WO2012015037A1 - 抗菌活性増強剤 - Google Patents
抗菌活性増強剤 Download PDFInfo
- Publication number
- WO2012015037A1 WO2012015037A1 PCT/JP2011/067488 JP2011067488W WO2012015037A1 WO 2012015037 A1 WO2012015037 A1 WO 2012015037A1 JP 2011067488 W JP2011067488 W JP 2011067488W WO 2012015037 A1 WO2012015037 A1 WO 2012015037A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- lactoferrin
- antibacterial activity
- milk
- protein
- hydrolyzate
- Prior art date
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- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3526—Organic compounds containing nitrogen
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/19—Dairy proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/20—Milk; Whey; Colostrum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Definitions
- the present invention relates to an antibacterial activity enhancer of lactoferrin or a lactoferrin hydrolyzate containing a protein derived from milk as an active ingredient.
- Lactoferrin is an iron-binding protein contained in tears, saliva, peripheral blood, milk, etc. in living organisms, and is harmful to harmful microorganisms such as Escherichia coli, Candida yeast, and Clostridium bacteria. It is known that antibacterial action is exhibited (Non-patent Document 1). It is also known to have antibacterial activity against staphylococci and enterococci at a concentration of 0.5 to 30 mg / ml (Non-patent Document 2).
- Patent Document 1 a composition for inhibiting the adhesion of pathogenic E. coli to digestive tract epithelial cells containing a lactoferrin degradation product is known (Patent Document 1).
- lactoferricin registered trademark
- Non-Patent Documents 3 and 4 methods for enhancing antibacterial activity by combining the above-mentioned lactoferrin and lactoferrin degradation products with components other than lactoferrin have been studied.
- an antibacterial agent containing a compound selected from the group consisting of a degradation product of lactoferrin, a lactoferrin-related antibacterial peptide, or any mixture thereof and a casein degradation product as an active ingredient is known (Patent Document 2). ).
- an antibacterial agent comprising a compound selected from the group consisting of a degradation product of lactoferrin, a lactoferrin-related antibacterial peptide, and any mixture thereof, and a protein that binds a metal (patent) Reference 3).
- Proteins that bind metals include lactoferrins, transferrin, conalbumin, and casein phosphopeptides.
- Patent Document 4 an antibacterial agent containing a degradation product of lactoferrin and / or an antibacterial peptide of this degradation product and lysozyme as active ingredients is described (Patent Document 4).
- composition comprising an enzymatic degradation product of lactoferrin, an antibacterial peptide obtained from the enzymatic degradation product of lactoferrin, or a chemically synthesized peptide containing a part of the amino acid sequence of lactoferrin and an emulsion of fatty acids is known.
- Patent Document 5 a composition comprising an enzymatic degradation product of lactoferrin, an antibacterial peptide obtained from the enzymatic degradation product of lactoferrin, or a chemically synthesized peptide containing a part of the amino acid sequence of lactoferrin and an emulsion of fatty acids is known.
- Patent Document 6 an antibacterial agent containing a new quinolone antibacterial agent and a lactoferrin hydrolyzate or an antibacterial peptide derived from a lactoferrin hydrolyzate as an active ingredient is known (Patent Document 6).
- Angiogenin which is known to be contained in milk and the like, is a protein thought to be involved in angiogenesis, but its function is unclear. It has been reported that some proteins classified into angiogenin have antibacterial activity. For example, human angiogenin has antibacterial activity against Candida albicans and Enterococcus faecalis, mouse angiogenin-1 has antibacterial activity against Candida albicans and Streptococcus pneumoniae, mouse angiogenin-4 has antibacterial activity against Enterococcus faecalis and Listeria monocytogenes However, depending on the type of angiogenin and the type of target microorganism, antibacterial activity is often not exhibited (Non-patent Document 5).
- angiogenin is known to have limited antibacterial activity, but milk-derived proteins such as angiogenin are more effective than lactoferrin or lactoferrin hydrolysates than their own antibacterial activity. It is not known to have an effect of enhancing the activity.
- lactoferrins lactoferrin and lactoferrin hydrolysates
- lactoferrins lactoferrin hydrolysates
- the present inventors paid attention to the fact that the protein fraction in the course of purification obtained in the production process of lactoferrin significantly enhances the antibacterial activity of lactoferrin, and conducted intensive studies on the protein components.
- the first invention of the present application that solves the above-mentioned problems is a lactoferrin or lactoferrin hydrolyzate antibacterial activity enhancer comprising a milk-derived protein having the following properties as an active ingredient: (A) It has an action of enhancing the antibacterial activity of lactoferrin or lactoferrin hydrolyzate. (B) The molecular weight is 30 kDa or less. (C) The isoelectric point is 9.0 to 11.0.
- the protein derived from milk contains lactoferrin or a lactoferrin hydrolyzate in the quantity of 2.0 mass% or less.
- the first invention of the present application has a preferred embodiment in which the milk-derived protein is Angiogenin-1 and / or Angiogenin-2.
- the first invention of the present application has a preferable embodiment in which the milk-derived protein itself does not have antibacterial activity against Escherichia coli.
- the milk-derived protein enhances the antibacterial activity of E. coli of lactoferrin or lactoferrin hydrolyzate by 7 times or more by using the protein and lactoferrin or lactoferrin hydrolyzate in combination. Having a property is a preferred embodiment.
- the second invention of the present application contains the antibacterial activity enhancer according to the first invention of the present application, and one or more selected from the group consisting of lactoferrin and a lactoferrin hydrolyzate, and the lactoferrin and the lactoferrin hydrolyzate It is an antibacterial agent composition whose mass ratio of the protein derived from the milk in the antibacterial activity enhancer with respect to is 1/80 or more.
- the third invention of the present application is an antibacterial drug containing the antibacterial agent composition according to the second invention of the present application.
- the fourth invention of the present application is a food or drink containing the antibacterial agent composition according to the second invention of the present application.
- the fifth invention of the present application is a feed containing the antibacterial composition according to the second invention of the present application.
- 6th invention of this application is use of the protein derived from the milk which has the following property in manufacture of the antibacterial activity enhancer of lactoferrin or a lactoferrin hydrolyzate: (A) It has an action of enhancing the antibacterial activity of lactoferrin or lactoferrin hydrolyzate. (B) The molecular weight is 30 kDa or less. (C) The isoelectric point is 9.0 to 11.0. 6th invention of this application makes it the preferable aspect that the said protein derived from milk contains lactoferrin or lactoferrin hydrolyzate in the quantity of 2.0 mass% or less.
- the sixth invention of the present application has a preferred embodiment in which the milk-derived protein is Angiogenin-1 and / or Angiogenin-2. Furthermore, the sixth invention of the present application has a preferred embodiment in which the milk-derived protein itself does not have antibacterial activity against Escherichia coli. Furthermore, in the sixth invention of the present application, the milk-derived protein enhances the antibacterial activity of E. coli of lactoferrin or lactoferrin hydrolyzate by 7 times or more by using the protein in combination with lactoferrin or lactoferrin hydrolyzate. Having a property is a preferred embodiment.
- the seventh invention of the present application is: (a) a step of bringing a milk raw material into contact with a cation exchange resin and subjecting the protein in the milk raw material to an adsorption treatment on the cation exchange resin; and (b) a cation exchange resin after the adsorption treatment.
- the method of manufacturing is: (A) It has an action of enhancing the antibacterial activity of lactoferrin or lactoferrin hydrolyzate. (B) The molecular weight is 30 kDa or less. (C) The isoelectric point is 9.0 to 11.0.
- FIG. 1 shows the primary structure of human lactoferricin (amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 2).
- 1 shows the primary structure of bovine lactoferricin (amino acid sequence: SEQ ID NO: 3).
- the identification result by the mass spectrometry of Angiogenin-1 obtained from the measurement of the same spot by two-dimensional electrophoresis is shown.
- bovine angiogenin-1 (SEQ ID NO: 4)
- middle row is bovine angiogenin-1 precursor (SEQ ID NO: 5) with 23 amino acids added before SEQ ID NO: 4
- lower row is SEQ ID NO: 4.
- It is an amino acid sequence of bovine angiogenin-1 precursor (SEQ ID NO: 6) to which 23 amino acids partially different from SEQ ID NO: 5 were added in advance.
- the underlined portion indicates a portion hit in the sequence of bovine angiogenin-1 by database search based on the results of mass spectrometry. The identification result by the mass spectrometry of angiogenin-2 obtained from the measurement of the same spot by two-dimensional electrophoresis is shown.
- the upper row is bovine angiogenin-2 (SEQ ID NO: 7), and the lower row is the amino acid sequence of bovine angiogenin-2 (SEQ ID NO: 8) in which 24 amino acids are added before SEQ ID NO: 7.
- the underlined portion indicates a portion hit in the sequence of bovine angiogenin-2 by database search based on the results of mass spectrometry.
- the antibacterial activity enhancer of the present invention has an effect of enhancing the antibacterial activity inherent to lactoferrin or lactoferrin hydrolyzate.
- the antibacterial activity enhancer of the present invention may be composed only of milk-derived proteins described below. However, as long as the milk-derived protein is included as an active ingredient, other ingredients such as the formulation described below can be used. The component used may be included.
- the antibacterial activity enhancer of the present invention is a protein derived from milk other than angiogenin-1 and / or angiogenin-2 as long as it contains angiogenin-1 and / or angiogenin-2 as an active ingredient (however, lactoferrin
- the purity of Angiogenin-1 and / or Angiogenin-2 with respect to the protein contained in the antibacterial activity enhancer is preferably 0.002% or more, more preferably 0.01 % Or more, more preferably 0.1% or more, and particularly preferably 1.0% or more.
- the amount of lactoferrin mixed will be described later.
- the antibacterial activity enhancer of this invention may mix with lactoferrin or a lactoferrin hydrolyzate, and may mix with powder. When powdered and mixed, the operation becomes easy when a tablet, troche or the like is formulated.
- the antibacterial activity enhancer of the present invention is that the milk-derived protein as an active ingredient has no antibacterial activity or has an antibacterial activity lower than that of lactoferrin or lactoferrin hydrolyzate even if it has antibacterial activity Or having a limited antimicrobial spectrum. And even if the antibacterial activity enhancer of the present invention itself does not have antibacterial activity, it acts to increase the antibacterial activity of lactoferrin or lactoferrin hydrolyzate when used or mixed with lactoferrin or lactoferrin hydrolyzate.
- the antibacterial activity enhancer of the present invention itself has antibacterial activity
- the antibacterial activity enhancer and the lactoferrin or lactoferrin hydrolyzed when used together or mixed with lactoferrin or lactoferrin hydrolyzate are higher than the combined antibacterial activity of each degradation product. “Enhancement of antibacterial activity” includes any of the above cases.
- lactoferrin or a lactoferrin degradation product to which an antibacterial activity enhancer of one form of the present invention is added is compared with that of lactoferrin or a lactoferrin degradation product to which the antibacterial activity enhancer is not added, as shown in this test example described later.
- the number of E. coli can be reduced to at least 1/7 or less.
- the amount of E. coli represented by CFU (colony forming unit) can be reduced to 1/7 or less.
- the effect of lactoferrin or a lactoferrin degradation product to which an antibacterial activity enhancer of another form of the present invention is added is at least the number of E. coli cells compared to lactoferrin or a lactoferrin degradation product to which no antibacterial activity enhancer is added. It can be reduced to 1/35 or less.
- the enhancement of antibacterial activity in the antibacterial activity enhancer of the present invention means that antibacterial activity against Escherichia coli possessed by a specific amount of lactoferrin or lactoferrin hydrolyzate by using the antibacterial activity enhancer in combination with lactoferrin or lactoferrin hydrolyzate. Is preferably 7 times or more, more preferably 35 times or more.
- the specific number of lactoferrin or lactoferrin hydrolyzate reduces the number of microorganisms to 1 / n, and lactoferrin or lactoferrin hydrolyzate and antibacterial activity If the number of microorganisms is reduced to 1 / m (where m> n) when the enhancer is used in combination, the antibacterial activity of lactoferrin or lactoferrin hydrolyzate is enhanced to m / n.
- the degree of enhancement is approximated by m / n if it is sufficiently lower than the antibacterial activity of lactoferrin or lactoferrin hydrolyzate.
- the evaluation of the degree of enhancement of antibacterial activity is preferably performed under the conditions showing the highest antibacterial activity when various amounts of the antibacterial activity enhancer of the present invention are added to a certain amount of lactoferrin or a lactoferrin hydrolyzate. .
- the antibacterial activity when the antibacterial activity enhancer and lactoferrin or lactoferrin hydrolyzate are used in combination is the same amount of antibacterial activity enhancer and lactoferrin or lactoferrin hydrolyzed respectively. It is a premise that the degradation products are higher than the combined antibacterial activities.
- the antibacterial activity enhancer of the present invention can also be used as a pharmaceutical composition for enhancing antibacterial action, an antibacterial action enhancer, and an additive for enhancing antibacterial action.
- the term “antibacterial” includes any concept of suppressing the growth of microorganisms (antibacterial), reducing microorganisms by removing microorganisms (disinfection), and killing microorganisms (sterilization). Therefore, the antibacterial activity enhancer of the present invention can be paraphrased as a disinfection aid or a bactericidal aid. Similarly, the antibacterial activity enhancer of the present invention can be paraphrased as a disinfecting composition or a bactericidal composition.
- the microorganism to be enhanced antibacterial activity is not particularly limited as long as lactoferrin or a lactoferrin hydrolyzate exhibits an antibacterial action, for example, Escherichia, Salmonella, Listeria, Bacillus, Examples include Gram-negative bacteria such as Cronobacter, Klebsiella, and Pseudomonas, Gram-positive bacteria such as Staphylococcus and Streptococcus, and fungi such as Candida yeast.
- the milk-derived protein that is the active ingredient of the present invention is a processed product such as colostrum, transitional milk, regular milk, end milk, etc. of mammals (eg, human, cow, buffalo, horse, goat, sheep, etc.). It can be prepared by cation exchange treatment or ultrafiltration treatment using skim milk as a milk raw material.
- the milk-derived protein can be prepared without particular limitation as long as the whey protein is contained in the milk raw material.
- the protein derived from milk is not limited to a protein obtained using milk or a dairy product as a raw material, as long as it has a structure equivalent to the protein contained in milk, or a protein obtained from body fluids or tissues of animals other than milk, or It may be a chemically synthesized product or a recombinant protein.
- Angiogenin-1 and Angiogenin-2 or fragments thereof are not limited to those having any amino acid sequence of SEQ ID NOs: 4 to 8, and may be conservative variants thereof.
- substitution or deletion of one or several, for example 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acid residues in the amino acid sequences of SEQ ID NOs: 4 to 8 A protein having a sequence containing insertion, addition, or inversion, or an amino acid sequence having a homology of 80% or more, preferably 90% or more, more preferably 95% or more with the amino acid sequence of SEQ ID NOs: 4 to 8 And a protein having an action of enhancing the antibacterial activity of lactoferrin or a lactoferrin hydrolyzate.
- lactoferrin that is the target of enhancement of antibacterial activity includes commercially available products, mammals (eg, humans, cows, buffalos, horses, goats, sheep, etc.) colostrum, transitional milk, normal milk, end milk, etc. Lactoferrin separated from these processed products, such as skim milk and whey by conventional methods such as ion exchange chromatography, apolactoferrin from which iron is removed from lactoferrin by conventional methods, metals such as iron, copper, zinc, manganese, etc. Can be used either partially or fully chelated metal unsaturated lactoferrin or metal saturated lactoferrin. In addition, recombinant fungi obtained by recombinant DNA technology, human lactoferrin produced by recombinant dairy cows (transgenic cows) and the like can also be used in the present invention.
- the lactoferrin hydrolyzate to be enhanced in antibacterial activity may be a commercially available product or a hydrolyzate obtained by hydrolyzing the lactoferrin of the present invention with an acid or an enzyme. It can be obtained by the method described in the invention of Hei 3-171736.
- the lactoferrin hydrolyzate to be enhanced in antibacterial activity includes a lactoferrin-derived peptide isolated or purified from the lactoferrin hydrolyzate.
- lactoferrin-derived peptide for example, lactoferricin (registered trademark) can be exemplified.
- FIG. 3 shows the primary structure (amino acid sequence: SEQ ID NO: 1, 2) of human lactoferricin (lactoferricin H), and
- FIG. 4 shows the primary structure of bovine lactoferricin (lactoferricin B) (amino acid sequence: SEQ ID NO: 3) are shown respectively.
- lactoferrin When hydrolyzing lactoferrin with an acid, lactoferrin is used as an aqueous solution, an inorganic acid or an organic acid is added thereto, and the mixture is heated to a predetermined temperature for a predetermined time for hydrolysis.
- lactoferrin When hydrolyzing with an enzyme, lactoferrin is made into an aqueous solution, adjusted to an optimum pH of the enzyme to be used, and an enzyme such as pepsin or trypsin is added thereto, and the mixture is kept at a predetermined temperature for a predetermined time for hydrolysis. After hydrolysis, the enzyme is inactivated by a conventional method.
- the degradation product obtained by hydrolysis with acid or enzyme is a mixture of degradation products having various molecular weights and containing peptides having antibacterial properties.
- the degree of degradation by hydrolysis is preferably in the range of 6 to 20%.
- the degree of decomposition is calculated by the following formula from these values by measuring the total nitrogen of the sample by the Kjeldahl method and the formol-type nitrogen of the sample by the formol titration method.
- the reaction solution obtained by hydrolysis with an acid or enzyme (a solution of a lactoferrin degradation product) is cooled by a conventional method, neutralized, desalted, and decolorized by a conventional method as necessary, and further if necessary.
- the obtained solution is used as it is, as a concentrated solution, or as a powder dried after concentration. Further, as described above, an antibacterial peptide such as lactoferricin may be isolated from the hydrolyzate.
- the active ingredient of the antibacterial activity enhancer in the present invention is composed of one or a plurality of protein components derived from milk, and these one or more components enhance the antibacterial activity of lactoferrin or a lactoferrin degradation product.
- the “milk-derived protein” as an active ingredient may be a single protein or a mixture of two or more proteins as long as it has an action of enhancing the antibacterial activity of lactoferrin or a lactoferrin degradation product. .
- the inventors of the present invention have a milk-derived protein that is a component that enhances antibacterial activity, at least 1) has an action of enhancing the antibacterial activity of lactoferrin or lactoferrin hydrolyzate, and 2) has a molecular weight of 30 kDa or less, And 3) it has been determined that the component satisfies the isoelectric point of 9.0 to 11.0.
- the milk-derived protein may contain lactoferrin or a lactoferrin hydrolyzate with a content of 2.0% by mass or less based on the total amount of milk-derived protein.
- the milk-derived protein that can be the active ingredient of the present invention preferably contains no lactoferrin or lactoferrin hydrolyzate, but the ratio of lactoferrin or lactoferrin hydrolyzate in the milk-derived protein of the active ingredient However, if the mass ratio is 2.0% or less, it is confirmed that the milk-derived protein as a whole does not exhibit antibacterial activity.
- Example 1 it was estimated that antibacterial activity was enhanced by any one component or a combination of a plurality of components contained in a spot obtained by two-dimensional electrophoresis. And it has been confirmed that angiogenin-1 or a mixture of angiogenin-1 and angiogenin-2 enhances the antibacterial activity of lactoferrin or lactoferrin hydrolyzate.
- the protein derived from milk that is an active ingredient of the antibacterial activity enhancer of the present invention include Angiogenin-1 (Angiogenin-1), Angiogenin-2 (Angiogenin-2), and mixtures thereof.
- the isoelectric points predicted from the amino acid sequences of Angiogenin-1 and Angiogenin-2 derived from bovine are 9.1 and 9.8-10, respectively.
- Bovine-derived angiogenin-1 and angiogenin-2 have substantially no antibacterial activity against E. coli.
- the milk-derived protein may be a dimer or fragment of angiogenin-1 and / or angiogenin-2, or a mixture thereof as long as it has an effect of enhancing the antibacterial activity of lactoferrin or lactoferrin hydrolyzate. Good.
- the milk-derived protein: lactoferrin or lactoferrin hydrolyzate (mass ratio) in the antibacterial activity enhancer is preferably 1:80 to The ratio is 1: 1, more preferably 1:80 to 1:10, more preferably 1:60 to 1:10, and still more preferably 1:40 to 1:10.
- lactoferrin registered trademark
- lactoferrin a lactoferrin-derived peptide, among lactoferrin degradation products
- Protein lactoferricin is preferably used in a ratio of 1: 1 to 4: 1
- the antibacterial agent composition of the present invention contains the antibacterial activity enhancer of the present invention and one or more selected from the group consisting of lactoferrin and lactoferrin hydrolysates, and lactoferrin and / or lactoferrin hydrolysates.
- the mass ratio of the protein derived from milk in the antibacterial activity enhancer is 1/80 or more.
- the mass ratio of the protein derived from milk in the antibacterial activity enhancer with respect to lactoferrin and lactoferrin hydrolyzate is not particularly limited as long as it is 1/80 or more, preferably 1/40 or more, more preferably 1/20 or more. is there.
- the upper limit of the mass ratio of the milk-derived protein in the antibacterial activity enhancer with respect to lactoferrin and lactoferrin hydrolyzate is not particularly limited, and for example, 1/1 or less, 1/2 or less, 1/5 or less, or 1/10 High antibacterial activity can be expected even below.
- the mass ratio of angiogenin and lactoferrin contained in normal whey is approximately 1: 200.
- the antibacterial agent composition of the present invention can be blended in pharmaceuticals, foods and drinks, feeds, cosmetics, quasi drugs and the like.
- the medicament of the present invention is characterized by containing the antibacterial agent composition of the present invention, or the antibacterial activity enhancer of the present invention and lactoferrin or a lactoferrin hydrolyzate.
- the form of the pharmaceutical of the present invention is not particularly limited, and can be formulated and administered in various modes by a known method, and can be orally administered in a preferred embodiment.
- the antibacterial activity enhancer of the present invention and lactoferrin or lactoferrin hydrolyzate are used in a mixed state.
- the antibacterial activity enhancer of the present invention can be produced by formulating the antibacterial activity enhancer of the present invention and lactoferrin or a lactoferrin hydrolyzate using any additive such as a pharmaceutically acceptable excipient.
- the content of lactoferrin or lactoferrin hydrolyzate in the formulation is not particularly limited. Although it is considered that there is almost no side effect due to the antibacterial activity enhancer of the present invention and lactoferrin or lactoferrin hydrolyzate, the content of the composition consisting of the antibacterial activity enhancer and lactoferrin or lactoferrin hydrolyzate in pharmaceuticals is 0.1 to 60% by mass, preferably 10 to 50% by mass.
- the content of the composition comprising the antibacterial activity enhancer and lactoferricin (registered trademark) in the pharmaceutical is usually 0.01 to 6% by mass, preferably 1 to 5% by mass.
- the amount ratio of the antibacterial activity enhancer, lactoferrin, and lactoferrin hydrolyzate is the same as described for the antibacterial composition.
- additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, solvents for injections, and the like can be used.
- Specific preparations include tablets (including sugar-coated tablets, enteric-coated tablets, buccal tablets), powders, capsules (including enteric capsules and soft capsules), granules (including those coated), pills,
- Illustrative examples include troches, encapsulated liposomes, solutions, and pharmaceutically acceptable sustained release formulations.
- Carriers and excipients used in these preparations include lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, gentian powder, etc.
- Examples thereof include starch, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, ethyl cellulose, methyl cellulose, carboxymethyl cellulose and the like.
- disintegrants examples include starch, agar, gelatin powder, sodium carboxymethylcellulose, calcium carboxymethylcellulose, crystalline cellulose, calcium carbonate, sodium bicarbonate, and sodium alginate.
- a lubricant magnesium stearate, hydrogenated vegetable oil, and macrogol, etc.
- a colorant red No. 2, yellow No. 4, and blue No. 1, etc. that are allowed to be added to pharmaceuticals, Each can be illustrated.
- Tablets and granules include sucrose, hydroxypropylcellulose, purified shellac, gelatin, sorbitol, glycerin, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, methyl methacrylate, It can also be coated with a methacrylic acid polymer or the like.
- the pharmaceutical composition to be used in combination may be contained as an active ingredient in the drug of the present invention, or may be commercialized as a separate drug without being contained in the drug of the present invention.
- a pharmaceutical composition for antibacterial such as toothpaste and oral cleaning solution can be exemplified.
- the food or drink of the present invention is characterized by containing the antibacterial agent composition of the present invention, or the antibacterial activity enhancer of the present invention and lactoferrin or a lactoferrin hydrolyzate.
- the content of the composition comprising the antibacterial activity enhancer and lactoferrin or lactoferrin hydrolyzate in the food and drink of the present invention is 0.1 to 60% by mass, preferably 10 to 50% by mass.
- the content of the composition comprising the antibacterial activity enhancer and lactoferricin (registered trademark) in the food and drink is usually 0.01 to 6% by mass, preferably 1 to 5% by mass.
- the amount ratio of the antibacterial activity enhancer, lactoferrin, and lactoferrin hydrolyzate is the same as described for the antibacterial composition.
- the form of the food / beverage product of the present invention is not particularly limited.
- soft drinks, milk drinks and the like in which lactoferrin, lactoferrin hydrolyzate powder, aqueous solutions thereof (syrups, etc.) and the like are blended with the antibacterial activity enhancer of the present invention.
- concentrated concentrates of these beverages and powders for preparation dairy products such as processed milk and fermented milk; infant formula milk; enteral nutrition; functional foods, etc., and carbonated beverages, nutritional beverages, fruit beverages Beverages such as concentrated concentrates of these beverages and preparation powders; Ice cream, ice sherbet, shaved ice and other frozen desserts; buckwheat, udon, harusame, gyoza skin, cucumber skin, Chinese noodles, instant noodles, etc.
- Noodles candy, chewing gum, candy, gum, chocolate, tablet confectionery, snack confectionery, biscuits, jelly, jam, cream, baked confectionery, etc .
- kamabo Processed fishery and livestock products such as ham, sausage; salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing and other fat and oil processed foods; sauces, sauces and other seasonings; soup, stew, salad, sugar beet , Pickles, bread and the like.
- Such foods and beverages include, for example, lactoferrin (including forms such as lactoferrin powder and lactoferrin aqueous solution (syrup, etc.), the antibacterial activity enhancer of the present invention, and sugars such as dextrin and starch; gelatin, soy protein, It can be produced by blending proteins such as corn protein; amino acids such as alanine, glutamine and isoleucine; polysaccharides such as cellulose and gum arabic; oils and fats such as soybean oil and medium chain fatty acid triglycerides. Moreover, liquid, a tablet-like supplement etc. can be illustrated as a suitable shape of food-drinks.
- the feed of the present invention is characterized by containing the antibacterial agent composition of the present invention, or the antibacterial activity enhancer of the present invention and lactoferrin or a lactoferrin hydrolyzate.
- the content of the composition comprising the antibacterial activity enhancer and lactoferrin or lactoferrin hydrolyzate in the feed of the present invention is 0.1 to 60% by mass, preferably 10 to 50% by mass.
- the content of the composition comprising the antibacterial activity enhancer and lactoferricin (registered trademark) in the feed is usually 0.01 to 6% by mass, preferably 1 to 5% by mass.
- the amount ratio of the antibacterial activity enhancer, lactoferrin, and lactoferrin hydrolyzate is the same as described for the antibacterial composition.
- the form of the feed of the present invention is not particularly limited.
- lactoferrin, lactoferrin hydrolyzate powder, aqueous solutions thereof (syrups, etc.) and the like can be blended with the antibacterial activity enhancer of the present invention.
- Inventive antibacterial activity enhancer and lactoferrin cereals such as corn, wheat, barley, rye, milo; vegetable oils such as soybean oil cake, rapeseed oil cake, coconut oil cake, linseed oil cake; Potatoes such as corn gluten meal, corn jam meal; animal feeds such as fish meal, skim milk powder, whey, yellow grease, tallow; yeasts such as torula yeast and brewer's yeast; tribasic calcium phosphate; It can be produced by blending mineral feed such as calcium carbonate; fats and oils; simple amino acids; Examples of the form of the feed include pet food, livestock feed, and fish feed.
- the antibacterial activity enhancer of the present invention can be produced, for example, by a method having the following steps. That is, (a) the milk raw material is brought into contact with a cation exchange resin and the protein in the milk raw material is adsorbed on the cation exchange resin, (b) the cation exchange resin after the adsorption treatment is washed and eluted. A step of eluting a protein fraction having an isoelectric point of 9.0 to 11.0 by passing a solvent, and (c) filtering the eluted protein fraction through an ultrafiltration membrane having a fractional molecular weight of 30 kDa.
- a method for producing an antibacterial activity enhancer of lactoferrin or a lactoferrin hydrolyzate comprising the milk-derived protein as an active ingredient comprising the step of preparing a milk-derived protein having the following properties: .
- the permeate is passed through an ultrafiltration membrane having a fractional molecular weight of 10 kDa, and a concentrated solution of 10 kDa or more is collected, Can also be included.
- the method for producing the antibacterial activity enhancer of the present invention is partly in common with the usual method for producing lactoferrin and lactoperoxidase, but differs in that it includes the steps (b) to (d).
- the antibacterial activity enhancer of the present invention is different from lactoferrin and lactoperoxidase in terms of molecular weight. Furthermore, the manufacturing method of the antibacterial activity enhancer of this invention is demonstrated in detail below.
- the milk raw material is brought into contact with a cation exchanger to perform an adsorption treatment.
- the milk raw material is not particularly limited as long as it contains the protein having the antibacterial activity enhancing action, and the raw milk of mammals (for example, human, cow, buffalo, horse, goat, sheep etc.), processed milk such as skim milk, Examples include fractions such as whey and milk produced by recombinant cows (transgenic cows).
- the milk may be any of colostrum, transitional milk, regular milk, terminal milk, and the like.
- the cation exchanger those having weakly acidic groups or strong acidic groups as ion exchange groups are used.
- an exchange group having an intended function as an ion exchange group can be arbitrarily selected.
- Examples of weakly acidic ion exchange groups include carboxyl groups and carboxymethyl groups.
- Examples of the strongly acidic ion exchange group include a sulfopropyl group.
- the cation exchanger is not particularly limited, and can be arbitrarily selected as necessary.
- preferred cation exchangers are those in which weakly acidic or strongly acidic ion exchange groups are introduced into porous particles made of cross-linked polysaccharides (agarose, dextran, cellulose, etc.), hydrophilic silica gel, synthetic polymer, etc. Etc.
- weakly acidic ion exchangers include Sepabeads FP-CM13 (manufactured by Mitsubishi Chemical, exchange group: carboxymethyl group) and CM-Sephadex C-50 (manufactured by GE Healthcare, exchange group: carboxymethyl).
- CM-Sepharose-FF manufactured by GE Healthcare
- exchange group carboxymethyl group
- strongly acidic ion exchangers include SP-Sepharose FF (manufactured by GE Healthcare) and SP-Sepharose big beads (manufactured by GE Healthcare).
- the shape, size, surface condition, material, etc. of the resin of the cation exchanger are arbitrary and can be selected as necessary.
- Examples of usage forms include pre-packed columns already filled with an ion exchanger resin, and those packed with a cation exchanger resin bead in a medium such as a column. In these cases, it is preferable from the viewpoint of simplicity to use one kind of cation exchanger in combination with one medium. However, if necessary, chromatography may be performed by connecting a plurality of media filled with resin beads in series or in parallel.
- the shape of the medium can be selected according to need, but it is preferable that it is easy to clean and gives many contact surfaces to the resin beads contained therein, and the inner wall should be a flat surface without irregularities. preferable.
- a shape having a circular surface such as a cylindrical shape (columnar shape, rod shape) or a conical shape can be preferably used as the shape of the medium.
- the material of the medium can be arbitrarily selected, but preferably stainless steel, glass, polypropylene, polyethylene, polyethylene terephthalate, polycarbonate, acrylic resin or the like can be used.
- the size of the medium may be appropriately selected depending on the processing scale, and can be arbitrarily selected from a scale of several cubic centimeters to several cubic meters, for example.
- Examples of commercially available prepacked columns that can be preferably used in the present invention include Hiprep CMFF 16/10 (GE Healthcare, exchange group: carboxymethyl group), Hipprep SPFF 16/10 (GE Healthcare, exchange group: sulfo). Propyl group), TOYOPEARL CM-650 series (produced by Tosoh Corporation, exchange group: carboxymethyl group), TOYOPEARL SP-650 series (produced by Tosoh Corporation, exchange group: sulfopropyl group) and the like.
- the adsorption treatment (contact) between the milk raw material and the cation exchanger can be arbitrarily selected.
- the adsorption treatment can be performed by a method such as a batch type stirring method or a column continuous method. Any treatment can be performed as long as the milk raw material and the cation exchanger can be sufficiently brought into contact with each other.
- One process may be performed, or a plurality of adsorption processes may be combined.
- a large amount of cation exchanger is used. If a large amount of cation exchanger is desired, a large amount of milk material is used. Is appropriate.
- the mixing volume ratio of the milk raw material and the cation exchanger in the batch method can be arbitrarily adjusted according to the adsorption capacity of the cation exchanger and a specific adsorption treatment method.
- the characteristic of a cation exchanger is divided roughly into two types, a hard type and a soft type, any type can be used in the present invention.
- the hard type the volume of the ion exchanger itself hardly changes depending on the ionic strength or pH, and the volume of the cation exchanger itself hardly changes even if the pressure applied to the cation exchanger changes due to a change in flow rate or the like. . Therefore, the hard type is more suitable for the column continuous method in which the cation exchanger is held in the column and passed at a high flow rate.
- the volume of the cation exchanger changes greatly depending on the ionic strength or pH, and when the pressure applied to the cation exchanger changes due to a change in flow rate, the volume of the cation exchanger itself increases. Change easily. For this reason, it is difficult to hold a soft-type cation exchanger in the column and let it flow at a high flow rate. In particular, when skimmed milk or whey is passed, a greater pressure loss occurs in the cation exchanger layer than when other salt solutions are passed. Therefore, the soft type cation exchanger is suitable only for the batch method.
- the milk raw material may freeze or increase in viscosity, and if it exceeds 60 ° C., the protein in the milk, for example, the base in the milk. Since the protein may be denatured, it is preferably carried out in the range of 0 to 60 ° C. Even at 25 to 60 ° C., when a long time is required for adsorption, proteins in milk, for example, basic proteins in milk may be gradually denatured. It is preferable to be performed at 25 ° C. In addition, when unsterilized milk raw materials are used, it is desirable to carry out at 0 to 10 ° C. in order to prevent bacterial growth.
- Conditions for the adsorption treatment time (contact time) of the milk raw material and the cation exchanger can be appropriately selected in consideration of the temperature during the adsorption treatment, the adsorption treatment method employed (batch type or column continuous method), etc. .
- the adsorption treatment time of the milk raw material and the cation exchanger is preferably 1 minute to 24 hours, and more preferably 10 minutes to 6 hours.
- the linear flow rate is preferably 10 cm / h or more and 1000 cm / h or less.
- the cation exchanger after the adsorption treatment is washed.
- the cleaning liquid at this time it is possible to use a salt aqueous solution having a low ionic strength of less than 0.07, or a buffer solution in a neutral or weakly acidic region. Is preferred.
- the milk material used may be washed and removed by any method.
- the cation exchanger may be moved from the container containing the milk raw material and the cation exchanger and washed in another place, or the milk raw material is moved from the container and then the cation exchanger is placed in the container. You may wash with.
- the elution solvent is brought into contact with the cation exchanger after the washing treatment.
- the target protein is eluted from the cation exchanger into the elution solvent to obtain an eluate.
- the elution solvent used in this step preferably has an ionic strength of 0.07 to 1.7, more preferably 0.16 to 1.0, and 0.25 to 0.6. The following ranges are particularly preferred. Thereby, it is possible to obtain an elution fraction having an isoelectric point of 9.0 to 11.0.
- a neutral or weakly acidic buffer solution having an ionic strength adjusted to the above range can also be used.
- an aqueous solution salt solution
- the salts that can be used in the present application can be arbitrarily selected from one or a combination of two or more as required.
- a preferable salt solution is an aqueous solution of a salt composed of one or a mixture of two or more selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and the like, specifically, 1 to 5% chloride.
- a salt composed of one or a mixture of two or more selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and the like, specifically, 1 to 5% chloride.
- Examples include sodium solutions, preferably 1.5 to 3.5% sodium chloride solution, and the like.
- membrane treatment is performed on the obtained eluate by a membrane separation method using an ultrafiltration membrane. This concentrates the eluate and produces a precipitate in the eluate.
- the operation method of the ultrafiltration membrane is the normal ultrafiltration method in which water and components having a molecular weight equal to or lower than the molecular weight cut off are permeated and the same amount of water as the permeate that has permeated the membrane.
- a flowing water filtration method diafiltration
- either method can be used in the present invention.
- the latter flowing water filtration method is more preferable in that it can be desalted at the same time as concentration and can highly remove low molecular weight components from the retentate.
- any commercially available ultrafiltration membrane can be used. Specific examples include IRIS3038 membrane and IRIS3072 membrane (both manufactured by Rhone-Poulenc), GR-61pp membrane (manufactured by DDS), etc., both of which have a fractional molecular weight of 30 kDa (molecular weight of 30 kDa cut off). It has a certain property.
- the material of the ultrafiltration membrane can be used regardless of whether it is made of an organic material or an inorganic material, and may be selected in consideration of cost and versatility.
- the eluate temperature during the ultrafiltration treatment can be carried out if it is lower than the heat resistant temperature of the membrane used (for example, 80 ° C. or lower for the GR-61pp membrane). However, from the viewpoint of preventing protein denaturation, it is 0 to Examples include 60 ° C., such as 25 to 60 ° C., or 0 to 25 ° C.
- the pressure at the time of ultrafiltration can be any pressure as long as it is below the pressure limit of the membrane to be used (for example, 0.6 MPa or less for the GR-61pp membrane). Since use near the breakdown voltage limit may shorten the life of the film, it is preferably performed at 2/3 or less of the breakdown voltage limit (for example, 04 MPa or less for a GR-61pp film).
- the ultrafiltration membrane module to be used may be any type such as a tube type, a hollow fiber type, a flat membrane type, and a spiral type. However, in the case of a hollow fiber type internal pressure type module or the like, there is a possibility that the flow path is clogged when a precipitate is generated in the hollow fiber.
- the antibacterial activity enhancement test measures the antibacterial activity of a sample obtained by mixing the antibacterial activity enhancer of the present invention with lactoferrin or a lactoferrin degradation product. As a control, antibacterial activity was also measured for a sample containing only lactoferrin or a lactoferrin degradation product.
- the specific test procedure is as follows.
- Sample preparation At least the following samples were prepared. In the test for lactoferrin, two types of samples 2 having different concentrations of the antibacterial activity enhancer were prepared. Sample 1: Mixture of lactoferrin or lactoferrin degradation product, etc. 2: Lactoferrin or lactoferrin degradation product, etc., and antibacterial activity enhancer Sample 3: Antibacterial activity enhancer
- E. coli K-12 was inoculated into a 5 mL polystyrene tube and pre-cultured in a 37 ° C. incubator for 6 hours to obtain a test bacterial solution. Then, precultured E. coli was added to 10 6 cells / mL along with the above sample on a 96-well plate, and cultured at 37 ° C. for a set time.
- the culture solution containing Escherichia coli is serially diluted with 0.85% physiological saline, plated on an agar medium, and then the number of colonies (CFU: Colony Forming Units) generated after culturing for a set time at 37 ° C. was measured.
- CFU Colony Forming Units
- Example 1 the antibacterial activity enhancer of the present invention was produced.
- Test method CM-Sephadex C-50 manufactured by GE Healthcare
- 20 liters of non-sterilized skim milk was passed through a column packed with 170 mL of cation exchange resin to adsorb milk protein onto the exchange resin.
- the cation exchange resin in the column was sufficiently washed with deionized water, and 0.27 M sodium chloride (manufactured by Kokusan Chemical Co., Ltd.) was passed through to elute the protein adsorbed on the resin.
- 160 mg of freeze-dried protein powder was dissolved in 10 mL of 0.5 M sodium chloride, and centrifuged (4,000 ⁇ g) using a centrifugal ultrafiltration membrane (trade name: Amicon Ultra, manufactured by Millipore) with a molecular weight cut off of 30 kDa. 15 minutes), and the permeate side basic protein fraction was recovered. Further, after adding 10 mL of 0.5 M sodium chloride to the centrifugal ultrafiltration membrane and performing centrifugation (4,000 ⁇ g, 15 minutes), the permeate-side basic protein fraction obtained was obtained. It was collected.
- a centrifugal ultrafiltration membrane trade name: Amicon Ultra, manufactured by Millipore
- This basic protein fraction and the previously obtained basic protein fraction were mixed, and desalted and concentrated with a centrifugal ultrafiltration membrane (trade name: Amicon Ultra, manufactured by Millipore) having a molecular weight cut off of 10 kDa.
- the fraction on the concentrate side was obtained as the antibacterial activity enhancer of the present invention.
- the molecular weight distribution of the antibacterial activity enhancer of the present invention by two-dimensional electrophoresis was 8-30 kDa, and the isoelectric point was 9.0-11.0.
- Example 2 To 95 g of bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.), 5 g of the antibacterial activity enhancer produced by the same method as in Example 1 was added and mixed uniformly to produce an antibacterial drug with enhanced lactoferrin antibacterial activity.
- Example 3 As a lactoferrin degradation product, bovine lactoferrin used in Example 2 was adjusted to pH 5 or lower, reacted with 3% pepsin, enzymatically digested, concentrated on a reverse osmosis membrane, and then freeze-dried, resulting in degradation of lactoferrin A product powder was used. 5 g of the antibacterial activity enhancer produced by the same method as in Example 1 was added to 95 g of the lactoferrin degradation product powder and mixed uniformly to produce an antibacterial drug with enhanced antibacterial activity of the lactoferrin degradation product.
- Example 4 To 95 g of bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.), 5 g of the antibacterial activity enhancer produced by the same method as in Example 1 was added and mixed uniformly. To produce fermented milk food.
- Example 1 Test Method In order to specify the components of the antibacterial activity enhancer obtained in Example 1, two-dimensional electrophoresis was performed using a ZOOM IPG Runner System (manufactured by Invitrogen). In the first dimension, ZOOM Strip pH 3-10NL having a pH range of 3 to 10 was used. In the second dimension, NuPAGE4-12% Bis-Tris ZOOM Gel was used. And it dye
- the antibacterial activity enhancer of the present invention had a molecular weight distribution of 8 to 30 kDa and an isoelectric point of 9.0 to 11.0. Furthermore, the stained gel obtained by two-dimensional electrophoresis was photographed with a densitograph (manufactured by ATTO) and subjected to relative quantification using analysis software.
- the obtained spot was cut out from the gel, the target protein was decolored, subjected to in-gel digestion by reductive alkylation and trypsin treatment, and mass spectrometry was performed with a MALDI-TOF / MS apparatus (trade name: Autoflex II, manufactured by BRUKER)
- the protein was identified by searching the database (http://www.ncbi.nlm.nih.gov/) of NCBI (National Center for Biotechnology information) using MASCOT software manufactured by Matrix Science.
- FIG. 1 shows the results of two-dimensional electrophoresis.
- the vertical axis indicates the molecular weight
- the horizontal axis indicates the isoelectric point.
- Table 1 shows the molecular weight of each spot shown in FIG. 1 correspond to the numbers of “Spot No.” in Table 1 and spots [1] to [7] in the specification.
- Spot [1] was revealed to be a protein derived from lactoferrin.
- Spots [2], [4], [6], and [7] are a protein monomer, dimer, known as Angiogenin-1, as shown in FIG. Body, fragment, etc.
- the spot [3] was found by a database search to be derived from a protein known as Angiogenin-2 as shown in FIG. However, the type of protein has not been identified for spot [5].
- the antibacterial activity enhancer of the present invention is a milk protein having a molecular weight of 30 kDa or less including components of spots [2] to [7] and an isoelectric point of 9.0 to 11.0. It became clear that there was. Further, the content of a substance derived from lactoferrin or a lactoferrin degradation product in the antibacterial activity enhancer of the present invention was measured. An analysis diagram using a densitometer is shown in FIG. 2, and an analysis result is shown in Table 2.
- Test Example 2 The purpose of this test was to confirm that the antibacterial activity enhancer of the present invention enhances the antibacterial activity of lactoferrin.
- the antibacterial activity enhancer obtained in Example 1 was used after diluted with deionized water.
- Sample 1 Bovine lactoferrin (2 mg / mL)
- Sample 2 Mixture of bovine lactoferrin (2 mg / mL) and antibacterial activity enhancer (160 ⁇ g / mL).
- Sample 3 Mixture of bovine lactoferrin (2 mg / mL) and antibacterial activity enhancer (32 ⁇ g / mL).
- Sample 4 Antibacterial activity enhancer (32 ⁇ g / mL)
- the culture solution containing Escherichia coli is serially diluted with 0.85% physiological saline, plated on an agar medium (Rapid Media RM-SPC, manufactured by Morinaga Milk Industry Co., Ltd.), and then cultured at 37 ° C. for 16 hours.
- the antibacterial activity of each sample was evaluated by measuring the number of colonies formed later (CFU: Colony Forming Units).
- CFU Colony Forming Units
- Results Table 3 shows the results of Test Example 2.
- the significance test is P ⁇ 0.001.
- the number of bacteria increased to 1.1 ⁇ 10 8 CFU / mL after 17 hours, whereas in the sample 1 containing lactoferrin, the number of bacteria decreased from the number of initial bacteria. The range was 6 ⁇ 10 5 CFU / mL.
- Test Example 3 The purpose of this test was to confirm that the antibacterial activity enhancer of the present invention enhances the antibacterial activity of the lactoferrin hydrolyzate.
- sample As a lactoferrin degradation product, bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) used in Test Example 2 was adjusted to pH 5 or lower, reacted with 3% pepsin, enzymatically digested, and concentrated with a reverse osmosis membrane. Thereafter, a lactoferrin degradation product powder obtained by freeze-drying was used. The lactoferrin degradation product powder was diluted with deionized water. On the other hand, the antibacterial activity enhancer obtained in Example 1 was used after diluted with deionized water.
- Sample 5 Lactoferrin degradation product (400 ⁇ g / mL)
- Sample 6 Mixture of lactoferrin degradation product (400 ⁇ g / mL) and antibacterial activity enhancer (32 ⁇ g / mL).
- Sample 7 Antibacterial activity enhancer (32 ⁇ g / mL)
- Test method was the same as in Test Example 2. In addition to samples 5-7, E. coli cultured in medium alone was used as a control.
- Results Table 4 shows the results of Test Example 3.
- the number of bacteria increased to 6.7 ⁇ 10 7 CFU / mL after 17 hours.
- the number of bacteria decreased to 8.6 ⁇ 10 4 CFU / mL, but in sample 7 to which an antibacterial activity enhancer was further added, the number of bacteria was 2.5 ⁇ 10 2 CFU / mL.
- the number of bacteria was significantly reduced as compared with Sample 5.
- the antibacterial activity of sample 6 was about 340 times or more of the antibacterial activity of sample 5.
- Test Example 4 The purpose of this test was to confirm that the antibacterial activity enhancer of the present invention enhances the antibacterial activity of lactoferrin (registered trademark), which is a lactoferrin hydrolyzate.
- Lactoferrin registered trademark
- Test Example 2 Sample preparation Bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) used in Test Example 2 was hydrolyzed with pepsin, purified by high-performance liquid chromatography, and freeze-dried bovine lactoferricin powder. Using. Bovine lactoferricin powder was used after diluting with deionized water. On the other hand, the antibacterial activity enhancer obtained in Example 1 was used after diluted with deionized water.
- sample 8 Bovine lactoferricin (16 ⁇ g / mL)
- Sample 9 Mixture of bovine lactoferricin (16 ⁇ g / mL) and antibacterial activity enhancer (32 ⁇ g / mL).
- Sample 10 Antibacterial activity enhancer (32 ⁇ g / mL)
- E. coli cultured in medium alone was used as a control.
- Results Table 5 shows the results of Test Example 4. As a result, in the control cultured with only the medium, the number of bacteria increased to 2.9 ⁇ 10 6 CFU / mL after 2 hours.
- the number of bacteria decreased to 8.8 ⁇ 10 3 CFU / mL, but in the sample 9 further added with the antibacterial activity enhancer, the number of bacteria was 2.5 ⁇ 10 6. It was 2 CFU / mL or less, and the number of bacteria was significantly reduced.
- the antibacterial activity of sample 9 was about 35 times or more the antibacterial activity of sample 8.
- Example 5 In the same manner as in Example 1, unsterilized skim milk was passed through a CM-Sephadex C-50 column, and the cation exchange resin in the column was thoroughly washed with deionized water. Then, 1.7 M sodium chloride was passed through to elute the protein fraction adsorbed on the resin.
- the eluted protein fraction was dissolved in a citrate buffer (pH 5.2) containing 0.5 M sodium chloride, and separated by HPLC using a gel filtration carrier TSKgel G2000SW (manufactured by Tosoh Corporation).
- a gel filtration carrier TSKgel G2000SW manufactured by Tosoh Corporation.
- FIG. 5 shows an elution curve of gel filtration HPLC.
- the peak ⁇ includes two components, a minor band having a molecular weight of about 17 kDa and a major band having a molecular weight of about 15 kDa.
- the peak ⁇ contains one component of a major band having a molecular weight of about 15 kDa.
- the band was cut out from the gel, decolorized, subjected to in-gel digestion by reductive alkylation and trypsin treatment, mass-analyzed with a MALDI-TOF / MS apparatus (trade name: Autoflex II, manufactured by BRUKER), and manufactured by Matrix Science.
- MALDI-TOF / MS apparatus trade name: Autoflex II, manufactured by BRUKER
- Matrix Science manufactured by Matrix Science.
- Bovine lactoferrin manufactured by Morinaga Milk Industry Co., Ltd.
- Angiogenin-1 and Angiogenin-2 separated by the method described in Example 5 were used after diluting with deionized water.
- the following samples 11 to 15 were prepared using angiogenin-1 and angiogenin-2 separated from the bovine lactoferrin.
- Sample 11 bovine lactoferrin (2 mg / mL)
- Sample 12 Mixture of bovine lactoferrin (2 mg / mL) and Angiogenin-1 (32 ⁇ g / mL)
- Sample 13 Mixture of bovine lactoferrin (2 mg / mL) and Angiogenin-1 and Angiogenin-2 ( Mixture of 32 ⁇ g / mL each)
- Sample 14 Angiogenin-1 (32 ⁇ g / mL)
- Sample 15 A mixture of angiogenin-1 and angiogenin-2 (each 32 ⁇ g / mL) Further, using 1% Bacto petone medium (Becton Dickinson), Escherichia coli K-12 strain in a 5 mL polystyrene tube (NBRC 3301) was inoculated to 10 6 CFU / mL and pre-cultured in an incubator at 37 ° C. for 6 hours to obtain a test bacterial solution.
- Bacto petone medium Be
- Test method A test bacterial solution was added to 10 6 CFU / mL together with the above-mentioned sample on a 96-well plate and cultured at 37 ° C for 17 hours. After culturing for 17 hours, the culture solution containing E. coli was serially diluted with 0.85% physiological saline, plated on an agar medium (Rapid Media RM-SPC, manufactured by Morinaga Milk Industry Co., Ltd.), and then incubated at 37 ° C. for 16 hours. The antibacterial activity (CFU: Colony Forming Units) of each sample was evaluated by culturing for a period of time and measuring the number of colonies generated on the medium. In addition to the samples 11 to 15, a control obtained by culturing E. coli only with the medium solution was used as a control.
- CFU Colony Forming Units
- Test results Table 6 shows the results of this test. As a result, in the control cultured only in the medium, the number of bacteria increased to 1.3 ⁇ 10 8 CFU / mL after 17 hours, whereas in the sample 11 containing lactoferrin, the number of bacteria decreased from the initial number. Of 2.9 ⁇ 10 5 CFU / mL.
- the antibacterial activity of sample 12 and sample 13 was about 12 times or more and about 7 times or more of the antibacterial activity of sample 11, respectively.
- sample 16 Bovine lactoferrin (8 mg / mL)
- Sample 17 Bovine lactoferrin (4 mg / mL)
- Sample 18 Bovine lactoferrin (2 mg / mL)
- Sample 19 Bovine lactoferrin (1 mg / mL)
- Sample 20 Bovine lactoferrin (0.5 mg / mL)
- Sample 21 Bovine lactoferrin (0.25 mg / mL)
- Sample 22 bovine lactoferrin (0.125 mg / mL)
- Samples 23 to 29 were prepared by adding an antibacterial activity enhancer (32 ⁇ g / mL) to each of samples 16 to 22.
- a sample 30 containing only an antibacterial activity enhancer 32 ⁇ g / mL was prepared.
- PYG medium with 1% Bacto peptone, manufactured by 0.05% yeast extract (Becton Dickinson Co.), 1% Glucose (manufactured by Wako Pure Chemical Industries, Ltd.)
- polystyrene tubes of 5 mL 10 6 cells of Streptococcus mutans JCM5705T
- the inoculum was inoculated to 37 mL / mL and cultured in a 37 ° C. incubator for 6 hours to obtain a test bacterial solution.
- Candida albicans TIMM1768 to 10 6 cells / mL in a 5 mL polystyrene tube using Sabouraud medium (1% Bacto peptone, 2% Glucose), and culture for 6 hours in a 37 ° C incubator. The bacterial solution for testing was used.
- Sabouraud medium 1% Bacto peptone, 2% Glucose
- test bacterial solution was added to a 96-well plate together with the sample so as to be 10 6 cells / mL, and cultured at 37 ° C. for 17 hours. After culturing for 17 hours, absorbance at 630 nm was measured with a microplate reader (Corona Electric Co., Ltd.).
- a culture in which bacteria were cultured only with the medium solution was used as a control.
- the minimum lactoferrin concentration at which the growth of the fungus is significantly suppressed and the absorbance is 50% or less relative to the absorbance of the control is defined as the minimum inhibitory concentration (MIC), and lactoferrin with and without added antibacterial activity enhancer. From the comparison with the MIC concentration, the effect of antibacterial activity enhancer against various bacteria was evaluated.
- MIC minimum inhibitory concentration
- Table 7 shows the results of this test.
- LF represents lactoferrin and MIC represents the minimum inhibitory concentration.
- Cronobacter sakazaki ATCC12868, Salmonella enteritidis IID604, Klebsiella pneumoniae JCM1662T, Pseudmonas aeruginosa MMI603, Staphylococcus aureus JCM2151 and Staphylococcus epidermidis Joc2oc As a result of comparison with samples (samples 23 to 29) in which 32 ⁇ g / mL of an antibacterial activity enhancer was further added to lactoferrin, a clear decrease in MIC was observed.
- MIC was reduced to 1/2 or less of that obtained when lactoferrin alone was acted by adding an antibacterial activity enhancer to lactoferrin.
- the fungus growth inhibitory effect was not recognized in any of the fungi, as in Escherichia coli O111.
- Sample 31 Bovine lactoferricin (32 ⁇ g / mL)
- Sample 32 Bovine lactoferricin (16 ⁇ g / mL)
- Sample 33 Bovine lactoferricin (8 ⁇ g / mL)
- Sample 34 Bovine lactoferricin (4 ⁇ g / mL)
- Sample 35 Bovine lactoferricin (2 ⁇ g / mL)
- Samples 36 to 40 were prepared by adding an antibacterial activity enhancer (32 ⁇ g / mL) to each of samples 31 to 35.
- a sample 41 containing only an antibacterial activity enhancer 32 ⁇ g / mL was prepared.
- PYG medium with 1% Bacto peptone, manufactured by 0.05% yeast extract (Becton Dickinson Co.), 1% Glucose (manufactured by Wako Pure Chemical Industries, Ltd.)
- polystyrene tubes of 5 mL 10 6 cells of Streptococcus mutans JCM5705T
- the inoculum was inoculated to 37 mL / mL and cultured in a 37 ° C. incubator for 6 hours to obtain a test bacterial solution.
- Candida albicans TIMM1768 to 10 6 cells / mL in a 5 mL polystyrene tube using Sabouraud medium (1% Bacto peptone, 2% Glucose), and culture for 6 hours in a 37 ° C incubator. The bacterial solution for testing was used.
- Sabouraud medium 1% Bacto peptone, 2% Glucose
- test bacterial solution was added to a 96-well plate together with the sample so as to be 10 6 cells / mL, and cultured at 37 ° C. for 17 hours. After culturing for 17 hours, absorbance at 630 nm was measured with a microplate reader (Corona Electric Co., Ltd.). In addition to the samples 31 to 41, a culture in which bacteria were cultured only with the medium solution was used as a control.
- the minimum lactoferrin concentration at which the growth of the fungus is significantly suppressed and the absorbance is 50% or less relative to the absorbance of the control is defined as the minimum inhibitory concentration (MIC), and lactoferrin with and without added antibacterial activity enhancer. From the comparison with the MIC concentration, the effect of antibacterial activity enhancer against various bacteria was evaluated.
- MIC minimum inhibitory concentration
- Table 8 shows the results of this test.
- LFcin represents lactoferrin and MIC represents the minimum inhibitory concentration.
- the MIC can be reduced to 1/2 or less of that obtained when lactoferrin is used alone. Declined.
- the sample to which only the antibacterial activity enhancer (sample 41) was added in any of the fungi, as in the case of Escherichia coli O111, no obvious bacterial growth inhibition was observed.
- the antibacterial activity enhancer of the present invention can remarkably improve the antibacterial activity of lactoferrin or lactoferrin hydrolyzate by adding to the antibacterial agent containing lactoferrin or lactoferrin hydrolyzate as an active ingredient.
- the antibacterial activity enhancer of the present invention exerts an effect only by mixing a very small amount with respect to lactoferrin or lactoferrin hydrolyzate, so that it does not affect the composition and physical properties of the product, food and drink and feed It can be blended in pharmaceuticals, cosmetics, etc.
- the antibacterial activity enhancer of the present invention is obtained from milk-derived components, there is almost no worry about side effects, and it can be taken daily with peace of mind.
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Abstract
Description
この他にも、ラクトフェリンのペプシン消化物から得られるラクトフェリン由来ペプチドであるラクトフェリシン(登録商標)が抗菌活性を有することが知られている(非特許文献3、4)。
さらには、上記ラクトフェリンやラクトフェリン分解物にラクトフェリン類以外の成分を組み合わせて、抗菌活性を増強する方法が検討されている。
上記のように、アンジオジェニンが限定的な抗菌活性を有することは知られているが、アンジオジェニンのような乳由来の蛋白質が、それ自身の抗菌活性以上にラクトフェリン又はラクトフェリン加水分解物の抗菌活性を増強する作用を有することは知られていない。
しかしながら、ラクトフェリン類の抗菌効果を顕著な程度に高める組み合わせはほとんど知られていない。
さらには、日常的な摂取による副作用がなく、安全性の高い抗菌剤が求められていた。
(A)ラクトフェリン又はラクトフェリン加水分解物の抗菌活性を増強する作用を有する。
(B)分子量が30kDa以下である。
(C)等電点が9.0~11.0である。
また、本願第一の発明は、乳由来の蛋白質が、アンジオジェニン-1(Angiogenin-1)及び/又はアンジオジェニン-2(Angiogenin-2)であることを好ましい態様としている。
さらに、本願第一の発明は、前記乳由来の蛋白質が、それ自体では大腸菌に対する抗菌活性を有さないことを好ましい態様としている。 さらに、本願第一の発明は、前記乳由来の蛋白質が、該蛋白質とラクトフェリン又はラクトフェリン加水分解物とを併用することにより、ラクトフェリン又はラクトフェリン加水分解物の大腸菌に対する抗菌活性を7倍以上に増強する性質を有することを好ましい態様としている。
(A)ラクトフェリン又はラクトフェリン加水分解物の抗菌活性を増強する作用を有する。
(B)分子量が30kDa以下である。
(C)等電点が9.0~11.0である。
本願第六の発明は、前記乳由来の蛋白質が、ラクトフェリン又はラクトフェリン加水分解物を2.0質量%以下の量で含有することを好ましい態様としている。
また、本願第六の発明は、乳由来の蛋白質が、アンジオジェニン-1(Angiogenin-1)及び/又はアンジオジェニン-2(Angiogenin-2)であることを好ましい態様としている。
さらに、本願第六の発明は、前記乳由来の蛋白質が、それ自体では大腸菌に対する抗菌活性を有さないことを好ましい態様としている。
さらに、本願第六の発明は、前記乳由来の蛋白質が、該蛋白質とラクトフェリン又はラクトフェリン加水分解物とを併用することにより、ラクトフェリン又はラクトフェリン加水分解物の大腸菌に対する抗菌活性を7倍以上に増強する性質を有することを好ましい態様としている。
(A)ラクトフェリン又はラクトフェリン加水分解物の抗菌活性を増強する作用を有する。
(B)分子量が30kDa以下である。
(C)等電点が9.0~11.0である。
本発明の抗菌活性増強剤は、ラクトフェリン又はラクトフェリン加水分解物が本来的に有する抗菌活性を増強する効果を有するものである。
本発明の抗菌活性増強剤は、以下に記載する乳由来の蛋白質のみからなるものであってもよいが、該乳由来の蛋白質を有効成分として含む限り、他の成分、例えば後述する製剤化に用いられる成分を含んでいてもよい。また、本発明の抗菌活性増強剤は、アンジオジェニン-1及び/又はアンジオジェニン-2を有効成分として含む限り、アンジオジェニン-1及び/又はアンジオジェニン-2以外の乳由来の蛋白質(但し、ラクトフェリンを除く)を含んでいてもよいが、抗菌活性増強剤中に含まれる蛋白質に対するアンジオジェニン-1及び/又はアンジオジェニン-2の純度は、好ましくは0.002%以上、より好ましくは0.01%以上、さらに好ましく0.1%以上、特に好ましくは1.0%以上である。ラクトフェリンの混在量については後述する。
また、本発明の抗菌活性増強剤は、ラクトフェリン又はラクトフェリン加水分解物と液体同士で混合してもよく、粉末同士で混合してもよい。粉末化して混合することで、錠剤、トローチ等の製剤化の際に操作が容易となる。
また、本発明の他の形態の抗菌活性増強剤を添加したラクトフェリン又はラクトフェリン分解物の効果は、抗菌活性増強剤を添加していないラクトフェリン又はラクトフェリン分解物と比較して、大腸菌の菌数を少なくとも1/35以下に低減することができる。
ある微生物に対して抗菌活性増強剤が抗菌活性を有さない場合は、特定量のラクトフェリン又はラクトフェリン加水分解物により微生物の菌数が1/nに低減し、ラクトフェリン又はラクトフェリン加水分解物と抗菌活性増強剤を併用したときに微生物の菌数が1/m(但しm>n)に低減すれば、ラクトフェリン又はラクトフェリン加水分解物が有する抗菌活性はm/nに増強されたことになる。抗菌活性増強剤が前記微生物に対する抗菌活性を有している場合であっても、ラクトフェリン又はラクトフェリン加水分解物が持つ抗菌活性よりも十分に低い場合は、増強の程度はm/nで近似される。
抗菌活性の増強の程度の評価は、一定量のラクトフェリン又はラクトフェリン加水分解物に、本発明の抗菌活性増強剤を種々の量で加えたときに、最も高い抗菌活性を示す条件で行うことが好ましい。但し、抗菌活性増強剤が抗菌活性を有している場合は、抗菌活性増強剤とラクトフェリン又はラクトフェリン加水分解物を併用したときの抗菌活性が、各々同じ量の抗菌活性増強剤及びラクトフェリン又はラクトフェリン加水分解物の各々の抗菌活性を合わせたよりも高いことが前提である。
なお、本発明の抗菌活性増強剤は、抗菌作用増強用医薬組成物、抗菌作用増強剤、及び抗菌作用増強用添加剤として使用することも可能である。
従って、本発明の抗菌活性増強剤は、除菌補助剤又は殺菌補助剤と言い換えることができる。同様に、本発明の抗菌活性増強剤は、除菌組成物又は殺菌組成物と言い換えることができる。
本発明の有効成分である乳由来の蛋白質は、哺乳類(例えば、ヒト、ウシ、水牛、ウマ、ヤギ、ヒツジ等)の初乳、移行乳、常乳、末期乳等、これらの処理物である脱脂乳を乳原料として用いて陽イオン交換処理や限外膜濾過処理によって調製することができるものである。
また、前記乳由来の蛋白質は、乳原料中にホエイ(乳清)蛋白質が含まれるものであれば特に制限なく調製することができる。
さらに、前記乳由来の蛋白質は、乳や乳製品を原料として得られる蛋白質に限られず、乳に含まれる蛋白質と同等の構造を有する限り、乳以外の動物の体液や組織から得られる蛋白質、又は、化学合成品もしくは組換え蛋白質であってもよい。また、アンジオジェニン-1及びアンジオジェニン-2又はそのフラグメントは、配列番号4~8のいずれかのアミノ酸配列を有するものに限られず、それらの保存的バリアントであってもよい。保存的バリアントとしては、配列番号4~8のアミノ酸配列において、1又は数個、例えば1~10個、好ましくは1~5個、より好ましくは1~3個のアミノ酸残基の置換、欠失、挿入、付加、又は逆位等を含む配列を有する蛋白質、又は、配列番号4~8のアミノ酸配列と80%以上、好ましくは90%以上、より好ましくは95%以上の相同性を有するアミノ酸配列を有する蛋白質であって、ラクトフェリン又はラクトフェリン加水分解物の抗菌活性を増強する作用を有する蛋白質が挙げられる。
本発明において抗菌活性の増強の対象となるラクトフェリンは、市販品の他、哺乳類(例えば、ヒト、ウシ、水牛、ウマ、ヤギ、ヒツジ等)の初乳、移行乳、常乳、末期乳等、これらの処理物である脱脂乳、ホエイ等からイオン交換クロマトグラフィー等の常法により分離したラクトフェリン、ラクトフェリンから常法により鉄を除去したアポラクトフェリン、アポラクトフェリンに鉄、銅、亜鉛、マンガン等の金属を一部又は完全にキレートさせた金属不飽和ラクトフェリン、または金属飽和ラクトフェリンのいずれであっても使用することができる。
また、組換えDNA技術により得られる組換え真菌、組換え乳牛(トランスジェニック・カウ)等により生産されるヒト・ラクトフェリン等も、同様に本発明に使用することができる。
本発明において抗菌活性の増強の対象となるラクトフェリン加水分解物は、市販品の他、本発明のラクトフェリンを酸又は酵素で加水分解して得られる加水分解物を使用することができ、例えば特願平3-171736号の発明に記載された方法によって得ることができる。
なお、本発明において抗菌活性の増強の対象となるラクトフェリン加水分解物とは、当該ラクトフェリン加水分解物から単離又は精製されたラクトフェリン由来ペプチドも含まれるものである。
ラクトフェリン由来ペプチドとしては、例えば、ラクトフェリシン(登録商標)を例示することが可能である。
図3は、ヒト・ラクトフェリシン(ラクトフェリシンH)の一次構造(アミノ酸配列:配列番号1、2)、図4はウシ・ラクトフェリシン(ラクトフェリシンB)の一次構造(アミノ酸配列:配列番号3)をそれぞれ示す。
本発明における抗菌活性増強剤の有効成分は、乳由来の単一又は複数の蛋白質成分からなっており、これらの一成分または複数の成分が、ラクトフェリン又はラクトフェリン分解物の抗菌活性を増強する作用効果を有している。
有効成分である「乳由来の蛋白質」は、ラクトフェリン又はラクトフェリン分解物の抗菌活性を増強する作用を有する限り、単一の蛋白質であってもよく、2種以上の蛋白質の混合物であってもよい。
乳由来の蛋白質は、ラクトフェリン又はラクトフェリン加水分解物の抗菌活性を増強する作用を有する限り、アンジオジェニン-1及び/又はアンジオジェニン-2の二量体、もしくはフラグメント、又はそれらの混合物であってもよい。
本発明の抗菌活性増強剤は、ラクトフェリン又はラクトフェリン分解物に対して使用する場合、抗菌活性増強剤中の乳由来の蛋白質:ラクトフェリン又はラクトフェリン加水分解物(質量比)は、好ましくは1:80~1:1、より好ましくは1:80~1:10、より好ましくは1:60~1:10、さらに好ましくは1:40~1:10である。
本発明の抗菌剤組成物は、本発明の抗菌活性増強剤と、ラクトフェリン、及びラクトフェリン加水分解物からなる群から選択される1又は2以上とを含有し、ラクトフェリン、及び/又はラクトフェリン加水分解物に対する抗菌活性増強剤中の乳由来の蛋白質の質量比が1/80以上であることを特徴としている。ラクトフェリン、及びラクトフェリン加水分解物に対する抗菌活性増強剤中の乳由来の蛋白質の質量比は、1/80以上であれば特に制限されないが、好ましくは1/40以上、より好ましくは1/20以上である。
ラクトフェリン、及びラクトフェリン加水分解物に対する抗菌活性増強剤中の乳由来の蛋白質の質量比の上限は特に制限されないが、例えば、1/1以下、1/2以下、1/5以下、又は1/10以下でも高い抗菌活性が期待できる。
尚、通常のホエイ中に含まれるアンジオジェニンとラクトフェリンの質量比は、およそ1:200である。
本発明の抗菌剤組成物は、医薬品、飲食品、飼料、化粧品、医薬部外品等に配合することができる。
本発明の医薬品は、本発明の抗菌剤組成物、又は、本発明の抗菌活性増強剤とラクトフェリン又はラクトフェリン加水分解物を含有することを特徴としている。
本発明の医薬品の形態としては特に制限されず、公知の方法により種々の態様に製剤化して投与することができ、好適な実施の態様において経口投与することができる。製剤化する際には、本発明の抗菌活性増強剤とラクトフェリン又はラクトフェリン加水分解物を混合した状態で使用される。
製剤化する場合、製剤中のラクトフェリン又はラクトフェリン加水分解物の含有量は特に限定されるものではない。基本的に本発明の抗菌活性増強剤とラクトフェリン又はラクトフェリン加水分解物による副作用はほとんどないと考えられるが、医薬品中における抗菌活性増強剤とラクトフェリン又はラクトフェリン加水分解物からなる組成物の含有量としては、0.1~60質量%、好ましくは10~50質量%である。そして、医薬品中における抗菌活性増強剤とラクトフェリシン(登録商標)からなる組成物の含有量としては、通常0.01~6質量%、好ましくは1~5質量%である。
抗菌活性増強剤と、ラクトフェリン、及びラクトフェリン加水分解物との量比は、抗菌剤組成物について記載したのと同様である。
なお、抗菌用の医薬組成物としては、歯みがきや口腔内洗浄液等の医薬品又は医薬部外品等を例示することができる。
本発明の飲食品は、本発明の抗菌剤組成物、又は、本発明の抗菌活性増強剤とラクトフェリン又はラクトフェリン加水分解物を含有することを特徴としている。
本発明の飲食品中における抗菌活性増強剤とラクトフェリン又はラクトフェリン加水分解物からなる組成物の含有量としては、0.1~60質量%、好ましくは10~50質量%である。そして、飲食品中における抗菌活性増強剤とラクトフェリシン(登録商標)からなる組成物の含有量としては、通常0.01~6質量%、好ましくは1~5質量%である。
抗菌活性増強剤と、ラクトフェリン、及びラクトフェリン加水分解物との量比は、抗菌剤組成物について記載したのと同様である。
また、飲食品の好適な形状としては、液状、タブレット状のサプリメント等を例示することができる。
本発明の飼料は、本発明の抗菌剤組成物、又は、本発明の抗菌活性増強剤とラクトフェリン又はラクトフェリン加水分解物を含有することを特徴としている。
本発明の飼料中における抗菌活性増強剤とラクトフェリン又はラクトフェリン加水分解物からなる組成物の含有量としては、0.1~60質量%、好ましくは10~50質量%である。そして、飼料中における抗菌活性増強剤とラクトフェリシン(登録商標)からなる組成物の含有量としては、通常0.01~6質量%、好ましくは1~5質量%である。
抗菌活性増強剤と、ラクトフェリン、及びラクトフェリン加水分解物との量比は、抗菌剤組成物について記載したのと同様である。
本発明の抗菌活性増強剤は、例えば、以下の工程を有する方法により製造することができる。
すなわち、(a)乳原料を陽イオン交換樹脂に接触させて乳原料中の蛋白質を陽イオン交換樹脂に吸着処理する工程、(b)前記吸着処理後の陽イオン交換樹脂を洗浄処理し、溶出溶媒を通液して、等電点が9.0~11.0の蛋白質画分を溶出する工程、(c)前記溶出した蛋白質画分を分画分子量30kDaの限外濾過膜により膜濾過して、下記性質を有する乳由来の蛋白質を調製する工程、を有することを特徴とする、前記乳由来の蛋白質を有効成分とするラクトフェリン又はラクトフェリン加水分解物の抗菌活性増強剤を製造する方法である。
(A)ラクトフェリン又はラクトフェリン加水分解物の抗菌活性を増強する作用を有する。
(B)分子量が30kDa以下である。
(C)等電点が9.0~11.0である。
すなわち、本発明の抗菌活性増強剤は、分子量の点でラクトフェリンやラクトパーオキシダーゼとは異なっている。
さらに、本発明の抗菌活性増強剤の製造方法について、以下に詳細に説明する。
乳原料としては、前記抗菌活性増強作用を有する蛋白質を含む限り特に制限されず、哺乳類(例えば、ヒト、ウシ、水牛、ウマ、ヤギ、ヒツジ等)の生乳の他、脱脂乳等の加工乳、ホエイ等の分画物、組換え乳牛(トランスジェニック・カウ)等により生産される乳等が挙げられる。乳は、初乳、移行乳、常乳、末期乳等のいずれであってもよい。
本発明に好ましく使用することができる市販のプレパックカラムとしては、Hiprep CMFF16/10(GEヘルスケア社製、交換基:カルボキシメチル基)、Hiprep SPFF16/10(GEヘルスケア社製、交換基:スルホプロピル基)、TOYOPEARL CM-650シリーズ(東ソー社製、交換基:カルボキシメチル基)、TOYOPEARL SP-650シリーズ(東ソー社製、交換基:スルホプロピル基)等が挙げられる。
硬質タイプは、イオン強度またはpHによってイオン交換体自身の体積はほとんど変化せず、また流速の変化等により陽イオン交換体にかかる圧力が変化しても陽イオン交換体自体の体積はほとんど変わらない。したがって、硬質タイプは、陽イオン交換体をカラム内に保持して高流速で通液するカラム連続法により適している。
なお、前記のセパビーズFP-CM13(三菱化学社製)、CM-セファロースFF(GEヘルスケア社製)、SP-セファロースFF(GEヘルスケア社製)、SP-セファロースビッグビーズ(GEヘルスケア社製)は硬質タイプであり、CM-セファデックスC-50(アマシャム社製)は軟質タイプである。
この工程で使用する溶出溶媒は、イオン強度が0.07以上1.7以下の範囲のものが好ましく、0.16以上1.0以下の範囲のものがより好ましく、0.25以上0.6以下の範囲のものが特に好ましい。これにより、等電点が9.0~11.0の溶出画分を得ることが可能である。
なお、前者の通常の限外濾過法を用いた場合には、限外濾過の後に、透析やゲル濾過等の方法で脱塩処理を行うことが好ましい。
限外濾過膜の材質は有機材料製又は無機材料製の何れであっても使用可能であり、コスト面、汎用性等を考慮して選択すればよい。
限外濾過処理時の溶出液の温度は、使用する膜の耐熱温度以下であれば(例えばGR-61pp膜なら80℃以下)実施可能であるが、蛋白質の変性を防ぐ観点からは、0~60℃、例えば25~60℃、又は0~25℃が挙げられる。
使用する限外濾過膜モジュールは、管型、中空糸型、平膜型、スパイラル型等いずれの形式でも可能である。ただし、中空糸型内圧式等のモジュールでは、中空糸内に沈澱物が生成した場合に流路閉塞を起こす可能性があるので、圧力等を考慮して行うことが好ましい。
まず、実施例及び試験例で使用した手法及び試験法について記載する。
本発明の抗菌活性増強剤の成分組成の特定のために、ZOOM IPG Runner System(Invitrogen社製)を用いて二次元電気泳動を行った。第一次元目は、pH範囲3~10のZOOM Strip pH3-10NLを用いた。第二次元目は、NuPAGE4-12%Bis-Tris ZOOM Gelを用いた。そして、SimplyBlue SafeStain(Invitrogen社製)で染色した。
二次元電気泳動により得られた染色後のゲルをデンシトグラフ(ATTO社製)で撮影し、解析ソフトによる相対的定量を行った。得られたスポットをゲルから切り出し、目的蛋白を脱色し、還元アルキル化及びトリプシン処理によるゲル内消化を行い、MALDI-TOF/MS装置(商品名:AutoflexII、BRUKER社製)にて質量分析し、Matrix Science社製のMASCOTソフトウェアを使用して、NCBI(National Center for Biotechnology information)のデータベース(http://www.ncbi.nlm.nih.gov/)検索により蛋白質を同定した。
抗菌活性増強試験は、本発明の抗菌活性増強剤をラクトフェリン又はラクトフェリン分解物に混合した試料について、抗菌活性を測定するものである。対照として、ラクトフェリン又はラクトフェリン分解物のみの試料についても抗菌活性を測定した。
具体的な試験の手順は下記の通りである。
少なくとも下記の試料を調製した。ラクトフェリンの試験では、試料2について抗菌活性増強剤の濃度が異なる2種類を調製した。
試料1:ラクトフェリン又はラクトフェリン分解物等
試料2:ラクトフェリン又はラクトフェリン分解物等、及び抗菌活性増強剤の混合物
試料3:抗菌活性増強剤
1%のBacto peptone培地を用い、5mLのポリスチレンチューブに大腸菌K-12株を植菌し、37℃のインキュベーター内で6時間前培養し、試験用菌液とした。そして、96穴プレート上にて、上記の試料と共に、前培養した大腸菌を106cells/mLになるよう添加し、37℃にて設定時間培養した。設定時間培養後に大腸菌を含む培養液を0.85%生理食塩水にて段階希釈し、寒天培地上にプレーティングした後、37℃、設定時間培養後に生じたコロニー数(CFU:Colony Forming Units)を測定した。
なお、上記試料の他、培地のみで大腸菌を培養したものをコントロールとした。
各試料のCFUの値から抗菌活性を評価した。抗菌活性の増強の程度については、試験後に残存した大腸菌数(CFU)同士を比較して算出した。例えば、試験後の試料1のCFUが100で、試料2のCFUが10のとき、試料2の抗菌活性は、試料1の10倍であるとすることができる。
本実施例では、本発明の抗菌活性増強剤を製造した。
(1)試験方法
陽イオン交換樹脂としてCM-セファデックス(CM-Sephadex) C-50(GEヘルスケア社製)を用いた。陽イオン交換樹脂170mLを充填したカラムに、未殺菌の脱脂乳20リットルを通液して交換樹脂に乳蛋白質を吸着させた。
次いで、カラム内の陽イオン交換樹脂を脱イオン水で充分洗浄し、0.27M塩化ナトリウム(国産化学株式会社製)を通液して、樹脂に吸着した蛋白質を溶出させた。
続いて、溶出溶媒として1.7M塩化ナトリウムを通液し、樹脂に吸着した蛋白質画分を溶出させた。この溶出液を分画分子量10kDaの限外濾過膜処理により脱塩し、常法にて濃縮し、凍結乾燥して蛋白質画分の粉末を得た。
二次元電気泳動による、本発明の抗菌活性増強剤の分子量分布は、8~30kDaであり、等電点は9.0~11.0であった。
ウシラクトフェリン(森永乳業社製)95gに対して、実施例1と同一の方法により製造した抗菌活性増強剤を5g添加して均一混合し、ラクトフェリンの抗菌活性を増強した抗菌用医薬品を製造した。
ラクトフェリン分解物として、実施例2で使用したウシラクトフェリンをpH5以下に調製し、3%ペプシンと反応させて酵素消化し、逆浸透膜にて濃縮した後、凍結乾燥して得られた、ラクトフェリン分解物粉末を用いた。
上記ラクトフェリン分解物粉末95gに対して、実施例1と同一の方法により製造した抗菌活性増強剤を5g添加して均一混合し、ラクトフェリン分解物の抗菌活性を増強した抗菌用医薬品を製造した。
ウシラクトフェリン(森永乳業社製)95gに対して、実施例1と同一の方法により製造した抗菌活性増強剤を5g添加して均一混合し、混合物の添加量が10質量%となるように発酵乳に添加して発酵乳食品を製造した。
[試験例1]
本実施例では、本発明の抗菌活性増強剤を製造し、成分の特定を行った。
実施例1で得た抗菌活性増強剤の成分を特定するために、ZOOM IPG Runner System(Invitrogen社製)を用いて二次元電気泳動を行った。第一次元目は、pH範囲3~10のZOOM Strip pH3-10NLを用いた。第二次元目は、NuPAGE4-12%Bis-Tris ZOOM Gelを用いた。そして、SimplyBlue SafeStain(Invitrogen社製)で染色した。
さらに、二次元電気泳動により得られた染色後のゲルをデンシトグラフ(ATTO社製)で撮影し、解析ソフトによる相対的定量を行った。得られたスポットをゲルから切り出し、目的蛋白を脱色し、還元アルキル化及びトリプシン処理によるゲル内消化を行い、MALDI-TOF/MS装置(商品名:AutoflexII、BRUKER社製)にて質量分析し、Matrix Science社製のMASCOTソフトウェアを使用して、NCBI(National Center for Biotechnology information)のデータベース(http://www.ncbi.nlm.nih.gov/)検索により蛋白質を同定した。
二次元電気泳動の結果を図1に示す。図1において、縦軸が分子量を示し、横軸が等電点を示している。図1に示した各スポットの分子量と同定された蛋白質の種類は表1の通りである。図1に矢印とともに示した丸数字は、表1の「スポットNo.」の数字および明細書中スポット[1]~[7]に対応している。
スポット[1]はラクトフェリン由来の蛋白質であることが明らかになった。スポット[2]、[4]、[6]、[7]は、データベース検索により、図6において表されるとおり、アンジオジェニン-1(Angiogenin-1)として知られる蛋白質の単量体、二量体、断片等であった。また、スポット[3]は、データベース検索により、図7において表されるとおり、アンジオジェニン-2(Angiogenin-2)として知られる蛋白質に由来することが明らかになった。
しかし、スポット[5]については蛋白質の種類が同定されていない。
また、本発明の抗菌活性増強剤におけるラクトフェリン又はラクトフェリン分解物に由来する物質の含有量を測定した。デンシトメーターによる解析図を図2に示し、解析結果を表2に示した。
図2において、[1]~[4]のピーク面積の合計を100とすると、本発明の抗菌活性増強剤に含まれるラクトフェリン由来物質の量は1.44質量%であることが明らかになった。
なお、本発明の抗菌活性増強剤には[1]~[4]以外にもスポットが存在することから、実際のラクトフェリンに由来する成分の含有量は1.44質量%よりもさらに低いことが予想される。
従って、本発明の抗菌活性増強剤は、その成分中のラクトフェリン又はラクトフェリン分解物に由来する成分の含有量が2.0質量%以下のものである。
本試験は、本発明の抗菌活性増強剤がラクトフェリンの抗菌活性を増強することを確認することを目的とした。
(1)試料の調製
ラクトフェリンとしてウシラクトフェリン(森永乳業社製)を脱イオン水で希釈して使用した。
一方、実施例1で得られた抗菌活性増強剤を脱イオン水で希釈して使用した。
試料1:ウシラクトフェリン(2mg/mL)
試料2:ウシラクトフェリン(2mg/mL)及び抗菌活性増強剤(160μg/mL)の混合物。
試料3:ウシラクトフェリン(2mg/mL)及び抗菌活性増強剤(32μg/mL)の混合物。
試料4:抗菌活性増強剤(32μg/mL)
1%のBacto peptone培地(Becton Dickinson社製)を用い、5mLのポリスチレンチューブに大腸菌K-12株(NBRC 3301)を106CFU/mLとなるよう植菌し、37℃のインキュベーター内で6時間前培養し、試験用菌液とした。そして、96穴プレート上にて上記の試料と共に前培養した大腸菌を106CFU/mLになるよう添加し、37℃にて17時間培養した。17時間培養後に大腸菌を含む培養液を0.85%生理食塩水にて段階希釈し、寒天培地(ラピッドメディアRM-SPC、森永乳業社製)上にプレーティングした後、37℃、16時間培養後に生じたコロニー数(CFU: Colony Forming Units)を測定することにより、各試料の抗菌活性を評価した。
なお、試料1~4の他、上記培地のみで大腸菌を培養したものをコントロールとした。
表3に試験例2の結果を示す。表3において、有意差検定はP<0.001である。培地のみで培養したコントロールでは17時間後に菌数が1.1×108CFU/mLに増加したのに対し、ラクトフェリンを含む試料1では、菌数が初菌数よりも低減して、3.6×105CFU/mLの範囲となった。
本試験結果から、ラクトフェリンと本発明の抗菌活性増強剤の組み合わせにより、顕著な抗菌活性の増加が認められた。
本試験は、本発明の抗菌活性増強剤がラクトフェリン加水分解物の抗菌活性を増強することを確認することを目的とした。
(1)試料の調製
ラクトフェリン分解物として、試験例2で使用したウシラクトフェリン(森永乳業社製)をpH5以下に調製し、3%ペプシンと反応させて酵素消化し、逆浸透膜にて濃縮した後、凍結乾燥して得られた、ラクトフェリン分解物粉末を用いた。
ラクトフェリン分解物粉末は、脱イオン水で希釈して使用した。
一方、実施例1で得られた抗菌活性増強剤を脱イオン水で希釈して使用した。
試料5:ラクトフェリン分解物(400μg/mL)
試料6:ラクトフェリン分解物(400μg/mL)及び抗菌活性増強剤(32μg/mL)の混合物。
試料7:抗菌活性増強剤(32μg/mL)
試験例2と同様の試験方法にて試験した。試料5~7の他、培地のみで大腸菌を培養したものをコントロールとした。
試験例3の結果を表4に示す。
培地のみで培養したコントロールでは17時間後に菌数が6.7×107CFU/mLに増加した。ラクトフェリン分解物400μg/mLを含む試料5では菌数が8.6×104CFU/mLと減少したが、さらに抗菌活性増強剤を添加した試料7では菌数が2.5×102CFU/mL以下となり、試料5と比較して顕著な菌数の減少が見られた。ここで、試料5と試料6の菌数の比較によれば、試料6の抗菌活性は試料5の抗菌活性の約340倍以上であった。
本試験結果から、ラクトフェリン分解物と本発明の抗菌活性増強剤の組み合わせにより、顕著な抗菌活性の増加が認められた。
本試験は、本発明の抗菌活性増強剤がラクトフェリン加水分解物であるラクトフェリシン(登録商標)の抗菌活性を増強することを確認することを目的とした。
(1)試料の調製
試験例2で使用したウシラクトフェリン(森永乳業社製)をペプシンで加水分解し、高速液体クロマトグラフィーにて精製し、凍結乾燥して得られたウシ・ラクトフェリシン粉末を用いた。
ウシ・ラクトフェリシン粉末は、脱イオン水で希釈して使用した。
一方、実施例1で得られた抗菌活性増強剤を脱イオン水で希釈して使用した。
試料8:ウシ・ラクトフェリシン(16μg/mL)
試料9:ウシ・ラクトフェリシン(16μg/mL)及び抗菌活性増強剤(32μg/mL)の混合物。
試料10:抗菌活性増強剤(32μg/mL)
培養時間を2時間に変更したことを除き、試験例2と同様の試験方法にて試験した。試料8~10の他、培地のみで大腸菌を培養したものをコントロールとした。
試験例4の結果を表5に示す。その結果、培地のみで培養したコントロールでは2時間後に菌数が2.9×106CFU/mLに増加した。
ここで、試料8と試料9の菌数の比較によれば、試料9の抗菌活性は試料8の抗菌活性の約35倍以上であった。
本試験結果から、ウシ・ラクトフェリシンと本発明の抗菌活性増強剤の組み合わせにより、顕著な抗菌活性の増加が認められた。
実施例1と同様にして、未殺菌の脱脂乳をCM-セファデックスC-50カラムに通液し、カラム内の陽イオン交換樹脂を脱イオン水で充分洗浄し、0.27M塩化ナトリウム、御及び、1.7M塩化ナトリウムを通液して、樹脂に吸着した蛋白質画分を溶出させた。
また、各々のピークα、βについて、SDS電気泳動により分析した結果、図5に示されるとおり、ピークαには、分子量約17kDaのマイナーバンドと約15kDaのメジャーバンドの2成分が含まれていることが確認された。また、ピークβには、分子量約15kDaのメジャーバンドの1成分が含まれていることが確認された。
バンドをゲルから切り出し、脱色して、還元アルキル化及びトリプシン処理によるゲル内消化を行い、MALDI-TOF/MS装置(商品名:AutoflexII、BRUKER社製)にて質量分析し、Matrix Science社製のMASCOTソフトウェアを使用して、NCBI(National Center for Biotechnology information)のデータベース(http://www.ncbi.nlm.nih.gov/)検索により蛋白質を同定した結果、ピークα、βのいずれにも含まれる約15kDaの成分はアンジオジェニン-1であることが判明し、ピークαに含まれる約17kDaの成分はアンジオジェニン-2であることが判明した。
本試験は、アンジオジェニン-1及びアンジオジェニン-2がラクトフェリンの抗菌活性を増強する作用を有することを確認するために行った。
ラクトフェリンとしてウシ・ラクトフェリン(森永乳業社製)を脱イオン水で希釈して使用した。
一方、実施例5に記載された方法により分離したアンジオジェニン-1及びアンジオジェニン-2を脱イオン水で希釈して使用した。
前記のウシ・ラクトフェリンと分離したアンジオジェニン-1及びアンジオジェニン-2を用いて、以下の試料11~15を調製した。
・試料11:ウシ・ラクトフェリン(2mg/mL)
・試料12:ウシ・ラクトフェリン(2mg/mL)及びアンジオジェニン-1(32μg/mL)の混合物
・試料13:ウシ・ラクトフェリン(2mg/mL)並びに、アンジオジェニン-1及びアンジオジェニン-2の混合物(各32μg/mL)の混合物
・試料14:アンジオジェニン-1(32μg/mL)
・試料15:アンジオジェニン-1及びアンジオジェニン-2の混合物(各32μg/mL)の混合物
さらに、1%のBacto peptone培地(Becton Dickinson社製)を用い、5mLのポリスチレンチューブに大腸菌K-12株(NBRC 3301)を106CFU/mLとなるよう植菌し、37℃のインキュベーター内で6時間前培養して、試験用菌液とした。
96穴プレート上にて前記の試料とともに試験用菌液を106CFU/mLとなるよう添加し、37℃にて17時間培養した。
17時間培養した後に、大腸菌を含む培養液を0.85%生理食塩水にて段階希釈し、寒天培地(ラピッドメディアRM-SPC、森永乳業社製)上にプレーティングした後、37℃で16時間培養して、培地上に生じたコロニー数を測定することにより、各試料の抗菌活性(CFU:Colony Forming Units)を評価した。
なお、試料11~15の他に、前記培地溶液のみで大腸菌を培養したものをコントロールとした。
本試験の結果を表6に示す。
その結果、培地のみで培養したコントロールでは、17時間後に菌数が1.3×108CFU/mLに増加したのに対し、ラクトフェリンを含む試料11では、菌数が初菌数よりも低減して、2.9×105CFU/mLの範囲となった。
本試験は、本発明の抗菌活性増強剤がラクトフェリンの種々の菌に対する抗菌活性を増強する作用を有することを確認するために行った。
ラクトフェリンとして、ウシラクトフェリン(森永乳業社製)を脱イオン水にて希釈して使用した。一方、実施例1に記載された方法により製造された抗菌活性増強剤(Angiogenin-1, Angiogenin-2を含有)を脱イオン水で希釈して使用した。
試料16:ウシラクトフェリン(8mg/mL)
試料17:ウシラクトフェリン(4mg/mL)
試料18:ウシラクトフェリン(2mg/mL)
試料19:ウシラクトフェリン(1mg/mL)
試料20:ウシラクトフェリン(0.5mg/mL)
試料21:ウシラクトフェリン(0.25mg/mL)
試料22:ウシラクトフェリン(0.125mg/mL)
また、試料16~22各々に抗菌活性増強剤(32μg/mL)を添加した、試料23~29を調製した。
また、抗菌活性増強剤(32μg/mL)のみの試料30を調製した。
また、同様にPYG培地(1%Bacto peptone、0.05%yeast extract(Becton Dickinson社製)、1%Glucose(和光純薬社製))を用い、5mLのポリスチレンチューブに、Streptococcus mutans JCM5705Tを106cells/mLになるよう植菌し、37℃のインキュベーターにて6時間培養して、試験用菌液とした。また、Sabouraud培地(1% Bacto peptone、2% Glucose)を用い、5mLのポリスチレンチューブに、Candida albicans TIMM1768を106cells/mLになるよう植菌し、37℃のインキュベーターにて6時間培養して、試験用菌液とした。
96穴プレート上にて前記の試料とともに、前記の試験用菌液を106cells/mLとなるようにそれぞれ添加し、37℃にて17時間培養した。
17時間培養した後に、マイクロプレートリーダー(コロナ電気社製)にて630nmの吸光度を測定した。なお、試料16~30の他に、前記培地溶液のみで菌を培養したものをコントロールとした。菌の発育が有意に抑制され、コントロールの吸光度に対して50%以下の吸光度を示す最小のラクトフェリン濃度を、最小発育阻止濃度(MIC)とし、抗菌活性増強剤を添加したラクトフェリンと未添加のラクトフェリンのMIC濃度との比較から、種々の菌に対する抗菌活性増強剤による菌の発育抑制の増強作用を評価した。
本試験の結果を表7に示す。表7中、LFはラクトフェリンを、MICは最小阻止濃度を各々表す。
一方、抗菌活性増強剤のみ(試料30)では菌の発育抑制は全く見られず、抗菌活性増強剤のMICは32μg/mL以上であった。
本試験は、本発明の抗菌活性増強剤がラクトフェリシンの種々の菌に対する抗菌活性を増強する作用を有することを確認するために行った。
ラクトフェリシンとして、ウシラクトフェリン(森永乳業社製)をペプシンで加水分解し、高速液体クロマトグラフィーにて精製し、凍結乾燥して得られたラクトフェリシン粉末を用いた。ラクトフェリシン粉末は脱イオン水にて希釈して使用した。一方、実施例1に記載された方法により製造された抗菌活性増強剤(Angiogenin-1, Angiogenin-2を含有)を脱イオン水で希釈して使用した。
試料31:ウシラクトフェリシン(32μg/mL)
試料32:ウシラクトフェリシン(16μg/mL)
試料33:ウシラクトフェリシン(8μg/mL)
試料34:ウシラクトフェリシン(4μg/mL)
試料35:ウシラクトフェリシン(2μg/mL)
また、試料31~35各々に抗菌活性増強剤(32μg/mL)を添加した、試料36~40を調製した。
また、抗菌活性増強剤(32μg/mL)のみの試料41を調製した。
96穴プレート上にて前記の試料とともに、前記の試験用菌液を106cells/mLとなるようにそれぞれ添加し、37℃にて17時間培養した。
17時間培養した後に、マイクロプレートリーダー(コロナ電気社製)にて630nmの吸光度を測定した。なお、試料31~41の他に、前記培地溶液のみで菌を培養したものをコントロールとした。菌の発育が有意に抑制され、コントロールの吸光度に対して50%以下の吸光度を示す最小のラクトフェリン濃度を、最小発育阻止濃度(MIC)とし、抗菌活性増強剤を添加したラクトフェリンと未添加のラクトフェリンのMIC濃度との比較から、種々の菌に対する抗菌活性増強剤による菌の発育抑制の増強作用を評価した。
本試験の結果を表8に示す。表8中、LFcinはラクトフェリンを、MICは最小阻止濃度を各々表す。
一方、抗菌活性増強剤のみ(試料41)では菌の発育抑制は全く見られず、抗菌活性増強剤のMICは32μg/mL以上であった。
一方、抗菌活性増強剤のみを添加した試料(試料41)では、いずれの菌においてもEscherichia coli O111と同様に、明らかな菌の発育阻止は認められなかった。
Claims (15)
- 下記性質を有する乳由来の蛋白質を有効成分とする、ラクトフェリン又はラクトフェリン加水分解物の抗菌活性増強剤:
(A)ラクトフェリン又はラクトフェリン加水分解物の抗菌活性を増強する作用を有する。
(B)分子量が30kDa以下である。
(C)等電点が9.0~11.0である。 - 前記乳由来の蛋白質は、ラクトフェリン又はラクトフェリン加水分解物を2.0質量%以下の量で含有する、請求項1に記載の抗菌活性増強剤。
- 前記乳由来の蛋白質が、アンジオジェニン-1(Angiogenin-1)及び/又はアンジオジェニン-2(Angiogenin-2)である、請求項1又は2に記載の抗菌活性増強剤。
- 前記乳由来の蛋白質は、それ自体では大腸菌に対する抗菌活性を有さない、請求項1~3のいずれか一項に記載の抗菌活性増強剤。
- 前記乳由来の蛋白質は、該蛋白質とラクトフェリン又はラクトフェリン加水分解物とを併用することにより、ラクトフェリン又はラクトフェリン加水分解物の大腸菌に対する抗菌活性を7倍以上に増強する性質を有する、請求項1~4のいずれか一項に記載の抗菌活性増強剤。
- 請求項1~5のいずれか一項に記載の抗菌活性増強剤と、ラクトフェリン、及びラクトフェリン加水分解物からなる群から選択される1又は2以上とを含有し、ラクトフェリン、及びラクトフェリン加水分解物に対する抗菌活性増強剤中の乳由来の蛋白質の質量比が1/80以上である、抗菌剤組成物。
- 請求項6に記載の抗菌剤組成物を含有する抗菌用医薬品。
- 請求項6に記載の抗菌剤組成物を含有する飲食品。
- 請求項6に記載の抗菌剤組成物を含有する飼料。
- ラクトフェリン又はラクトフェリン加水分解物の抗菌活性増強剤の製造における、下記性質を有する乳由来の蛋白質の使用:
(A)ラクトフェリン又はラクトフェリン加水分解物の抗菌活性を増強する作用を有する。
(B)分子量が30kDa以下である。
(C)等電点が9.0~11.0である。 - 前記乳由来の蛋白質は、ラクトフェリン又はラクトフェリン加水分解物を2.0質量%以下の量で含有する、請求項10に記載の使用。
- 前記乳由来の蛋白質が、アンジオジェニン-1(Angiogenin-1)及び/又はアンジオジェニン-2(Angiogenin-2)である、請求項10又は11に記載の使用。
- 前記乳由来の蛋白質が、それ自体では大腸菌に対する抗菌活性を有さない、請求項10~12のいずれか一項に記載の使用。
- 前記乳由来の蛋白質は、該蛋白質とラクトフェリン又はラクトフェリン加水分解物とを併用することにより、ラクトフェリン又はラクトフェリン加水分解物の大腸菌に対する抗菌活性を7倍以上に増強する性質を有する請求項10~13のいずれか一項に記載の使用。
- (a)乳原料を陽イオン交換樹脂に接触させて乳原料中の蛋白質を陽イオン交換樹脂に吸着処理する工程、
(b)前記吸着処理後の陽イオン交換樹脂を洗浄処理し、溶出溶媒を通液して、等電点が9.0~11.0の蛋白質画分を溶出する工程、
(c)前記溶出した蛋白質画分を分画分子量30kDaの限外濾過膜により膜濾過して、下記性質を有する乳由来の蛋白質を調製する工程、
を有することを特徴とする、前記乳由来の蛋白質を有効成分とするラクトフェリン又はラクトフェリン加水分解物の抗菌活性増強剤を製造する方法:
(A)ラクトフェリン又はラクトフェリン加水分解物の抗菌活性を増強する作用を有する。
(B)分子量が30kDa以下である。
(C)等電点が9.0~11.0である。
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US13/813,240 US20130129707A1 (en) | 2010-07-30 | 2011-07-29 | Antimicrobial activity enhancing agent |
JP2012526593A JPWO2012015037A1 (ja) | 2010-07-30 | 2011-07-29 | 抗菌活性増強剤 |
AU2011283472A AU2011283472B2 (en) | 2010-07-30 | 2011-07-29 | Antimicrobial activity enhancing agent |
EP11812626.7A EP2599493A4 (en) | 2010-07-30 | 2011-07-29 | MEDIUM FOR INCREASING ANTIMICROBIAL EFFECT |
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EP (1) | EP2599493A4 (ja) |
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JP2015536905A (ja) * | 2012-10-08 | 2015-12-24 | マレー・ゴールバーン・コー−オペラティヴ・カンパニー・リミテッド | 乳汁からラクトフェリンを精製するための改良されたプロセスおよびその製品 |
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