JP2017149692A - Composition for biological defense and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel composition having an excellent biological defense action.SOLUTION: According to the present invention, there is provided a biological defense composition comprising a protein or peptide having a biological defense action. The composition comprises a rice bran protein hydrolysate or an amino acid sequence of any one of the following (A) to (C) with 600 or less amino acid residues: (A) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 24; (B) an amino acid sequence having a conservative substitution or deletion of one to several amino acids in the amino acid sequence of (A); and (C) an amino acid sequence consisting of at least four consecutive amino acids in the amino acid sequence of (A) or (B).SELECTED DRAWING: None

Description

  The present invention relates to a biodefense composition and food / beverage products, food / beverage product additives, pharmaceuticals, quasi-drugs, cosmetics and feeds containing the same.

  As a person ages, oral functions such as pronunciation, mastication, swallowing, and salivation decrease. In particular, when salivary secretion decreases, oral diseases such as periodontal disease, stomatitis, caries (cavities) and bad breath increase. In addition, the skin barrier function and moisturizing function are reduced by skin diseases and injuries. In addition, infants and the elderly have low immune functions and are susceptible to infection with pathogenic bacteria. The decline of these functions is a serious issue for the maintenance and promotion of public health.

  Until now, as antibacterial components or bactericidal components added to oral care products and the like, organic solvents such as ethanol, antibiotics, and the like have been proposed (for example, see Patent Document 1). However, the oral care product using the organic solvent has a problem that it is not physically acceptable and cannot be used for infants. In the oral care product using the antibiotics, resistant bacteria are caused by long-term use. There is a problem of appearing.

  On the other hand, various organisms such as animals, plants, insects, and microorganisms have self-defense mechanisms for self-protection against the invasion of pathogenic microorganisms from the outside world, such as polysaccharide degrading enzymes and lytic enzymes. The organism itself produces antibacterial peptides consisting of about 10 to about 50 proteins and amino acids. These antibacterial components are expected to be used as antibacterial agents for oral cavity because they have a wide range of antibacterial activity as compared with the above-mentioned antibiotics and have the property of hardly causing resistant bacteria.

As a biological antibacterial agent, for example, oryzastatin, an antibacterial protein derived from rice, is known. However, although oryzasistatin is known to inhibit gingipaine of periodontal disease-causing bacteria (such as Porphyromonas gingivalis ), it does not directly exhibit antibacterial activity against the cells.
Although there are homologues of known antibacterial proteins such as defensin in the rice genome, its contribution to the defense of rice is unknown.

  It has been reported that a fragment consisting of a specific protein or a part thereof has an infection protective action, a biological defense action and the like (for example, Patent Documents 1 and 2).

  However, the present situation is that the development of a composition that is derived from a plant and has an excellent biological defense action is still desired.

JP 2013-60416 A JP 2014-237626 A

  An object of the present invention is to provide a novel composition having an excellent biophylaxis effect. Since the composition in the present invention does not have hemolytic activity, it can be used safely.

Means for solving the problems are as follows. That is,
[1] A composition for biological defense containing a rice bran protein enzyme hydrolyzate.
[2] The composition for biological defense according to [1], wherein the rice bran protein enzyme hydrolyzate contains a peptide having a molecular weight of 1000 to 3000 and an isoelectric point of 10 or more.
[3] Rice bran protein enzyme hydrolyzate contains an amino acid sequence of any of the following (A) to (C), the number of amino acid residues is 600 or less, and contains a protein or peptide having a biological defense action The composition for biological defense according to [1] or [2].
(A) An amino acid sequence represented by any one of SEQ ID NOs: 1 to 24 (B) An amino acid sequence having a conservative substitution or deletion of one to several amino acids in the amino acid sequence of (A) (C) (A) Or amino acid sequence consisting of at least four consecutive amino acids in the amino acid sequence of (B) or [B], wherein the enzyme is one or more selected from the group consisting of pepsin, trypsin, chymotrypsin and papain [1] to [3] The composition for biological defense according to any one of the above.
[5] A composition for biological defense comprising an amino acid sequence of any of the following (A) to (C), having a number of amino acid residues of 600 or less and having a protein or peptide having a biological defense action.
(A) An amino acid sequence represented by any one of SEQ ID NOs: 1 to 24 (B) An amino acid sequence having a conservative substitution or deletion of one to several amino acids in the amino acid sequence of (A) (C) (A) ) Or amino acid sequence consisting of at least four consecutive amino acids in the amino acid sequence of (B) [6] The above [1] to [5], wherein the biological defense is one or more selected from the group consisting of antibacterial, anti-inflammatory and wound healing ] The composition for biological defense in any one of.
[7] A food / beverage product, a food / beverage product additive, a pharmaceutical product, a quasi-drug, a cosmetic product or a feed containing the biodefense composition according to any one of [1] to [6].

  ADVANTAGE OF THE INVENTION According to this invention, the novel composition which has the outstanding biological defense effect, and its use (food / beverage products, food / beverage product additives, a pharmaceutical, a quasi-drug, cosmetics, feed, etc.) can be provided.

FIG. 1 shows a fraction obtained by isoelectric focusing of a hydrolyzate of rice bran protein using pepsin. FIG. 2 shows fractionation by isoelectric focusing of a rice bran protein hydrolyzate using an equal mass mixture of trypsin and chymotrypsin. FIG. 3 is a graph showing antibacterial activity against Porphyromonas gingivalis of a fraction obtained by isoelectric focusing of a sample obtained by hydrolyzing rice bran protein using pepsin. FIG. 4 is a graph showing the antibacterial activity against fractions of Streptococcus mutans obtained by isoelectric focusing of a sample obtained by hydrolyzing rice bran protein using pepsin. FIG. 5 is a graph showing the antibacterial activity against Propionibacterium acnes of a fraction obtained by isoelectric focusing of a sample obtained by hydrolyzing rice bran protein using pepsin. FIG. 6 is a graph showing antibacterial activity against Candida albicans of a fraction obtained by isoelectric focusing of a sample obtained by hydrolyzing rice bran protein using pepsin. FIG. 7 is a graph showing the antibacterial activity against Porphyromonas gingivalis of a fraction obtained by isoelectric focusing of a sample obtained by hydrolyzing rice bran protein using an equal mass mixture of trypsin and chymotrypsin. FIG. 8 is a graph showing the antibacterial activity against fractions of Streptococcus mutans obtained by isoelectric focusing of a sample obtained by hydrolyzing rice bran protein using an equimolar mixture of trypsin and chymotrypsin. FIG. 9 is a graph showing the antibacterial activity against Propionibacterium acnes of a fraction obtained by isoelectric focusing of a sample obtained by hydrolyzing rice bran protein using an equal mass mixture of trypsin and chymotrypsin. FIG. 10 is a graph showing the antibacterial activity against Candida albicans of a fraction obtained by isoelectric focusing of a sample obtained by hydrolyzing rice bran protein using an equal mass mixture of trypsin and chymotrypsin. FIG. 11 is a graph showing the tube formation promotion activity of a synthetic peptide. FIG. 12 is a graph showing the tube formation promotion activity of a synthetic peptide.

  The present invention provides a biodefense composition containing rice bran protein enzyme hydrolyzate.

[Rice bran protein enzyme hydrolyzate]
A rice bran protein enzyme hydrolyzate is obtained by hydrolyzing rice bran protein with an enzyme.
The rice bran used as a raw material can be obtained by milling brown rice. The rice bran preferably has a low white rice content. The rice bran is preferably a rice bran obtained from milled rice having a rice milling ratio of 85% or more, more preferably a rice bran obtained from milled rice having a rice milling ratio of 90% or more, and more preferably a rice bran obtained from milled rice having a rice milling ratio of 95% or more. It is. The rice bran contains defatted rice bran, and the defatted rice bran, which is a residue obtained by extracting rice oil from the rice bran, can be suitably used as a raw material for the rice bran extract in the present invention.
Examples of the enzyme include pepsin, trypsin, chymotrypsin, papain and the like. It is preferable to hydrolyze the rice bran protein at an optimum pH and temperature depending on the enzyme used. The rice bran protein enzyme hydrolyzate may have a molecular weight of, for example, 1000 to 3000, 1000 to 2000, 2000 to 3000, or 1500 to 2500. The rice bran protein enzyme hydrolyzate preferably has a positive net charge at pH 7, more preferably +1 to 5. The rice bran protein enzyme hydrolyzate preferably has an isoelectric point of, for example, 10 or more, and more preferably 10.5 or more.

[Protein or peptide]
The rice bran protein enzyme hydrolyzate includes, for example, any of the following amino acid sequences (A) to (C), the number of amino acid residues is 600 or less, and a protein or peptide having a biological defense action (hereinafter referred to as the present invention) (Also referred to as protein or peptide in the invention).
(A) An amino acid sequence represented by any one of SEQ ID NOs: 1 to 24 (B) An amino acid sequence having a conservative substitution or deletion of one to several amino acids in the amino acid sequence of (A) (C) (A) ) Or (B) amino acid sequence consisting of at least 4 consecutive amino acids

  SEQ ID NOs: 1 to 24 are shown below. In the parentheses next to SEQ ID NOs: 1 to 12, “Gene Identity and protein name of protein registered in National Center for Biotechnology Information (NCBI)” is shown.

  The lower limit of the number of amino acids of the protein or peptide in the present invention is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, It may be 15 or 16. The upper limit of the number of amino acids of the protein or peptide in the present invention is 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, It may be 50, 30, or 20.

  In the present invention, “several” of “having conservative substitution or deletion of one to several amino acids” means, for example, 10, 9, 8, 7, 6, 5, 4 3, 2, etc.

  In the present invention, “conservative substitution of amino acids” refers to substitution between amino acids in each group of Table 1 below. Among these, preferred conservative substitutions of amino acids include substitution between aspartic acid and glutamic acid, substitution between arginine, lysine and histidine, substitution between tryptophan and phenylalanine, phenylalanine and valine. And the like, substitution between leucine, isoleucine and alanine, substitution between glycine and alanine, and the like.

The protein or peptide in the present invention may be a derivative. In the derivative, the C-terminus of a protein or peptide represented by a specific amino acid sequence is any of a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ), or an ester (—COOR). Also good. As R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, for example, phenyl, α - _{C 6-12} aryl group such as naphthyl, for example, benzyl, _{C 7 - 14} aralkyl such as α- naphthyl _{-C 1-2} alkyl group such as a phenyl _{-C 1-2} alkyl or α- naphthylmethyl such phenethyl In addition to the group, a pivaloyloxymethyl group, which is widely used as an oral ester, can be mentioned. The amides, amido, C _1-6 one or amide substituted with two, one or two with an amide substituted with a C _1-6 alkyl group substituted by a phenyl group of an alkyl group, Examples thereof include amides that contain a nitrogen atom of an amide group to form a 5- to 7-membered azacycloalkane.
When the protein or peptide of the present invention has a carboxyl group or a carboxylate other than the C-terminus, those in which those groups are amidated or esterified are also included in the protein or peptide of the present invention. When the protein or peptide of the present invention has an amino group other than the N-terminus, the protein or peptide of the present invention also includes an amino group that is amidated.

In the derivatives, the N-terminal amino group is protected with a protecting group (for example, a C _1-6 acyl group such as a C _2-6 alkanoyl group such as formyl group or acetyl), and the N-terminal side is in vivo. Appropriate protection of glutamyl group cleaved and produced by pyroglutamine oxidation, and substituents on the side chain of amino acids in the molecule (for example, —OH, —SH, amino group, imidazole group, indole group, guanidino group, etc.) Those protected with a group (for example, a C _1-6 acyl group such as a C _2-6 alkanoyl group such as formyl group and acetyl) are also included.

The side chain of the amino acid constituting the protein or peptide derivative of the present invention may be modified with an arbitrary substituent. Although a substituent is not specifically limited, For example, a fluorine atom, a chlorine atom, a cyano group, a hydroxyl group, a nitro group, an alkyl group, a cycloalkyl group, an alkoxy group, an amino group, a phosphate group etc. are mentioned. Further, the substituent on the side chain may be protected with a protecting group.
The protein or peptide in the present invention may contain a D-amino acid or an unnatural amino acid as long as the properties of the original protein or peptide are retained. In addition, the protein or peptide of the present invention may be linked to another substance to the protein or peptide as long as the properties of the original protein or peptide are retained. Examples of other substances that can be linked to proteins or peptides include other proteins or peptides, lipids, sugars or sugar chains, acetyl groups, natural or synthetic polymers, and the like. In addition, the protein or peptide of the present invention may be subjected to modifications such as glycosylation, side chain oxidation, phosphorylation, etc., as long as the properties of the original protein or peptide are retained.
These techniques have been well established in the past, and may be followed in the present invention. In addition, the protecting group in the present invention may be any as long as it seals the reactivity of the group to be protected, and the protein or peptide of the present invention is administered to a living body while having a protecting group. May be.

  The protein or peptide of the present invention may form a salt, and a physiologically acceptable salt is preferred as the salt. Examples of physiologically acceptable salts include salts with acids such as hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, tartaric acid, maleic acid, fumaric acid, oxalic acid, malic acid, citric acid, oleic acid, and palmitic acid; sodium Salts of alkali metals or alkaline earth metals such as potassium, calcium, or aluminum hydroxides or carbonates; salts with organic bases such as triethylamine, benzylamine, diethanolamine, t-butylamine, dicyclohexylamine, arginine Etc.

The protein or peptide in the present invention is not only produced by hydrolyzing rice bran protein, but also according to a known general protein or peptide synthesis protocol, a solid phase synthesis method (Fmoc method, Boc method) or a liquid phase It can also be produced by synthetic methods. Moreover, it can also manufacture using the transformant which introduce | transduced the expression vector containing DNA which codes the protein or peptide in this invention. In addition, a protein or peptide is obtained using a transformant introduced with an expression vector containing a DNA encoding a protein or peptide partially containing the protein or peptide of the present invention, and this is used as an appropriate protease, peptidase, etc. It can also be produced by cleaving with a protein hydrolase. It can also be produced by a method using an in vitro transcription / translation system.
That is, the present invention also includes a biodefense composition comprising an amino acid sequence of any of the following (A) to (C), having a number of amino acid residues of 600 or less and containing a protein or peptide having a biodefense action: Include.
(A) An amino acid sequence represented by any one of SEQ ID NOs: 1 to 24 (B) An amino acid sequence having a conservative substitution or deletion of one to several amino acids in the amino acid sequence of (A) (C) (A) ) Or an amino acid sequence consisting of at least four consecutive amino acids in the amino acid sequence of (B) As the explanation for the protein or peptide, the protein in the biological defense composition containing the rice bran protein enzyme hydrolyzate of the present invention or It may be synonymous with a peptide.

(Bioprotective action)
In the present invention, biological defense is not particularly limited, and examples thereof include antibacterial, anti-inflammatory and wound healing. Having antibacterial activity is, for example, P.I. gram negative bacteria such as gingivalis ATCC 33277, mutans JCM 5705, P.M. gram-positive bacteria such as C. acnes JCM 6473, C.I. The sample is added to a culture medium of fungi such as albicans NBRC 1385, adenosine triphosphate (ATP) derived from live bacteria is quantified, and the bacterial growth inhibition rate (%) obtained by comparing with that of the control group This can be confirmed by calculation. In the present invention, the growth inhibition rate of any one or more of the above bacteria is preferably 10% or more, more preferably 20% or more, more preferably 30% or more, and 40% or more. More preferably, it is 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, and 90% or more. More preferably.

  It has an anti-inflammatory action by using, for example, “Endospecy ES-50M Set” (manufactured by Seikagaku Corporation) and “Endotoxin Standard CSE-L Set” (manufactured by Seikagaku Corporation), etc. It can be confirmed by calculating the endotoxin neutralizing activity (%) obtained by evaluating the neutralizing activity and comparing it with that of the control group. In the present invention, the endotoxin neutralizing activity (%) is preferably 1% or more, preferably 10% or more, more preferably 20% or more, and more preferably 30% or more, More preferably, it is 40% or more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, further preferably 80% or more, 90% or more is particularly preferable.

  For example, HUVEC (Human Umbilical Vein Endothelial Cell, Kurashiki Boseki Co., Ltd., KE-4109) has a wound healing action. It is possible to confirm by calculating the tube formation promoting activity (%) obtained by measuring the total value of and comparing with that of the control group. In the present invention, the lumen formation promoting activity (%) is preferably 101% or more, preferably 103% or more, more preferably 110% or more, and more preferably 120% or more. 125% or more, more preferably 130% or more, still more preferably 135% or more, and particularly preferably 140% or more.

Since the composition of the present invention does not have hemolytic activity, it can be used safely. The absence of hemolytic activity can be confirmed, for example, by adding a sample to erythrocytes, measuring the absorbance at 405 nm, and calculating the hemolytic activity (%) from the following formula. In the present invention, the hemolytic activity (%) is preferably 10% or less, preferably 8% or less, more preferably 7% or less, more preferably 6% or less, and more preferably 5%. It is more preferably at most 4, more preferably at most 4%, particularly preferably at most 3%.
Hemolytic activity (%) = (A _S −A ₀ ) × 100 / (A _T −A ₀ )
_{(A 0} the absorbance when no additive, A _s represents absorbance upon addition of each sample, and _{A T} is the absorbance upon addition of 0.1 wt% TritonX-100, respectively.)

[Food and drink, food and drink additives, pharmaceuticals, quasi drugs, cosmetics and feed]
The food / beverage products, food / beverage product additives, pharmaceuticals, quasi-drugs, cosmetics or feed of the present invention contain the biological defense composition.

The food and drink is not particularly limited, for example, various soft drinks, fruit juice drinks, Japanese and Western sweets, dairy products and other livestock processed products, processed fruit products, processed vegetable products, processed grain products, processed fish products, There are many foods and drinks, such as so-called various health foods mainly composed of seasonings and vitamins. The food / beverage products of this invention are suitable as food / beverage products for biological defense.
Foods and drinks include health foods, functional foods, foods for specified health use for preventing dental caries, foods for the sick, and supplements. The form of the food or drink is not particularly limited. For example, beverages such as tea drinks, soft drinks, carbonated drinks, nutrition drinks, fruit drinks, lactic acid drinks, noodles such as buckwheat, udon, Chinese noodles, instant noodles, strawberries, throat protection, removal of bad breath, imparting a refreshing feeling, etc. Candy having a function such as prevention of caries, removal of bad breath, refreshing feeling, drowsiness prevention, chocolate, snacks, biscuits, jelly, jam, cream, baked goods, bread and other confectionery and breads, kamaboko, ham, Seafood and livestock processed foods such as sausages, dairy products such as processed milk and fermented milk, salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing and other fats and oils processed foods, sauces, sauces and other seasonings Retort pouch foods such as curry, stew, rice cakes, rice cakes, miscellaneous foods, ice cream, sorbets, shaved ice, etc. Rukoto can. Supplements can be provided in the form of tablets, granules, powders, drinks, and the like. The form of the food or drink additive is not particularly limited, and examples thereof include liquid, paste, powder, flakes, and granules. The food-drinks additive of this invention can be manufactured according to the manufacturing method of a general food-drinks additive.
In general, for example, in a human body weighing about 60 kg, the protein or peptide of the present invention is ingested at about 0.01 to 1000 mg, preferably about 0.1 to 100 mg, more preferably about 0.5 to 50 mg per day. Also good.

  The drug or quasi-drug is not particularly limited, but it is used for treatment of periodontal disease, bactericidal coating, medicinal throat spray, troche, medicinal throat, stomatitis treatment, medicinal troche, mouthwash, toothpaste (tooth Toothpaste prevention toothpaste, bad breath prevention toothpaste, etc.), mouthwash (mouthwash, dental rinse, etc.), denture cleaner, bandage, acne treatment and / or prevention agent, opportunistic infection prevention and / or treatment agent Etc. are preferable.

  The pharmaceutical or quasi-drug of the present invention contains the protein or peptide of the present invention as a component, and is pharmaceutically acceptable according to a known method for producing a pharmaceutical preparation (for example, a method described in the Japanese Pharmacopoeia). A suitable carrier or additive can be added to prepare a preparation. Examples of the preparation include tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (including soft capsules and microcapsules), Lozenges, syrups, solutions, emulsions, suspensions, controlled-release preparations (eg, immediate-release preparations, sustained-release preparations, sustained-release microcapsules), aerosols, films (eg, orally disintegrating films) , Oral mucosa film), injection (eg, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), drip, transdermal preparation, ointment, lotion, patch, Oral or parenteral preparations such as suppositories (eg, rectal suppositories, vaginal suppositories), pellets, nasal preparations, pulmonary preparations (inhalants), eye drops and the like can be mentioned. What is necessary is just to set suitably about the mixture ratio of a carrier or an additive based on the range normally employ | adopted in the field | area of a pharmaceutical or a quasi-drug. Carriers or additives that can be blended are not particularly limited. For example, various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases, excipients, binders, pH adjusters, disintegrants, absorption Various additives such as an accelerator, a lubricant, a colorant, a corrigent, and a fragrance are included.

Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavorings such as peppermint, red oil and cherry. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection can be prepared according to normal pharmaceutical practice (for example, dissolving or suspending an active ingredient in a solvent such as water for injection or natural vegetable oil). As an aqueous solution for injection, for example, isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80 ^™ , HCO-50) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. Buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), storage You may mix | blend with an agent (for example, benzyl alcohol, phenol, etc.), antioxidant, etc.

Since the preparation thus obtained is safe and has low toxicity, for example, it is administered to humans and other mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) can do.
The medicament of the present invention varies depending on the dosage form, administration method, carrier, etc., but the protein or peptide of the present invention is usually 0.01 to 100% (w / w), preferably 0.1 to 95, based on the total amount of the preparation. It can manufacture in accordance with a conventional method by adding in the ratio of% (w / w).
The dose varies depending on the administration subject, symptom, administration route and the like, but in the case of oral administration, for example, generally about 0.01 to 1000 mg, preferably about 0.1, per day for a human body weight of about 60 kg. 1 to 100 mg, more preferably about 0.5 to 50 mg. The total daily dose may be a single dose or divided doses. The medicament or quasi-drug of the present invention may contain other active ingredients (for example, active ingredients known as bioprotective agents such as orizastatin).

  Examples of the cosmetics include face wash, cleansing, lotion, milky lotion, beauty serum, spot care, moisture, massage pack, makeup base, foundation, face powder, body cream, body lotion, body massage care cream, suntan, Sunscreen, bath products, body shampoo, lip balm, spray, rinse, conditioner, rinse in shampoo, in bath treatment, out bath treatment, aerosol products, deodorant, fragrance, deodorant, bath preparation, anti-aging agent, acne Listed products, whitening agents, etc. The cosmetics of the present invention are components generally used as cosmetics other than the protein or peptide of the present invention, for example, surfactants, moisturizers, animal and plant derived fats and oils, silicones, higher alcohols, lower alcohols, animal and plant derived extracts, Ultraviolet absorbers, anti-inflammatory agents, metal sequestering agents, vitamins, antioxidants, thickeners, preservatives, bactericides, pH adjusters, colorants, various fragrances, and the like can be appropriately blended depending on the purpose.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these.

[Example 1: Preparation and purification of rice bran protein enzyme hydrolyzate and peptide identification]
1. Preparation of rice bran protein-derived peptide Weigh 3.00 g of rice bran protein (Tsuno-RBP ^TM 55, Tsukino Food Industry Co., Ltd.) in a beaker, add 60 mL of ultrapure water, and suspend using homogenizer POLYTRON (KINEMATICA). The liquid was homogenized. Next, the suspension was homogenized using Spectra / Por (registered trademark) Dialysis Membrane, MWCO: 6,000-8,000 Da (132655: Spectrum Laboratories, Inc.) overnight, and then the small molecule was dialyzed overnight. Ingredients were removed. Thereafter, the dialyzed suspension was taken out into an Erlenmeyer flask, and an equal mass mixture of trypsin (T0303-1G: Sigma-Aldlich) and chymotrypsin (C4129-1G: Sigma-Aldrich), pepsin (P7012-1G: Sigma-Aldlich), Alternatively, papain (164-00172: Wako Pure Chemical Industries, Ltd.) was added so that the weight ratio with the rice bran protein was 2% (w / w). The temperature of the resulting suspension is adjusted using a thermostatic bath so that the equimolar mixture of trypsin and chymotrypsin is 37 ° C, pepsin is 37 ° C, or papain is 50 ° C. Went. After the reaction, in order to stop the reaction, a heat treatment was performed at 90 ° C. for 10 minutes to inactivate the protease. However, when pepsin was used, it was deactivated by adding 5M NaOH and raising the pH without performing the deactivation operation by heating. After inactivating the protease, the suspension was dispensed into a centrifuge tube and centrifuged at 10,000 × g and 4 ° C. for 30 minutes. The supernatant obtained by centrifugation is subjected to dialysis again using Spectra / Por (registered trademark) Dialysis Membrane, MWCO: 500 to 1,000 (131096, Spectrum Laboratories, Inc.). Low molecular components were removed. The dialysate was collected in an assist tube, frozen at −80 ° C., and then freeze-dried using a freeze dryer (FDU-2100, EYELA).

2. Fractionation of rice bran protein-derived peptide by isoelectric focusing electrophoresis 200 mg of rice bran protein enzymatic hydrolyzate prepared as described above was subjected to preparative isoelectric focusing apparatus (Rotofor (registered trademark) 170-2950 And fractionated into 20 fractions using Bio-Rad). That is, 200 mg of the hydrolyzate was dissolved in 50 mL of ultrapure water to obtain a sample solution. Separation was performed at 12 W for 150 minutes. After the electrophoresis was completed, each fraction was collected, measured for pH, frozen at −80 ° C., lyophilized using a lyophilizer, and the collected weight was measured.

3. Purification of peptide derived from rice bran protein by reverse phase chromatography Reversed phase chromatography is performed using a pump (LC-10ATvp: Shimadzu Corporation), an online degasser (DGU-12A: Shimadzu Corporation), a column oven (CTO-10Avp: Shimadzu Corporation), A high-performance liquid chromatography apparatus connected with a detector (SPD-10AVvp: Shimadzu Corporation), a fraction collector (FRC-10A: Shimadzu Corporation), and a system controller (SCL-10Avp: Shimadzu Corporation) was used. Software (LabSolutions, Shimadzu Corporation) was used for chromatographic operations and data analysis. Separation columns are CAPCELL PAK C-18 (column: diameter 10 mm x length 150 mm, particle size 5 μm, SHISEIDO: 90603) and Inertsil WP300 C8 (column: diameter 10 x length 150 mm, particle size 5 μm, GL Science Inc .: 5020-85735). The eluate was prepared using acetonitrile (catalog number: 1.00030.4000, Merck Ltd.), trifluoroacetic acid (34833-92: Nacalai Tesque) and ultrapure water. As eluent A, 0.1% (v / v) trifluoroacetic acid was used, and as eluent B, 80% (v / v) acetonitrile containing 0.1% (v / v) trifluoroacetic acid was used.
The concentration of eluate B is increased linearly from the initial concentration of 0% (v / v) at a rate of 1% (v / v) per minute and becomes 70% (v / v) after 70 minutes. Furthermore, the time program was set so that it might become 100% (v / v) between 70 and 90 minutes, and it eluted. The flow rate was 2.0 mL / min, the eluate was collected every 30 seconds after 7 minutes from the start of elution, and the absorbance at a wavelength of 210 nm was measured to detect a peak. Each peak fraction was frozen at −80 ° C. and lyophilized using a lyophilizer (FDU-2100, EYELA).
The fraction of each peak was purified again using the same separation column to obtain a single peak.

4). Identification of rice bran protein enzyme hydrolyzate (peptide) using matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF / MS) α-cyano as a matrix for peptide mass (MS) analysis -4-hydroxy-cinnamic acid [HCCA] (# 201344: Bruker Daltonics) and Peptide calibration standard II (# 222570: Bruker Daltonics) were used as calibration reagents. Mix 500 μL of TA solution (100% (v / v) acetonitrile: 0.1% (v / v) trifluoroacetic acid = buffer mixed at a ratio of 1: 2 [v / v]) with about 1 cup of HCCA. Then, it was dissolved by sonication for 10 minutes to prepare an HCCA saturated solution (matrix solution). Centrifuge the prepared matrix solution at 9,000 xg for 10 minutes, and in a sample tube for MS (Eppendorf), dissolve 1 mL sample solution and 4 μL matrix in 2 mL 80% (v / v) acetonitrile. The supernatant of the solution was mixed. 1 μL of the prepared 5 μL matrix mixed sample was spotted on a mass spectrometric plate (MTP 384 target plate ground steel TF; Bruker Daltonics) and left to dry. For calibration of molecular weight, a calibration reagent was prepared in the same manner as the sample and spotted.
After the spotted sample and the calibration reagent were dried, MALDI-TOF / MS and MS / MS analysis were performed using Auto Flex-III (registered trademark) (Bruker Daltonics). The MS-Range was adjusted in the range of m / z 800-43,000, and the potential of the detector was scanned as 1300-1800 V. The obtained MS or MS / MS spectrum was processed using the processing software flexAnalysis (registered trademark) (Bruker Daltonics) and biotools (registered trademark) (Bruker Daltonics), and then the analysis software Mascot search (registered) Trademark) (Matrix Science Ltd.) and collated with NCBI database (http://www.ncbi.nlm.nih.gov/BLAST/fasta.shtml, dated January 27, 2015) I did a search. At this time, “none”, which does not select a specific enzyme, was selected and searched as a digestive enzyme of the sample. Table 2 shows rice bran protein hydrolyzate (peptide) when pepsin is used as an enzyme. Table 3 shows rice bran protein hydrolyzate (peptide) when an equal mass mixture of trypsin and chymotrypsin is used as an enzyme.

In the above table, the alphabet at the right end of the Sequence column indicates that the peptide was obtained from the same protein (see Protein source column).

In the above table, the alphabet at the right end of the Sequence column indicates that the peptide was obtained from the same protein (see Protein source column).

[Example 2: Antibacterial activity]
1. Each fraction fractionated by isoelectric focusing in antibacterial test or the identified peptides contained in the fractions shown in Tables 2 and 3 were added to the medium, and the test bacteria shown in Table 4 were cultured.

P. gingivalis ATCC 33277 is a modified GAM medium (1 g, peptone 5.0 g, soybean peptone 3.0 g, proteose peptone 5.0 g, digested serum powder 10.0 g, yeast extract powder 2. meat extract powder 2. 2 g, liver extract powder 1.2 g, glucose 0.5 g, soluble starch 5.0 g, L-tryptophan 0.2 g, L-cysteine hydrochloride 0.3 g, sodium thioglycolate 0.3 g, L-arginine 1.0 g , Vitamin K 10.005 g, Hemin 0.01 g, Potassium dihydrogen phosphate 2.5 g, Sodium chloride 3.0 g, pH 7.3), and then left to stand at 37 ° C. for 48 hours under absolute anaerobic conditions.
S. mutans JCM 5705 is a BHI medium (1 L, pork brain extract powder 4.0 g, pork heart extract powder 4.0 g, peptone 17.5 g, glucose 2.0 g, sodium chloride 5.0 g, disodium hydrogen phosphate 2. 5 g, pH 7.2) was used for static culture at 37 ° C. for 6 hours under facultative anaerobic conditions.
P. acnes JCM 6473 is a GAM medium (1 g of peptone 10.0 g, soybean peptone 3.0 g, proteose peptone 10.0 g, digested serum powder 13.5 g, yeast extract 5.0 g, meat extract 2.2 g, liver extract 1.2 g, glucose 3.0 g, potassium dihydrogen phosphate 2.5 g, sodium chloride 3.0 g, soluble starch 5.0 g, L-cysteine hydrochloride 0.3 g, sodium thioglycolate 0.3 g, pH 7.1 ) For 24 hours at 37 ° C. under facultative anaerobic conditions.
C. albicans NBRC 1385 medium is YM medium (1 g, glucose 10.0 g, peptone 5.0 g, yeast extract 3.0 g, malt extract 1.0 g, pH 6.2) under a facultative anaerobic condition. The culture was stationary at 24 ° C. for 24 hours.
The antibacterial activity test against the test bacteria was performed according to the following procedure. After inoculating 80 μL of the culture solution of each test bacterium in the same medium as used for culturing the test bacterium, the culture was placed in an incubator set at a predetermined temperature and cultured for a predetermined time. Then, it was transplanted to a new medium and further cultured for a predetermined time. The obtained preculture solution was serially diluted to prepare a bacterial solution of OD ₆₅₀ = 5.0 × 10 ⁻⁵ .
Next, a reservoir was prepared, and 300 μL of a 1.33-fold concentration medium and 100 μL of each peptide solution (the concentration of each fraction fractionated by isoelectric focusing was adjusted to 3 mg / mL in each lane. The concentration of the chemically synthesized peptide was adjusted to 300 μM or 500 μM.), 100 μL of the bacterial solution was added and mixed well.
The same amount of sterilized water was added to the control and the blank instead of the peptide solution, and the same amount of each medium having a 1-fold concentration was added to the blank instead of the bacterial solution. 100 μL each of the peptide solution added, control and blank was dispensed into wells of a 96-well culture plate (# 3595, Corning), and cultured under the same conditions as the culture conditions for each of the test bacteria.

2. Method for evaluating antibacterial activity The antibacterial activity of each peptide was evaluated by quantifying ATP derived from live bacteria.
ATP derived from viable bacteria was quantified by a luciferin-luciferase luminescence method using BacTiter · Glo (registered trademark) Microbiological Cell Viability Assay Kit (manufactured by Promega). ATP was extracted from the microorganism as follows, and the amount of microorganism was measured from the luminescence intensity corresponding to the amount of ATP.
Specifically, first, 10 μL of lucifer ATP elimination reagent (manufactured by Kikkoman Co., Ltd.) is added to 100 μL of the test bacterium culture solution dispensed to each well of the culture plate, and the mixture is stirred for 10 minutes. ATP present in the sample was decomposed and erased to prepare a sample.
Next, 50 μL of the sample was added to 50 μL of the ATP luminescence reagent dispensed in advance in each well of a 96-well plate (OptiPlate-96, manufactured by PerkinElmer). The ATP emission intensity (emission wavelength 560 nm) derived from the viable bacteria in each well was measured as a relative light unit (RLU) using a microplate reader 1420 (Multilabel Counter Arvo (registered trademark) MX, manufactured by PerkinElmer). One sample was measured three times, and the measurement was performed at the initial logarithmic growth of each bacterium. The antibacterial activity was calculated by calculating the growth inhibition rate by setting the RLU of the control containing no antibacterial component as 100%, obtaining the RLU at each concentration.

3. Chemical synthesis of the identified peptide and measurement of its antibacterial activity Among the identified peptides contained in each fraction, 12 types of peptides (SEQ ID NOs: 13 to 24) were chemically synthesized. Each peptide was synthesized using a peptide synthesizer (PSSM-8, manufactured by Shimadzu Corporation) and equipped with a column (Cadenza CD-C18, manufactured by Intact Inc.) HPLC (10A system, manufactured by Shimadzu Corporation). And purified under the following purification conditions. The antibacterial activity of these peptides was measured by the same method as described above.
<Purification conditions>
Solvent A: acetonitrile containing 0.1% (w / v) trifluoroacetic acid Solvent B: water containing 0.1% (w / v) trifluoroacetic acid Flow rate: 1.0 mL / min Wavelength: 220 nm
・ Injection volume: 20μL
Gradient conditions: 0.01 minutes (10% by volume of solvent A, 90% by volume of solvent B) → 25.0 minutes (35% by volume of solvent A, 65% by volume of solvent B) → 25.1 minutes (100% by volume of solvent A) , Solvent B 0% by volume) → 30 minutes (stop)

[Example 3: Measurement of endotoxin neutralizing activity of synthetic peptide]
Endotoxin neutralizing activity was evaluated using “Endspecy ES-50M set” (manufactured by Seikagaku Corporation) and “Endotoxin standard CSE-L set” (manufactured by Seikagaku Corporation).
Twelve kinds of synthetic peptides (SEQ ID NOs: 13 to 24) were used. To each well of a 96-well plate (# 3595 multiple well plate (flat bottom), Corning), 25 μL of endotoxin standard (0.10 EU / mL) and peptide solution (final concentrations 1 μM and 10 μM) were added at 37 ° C. Incubated for 30 minutes or 35 minutes with shaking. Next, 50 μL of LAL reagent contained in the set was added to each well and incubated at 37 ° C. for 10 minutes with shaking. Thereafter, absorbance at a wavelength of 405 nm was measured using a microplate reader (2030 Arvo X, manufactured by PerkinElmer). What added distilled water (endotoxin free) instead of the peptide solution was used as a control, and the relative value when the absorbance of the control (0 μM) was 100% was defined as endotoxin neutralizing activity.

[Example 4: Measurement of wound healing activity of synthetic peptide]
The wound healing action of the synthetic peptide was evaluated based on the lumen formation promoting action of HUVEC (human umbilical vein endothelial cells, Kurashiki Boseki Co., Ltd., KE-4109).
After culturing HUVEC for 3 to 4 days using a 96-well plate (# 3595, Corning) until it becomes confluent, it was washed with HEPES buffer (Kurashiki Boshoku Co., Ltd., HK-3320) to remove impurities. Removed. Next, it was treated with trypsin / EDTA (manufactured by Kurashiki Boseki Co., Ltd., HK-3120) for 3 minutes, and the peeled HUVECs were collected and added to a trypsin neutralizing solution (Kurashiki Boseki Co., Ltd., HK-3220). After centrifuging this cell suspension at 800 rpm for 5 minutes, the supernatant was removed, and HuMedia-EG2 (Kurashiki Boseki Co., Ltd., KE-2150S) was added to adjust the cell concentration to 2.0 × 10 ⁵ cells / mL. Adjusted. The obtained cell suspension and each peptide were mixed at a ratio of 1: 1 (v / v).
In this experiment, LL-37 (Leu-Leu-Gly-Asp-Phe-Phe-Arg-Lys-Ser-Lys-Glu-Lys-Ile- known as a biological defense peptide having human wound healing action) Gly-Lys-Glu-Phe-Lys-Arg-Ile-Val-Gln-Arg-Ile-Lys-Asp-Phe-Leu-Arg-Asn-Leu-Val-Pro-Arg-Thr-Glu-Ser: peptide sequence No. 25 / manufactured by Peptide Laboratories, Inc., 4445-s) was used as a positive control to evaluate the tube formation promotion action of the synthetic peptide. In addition, LL-37 is a biological defense peptide having a human wound healing action. Carretero, M.M. J. et al. Escamez, M.M. Garcia, B.A. Duarte, A.D. Holguin, L.H. Retamosa, J. et al. L. Jorcano, M.M. del Rio, and F.M. Larcher: In vitro and in vivo wound healing promoting of human catalystin LL-37. Journal of Investigative Dermatology, (2008) Vol. 128, pp. 233-236. 2) R.A. Ramos, J .; P. Silva, A.M. C. Rodrigues, R Costa, L. Guardao, F.A. Schmitt, R.A. Soares, M.M. Vilanova, L.M. Domingues, and M.M. Gama: Wound healing activity of the human antimicrobial peptide LL37. Peptides, (2011) Vol. 32, pp. 1469-1476. It is described in.

Since Matrigel (Becton Deckinson and Company, 354234) hardens at room temperature and becomes a liquid state at 4 ° C., 40 μL of Matrigel was first added to a 96-well plate (# 3595, Corinig) on ice. After the added Matrigel was incubated at 37 ° C. for 30 minutes, a cell suspension and a mixed solution (100 μL) of each peptide or LL-37 prepared in advance were added and cultured for 15 hours.
The luminal structure formed by HUVEC in Matrigel was observed with a microscope at a magnification of 40 times and photographed. Further, a range of 300 × 400 pixels was extracted from the obtained image, the total value of the lengths of the cells having the formed luminal structure was measured, and the wound healing action of each peptide was evaluated.

[Example 5: Measurement of hemolytic activity of synthetic peptide]
The hemolytic activity of the peptide was tested using sheep defibrillated sterile blood (0102-1, manufactured by Nippon Biotest Laboratories, Inc., hereinafter also referred to as “red blood cells”).
In a microtube, 40 μl of red blood cells were suspended in a phosphate buffer solution (PBS: pH 7.2) containing 960 μl of physiological saline so as to be 4% by volume to obtain a suspension. The suspension was centrifuged at 5,000 rpm for 5 minutes, the supernatant was removed, and 960 μL of PBS was newly added to resuspend red blood cells. After this operation was repeated three times, the obtained red blood cells were used as samples. A solution of 12 kinds of peptides (SEQ ID NOs: 13 to 24) diluted to an arbitrary concentration, or 0.1% by weight Triton X-100 (595-13135, manufactured by Wako Pure Chemical Industries, Ltd.) in a 96-well plate (# 3595, 50 μl was dispensed into each well of Corning). Next, 50 μL of 4% by volume of erythrocyte suspension was dispensed into each well, followed by incubation at 37 ° C. for 1 hour. Thereafter, centrifugation was performed at 4,000 rpm for 10 minutes. 50 μL of the supernatant obtained by centrifugation was added to wells in which 50 μL of PBS or water had been dispensed in advance. The absorbance at 405 nm of each well was measured using a microplate reader (2030 Arvo ™ X, manufactured by PerkinElmer). The hemolysis activity was evaluated using the following formula, assuming that the absorbance when the peptide was not added was 0% and the absorbance when 0.1 mass% Triton X-100 was added instead of the peptide was 100%.
Hemolytic activity (%) = (A _peptide −A ₀ ) × 100 / (A _{Triton X−100} −A ₀ )
Here, A ₀ represents the absorbance when no addition was made, A _peptide represents the absorbance when each peptide was added, and A _{Triton X-100} represents the absorbance when 0.1 mass% Triton X-100 was added.

The results are shown in FIGS. In Table 5, “ND” indicates “not detected”, and “−” indicates “not measured” or “not measured”.
<Antimicrobial activity>

Fraction (Figures 3-10)
As apparent from FIGS. 3 to 6, the rice bran protein hydrolyzate (fraction) using pepsin detected antibacterial activity in the fraction having a high isoelectric point. That is, no. Fractions 18-20 are gingivalis ATCC 33277 and P. gingivalis. It showed strong antibacterial activity against acnes JCM 6473. No. Twenty fractions were obtained from S. gram of Gram-positive bacteria. mutans JCM 5705 and the opportunistic fungus C. Albicans NBRC 1385 also showed strong antibacterial activity.
On the other hand, as apparent from FIGS. 7 to 10, rice bran protein hydrolyzate (fraction) using an equal mass mixture of trypsin and chymotrypsin has a high isoelectric point. 20 fractions gingivalis ATCC 33277 and P. gingivalis. It showed strong antibacterial activity against acnes JCM 6473.
Synthetic peptides (Table 5)
As is clear from Table 5, both peptides were P.A. Antibacterial activity was demonstrated against gingivalis ATCC 33277. In addition, the peptide of SEQ ID NO: 16 is S. gram of Gram-positive bacteria. mutans JCM 5705, and P.M. acnes JCM 6473, and fungal C.I. All showed antibacterial activity against albicans NBRC 1385.
The peptide of SEQ ID NO: 20 is a typical periodontal disease, P. gingivalis ATCC 33277, P. acne causing bacteria. acnes JCM 6473, and C. Antibacterial activity was shown against albicans NBRC 1385.

<Anti-inflammatory effect>
As is apparent from Table 5, it was shown that 10 kinds of peptides of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 24 neutralize endotoxin in a concentration-dependent manner. In particular, the neutralizing activity of SEQ ID NOs: 15, 16, and 20 is equivalent to that of polymyxin B (P1004-1, manufactured by Sigma; a medical antibacterial agent, which exhibits a neutralizing activity of about 50% at 0.1 μM). Met.

<Injury healing action>
The lumen formation promoting action of each peptide and LL-37 on HUVEC is shown in FIGS. Further, Table 5 summarizes the ratio of the cell length when the peptide was added to the cell length of the control not added with the peptide. As is apparent from FIGS. 11 and 12 and Table 5, eight of the 12 peptides (SEQ ID NOs: 13, 14, 16, 17, 18, 19, 22, and 24) are in the same concentration range as LL-37. It showed a lumen formation promoting action, and the action was concentration dependent. Further, as a result of microscopic observation, when 10 μM peptide was added, as in the case of 10 μM LL-37, cell proliferation was promoted compared to the case where no peptide was added, and the luminal structure It was found that the length of the cells that had been increased. Therefore, since eight types of peptides (SEQ ID NOs: 13, 14, 16, 17, 18, 19, 22, and 24) have the action of promoting the formation of lumens of HUVEC in the same manner as LL-37, they have a wound healing action. I found out.

<Hemolytic activity>
Melittin (511-97531, manufactured by Serva Electrophoresis), an antibacterial peptide in bee venom having strong antibacterial activity but also strong hemolytic activity, showed 94% hemolysis activity even at 10 μM. On the other hand, as is apparent from Table 5, the peptide showed almost no hemolytic activity at a concentration of 500 μM. Therefore, it was confirmed that the peptide does not show hemolytic activity in a concentration range showing antibacterial activity, anti-inflammatory activity, and wound healing action.

Claims (7)

  A composition for biological defense containing a rice bran protein enzyme hydrolyzate.   The composition for biological defense according to claim 1, wherein the rice bran protein enzyme hydrolyzate contains a peptide having a molecular weight of 1000 to 3000 and an isoelectric point of 10 or more. The rice bran protein enzyme hydrolyzate contains an amino acid sequence of any of the following (A) to (C), the number of amino acid residues is 600 or less, and contains a protein or peptide having a biological defense action. Or the bioprotective composition according to 2.
(A) An amino acid sequence represented by any one of SEQ ID NOs: 1 to 24 (B) An amino acid sequence having a conservative substitution or deletion of one to several amino acids in the amino acid sequence of (A) (C) (A) ) Or (B) amino acid sequence consisting of at least 4 consecutive amino acids   The composition for biological defense according to any one of claims 1 to 3, wherein the enzyme is one or more selected from the group consisting of pepsin, trypsin, chymotrypsin and papain. A composition for biological defense comprising an amino acid sequence of any of the following (A) to (C), having a number of amino acid residues of 600 or less, and comprising a protein or peptide having a biological defense action.
(A) An amino acid sequence represented by any one of SEQ ID NOs: 1 to 24 (B) An amino acid sequence having a conservative substitution or deletion of one to several amino acids in the amino acid sequence of (A) (C) (A) ) Or (B) amino acid sequence consisting of at least 4 consecutive amino acids   The composition for biological defense according to any one of claims 1 to 5, wherein the biological defense is one or more selected from the group consisting of antibacterial, anti-inflammatory and wound healing.   A food / beverage product, a food / beverage product additive, a pharmaceutical product, a quasi-drug, a cosmetic product or a feed containing the biodefense composition according to claim 1.