WO2024071404A1 - Nouveau peptide et composition antioxydante - Google Patents

Nouveau peptide et composition antioxydante 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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English (en)
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/fr

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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

La présente invention aborde le problème de la fourniture d'un peptide antioxydant applicable aux aliments et d'une composition antioxydante le contenant. La présente invention concerne un peptide comprenant la séquence d'acides aminés Gln-Pro-Glu-Val-Met et une composition antioxydante contenant ledit peptide.
PCT/JP2023/035715 2022-09-30 2023-09-29 Nouveau peptide et composition antioxydante WO2024071404A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0641191A (ja) * 1992-03-04 1994-02-15 Calpis Food Ind Co Ltd:The ペプチド及びこれを含む生理活性剤
JP2013005763A (ja) * 2011-06-24 2013-01-10 Calpis Co Ltd 脳機能改善用ペプチドの酵素的製造方法
CN106554387A (zh) * 2015-09-25 2017-04-05 中国科学院大连化学物理研究所 具有ace和dpp-iv双重抑制活性的九肽及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0641191A (ja) * 1992-03-04 1994-02-15 Calpis Food Ind Co Ltd:The ペプチド及びこれを含む生理活性剤
JP2013005763A (ja) * 2011-06-24 2013-01-10 Calpis Co Ltd 脳機能改善用ペプチドの酵素的製造方法
CN106554387A (zh) * 2015-09-25 2017-04-05 中国科学院大连化学物理研究所 具有ace和dpp-iv双重抑制活性的九肽及其应用

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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