JP2021052597A - Cholesterol absorption control agent - Google Patents

Abstract

To provide peptide having a cholesterol absorption control action, preferably peptide having a cholesterol absorption control action capable of being simply produced from protein (preferably food protein) by making use of enzymolysis.SOLUTION: The peptide includes 4 to 6 amino acid residue containing basic amino acid residue.SELECTED DRAWING: None

Description

The present invention relates to a cholesterol absorption inhibitor and the like.

Cholesterol in vivo is taken up by micelles of bile acids and then absorbed through the intestinal wall. In order to prevent abnormal lipid metabolism such as hypercholesterolemia, it is desirable to suppress cholesterol absorption.

The present inventor previously investigated the bile acid-binding activity of 500 types of screening array libraries, and when the results were PC-plotted by principal component analysis, a highly active (high-bile acid-binding) peptide was found in a specific region. It was revealed that the peptides were concentrated, and that with 500 novel 4-residue peptides and new 6-residue peptides, the peptides present in the high-functioning region became highly active on average (non-). Patent Document 1).

Ito et al., Journal of Bioscience and Bioengineering, 127 (3), 336-371 (2019)

While conducting research on peptides having an inhibitory effect on cholesterol absorption, the present inventor considered that such peptides are desirable to be enzymatic decomposition products of proteins from the viewpoint of safety. However, in the prior art such as Non-Patent Document 1, it is up to whether or not a peptide having a sequence having a cholesterol absorption inhibitory action is present in the protein and whether or not such a peptide can be excised by enzymatic decomposition. did not understand.

An object of the present invention is to provide a peptide having an inhibitory effect on cholesterol absorption. Preferably, it is an object of the present invention to provide a peptide having a cholesterol absorption inhibitory action, which can be easily produced from a protein (preferably a protein in a food) by utilizing enzymatic decomposition.

As a result of diligent research in view of the above problems, the present inventor has found that a peptide consisting of 4 to 6 amino acid residues including basic amino acid residues has a cholesterol absorption inhibitory effect. The present inventor has also found that such a protein can be easily obtained by degrading the protein with an enzyme such as an enzyme having an activity of producing a peptide containing a basic amino acid residue. The present inventor has completed the present invention as a result of further research based on these findings. That is, the present invention includes the following aspects.

Item 1. A cholesterol absorption inhibitor containing a peptide consisting of 4 to 6 amino acid residues including a basic amino acid residue.

Item 2. Item 2. The cholesterol absorption inhibitor according to Item 1, wherein the basic amino acid residue is at least one selected from the group consisting of an arginine residue and a lysine residue.

Item 3. Item 2. The cholesterol absorption inhibitor according to Item 1 or 2, which contains an enzymatically decomposed product of a protein as the peptide.

Item 4. Item 3. The cholesterol absorption inhibitor according to Item 3, wherein the enzyme is an enzyme having an activity of producing a peptide containing a basic amino acid residue.

Item 5. Item 3. The cholesterol absorption inhibitor according to Item 3 or 4, wherein the protein is a food protein.

Item 6. Item 2. The cholesterol absorption inhibitor according to any one of Items 3 to 5, wherein the protein is a rice protein.

Item 7. Item 2. The cholesterol absorption inhibitor according to any one of Items 1 to 6, which contains a plurality of types of the peptides having different amino acid sequences from each other.

Item 8. As the peptide
For (a) peptides consisting of any of the amino acid sequences of SEQ ID NOs: 11, 19, 23, 27, and 39, and (b) for any of the amino acid sequences of SEQ ID NOs: 11, 19, 23, 27, and 39. Selected from the group consisting of a peptide consisting of an amino acid sequence in which one or more amino acid residues are substituted, deleted, added or inserted, and having a cholesterol-containing micelle decay promoting activity and / or a cholesterol-containing micelle formation inhibitory activity. Item 2. The cholesterol absorption inhibitor according to any one of Items 1 to 7, which contains at least one type.

Item 9. As the peptide
(A) A peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1-43, and (b) one or more amino acid residues substituted or deleted from any of the amino acid sequences of SEQ ID NOs: 1-43. Any of Items 1 to 7, which comprises an added or inserted amino acid sequence and contains at least one selected from the group consisting of peptides having cholesterol-containing micelle disintegration promoting activity and / or cholesterol-containing micelle formation inhibitory activity. The cholesterol absorption inhibitor described in.

Item 10. Item 2. The cholesterol absorption inhibitor according to any one of Items 1 to 9, which is used for promoting cholesterol-containing micelle disintegration and / or suppressing cholesterol-containing micelle formation.

Item 11. Item 2. The cholesterol absorption inhibitor according to any one of Items 1 to 10, which is a food additive or a food composition.

Item 12. A peptide consisting of any of the amino acid sequences of SEQ ID NOs: 11, 19, 23, 27, and 39.

Item 13. A peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 11 and 13 to 43.

Item 14. A method for producing a peptide composition, which comprises a step of decomposing a protein with an enzyme having an activity of producing a peptide containing a basic amino acid.

Item 15. Item 4. The production method according to Item 14, wherein the peptide composition is a cholesterol absorption inhibitor.

According to the present invention, it is possible to provide a peptide having a cholesterol absorption inhibitory action, a cholesterol absorption inhibitor containing the peptide, a simple method for producing the peptide, and the like.

The results of a micellar disintegration test for a milk protein-derived peptide are shown (Example 2). The vertical axis indicates the amount of cholesterol present in the supernatant at the end of the test operation (after centrifugation) (= the amount of cholesterol present as micelles in the supernatant), and the horizontal axis indicates the final concentration of the test peptide. The test peptide is shown above each graph. The results of a micellar disintegration test for a milk protein-derived peptide are shown (Example 2). The notation in the graph is the same as in FIG. The results of a micellar disintegration test for a milk protein-derived peptide are shown (Example 2). The notation in the graph is the same as in FIG. The results of a micellar disintegration test for a milk protein-derived peptide are shown (Example 2). The notation in the graph is the same as in FIG. The results of a micellar disintegration test for a milk protein-derived peptide are shown (Example 2). The notation in the graph is the same as in FIG. The results of a micellar disintegration test for a glutelin-derived peptide are shown (Example 2). The notation in the graph is the same as in FIG. The results of a micellar disintegration test for a glutelin-derived peptide are shown (Example 2). The notation in the graph is the same as in FIG. The results of a micellar disintegration test for a glutelin-derived peptide are shown (Example 2). The notation in the graph is the same as in FIG. The results of a micellar disintegration test for a glutelin-derived peptide are shown (Example 2). The notation in the graph is the same as in FIG. The results of a micellar disintegration test for a glutelin-derived peptide are shown (Example 2). The notation in the graph is the same as in FIG. The results of the micellar disintegration test for the glutelin-derived peptide and casein hydrolyzate are shown (Example 2). The notation in the graph is the same as in FIG. The results of a micelle formation inhibition test for a milk protein-derived peptide are shown (Example 3). The notation in the graph is the same as in FIG. The results of a micelle formation inhibition test for a milk protein-derived peptide are shown (Example 3). The notation in the graph is the same as in FIG. The results of a micelle formation inhibition test for a milk protein-derived peptide are shown (Example 3). The notation in the graph is the same as in FIG. The results of a micelle formation inhibition test for a glutelin-derived peptide are shown (Example 3). The notation in the graph is the same as in FIG. The results of a micelle formation inhibition test for a glutelin-derived peptide are shown (Example 3). The notation in the graph is the same as in FIG. The results of a micelle formation inhibition test for a glutelin-derived peptide are shown (Example 3). The notation in the graph is the same as in FIG. The results of a micelle formation inhibition test on a glutelin-derived peptide and casein hydrolyzate are shown (Example 3). The notation in the graph is the same as in FIG.

In the present specification, the expressions "contains" and "contains" include the concepts of "contains", "contains", "substantially consists" and "consists of only".

As used herein, amino acids include both natural and artificial amino acids.

In the present specification, the "amino acid residue" of various peptides is a structural unit of amino acids constituting the peptide, and the hydrogen atom is removed from the amino acid for the main chain amino group and / or the main chain. Regarding the carboxyl group of, it represents a group obtained by removing -OH.

In one aspect of the present invention, cholesterol absorption comprising a peptide consisting of 4 to 6 amino acid residues including a basic amino acid residue (sometimes referred to as "the peptide of the present invention" in the present specification). It relates to an inhibitor (sometimes referred to as "the agent of the present invention" in the present specification). This will be described below.

The basic amino acid residue is not particularly limited as long as it is an amino acid residue having a basic side chain. The basic side chain is not particularly limited, and examples thereof include an amino group, a side chain having a group obtained by removing one hydrogen atom from an aromatic ring which is an amine, and the like. The basic amino acid residue is, for example, an amino acid residue having an isoelectric point of more than 7, preferably an amino acid residue having an isoelectric point of 7.5 or more. Specific examples of the basic amino acid residue include arginine residue, lysine residue, histidine residue and the like, and preferably arginine residue, lysine residue and the like. As the basic amino acid residue, one type alone may be adopted, or two or more types may be adopted in combination.

The number of basic amino acid residues in the peptide of the present invention is not particularly limited. The number is, for example, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

The position of the basic amino acid residue in the peptide of the present invention is not particularly limited. Basic amino acid residues can be present, for example, at the N-terminus, C-terminus, or other positions. In one aspect of the present invention, it is preferable that the C-terminal amino acid residue of the peptide of the present invention is a basic amino acid residue from the viewpoint that it can be easily produced from a protein by using enzymatic decomposition.

The number of amino acid residues of the peptide of the present invention is 4 to 6 amino acid residues, and is not particularly limited as long as this is the case. The number of residues is, for example, 4, 5, 6, 4-5, or 5-6.

Specifically, as the peptide of the present invention, for example,
(A) A peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1-43, and (b) one or more amino acid residues substituted or deleted from any of the amino acid sequences of SEQ ID NOs: 1-43. Examples thereof include peptides having an added or inserted amino acid sequence and having cholesterol-containing micelle disintegration promoting activity and / or cholesterol-containing micelle formation inhibitory activity.

The cholesterol-containing micelle disintegration-promoting activity is measured by the method described in Example 2 below. The cholesterol-containing micelle formation inhibitory activity is measured by the method described in Example 3 described later.

In one aspect of the invention, the 50% disintegration concentration of cholesterol-containing micelles and / or the 50% inhibition concentration of cholesterol-containing micelles when peptide (b) is used has the corresponding SEQ ID NO: (eg, peptide (b). In the case of a peptide in which the amino acid is mutated with respect to SEQ ID NO: 1, the concentration when the peptide (a) of SEQ ID NO: 1) is used, for example, 10 times or less, 7 times or less, 5 times or less, 3 times or less, 2 It is preferably 2 times or less, or 1.5 times or less.

In the present specification, the 50% disintegration concentration of cholesterol-containing micelles is measured by the method described in Example 2 below. From the results obtained by this method, the peptide concentration when the cholesterol content of the supernatant is 50% is calculated according to a conventional method, and the obtained value is used as the 50% disintegration concentration.

In the present specification, the 50% inhibitory concentration of cholesterol-containing micelle formation is measured by the method described in Example 3 below. From the results obtained by this method, the peptide concentration when the cholesterol content of the supernatant is 50% is calculated according to a conventional method, and the obtained value is used as the 50% inhibitory concentration.

Among the peptides (a) and (b), the peptide (a) is preferable.

The "plurality" of the peptide (b) is not particularly limited. The "plurality" is, for example, 2 to 3 or 2.

The amino acid mutation in peptide (b) is preferably a substitution, more preferably a conservative substitution.

Conservative substitution means that the amino acid residue is replaced with an amino acid residue having a side chain of similar properties. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is a conservative substitution technique. Other amino acid residues with acidic side chains such as aspartic acid and glutamic acid; amino acid residues with non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine; alanine, valine, leucine, isoleucine, Amino acid residues with non-polar side chains such as proline, phenylalanine, methionine and tryptophan; amino acid residues with β-branched side chains such as threonine, valine and isoleucine, and aromatic side chains such as tyrosine, phenylalanine, tryptophan and histidine. Substitution between amino acid residues is also a conservative substitution.

Among SEQ ID NOs: 1-43, SEQ ID NOs: 11, 19, 23, 27, 39 and the like are preferably mentioned from the viewpoint of cholesterol-containing micelle disintegration promoting activity and / or cholesterol-containing micelle formation inhibitory activity.

In one aspect of the present invention, among SEQ ID NOs: 1 to 43, SEQ ID NOs: 1 to 11 and 13 to 43 are preferable.

The present invention relates to the above-mentioned peptides (a) and peptides (b) in one aspect thereof. These peptides are preferably isolated, concentrated, or purified peptides.

The peptides of the present invention also include those in which terminal amino acid residues are chemically modified as long as the cholesterol-containing micelle disintegration promoting activity and / or the cholesterol-containing micelle formation inhibitory activity is not significantly reduced.

The peptides of the invention, C-terminal, carboxyl group (-COOH), a carboxylate (-COO ^-), those amide (-CONH ₂₎ or ester (-COOR), encompasses the like.

_{Here, R in the ester is, for example, a C 1-6} alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl; for example, a C _3-8 cycloalkyl group such as cyclopentyl, cyclohexyl; for example, phenyl. _{, C 6-12} aryl groups such as α-naphthyl; for example, phenyl-C _{1-2 alkyl groups such as benzyl, phenethyl; C 7-} such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl. ₁₄ Alkyl group; Pivaloyloxymethyl group etc. are used.

Further, in the peptide of the present invention, the amino group on the main chain of the N-terminal amino acid residue is a protective group (for example, a C _1-6 acyl group such _{as C 1-6 alkanoyl such as a formyl group or an acetyl group).} It also includes protected substances, myritoylation, pyroglutamylation, methylation, etc.

The peptide of the present invention includes those in which amino acid residues other than the terminal are chemically modified as long as the cholesterol-containing micelle disintegration promoting activity and / or the cholesterol-containing micelle formation inhibitory activity is not significantly reduced. The peptides of the invention do not include those in which amino acid residues other than the terminal (particularly basic amino acid residues) are chemically modified. Examples of the chemical modification in this case include amidation and esterification of a carboxyl group; protection of an amino group by a protecting group and the like. The esterification and protecting group are the same as those for the above-mentioned chemical modification of the terminal.

In the present specification, "as long as the cholesterol-containing micelle disintegration promoting activity and / or cholesterol-containing micelle formation inhibitory activity is not significantly reduced" means, for example, 50% disintegration concentration of cholesterol-containing micelles and / or cholesterol-containing micelle formation. It means that the 50% inhibition concentration of is, for example, 10 times or less, 7 times or less, 5 times or less, 3 times or less, 2 times or less, or 1.5 times or less of the concentration when the peptide before chemical modification is used. To do.

The peptides of the invention also include the form of salts with acids or bases. The salt is not particularly limited, and either an acidic salt or a basic salt can be adopted. Examples of acidic salts include inorganic acid salts such as hydrochlorides, hydrobromide, sulfates, nitrates, phosphates; acetates, propionates, tartrates, fumarates, maleates, apples. Organic acid salts such as acid salts, citrates, methane sulfonates and paratoluene sulfonates; amino acid salts such as asparaginates and glutamates can be mentioned. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt.

The peptides of the invention also include the form of solvates. The solvent is not particularly limited, and examples thereof include water, ethanol, glycerol, acetic acid and the like.

As the peptide of the present invention, one type alone may be adopted, or two or more types may be adopted in combination. The agent of the present invention is preferably a plurality of types (for example, 2 types or more, 3 types or more, 4 types or more, 5 types or more, 6 types or more, 7 types or more, 8 types or more, 9 types or more, 10 types or more). It contains the peptides of the invention (preferably, a plurality of peptides of the present invention having different amino acid sequences from each other).

The agent of the present invention also includes cases where the peptide contains a peptide other than the peptide of the present invention. The content of the peptide of the present invention in the agent 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, still more preferably 70% by mass or more, based on 100% by mass of the peptide. , More preferably 80% by mass or more, particularly more preferably 90% by mass or more, and particularly preferably 95% by mass or more.

The peptide of the present invention is preferably an enzymatically decomposed product of a protein from the viewpoint that it can be easily produced. In one aspect thereof, the present invention relates to a simple method for producing a peptide (or an agent of the present invention) of the present invention.

The protein is not particularly limited as long as it contains a basic amino acid residue. From the viewpoint of safety, the protein is preferably a food protein. Specific examples of the protein include milk protein (for example, casein, sodium caseinate, MPC (Milk Protein Concentrate), α-casein, β-casein, κ-casein, lactoalbumin, etc., decomposition products thereof, etc.), soybean protein. (For example, glycinin, β-conglycinin, etc.), grain (for example, rice, wheat, etc.) protein (for example, gluten, gluazine, glutelin, etc.), livestock protein (for example, muscle structure protein, myosin, actin, etc.), fish meat protein (for example, muscle fiber protein, etc.) , Actomyosin, myosin, actin, etc.), chicken egg protein (eg, egg white albumin, egg yolk lipoprotein, etc.), pig skin protein (eg, gelatin, etc.) and the like.

The enzyme is not particularly limited as long as it has proteolytic activity, and examples thereof include aspartic protease, serine protease, cysteine protease, metalloprotease, and threonine protease.

Examples of aspartic proteases include pepsin, renin, cathepsin D, cathepsin E, napsin, β-secretase, γ-secretase, signal peptide peptidase, HIV protease, HTLV protease, NS3A protease, plasmepsin, suspase, chymosin and the like.

Serine proteases include, for example, dipeptidyl peptidase 4, trypsin, chymotrypsin, plasmin, thrombin, factors Xa and other blood coagulation / fibrinolytic systems and co-systems and their regulatory system factors, neutrophil elastase, subtilisin, furin, PACE4, etc. PC2, PC7, Kexin, Cucumisin, Ranchbiotic peptidase, Thermitase, Acrosin, Kallikrein, Urokinase, Granteam, Tryptase, Chimase, Catepsin A, Prolylaminopeptidase, P-type signal peptidase, Proteospecific antigen, HCMV protease, V8 protease Protease K and the like can be mentioned.

Examples of cysteine proteases include cathepsins such as cathepsin B, cathepsin H, cathepsin L, cathepsin S, and cathepsin K, legmain, angiotensin converting enzyme, bleomycin hydrolase, carpine, caspase, ER-60, papain, and coronavirus 3CL protease. , Cathepsin, TEV protease, HRV3C protease and the like.

Examples of metalloproteases include ADAM, matrix metalloproteinase, thermosin, neprilysin, carboxypeptidase, endoserin converting enzyme, KELL antigen, bone-forming factor-1, mepurin, serralysin, PAPP, mitochondrial processing protease, insulin degrading enzyme, aminopeptidase, prenyl. Proteases and the like can be mentioned.

Examples of the threonine protease include proteasome and γ-glutamyl transferase.

As the enzyme, one type can be adopted alone, or two or more types can be adopted in combination.

The combination of protein and enzyme can be determined, for example, as follows. That is, it can be determined by a method of searching the amino acid sequence of a protein for a peptide sequence consisting of 4 to 6 amino acid residues including basic amino acid residues, which is generated when degraded by an enzyme. This makes it possible to obtain a protein / enzyme combination that gives a peptide sequence consisting of 4 to 6 amino acid residues including basic amino acid residues. The amino acid sequence of the protein and the cleavage specificity of the enzyme can be easily determined according to known information.

Among the enzymes, an enzyme having an activity of producing a peptide containing a basic amino acid residue is preferable from the viewpoint that the peptide of the present invention can be produced more easily. Specifically, for example, a basic amino acid residue (or a sequence containing it) in a protein is recognized, and an N-terminal peptide bond or a C-terminal peptide bond of the residue (or a sequence containing the same), or a C-terminal peptide bond, or Examples thereof include enzymes having an activity of cleaving the peptide bond in the sequence. Examples of such an enzyme include trypsin.

An enzymatically degraded product of a protein can be obtained by a method including the step of degrading a protein with an enzyme. The degradation can be specifically carried out, for example, by incubating a reaction solution containing a protein and an enzyme. The composition of the reaction solution, reaction temperature, reaction time, protein concentration, enzyme concentration, presence / absence of additives and their types can be appropriately set according to the types of proteins and enzymes.

After the step of degrading the protein with an enzyme, it is preferable to further carry out a step of purifying a peptide consisting of 4 to 6 amino acid residues including basic amino acid residues. In one aspect of the present invention, it is preferable to use the "enzymatic degradation product of protein" obtained through such purification as the peptide of the present invention.

The purification method is not particularly limited as long as it is a method capable of concentrating the peptide of the present invention (that is, increasing the concentration of the peptide of the present invention in the entire peptide). Examples of the purification method include purification with silica gel, purification with synthetic adsorption resin, normal phase partition chromatography, reverse phase partition chromatography, anion exchange chromatography, desalting by electrodialysis, molecular weight by ultrafiltration membrane, etc. It can be purified by drawing, size exclusion chromatography, affinity chromatography and the like. As the purification method, one type alone may be adopted, or two or more types may be used in combination.

The agent of the present invention is for use in suppressing cholesterol absorption. Cholesterol absorption suppression means suppression of cholesterol absorption in the intestinal tract. Cholesterol absorption suppression is preferably carried out through promotion of cholesterol-containing micelle disintegration and / or suppression of cholesterol-containing micelle formation. As a result, the effect can be exhibited earlier than in the case of suppressing cholesterol absorption through, for example, gene expression regulation, so that the timing of ingestion can be flexibly set. Moreover, since it is not necessary to absorb the peptide of the present invention into the intestinal wall, the ingestion form and the like can be flexibly set. From these viewpoints, the agent of the present invention is preferably used for promoting cholesterol-containing micelle disintegration and / or suppressing cholesterol-containing micelle formation.

The agent of the present invention can be used in various fields, for example, as a food additive, a food composition (including a health promoter, a nutritional supplement (supplement, etc.)), a medicine, and the like.

The agents of the present invention are usually taken orally, but are not limited thereto.

The form of the agent of the present invention is not particularly limited, and can take a form usually used in each application depending on the application.

The form of the agent of the present invention is, for example, a tablet (intraoral disintegrating tablet, chewable tablet, effervescent tablet, troche) when the use is a food additive, a medicine, a health enhancer, a dietary supplement (supplement, etc.). Includes agents, jelly-like drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), jelly Examples include agents.

In the form of the agent of the present invention, when the use is a food composition, liquid, gel or solid foods such as ramen, hamburger, fried food, juice, soft drink, tea, soup, soy milk and other beverages, salad oil , Butter and other edible oils, dressings, yogurt, jelly, pudding, sprinkles, soymilk for childcare, cake mix, powdered or liquid dairy products, bread, cookies and the like.

The agent of the present invention may further contain other components, if necessary. The other ingredients are not particularly limited as long as they can be blended in food additives, food compositions, medicines, health enhancers, dietary supplements (supplements, etc.), and the like, but are not particularly limited, for example, bases and carriers. , Solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, colorants, fragrances, chelating agents and the like.

The content of the peptide (active ingredient) of the present invention in the agent of the present invention depends on the intended use, mode of use, state of application, etc., and is not limited, but is, for example, 0.0001 to 100% by mass, preferably 0.0001 to 100% by mass. It can be 0.001 to 50% by mass.

The amount of the agent of the present invention applied (for example, administration, ingestion, inoculation, etc.) is not particularly limited as long as it is an effective amount that exerts the effect, and is usually, as a dry weight of the peptide of the present invention, generally per day. The body weight is 0.1 to 10000 mg / kg. The above application amount is preferably applied once a day or more (for example, 1 to 3 times), and can be appropriately increased or decreased depending on the age, pathological condition, and symptoms.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

Reference example 1. Bile acid binding amount measurement using peptide array
180 kinds of random peptides (4 residues: 90 kinds, 6 kinds: 90 kinds) were designed, and the peptides were synthesized on a cellulose membrane by the F-moc solid-phase synthesis method (spot volume; linker 0.3 μL, amino). acid 0.5 μL). After final deprotection, the membrane was washed with PBS for 1 day.

Using a 1% BSA (019-15123, Wako Pure Chemical Industries, Osaka) in PBS solution to block the peptide array, place each membrane in a square petri dish (AW2000, Eiken Kagaku, Tokyo). 30 ml of solution was added per sheet, and the mixture was shaken at 4 ° C. for 12 h and 50 rpm. The blocked membrane was washed once with PBS (30 ml, 5 min, room temperature).

After washing, a 10 μg / mL taurocholic acid solution (T-4009, Sigma, USA) was poured into a 30 ml membrane and shaken at 37 ° C. for 1 h at 50 rpm to bind the peptide and taurocholic acid. It was then washed once with PBS (30 ml, 5 min, room temperature).

To detect taurocholic acid, a primary antibody, taurocholic acid antibody (FKA502, Cosmobio, Tokyo), was dissolved in 1 ml of sterile water to obtain an antibody solution. Cholic acid antibody: A 0.25% BSA solution was dissolved at a ratio of 1: 200, which was poured into a square petri dish and shaken at 37 ° C. for 1 h at 50 rpm to react with the membrane and the antibody. It was then washed once with 0.05% -Tween 20 TBS (30 ml, 5 min, room temperature).

For fluorescence observation, prepare a secondary antibody solution at a ratio of Alexa Fluor 488 goat anti-rabbit IgG (H + L) (A11008, invitrogen, USA): PBS = 1: 500, and apply this to a new sterile No. 2 square chalet 30. Pour ml at a time. The washed peptide array was gently immersed therein, covered with aluminum foil, and then shaken at 37 ° C. for 1 h and 50 rpm to react with the secondary antibody. It was then washed once with 0.05% -Tween 20 TBS (30 ml, 5 min, room temperature).

The spots of the peptide array after the reaction were detected by a fluoro-image analyzer (Typhoon FLA 9500, Fuji Film Co., Ltd., Tokyo) (excitation wavelength 495 nm, fluorescence wavelength 519 nm). The obtained image data was analyzed using Image Quant software (Fuji Photo Film Co., Ltd.), and the fluorescence intensity of each spot was quantified. In order to subtract the value of non-specific adsorption of the secondary antibody, a peptide array of the same lot was prepared under the reaction conditions of only the secondary antibody without adding the primary antibody, and the fluorescence intensity value was obtained in the same manner.

For each of the 4-residue peptide and 6-residue peptide, all peptides (All 90 peptides), peptides having basic amino acid residues (Peptides with R or K) and other peptides (The others), acidic amino acid residues Peptides with D or E and other peptides (The others), and peptides with hydrophobic amino acid residues (Peptides with I, L or V) and other peptides (The others). The average value and standard deviation (SD) of (Fluorescence intensities) were calculated. The results are shown in Table 1.

表１に示されるとおり、塩基性アミノ酸を有するペプチドは、塩基性アミノ酸を有しないペプチドと比較して、胆汁酸結合活性が高かった。

As shown in Table 1, peptides having basic amino acids had higher bile acid binding activity than peptides not having basic amino acids.

Example 1. From the viewpoint of safety, it is desirable that the peptide that can be used for designing peptides containing basic amino acids and suppressing synthetic cholesterol absorption is an enzymatic degradation product of a protein. Therefore, we focused on trypsin, which is an endopeptidase that can selectively cleave the peptide bond on the C-terminal side of the lysine residue and arginine residue of the protein. It is a tripsin-degrading peptide derived from milk protein (Bos taurus: αS1-casein, αS2-casein, β-casein, κ-casein, α-lactalbumin, and β-lactoglobulin) or rice glutelin (Oryza sativa sbsp. Japonica: Glutelin). The 4- to 6-residue peptides were identified from the amino acid sequences on the database and synthesized according to a conventional method. The amino acid sequences (one-letter notation) of the synthesized peptides are shown in Tables 2 and 3. In the table, the numbers in parentheses next to the amino acid sequence indicate the SEQ ID NOs in the sequence listing.

Example 2. Micelle Disintegration Test The disintegration activity of the peptide synthesized in Example 1 against cholesterol-containing micelles was measured. Specifically, it was carried out as follows.

Test solutions (composition: Na 6.6 mM taurocholic acid, 0.5 mM cholesterol (193.3 mg / L), 1 mM oleic acid, 5 mM monooleine, 0.6 mM phosphatidylcholine, PBS (pH 7.4)) were prepared. The test solution was ultrasonically treated for 30 minutes and then incubated at 37 ° C. for 24 hours to form micelles. The test peptide was added to the test solution to a predetermined concentration (final concentration: 2, 4, 6, 8, or 10 mg / mL) and incubated at 37 ° C. for 1 hour. After centrifugation (10,000 g, 20 minutes), filter with a 0.45 μm filter (ADVANTEC, 13HP045AN), and check the cholesterol level in the filtrate with a cholesterol kit using cholesterol oxidase (Fuji Film Wako Pure Chemical, Cholesterol E-test). Wako) was used for measurement. The amount of cholesterol in the supernatant indicates the amount of cholesterol present as micelles in the supernatant. That is, when the test peptide has micellar disintegration activity, the amount of cholesterol in the supernatant is reduced as compared with the case where the test peptide is not added.

The results of milk protein-derived peptides are shown in FIGS. 1 to 5, and the results of glutelin-derived peptides are shown in FIGS. 6 to 11. Further, FIG. 11 shows data when a casein hydrolyzate (Casein hydrolysate (acid hydrolyzed), Cat No. 102245) manufactured by MERCK Co., Ltd. was used as the test peptide.

Example 3. Micelle formation inhibition test The cholesterol-containing micelle formation inhibitory activity of the peptide synthesized in Example 1 was measured. Specifically, it was carried out as follows.

A test solution (composition: 0.5 mM cholesterol (193.3 mg / L), 1 mM oleic acid, 5 mM monooleine, 0.6 mM phosphatidylcholine, PBS (pH 7.4)) was prepared. Taurocholic acid Na and the test peptide were added to the test solution to a predetermined concentration (taurocholic acid Na final concentration: 6.6 mM) (test peptide final concentration: 2, 4, 6, 8, or 10 mg / mL). After treating with ultrasound for 30 minutes, it was incubated at 37 ° C. for 24 hours to form micelles. After centrifugation (10,000 g, 20 minutes), the mixture was filtered through a 0.45 μm filter (ADVANTEC, 13HP045AN), and the cholesterol content of the filtrate was measured in the same manner as in Example 2. The amount of cholesterol in the supernatant indicates the amount of cholesterol present as micelles in the supernatant. That is, when the test peptide has a micelle formation inhibitory activity, the amount of cholesterol in the supernatant is reduced as compared with the case where the test peptide is not added.

The results of milk protein-derived peptides are shown in FIGS. 12-14, and the results of glutelin-derived peptides are shown in FIGS. 15-18. Further, FIG. 18 shows data when a casein hydrolyzate (Casein hydrolysate (acid hydrolyzed), Cat No. 102245) manufactured by MERCK Co., Ltd. was used as the test peptide.

Claims (16)

A cholesterol absorption inhibitor containing a peptide consisting of 4 to 6 amino acid residues including a basic amino acid residue. The cholesterol absorption inhibitor according to claim 1, wherein the basic amino acid residue is at least one selected from the group consisting of an arginine residue and a lysine residue. The cholesterol absorption inhibitor according to claim 1 or 2, which contains an enzymatically decomposed product of a protein as the peptide. The cholesterol absorption inhibitor according to claim 3, wherein the enzyme is an enzyme having an activity of producing a peptide containing a basic amino acid residue. The cholesterol absorption inhibitor according to claim 3 or 4, wherein the protein is a food protein. The cholesterol absorption inhibitor according to any one of claims 3 to 5, wherein the protein is a rice protein. The cholesterol absorption inhibitor according to any one of claims 1 to 6, which contains a plurality of types of the peptides having different amino acid sequences from each other. As the peptide
For (a) peptides consisting of any of the amino acid sequences of SEQ ID NOs: 11, 19, 23, 27, and 39, and (b) for any of the amino acid sequences of SEQ ID NOs: 11, 19, 23, 27, and 39. Selected from the group consisting of a peptide consisting of an amino acid sequence in which one or more amino acid residues are substituted, deleted, added or inserted, and having a cholesterol-containing micelle decay promoting activity and / or a cholesterol-containing micelle formation inhibitory activity. The cholesterol absorption inhibitor according to any one of claims 1 to 7, which contains at least one type. As the peptide
(A) A peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1-43, and (b) one or more amino acid residues substituted or deleted from any of the amino acid sequences of SEQ ID NOs: 1-43. Any of claims 1 to 7, which comprises at least one selected from the group consisting of an added or inserted amino acid sequence and consisting of a peptide having a cholesterol-containing micelle disintegration promoting activity and / or a cholesterol-containing micelle formation inhibitory activity. The cholesterol absorption inhibitor described in Crab. The cholesterol absorption inhibitor according to any one of claims 1 to 9, which is used for promoting cholesterol-containing micelle disintegration and / or suppressing cholesterol-containing micelle formation. The cholesterol absorption inhibitor according to any one of claims 1 to 10, which is a food additive or a food composition. A peptide consisting of any of the amino acid sequences of SEQ ID NOs: 11, 19, 23, 27, and 39. A peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 11 and 13 to 43. A method for producing a peptide composition, which comprises a step of decomposing a protein with an enzyme having an activity of producing a peptide containing a basic amino acid. The production method according to claim 14, wherein the peptide composition is a cholesterol absorption inhibitor. A method for searching a peptide sequence consisting of 4 to 6 amino acid residues including basic amino acid residues, which is generated when degraded by an enzyme, from the amino acid sequence of a protein.

Images