Secondary bile acid absorption inhibitor

Abstract

To provide a novel secondary bile acid absorption inhibitor.SOLUTION: A secondary bile acid absorption inhibitor comprises at least one selected from the group consisting of a peptide or a protein containing any amino acid sequence among specific 42 amino acid sequences, wherein the peptide or protein may comprise, as an active ingredient, a peptide or protein contained in a pepsin digest of soy protein and / or an indigestible protein in soy protein.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a secondary bile acid absorption inhibitor. [Background technology]

[0002] Patent Document 1 is the technology of the present applicant, and discloses a cholesterol-lowering protein hydrolysate containing peptides having a molecular weight of 200 to 1500 obtained by hydrolyzing a protein with an enzyme.

[0003] Patent Document 2 is the applicant's technology and discloses a cholesterol-lowering substance which is a water-insoluble fraction obtained by removing a water-soluble fraction from an enzymatic hydrolyzate of a protein, which is soluble in an SDS solution and has a molecular weight of 1,000 to 150,000 after solubilization in the SDS solution.

[0004] Patent Document 3 is the applicant's technology, and discloses that a peptide obtained from an enzymatic hydrolysis product of soybean oleosin protein binds to primary bile acids, disrupts primary bile acid micelles, and inhibits the absorption of primary bile acids. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Published Patent Application No. 60-011425 [Patent Document 2] International Publication No. WO89 / 001495 [Patent Document 3] Published Patent Application JP2014-100075 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a novel secondary bile acid absorption inhibitor. [Means for solving the problem]

[0007] The present inventors have found that a peptide consisting of specific amino acid residues binds to secondary bile acids (DCA, etc.). The present invention includes the following secondary bile acid absorption inhibitors.

[0008] Section 1. A secondary bile acid absorption inhibitor comprising at least one selected from the group consisting of peptides and proteins comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 42.

[0009] Section 2. 2. The secondary bile acid absorption inhibitor according to item 1, comprising at least one selected from the group consisting of peptides or proteins comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12.

[0010] Section 3. 2. The secondary bile acid absorption inhibitor according to 1, comprising a plurality of types of the peptides or proteins having different amino acid sequences.

[0011] Section 4. The peptide or the protein is peptic digest of soy protein, and / or Resistant proteins in soybean protein, 2. The secondary bile acid absorption inhibitor according to claim 1, comprising as an active ingredient a peptide or protein contained in

[0012] Section 5. 2. The secondary bile acid absorption inhibitor according to Item 1, wherein the secondary bile acid is at least one secondary bile acid selected from the group consisting of deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and hyodeoxycholic acid (HDCA).

[0013] Section 6. 2. The secondary bile acid absorption inhibitor according to item 1, which is a food additive or a food composition.

[0014] Secondary bile acids (DCA, etc.) converted by microorganisms from primary bile acids (CA, etc.) are thought to be the cause of colon cancer. The peptide of the present invention, which is composed of specific amino acid residues, binds to secondary bile acids, increases the content of secondary bile acids in feces, and plays a role in excreting them from the body, which is expected to eliminate the cause of colon cancer. Effect of the Invention

[0015] The present invention can provide a novel secondary bile acid absorption inhibitor. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 shows a micelle-disrupting activity evaluation system using a 96-well plate. [Diagram 2] FIG. 2 shows the correlation between the 96-well plate evaluation method and the conventional method. [Diagram 3] FIG. 3 shows the DC50 calculation results using the 96-well plate evaluation method, A) concentration dependency using the 96-well evaluation method, and B) results using the conventional method. [Figure 4] FIG. 4 shows the micelle-disrupting activity of 42 peptides. [Diagram 5] FIG. 5 shows the results of an Ala scan of the VRFPLL peptide. [Figure 6] FIG. 6 shows the micelle-disrupting activity of real-length peptides. [Figure 7] FIG. 7 shows the mapping results of peptides identified by LC-MS / MS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention will be described in detail below. The embodiments of the present invention are intended to allow a better understanding of the gist of the invention, and do not limit the contents of the invention unless otherwise specified.

[0018] In this specification, the terms "comprise" and "containing" are concepts which encompass all of "comprise," "consist essentially of," and "consist of."

[0019] In this specification, when a numerical range is indicated as "A to B," it means "A or more and B or less."

[0020] In this specification, the designations parts, % etc. are generally used.

[0021] In this specification, the terms "parts by mass," "parts by weight," "% by mass," and "% by weight (wt%)" are generally used.

[0022] The present invention includes the following secondary bile acid absorption inhibitors:

[0023] [1] Secondary bile acid absorption inhibitors (1-1) Peptide or protein containing the amino acid sequence of SEQ ID NO: 1 to 42 Bile acids biosynthesized in the liver are called primary bile acids, and typical bile acids in humans are cholic acid, chenodeoxycholic acid, etc. Some of the primary bile acids are converted by intestinal bacteria in the intestinal tract, and the metabolites produced by these bacteria are called secondary bile acids, and include deoxycholic acid, lithocholic acid, etc.

[0024] Secondary bile acids, which are converted from primary bile acids by microorganisms, are thought to be the cause of colorectal cancer.

[0025] Primary bile acids and secondary bile acids are absorbed into the body by forming micelles. By ingesting a peptide or protein containing any of the amino acid sequences of SEQ ID NOs: 1 to 42, it exhibits secondary bile acid-containing micelle disintegration promoting activity and secondary bile acid-containing micelle formation inhibitory activity, suppressing absorption into the body, increasing the secondary bile acid content in feces, and excreting them from the body. Soybean peptides or soybean proteins containing any of the amino acid sequences of SEQ ID NOs: 1 to 42 bind to secondary bile acids and play a role in excreting them from the body.

[0026] The secondary bile acid absorption inhibitor of the present invention contains at least one selected from the group consisting of peptides or proteins containing any of the amino acid sequences of SEQ ID NOs: 1 to 42, in that it has good secondary bile acid binding ability. The secondary bile acid absorption inhibitor preferably contains a plurality of the peptides or proteins having different amino acid sequences.

[0027] The secondary bile acid of the secondary bile acid absorption inhibitor is preferably at least one secondary bile acid selected from the group consisting of deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and hyodeoxycholic acid (HDCA).

[0028] The term "amino acid" includes both natural and artificial amino acids. The term "amino acid residue" of a peptide or protein refers to a structural unit of an amino acid that constitutes a peptide or protein, and refers to a group formed by removing a hydrogen atom from the main chain amino group and / or removing an -OH from the main chain carboxyl group of an amino acid.

[0029] The secondary bile acid absorption inhibitor contains at least one selected from the group consisting of peptides or proteins having the amino acid sequence of any of SEQ ID NOs: 1 to 42, preferably any of SEQ ID NOs: 1 to 12, in that it has good secondary bile acid binding ability.

[0030] [ka]

[0031] The secondary bile acid absorption inhibitor preferably contains a minimum of 6 residues and a maximum of 24 residues of any of the amino acid sequences (amino acid residues of peptides) of SEQ ID NOs: 1 to 42, in order to have good secondary bile acid (DCA, etc.) binding ability.

[0032] (1-2) Peptide or protein The protein is preferably an edible protein. The edible protein is a protein that is abundantly contained in food materials, and is preferably a milk protein (e.g., casein, sodium caseinate, MPC (Milk Protein Concentrate), α-casein, β-casein, κ-casein, lactalbumin, etc., and their decomposition products), a soy protein (e.g., glycinin, β-conglycinin, soybean lipid-associated protein, etc.), a cereal (e.g., rice, wheat, etc.) protein (e.g., gluten, gluadin, glutelin, glutenin storage protein, etc.), a livestock meat protein (e.g., muscle structural protein, myosin, actin, etc.), a fish meat protein (e.g., muscle fiber protein, actomyosin, myosin, actin, etc.), a chicken egg protein (e.g., egg white albumin, egg yolk lipoprotein, etc.), a pig skin protein (e.g., gelatin, etc.), etc.

[0033] The peptide or protein is preferably a soybean peptide or soybean protein, since it has good secondary bile acid binding ability.

[0034] The peptide or protein used as an active ingredient is a peptide or protein contained in a pepsin digest of soybean protein, which has good secondary bile acid binding ability.

[0035] The peptide or protein used as the active ingredient is a peptide or protein contained in indigestible protein (including peptides excised in post-treatment) in soybean protein, which has good secondary bile acid binding ability.

[0036] The peptide or protein is preferably an isolated, enriched, or purified peptide or protein.

[0037] The peptide or protein also includes those in which the terminal amino acid residues and / or non-terminal amino acid residues have been chemically modified, so long as the secondary bile acid-containing micelle disintegration-promoting activity is not significantly reduced.

[0038] Peptides or proteins also include those in which the C-terminus is a carboxyl group (-COOH), a carboxylate (-COO-), an amide (-CONH2) or an ester (-COOR).

[0039] R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1-6 Alkyl groups; for example, cyclopentyl, cyclohexyl, etc. 3-8 Cycloalkyl groups; for example, C phenyl, α-naphthyl, etc. 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl group; α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14 Aralkyl groups; pivaloyloxymethyl groups, and the like.

[0040] In the case of a peptide or protein, the amino group on the main chain of the N-terminal amino acid residue is protected by a protecting group (e.g., a formyl group, an acetyl group, or the like). 1-6 Alkanoyl C 1-6 These also include those protected with an acyl group, myristoylation, pyroglutamylation, methylation, etc. The peptides and proteins of the present invention also include salt forms with acids or bases. The salt is not particularly limited, and either an acid salt or a basic salt can be used.

[0041] Examples of acid salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, paratoluenesulfonate, etc.; amino acid salts such as aspartate, glutamate, etc.

[0042] Examples of basic salts include alkali metal salts such as sodium salts, potassium salts, etc.; alkaline earth metal salts such as calcium salts, magnesium salts, etc.; and the like.

[0043] The peptide or protein also includes a form of a solvate. The solvent is not particularly limited, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0044] The peptide or protein may be used alone or in combination of two or more. The peptide or protein preferably contains multiple (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more) types of peptides or proteins (preferably, multiple types of the peptides of the present invention having different amino acid sequences).

[0045] The peptide or protein is a peptide or protein consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 42, and a peptide or protein consisting of an amino acid sequence in which one or more amino acid residues have been substituted, deleted, added or inserted relative to any of the amino acid sequences of SEQ ID NOs: 1 to 42, and having secondary bile acid-containing micelle disintegration-promoting activity and / or secondary bile acid-containing micelle formation-inhibiting activity. The peptide or protein may have an amino acid sequence in which one or more amino acid residues have been substituted, deleted, added or inserted relative to any of the amino acid sequences of SEQ ID NOs: 1 to 42, as long as the secondary bile acid-containing micelle disintegration-promoting activity is not significantly reduced.

[0046] (1-3) Activity of promoting the disintegration of secondary bile acid-containing micelles and activity of inhibiting the formation of secondary bile acid-containing micelles The secondary bile acid-containing micelle disintegration-promoting activity and secondary bile acid-containing micelle formation-inhibiting activity exhibited by the secondary bile acid absorption inhibitor of the present invention are measured by the method described in the Examples below.

[0047] Inhibition of secondary bile acid absorption means inhibition of absorption of secondary bile acid in the intestinal tract. Inhibition of secondary bile acid absorption is preferably achieved through promotion of disintegration of secondary bile acid (cholesterol)-containing micelles and / or inhibition of formation of secondary bile acid (cholesterol)-containing micelles. This allows the timing of intake to be flexibly set. The secondary bile acid absorption inhibitor is preferably used for promotion of disintegration of secondary bile acid-containing micelles and / or inhibition of formation of secondary bile acid-containing micelles.

[0048] As used herein, "as long as the secondary bile acid-containing micelle disintegration-promoting activity and / or secondary bile acid-containing micelle formation-inhibiting activity is not significantly reduced" means that the 50% disruption concentration of secondary bile acid-containing micelles and / or the 50% inhibition concentration of secondary bile acid-containing micelle formation 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.

[0049] As long as the activity of promoting the collapse of secondary bile acid-containing micelles is not significantly decreased, when a peptide or protein in which a terminal amino acid residue and / or a non-terminal amino acid residue has been chemically modified, or a peptide or protein having an amino acid sequence in which one or more amino acid residues have been substituted, deleted, added or inserted relative to any of the amino acid sequences of SEQ ID NOs: 1 to 42 is used, the 50% collapse concentration of secondary bile acid-containing micelles is preferably 3 times or less, 2 times or less, 1.5 times or less, 1.3 times or less, 1.2 times or less, or 1.1 times or less of the concentration when the corresponding amino acid sequence of SEQ ID NOs: 1 to 42 is used. The lower limit of this concentration is preferably 0.1 times or more, 0.2 times or more, 0.4 times or more, 0.6 times or more, 0.8 times or more, 0.9 times or more, or 0.95 times or more of the concentration when the corresponding amino acid sequence of SEQ ID NOs: 1 to 42 is used.

[0050] In the present specification, the 50%-destruction concentration of secondary bile acid-containing micelles is measured by the method described in the Examples below. From the results obtained by this method, the peptide concentration when the amount of secondary bile acid in the supernatant is 50% is calculated according to a conventional method, and the obtained value is defined as the 50%-destruction concentration.

[0051] [2] Method for producing secondary bile acid absorption inhibitor (peptide or protein) The peptide or protein contained in the secondary bile acid absorption inhibitor of the present invention can be produced by various methods.

[0052] (2-1) Solid phase synthesis method The peptide or protein can be produced, for example, by solid phase synthesis.

[0053] (2-2) Enzymatic Decomposition of Proteins The peptide or protein is preferably an enzymatic hydrolysis product of a protein, from the viewpoint of easy production. When the peptide or protein is a peptide consisting of a partial amino acid sequence of an edible protein, it can be obtained by enzymatic hydrolysis of the protein.

[0054] The enzyme has proteolytic activity, and is preferably an aspartic acid protease (preferably pepsin), a serine protease, a cysteine ​​protease, a metalloprotease, a threonine protease, or the like.

[0055] The aspartic acid protease is preferably pepsin, renin, cathepsin D, cathepsin E, napsin, β-secretase, γ-secretase, signal peptide peptidase, HIV protease, HTLV protease, NS3A protease, plasmepsin, saspase, chymosin, or the like.

[0056] Preferred examples of serine proteases include dipeptidyl peptidase 4, trypsin, chymotrypsin, plasmin, thrombin, factor Xa, and other factors of the blood coagulation / fibrinolysis system and the complement system and their control 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] Preferred 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] The metalloprotease is preferably 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, prenyl protease, or the like.

[0059] Preferred examples of the threonine protease include proteasome and gamma glutamyltransferase.

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

[0061] The combination of edible protein and enzyme is determined, for example, by a method of searching for the amino acid sequence of SEQ ID NO: 1 to 42 contained in the peptide or protein in the secondary bile acid absorption inhibitor of the present invention, which is generated when the amino acid sequence of the edible protein is decomposed with an enzyme. This makes it possible to obtain an edible protein / enzyme combination that provides the amino acid sequence of SEQ ID NO: 1 to 42 contained in the peptide or protein in the secondary bile acid absorption inhibitor 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] A method including a step of decomposing a protein with an enzyme can produce an enzymatic decomposition product of a protein. Specifically, the decomposition is 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. are appropriately set according to the types of protein and enzyme.

[0063] After the step of hydrolyzing proteins with an enzyme, a step of purifying the peptide or protein contained in the secondary bile acid absorption inhibitor of the present invention is preferably further carried out. The purification method is not particularly limited as long as it is a method that can concentrate the peptide or protein (i.e., can increase the peptide or protein contained in the secondary bile acid absorption inhibitor of the present invention in the whole peptide or protein).

[0064] The purification method preferably includes purification using silica gel, purification using a synthetic adsorption resin, normal phase partition chromatography, reverse phase partition chromatography, anion exchange chromatography, desalting by electrodialysis or the like, molecular weight fractionation using an ultrafiltration membrane or the like, size exclusion chromatography, affinity chromatography, or the like.

[0065] The peptide or protein contained in the secondary bile acid absorption inhibitor of the present invention is preferably purified by utilizing its secondary bile acid binding ability (for example, by utilizing a carrier carrying a secondary bile acid).

[0066] The purification method may be a single method or a combination of two or more methods.

[0067] [3] Food additives and food compositions (3-1) Food type The secondary bile acid absorption inhibitor of the present invention contains a peptide or protein containing any of the amino acid sequences of SEQ ID NOs: 1 to 42, and has a secondary bile acid absorption inhibitory effect. The peptide or protein can be used as an active ingredient of the secondary bile acid absorption inhibitor. The peptide or protein preferably contains as an active ingredient a peptide or protein contained in a pepsin digest of soybean protein and a resistant protein in soybean protein.

[0068] By ingesting pepsin digests of soybean protein and the resistant proteins in soybean protein, the absorption of secondary bile acids is suppressed and they are excreted from the body.

[0069] The food additive and food composition of the present invention contain the secondary bile acid absorption inhibitor of the present invention, and may contain a peptide or protein other than the peptide or protein contained in this secondary bile acid absorption inhibitor.

[0070] The content of the secondary bile acid absorption inhibitor of the present invention in the food additive and food composition of the present invention is, for example, 0.0001% by mass to 100% by mass, preferably 10% by mass or more, 30% by mass or more, more preferably 50% by mass or more, 70% by mass or more, even more preferably 80% by mass or more, 90% by mass or more, particularly preferably 95% by mass or more, or may be 100% by mass, relative to the total amount (100% by mass) of the food additive and food composition.

[0071] When the secondary bile acid absorption inhibitor is used for purposes such as food additives, health promotion agents, nutritional supplements (supplements, etc.), the form of the secondary bile acid absorption inhibitor is preferably a tablet (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly-like drops, etc.), pill, granule, fine granule, powder, hard capsule, soft capsule, dry syrup, liquid (including drinks, suspensions, and syrups), jelly, etc.

[0072] When the secondary bile acid absorption inhibitor is used in the form of a food composition, it is preferably in the form of liquid, gel or solid food, for example, ramen, hamburgers, fried foods, juice, soft drinks, tea, soup, beverages such as soy milk, edible oils and fats such as salad oil and butter, dressings, yogurt, jelly, pudding, sprinkles, powdered milk for infants, cake mixes, powdered or liquid dairy products, bread, cookies, etc.

[0073] The secondary bile acid absorption inhibitor may further contain other components as necessary. The other components are components that can be blended into food additives, food compositions, health promotion agents, nutritional supplements (supplements, etc.), etc., such as bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, colorants, flavors, chelating agents, etc.

[0074] The amount of the agent of the present invention or the composition of the present invention to be applied (e.g., ingested) may be an effective amount that exerts its effect, and is generally 0.01 mg / kg body weight to 1,000 mg / kg body weight per day as the dry weight of the peptide of the present invention. The lower limit of the application amount is, for example, 0.1 mg / kg body weight, 1 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight, 20 mg / kg body weight, 50 mg / kg body weight, 100 mg / kg body weight, 200 mg / kg body weight, or 500 mg / kg body weight. This application amount is preferably applied once or more times a day (e.g., 1 to 3 times), and may be increased or decreased as appropriate according to age, pathology, and symptoms.

[0075] The secondary bile acid absorption inhibitor of the present invention includes embodiments such as a food additive and a food composition (including a health promoter and a nutritional supplement (such as a supplement)). The secondary bile acid absorption inhibitor of the present invention is preferably taken orally.

[0076] (3-2) Health functional foods The secondary bile acid absorption inhibitor, food additive, and food composition of the present invention are useful as health functional foods such as foods for specified health uses and foods with functional claims.

[0077] By ingesting the secondary bile acid absorption inhibitor, food additive, and food composition of the present invention, for example, as soy protein (β-conglycinin) or soy milk, it is expected that the absorption of secondary bile acids will be suppressed and the excretion of secondary bile acids from the body will be increased. By ingesting the secondary bile acid absorption inhibitor, food additive, and food composition of the present invention, it is expected that the absorption of secondary bile acids, which are thought to be the cause of colon cancer, will be suppressed.

[0078] The secondary bile acid absorption inhibitor, food additive, and food composition of the present invention are foods that regulate the intestinal environment and are useful for improving dietary habits. EXAMPLES

[0079] The present invention will be described in detail below with reference to examples.

[0080] The present invention is not limited to these examples.

[0081] [1] Analysis of secondary bile acid-binding peptides using peptide arrays We conducted a comprehensive search and sequence feature analysis of peptides derived from eight soy protein sequences that exhibit secondary bile acid binding ability. We designed a peptide library from the amino acid sequences of soy protein, and narrowed down the candidate peptides by evaluating the binding activity of secondary bile acids to solid-phase synthesis peptide arrays. We then synthesized the peptides and evaluated the binding strength of free peptides using the solubilization of cholesterol by secondary bile acid micelles as an indicator.

[0082] The difference in binding ability of the peptide to conjugated and unconjugated bile acids was confirmed.

[0083] Free peptides were synthesized for 42 types of 6-residue peptides, and the strength of binding was evaluated in a bile acid micelle disruption test. A system was constructed in which the following bile acids could be evaluated in a 96-well plate: sodium deoxycholate (DCA), sodium cholate (CA), sodium taurodeoxycholate hydrate (TDCA), and sodium taurocholic acid sodium salt (TCA).

[0084] The compounds were classified by clustering into 18 groups that disrupted only DCA micelles, 10 groups that also showed disruptive activity against CA micelles, 6 groups that showed slight activity against TDCA and TCA micelles, 4 groups that showed activity against all four types of micelles, and 4 groups that showed little activity against any of the micelles.

[0085] Based on the results of LC-MS / MS analysis of soy protein pepsin hydrolysate (SPH), 10 sequences containing 42 peptides were synthesized from 1,354 soluble peptides, and their micelle-disrupting activity was examined. The effect of the added residues on the DCA micelle-disrupting activity was not significant, and the DCA micelle-disrupting activity as the responsible residue was maintained even when the residues were lengthened.

[0086] 1. Experimental Method 1-1. Bile acid micelle-disrupting activity test (conventional method) <Day 1> (1) Preparation of cholesterol solution (1-1) Weigh out 3.89 mg of cholesterol, 8.808 mg of phosphatidylcholine, 9.7 μl of oleic acid, and 37.9 μl of monoolein into a centrifuge tube. (1-2) Dissolve in 1 ml of methanol and homogenize the lipids. (1-3) Place in a vacuum degassing apparatus for 12 hours or more to completely remove the methanol.

[0087] <Day 2> (2) Preparation of DCA micelle solution (2-1) Weigh out 54.7 mg of DCA into a centrifuge tube and dissolve it in 20 ml of PBS (Phosphate-Buffered Saline). If using other bile acids, standardize the molar amount; for example, change to 70.9 mg for TCA. (2-2) Add the DCA solution to the cholesterol solution and sonicate for 3 minutes. (2-3) Incubate at 37℃ for 2 to 6 hours.

[0088] (3) Peptide addition (3-1) A centrifuge tube was prepared and the peptide powder was weighed out to obtain the desired concentration (6.0 mg / ml). 50 When evaluating the above, the peptide concentrations are set to six conditions: 0, 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, 4.0 mg / ml, and 6.0 mg / ml. (3-2) Add DCA micelle solution to the desired concentration. (3-3) Sonicate for 3 minutes. (3-4) Incubate at 37℃ with shaking at 140 rpm for 2 to 6 hours.

[0089] (4) Quantification of supernatant cholesterol content (4-1) Centrifuge the precipitate and the supernatant at 37°C, 20 min, and 4,000 rpm. (4-2) Pass the supernatant through a 0.2 μm filter. (4-3) Add 500 μl of the supernatant to a centrifuge tube containing 3 ml of the color reagent from the cholesterol analysis kit. (4-4) Incubate in a 37℃ incubator for 3 minutes. (4-5) Centrifuge at 37°C, 20 min, and 4,000 rpm to remove the supernatant. (4-6) Measure the absorbance at 600 nm.

[0090] 1-2. Bile acid micelle disintegration test (96-well evaluation system) <Day 1> (1) Preparation of cholesterol solution (1-1) Cholesterol, phosphatidylcholine, oleic acid, and monoolein were weighed out in the order shown in Table 1 (Preparation of cholesterol solution) and placed in a 15 ml centrifuge tube. (1-2) A specified amount of methanol was added and vortexed to dissolve. (1-3) 1 ml of the methanol solution was dispensed into 50 ml centrifuge tubes. (1-4) Place in a vacuum degassing apparatus for 12 hours or more to completely remove the methanol.

[0091] Cholesterol, CAS57-88-5, molecular weight 386.66 Phosphatidylcholine, CAS8002-43-5, molecular weight 786.129 Oleic acid, CAS112-80-1, molecular weight 282.46, density 0.89 [g / ml] Monoolein, CAS111-03-5, molecular weight 356.54, density 0.94 [g / ml]

[0092] [Table 1]

[0093] <Day 2> (2) Preparation of bile acid micelle solution (2-1) As shown in Table 2 (amount of bile acid in the bile acid micelle solution), the bile acid was weighed out and placed in the centrifuge tubes prepared in (1) to prepare each of the four types of bile acid. (2-2) 10 ml of PBS was added and the mixture was sonicated for 3 minutes. (2-3) Incubated at 37°C for 2 hours.

[0094] DCA: Sodium Deoxycholate, CAS302-95-4, molecular weight 414.55 CA: Sodium Cholate, CAS361-09-1, molecular weight 430.55 TDCA: Sodium taurodeoxycholate hydrate, CAS207737-97-1, molecular weight 521.69 TCA: Taurocholic acid sodium salt, CAS345909-26-4, molecular weight 537.68

[0095] [Table 2]

[0096] (3) Preparation of peptide solution buffer (3-1) Four types of bile acids were weighed out as shown in Table 3 (Preparation of Peptide Dissolution Buffer) and placed together in one centrifuge tube. (3-2) 10 ml of PBS was added and the mixture was sonicated for 3 minutes. (3-3) 100 μl of the (3-2) solution was added to 19.9 ml of PBS (200-fold dilution).

[0097] [Table 3]

[0098] (4) Preparation of peptide solution (4-1) A microtube was prepared and the required amount of peptide powder was weighed out. (4-2) The buffer prepared in (3) was added to the mixture to obtain the desired concentration (6.0 mg / ml). (4-3) Vortex to dissolve.

[0099] (5) Micelle collapse reaction in 96-well plate (5-1) A v-bottom 96-well plate was prepared, and the bile acid solution prepared in (2) was dispensed into each well in an amount of 100 μl. (5-2) 100 μl of the peptide solution prepared in (4) was added to (5-1). (5-3) The well plate was sealed and fixed in an incubator shaker. (5-4) The mixture was incubated at 37°C for 2 hours while shaking at 140 rpm.

[0100] (6) Quantification of Supernatant Cholesterol Content (6-1) The precipitate and the supernatant were centrifuged at 37°C, 20 minutes, and 4,000 rpm. (6-2) 100 μl of the supernatant was collected and transferred to a 96-well plate filter. (6-3) The pressure was reduced, and the liquid that passed through the filter was collected in a v-bottom 96-well plate. (6-4) 40 μl of the solution (6-3) was added to a 96-well plate containing 240 μl of the color-developing reagent of the cholesterol analysis kit. (6-5) Incubated at 37℃ for 3 minutes. (6-2) The absorbance was measured at 600 nm.

[0101] 2. Results and Discussion 2-1. Construction of a 96-well multi-sample evaluation system capable of evaluating bile acid micelle-disrupting activity The conventional method was to prepare cholesterol-containing bile acid micelles in advance by ultrasonication in a centrifuge tube, add the peptide, and then ultrasonicate again to try to reform the micelles, after which the precipitate was removed and the cholesterol dissolved and suspended in the supernatant was analyzed using an assay kit. The conventional method can simultaneously measure not only the micelle disruption activity of the added peptide, but also the activity of inhibiting micelle reformation.

[0102] However, in order to increase throughput so that multiple samples and conditions can be evaluated simultaneously, we investigated a method using a 96-well plate (96-well evaluation system). The outline is shown in Figure 1 (96-well plate micelle disruption activity evaluation system).

[0103] A small amount of a mixture of four bile acids is added to the peptide to be tested to dissolve it, and a micellar solution containing cholesterol that has been prepared in advance by ultrasonic treatment is added to the wells and left at 37°C for 2 hours. The supernatant is then filtered using a plate centrifuge to recover the supernatant's cholesterol concentration, which is then analyzed using an assay kit.

[0104] In the absence of micelle disrupting activity, the micelle formation rate is high and the cholesterol concentration in the supernatant is high, whereas in the presence of micelle disrupting activity, the cholesterol concentration is low. The cholesterol concentration in the supernatant was expressed as micelle solubility (%).

[0105] Of the 18 peptides, 16 that were easy to synthesize were used to compare with the conventional method. Although there were some discrepancies for CA micelles, there was about a 90% correlation for other bile acids, and it was determined that this method could be used as an evaluation method (Figure 2 (Correlation between the 96-well plate evaluation method and the conventional method)).

[0106] In the conventional method, ultrasonic treatment is performed after adding peptides. CA has high micelle stability, so it is difficult for it to collapse even in the presence of CA-bound peptides, and it is thought that some micelles are destroyed by ultrasonic treatment.

[0107] Can concentration dependence be observed in a 96-well evaluation system? Furthermore, can the half-life value of micelle solubility (DC 50 ) can be calculated, the concentration dependence of the physiologically active peptide VRFPLL (SEQ ID NO: 34) on the micelle-disintegrating activity using four types of bile acids was examined.

[0108] Figure 3 (DC in 96-well plate evaluation method) 50 As shown in the calculation results, A) concentration dependence in the 96-well evaluation method, and B) results in the conventional method, concentration dependence was observed, and the DC 50A value almost equivalent to the above (approximately 2 mg / ml) was obtained (2.76 mg / ml).

[0109] 2-2. Micelle-disrupting activity of 42 peptides In the DCA binding experiment, (1) 10 peptides with high DCA-binding fluorescence intensity (signal intensity) and (2) 10 peptides with small standard deviation (SD) were selected from the high DCA-binding sequences. Free peptides were synthesized for a total of 18 types of 6-residue peptides, excluding overlaps, from (1) and (2), and the binding strength was evaluated in a bile acid micelle disruption test.

[0110] Furthermore, 42 types of 6-residue peptides that satisfied the DCA binding fluorescence intensity ≧Average+2SD were targeted. The obtained micelle solubilities are summarized in Table 4 (micelle disruption activity of 42 types of peptides).

[0111] [Table 4(1)]

[0112] [Table 4(2)]

[0113] The results of clustering using this result are shown in the color scale in Figure 4 (clustering analysis results regarding the micelle-disrupting activity of 42 peptides). The sequence numbers of the sequences of the 42 peptides in Figure 4 are shown in SEQ ID NOs: 1 to 42 above.

[0114] They were divided into 18 groups that disrupted only DCA micelles, 10 groups that also showed disruptive activity against CA micelles, 6 groups that showed slight activity against TDCA and TCA micelles, 4 groups that showed activity against all four types of micelles, and 4 groups that showed little activity against those micelles.

[0115] 2-3. Determining Responsible Residues by Ala Scanning To determine the important residues in the sequence, Ala-substituted peptides of the VRFPLL peptide (SEQ ID NO: 34) were synthesized and their effects on micelle-disintegrating activity were examined. The results are shown in Figure 5 (Ala scan results of the VRFPLL peptide).

[0116] All residue substitutions slightly decreased the disruptive activity of TCA micelles. Substitution of the second residue R with A resulted in a slight disruptive activity against TDCA micelles. No clear decrease in activity was observed due to residue substitution. This suggests that the physical properties of the entire peptide chain are important and that substitution of a single residue does not affect the activity.

[0117] 2-4. Micelle-disrupting activity of long-residue, highly active peptides identified by LC-MS / MS Soy protein was hydrolyzed with pepsin for 3 hours, and the soluble fraction of the resulting soy protein pepsin hydrolysate (SPH) was analyzed by LC-MS / MS to identify peptides. 1,354 peptides were identified. Among them, peptides with sequences matching 42 highly active peptides are shown in Table 5 (sequences containing highly active peptides among the 1,354 peptides identified by LC-MS / MS).

[0118] The sequences that were evaluated for synthesis are marked with an X in the Check column. Peptides with 6 to 30 residues were picked up. These are real soluble peptides contained in SPH, and can be said to be peptides that have been lengthened from 6-residue peptides.

[0119] [Table 5(1)]

[0120] [Table 5(2)]

[0121] Ten sequences were selected from the peptides in Table 5, and the effect of increasing the residue length on micelle-disrupting activity was examined. The results are shown in Figure 6 (micelle-disrupting activity of actual peptides with increased residue length). The sequence numbers of the sequences in Examples 1 to 10 in Figure 6 are shown in Table 5.

[0122] None of the peptides exhibited a loss of activity against DCA micelles.

[0123] Peptide No. 1 (SEQ ID NO: 45) showed a significant improvement in DCA activity simply by adding K to the N-terminus.

[0124] Peptides No. 4 (SEQ ID NO: 62), No. 6 (SEQ ID NO: 68) and No. 10 (SEQ ID NO: 88) showed a decrease in DCA activity of about 20%.

[0125] Peptides No. 2 (SEQ ID NO: 52), No. 3 (SEQ ID NO: 53) and No. 4 (SEQ ID NO: 62) were newly found to have CA activity.

[0126] No. 8 (SEQ ID NO: 84) is a nine-residue peptide consisting of two highly active peptides (FGSLRK and LRKNAM) linked together, but it has lost its CA activity, and the results were similar to the properties of LRMNAM.

[0127] No. 2 (SEQ ID NO: 52) and No. 9 (SEQ ID NO: 86) have similar C-terminal sequences and similar activity trends, indicating that the C-terminal sequence is important. On the other hand, no activity was improved with TDCA or TCA.

[0128] It was concluded that even if the residues are lengthened, DCA activity tends to be maintained as the responsible residues.

[0129] [2] Peptide sequence analysis of indigestible proteins (1) Preparation of resistant protein 0.2% by mass of protease (Amano Enzyme, product name "Protin SD-AY10") was added to a 10% by mass aqueous solution of isolated soy protein (manufactured by Fuji Oil Co., Ltd., product name "Fujipro-R"), and an enzymatic decomposition reaction was carried out at 50°C for 5 hours. After the reaction, the mixture was heated at 100°C for 5 minutes to inactivate the enzyme, cooled to room temperature, and then centrifuged at 8,000G for 10 minutes to obtain an insoluble fraction (HMF), which is a resistant protein.

[0130] This HMF fraction was diluted with water to a solid content of 10% by mass, and then homogenized twice at 15 MPa using a high-pressure homogenizer (manufactured by APV Corp.) to prepare hydrophilized HMF with improved hydrophilicity. The obtained hydrophilized HMF was freeze-dried and then used in the subsequent steps.

[0131] (2) Enzymatic treatment of resistant proteins To 1 mg of hydrophilized HMF, 1 mL of 50 mM Tris-HCl buffer (pH 8.0) containing 8 M urea was added to solubilize the protein. 200 μL of the protein solution was weighed out, and Lys-C (VA117A: Promega), an enzyme that specifically cleaves the C-terminus of lysine residues, was added so that the ratio (weight ratio) of protein to enzyme was 200:1, and the reaction was carried out at 37°C for 16 hours. Formic acid (063-05895: Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 0.1 mass% (v / v) to stop the enzyme reaction.

[0132] 100 μL of the enzyme reaction solution of the protein was sealed in a Micro Float-A-Lyzer(R), MWCO: 0.1-0.5 kD (F235049: REPLIGEN), and dialysis was performed using 100 mM phosphate buffer as the external dialysis solution to remove urea and exchange the buffer.

[0133] The dialysate was collected, and Glu-C (V165A: Promega), an enzyme that specifically cleaves the C-terminus of aspartic acid or glutamic acid residues, was added so that the ratio (weight ratio) of protein to enzyme was 200:1, and the reaction was carried out for 16 hours at 37° C. Formic acid was added to a final concentration of 0.1% by mass (v / v) to terminate the enzyme reaction.

[0134] (3) Sequence analysis of peptides in indigestible proteins using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS) The prepared solution of the protein enzymatic digest was used as a sample, and LC-ESI-QTOF-MS and MS / MS analyses were performed using Vion IMS QTof (Waters). The MS-Range was adjusted to the range of m / z 100 to 2,000, and the detector potential was set to 1.0 kV. The obtained MS or MS / MS spectra were processed using the UNIFI Scientific Information System (Waters), and then collated with the UniProt database (https: / / www.uniprot.org / ) using UNIFI Peptide Map (Waters) to search for candidate peptides.

[0135] Eight subunits of soybean proteins β-conglycinin and glycinin were used as reference sequences (Uniprot IDs: P0DO16, P11827, P25974, P04776, P11828, P04405, Q39922, P02858).

[0136] At this time, the search was performed by selecting "none" which does not select any specific enzyme as the digestive enzyme of the sample. As a result of the data analysis, the partial sequences containing the 6-residue peptide exhibiting DCA binding activity present in the hydrophilized HMF that were identified are shown in Table 6 below (Partial sequences containing the 6-residue peptide exhibiting DCA binding activity).

[0137] [Table 6]

Claims

1. A secondary bile acid absorption inhibitor comprising at least one selected from the group consisting of peptides or proteins comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 42.

2. 2. The secondary bile acid absorption inhibitor according to claim 1, comprising at least one selected from the group consisting of peptides or proteins comprising any of the amino acid sequences of SEQ ID NOs: 1 to 12.

3. 2. The secondary bile acid absorption inhibitor according to claim 1, comprising a plurality of types of the peptides or proteins having different amino acid sequences.

4. The peptide or the protein is peptic digest of soy protein, and / or Resistant proteins in soybean protein, 2. The secondary bile acid absorption inhibitor according to claim 1, comprising as an active ingredient a peptide or protein contained in

5. The secondary bile acid absorption inhibitor according to claim 1, wherein the secondary bile acid is at least one secondary bile acid selected from the group consisting of deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and hyodeoxycholic acid (HDCA).

6. 2. The secondary bile acid absorption inhibitor according to claim 1, which is a food additive or a food composition.

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