JP2010270014A - Nisin stabilizer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for stabilizing lantibiotic, especially nisin and a germicidal composition. <P>SOLUTION: The germicidal composition is a composition containing an antimicrobially effective amount of lantibiotic, and comprises 10-100,000 pts.wt. (preferably 50-50,000 pts.wt., more preferably 100-10,000 pts.wt.) of thioctic acid or a salt thereof acceptable from the viewpoint of medical treatment or food sanitation based on 1 pt.wt. of lantibiotic. Lantibiotic is stably kept even in an oily base and in the presence of a surfactant by using thioctic acid. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for stabilizing a lantibiotic represented by nisin. The present invention can be used for the stabilization of lantibiotics contained in sterilizing / sterilizing compositions, cleaning compositions and the like. The present invention is useful in hygiene management and cleaning in food and medicine.

  Bacteriocin, which is a protein antibacterial substance produced by a specific bacterial strain and active against other bacterial strains, does not induce resistant bacteria like antibiotics and is degraded in the human body. Therefore, there are high expectations for the use of bacteriocin in the fields of medical care, poultry farming, aquaculture, etc., where countermeasures against resistant bacteria are important issues, as well as in the food field where consumer health, natural / safety orientation is increasing.

  Nisin is a bacteriocin produced by lactic acid bacteria (Non-Patent Document 1), and is an antibacterial peptide consisting of 34 amino acid residues including abnormal amino acids such as unsaturated amino acids and lanthionine. In addition to being incorporated into foods, nisin has been studied for use in an effective form in the medical and hygiene fields (for example, Patent Document 1 and Patent Document 2).

  When nisin is used for the purpose of sterilization and sterilization, the desired effect is exhibited even at a relatively low concentration because of its high antibacterial activity. However, it is generally difficult to keep proteins and peptides stable at room temperature, particularly in dilute solutions for long periods of time. In this regard, there has been an attempt to stabilize a composition containing a lanthionine-containing bacteriocin such as nisin by containing a thioether compound, particularly methionine (Patent Document 1).

  In recent studies, in nisin treated with hydrogen peroxide under acidic conditions, lanthionine, methionine and histidine are oxidized, and oxidation of lipid II binding site (N-terminal site), which is the site of nisin action. Has been reported to affect antibacterial activity (Non-patent Document 2).

  On the other hand, thioctic acid (α-lipoic acid) is considered to be used for imparting antioxidant properties to oily substances such as edible fats and oils, unsaturated fatty acids, carotenoids, phospholipids and oil-soluble vitamins. (Patent Document 4).

Japanese Unexamined Patent Publication No. 2007-99809 WO94 / 39842 (Japanese Patent Publication No. 11-507363) WO94 / 13143 (Japanese Patent Publication No. 8-51716, Japanese Patent No. 3692385) JP 2008-13630 A

Norihiro Sashihara et al; Bacteriocin produced by lactic acid bacteria and its application; Japanese Journal of Lactic Acid Bacteria, Vol.10, No.1, p2-18 (1999) Appl. Environ. Microbiol. 75 (5), p.1381-1387 (2009)

  It cannot be said that the methionine itself has an action to prevent oxidation. In addition, according to the study by the present inventors, certain antioxidants have problems such as insufficient stabilization effect when incorporated into nisin, and conversely, nisin activity is significantly reduced. Further, methionine that does not dissolve in an organic solvent may not be able to sufficiently exhibit a stabilizing effect in an oil-based formulation.

  The present inventors have studied various agents for stabilizing nisin. They have found that thioctic acid is particularly effective for this purpose. Further, the present inventors have found that thioctic acid is effective even in oily formulations and surfactants in solution, thereby completing the present invention.

The present invention provides the following.
1) A composition comprising an antibacterial effective amount of lantiobiotic, which is 10-100,000 parts by weight (preferably 50-50,000 parts by weight, more preferably 100-10,000 parts by weight) per 1 part by weight of lantiobiotic ) Thioctic acid or a medical or food hygiene acceptable salt thereof.
2) Lantiobiotic, and thioctic acid or its medically or food hygienically acceptable salt, the concentration of lantiobiotic is 25-200,000 IU / ml, preferably 100-100,000 IU / ml, more preferably 500 to 50,000 IU / ml), and the concentration of thioctic acid is 0.001 to 10% by weight (preferably 0.005 to 2% by weight, more preferably 0.01 to 1% by weight).
A liquid or semi-solid composition.
3) The composition according to 1) or 2), further comprising a surfactant.
4) The composition according to 1) or 2), further comprising an alcohol, wherein the concentration of the alcohol is 1 to 80% by weight (preferably 10 to 60% by weight, more preferably 20 to 40% by weight).
5) The composition according to 1) or 2), wherein the lantibiotic and thioctic acid are dissolved in an oily base.
6) Lantiobiotic stabilizer containing thioctic acid.
7) A method for stabilizing a lantibiotic, comprising using thioctic acid.
8) A method for sterilizing a subject using lantiobiotic and thioctic acid.

  The composition of the present invention contains lantibiotic, and also contains thioctic acid or a medically or food hygienically acceptable salt as a component for lantibiotic stabilization.

  In the present invention, the term lantibiotic is a bacteriocin produced by lactic acid bacteria, unless otherwise indicated, and includes an abnormal amino acid such as an unsaturated amino acid, lanthionine, or 3-methyllanthionine, and a small molecule of 5 kDa or less An antibacterial peptide. Lantibiotics include nisin, lactisin, citricin, lactosine. In the present invention, “nisin” includes nisin A, nisin Z, and nisin Q unless otherwise specified. Of the lantibiotics, nisin, preferably nisin A or Z, and more preferably nisin A is used in the composition of the present invention.

Nisin A is produced by the lactic acid bacterium Lactococcus lactis and has already been used as a food additive in more than 50 countries including Europe and America. Nisin Z is a bacteriocin similar to nisin A. Of the 34 amino acid residues constituting nisin A, the 27th from the N-terminus is nisin A, whereas nisin Z is an asparagine residue. It differs only in that it is a residue.

  In this specification, among the lantibiotics, nisin or nisin A may be described as an example, but the description also applies to other lantibiotics unless otherwise specified.

  Thioctic acid (also known as α-lipoic acid, lipoic acid, IUPAC name: 5-[(3R) -dithiolan-3-yl] pentanoic acid) has two optical isomers, and those derived from natural products Have the following structure.

  In the present invention, “thioctic acid” may be either one of two optical isomers or a mixture in an appropriate ratio. Thioctic acid is contained in many foods such as spinach, red meat, broccoli, liver and tomatoes, and is also used as a medicine for the treatment of liver disease and extreme fatigue (such as diabetic peripheral neuropathy). ing.

  The medically or food hygienically acceptable salt is, for example, a base selected from thioctic acid, alkali metals, alkaline earth metals, ammonium hydroxide, basic amino acids (eg arginine, lysine, methionine, cystine, ornithine) And salt. Pharmaceutically or food acceptable salts are preferred examples of medical or food hygiene acceptable salts.

  In the present specification, thioctic acid or its medical or food hygiene-acceptable salt may be explained by taking thioctic acid as an example. This also applies to food hygiene acceptable salts.

  According to the study by the present inventors, the nisin-containing alcohol preparation containing thioctic acid and the preparation containing no thioctic acid were stored at 45 ° C. for 6 days. As a result, the nisin activity of the preparation containing no thioctic acid was greatly reduced. On the other hand, it was found that nisin activity was retained in the added preparation.

  Nisin oxidation in ethanol is due to oxidation of the sulfur part of the methionine residue constituting nisin to methionine sulfide (monoxide) and methionine sulfone (dioxide). The effect of oxidation of methionine residues on antibacterial activity is not clear, but can be an indicator of nisin oxidation. On the other hand, when the HPLC chart of the above-described alcohol preparation added with thioctic acid is viewed after a certain period of time, the decomposition product or by-product of thioctic acid overlaps around the peak of nisin oxide, and thioctic acid is caused. The degree of inhibition of nisin oxide formation could not be confirmed (see Example 2 of the present specification).

  The mechanism by which thioctic acid stabilizes nisin is not clear, and many antioxidants subjected to the test by the present inventors did not show the blending effect.

  The present inventors have confirmed that methionine also has a high effect of stabilizing nisin, but methionine itself has no antioxidant effect. It is presumed that the addition of methionine suppresses the oxidation of methionine residues present in nisin.

FIG. 1 is a graph showing the results of HPLC analysis of the effect of adding a stabilizer to nisin. DL-methionine 98 +% (Ti) manufactured by Wako Pure Chemical Industries, Ltd. was used as a stabilizer. FIG. 2A is a graph showing experimental results for screening for nisin stabilizers. FIG. 2B is a graph showing experimental results for screening for nisin stabilizers. FIG. 3 is a graph showing the results of analyzing the effect of adding a stabilizer to nisin by HPLC. FIG. 4 is a graph showing the effect of adding a stabilizer to nisin in an oily ointment. FIG. 5 is a graph showing the effect of adding a stabilizer to nisin in an emulsion. FIG. 6 is a graph showing the effect of adding a stabilizer to nisin in a surfactant solution.

  In the composition of the present invention, when nisin is used as the lantibiotic, nisin can be obtained by culturing and purifying lactic acid bacteria by a known method. For example, after cultivating lactic acid bacteria in the MRS medium (MRS medium, manufactured by Oxoid), the culture supernatant is treated with Amberlite (Amberlite XAD-16, manufactured by Sigma) to adsorb nisin. Amberlite was washed with distilled water and 40% ethanol, and then nisin was eluted with 70% isopropanol containing 0.1% trifluoroacetic acid. Nisin refined product can be obtained by subjecting it to the company. If necessary, the degree of purification can be further increased by subjecting to reverse phase chromatography (see Biosci. Biotechnol. Biochem., 67 (7), p1616-1619, 2003). Commercial products such as Nisaplin (trademark, manufactured by Danisco Co., Ltd.) can also be purchased. Nisuprin is a mixture of nisin and sodium chloride derived from Lactococcus lactis subsp. Lactis and contains components derived from non-fat milk or sugar medium. If necessary, the degree of purification can be increased by the above purification method.

  In the present invention, the term “bactericidal effective (amount)” with respect to a lantibiotic means an amount or concentration at which the lantibiotic can exert its bactericidal activity, unless otherwise indicated. “Sterilization” as used in the present invention includes not only killing microorganisms but also stopping, inhibiting or delaying the growth of microorganisms.

  The “bactericidal effective amount” of lantibiotic in the present invention usually refers to a concentration of about 25 IU / ml or more, but in combination with an appropriate surfactant (Patent Document 1), the activity of lantibiotic is enhanced. In some cases, it may be lower. The concentration of the antibacterial effective lantiobiotic is, for example, 25 to 200,000 IU / ml, preferably 100 to 100,000 IU / ml, more preferably 500 to 50,000 IU / ml.

  The amount or activity of the lantibiotic can be expressed as an activity value (IU) or weight by HPLC area ratio based on the standard, and can also be measured by the minimum inhibition circle concentration assay. For nisin, as described in JP-A-2002-369672, nisin A manufactured by Sigma and nisin Z (1 μg = 40 IU) manufactured by ICN Biomedical Inc. were used as standard products, and the activity was determined by comparing the peak areas of HPLC. In addition, as described in the Food Additive Analysis Method in Food (Ministry of Health, Labor and Welfare), the Niisin standard distributed by the Japan Standards Association is used to compare the peak areas of HPLC. It can be measured by the minimum inhibition circle concentration test method. One unit of nisin titer corresponds to 0.025 μg of the antimicrobial polypeptide containing nacin A. In the examples of the present specification, Nisin A manufactured by Ohm Dairy Co., Ltd. is used, and the activity of this purified Nisin product (aqueous solution) is 50 to 100 kIU / mL. A more detailed approach is also described in the examples herein.

  The composition of the present invention contains thioctic acid in an amount sufficient to stabilize the lantiobiotic relative to 1 part by weight of the lantiobiotic.

  In the present invention, whether or not the amount of thioctic acid is sufficient can be determined by the amount of lantiobiotic or the activity remaining in the composition after storage for a certain period of time. Since thioctic acid is considered to have a strong sulfur odor by blending a high concentration, the upper limit of the thioctic acid content may be determined from such a viewpoint. Illustrating a specific amount, the concentration of thioctic acid is 10 to 100,000 parts by weight, preferably 50 to 50,000 parts by weight, more preferably 100 parts per 1 part by weight of the above-mentioned sterilically effective lantiobiotic. ~ 10,000 parts by weight. When the composition of the present invention is a liquid or semi-solid composition, the concentration of thioctic acid can be 0.001 to 10% by weight, preferably 0.005 to 2% by weight, more preferably 0.01 to 1% by weight. . When a salt of thioctic acid is used instead of thioctic acid, the amount corresponding to thioctic acid is set within this range.

  The composition of the present invention may contain components other than thioctic acid, such as methionine, for the stabilization of lantiobiotics. When methionine and thioctic acid are used, the concentration thereof can be 10 to 100,000 parts by weight, preferably 50 to 50,000 parts by weight, more preferably 100 to 10,000 parts by weight. When the composition of the present invention is a liquid or semi-solid composition, the total concentration can be 0.001 to 10% by weight, preferably 0.005 to 2% by weight, more preferably 0.01 to 1% by weight.

  The combined use of thioctic acid and methionine can exhibit a synergistic stabilizing effect that cannot be achieved by itself.

The sterilizing performance of the composition of the present invention can be determined based on, for example, an evaluation result obtained by antibacterial evaluation (MBC modified method). The MBC method is a method for measuring the minimum bactericidal concentration. In the MBC modified method, various conditions such as culture conditions, bactericidal agent concentration, contact time, contact temperature, and test bacteria concentration are arbitrarily set based on the MBC method. And the number of surviving bacteria under the conditions is measured. For example, after culturing a test bacterium (eg, E. coli) in SCD broth medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 37 ° C. for 24 hours, the culture solution is diluted with sterilized purified water to adjust the number of bacteria (10 ⁷ cfu / mL). The diluted culture solution (100 μL) is added to the test solution (Nisin-containing composition, 10 mL) and mixed to bring the test solution into contact with the test bacteria at room temperature (30 seconds). 100 μL) is taken and diluted with a medium containing an inactivating agent (SCDLP medium, 10 mL). The number of bacteria surviving in the medium containing the inactivating agent is cultured by the pour plate method (37 ° C., 24 hours), the colonies that appear are counted, and the following calculation formula:
Bactericidal rate (%) = [1− (number of surviving bacteria in test solution / number of test bacteria)] × 100
To calculate the sterilization rate.

  In the modified MBC method, conditions such as fungus concentration, contact time, contact temperature, and test bacteria concentration can be appropriately changed according to the test purpose, and the medium and culture conditions are appropriately changed depending on the type of the test bacteria. be able to. Moreover, it is also possible to culture | cultivate the culture medium containing an inactivation agent, and to determine qualitatively the presence or absence of the microbe to survive in the said culture medium, and the grade of growth based on the turbidity of the culture medium after culture | cultivation.

  In the above MBC modified method, if the sterilization rate of the microorganism is 90% or more, it can be determined that the composition has a sterilizing effect. The sterilization rate required varies depending on the field of use, application, and usage, but the sterilization rate by the composition is, for example, preferably 90 to 100%, more preferably 99 to 100%, and further preferably 99.9 to 100%. is there. 99.9% sterilization rate is sufficient for general disinfectant and environmental sterilization applications, but 99.999% or more is required for sterilization of aseptic equipment that comes in contact with food. Sometimes it is done.

  The composition of the present invention may contain components other than lantiobiotic and thioctic acid. For example, surfactants (emulsifiers, dispersants (anti-redeposition agents)), wetting agents, flavoring agents, fragrances, abrasives, lubricants, chelating agents, solvents, humectants, antiseptics (antibacterial agents), stable An agent, a cleaning enzyme, a viscosity modifier, an antiseptic, an antifungal agent, a colorant, a fragrance and the like may be blended.

  As the surfactant, nonionic surfactant: for example, polyethoxylated sorbitol ester, polycondensate of ethylene oxide and propylene oxide (poloxamer), propylene glycol condensate, polyethoxylated hydrogenated castor oil, sorbitan aliphatic Esters; amphoteric surfactants: e.g., long chain imidazoline derivatives, long chain alkylbetaines, long chain alkylamidoalkylbetaines; cationic surfactants: e.g., D, L-2-pyrrolidone of N-cocoyl-L-ethyl alginate Examples include 5-carboxylate, cocamidopropyl PG dimonium chloride phosphate, and lauramidopropyl PG dimonium chloride phosphate.

  When a surfactant is blended, one that does not reduce the bactericidal efficacy of the active ingredient is good. In addition, those acceptable for medical or food hygiene are preferable, and examples thereof include surfactants that are permitted as food additives. Specifically, glycerol fatty acid ester, polyglycerol ester (diglycerol monocaprate, diglycerol monolaurate, diglycerol monostearate, diglycerol monooleate, decaglycerol monolaurate, decaglycerol monostearate, hexaglycerol condensation Ricinoleate), organic acid monoglyceride (acetic acid monoglyceride, citric acid monoglyceride, citric acid monoglyceride, diacetyltartaric acid monoglyceride, succinic acid monoglyceride), sorbitan fatty acid ester, propylene glycol fatty acid ester, lecithin (lecithin, enzymatically decomposed lecithin), sucrose stearate , Sucrose palmitate, sucrose myristic ester, sucrose oleate, sucrose laurate , Sucrose behenate, sucrose erucate sucrose mixed fatty acids (oleic acid, palmitic acid, stearic acid) esters.

  The amount of the surfactant can be appropriately designed by those skilled in the art. For example, when the composition of the present invention is a liquid or semi-solid composition, it is 1 to 50% by weight, preferably 1 -30% by weight, more preferably 5-25% by weight.

Examples of the chelating agent include EDTA, citric acid, disodium hydrogen citrate and the like. The amount of the chelating agent can be appropriately designed by those skilled in the art. For example, when the composition of the present invention is a liquid or semi-solid composition, 0.1 to 10% by weight, preferably
You may mix | blend with the composition of this invention 0.1 to 5weight%, More preferably, 0.5 to 2weight%.

  When nisin is used as a lantibiotic, gram-negative bacteria can be sterilized by itself, so that a wide range of bactericidal activity can be exhibited without including a chelating agent in the composition. By adding in an amount, bactericidal properties against gram-negative bacteria such as E. coli can be enhanced. It may be preferable to add a bacterial membrane permeability enhancer such as citric acid instead of a chelating agent.

  Examples of the wetting agent include glycerin, sorbitol, propylene glycol, and polyethylene glycol. The amount of the wetting agent can be appropriately designed by those skilled in the art. For example, when the composition of the present invention is a liquid or semi-solid composition, 0.5 to 50% by weight, preferably 1 to You may mix | blend with the composition of this invention in 10 weight%, More preferably, 2-5 weight%.

  Examples of the abrasive include calcium carbonate, calcium phosphate, calcium pyrophosphate, insoluble sodium metaphosphate, sodium aluminosilicate, alumina, hydrogenated alumina, zinc orthophosphate, plastic particles, and silica. A person skilled in the art can appropriately design the blending amount of the abrasive. For example, when the composition of the present invention is a liquid or semi-solid composition, 0.1 to 70% by weight, preferably 10 to 10%. You may mix | blend with the composition of this invention by 50 weight%, More preferably, 20-50 weight%.

  Antibacterial agents include cationic antimicrobial agents: quaternary ammonium compounds such as benzalkonium chloride, dodecyltrimethylammonium chloride, benzyldimethylstearylammonium chloride, cetyltrimethylammonium bromide, benzethonium chloride, methylbenzethonium chloride; : Chlorhexidine, alexidine, chlorhexidine digluconate and chlorhexidine acetate, cetylpyridinium chloride, hexetidine citrate, triclosan, phenyl salicylate, gramidicin, tyrosricin, chlorhexidine digluconate, triclosan, cetylpyridinium chloride; Fungal agents: miconazole, triconazole; The amount of the bactericide can be appropriately designed by those skilled in the art. For example, when the composition of the present invention is a liquid or semi-solid composition, it is 1 to 15% by weight, preferably 5 to 5%. You may mix | blend with the composition of this invention at 15 weight%, More preferably, 5-10 weight%.

  Examples of the viscosity modifier include thickeners: methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methylcellulose. The amount of the viscosity modifier can be appropriately designed by those skilled in the art. For example, when the composition of the present invention is a liquid or semi-solid composition, 0.05 to 5% by weight, preferably 0.1 It may be added to the composition of the present invention at ˜2 wt%, more preferably 0.5-2 wt%.

The composition of the present invention can also be configured using a hydrophilic base (solvent), and can also be configured using an oil-based base. For the purpose of assisting, ethanol or other alcohols may be included. The concentration of the alcohol can be 0.1 to 99% by weight, preferably 1 to 50% by weight.

Examples of oily bases include vegetable oils and fats such as olive oil, soybean oil, rapeseed oil, sunflower oil, palm oil, palm oil, corn oil, peanut oil; animal oils and fats such as pork fat ( Lard), beef tallow (Het); plant processed oil and fat; edible hardened oil; beeswax; In addition, in the above-mentioned Patent Document 3, methionine proposed to be used for stabilizing a composition containing a bacteriocin containing lanthionine such as nisin is soluble in water (3 g / 100 g, H ₂ O (0 ° C), 5.6 g / 100 g H ₂ O (30 ° C)) but hardly soluble in organic solvents. Thus, it may be difficult to use in formulations where the base is oily. On the other hand, thioctic acid does not have such a drawback. Thioctic acid is insoluble in water (soluble in dilute alkaline water) but easily dissolved in ethanol and methanol.

  The composition of the present invention may be liquid, semi-solid, or solid, and may be a liquid, an emulsion (emulsion), a suspension, a gel, a cream, an ointment, and various types impregnated with them. It can be a dosage form.

  The concentration and pH of each component of the composition of the present invention may be adjusted to a range suitable for sterilization, but such concentration and pH are not appropriate for handling such as transportation and storage or storage stability. Where possible, a kit may be constructed such that a composition at a concentration and pH suitable for use is prepared at the time of use. The kit may contain, for example, one or a plurality of premixes, solvents and the like containing each component alone or in combination in a storage effective form. A premix refers to a single unit or composition of a specific amount or blending ratio for use by dissolving, diluting, and / or mixing at the time of use.

  When the composition of the present invention is liquid, the pH is preferably weakly acidic, i.e., about 3.0 to 6.0 from the viewpoint of low skin irritation, and preferably from about nisin storage stability. It can be 5.5 or less, more preferably about 4.5 or less. Considering the stability of the surfactant as a whole, the pH of the composition of the present invention that is liquid as a whole is about 4.0 to 6.0, preferably about 4.5 to 5.8, more preferably about 5.0 to 5.6 (eg about 5.5). be able to. From the viewpoint of the bactericidal effect on gram-positive bacteria, the pH can be, for example, about 4.0 to 10.0, preferably about 4.5 to 8.0, more preferably about 5.0 to 7.5. From the viewpoint of effect, the pH can be about 4.0 to 8.0, preferably about 4.5 to 7.0, more preferably about 5.0 to 6.5. The composition of the present invention exhibits excellent bactericidal activity against microorganisms in a specific pH range. The specific pH range is, for example, pH 4.0 to 7.0, preferably pH 5.0 to 6.5. In this pH range, when the composition of the present invention is contacted with a microorganism, both gram-positive bacteria and gram-negative bacteria can be sterilized.

  When the composition of the present invention is in liquid form, more specific forms include a spray container, a push container (a type that comes out with liquid, a type that comes out with foam), and a refill bottle. be able to. Other specific forms include oral cleaning / rinsing agents, dentifrices, dipping agents, breast injecting agents, wipes, paper towels, wet tissues and the like.

  The composition of the present invention can be used for any subject in need of sterilization of microorganisms. For example, parts of the body of animals including humans (oral cavity, teeth and periodontal, nasal cavity, fingers, face, feet, etc.), appliances / machines (medical instruments, cooking utensils, hygiene instruments, nursing supplies, baby products, toys, Food production machines, medical equipment, dining tables, desks, chairs, home furnishings, etc.), places (household and commercial kitchens, food processing factories, food material production factories, houses, hotels, restaurants, etc., medical facilities, hospital rooms , Floor, workbench, etc.) can be used to microbiologically clean.

  The composition of the present invention comprises a Gram-positive bacterium such as Staphylococcus, Streptococcus, Lactobacillus, preferably Staphylococcus aureus, Staphylococcus -It can be used for sterilization of microorganisms belonging to Staphylococcus epidermis, Streptococcus mutans, and Lactobacillus plantarum. The composition of the present invention is used for sterilizing microorganisms belonging to Gram-negative bacteria such as Escherichia, Bacillus, preferably Escherichia coli, Bacillus subtilis. can do. In addition, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-resistant enterococci (VRE), penicillin-resistant pneumococci (PRSP), multidrug-resistant Pseudomonas aeruginosa (MDRP), carbapenem-resistant Pseudomonas aeruginosa It can be used for sterilization of antibiotic-resistant bacteria such as bacteria, carbapenem-resistant Serratia, third-generation cephalosporin-resistant Escherichia coli, third-generation cephalosporin-resistant Klebsiella pneumoniae, multi-drug-resistant Acinetobacter.

(1) Experimental method and experimental conditions [Screening of stabilizers]
A preparation was prepared by mixing ethyl alcohol adjusted to a concentration of 25% (w / w) with nisin A purified product (Lot.070601), ultrapure water and a screening target substance provided by Ohm Dairy Co., Ltd. Nisin A was prepared to contain 4,000 IU / mL. The amount of the substance to be screened was adjusted based on the 0.1% (w / w) concentration and considering the solubility of the substance to be screened. Storage stability was stored for 6-10 days under accelerated test conditions (45 ° C.), and nisin stability in the test samples was quantified by HPLC.

[Nisin determination]
Nisin in the test sample was analyzed by HPLC (Hitachi EZ Chrom Elite HPLC L-2000). Analysis was performed by reversed-phase HPLC using a column (Shodex Asahipak ODP-50 6E) and a guard column (Shodex Asahipak ODP-50G 6A). The column temperature was set to 25 ° C. Development was performed at a flow rate of 1.0 mL / min using a mixed system.

[Preparation of alcohol preparation]
Formulation of nisin refined product nisin A refined product (Lot.070601) provided by Ohm Dairy Co., Ltd., purified water and stabilizer selected by screening in ethyl alcohol adjusted to 25% (w / w) concentration It was created. The alcohol preparation was prepared to contain 4,000 IU / mL of nisin A. Storage stability was stored for 0-30 days under accelerated test conditions (45 ° C.), and nisin stability in the test sample was quantitatively evaluated by HPLC.

[Bactericidal test]
The bactericidal activity test was measured by a method for quantifying the number of surviving bacteria. Escherichia coli NBRC3972 and Staphylococcus aureus NBRC 12732 were used as test bacteria . The bacterial solution was added to the test sample, allowed to react for a certain period of time, and this sample was added to a double concentration SCDLP broth medium. The number of bacteria was measured by the pour plate method for this sample, and the bactericidal effect of the prototype was confirmed.

[Antimicrobial durability test]
By measuring the antibacterial activity of the film formed on the hard surface after spraying the formulation, the durability of the antibacterial activity expected as a feature of this formulation was verified. First, the prototype was dried at room temperature under reduced pressure to form an antibacterial film. Next, the bacterial solution was dropped, and the antibacterial film and the bacterial solution were brought into contact for a predetermined period. This sample was subjected to bacterial count by the pour plate method to evaluate antibacterial activity. The test bacteria used were Escherichia coli NBRC3972, Staphylococcus aureus NBRC 12732, Bacillus subtillis ATCC6633.

(2) Results [Additive effect of stabilizer]
Nisin determination method by HPLC was introduced, and the stability of nisin in alcohol blending system and the effect of adding stabilizer were analyzed by HPLC. When the state of nisin A in the alcohol preparation was analyzed, it was confirmed that a decrease in nisin A activity was caused.

  An alcohol product prototype was prototyped and a storage test was performed under acceleration conditions (40 ° C). In FIG. 1, as an example, the state of nisin on the 11th day from the start of storage is compared. Nisin A decreased and nisin oxide increased in the blended system to which no stabilizer was added, but it was found that the stability of nisin A was improved by the addition of the stabilizer.

[Screening for stabilizers]
The effect of adding the stabilizer in the previous section suggested the applicability of the existing technology related to nisin formulation. Therefore, in the next examination item, screening was conducted for a stabilizer that can be used in combination with nisin and that is effective in an alcohol blending system.

  Screening of nisin stabilizers in the alcohol blending system was carried out on 21 types that could be blended (solubilized) in the model formulation according to the model formulation shown in Table 1. The numerical values in the table all indicate% by weight. The test was performed in four steps, but a control test group (Cont) in which no stabilizer was added was set for each test, and used as a criterion for judging the effect of adding the stabilizer. That is, compared to the control test group, the stability of nisin is improved, and the stabilizer having an activity remaining rate of 80% or more is evaluated as excellent (◎), and the nisin activity remaining rate is 10% or more compared to the control test group. Evaluation of improved stabilizer as good (○), evaluation of stabilizer with improved nisin activity remaining less than 0-10% (△), stabilizer with nisin activity lower than control test group The evaluation was impossible (x). Table 1 shows the judgment results.

  FIG. 2 (A) and FIG. 2 (B) show nisin activity and nisin residual activity in screening. Storage stability was evaluated by quantitatively evaluating nisin stability in a test sample in an accelerated test under severe conditions (45 ° C.). FIG. 2 shows a comparison of nisin activity on day 0, 7 or 10 after the start of storage.

  From the screening results, methionine (Ao01) and thioctic acid (Ao02) were obtained as stabilizers for excellent evaluation (◎). These showed 92% and 83.5% nisin residual activity after storage (D + 6). EDTA-2Na (Ao13) and BHA (Ao15) were obtained as good evaluation (◯) stabilizers. These had decreased nisin activity after storage (D + 7), but their residual activity exceeded 10% or more in comparison with the control group. In addition, tocopherol (Ao03), tocopherol acetate (Ao04), curcumin (Ao08), and lactic acid (Ao21) were obtained as evaluable (Δ) stabilizers.

  A well-evaluated or evaluable stabilizer is inadequate in stabilizing nisin, but it is estimated that there is little adverse effect on the mutual stability with nisin. For this reason, it is thought that it can be used as a component in a nisin compounding system. On the other hand, as for other stabilizers (13 types), an apparent decrease in activity was observed after the storage test as compared with the control group, and it is considered unsuitable for use as a component in the nisin combination system. For example, cysteine hydrochloride (Ao05), ascorbic acid Na (Ao12), glutathione (Ao17) and the like have remarkable nisin activity after the storage test, and are considered unsuitable for use in a nisin-containing system.

(1) Experimental method and conditions [Nisin determination]
Nisin A in the test sample was analyzed by HPLC (Hitachi EZ Chrom Elite HPLC L-2000). Analysis was performed by reversed-phase HPLC using a column (Shodex Asahipak ODP-50 6E) and a guard column (Shodex Asahipak ODP-50G 6A). The column temperature was set to 25 ° C. Development was performed at a flow rate of 1.0 mL / min using a mixed system.

[Preparation of alcohol preparation containing thioctic acid]
A preparation was prepared by blending ethyl alcohol prepared to a concentration of 25% (w / w) with nisin A purified product (Lot.070601), purified water and thioctic acid provided by Ohm Dairy Co., Ltd. Nisin A was prepared to contain 4,000 IU / mL and 0.1% (w / w) of thioctic acid. Storage stability was stored for 6 days under accelerated test conditions (45 ° C.), and nisin A stability in the test sample was quantitatively evaluated by HPLC.

(2) Results [Addition effect of thioctic acid]
Nisin determination method by HPLC was introduced and the stability of nisin and the effect of addition of thioctic acid in alcohol blending system were analyzed by HPLC. When the state of nisin A in the alcohol preparation was analyzed, it was confirmed that a decrease in nisin A activity was caused.

  An alcohol product prototype was prototyped and a storage test was performed under acceleration conditions (40 ° C). In FIG. 3, as an example, the state of nisin on the sixth day after the storage is compared. In the blended system without addition of thioctic acid, nisin A decreased and nisin oxide increased, but it was found that the addition of thioctic acid improves the stability of nisin A. As a result of calculating the residual activity of nisin A on the basis of the 0th day from the start of the storage, it was 91.2% in the thioctic acid-containing system and 55.7% in the additive-free system. From this, it was confirmed that the activity of nisin A was retained by blending thioctic acid.

  In order to investigate the stabilizing effect in the oily ointment, nisin and a stabilizer were blended in the ointment, and a storage test was conducted. A bioassay was performed after storage at 45 ° C. for 4 days to measure the antibacterial activity of nisin.

(1) Experimental method
i) Preparation of ointment Purified nisin A (Ohm Milk Industry Co., Ltd.) was mixed with hydrophilic petrolatum prepared by adding a stabilizer at 7: 3, and a storage test was performed at 45 ° C. Stabilizers are (1) additive-free, (2) thioctic acid (manufactured by Wako Pure Chemical Industries, Ltd.), (3) methionine (manufactured by Alps Pharmaceutical Industries, Ltd.), (4) tocopherol (Wako Pure Chemical Industries, Ltd.) )), (5) Thioctic acid and methionine were added so as to be 0.1% of the total amount including nisin. However, 0.1% of each test sample (5) was added.

ii) Antibacterial activity test Ointment was added to 0.1% Tween80 (manufactured by Wako Pure Chemical Industries, Ltd.) and stirred to elute nisin, and the eluate was subjected to bioassay. Lactobacillus plantarum ATCC14917 ^T was used as a test bacterium.

(2) Results and discussion In the presence or absence of a stabilizer, the effect was observed in all test samples compared to no addition. In particular, the test sample (2) to which thioctic acid was added showed excellent stability compared to other stabilizers. In addition, in the test sample (5) to which thioctic acid and methionine were added, the stability of nisin was improved as compared with the addition of thioctic acid alone (table below and FIG. 4).

  In order to investigate the stabilizing effect in oil, an emulsion in which nisin and a stabilizer were added to the oil was prepared, and nisin in the emulsion was quantified.

(1) Experimental method
i) Preparation of emulsion Emulsions are made of soybean oil (Wako Pure Chemical Industries, Ltd.) and an equal amount of purified nisin (Ohm Dairies Co., Ltd.). (Wako Pure Chemical Industries, Ltd.)), (2) Methionine (Alps Yakuhin Kogyo Co., Ltd.) added, (3) Thioctic acid and methionine 0.5% added, (4) No additive Was made by stirring.

ii) Quantification of nisin The emulsion was crushed by centrifugation, only nisin was collected, and the nisin state (oxidation / activity) was quantified by HPLC.

  Nisin in the test sample was analyzed by HPLC (Hitachi EZ Chrom Elite HPLC L-2000). Analysis was performed by reversed-phase HPLC using a column (Shodex Asahipak ODP-50 6E) and a guard column (Shodex Asahipak ODP-50G 6A), the column temperature was set to 25 ° C., and acetonitrile and 0.05% TFA were used as the solvent system. Development was performed at a flow rate of 1.0 mL / min using a mixed system.

(2) Results and discussion The remaining nisin activity in the test sample (4) with no stabilizer added was 79.0%. The test sample (1) to which thioctic acid was added was 93.3%, the test sample (2) to which methionine was added was 93.7%, and the test sample (3) to which both were added was 94.3%, and an excellent stabilizing effect was obtained. In addition, although the stabilization effects of the test samples (1) and (2) were equivalent, the test sample (3) combined with both resulted in further suppression of oxidation (table below and FIG. 5).

  In order to examine the stabilizing effect in the surfactant solution (final concentration 10%), nisin and a stabilizer were added to the surfactant solution, and the antibacterial activity of nisin in the test sample was measured and quantified.

(1) Experimental method
i) Preparation of test sample A stabilizer and purified nisin (manufactured by Ohm Dairies) were added to the surfactant solution, and a storage test was performed at 45 ° C. Stabilizers are (1) additive-free, (2) thioctic acid (manufactured by Wako Pure Chemical Industries, Ltd.), (3) methionine (manufactured by Alps Pharmaceutical Co., Ltd.), (4) thioctic acid and methionine, nisin It was added so that it might become 0.1% of the whole quantity containing. However, 0.05% of each test sample (4) was added.

ii) Antibacterial activity test A bioassay was performed using a test sample. Lactobacillus plantarum ATCC14917 ^T was used as a test bacterium. As a control, purified nisin used in preparing the test sample was also tested at the same time.

iii) Quantification of Nisin Nisin in the test sample was analyzed by HPLC (Hitachi EZ Chrom Elite HPLC L-2000). Analysis was performed by reversed-phase HPLC using a column (Shodex Asahipak ODP-50 6E) and a guard column (Shodex Asahipak ODP-50G 6A). The column temperature was set to 25 ° C. Development was performed at a flow rate of 1.0 mL / min using a mixed system.

(2) Results and discussion
i) Antibacterial activity test As a surfactant, Newpol PE-64 (manufactured by Sanyo Kasei Kogyo Co., Ltd.), which is known to have poor stability of nisin when no stabilizer was used, was used. The main component is a nonionic surfactant polyoxyethylene polyoxypropylene block polymer. The prescriptions and evaluation results of the test samples are shown in the table below.

  It was found that the test sample (1) to which no stabilizer was added had decreased nisin activity. The MIC value of the test sample (2) with thioctic acid added is 8.7 IU / mL, and the test sample (3) with methionine added is 12 IU / mL, which is a slight difference, but the effect of adding thioctic acid is excellent. It became the result. Moreover, the MIC value of the test sample (4) to which both were added was 4.9 IU / mL, and it can be assumed that the synergistic effect of thioctic acid and methionine was obtained.

ii) Determination of nisin Nisin (non-oxidized type) in the test sample (1) to which no stabilizer was added was lowered to the detection limit value or less and could not be quantified. The residual rate of test sample (2) with thioctic acid added is 71.5%, test sample (3) with methionine added is 59.4%, and the addition effect of thioctic acid is superior to methionine in surfactant solution Was confirmed. Moreover, the residual rate of the test sample (4) to which both were added was 82.0%, and it was found that nisin could be further stabilized by addition of thioctic acid alone. It is thought that the synergistic effect of both stabilizers was obtained. This result is consistent with the antimicrobial activity test result (Fig. 6).

  Prepare nisin-containing fungicide with the following formula.

Alcohol preparation:
Ethanol 25% (w / w)
Nisin 4000IU / mL
Thioctic acid 0.1% (w / w)
ointment:
Hydrophilic Vaseline Nisin 15000IU / mL (30% against hydrophilic petrolatum)
Thioctic acid 1% (w / w)
Disinfectant with surfactant:
New Pole PE-64 10% (w / w)
Nisin 5000IU / mL
Thioctic acid 0.1% (w / w)

  A nisin-containing fungicide can be prepared with the following formulation.

Hand cleaner:
A safe hand cleaner composed of food additives and cosmetic ingredients.
Use: Skin cleansing, disinfection, disinfection Concentration: Stock solution usage: Foam pump may be used.
Take a fixed amount of stock solution (bubbles), wash well, and rinse.

Cleaning composition for food and tableware:
A very safe food and dishwashing detergent composition, where all ingredients are composed of food additives.
Use: Sterilization of vegetables, etc. Washing concentration of dishes: Stock solution-200 times Usage: Use diluted after use.

Environmental cleaning antibacterial composition:
A very safe environmental cleaning antibacterial agent with all ingredients composed of food additives.
Use: Washing and disinfecting floors, walls, handrails, etc. Concentration: Undiluted solution Usage method: Soaked in cloth, etc. Rinse is unnecessary.

Preparation procedure (It is recommended to prepare immediately before use.)
1) Heat activator solution components other than Crewat N in purified water and cool to room temperature. Clewat N is added and dissolved to prepare the activator solution.
2) Prepare 40,000 IU / ml nisin solution.
3) Mix the surfactant solution and nisin solution and adjust the pH to 5.0 with NaOH.

Claims (8)

  A composition comprising an antibacterial effective amount of lantiobiotic, which is 10 to 100,000 parts by weight (preferably 50 to 50,000 parts by weight, more preferably 100 to 10,000 parts by weight) with respect to 1 part by weight of the lantiobiotic A sterilizing composition comprising thioctic acid or a salt acceptable in medical or food hygiene. Lantibiotic, and thioctic acid or a medically or food hygiene acceptable salt thereof,
The concentration of lantibiotic is 25-200,000 IU / ml, preferably 100-100,000 IU / ml, more preferably 500-50,000 IU / ml;
The concentration of thioctic acid is 0.001 to 10% by weight (preferably 0.005 to 2% by weight, more preferably 0.01 to 1% by weight).
A liquid or semi-solid composition.   The composition according to claim 1 or 2, further comprising a surfactant.   The composition according to claim 1 or 2, further comprising an alcohol, wherein the concentration of the alcohol is 1 to 80% by weight (preferably 10 to 60% by weight, more preferably 20 to 40% by weight).   The composition according to claim 1 or 2, wherein the lantibiotic and thioctic acid are dissolved in an oily base.   Lantiobiotic stabilizer containing thioctic acid.   A method of stabilizing a lantibiobiotic comprising using thioctic acid.   A method of sterilizing a subject using lantiobiotic and thioctic acid.

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