioxidant power,""supports antioxidant function," and the like.
　なお、前記＜ペプチド＞、＜カゼイン分解物＞、＜抗酸化用組成物＞の項において説明された事項は、本発明の飲食品の説明に全て適用され得る。 The matters explained in the above sections "Peptides", "Casein hydrolysate", and "Antioxidant composition" can all be applied to the description of the food and beverage of the present invention.
＜医薬品＞
　本発明の組成物は、医薬品の態様とすることもできる。
　すなわち、本発明の一態様は、本発明の組成物を含む、医薬品（以下、「本発明の医薬品」ということがある）に関する。本発明の医薬品は、ペプチドQPEVMを含有させること以外は、通常医薬品に用いられる原料を用いて通常の方法によって製造することができる。
　本発明の医薬品は、非健常者を対象に投与され、上記の治療目的として使用されうる。
　医薬品の投与経路は、経口または非経口のいずれでもよいが経口が好ましい。また、非経口摂取（投与）としては、経皮、静注、直腸投与、吸入等が挙げられる。
　医薬品の形態としては、投与方法に応じて、適宜所望の剤形に製剤化することができる。例えば、経口投与の場合、散剤、顆粒剤、錠剤、カプセル剤等の固形製剤；溶液剤、シロップ剤、懸濁剤、乳剤等の液剤等に製剤化することができる。また、非経口投与の場合、座剤、軟膏剤、注射剤等に製剤化することができる。
　製剤化に際しては、本発明のペプチドの他に、通常製剤化に用いられているpH調整剤、着色剤等の成分を用いることができる。また、他の薬効成分や、公知のまたは将来的に見出される抗酸化作用を有する成分や、他の医薬を併用することも可能である。
　加えて、製剤化は剤形に応じて適宜公知の方法により実施できる。製剤化に際しては、適宜、通常製剤化に用いる担体を配合して製剤化してもよい。かかる担体としては、賦形剤、結合剤、崩壊剤、滑沢剤、安定剤、矯味矯臭剤等が挙げられる。 <Drugs>
The composition of the present invention may also be in the form of a pharmaceutical product.
That is, one aspect of the present invention relates to a pharmaceutical product (hereinafter, sometimes referred to as "the pharmaceutical product of the present invention") that contains the composition of the present invention. The pharmaceutical product of the present invention can be manufactured by a conventional method using raw materials that are generally used for pharmaceutical products, except that it contains the peptide QPEVM.
The pharmaceutical agent of the present invention can be administered to non-healthy subjects and used for the above-mentioned therapeutic purposes.
The route of administration of pharmaceuticals may be either oral or parenteral, with oral being preferred. Parenteral intake (administration) includes transdermal, intravenous, rectal, inhalation, etc.
The pharmaceutical form can be formulated into a desired dosage form depending on the administration method.For example, in the case of oral administration, it can be formulated into solid preparations such as powders, granules, tablets, and capsules; liquid preparations such as solutions, syrups, suspensions, and emulsions.In addition, in the case of parenteral administration, it can be formulated into suppositories, ointments, injections, and the like.
In the formulation, in addition to the peptide of the present invention, components that are usually used in formulations, such as a pH adjuster, a colorant, etc., can be used. It is also possible to use other medicinal ingredients, known or future components with antioxidant activity, and other medicines in combination.
In addition, formulation can be carried out by a known method according to the dosage form. When formulating, a carrier usually used in formulation may be appropriately blended. Such carriers include excipients, binders, disintegrants, lubricants, stabilizers, flavorings, etc.
　前記賦形剤としては、例えば、乳糖、白糖、ブドウ糖、マンニット、ソルビット等の糖誘導体；トウモロコシデンプン、馬鈴薯デンプン、α-デンプン、デキストリン、カルボキシメチルデンプン等のデンプン誘導体；結晶セルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム等のセルロース誘導体；アラビアゴム；デキストラン；プルラン；軽質無水珪酸、合成珪酸アルミニウム、メタ珪酸アルミン酸マグネシウム等の珪酸塩誘導体；リン酸カルシウム等のリン酸塩誘導体；炭酸カルシウム等の炭酸塩誘導体；硫酸カルシウム等の硫酸塩誘導体等が挙げられる。 The excipients include, for example, sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, and calcium carboxymethyl cellulose; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; and sulfate derivatives such as calcium sulfate.
　前記結合剤としては、例えば、上記賦形剤の他、ゼラチン；ポリビニルピロリドン；マクロゴール等が挙げられる。 The binder may be, for example, gelatin, polyvinylpyrrolidone, macrogol, etc., in addition to the above excipients.
　前記崩壊剤としては、例えば、上記賦形剤の他、クロスカルメロースナトリウム、カルボキシメチルスターチナトリウム、架橋ポリビニルピロリドン等の化学修飾されたデンプンまたはセルロース誘導体等が挙げられる。 The disintegrants include, for example, the above-mentioned excipients, as well as chemically modified starch or cellulose derivatives such as croscarmellose sodium, sodium carboxymethyl starch, and cross-linked polyvinylpyrrolidone.
　前記滑沢剤としては、例えば、タルク；ステアリン酸；ステアリン酸カルシウム、ステアリン酸マグネシウム等のステアリン酸金属塩；コロイドシリカ；ビーガム、ゲイロウ等のワックス類；硼酸；グリコール；フマル酸、アジピン酸等のカルボン酸類；安息香酸ナトリウム等のカルボン酸ナトリウム塩；硫酸ナトリウム等の硫酸塩類；ロイシン；ラウリル硫酸ナトリウム、ラウリル硫酸マグネシウム等のラウリル硫酸塩；無水珪酸、珪酸水和物等の珪酸類；デンプン誘導体等が挙げられる。 The lubricants include, for example, talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as veegum and galangal; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylates such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; starch derivatives, etc.
　前記安定剤としては、例えば、メチルパラベン、プロピルパラベン等のパラオキシ安息香酸エステル類；クロロブタノール、ベンジルアルコール、フェニルエチルアルコール等のアルコール類；塩化ベンザルコニウム；無水酢酸；ソルビン酸等が挙げられる。 Examples of the stabilizer include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; and sorbic acid.
　前記矯味矯臭剤としては、例えば、甘味料、酸味料、香料等が挙げられる。 Examples of the flavoring agent include sweeteners, acidulants, and fragrances.
　なお、経口投与用の液剤の場合に使用する担体としては、水等の溶剤、矯味矯臭剤等が挙げられる。 In the case of liquid preparations for oral administration, examples of carriers to be used include solvents such as water, flavoring agents, etc.
　なお、前記＜ペプチド＞、＜カゼイン分解物＞、＜抗酸化用組成物＞の項において説明された事項は、本発明の医薬品の説明に全て適用され得る。 The matters explained in the above sections "Peptides", "Casein hydrolysate", and "Antioxidant composition" can all be applied to the explanation of the pharmaceutical product of the present invention.
　本発明の医薬品を摂取するタイミングは、例えば食前、食後、食間、就寝前等特に限定されない。 The timing of taking the pharmaceutical of the present invention is not particularly limited, and may be, for example, before or after meals, between meals, or before going to bed.
　以下、実施例により、本発明をさらに具体的に説明するが、本発明はその要旨を超えない限り、これらの実施例に限定されるものではない。 The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples as long as they do not depart from the gist of the invention.
（ペプチド組成物の調製）
　牛乳由来のカゼインナトリウム（Tatua Dairy Company社製）10gに水90gを加え、よく分散させ、水酸化ナトリウムを添加して溶液のpHを7.0に調整したうえでカゼインを完全に溶解し、濃度約10質量%のカゼイン水溶液を調製した。該カゼイン水溶液を85℃で10分間加熱殺菌し、50℃に温度調整し、スミチームLP（新日本化学工業社製）75000活性単位を添加して、7時間反応させた。その後、80℃で7分間加熱して酵素を失活させて酵素反応を停止し、10℃に冷却し、カゼイン加水分解物溶液を得た。該加水分解物溶液を濃縮後、凍結乾燥し、平均分子量350Da、遊離アミノ酸比率が15質量%のペプチド組成物Aを調製した。
　カゼインナトリウムを原料として、平均分子量、遊離アミノ酸比率が異なるペプチド組成物B（平均分子量1000Da、遊離アミノ酸比率<5質量%）およびC（平均分子量190Da、遊離アミノ酸比率>60質量%）を調製した。 Preparation of Peptide Compositions
90g of water was added to 10g of sodium caseinate (manufactured by Tatua Dairy Company) derived from milk, and the mixture was thoroughly dispersed. Sodium hydroxide was added to adjust the pH of the solution to 7.0, and then casein was completely dissolved to prepare an aqueous casein solution with a concentration of about 10% by mass. The aqueous casein solution was sterilized by heating at 85°C for 10 minutes, the temperature was adjusted to 50°C, and 75,000 activity units of Sumiteam LP (manufactured by Shin Nippon Chemical Industry Co., Ltd.) were added and reacted for 7 hours. The solution was then heated at 80°C for 7 minutes to inactivate the enzyme and stop the enzyme reaction, and cooled to 10°C to obtain a casein hydrolysate solution. The hydrolysate solution was concentrated and then freeze-dried to prepare peptide composition A with an average molecular weight of 350 Da and a free amino acid ratio of 15% by mass.
Using sodium caseinate as a raw material, peptide compositions B (average molecular weight 1000 Da, free amino acid ratio <5% by mass) and C (average molecular weight 190 Da, free amino acid ratio >60% by mass) with different average molecular weights and free amino acid ratios were prepared.
（抗酸化能の測定）
　ペプチド組成物A～Cを10 mg/mlとなるよう蒸留水で溶解させ、超音波処理の後、Millex-GV 0.22 μm PVDFフィルターに供し、透過液をサンプルとした。各サンプルの抗酸化活性を測定するため、H-ORAC測定キット（富士フイルム和光純薬株式会社）を用いて、キットのプロトコルに従ってORAC値を測定した。具体的には、以下のようにして測定を行った。 (Measurement of antioxidant capacity)
Peptide compositions A to C were dissolved in distilled water to a concentration of 10 mg/ml, and after ultrasonic treatment, the solution was passed through a Millex-GV 0.22 μm PVDF filter, and the permeate was used as a sample. To measure the antioxidant activity of each sample, the ORAC value was measured using an H-ORAC measurement kit (FUJIFILM Wako Pure Chemical Corporation) according to the kit's protocol. Specifically, the measurement was performed as follows.
（試薬の調製）
　Trolox（6-hyrodxy-2,5,7,8-tetoramethyl-chroman-2-carboxilic acid）をリン酸カリウム緩衝液に溶解してTrolox標準溶液とした。AAPH（2,2'-Azobis(2-amidinopropane)dihydrochloride）をリン酸カリウム緩衝液に溶解して活性酸素溶液とした（用時調製）。蛍光試薬（Sodium Fluorescein）をリン酸カリウム緩衝液に溶解して蛍光試薬溶液とした。 (Preparation of Reagents)
Trolox (6-hydroxy-2,5,7,8-tetoramethyl-chroman-2-carboxilic acid) was dissolved in potassium phosphate buffer to prepare a Trolox standard solution. AAPH (2,2'-Azobis(2-amidinopropane)dihydrochloride) was dissolved in potassium phosphate buffer to prepare an active oxygen solution (prepared just before use). Fluorescent reagent (sodium fluorescein) was dissolved in potassium phosphate buffer to prepare a fluorescent reagent solution.
（ORAC活性の測定）
　96wellプレートの各wellに、蛍光試液を115μL分注し、Trolox標準溶液又はサンプルを、各wellに35μLずつ添加した。また、バックグランドには、リン酸カリウム緩衝液を35μL添加した。SH-9000Lab（コロナ電気社）で蛍光強度（Excitation波長:485nm、Emission波長:535nm）を測定した。活性酸素溶液を各wellに50μL添加し、蛍光強度を2分ごとに90分間測定し、ORAC値を算出した。
　ORAC値はTrolox Equivalent（TE、トロロックス当量）にて評価した。
　結果を表１に示す。 (ORAC Activity Measurement)
115μL of the fluorescent test solution was dispensed into each well of a 96-well plate, and 35μL of Trolox standard solution or sample was added to each well. 35μL of potassium phosphate buffer was added as background. Fluorescence intensity (Excitation wavelength: 485nm, Emission wavelength: 535nm) was measured using SH-9000Lab (Corona Electric Co., Ltd.). 50μL of active oxygen solution was added to each well, and the fluorescence intensity was measured every 2 minutes for 90 minutes, and the ORAC value was calculated.
The ORAC value was evaluated using Trolox Equivalent (TE).
The results are shown in Table 1.
（抗酸化ペプチドの同定）
　ペプチド組成物A～Cのうち、ペプチド組成物Aに高いORAC値を示したため、ペプチド組成物Aに含まれる抗酸化ペプチドを同定した。ペプチド組成物AをCD-C18カラム（Cadenza社；250×10mm）を用いて逆相HPLCに供した。クロマトグラフィーは、溶媒（A液：0.1%トリフルオロ酢酸水溶液；B液：0.1%トリフルオロ酢酸アセトニトリル溶液）、濃度勾配（B液2%（0.0分）-B液25%（30.0分）-B液50%（40.0分）-B液80%（43.0分）-B液2%（53.1分）-STOP（60.1分））、流速3 ml/min、検出波長215nmの条件で行った。15秒ごとに溶出液を捕集し、遠心エバポレーターで濃縮乾固することで、分画サンプルを得た。同様に分画を繰り返し、強い抗酸化活性を示すピークを分取した。Q Exactive Orbitrap（Thermo Fisher Scientific社）を用いて当該ピークに含まれるペプチドを解析したところ、Gln-Pro-Glu-Val-Met（QPEVM）であることが確認された。
　なお、ペプチド組成物BおよびCにはいずれもGln-Pro-Glu-Val-Met（QPEVM）が含有されていないことが確認された。 (Identification of antioxidant peptides)
Among peptide compositions A to C, peptide composition A showed a high ORAC value, so the antioxidant peptide contained in peptide composition A was identified. Peptide composition A was subjected to reverse-phase HPLC using a CD-C18 column (Cadenza; 250 x 10 mm). Chromatography was performed under the following conditions: solvent (liquid A: 0.1% trifluoroacetic acid aqueous solution; liquid B: 0.1% trifluoroacetic acid acetonitrile solution), concentration gradient (liquid B 2% (0.0 min) - liquid B 25% (30.0 min) - liquid B 50% (40.0 min) - liquid B 80% (43.0 min) - liquid B 2% (53.1 min) - STOP (60.1 min)), flow rate 3 ml/min, detection wavelength 215 nm. The eluate was collected every 15 seconds and concentrated to dryness using a centrifugal evaporator to obtain a fractionated sample. Fractionation was repeated in the same manner, and the peak showing strong antioxidant activity was separated. The peptide contained in this peak was analyzed using a Q Exactive Orbitrap (Thermo Fisher Scientific) and was confirmed to be Gln-Pro-Glu-Val-Met (QPEVM).
It was confirmed that neither peptide composition B nor peptide composition C contained Gln-Pro-Glu-Val-Met (QPEVM).
（合成ペプチドによる抗酸化活性確認）
　固相合成法によりペプチドQPEVMを得た。QPEVMを1 mg/mlとなるよう蒸留水で溶解し、超音波処理に供した後、当該ペプチド溶液の抗酸化活性をH-ORAC測定キット（富士フイルム和光純薬株式会社）を用いて測定した。その結果、QPEVMのORAC値は1.457 μmol TE/molであった。 (Confirmation of antioxidant activity using synthetic peptides)
Peptide QPEVM was obtained by solid-phase synthesis. QPEVM was dissolved in distilled water to a concentration of 1 mg/ml and subjected to ultrasonic treatment. The antioxidant activity of the peptide solution was measured using an H-ORAC measurement kit (FUJIFILM Wako Pure Chemical Corporation). As a result, the ORAC value of QPEVM was 1.457 μmol TE/mol.
　結果を表１に示す。 The results are shown in Table 1.
　以上のとおり、カゼイン由来のペプチドQPEVMが抗酸化作用を有することが明らかとなった。また、同ペプチドを含有する組成物も抗酸化作用を有することが明らかとなった。 As described above, it has become clear that the casein-derived peptide QPEVM has antioxidant properties. It has also become clear that a composition containing the same peptide also has antioxidant properties.
［製造例１］
　本発明のカゼイン分解物を含む調製粉乳の製造方法を下記に示す。 [Production Example 1]
The method for producing powdered milk containing the casein hydrolysate of the present invention is described below.
　上記実施例の方法に準じて調製したペプチド組成物Aを6kg、脱塩牛乳乳清蛋白質粉末（ミライ社製）10kg、ラクトース（ミライ社製）48kg、ミネラル混合物（富田製薬社製）920g、ビタミン混合物（田辺製薬社製）32g、及びラクチュロース（森永乳業社製）500gを温水300kgに溶解し、更に90℃で10分間加熱溶解し、調製脂肪（太陽油脂社製）28kgを添加して均質化する。その後、殺菌、濃縮工程を行って、噴霧乾燥し、調製粉乳約95kgを調製する。このようにして得られた調製粉乳を対象に投与することにより、対象における抗酸化効果が期待できる。また、得られる調製粉乳の酸化による風味劣化、色調変化の防止効果が期待できる。 6 kg of peptide composition A prepared according to the method of the above example, 10 kg of desalted milk whey protein powder (Mirai Co., Ltd.), 48 kg of lactose (Mirai Co., Ltd.), 920 g of mineral mixture (Tomita Pharmaceutical Co., Ltd.), 32 g of vitamin mixture (Tanabe Pharmaceutical Co., Ltd.), and 500 g of lactulose (Morinaga Milk Industry Co., Ltd.) are dissolved in 300 kg of warm water, and further heated and dissolved at 90°C for 10 minutes, and 28 kg of prepared fat (Taiyo Yushi Co., Ltd.) is added and homogenized. After that, sterilization and concentration processes are performed, and the mixture is spray-dried to prepare about 95 kg of prepared powdered milk. By administering the prepared powdered milk obtained in this way to a subject, an antioxidant effect in the subject can be expected. In addition, an effect of preventing flavor deterioration and color change due to oxidation of the obtained prepared powdered milk can be expected.
［製造例２］
　本発明のカゼイン分解物を含む飲料の製造方法を下記に示す。 [Production Example 2]
A method for producing a beverage containing the casein hydrolysate of the present invention is described below.
　上記実施例の方法に準じて調製したペプチド組成物A 40gを360gの精製水に溶解し、クエン酸でpHを3.6に調整してペプチド組成物溶液を得る。当該ペプチド組成物溶液、砂糖110g、乳酸カルシウム2g、乳化剤1g、及び大豆多糖類10gを添加し、精製水を加えて全量を0.8Lとし、80℃で加熱後、冷却して酸性飲料を得る。このようにして得られた飲料を対象に投与することにより、対象における抗酸化効果が期待できる。また、得られる飲料の酸化による風味劣化、色調変化の防止効果が期待できる。 40 g of peptide composition A prepared according to the method of the above Example is dissolved in 360 g of purified water, and the pH is adjusted to 3.6 with citric acid to obtain a peptide composition solution. The peptide composition solution, 110 g of sugar, 2 g of calcium lactate, 1 g of emulsifier, and 10 g of soybean polysaccharides are added, and purified water is added to make the total volume 0.8 L. The mixture is heated to 80°C and then cooled to obtain an acidic beverage. By administering the beverage obtained in this manner to a subject, an antioxidant effect in the subject can be expected. In addition, the beverage obtained can be expected to have an effect of preventing deterioration in flavor and color change due to oxidation.
［製造例３］
　本発明のカゼイン分解物を含む医薬品の製造方法を下記に示す。 [Production Example 3]
The method for producing a pharmaceutical containing the casein hydrolysate of the present invention is described below.
　上記実施例の方法に準じて調製したペプチド組成物A、及び結晶セルロースを撹拌造粒機に投入し混合する。その後、精製水を加え造粒し、造粒物を乾燥し造粒物（医薬品組成物）を得る。このようにして得られた医薬品を対象に投与することにより、対象における抗酸化効果が期待できる。また、得られる医薬品の酸化による劣化、色調変化の防止効果が期待できる。 Peptide composition A prepared according to the method of the above example and crystalline cellulose are placed in an agitator granulator and mixed. Purified water is then added to granulate, and the granules are dried to obtain a granule (pharmaceutical composition). By administering the pharmaceutical product obtained in this manner to a subject, an antioxidant effect in the subject can be expected. In addition, the obtained pharmaceutical product can be expected to have an effect of preventing deterioration and color change due to oxidation.
　本発明は、飲食品、健康食品、機能性食品、サプリメント、医薬品等の分野で有用である。 The present invention is useful in the fields of food and beverages, health foods, functional foods, supplements, pharmaceuticals, etc.
＜配列表の説明＞
配列番号１：本発明のペプチドのアミノ酸配列 <Explanation of sequence listing>
SEQ ID NO: 1: Amino acid sequence of the peptide of the invention

Claims (5)

  1. 　アミノ酸配列Gln-Pro-Glu-Val-Metを含むペプチド。 A peptide containing the amino acid sequence Gln-Pro-Glu-Val-Met.
  2. 　請求項１に記載のペプチドを含有する、カゼイン分解物。 A casein hydrolysate containing the peptide described in claim 1.
  3. 　請求項１に記載のペプチド、または請求項２に記載のカゼイン分解物を含有する、抗酸化用組成物。 An antioxidant composition containing the peptide described in claim 1 or the casein hydrolysate described in claim 2.
  4. 　請求項３に記載の組成物を含む、飲食品。 A food or drink comprising the composition described in claim 3.
  5. 　請求項３に記載の組成物を含む、医薬品。 A pharmaceutical comprising the composition according to claim 3.
PCT/JP2023/035715 2022-09-30 2023-09-29 Novel peptide, and antioxidant composition WO2024071404A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0641191A (en) * 1992-03-04 1994-02-15 Calpis Food Ind Co Ltd:The Peptide and physiological activator containing the same
JP2013005763A (en) * 2011-06-24 2013-01-10 Calpis Co Ltd Enzymatic production method for brain-function-improving peptide
CN106554387A (en) * 2015-09-25 2017-04-05 中国科学院大连化学物理研究所 Nonapeptide with ACE and DPP-IV dual restraining activities and its application

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0641191A (en) * 1992-03-04 1994-02-15 Calpis Food Ind Co Ltd:The Peptide and physiological activator containing the same
JP2013005763A (en) * 2011-06-24 2013-01-10 Calpis Co Ltd Enzymatic production method for brain-function-improving peptide
CN106554387A (en) * 2015-09-25 2017-04-05 中国科学院大连化学物理研究所 Nonapeptide with ACE and DPP-IV dual restraining activities and its application

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FLORIAN BAUM: "Analysis of the Endogenous Peptide Profile of Milk: Identification of 248 Mainly Casein-Derived Peptides", JOURNAL OF PROTEOME RESEARCH, AMERICAN CHEMICAL SOCIETY, vol. 12, no. 12, 6 December 2013 (2013-12-06), pages 5447 - 5462, XP093154737, ISSN: 1535-3893, DOI: 10.1021/pr4003273 *

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