Peptide and composition

Abstract

The present invention provides: a peptide having excellent heat resistance while having a function for treating, preventing, or ameliorating mood disorders; and a composition comprising said peptide. The present invention pertains to: a peptide comprising an amino acid sequence that shares a sequence identity of at least 90% with the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2; the aforementioned peptide used to treat, prevent, or ameliorate mood disorders; a composition comprising one among the peptides; the aforementioned composition which is a food and drink or a pharmaceutical product; and the aforementioned composition, wherein the food and drink is a baked food, a retort food, a beverage, or a jelly.

Description

Peptides and Compositions

[0001] The present disclosure relates to peptides and compositions.

[0002] Reflecting the stressful nature of today's society, the increase in mood disorders, such as tension, anxiety, loss of motivation, and depression, has become a problem. Anxiety, one of the causes of mood disorders, is essentially necessary as a warning to avoid danger in the body, but excessive anxiety is involved in the onset of mood disorders and the progression of symptoms, and there is a need for the development of foods and medicines that can alleviate anxiety.

[0003] For example, Patent Document 1 describes that a specific dipeptide is suitable as an anti-anxiety drug, etc. Patent Document 2 describes that among oligopeptides derived from rice endosperm protein (REP) contained in a rice endosperm protein hydrolysate, a peptide having a specific amino acid sequence is a peptide that can treat, prevent, or ameliorate mood disorders.

[0004] International Publication No. WO 2013 / 129220 International Publication No. WO 2019 / 216307

[0005] In general, peptides have low heat resistance and are generally stored refrigerated or frozen, and are known to lose their functionality at room temperature or under heated conditions. On the other hand, many foods and beverages are cooked using various methods, such as baking, boiling, steaming, and frying. Furthermore, to avoid the risk of spoilage and microbial contamination, many products are heat-sterilized before being distributed to the market. For this reason, many peptides used as ingredients in foods and beverages are decomposed by heat treatment during the manufacturing process, resulting in the inability to fully demonstrate their functionality.

[0006] The problem to be solved by the embodiments of the present disclosure is to provide a peptide that has excellent heat resistance while having the function of treating, preventing, or ameliorating mood disorders, and a composition containing the peptide.

[0007] Specific means for solving the above problems include the following embodiments. [1] A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. [2] A peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. [3] The peptide according to [1] or [2] above, which is used for treating, preventing, or ameliorating mood disorders. [4] The peptide according to [1] or [2] above, which has a survival rate of 90% or more after heat treatment at 85°C. [5] The peptide according to [1] or [2] above, which has a survival rate of 50% or more after heat treatment at 120°C. [6] The peptide according to [3] above, which has a survival rate of 90% or more after heat treatment at 85°C. [7] The peptide according to [3] above, which has a survival rate of 50% or more after heat treatment at 120°C. [8] A composition comprising the peptide according to any one of [1] to [7] above. [9] The composition according to [8] above, which is a food or drink or a pharmaceutical product.

[10] The composition according to [9], wherein the food or drink is a baked food, a retort food, a beverage, or a jelly.

[11] A food additive for cooking with heat, the main component of which is the peptide according to any one of [1] to [7].

[12] A pharmaceutical additive, the main component of which is the peptide according to any one of [1] to [7].

[0008] According to an embodiment of the present disclosure, there are provided a peptide having excellent heat resistance while having the function of treating, preventing, or ameliorating a mood disorder, and a composition containing the peptide.

[0009] The peptides and compositions according to the present disclosure will be described in detail below. The following description of the requirements may be based on representative embodiments of the present disclosure, but the present disclosure is not limited to such embodiments and can be implemented with appropriate modifications within the scope of the present disclosure.

[0010] In the present disclosure, the term "mood disorder" refers to a state in which a person feels distressed or experiences some kind of disruption in daily life due to a change in mood or emotion. Specifically, the term "mood disorder" refers to one or more of tension, anxiety, loss of motivation, depression, depressive symptoms, and symptoms associated with these.

[0011] In the present disclosure, "treatment" means, for example, delaying the progression of a mood disorder and curing symptoms. "Prevention" means, for example, suppressing or delaying the onset of a mood disorder. "Amelioration" means, for example, alleviating or reducing symptoms of a mood disorder.

[0012] In the present disclosure, a "peptide consisting of the amino acid sequence represented by SEQ ID NO: N (N is an integer of 1 or more)" may be referred to as "peptide N." Hereinafter, the amino acid sequence will be written from the N-terminus to the C-terminus, with the N-terminus at the left end.

[0013] In the present disclosure, the term "major component" refers to the component that is contained in the largest amount.

[0014] In the present disclosure, a numerical range indicated using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits, respectively. In the numerical ranges described in stages in the present disclosure, the upper or lower limit described in a certain numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Furthermore, in the numerical ranges described in the present disclosure, the upper or lower limit described in a certain numerical range may be replaced with a value shown in the examples.

[0015] <Peptide> The peptide according to the present disclosure consists of an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. SEQ ID NO: 1: LPEGQSQSQK SEQ ID NO: 2: pEQFLPEGQSQSQK (In SEQ ID NO: 2, pE represents a pyroglutamic acid residue.)

[0016] The peptide according to the present disclosure is particularly preferably a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. Hereinafter, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 may be referred to as "peptide 1," and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 may be referred to as "peptide O1."

[0017] From the viewpoint of improving heat resistance, the peptide according to the present disclosure preferably has a survival rate of 90% or more after heat treatment at 85°C, more preferably 95% or more, and even more preferably 98% or more.

[0018] (Procedure for measuring the residual rate after heat treatment at 85°C) A peptide solution is prepared by mixing the peptide to be measured with distilled water. At this time, the peptide concentration in the peptide solution is adjusted to 10 ng / mL, and 5 mL of the peptide solution is prepared. The peptide solution is left to stand in a dry bath at 85°C for 30 minutes for heat treatment. A measurement sample is prepared from the heat-treated peptide solution using the procedure described below in "Pretreatment". The peak area derived from the precursor ion is measured in SIM mode using an LC-MS system (LC conditions and MS conditions are shown below) having a liquid chromatograph in the separation section and a high-resolution mass spectrometer in the detection section, and this is the measured value after heat treatment. The peak area derived from the precursor ion is measured using the same procedure as above, except that heat treatment is not performed, and this is the measured value before heat treatment. The percentage of the measured value after heat treatment, assuming the measured value before heat treatment to be 100 (i.e., {(measured value after heat treatment) / (measured value before heat treatment)} × 100), is calculated, and this value is the residual rate (unit: %).

[0019] - Pretreatment - The peptide solution was centrifuged at 15,000 rpm for 15 minutes to collect the supernatant, which was then diluted 10-fold (by mass) with ultrapure water to obtain a dispersion. The dispersion was then filtered through a syringe filter (Mycles (registered trademark) SLLHH04NL 0.45 μm / 4 mm) to clarify it and use it as a measurement sample.

[0020] -LC Conditions- Apparatus: Vanquish UHPLC System Column: Thermo Fischer Scientific Acclaim 120 C18 3 μm 120 Å (2.1 × 100 mm) Column temperature: 40°C Autosampler temperature: 4°C Injection volume: 10 μL Mobile phase: (A) Ultrapure water with 0.1% formic acid (B) Acetonitrile gradient with 0.1% formic acid: (B) changed from 2% to 35% (0 min to 10 min), (B) changed from 35% to 95% (10 min to 11 min), (B) kept constant at 95% (11 min to 13 min), (B) changed from 95% to 2% (13 min to 13.5 min), (B) changed from 2% to 2% (13.5 min to 20 min) Flow rate: 300 μL / min

[0021] -MS Conditions- Apparatus: OrbiTrap Exploris 120 (Thermo Fischer Scientific) Ion source type: H-ESI Spray pressure: 4500 V Sheath gas: 40 Aux gas: 10 Ion transfer tube temperature: 300°C Vaporizer temperature: 400°C Resolution: 60000 Polarity: positive

[0022] From the viewpoint of improving heat resistance, the peptide according to the present disclosure preferably has a residual rate of 40% or more, more preferably 50% or more, and even more preferably 60% or more after heat treatment at 120°C. The upper limit of the residual rate of the peptide according to the present disclosure after heat treatment at 120°C is not particularly limited, but may be 90% or less, 80% or less, or 70% or less. The residual rate of the peptide according to the present disclosure after heat treatment at 120°C may be 40% or more and 90% or less, 50% or more and 80% or less, or 60% or more and 70% or less.

[0023] (Procedure for measuring the residual rate after heat treatment at 120°C) The measurement was carried out using the same procedure as described above (Procedure for measuring the residual rate after heat treatment at 85°C), except that the heat treatment conditions were changed to 15 minutes in an autoclave with an internal temperature of 120°C.

[0024] The peptide according to the present disclosure may be a peptide consisting of an amino acid sequence in which one amino acid residue has been added, deleted, or substituted in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 (hereinafter, such peptides may also be simply referred to as "sequence-altered peptides"). Sequence-altered peptides include peptides consisting of an amino acid sequence in which one amino acid residue has been added, deleted, or substituted in a region other than the C-terminal six amino acids (i.e., QSQSQK) of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. The peptide according to the present disclosure is preferably a peptide in which the amino acid sequence in the region other than the C-terminal six amino acids (i.e., QSQSQK) is conserved and which consists of an amino acid sequence that has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

[0025] Specific examples of peptides according to the present disclosure include the peptides shown in Table 1.

[0026]

[0027] From the viewpoint of heat resistance and the effect of treating, preventing, or ameliorating mood disorders, the peptide according to the present disclosure is preferably a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

[0028] The peptides according to the present disclosure are preferably used for the treatment, prevention, or amelioration of mood disorders. As shown in the Examples below, it has been shown that ingesting the peptides according to the present disclosure can treat, prevent, or ameliorate mood disorders. Therefore, the peptides according to the present disclosure can be preferably used for the treatment, prevention, or amelioration of mood disorders, and are particularly preferably used for the treatment, prevention, or amelioration of one or more symptoms selected from the group consisting of tension, anxiety, loss of motivation, depression, depressive mood disorders, and symptoms associated therewith.

[0029] Peptides according to the present disclosure can be obtained by chemical synthesis or by hydrolysis of naturally occurring proteins or polypeptides.

[0030] Examples of chemical synthesis methods include known peptide synthesis methods. Specific examples include liquid phase and solid phase methods, which are methods commonly used in peptide synthesis. More specific examples include the Fmoc method and the Boc method. The synthesized peptide may be purified. Examples of purification methods include methods using ion exchange chromatography, reverse phase liquid chromatography, affinity chromatography, etc.

[0031] The natural protein or polypeptide to be hydrolyzed is preferably a protein of a food material, and preferably rice protein or a hydrolyzate thereof. The rice variety used as the raw material is not particularly limited and may be japonica (Oryza sativa subsp. japonica), indica (Oryza sativa subsp. indica), or javanica (Oryza sativa subsp. javanica). Furthermore, rice proteins from various varieties of japonica rice, such as Nipponbare, Koshihikari, and Sasanishiki, can be used. Examples of hydrolysis methods include methods using hydrolases and methods using strong acids or strong bases.

[0032] In the method using hydrolases, hydrolases derived from animals, plants, or microorganisms (trypsin, chymotrypsin, papain, pepsin, carboxypeptidase, thermolysin, etc.) can be used. Microorganisms that can be used as food (e.g., edible yeasts such as baker's yeast and brewer's yeast) may also be used as hydrolases. The conditions for hydrolysis using hydrolases are not particularly limited, but the pH may be adjusted to an appropriate value depending on the enzyme used, and the reaction may be carried out at a temperature of approximately 30°C to 60°C for 30 minutes to 48 hours. The resulting hydrolysate (hydrolysis reaction product) contains the peptide according to the present disclosure. The peptide according to the present disclosure can be purified from the hydrolysate, and the resulting purified product can be used as the peptide according to the present disclosure. Furthermore, when the subject of hydrolysis is a food material, the resulting hydrolysate may be used directly as a composition according to the present disclosure.

[0033] In the method using a strong acid, for example, hydrochloric acid, nitric acid, sulfuric acid, etc. can be used. In the method using a strong base, for example, alkali metal hydroxides (sodium hydroxide, potassium hydroxide, lithium hydroxide, etc.), alkali metal carbonates (sodium carbonate, potassium carbonate, etc.), and alkali metal bicarbonates (sodium bicarbonate, potassium bicarbonate, etc.) can be used. The conditions for hydrolysis using a strong acid or strong base are not particularly limited, but the reaction may be carried out in water in the presence of a strong acid or a strong base at a temperature of 1°C to 100°C for 30 minutes to 48 hours. The obtained hydrolysate contains the peptide according to the present disclosure. The hydrolysate can be purified, and the obtained purified product can be used as the peptide according to the present disclosure. Alternatively, the hydrolysate may be used as is as a composition according to the present disclosure after adjusting the pH.

[0034] The amino acid sequences of peptides obtained by various methods can be analyzed by a protein sequencer that reads the amino acid sequence from the C-terminus by the Edman degradation method, LC-MS, or the like.

[0035] <Composition> The composition according to the present disclosure may contain only one type of peptide according to the present disclosure, or may contain two or more types of peptides according to the present disclosure. The composition according to the present disclosure preferably contains at least Peptide 1, and more preferably contains Peptide 1 and Peptide O1.

[0036] The composition according to the present disclosure may contain components other than the peptide according to the present disclosure. For example, when the peptide according to the present disclosure is a peptide prepared by degradation of an edible protein such as rice protein, the resulting degradation product is a mixture containing the peptide according to the present disclosure and other peptides. The degradation product can be used as is as the composition according to the present disclosure. When the composition according to the present disclosure contains a peptide other than the peptide according to the present disclosure, it is more preferable that the composition according to the present disclosure contain peptide 1, peptide O1, and peptide O2 (amino acid sequence of SEQ ID NO: 3: QQFLPEGQSQSQK) from the viewpoint of improving heat resistance.

[0037] The composition according to the present disclosure may be a rice protein (specifically, rice endosperm protein) hydrolysate. The rice protein is preferably hydrolyzed using a hydrolase. The hydrolysate obtained by this method contains at least Peptide 1, Peptide O1, and Peptide O2.

[0038] The content (mass ratio) of the peptide according to the present disclosure relative to the total amount of the composition according to the present disclosure is not particularly limited.When the peptide according to the present disclosure contained is a purified product, the content (mass ratio) of the peptide according to the present disclosure relative to the total amount of the composition according to the present disclosure is preferably 50% by mass or more, more preferably 70% by mass or more, even more preferably 80% by mass or more, and particularly preferably 90% by mass or more.When the peptide according to the present disclosure contained is an unpurified product such as a proteolysis product, the content (mass ratio) of the peptide according to the present disclosure relative to the total amount of the composition according to the present disclosure is preferably 0.01% by mass or more, more preferably 0.1% by mass or more, even more preferably 1% by mass or more, and even more preferably 10% by mass or more.

[0039] When the composition of the present disclosure contains peptide 1, peptide O1, and peptide O2, the total mass of peptide 1, peptide O1, and peptide O2 relative to the volume of the composition is preferably 10 μg / mL or more and 2000 μg / mL or less, more preferably 50 μg / mL or more and 1000 μg / mL or less, even more preferably 70 μg / mL or more and 500 μg / mL or less, and particularly preferably 100 μg / mL or more and 200 μg / mL or less.

[0040] When a composition according to the present disclosure comprises peptide 1, peptide O1, and peptide O2, the mass of peptide 1 relative to the total mass of peptide 1, peptide O1, and peptide O2 is preferably 30% by mass or more and 95% by mass or less, more preferably 40% by mass or more and 90% by mass or less, and even more preferably 50% by mass or more and 85% by mass or less. When a composition according to the present disclosure comprises peptide 1, peptide O1, and peptide O2, the mass of peptide O1 relative to the total mass of peptide 1, peptide O1, and peptide O2 is preferably 1% by mass or more and 50% by mass or less, more preferably 5% by mass or more and 40% by mass or less, and even more preferably 8% by mass or more and 30% by mass or less. When a composition according to the present disclosure comprises peptide 1, peptide O1, and peptide O2, the mass of peptide O2 relative to the total mass of peptide 1, peptide O1, and peptide O2 is preferably 1% by mass or more and 30% by mass or less, more preferably 5% by mass or more and 25% by mass or less, and even more preferably 8% by mass or more and 20% by mass or less.

[0041] The composition according to the present disclosure is preferably a food or beverage product or a pharmaceutical product. The peptide according to the present disclosure is a peptide with excellent heat resistance. Therefore, the composition according to the present disclosure is highly suitable for applications requiring heat treatment or heat sterilization (specifically, food or beverage products or pharmaceutical products). In the case of peptides with low heat resistance, heat treatment or heat sterilization decomposes much of the incorporated peptide, resulting in a low residual rate and a reduced amount of active ingredient in the resulting composition. In contrast, the composition according to the present disclosure contains a peptide with excellent heat resistance, and therefore, even if heat treatment or heat sterilization is performed during the manufacturing process, a composition with a high residual rate of active ingredient after manufacture can be produced. Therefore, the peptide according to the present disclosure is suitable, for example, as a food additive for heat cooking (an additive incorporated into a food or beverage product obtained by heat cooking) and as a main component of a pharmaceutical additive for which heat sterilization is important.

[0042] The heat treatment is not particularly limited and may be any of various common heat cooking methods such as baking, boiling, steaming, frying, dissolving, etc. Furthermore, the heat sterilization treatment may be any of retort sterilization (high-temperature, high-pressure sterilization) treatment, heat sterilization treatment at 60°C or higher, etc. The composition according to the present disclosure has high resistance to common heat cooking and is therefore suitable for use as a food or beverage.

[0043] The composition according to the present disclosure can be produced by a conventional method, except that the peptide according to the present disclosure is used as a raw material. The composition may be a food or drink, or a pharmaceutical product. In particular, the composition according to the present disclosure is preferably produced by subjecting a mixture containing the peptide according to the present disclosure to a heat treatment or heat sterilization treatment.

[0044] When the composition according to the present disclosure is a pharmaceutical product, the peptide according to the present disclosure can be prepared together with a carrier, diluent, or excipient as one of the following formulations: tablets (plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, etc.), capsules, troches, powders, fine granules, granules, liquids, suspensions, emulsions, pastes, creams, injections (including when incorporated into infusions such as amino acid infusions and electrolyte infusions), enteric-coated tablets, capsules, sustained-release formulations, etc.

[0045] The carrier, diluent, or excipient may be a substance commonly used in the pharmaceutical field that does not react with the peptide of the present disclosure, such as lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminum metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange resin, methylcellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, Veegum, titanium oxide, sorbitan fatty acid esters, sodium lauryl sulfate, glycerin, fatty acid glycerin esters, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white petrolatum, fluorocarbon, nonionic surfactant, propylene glycol, water, etc.

[0046] Tablets are preferred as the dosage form of pharmaceuticals containing the peptides according to the present disclosure, as they can fully exhibit the excellent heat resistance of the peptides according to the present disclosure. Tablets may have high internal temperatures due to the pressure applied during tableting, which may result in a low residual rate of active ingredients with low heat resistance. However, the peptides according to the present disclosure make it easier to obtain tablets with high residual rates.

[0047] When the composition according to the present disclosure is a food or drink, it can be prepared in any form, and examples thereof include the following: baked foods (biscuits, cookies, crackers, sables, florentines, galettes, langue de chat, biscotti, shortbread, madeleines, financiers, pound cake, bread, scones, jellies (e.g., jelly, agar, gummies, marshmallows, yokan, jelly drinks, etc.), retort foods, beverages (e.g., coffee, cocoa, juice, soft drinks, mineral drinks, tea drinks, green tea, black tea, oolong tea, dairy drinks, lactic acid bacteria drinks, yogurt drinks, carbonated drinks, etc.), gum, candy, frozen desserts (e.g., ice cream, popsicles, sorbet, shaved ice, etc.), and the like.

[0048] As a composition according to the present disclosure, preferred are foods and beverages produced under relatively severe heating conditions. Examples of such foods and beverages include baked foods, gel foods, and retort foods. For example, baked foods containing peptides according to the present disclosure can be obtained by baking dough kneaded with peptides according to the present disclosure in an oven or the like. Furthermore, gel foods can be produced by mixing peptides according to the present disclosure with other ingredients in a solution obtained by heating and melting a gelling agent such as agar or gelatin, followed by cooling. Other preferred compositions containing peptides according to the present disclosure are beverages that are subjected to heat sterilization, and in particular, neutral soft drinks (pH 5.0 to 7.6) that require relatively strong heat sterilization are preferred.

[0049] Compositions containing the peptides disclosed herein may be prepared as so-called health foods, functional foods, nutritional supplements, supplements, foods for specified health uses, foods with functional claims, foods for the sick, combined foods for the sick (a type of special-purpose food listed by the Ministry of Health, Labour and Welfare), or foods for the elderly (a type of special-purpose food listed by the Ministry of Health, Labour and Welfare).

[0050] The amount of the peptide according to the present disclosure in a composition containing the peptide according to the present disclosure can be appropriately set depending on the effect to be obtained, etc. For example, the peptide according to the present disclosure may be blended in an amount of preferably 0.01% by mass or more, more preferably 1.00% by mass or more, relative to the composition. Alternatively, the peptide according to the present disclosure may be blended in an amount of preferably 100% by mass or less, more preferably 90% by mass or less, relative to the composition.

[0051] The amount of components other than the peptide according to the present disclosure in a composition containing the peptide according to the present disclosure can be appropriately set depending on the type of the component, the form of the composition, the effect to be obtained, etc.

[0052] The method of administration of a composition containing a peptide according to the present disclosure is not particularly limited, and may be either oral administration or parenteral administration (injection, etc.). From the viewpoint of easily achieving the effects of the present disclosure, it is preferable that the composition containing a peptide according to the present disclosure is administered orally.

[0053] The dosage of the composition according to the present disclosure varies depending on the administration method, the condition and age of the subject, etc., but is, for example, preferably 0.01 mg / kg to 500 mg / kg, more preferably 0.05 mg / kg to 100 mg / kg, and even more preferably 0.1 mg / kg to 30 mg / kg per day for an adult, converted into the amount of the peptide according to the present disclosure. Within the above range, the higher the dosage, the more likely the effects of the present disclosure are to be achieved.

[0054] By administering and ingesting the composition according to the present disclosure to a subject animal, mood disorders in the subject animal can be treated, prevented, or improved. The administration method can be appropriately selected depending on the form of the composition. The number of administrations, administration interval, and dosage can be appropriately selected depending on the condition (symptoms, age, body weight, etc.) of the subject.

[0055] There are no particular limitations on the subjects of administration, and examples include humans and non-human mammals (dogs, cats, livestock (cows, pigs, sheep, goats, etc.)).

[0056] Next, the peptides and compositions of the present disclosure will be described in more detail with reference to examples, but the peptides and compositions of the present disclosure are not limited to the following examples.

[0057] Hereinafter, unless otherwise specified, "%" means "% by mass." In the following examples, the residual rates after heat treatment were measured using the same procedures as those described above (Procedure for measuring residual rates after heat treatment at 85°C) and (Procedure for measuring residual rates after heat treatment at 120°C). The precursor ions (m / z) were 551.2803 for peptide 1, 353.1799 for peptide C, 744.3597 for peptide O1, and 752.8732 for peptide O2.

[0058] Synthesis Example 1 Peptide 1 (LPEGQSQSQK), peptide O1 (pEQFLPEGQSQSQK), peptide C (QSQSQK), and peptide O2 (QQFLPEGQSQSQK) were synthesized by the Fmoc method and then purified by reverse-phase HPLC.

[0059] Example 1 The residual rates of peptide 1, peptide C, and peptide O2 after heat treatment were examined to evaluate their heat resistance. Specifically, each peptide was heat-treated at 85°C for 30 minutes or at 120°C for 15 minutes, and the residual rate (%) of each peptide was calculated using the following procedure (residual rate calculation procedure). The results are shown in Table 2.

[0060]

[0061] As shown in Table 2, peptide 1, whose N-terminus is other than glutamine residue, clearly had a higher residual rate and better heat resistance than peptide O2 and peptide C, whose N-terminus is glutamine residue.

[0062] Example 2: The residual ratio of a mixture of peptide 1, peptide O1, and peptide O2 after heat treatment was examined to evaluate heat resistance. Specifically, a composition containing peptide 1, peptide O1, and peptide O2 at a combined concentration of 110 μg / mL (i.e., a composition in which the combined mass of peptide 1, peptide O1, and peptide O2 relative to the volume of the composition was 110 μg / mL) was prepared and heated at 85°C for 30 minutes or 120°C for 15 minutes, and the residual ratio (%) of each peptide in each aqueous solution was determined. The ratio of peptide 1, peptide O1, and peptide O2 in the composition was approximately peptide 1:peptide O1:peptide O2 = 8:1:1 by mass. The results are shown in Table 3.

[0063]

[0064] As shown in Table 3, the composition containing the three peptides remained intact even after heat treatment, demonstrating high heat resistance. Peptide O1 also exhibited a high residual rate, similar to that of peptide 1, demonstrating its high heat resistance. Furthermore, although the reason for this is unclear, it was revealed that the residual rate of peptide O2 at 120°C was improved by the presence of peptide 1 and peptide O1 together, compared to when peptide O2 was present alone. Specifically, as shown in Example 1, the residual rate at 120°C when peptide O2 was present alone was 0%, whereas the residual rate of peptide O2 at 120°C improved to 63% when the composition contained peptide 1, peptide O1, and peptide O2. As shown in Table 3, even when the three peptides were mixed and heat-treated, peptide 1 and peptide O1, which have N-termini other than glutamine residues, exhibited significantly higher residual rates and better heat resistance than peptide O2, which has an N-termini residue.

[0065] [Example 3] A mixture of Peptide 1 and Peptide O2 was added to jelly, canned coffee drinks, and cookies, and the residual rate of each peptide in each food and drink was examined to evaluate heat resistance. The mixture of Peptide 1 and Peptide O2 used was a peptide mixture prepared by mixing Peptide 1 and Peptide O2 in a 2:1 (mass ratio).

[0066] <Jelly> After thoroughly mixing the powdered agar, the peptide mixture, and water, the resulting mixture was heated to a liquid temperature of 85°C and then further heated for 30 minutes to prepare a jelly liquid. The jelly liquid was then cooled and solidified to produce a jelly.

[0067] <Canned Coffee Drink> The peptide mixture was added to a coffee extract obtained by hot water extraction from roasted coffee beans and mixed in. The coffee extract was then filled into cans and heat sterilized at 120°C for 15 minutes to produce a canned coffee drink.

[0068] Cookies: Cookie dough was produced by mixing flour, sugar, butter, and eggs, adding the peptide mixture to the resulting dough, and further kneading until homogeneous. The resulting cookie dough was shaped and baked at 170°C for 18 minutes.

[0069] The residual rates of peptide 1 and peptide O2 in each food and drink were calculated based on the amount of peptide added as a raw material to each food and drink, which was set at 100%. The results are shown in Table 4.

[0070]

[0071] As shown in Table 4, when Peptide 1 was added to foods and beverages such as jellies and canned coffee drinks, it had a significantly higher residual rate and better heat resistance than Peptide O2. In particular, when Peptide O2 was added to the dough before baking, Peptide 1 retained 50% of its original amount even after baking.

[0072] Example 4 A tablet-type food containing 25 mg of rice endosperm protein (REP) hydrolysate (hereinafter referred to as a peptide-containing food) was prepared by the following procedure. The REP hydrolysate contained Peptide 1, Peptide O1, and Peptide O2.

[0073] First, rice endosperm protein was prepared using the method described in JP 2016-172693 A. The rice endosperm protein was dissolved in water to prepare an aqueous solution with a concentration of 20 mg / mL. Glucoamylase (Sumizyme®, manufactured by Shin-Nihon Chemical Co., Ltd.) was added to the aqueous solution, and the hydrolysis reaction was carried out at 50°C for 5 hours at pH 7.0. The reaction solution was boiled for 10 minutes to terminate the enzymatic reaction, and then dried using a freeze dryer to obtain the REP hydrolysate. The REP hydrolysate and excipients (reduced maltose starch syrup, crystalline cellulose, fine silicon dioxide, and calcium stearate) were mixed in the amounts listed in Table 5, and tablets were produced using a tablet press so that each tablet contained 250 mg of REP hydrolysate. The ratio of Peptide 1, Peptide O1, and Peptide O2 in the peptide-containing food was approximately Peptide 1:Peptide O1:Peptide O2 = 5:3:2 by mass.

[0074]

[0075] For comparison, a tablet-type food product containing no REP hydrolysate (hereinafter referred to as the comparative food product) was prepared. The manufacturing procedure for the tablet was the same as that for the peptide-containing food product, except that no REP hydrolysate was added, and instead, crystalline cellulose was added (i.e., the amount of rice endosperm protein hydrolysate was 0 mg / tablet, and the amount of crystalline cellulose was 140.5 mg / tablet).

[0076] Thirty-eight subjects (hereinafter referred to as the peptide group) were given two tablets of the peptide-containing food, and another 38 subjects (hereinafter referred to as the placebo control group) were given two tablets of the comparative food. Psychological surveys were conducted before and after the intake to evaluate whether or not mood disorders improved. The psychological survey was conducted using POMS2 (Profile of Mood Status 2nd edition for adults), and seven emotional states (anger / hostility (AH), confusion / bewilderment (CB), depression / depression (DD), fatigue / lethargy (FI), tension / anxiety (TA), vitality / vigor (VA), and friendliness (F)) were evaluated. The post-meal psychological survey was conducted 3 hours after eating the tablet-type food.

[0077] The results are shown in Table 6. In Table 6, the "p value" is the p value obtained by performing a two-sample t-test on the change in the placebo control group and the change in the peptide group. If this value is less than 0.05, the difference between the change in the placebo control group and the change in the peptide group is considered to be a significant difference.

[0078]

[0079] As shown in Table 6, the evaluation results showed a significant difference between the peptide group and the placebo control group in the change in the score for "tension / anxiety" out of the seven emotional states.

[0080] From the above, it has been shown that a tablet-type food containing REP hydrolysate reduces tension and anxiety, which are mood disorders. Therefore, it has been shown that a tablet-type food containing REP hydrolysate (i.e., the composition according to the present disclosure) can treat, prevent, or ameliorate mood disorders. For the same reasons, it has also been shown that peptide 1 and peptide O1 can treat, prevent, or ameliorate mood disorders.

[0081] The disclosure of Nutrients 2023, 15, 3491, published on August 7, 2023, is incorporated herein by reference in its entirety. All publications, patent applications, and technical standards mentioned herein are incorporated herein by reference to the same extent as if each individual publication, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO:

2.

2. A peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO:

2.

3. A peptide according to claim 1 or 2, which is used for the treatment, prevention or amelioration of mood disorders.

4. A peptide according to claim 1 or claim 2, which has a residual rate of 90% or more after heat treatment at 85°C.

5. A peptide according to claim 1 or claim 2, which has a residual rate of 50% or more after heat treatment at 120°C.

6. The peptide according to claim 3, which has a survival rate of 90% or more after heat treatment at 85°C.

7. The peptide according to claim 3, which has a residual rate of 50% or more after heat treatment at 120°C.

8. A composition comprising the peptide of claim 1 or claim 2.

9. The composition according to claim 8, which is a food, drink or pharmaceutical product.

10. The composition according to claim 9, wherein the food or drink is a baked food, a retort food, a beverage, or a jelly.

11. A food additive for cooking with heat, comprising as its main component the peptide described in claim 1 or claim 2.

12. A pharmaceutical additive comprising the peptide according to claim 1 or 2 as its main component.

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