peptide

Abstract

To provide a saltiness-expressing or -enhancing substance.SOLUTION: A peptide consists of 3 or 4 amino acids, comprising an amino acid sequence represented by formula (1): X1a-R-A (where X1a is an aliphatic amino acid) or an amino acid sequence represented by formula (2): R-A-X2a (where X2a is an aromatic amino acid or a basic amino acid).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to peptides and the like. [Background technology]

[0002] In recent years, excessive salt intake has become a global problem. While the World Health Organization (WHO) recommends a daily salt intake of 5 grams, most people currently consume between 9 and 12 grams of salt per day. In Japan, the Japanese Society of Hypertension (JSHY) announced the Tokyo Declaration for Salt Reduction in October 2019, which set a goal of "implementing six strategies aimed at reducing daily salt intake to 6 grams." Excessive salt intake is known to cause various health problems. Hypertension, one of the health risks of excessive salt intake, is one of the major causes of ischemic stroke and heart disease due to left ventricular hypertrophy.

[0003] To reduce salt intake, several methods have been used, including stealth reduction, multisensory application, and salty taste substitution / enhancement. Stealth reduction involves gradually reducing dietary salt content so that consumers do not notice the reduction in saltiness. However, once the amount of sodium chloride falls below a certain level, the brain's reward system is not stimulated, resulting in a loss of palatability and a decline in quality. Multisensory application compensates for the reduction in saltiness using yeast extract, spices, herbs, aromatic compounds, etc., but it is not possible to enhance saltiness depending on the food combination. The use of salty taste substitutes and salty taste enhancers aims to maintain a strong salty taste as a "palatable" taste while reducing salt content, and is currently attracting the most attention. Alkaline hydrochlorides such as potassium chloride and ammonium chloride have been reported as salty taste substitutes, but these substances are known to contain metallic, bitter, and astringent tastes in addition to saltiness. Salty taste enhancers such as L-arginine and L-aspartic acid have been reported. However, these salty taste enhancers are known to contain off-flavors (tastes and odors that are not appropriate for saltiness). For these reasons, their use is limited to baked foods and infant formula. Therefore, there is a need to explore new, highly active salty taste substitutes and enhancers.

[0004] Taste cells, present in large numbers on taste buds, are sensors that detect taste and transmit taste information. Human taste cells independently detect each of the five basic tastes (salty, sweet, bitter, sour, and umami), guiding us to avoid toxins and indigestible substances and to ingest nutritious foods. G protein-coupled receptors and ion channels are present on taste buds as taste receptors that specifically receive taste stimuli. The existence of epithelial sodium channels (ENaC) has been reported as one of the ion channels that mediate salty taste.

[0005] Peptides are substances in which 20 types of amino acids are linked by peptide bonds. Peptides are highly diverse; for example, there are 20 x 20 x 20 = 8,000 types of peptides in which three amino acids are linked together. Many peptides exhibit some kind of taste themselves, or have a transformative effect on the sense of taste, playing an important role in contributing to the deliciousness of food. Among these peptides are peptides that have a salty taste enhancing effect, and dipeptides such as RA have been reported to have a salty taste enhancing effect (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Xu, JJ et al. Scientific Reports. 7, 7483 (2017). DOI:10.1038 / s41598-017-07756-x Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a substance that expresses or enhances salty taste. [Means for solving the problem]

[0008] In view of the above problems, the present inventors have conducted extensive research and have found that a compound represented by the formula (1): X 1a -RA (wherein: X 1a represents an aliphatic amino acid) or an amino acid sequence represented by formula (2): RAX 2a (In the formula:X 2a The inventors have found that the above-mentioned problems can be solved by a peptide consisting of 3 or 4 amino acids, which contains an amino acid sequence represented by the formula (wherein r represents an aromatic amino acid or a basic amino acid). Based on this finding, the inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

[0009] Term 1. Formula (1):X 1a-RA (wherein: X 1a represents an aliphatic amino acid) or an amino acid sequence represented by formula (2): RAX 2a (In the formula:X 2a A peptide consisting of 3 or 4 amino acids containing the amino acid sequence shown in (wherein indicates an aromatic amino acid or a basic amino acid).

[0010] Section 2. Said X 1a The aliphatic amino acid represented by the formula: is V, I, L, G, or A, or 2a is F, Y, or W, and X 2a Item 2. The peptide according to Item 1, wherein the basic amino acid represented by the formula: is K, H, or R.

[0011] Item 3. The peptide according to Item 1 or 2, which comprises the amino acid sequence represented by formula (1).

[0012] Item 4. An amino acid sequence represented by formula (1) having an arbitrary amino acid X at the N-terminal side thereof 1b Item 4. The peptide according to any one of Items 1 to 3, comprising an amino acid sequence to which is added:

[0013] Section 5. Said X 1b Item 5. The peptide according to Item 4, wherein is an amide group-containing amino acid, a hydroxy group-containing amino acid, or an aromatic amino acid.

[0014] Section 6. Said X 1b is N or Q, and 1b is S or T, or 1b Item 6. The peptide according to Item 5, wherein the aromatic amino acid represented by the formula: is F, Y, or W.

[0015] Section 7. Said X 1a Item 7. The peptide according to any one of Items 3 to 6, wherein the aliphatic amino acid represented by the formula: is V, I, L, or G.

[0016] Item 8. The peptide according to any one of Items 1 to 7, comprising an amino acid sequence represented by any one of SEQ ID NOs: 10 to 12 and 20 to 23.

[0017] Item 9. An enzymatic hydrolysate of a protein or a purified product thereof, comprising the peptide according to any one of Items 1 to 8.

[0018] Item 10. A composition comprising the peptide according to any one of items 1 to 8.

[0019] Item 11. The composition according to Item 10, which is a solution, gel, powder, or tablet.

[0020] Item 12. A salty taste expressing or enhancing agent, comprising the peptide according to any one of items 1 to 8.

[0021] Item 13. The salty taste enhancer or enhancer according to Item 9, which is a preferred salty taste enhancer or enhancer.

[0022] Item 14. The salty taste expressing or enhancing agent according to Item 9 or 10, wherein the peptide comprises an enzymatic hydrolyzate of a protein.

[0023] Item 15. A food or drink containing the peptide according to any one of Items 1 to 8. [Effects of the Invention]

[0024] According to the present invention, a peptide having the effect of expressing or enhancing a salty taste can be provided. [Brief explanation of the drawings]

[0025] [Figure 1] 1 shows the results of measuring the salty taste expression or enhancement effect of peptides in group A (Test Example 1), where the vertical axis indicates salty taste response and the horizontal axis indicates the amino acid sequence of the peptide. [Figure 2] 1 shows the results of measuring the salty taste expression or enhancement effect of peptides in group B (Test Example 1), where the vertical axis indicates salty taste response and the horizontal axis indicates the amino acid sequence of the peptide. [Figure 3]1 shows the results of measuring the salty taste expression or enhancement effect of peptides in group C (Test Example 1), where the vertical axis indicates salty taste response and the horizontal axis indicates the amino acid sequence of the peptide. [Figure 4] 1 shows the results of measuring the salty taste expression or enhancement effect of peptides in group D (Test Example 1), where the vertical axis indicates salty taste response and the horizontal axis indicates the amino acid sequence of the peptide. [Figure 5] 1 shows the results of measuring the salty taste expression or enhancement effect of peptides in group E (Test Example 1), where the vertical axis indicates salty taste response and the horizontal axis indicates the amino acid sequence of the peptide. [Figure 6] 1 shows the results of measuring the salty taste expression or enhancement effect of peptides from group F (Test Example 1), where the vertical axis indicates salty taste response and the horizontal axis indicates the amino acid sequence of the peptide. [Figure 7] 1 shows the results of measuring the palatability of the peptides of the present invention (Test Example 2), where the vertical axis indicates palatability and the horizontal axis indicates sodium chloride concentration. [Figure 8] 1 shows the results of measuring the preference for the peptides of the present invention (Test Example 2), where the vertical axis indicates preference and the horizontal axis indicates the test sample. [Figure 9] 1 shows the results of measuring the preference for the peptides of the present invention (Test Example 2), where the vertical axis indicates preference and the horizontal axis indicates the test sample. DETAILED DESCRIPTION OF THE INVENTION

[0026] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "include," "consist essentially of," and "consist only of."

[0027] In this specification, amino acids in an amino acid sequence may be represented by single letter codes.

[0028] As used herein, the amino acids (amino acid residues) constituting an amino acid sequence are not particularly limited as long as they are amino acids capable of constituting a peptide, and include both natural and artificial amino acids. Examples of amino acids include aliphatic amino acids such as G, A, V, I, and L; sulfur-containing amino acids such as C and M; basic amino acids such as K, H, and R; amide group-containing amino acids such as N and Q; hydroxy group-containing amino acids such as S and T; aromatic amino acids such as F, Y, and W; acidic amino acids such as D and E; and imino acids such as P.

[0029] 1. Peptides In one aspect, the present invention provides a compound represented by formula (1): X 1a -RA (wherein: X 1a represents an aliphatic amino acid) or an amino acid sequence represented by formula (2): RAX 2a (In the formula:X 2a The present invention relates to a peptide consisting of 3 or 4 amino acids, which contains an amino acid sequence represented by the formula (A) and (B) (the amino acids represent aromatic amino acids or basic amino acids) (herein, this may be referred to as "the peptide of the present invention"). This will be explained below.

[0030] In formulas (1) and (2), the amino acids (=amino acid residues) constituting the peptide are represented by single letters. The "-" in formulas (1) and (2) indicates that the amino acids on both sides of it are linked by a peptide bond. In formulas (1) and (2) and the amino acid sequences, the left side indicates the N-terminus, and the right side indicates the C-terminus.

[0031] X 1a There are no particular limitations on the aliphatic amino acid represented by X, as long as it is an amino acid whose side chain is entirely hydrogen atoms or an aliphatic chain (such as an alkyl group). 1a Examples of the aliphatic amino acid represented by the formula (I) include V, I, L, G, A, etc., preferably V, I, L, G, etc., more preferably V.

[0032] X 2aThe aromatic amino acid represented by X is not particularly limited as long as it is an amino acid having a side chain containing an aromatic group (for example, a one- or two-membered aryl or heteroaryl (heteroatom is a nitrogen atom or the like) group which may be substituted with a hydroxyl group or the like). 2a Examples of aromatic amino acids represented by the formula (I) include F, Y, W, etc., and preferably F.

[0033] X 2a There are no particular limitations on the basic amino acid represented by X, as long as it is an amino acid having a basic side chain (for example, a side chain containing an amino group). 2a Examples of the basic amino acid represented by the formula (I) include K, H, and R, and preferably K.

[0034] The peptide of the present invention may be a peptide consisting of three amino acids having the amino acid sequence shown in formula (1) or formula (2), or may be a peptide consisting of four amino acids in which any amino acid is added to the N-terminal or C-terminal side of the amino acid sequence shown in formula (1) or formula (2).

[0035] From the viewpoint of salty taste expression or enhancement, the peptide of the present invention is preferably a peptide consisting of four amino acids. In this case, the amino acids added to the N-terminus or C-terminus of the amino acid sequence represented by formula (1) or formula (2) are not particularly limited and include, for example, aliphatic amino acids such as G, A, V, I, and L; sulfur-containing amino acids such as C and M; basic amino acids such as K, H, and R; amide group-containing amino acids such as N and Q; hydroxy group-containing amino acids such as S and T; aromatic amino acids such as F, Y, and W; acidic amino acids such as D and E; and imino acids such as P. While not intended to be restrictive, the results of Test Example 1 described below showed that increasing the number of amino acids from three to four improved activity regardless of the type of amino acid, and that increasing the number of amino acids to five rapidly decreased activity. Therefore, it is believed that a peptide consisting of four amino acids is optimal for the EnaC binding pocket.

[0036] From the viewpoint of salty taste expression or enhancement, the peptide of the present invention preferably contains the amino acid sequence represented by formula (1). In this case, from the viewpoint of salty taste expression or enhancement, the peptide of the present invention preferably contains any amino acid X on the N-terminal side of the amino acid sequence represented by formula (1). 1b It is more preferable that the amino acid sequence comprises an amino acid sequence comprising the following:

[0037] X 1b is preferably an amide group-containing amino acid, a hydroxy group-containing amino acid, or an aromatic amino acid.

[0038] X 1b There are no particular limitations on the amide group-containing amino acid represented by X, as long as it is an amino acid having a side chain containing an amide group (—C(═O)—NH—). 1b Examples of the amide group-containing amino acid represented by the formula (I) include N and Q, and N is preferred.

[0039] X 1b There are no particular limitations on the hydroxyl group-containing amino acid represented by X, as long as it is an amino acid having a side chain containing a hydroxyl group (preferably at the terminal). 1b Examples of the hydroxyl group-containing amino acid represented by the formula (I) include S and T, and preferably S.

[0040] X 1b The aromatic amino acid represented by X is not particularly limited as long as it is an amino acid having a side chain containing an aromatic group (for example, a one- or two-membered aryl or heteroaryl (heteroatom is a nitrogen atom or the like) group which may be substituted with a hydroxyl group or the like). 1b Examples of aromatic amino acids represented by the formula (I) include F, Y, W, etc., and preferably F.

[0041] In one aspect of the present invention, preferred amino acid sequences comprised by the peptides of the present invention include VRA (SEQ ID NO: 1), RAF (SEQ ID NO: 5), RAK (SEQ ID NO: 6), NVRA (SEQ ID NO: 10), SVRA (SEQ ID NO: 11), FVRA (SEQ ID NO: 12), MVRA (SEQ ID NO: 13), KVRA (SEQ ID NO: 14), PVRA (SEQ ID NO: 15), VRAN (SEQ ID NO: 16), NIRA (SEQ ID NO: 20), NLRA (SEQ ID NO: 21), NGRA (SEQ ID NO: 22), QGRA (SEQ ID NO: 23), etc. Among these, more preferred are amino acid sequences represented by any of SEQ ID NOs: 10 to 12 and 20 to 23.

[0042] The peptides of the present invention are preferably peptides consisting of a partial amino acid sequence in an edible protein. Whether a certain sequence a is a sequence within a protein can be easily determined by extracting amino acid sequences with the same number of residues as sequence a from the amino acid sequences of the protein (for example, from the 710 proteins in the edible protein database (URL: http: / / www.uwm.edu.pl / biochemia / index.php / en / biopep)), shifting the residues by one at a time, and examining whether a sequence corresponding to sequence a exists among them. The edible protein is not particularly limited as long as it is a protein that is abundant in food materials, and specific examples include milk proteins (e.g., casein, sodium caseinate, MPC (Milk Protein Concentrate), α-casein, β-casein, κ-casein, lactalbumin, and hydrolysates thereof), soy proteins (e.g., glycinin, β-conglycinin, convicilin, histone, and the like), cereal (e.g., rice, wheat, and the like) proteins (e.g., gluten, gluadin, glutelin, glutenin storage protein, and the like), meat proteins (e.g., muscle structural proteins, myosin, actin, and the like), fish meat proteins (e.g., muscle fiber proteins, actomyosin, myosin, actin, and the like), chicken egg proteins (e.g., ovalbumin, egg yolk lipoprotein, and the like), and pig skin proteins (e.g., gelatin, and the like).

[0043] The salty taste expression or enhancement effect of the peptide of the present invention, as measured by the method of Test Example 1 described below, is, for example, 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, or 5 times or more than the salty taste expression or enhancement effect of the dipeptide (RA).

[0044] The peptides of the present invention are preferably isolated, enriched, or purified peptides.

[0045] The peptides of the present invention also include those in which the terminal amino acid residues have been chemically modified, as long as the salty taste expression or enhancement effect is not significantly reduced.

[0046] The peptide of the present invention has a C-terminal carboxyl group (-COOH), a carboxylate group (-COO - ), amide (-CONH2) or ester (-COOR), etc.

[0047] Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1-6 Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl 3-8 Cycloalkyl groups such as phenyl and α-naphthyl 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl groups; α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0048] Furthermore, in the peptide of the present invention, the amino group on the main chain of the N-terminal amino acid residue is protected by a protecting group (e.g., a C group such as a formyl group or an acetyl group). 1-6 C such as alkanoyl 1-6 These also include those protected with an acyl group, myristoylated, pyroglutamylated, methylated, etc.

[0049] The peptides of the present invention may include those in which amino acid residues other than the terminal ones have been chemically modified, provided that the salty taste expression or enhancement effect is not significantly reduced. However, preferably, the peptides of the present invention do not include those in which amino acid residues other than the terminal ones have been chemically modified. Examples of chemical modifications in this case include amidation or esterification of carboxyl groups, and protection of amino groups with protecting groups. The esterification and protecting groups are similar to those described above for the chemical modification of the terminals.

[0050] The peptides of the present invention also include salts with acids or bases. The salts are not particularly limited, and both acidic and basic salts can be used. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0051] The peptide of the present invention also includes solvates. The solvent is not particularly limited, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0052] The peptides of the present invention may be used singly or in combination of two or more.

[0053] The peptides of the present invention can be produced by various methods. For example, the peptides of the present invention can be produced by solid-phase synthesis. Furthermore, when the peptides of the present invention are peptides consisting of a partial amino acid sequence of an edible protein, they can be obtained by enzymatic degradation of the protein.

[0054] The enzyme is not particularly limited as long as it has proteolytic activity, and examples thereof include aspartic acid proteases, serine proteases, cysteine ​​proteases, metalloproteases, and threonine proteases.

[0055] Examples of aspartic acid proteases include pepsin, renin, cathepsin D, cathepsin E, napsin, β-secretase, γ-secretase, signal peptide peptidase, HIV protease, HTLV protease, NS3A protease, plasmepsin, saspase, and chymosin.

[0056] Examples of serine proteases include factors of the blood coagulation / fibrinolysis system and the complement system, such as dipeptidyl peptidase 4, trypsin, chymotrypsin, plasmin, thrombin, and factor Xa, as well as their regulatory systems; neutrophil elastase, subtilisin, furin, PACE4, PC2, PC7, kexin, cucumisin, lantibiotic peptidase, thermitase, acrosin, kallikrein, urokinase, granzyme, tryptase, chymase, cathepsin A, prolyl aminopeptidase, P-type signal peptidase, prostate-specific antigen, HCMV protease, V8 protease, and protease K.

[0057] Examples of cysteine ​​proteases include cathepsins such as cathepsin B, cathepsin H, cathepsin L, cathepsin S, and cathepsin K, legumain, angiotensin-converting enzyme, bleomycin hydrolase, calpain, caspase, ER-60, papain, coronavirus 3CL protease, falcipain, TEV protease, and HRV3C protease.

[0058] Examples of metalloproteases include ADAM, matrix metalloprotease, thermolysin, neprilysin, carboxypeptidase, endothelin-converting enzyme, KELL antigen, bone morphogenetic protein-1, meprin, serralysin, PAPP, mitochondrial processing protease, insulin-degrading enzyme, aminopeptidase, and prenyl protease.

[0059] Examples of threonine proteases include proteasome and gamma glutamyltransferase.

[0060] The enzyme may be used alone or in combination of two or more.

[0061] The combination of an edible protein and an enzyme can be determined, for example, as follows. That is, it can be determined by searching for the amino acid sequence of the peptide of the present invention that is produced when the amino acid sequence of the edible protein is enzymatically degraded from the amino acid sequence of the edible protein. In this way, it is possible to obtain an edible protein / enzyme combination that provides the amino acid sequence of the peptide of the present invention. The amino acid sequence of the edible protein and the cleavage specificity of the enzyme can be easily determined according to publicly known information.

[0062] Enzymatic degradation products of proteins can be obtained by a method including a step of degrading proteins with enzymes. Specifically, the degradation can be carried out, for example, by incubating a reaction solution containing the protein and the enzyme. The composition of the reaction solution, reaction temperature, reaction time, protein concentration, enzyme concentration, the presence or absence of additives and their types, etc. can be appropriately set depending on the types of protein and enzyme.

[0063] After the step of hydrolyzing the protein with an enzyme, it is preferable to further carry out a step of purifying the peptide of the present invention.

[0064] The purification method is not particularly limited as long as it can concentrate the peptide of the present invention (i.e., can increase the concentration of the peptide of the present invention in the total peptide). Examples of purification methods include purification using silica gel, purification using a synthetic adsorption resin, normal-phase partition chromatography, reverse-phase partition chromatography, anion exchange chromatography, desalting using electrodialysis or the like, molecular weight fractionation using an ultrafiltration membrane or the like, size exclusion chromatography, affinity chromatography, etc. The peptide of the present invention can also be purified by utilizing its bile acid binding ability (for example, by using a carrier carrying bile acids). One type of purification method can be used alone, or two or more types can be used in combination.

[0065] 2.Applications In one aspect, the present invention relates to a composition containing the peptide of the present invention (the composition of the present invention). Note that the term "composition" refers to a mixture of multiple components.

[0066] The peptides of the present invention have the effect of expressing or enhancing a salty taste, more preferably the effect of expressing or enhancing a palatable salty taste (ENaC activation, ENaC-mediated salty taste expression or enhancement). Therefore, the peptides of the present invention can be used as an active ingredient of a salty taste expression or enhancement agent (the agent of the present invention). Expressing or enhancing a salty taste refers to expressing or enhancing a salty taste independent of sodium chloride. The salty taste expression or enhancement agent can impart or enhance a salty taste to an orally ingested substance. Addition of the peptides of the present invention can improve palatability. Furthermore, addition of the peptides of the present invention can improve palatability even in a state where an aversive salty taste is present (in the presence of high concentrations of sodium chloride).

[0067] The agent and composition of the present invention may contain a peptide other than the peptide of the present invention as a peptide. The content of the peptide of the present invention in the agent and composition of the present invention is, for example, 10% by mass or more, preferably 30% by mass or more, more preferably 50% by mass or more, even more preferably 70% by mass or more, still more preferably 80% by mass or more, particularly preferably 90% by mass or more, and particularly preferably 95% by mass or more, relative to 100% by mass of the peptide. The agent and composition of the present invention may contain an enzymatic hydrolysate of a protein or a purified product thereof as a peptide including the peptide of the present invention.

[0068] The agent and composition of the present invention can be used in various fields, for example, as food additives, foods and drinks, medicines, health enhancers, nutritional supplements (supplements, etc.), and the like.

[0069] The agent of the present invention and the composition of the present invention can be used, for example, by adding them to something that is taken orally, or by taking them orally as they are.

[0070] The form of the agent of the present invention and the composition of the present invention is not particularly limited, and may take a form that is normally used for each application depending on the application.

[0071] When the agent or composition of the present invention is used as a food additive, a medicine, a health-promoting agent, a nutritional supplement (such as a supplement), or the like, examples of the form of the agent or composition of the present invention include a solution, gel, powder, tablet (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, and the like), pill, granule, fine granule, powder, hard capsule, soft capsule, dry syrup, liquid (including drinks, suspensions, and syrups), jelly, etc.

[0072] The content of the peptide of the present invention in the agent or composition of the present invention depends on the intended use, mode of use, condition of the subject to be treated, etc., and is not limited to, but may be, for example, 0.0001 to 100% by mass, preferably 0.001 to 50% by mass. The lower limit of the content is, for example, 0.01%, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by mass.

[0073] When the peptide of the present invention is added to a food or drink, the target food or drink is not particularly limited and includes, for example, soups such as ramen, udon, soba, potage, consommé, bouillon, miso soup, clear soup, etc., noodles such as ramen, udon, soba, yakisoba, pasta, etc., bread, food seasonings such as soy sauce, miso, dressing, etc., seasoning powder for snacks, etc. The peptide may be added in an amount such that the concentration of the peptide of the present invention in the food or drink is, for example, 0.001 to 100 mM, 0.01 to 10 mM, or 0.05 to 2 mM. [Example]

[0074] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0075] In the following tests, comparisons between two groups were performed using Student's t-test, and a p-value of 0.05 or less was defined as a significant difference.

[0076] In the following tests, comparisons between multiple groups were evaluated using one-way analysis of variance (ANOVA) after testing for homogeneity of variance using the Bartlett test. Subsequently, multiple comparison tests were performed using the Tukey-Kramer method (hereafter referred to as the Tukey method). A p value of 0.05 or less was defined as a significant difference.

[0077] Test Example 1: Measurement of salty taste expression or enhancement (in vitro) A peptide having an amino acid sequence formed by adding an amino acid to a known salt taste expression or enhancing peptide (RA) was synthesized, and using cells expressing EnaC, which is a salt taste receptor, intracellular Ca 2+ was measured for salt taste expression or enhancing effect by imaging. Specifically, it was carried out as follows.

[0078] <1-1. Peptide> The amino acid sequences of the synthesized peptides are as follows.

[0079] <Group A: 1aa-RA> ·VRA (SEQ ID NO: 1) ·SRA (SEQ ID NO: 2) ·FRA (SEQ ID NO: 3) ·KRA (SEQ ID NO: 4).

[0080] <Group B: RA-1aa> ·RAF (SEQ ID NO: 5) ·RAK (SEQ ID NO: 6) ·RAV (SEQ ID NO: 7) ·RAN (SEQ ID NO: 8) ·RAP (SEQ ID NO: 9).

[0081] <Group C: 1aa-V-RA> [[ID=四十]]·NVRA (SEQ ID NO: 10) ·SVRA (SEQ ID NO: 11) ·FVRA (SEQ ID NO: 12)<U ·MVRA (SEQ ID NO: 13) ·KVRA (SEQ ID NO: 14) ·PVRA (SEQ ID NO: 15).

[0082] <Group D: V-RA-1aa> ·VRAN (SEQ ID NO: 16).

[0083] <Group E: 1aa-N-V-RA> ·VNVRA (SEQ ID NO: 17) ·DNVRA (SEQ ID NO: 18)<U ·PNVRA (SEQ ID NO: 19).

[0084] <F group: Amide group-containing amino acid - aliphatic amino acid - RA> ·NIRA (SEQ ID NO: 20) ·NLRA (SEQ ID NO: 21) ·NGRA (SEQ ID NO: 22) ·QGRA (SEQ ID NO: 23).

[0085] <1-2. Expression of salt receptor ENaC> <1-2-1. Replication of plasmid> Because the salty taste receptor epithelial sodium channel (ENaC) is composed of a trimer, we used three expression plasmids containing the sequences of each subunit: h-alpha-ENaC (83430, addgene, USA), h-beta-ENaC (83429, addgene, USA), and h-gamma-ENaC Myc (83428, addgene, USA). Plasmid replication was performed by transforming High Efficacy DH5α Competent cells (DH01-20, GMbiolab, Taiwan; hereafter referred to as competent cells) followed by culture and purification. 100 μL of competent cells stored at -80°C was thawed on ice, and 5 μL of the plasmid was added. The cells were then thawed on ice for 30 minutes. The cells were then heat-shocked by incubating in a 42°C incubator for 45 seconds and immediately returned to ice. 350 μL of room-temperature SOC medium was added to the tube, and the tube was inverted 10 times and then cultured at 200 rpm in a 37°C incubator for 1 hour. The cultured bacterial suspension was then suspended and plated on LB agar medium containing ampicillin (012-23303, Fujifilm Wako Pure Chemical Corporation, Osaka) and cultured overnight in a 37°C incubator. On the second day, a single colony was picked with a toothpick and inoculated into 2 mL of ampicillin-containing LB liquid medium in a Wasserman flask. Cultured overnight at 200 rpm in a 37°C incubator. On the third day, 50 mL of ampicillin-containing LB liquid medium was dispensed into a 200-mL Erlenmeyer flask, and 500 μL of the cultured bacterial suspension was inoculated into the flask. The culture was then expanded overnight at 200 rpm in a 37°C incubator. On the fourth day, the cloned plasmid was purified using the Genopure Plasmid Midi Kit (03143414001, Roche, Germany) according to the instructions in the manufacturer's manual, and then dissolved in 50 μL of TE buffer and stored at -80°C.

[0086] <1-2-2. Expression of salty taste receptor ENaC on the surface of HEK293 cells> HEK293 cells were plated in a 24-well cell culture plate (TR5002, True Line, USA; hereafter referred to as a 24-well plate) at a density of 7.2 × 10 4 The cells were seeded at 1000 cells / well and cultured for 24 hours in a CO2 incubator at 37°C under 5% CO2 and 95% air. The salty taste receptor ENaC was then expressed by lipofection. The Lipofectamine 3000 Transfection Kit (L3000-008, Invitrogen, USA) was used for lipofection. Two reagents were prepared for each well: DNA solution and Lipofectamine solution, as shown in Table 1. The Lipofectamine solution was added to the DNA solution, mixed by tapping, and incubated at room temperature for 15 minutes. Then, 50 μL / well of the solution was added to the cultured HEK293 cells. After lipofection, the cells were incubated for 24 hours in a CO2 incubator under 37°C, 5% CO2, and 95% air, and calcium imaging was performed.

[0087] [Table 1]

[0088] <1-3. Intracellular Ca 2+ Imaging> <1-3-1.Ca 2+ Preparation of imaging reagents Intracellular Ca 2+ Fluo-4 Direct was used to measure the concentration. TM Calcium Assay Kit (F10471, Invitrogen, USA) was used. TM is fluorescent Ca 2+ Indicator of intracellular Ca 2+ Fluo-4 Direct is added to the culture medium and emits fluorescence. TM The calcium reagent loading solution was prepared to be 2x concentrated. TMFluo-4 Direct contains 77 mg of water-soluble probenecid, which prevents extracellular outflow of TM Dissolved in 1 mL of calcium buffer. Fluo-4 Direct TM Fluo-4 Direct is added to the calcium assay reagent bottle. TM 10 mL of calcium buffer was added and dissolved by vortexing, then 200 μL of the prepared probenecid solution was added and the reagent was completely dissolved by vortexing, and the mixture was stored in a dark place.

[0089] <1-3-2. Intracellular Ca 2+ Imaging> Intracellular Ca 2+ The concentration was measured for HEK293 cells expressing the salty taste receptor ENaC by lipofection and HEK293 cells not expressing ENaC. The lipofected cells were transfected according to the method described in 1-2-2, and the untreated cells were transfected into a 24-well cell culture plate (TR5002, True Line, USA) at 7.2 × 10 4 After seeding at 100 cells / well, the cells were cultured for 48 hours in a CO2 incubator at 37°C under 5% CO2 and 95% air. After removing the medium, the cells were resuspended in 250 μl of DMEM medium (10% FBS, 1% PS) and 2× Fluo-4 Direct. TM250 μL of calcium reagent loading solution was added and incubated for 1 h in a CO2 incubator at 37°C under a 5% CO2, 95% air atmosphere. The medium and reagent were then removed, and the cells were washed twice with 500 μL of 15 mM HEPES / HBSS. After washing, 200 μL of 15 mM HEPES / HBSS was added and the cells were mounted on a fluorescence microscope (IX81, OLYMPUS, Tokyo). Images were taken using Metamorph imaging software with an exposure time of 250 ms and a magnification of 10x. Time-lapse observations (150 s intervals) were performed, and after 30 s, 50 μL of test solution (containing peptide and sodium chloride) was added to the medium to achieve final concentrations of 200 μM peptide and 200 mM sodium chloride. Fluorescence images were captured using a 488 nm laser. Changes in intracellular fluorescence intensity were measured by quantifying the fluorescence images at each time point using ImageJ. The image resolution was 1228 pixels x 1228 pixels, and each pixel was converted into a brightness value of 16 levels. The screen was scanned at each time and the sum of the pixels was calculated.

[0090] Intracellular Ca 2+ The imaging experimental data was evaluated using the area under the fluorescence intensity-time curve (AUC) for 120 seconds after sample addition, and expressed as the mean and standard deviation. The salty taste response was defined by the following equation:

[0091]

number

[0092] <1-4.Results> The results of Group A are shown in Figure 1, the results of Group B in Figure 2, the results of Group C in Figure 3, the results of Group D in Figure 4, the results of Group E in Figure 5, and the results of Group F in Figure 6. From Figures 1 to 6, the formula (1):X 1a -RA (wherein: X 1a represents an aliphatic amino acid) or an amino acid sequence represented by formula (2): RAX 2a (In the formula:X2a It was found that a peptide consisting of 3 or 4 amino acids containing an amino acid sequence represented by the formula (wherein represents an aromatic amino acid or a basic amino acid) can exhibit a high salty taste expression or enhancement effect.

[0093] Test Example 2. Measurement of palatability (in vivo) Preference was measured based on behavioral evaluation of the mice as follows.

[0094] <2-1. Mouse breeding> Eight 6-week-old male C57BL / 6JJcl mice (CLEA Japan, Tokyo; hereafter referred to as mice) were used for the animal behavioral tests. Each mouse was housed in a plastic cage (CL-0133, CLEA Japan, Tokyo; hereafter referred to as cage) lined with sawdust, with one mouse per cage. Each mouse was provided with free access to animal chow (CE-2, CLEA Japan, Tokyo; hereafter referred to as chow). The laboratory temperature was maintained at 23 ± 2°C, and the lights were set to turn on at 8:00 AM and off at 8:00 PM. For the first week, the mice were allowed free access to chow and ultrapure water (hereafter referred to as water) to allow them to acclimate to the environment.

[0095] <2-2. Preparation of intake sample> The sodium chloride aqueous solution (191-01665, Fujifilm Wako Pure Chemical Industries, Osaka) was prepared to have concentrations of 15 mM, 30 mM, 45 mM, 75 mM, and 150 mM. The peptide solution was prepared using the purchased NVRA peptide (Biologica, Aichi) to a final concentration of 0.2 mM.

[0096] <2-3. Two-bottle choice test> The two-bottle choice test was conducted under the conditions described in 2-1. The bottles used to present the samples were 10 mL Stripettes. TMSerological pipets (4488, Corning, USA; hereafter referred to as pipettes) were cut and fitted with 8 mm Φ × 65 mm stainless steel ball-ends (TD-100, Japan CLEA, Tokyo; hereafter referred to as pipettes). The pipettes and ends were secured with 8 mm silicone tubing (Cole Parmer, 06411-76, USA; hereafter referred to as silicone tubing). When samples were added, the pipettes were capped with Parafilm M 4 inches × 125 feet (Nikkei Seisakusho, PM-996, Osaka; hereafter referred to as Parafilm). The prepared sample was placed in one bottle and water in the other. The bottles containing the samples were weighed. They were then presented 4.0 cm apart. After 24 h, the weights of the bottles containing the samples were measured, and the bottles were swapped. After another 24 h, the weights of the bottles containing the samples were measured.

[0097] The experimental data for the two-bottle choice test were calculated by calculating the average intake per 24 h for each animal from the weight of the measured samples, and the intake and preference of the eight animals were shown as the mean and standard deviation. Preference was defined by the following formula:

[0098]

number

[0099] <2-4.Results> The results when a solution containing only sodium chloride was used are shown in Figure 7. It was found that preference decreased when high concentrations of sodium chloride solutions (50 mM, 150 mM) were used.

[0100] Figure 8 shows the results of a comparison between water and a solution containing only the peptide. Figure 9 also shows the results of a comparison between a high-concentration (150 mM) sodium chloride solution and a solution to which the peptide was further added. It was found that adding the peptide of the present invention to high-concentration saline significantly increased palatability. Since aversive salty tastes activate receptors other than ENaC, it is thought that adding the peptide of the present invention further activated ENaC, which produces a palatable salty taste, thereby increasing palatability.

[0101] Test Example 3: Search for peptide sequences that express or enhance salty taste The presence of NGRA and QGRA in protein sequences was searched using the following two protein databases: 1. Search the peptide database derived from edible proteins, which was originally constructed using BIOPEP-UWM (http: / / www.uwm.edu.pl / biochemia / index.php / pl / biopep), and link it to Uniprot. 2. Japanese public database: Protein Research Foundation: https: / / www.prf.or.jp / seqdb.html.

[0102] The results are shown below.

[0103] <ngra> 1. UniProtKB - P04405 (GLYG2_SOYBN) 2. SEQDB ID: 1313364A, Reference: 1313364 Name: glycinin A2B1a, Organism: Glycine max Front and rear sequence: IYALNGRALVQV Cleavage prediction: Pepsin (pH>2) I_Y_A_L_NGRA_LVQV (cleavage at position _) Other similar sequences 1. UniProtKB - P04776 (GLYG1_SOYBN) 1. UniProtKB - P11828 (GLYG3_SOYBN).

[0104] <qgra> 1. UniProtKB - P12615 (SSG1_AVESA) :393-936 2. SEQDB ID: 1605264A, Reference: 1605264 Name: 12S seed storage protein, Organism: Avena sativa (oats) Front and rear sequence: MIQGRARVQV Cleavage prediction: MI_QGRA_RV_QV for Proteinase K.

[0105] <qgra> 1. UniProtKB - P07730 (GLUA2_ORYSJ) :382-385 2. SEQDB ID: 1311273A, Reference: 1311273 Name: glutelin,Organism: Oryza sativa Front and rear arrangement: ITQGRAQVQV Cleavage prediction: I_T_QGRA_QV_QV for Proteinase K.

[0106] <qgra> 1. UniProtKB - Q09151 (GLUA3_ORYSJ):381-384 2. SEQDB ID: 1210248A, Reference: 1210248 Name: glutelin precursor,Organism: Oryza sativa Front and rear sequence: ITQGRARVQVV Cleavage prediction: Proteinase K I_T_QGRA_RV_QV_V.

[0107] As described above, it was found that NGRA and QGRA are present within the sequences of edible proteins (soybean conglycinin, rice glutelin, etc.) and can be excised with proteases (pepsin or proteinase K).< / qgra> < / qgra> < / qgra> < / ngra>

Claims

1. A peptide consisting of an amino acid sequence shown in any of SEQ ID NOs: 1, 5-6, 10-16, and 20-23.

2. The peptide according to claim 1, consisting of an amino acid sequence represented by any one of SEQ ID NOs: 10 to 12 and 20 to 23.

3. An enzymatic hydrolysate of a protein or a purified product thereof, which contains the peptide described in claim 1 or 2, and the content of the peptide in the enzymatic hydrolysate or purified product thereof is 0.0001 to 100 mass%.

4. A composition comprising the peptide described in claim 1 or 2, and wherein the content of the peptide in the composition is 0.0001 to 100% by mass.

5. 5. The composition of claim 4, which is a solution, gel, powder, or tablet.

6. A salty taste expressing or enhancing agent comprising the peptide according to claim 1 or 2.

7. The salty taste expressing or enhancing agent according to claim 6, which is an agent for expressing or enhancing a palatable salty taste.

8. The salty taste expressing or enhancing agent according to claim 6 or 7, wherein the peptide comprises an enzymatic hydrolyzate of a protein.

9. A food or beverage containing the peptide described in claim 1 or 2, wherein the concentration of the peptide in the food or beverage is 0.001 to 100 mM.

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