JP2011001352A - Bactericidally effective method for using nisin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition which operates on both of gram-positive bacteria and gram-negative bacteria and has broad-ranging biocidal activity against microorganisms; and a biocidal method for microorganisms.SOLUTION: The composition includes nisin, for example nisin A or nisin Z, in a content of 2-50,000 IU/mL, is adjusted to a pH of 4.0-10.5, may further include a surfactant, and is used as a bactericide against at least one kind of microorganisms being gram-negative or gram-positive. A kit for preparing the composition is also provided.

Description

  The present invention relates to a method for effective sterilization of nisin. More particularly, the present invention relates to the use of nisin at sterilizing effective pH conditions and compositions therefor.

  Nisin is an antibacterial peptide produced by lactic acid bacteria, but it is generally recognized as safe (generally recognized as safe: GRAS) in the United States, and is actually used as a preservative for cheese, etc. It has been approved as a food additive. Therefore, if nisin is used as an antibacterial agent, a highly safe antibacterial agent can be provided.

  Nisin is known to exhibit broad antibacterial activity against gram-positive bacteria but weak antibacterial activity against gram-negative bacteria. In this regard, reports that nisin is used in combination with a chelating agent such as ethylenediaminetetraacetic acid (EDTA) or an organic acid to increase antibacterial activity against gram-negative bacteria (Non-patent Document 1, Non-patent Document 2), There is a report that the antibacterial activity against Gram-negative bacteria decreases due to the presence of metal ions (Non-patent Document 3).

  On the other hand, in order for nisin to exhibit sufficient antibacterial activity, it is premised that nisin exists physically and stably. Regarding the stability of nisin, studies have been made on blending methionine as a stabilizer (Patent Document 1) and selecting an appropriate surfactant (Patent Document 2) when used in a cleaning composition. Nisin is also known to be most stable at pH 3.

Japanese translation of PCT publication No. 8-510716 JP 2007-99809 A

Applied and Environmental Microbiology, 58 (5), p.1786-88, 1992 International Journal of Food Microbiology, 21, p.305-314, 1994 International Journal of Antimicrobial Agents, 26, p.396-402, 2005

  According to the study of the present inventors, when EDTA was added to nisin, the antibacterial effect against Gram-negative bacteria was not obtained due to the presence of sodium ions, and when an organic acid was added, Although the antibacterial effect against gram-negative bacteria is high, there is a problem that the pH becomes very low and the practicality becomes poor. Furthermore, the antibacterial effect was hardly confirmed about what mixed EDTA and the organic acid in the liquid mixture of surfactant and nisin, and made pH weakly acidic.

  As a result of intensive studies in view of the above circumstances, the present inventors have found that the bactericidal activity of nisin is improved at pH 4.0 to 10.5. This improvement in antibacterial activity improves the bactericidal activity against gram-negative bacteria that have been thought to not work with nisin alone, in addition to improving the bactericidal activity of nisin against gram-positive bacteria. Also based on what to do. Moreover, it discovered that the bactericidal activity with respect to Gram positive bacteria further improved in pH 5.0-6.5, and the bactericidal activity with respect to Gram negative bacteria further improved in pH 4.0-6.5. From the above results, it was derived that nisin further improved the bactericidal activity against both gram positive bacteria and gram negative bacteria at pH 5.0 to 6.5. The present invention has been completed based on such findings.

The present invention provides the following.
1) A composition for sterilization of at least one gram-negative or gram-positive microorganism, adjusted to pH 4.0-10.5, containing nisin at 2 to 50,000 IU / mL, or for its preparation Kit.

2) The composition or kit according to 1), wherein the nisin is nisin A or nisin Z.
3) Nisin is nisin A, the pH of which is adjusted to 5.0 to 6.5, for sterilization of at least one gram-positive microorganism and at least one sterilization of a gram-negative microorganism. The composition or kit according to 1), which is for use.

  4) Gram-positive bacteria are Streptococcus mutans, Streptococcus sobrinus, Staphylococcus aureus, Staphylococcus derp 1), or a composition or kit according to 1), wherein the Gram-negative bacterium is selected from Escherichia coli, and the gram-negative bacterium is selected from Escherichia coli.

5) The composition or kit according to 1), further comprising a surfactant.
6) At least one kind of gram-positive or gram-negative microorganisms at pH 4.0 to 10.5 is sterilized by contacting them at a rate of 10 to 10,000 cfu per nisin at a rate of 10 to 10,000 cfu for 10 to 180 seconds. Sterilization method.

7) The sterilization method according to 6), wherein the nisin is nisin A or nisin Z.
8) Nisin is Nisin A, and at pH 5.0 to 6.5, a gram-positive bacterium (preferably a bacterium selected from Streptococcus, Staphylococcus, Lactobacillus, Propionibacterium, For sterilizing at least one species selected from Streptococcus mutans, Streptococcus sobrinas, Staphylococcus aureus, Staphylococcus epidermidis, Lactobacillus plantarum, Propionibacterium acnes), and The sterilization method according to 7), for sterilizing at least one gram-negative bacterium (preferably a bacterium selected from the genus Escherichia, more preferably a bacterium selected from Escherichia coli).

9) At pH 4.0-10.5, nisin 1 IU and microorganisms 10-10,000 cfu are contacted for 10-180 seconds to kill Gram-positive bacteria; at pH 4.0-8.0, nisin and microorganisms 10 Sterilize Gram-negative bacteria by contact for ~ 180 seconds;
Microbial sterilization method.

10) The sterilization method according to 8) or 9), further comprising a step of treating with a surfactant.
11) A method for improving the sterilizing power of nisin, wherein the pH is adjusted to 5.0 to 7.0.

  The “composition” as used herein is not particularly limited as long as it contains nisin. The composition can be in a form suitable for the application of the composition. For example, liquid, powder, granule, gel, solid, or a form immobilized on an appropriate carrier can be mentioned. In addition, ingredients known in the art of the present invention can be incorporated into the compositions of the present invention. For example, surfactants, emulsifiers, wetting agents, flavoring agents, fragrances, abrasives, lubricants, chelating agents, dispersants (anti-redeposition agents), solvents, humectants, bactericides (antibacterial agents), stabilization An agent, a cleaning enzyme, a viscosity modifier, an antiseptic, an antifungal agent, a colorant, and a fragrance can be appropriately selected and blended in the composition of the present invention. As one aspect, the above components may be blended so that the composition of the present invention can exert a cleaning action in addition to the bactericidal action. In addition, since nisin contained in the composition can sterilize gram-negative bacteria alone, it can exhibit a wide range of bactericidal activity even if the composition does not contain a chelating agent. Here, the chelating agent means a multidentate ligand that coordinates to a metal ion and gives a chelate compound. Examples thereof include polyaminocarboxylic acids such as EDTA and oxycarboxylic acids such as citric acid.

  The composition of the present invention can be widely used in various fields that require sterilization of microorganisms. For example, it can be used in the food field. As one aspect, the composition of the present invention can be used for sterilization of food poisoning microorganisms and food harmful microorganisms in household and commercial kitchens, cooking utensils, food processing factories, food material production factories and the like. In particular, in food-related industries where high hygiene management is required, sterilization of food poisoning microorganisms and food harmful microorganisms is an essential matter, and therefore an effective and safe sterilization method is strongly demanded. Here, diarrheagenic Escherichia coli (enterohemorrhagic Escherichia coli O157 and the like), Staphylococcus aureus (Salphylococcus aureus), Salmonella (Listeria), enteritis vibrio (Vibrio parahaemyticus), Campylobacter (Campylobacter) Campylobacter, Clostridium welchii, Yersinia enterocolitica, Bacillus cereus, Clostridium botulinum, Nagublio genus Death (Plesiomonas shigelloides) and the like. In addition to food poisoning microorganisms, food harmful microorganisms include lactic acid bacteria, Pseudomonas, Bacillus, Micrococcus, Vibrio, Serratia, Clostridium, and the like. It is done.

As another example, the composition of the present invention can be used to sterilize microorganisms present in a room such as a house, a hotel, a restaurant, furniture, a floor, and the like.
Another example is the use of the composition of the present invention in the medical field. In the medical field, a high level of hygiene management is required due to the treatment of human beings. In recent years, antibiotic-resistant bacteria such as 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, carbapenem resistant Serratia, third generation cephalosporin resistant E. coli, third generation Nosocomial infections caused by cephalosporin-resistant Klebsiella pneumoniae, multidrug-resistant Acinetobacter, Clostridium difficile have become a major problem. From such a viewpoint, the composition of the present invention can be widely used for sterilization of medical facilities.

  The composition of the present invention is suitable for prevention and treatment of skin diseases. Normally, a wide variety of microorganisms such as Staphylococcus epidermidis and acne bacteria exist on human skin, and skin diseases are caused by excessive growth of these microorganisms. Develops. Traditionally used disinfectants for the purpose of disinfecting these microorganisms present in the skin are skin irritants and contribute not only to the target bacteria but also to the repair of skin tissue, such as immune cells, fibers There are also problems such as toxicity to blast cells and epidermal cells. For example, acne (acne vulgaris), a common skin disease, can be caused by increased sebum secretion, obstruction of the follicular sebum line, and abnormal growth of Acne bacteria (Propionibacterium acnes). Are known. In the prevention and treatment of acne, antibiotics and exfoliating agents have been used in the past, but the use of antibiotics has the problem of causing side effects and resistant bacteria, and exfoliating exfoliated substances such as sulfur and sulfa drugs. The use of the agent has the problems of insufficient skin irritation and prevention or treatment of acne.

  For these reasons, there is a need for a composition for preventing or treating skin diseases that has low skin irritation, high specificity for target microorganisms, and high safety. The present invention can provide such a composition. For example, the composition of the present invention is extremely low in irritation to the skin and can effectively sterilize acne bacteria, so that a composition for preventing and treating acne can be provided.

Furthermore, the composition of the present invention is also suitable for sterilization of oral microorganisms. As used herein, “sterilization” includes not only killing microorganisms but also stopping, inhibiting or delaying the growth of microorganisms. Therefore, the “sterilizing composition” in the present specification means a composition suitable for sterilizing microorganisms. Here, whether or not the composition is suitable for sterilization can be determined, for example, based on an evaluation result by antibacterial evaluation (MBC modified method). The MBC method is a method for measuring the minimum bactericidal concentration. The MBC modified method is based on the MBC method and arbitrarily sets various conditions such as culture conditions, bactericide concentration, contact time, contact temperature, and test bacteria concentration. It is a method of setting and measuring the number of surviving bacteria under the conditions. For example, a test bacterium (for example, E. coli) is cultured in an SCD bouillon medium (manufactured by Nissui Pharmaceutical Co., Ltd.) for 24 hours at 37 ° C., and then 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, 5 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, 5 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 MBC modified method, various conditions such as bactericide concentration, contact time, contact temperature, and test bacteria concentration can be appropriately changed depending on the test purpose, and the medium and culture conditions are appropriately changed depending on the type of the test bacteria. be able to. In addition, after contacting the test solution with the test bacteria, culturing using a liquid medium containing an inactivating agent, based on the turbidity of the cultured medium, the presence or absence of bacteria remaining in the medium and the growth It is also possible to qualitatively determine the degree of. Here, the inactivating agent is a compound or composition that inactivates the bactericidal agent, and, for example, a surfactant that forms micelles and takes in a bactericidal component is widely used. In addition, a neutralizing agent such as an anionic surfactant or sodium thiosulfate may be used depending on the type of disinfectant. For example, the surfactant polysorbate can be used as an inactivating agent for nisin.

  In the 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 degree of sterilization effect 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 still more preferably 99.9 to 100%. 99.9% sterilization rate is sufficient for general disinfectant and environmental sterilization applications, but 99.999% or higher sterilization rate is required for sterilization of aseptic equipment in contact with food. Sometimes it is done.

Whether or not the composition of the present invention is suitable for sterilization can be determined using the amount of ATP flowing out of the microbial cell body as an index. Nisin exerts a bactericidal effect by acting on the cell membrane (Lipid II) of microorganisms and forming pores in the cell membrane. Therefore, the greater the amount of ATP that flows out of the cells from the formed pores, the higher the bactericidal activity. It can be judged. The amount of ATP can be measured, for example, by measuring the luminescence generated by the reaction between ATP and luciferin catalyzed by luciferase as a relative luminescence amount (relative light unit: RLU). The amount of relative luminescence can be measured by using a measuring device having a photomultiplier tube as a detector, for example, luminescence generated by the reaction. Such measurement may be performed using a commercially available measurement kit. Further, when the relationship between the ATP concentration in the sample and the relative light emission amount is plotted on a log-log graph, a good linear relationship is obtained between the two measured values, for example, in the range of 2 × 10 ⁻¹² to 2 × 10 ⁻⁸ M in ATP concentration. Therefore, it is also possible to create a calibration curve using an ATP standard reagent as necessary and convert the relative luminescence amount to the ATP concentration.

  The example of the judgment method which makes the parameter | index the quantity of ATP which flows out out of a microbial cell is shown. A microorganism belonging to the genus Propionibacterium is pre-cultured in an appropriate medium, the culture medium is inoculated into a new medium, swirled in an anaerobic environment, and this culture liquid is used as a test bacterial solution. By adding the test bacterial solution to the nisin solution, nisin and the microorganism are brought into contact for 3 minutes, and this solution is used as the test solution. On the other hand, a test bacterial solution is added to a solution not containing nisin, and the same treatment is used as a control test solution. A test solution and a control test solution are appropriately diluted with a buffer used for measurement, and the amount of ATP present outside the cells is measured as a relative luminescence amount using a commercially available kit. A value obtained by subtracting the relative luminescence amount of the control test solution from the relative luminescence amount of the test solution is defined as the amount of ATP flowing out of the microbial cells. When the amount of ATP is measured under the conditions shown in this example, if the relative light emission amount is 20,000 RLU or more, preferably 50,000 RLU or more, more preferably 100,000 RLU or more, the composition has a bactericidal effect. Judgment can be made.

  The microorganisms in this specification can be classified into Gram-positive bacteria and Gram-negative bacteria. Microorganisms belonging to Gram-positive bacteria and Gram-negative bacteria include, for example, Bergey's Manual Of Systematic Bacteriology: The Proteobacteria; The Alpha-, Beta-, Delta-, and Epsilonproteobacteria (Bergey's Manual of Systematic Bacteriology (Springer-Verlag)) George Garrity, Included in Don J. Brenner, Noel R. Krieg, James T. Staley. The composition of the present invention is a gram-positive bacterium such as Bacillus, Staphylococcus, Streptococcus, Lactobacillus, Clostridium, Listeria, Micrococcus, Propionibacterium. The genus, preferably Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, and Streptococcus mutans, Propionibacterium · To exert bactericidal activity against microorganisms belonging to the acnes. The composition of the present invention is a gram-negative bacterium such as Escherichia, Pseudomonas, Enterobacter, Klebsiella, Citrobacter, Proteus, Serratia. It exhibits bactericidal activity against microorganisms belonging to the genus Erwinia, Vibrio, Acinetobacter, preferably Escherichia coli.

  As used herein, “oral microorganism” means a microorganism that is or can exist in the oral cavity, among the microorganisms defined above. There are various types of oral microorganisms, for example, Streptococcus, Neisseria, Micrococcus, Lactobacillus, Spirochaets, and the like.

  The oral cavity Streptococcus, Streptococcus mutans, Streptococcus sobrinus (Streptococcus sobrinus) Streptococcus sanguis (Streptococcus sanguis), Streptococcus Michioru (Streptococcus mitior), Streptococcus salivarius (Streptococcus salivarius), Streptococcus Mireri (Streptococcus milleri) Etc. Examples of the genus Neisseria include Neiseria flava, Neiseria subflava, Neiseria sicca and the like.

  Examples of the oral Lactobacillus genus include Lactobacillus casei and Lactobacillus acidophilus. Examples of the genus Spirocheta include Treponema denticola, Treponema macredentium, Treponema oralis, and the like.

  Among oral microorganisms, those belonging to Gram-positive bacteria include, for example, Streptococcus, Peptococcus, Peptostreptoccus, Micrococcus, Actinomyces, Lactobacillus , Microorganisms belonging to the genus Bacterionema, Arachnia, and Bifidobacterium. For example, Streptococcus mutans and Streptococcus sobrinus can be mentioned as microorganisms belonging to the genus Streptococcus. These are known as caries-causing bacteria.

  On the other hand, among oral microorganisms, those belonging to Gram-negative bacteria include, for example, Neisseria, Branhamella, Veillonella, Bacteroides, Haemophilus, Spirocheta, and Serenomonas Included are microorganisms belonging to the genus (Selenomonas).

  The term “nisin” used in the present specification includes nisin A and nisin Z in the range unless otherwise specified. The structures of Nisin A and Nisin Z are similar. In nisin A, the 27th amino acid from the N-terminus of the polypeptide chain is histidine, but in nisin Z, it differs only in that it is asparagine. The nisin activity in the composition is not limited as long as it shows bactericidal activity against microorganisms, but is, for example, 2 to 50,000 IU / mL, preferably 10 to 10,000 IU / mL, more preferably 50 to 1,000 IU / mL. It is.

  The compositions of the present invention are particularly suitable for use in specific pH ranges. That is, when the composition is brought into contact with a microorganism in a specific pH range, it exhibits high bactericidal activity against the microorganism. Here, the bactericidal activity can be expressed by the bactericidal rate by the MBC method or the MBC modified method. The high bactericidal activity is, for example, preferably a sterilization rate of 90 to 100%, more preferably 99 to 100%, and further preferably 99.9 to 100%. The specific pH range may be, for example, pH 4.0 to 10.5, preferably pH 4.0 to 7.0, and more preferably pH 4.5 to 6.5. As one aspect, when the composition of the present invention is brought into contact with a microorganism at pH 4.0 to 10.5, preferably pH 4.0 to 7.0, more preferably pH 4.5 to 6.5, High bactericidal activity.

  Moreover, the composition of this invention can improve the bactericidal activity with respect to a specific microorganism group, for example, a Gram positive bacterium or a Gram negative bacterium, by changing pH at the time of contacting with a microorganism. In one embodiment, the composition of the present invention and Gram-positive bacteria are, for example, pH 4.0 to 10.5, preferably 4.5 to 8.0, more preferably 5.0 to 7.5. The product and the gram-negative bacterium can be contacted, for example, at pH 4.0 to 8.0, preferably 4.5 to 7.0, more preferably 5.0 to 6.5.

An example of but not limited to the contact pH of nisin for individual microorganisms is shown below:
For Staphylococcus aureus, pH 4.5-10.5, preferably pH 5.0-9.0, more preferably pH 7.0-8.0;
For Streptococcus mutans, pH 4.0-7.0, preferably pH 4.5-6.5, more preferably pH 4.5-6.0 (in the case of Nisin A, pH 4.5-5.5). Or in the case of Nisin Z, the pH may be 5.0 to 6.0.);
For Escherichia coli, pH 4.5 to 7.0, preferably pH 5.0 to 6.6, more preferably pH 6.0 to 6.5;
For Propionibacterium acnes, pH 4.0-8.0, preferably pH 4.0-7.0, more preferably pH 5.0-6.0.

  The present invention can also be applied to a method for sterilizing microorganisms. Here, in the said sterilization method, when "it is in pH 4.0-10.5" means pH at the time Nisin and microorganisms are contacting. The said pH means that it may be the same as that of the composition itself containing the nisin before contacting with microorganisms, and may differ. That is, the composition of the present invention whose pH is adjusted to 2.0 to 4.0 is adjusted so that the pH at the time of contact becomes 4.0 to 10.5 before or after contacting with the microorganism. The pH of the product can be adjusted. The pH can be adjusted, for example, with an alkali agent such as sodium hydroxide or potassium hydroxide.

  In the sterilization method of the present invention, nisin and microorganisms can be contacted at a specific ratio. For example, the number of microorganisms per nisin IU can be 10 to 10,000 cfu, preferably 10 to 5,000 cfu, more preferably 10 to 1,000 cfu.

  The contact time between nisin and the microorganism may be a time required for nisin to exert the bactericidal activity, and can be set based on, for example, the bactericidal rate calculated by the MBC method or the MBC modified method. The contact time can be set to, for example, 10 to 180 seconds, preferably 10 to 60 seconds, and more preferably 20 to 30 seconds. In applications where immediate effect is not required, the contact time may not be set, but may be set to, for example, 3 minutes to 24 hours, preferably 1 to 12 hours. Moreover, in the use in which the durability of an antimicrobial action is requested | required, you may make it act on microorganisms continuously by exchanging or adding nisin.

Here, cfu means a colony forming unit and is calculated based on the number of colonies formed on the agar medium and the dilution factor.
In the sterilization method of the present invention, conditions suitable for sterilization of Gram-positive bacteria and Gram-negative bacteria can be applied stepwise. For example, the microorganism is contacted with the composition of the present invention under conditions suitable for sterilization of Gram-positive bacteria, and further contacted with the composition of the present invention under conditions suitable for sterilization of Gram-negative bacteria. By this method, the sterilization rate of microorganisms can be further increased. It can be easily understood that the same effect can be obtained even if the order of these two processes is changed.

  The sterilization method of the present invention may include a step of treating with a surfactant in addition to the step of contacting nisin and a microorganism. This step can be performed, for example, before, after, or at the same time as the step of contacting nisin and the microorganism.

  The sterilization method of the present invention may also be a skin sterilization method including a step of applying to the skin a composition adjusted to pH 4.0 to 8.0 containing nisin at 2 to 50,000 IU / g. it can.

  The sterilization method of the present invention also includes a step of rinsing the oral cavity for a time effective for sterilization with a liquid adjusted to pH 4.0 to 7.0 containing nisin at 2 to 50,000 IU / mL. It can be a sterilization method in the oral cavity.

  According to the present invention, there is provided a method for improving the sterilizing power of nisin, wherein the pH is adjusted to 5.0 to 7.0.

Bactericidal activity of Nisin A against Streptococcus mutans Xc. The bar graph represents cfu and the line graph represents pH. Effect of pH on bactericidal activity of Nisin A against Streptococcus mutans Xc. ●: Nisin activity 2 IU / mL; ○: Nisin activity 10 IU / mL; ▲: Nisin activity 50 IU / mL; □: Nisin activity 100 IU / mL. Effect of pH on bactericidal activity of Nisin A and Nisin Z against Streptococcus mutans Xc. ●: Nisin A 50 IU / mL; □: Nisin Z 50 IU / mL. Effect of pH on bactericidal activity of Nisin A against Staphylococcus aureus IFO13276. △: Nisin A 0 IU / mL, contact time 30 seconds; ▲: Nisin A 500 IU / mL, contact time 30 seconds; ■: Nisin A 4,000 IU / mL, contact time 30 seconds; ○: Nisin A 0 IU / mL, contact Time: 60 minutes; ●: Nisin A 500 IU / mL, contact time 60 minutes; □: Nisin A 4,000 IU / mL, contact time 60 minutes. Effect of pH on bactericidal activity of Nisin A against Escherichia coli NBRC 3972. Δ: Nisin A 0 IU / mL, contact time 30 seconds; Δ: Nisin A 5,000 IU / mL, contact time 30 seconds; ■: Nisin A 20,000 IU / mL, contact time 30 seconds; ○: Nisin A 0 IU / mL , Contact time 60 minutes; ●: Nisin A 5,000 IU / mL, contact time 60 minutes; □: Nisin A 20,000 IU / mL, contact time 60 minutes. Effect of Nisin concentration on bactericidal activity of Nisin A against Staphylococcus aureus ATCC31885. □: contact pH not adjusted, contact time 30 seconds; ▲: contact pH unadjusted, contact time 60 minutes; ●: contact pH 5.0, contact time 30 seconds; ■: contact pH 5.0, contact time 60 minutes. Effect of Nisin concentration on bactericidal activity of Nisin A against Escherichia coli NBRC3982. ■: Contact pH not adjusted, contact time 30 seconds; □: Contact pH not adjusted, contact time 15 minutes; ▲: Contact pH 5.0, contact time 30 seconds; ●: Contact pH 5.0, contact time 15 minutes. Examination of contact time between Nisin A and Staphylococcus aureus. ●: Nisin activity 0 IU / mL, contact pH 5.0; ■: Nisin activity 1,000 IU / mL, contact pH 5.0; ▲: Nisin activity 4,000 IU / mL, contact pH 5.0; ○: Nisin activity 0 IU / mL Contact pH not adjusted; □: Nisin activity 1,000 IU / mL, contact pH not adjusted; Δ: Nisin activity 4,000 IU / mL, contact pH not adjusted. Examination of contact time between Nisin A and Escherichia coli NBRC3972. ●: Nisin activity 0 IU / mL, contact pH 5.0; ■: Nisin activity 5,000 IU / mL, contact pH 5.0; ▲: Nisin activity 20,000 IU / mL, contact pH 5.0; ○: Nisin activity 0 IU / mL □: Nisin activity 5,000 IU / mL, contact pH not adjusted; Δ: Nisin activity 20,000 IU / mL, contact pH unadjusted. Bactericidal activity of mouthwash containing Nisin A against Streptococcus mutans Xc, Streptococcus mutans JCM5707 and Streptococcus sobrinus AHT-K. Open bar graph: control; hatched bar graph: treatment with mouthwash adjusted to pH 3.0; black bar graph: treatment with mouthwash adjusted to pH 5.0. SM-Xc: Streptococcus mutans Xc, SM-JCM: Streptococcus mutans JCM5707; SB-AHT: Streptococcus sobrinus AHT-K. Effect of pH and nisin activity on the reaction of nisin A with target cells (Propionibacterium acnes). ○: Nisin activity 1,000 IU / mL; □: Nisin activity 500 IU / mL.

  The present invention provides a composition for sterilizing microorganisms containing nisin, which exhibit bactericidal activity against microorganisms. Nisin is a kind of bacteriocin, which is a low molecular protein consisting of 34 amino acid residues including lanthionine. Nisin in the present invention includes, for example, nisin A and nisin Z. Nisin can be obtained by culturing and purifying lactic acid bacteria by a known method. For example, after cultivating lactic acid bacteria in MR S medium (MRS medium, manufactured by Oxoid), the culture supernatant is treated with 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, and the eluted fraction was extracted with a cation exchange column (SP-Sepharose FF, GE Healthcare Bio). By offering it to Science Co., Ltd., a purified product of Nisin can be obtained. If necessary, the degree of purification may be further increased by subjecting it to reverse phase chromatography (see Biosci. Biotechnol. Biochem., 67 (7), p1616-1619, 2003). Moreover, you may purchase a commercial item, for example, Nisuprin (Nisaplin, a trademark, Danisco Co., Ltd. product). 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 may be increased by the above purification method.

The nisin activity in the composition is not limited as long as it shows bactericidal activity against microorganisms, but is, for example, 2 to 50,000 IU / mL, preferably 10 to 10,000 IU / mL, more preferably 50 to 1,000 IU / mL. . The activity of nisin can be calculated by comparing the peak areas of HPLC using nisin (1 μg = 40 IU, manufactured by ICN Biomedical Inc) as a standard according to the method described in JP-A-2002-369672. The HPLC conditions are shown below:
HPLC: Hitachi EZ Chrom Elite HPLC L-2000;
Column: Shodex Asahipak ODP-50 6E;
Guard column: Shodex Asahipak ODP-50G 6A;
Column temperature: 25 ° C;
Eluent: acetonitrile containing 0.05% TFA;
Flow rate: 1.0 mL / min.

  In the present specification, the bactericidal activity of nisin is a value calculated by the above MBC method or MBC modified method, unless otherwise indicated. The amount or activity of nisin can also be measured by a minimum inhibition circle concentration assay.

  You may mix | blend components other than nisin with the composition in this invention. 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: for example long chain imidazoline derivatives, long chain alkylbetaines, long chain alkylamidoalkylbetaines; cationic surfactants: for example D, L-2-pyrrolidone of N-cocoyl-L-ethyl alginate 5-carboxylate, cocamidopropyl PG dimonium chloride phosphate, 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 that are acceptable for medical or food hygiene are preferred, 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) and the like esters.

  A person skilled in the art can appropriately set the blending amount of the surfactant. For example, when the composition of the present invention is a liquid or semi-solid composition, 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 compounding amount of the chelating agent can be appropriately set 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%.

  Examples of the wetting agent include glycerin, sorbitol, propylene glycol, and polyethylene glycol. The amount of the wetting agent can be appropriately set 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 You may mix | blend with the composition of this invention 1 to 10weight%, More preferably, 2 to 5weight%.

  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. The blending amount of the abrasive can be appropriately set 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 70% by weight, You may mix | blend with the composition of this invention 10 to 50weight%, More preferably, 20 to 50weight%.

  Bactericides include cationic antimicrobial agents: quaternary ammonium compounds such as benzalkonium chloride, dodecyltrimethylammonium chloride, benzyldimethylstearylammonium chloride, cetyltrimethylammonium bromide, benzethonium chloride, methylbenzethonium chloride; antimicrobial agents : Chlorhexidine, alexidine, chlorhexidine digluconate, chlorhexidine acetate, cetylpyridinium chloride, hexetidine citrate, triclosan, phenyl salicylate, gramidicin, tyroslicin, cetylpyridinium chloride; antifungal agents: miconazole, triconazole; etc. Is given. The amount of the bactericide can be appropriately set 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 in 15 weight%, More preferably, 5 to 10 weight%.

  Examples of the viscosity modifier include thickeners: methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methylcellulose. The blending amount of the viscosity modifier can be appropriately set 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 to 2% by weight, more preferably 0.5 to 2% by weight, may be added to the composition of the present invention.

  The composition of the present invention can be used in any application where sterilization of microorganisms is required. For example, it can be used for sterilization of microorganisms existing in homes, commercial kitchens, cooking utensils, food processing factories, food material factories, houses, hotels, restaurants, indoors, furniture, and floors.

  As another example, it can be used for sterilization of medical facilities such as hospital rooms and medical devices. Specifically, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-resistant enterococci (VRE), penicillin-resistant pneumococci (PRSP), multidrug-resistant Pseudomonas aeruginosa (MDRP), carbapenem Can be used to sterilize antibiotic-resistant bacteria such as resistant Pseudomonas aeruginosa, carbapenem-resistant Serratia, third-generation cephalosporin-resistant Escherichia coli, third-generation cephalosporin-resistant Klebsiella pneumoniae, multidrug-resistant Acinetobacter, Clostridium difficile .

  The composition of the present invention can be used for skin sterilization. In addition to nisin, the composition used for the application includes, for example, a surfactant, an emulsifier, an oil component, a moisturizer, an anti-inflammatory agent, an astringent, a solvent, ethanol, an emollient, a stabilizer, a dispersant, an oxidation agent. An inhibitor, a chelating agent, a colorant, a fragrance, an ultraviolet absorber, a buffer, a preservative, and the like can be blended.

  When the composition of the present invention is used for the prevention / treatment of acne, it can be in various forms, for example, face wash, lotion, milky lotion, and serum. As one aspect thereof, a lotion for preventing / treating acne can be prepared as follows. Purified nisin (1,000 IU / mL), glycerin (3.0 wt%), and 1,3-butylene glycol (1.0 wt%) are dissolved in purified water. Further, ethanol (5.0% by weight) in which methyl paraoxybenzoate (0.05% by weight) is dissolved is added, and a desired pH, for example, pH 5. with hydrochloric acid (0.1N) or sodium hydroxide (0.1N). Adjust to 5. Instead of adjusting the pH with hydrochloric acid or sodium hydroxide, a buffering agent such as citric acid and sodium citrate may be added in advance to the purified water so that the lotion has the desired pH. In yet another embodiment, an acidic solution containing nisin and an alkaline solution containing other components may be prepared in advance, and a lotion having a target pH may be prepared by mixing both at the time of use.

  Furthermore, the composition of the present invention can be used for sterilization of oral microorganisms. In addition to nisin, the composition used for the application includes, for example, an antibacterial agent, an enzyme, an anti-inflammatory agent, a fluoride, an abrasive, a wetting agent, a foaming agent, a thickening agent, a flavoring agent (sweetening agent, fragrance, flavor), Coloring agents and preservatives can be blended. As one aspect thereof: a solution prepared by diluting purified nisin with sterilized purified water and adjusting nisin activity to 10,000 IU / mL, sorbitol or xylitol (5 wt%) and rosemary extract (0.1 wt%) And adjust to pH 3-5 with hydrochloric acid (0.1N) or sodium hydroxide (0.1N).

  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 10.5, preferably pH 4.0 to 7.0, and more preferably pH 5.0 to 6.5. In this pH range, when the composition of the present invention is contacted with microorganisms, both gram positive bacteria and gram negative bacteria can be sterilized.

  As another aspect of the present invention, the sterilizing composition of the present invention can be contacted with microorganisms in a pH range more suitable for sterilizing Gram positives. The pH range more suitable for sterilizing Gram-positive bacteria is, for example, pH 4.0 to 10.5, preferably pH 4.5 to 8.0, more preferably pH 5.0 to 7.5. When the composition of the present invention is brought into contact with the microorganism in such a pH range, the bactericidal activity against Gram-positive bacteria can be further increased.

  As another aspect of the present invention, the sterilizing composition of the present invention can be contacted with microorganisms in a pH range more suitable for sterilizing gram-negative bacteria. Suitable pH ranges for sterilizing Gram negative bacteria include, for example, pH 4.0-8.0, preferably pH 4.5-7.0, more preferably pH 5.0-6.5. When the composition of the present invention is brought into contact with a microorganism in such a pH range, the bactericidal activity against gram-negative bacteria can be further increased.

  In the present invention, the pH can be measured using, for example, a pH meter (Horiba F-52, electrode type 9611-10D, measurement temperature 25 ° C.). In addition, as the pH adjuster, for example, hydrochloric acid, an organic acid such as malic acid or citric acid as an acidic pH adjuster, sodium hydroxide, potassium hydroxide or the like as an alkaline pH adjuster is used. Can do. The density | concentration of a pH adjuster can be 0.001-1N, for example, Preferably it is 0.01-0.5N, More preferably, it can be 0.1-0.2N.

  Examples of the Gram-positive bacteria include Streptococcus microorganisms: Streptococcus mutans, Streptococcus sobrinas, Staphylococcus microorganisms: Staphylococcus aureus, and Propionibacterium microorganisms: Propionibacterium・ Acnes can be given. Among them, Streptococcus mutans and Streptococcus sobrinus are known as caries-causing bacteria, and Staphylococcus aureus is known as Staphylococcus aureus. Propionibacterium acnes is a fungus that exists in human and animal skin, and is known as a cause of skin diseases such as acne.

  Examples of the gram-negative bacterium include Escherichia genus: Escherichia coli. Escherichia coli is a bacterium that exists widely in various environments, and some of them are highly toxic.

  From the above, the composition of the present invention can be used for any object that requires sterilization of microorganisms. For example, parts of the body of animals including humans (oral cavity, teeth and periodontal, skin, nasal cavity, fingers, face, feet, etc.), appliances / machines (medical instruments, cooking utensils, sanitary instruments, care products, baby products, Toys, food manufacturing machines, medical equipment, dining tables, desks, chairs, home furnishings, etc.), places (household and commercial kitchens, food processing factories, food material manufacturing factories, houses, hotels, restaurants, etc., medical facilities , Hospital rooms, floors, worktables, etc.) can be used to microbiologically clean.

  The composition of the present invention can be used in a method for sterilizing microorganisms. For example, it can be used in a method for sterilizing microorganisms, which comprises contacting nisin and microorganisms at a certain rate for a predetermined time in a specific pH range. The specific pH range can be, for example, pH 4.0 to 10.5, preferably pH 4.0 to 7.0, and more preferably pH 5.0 to 6.5. The pH range for individual microorganisms is as described above. Nisin and microorganisms can be contacted at a ratio of, for example, 10 to 10,000 cfu of microorganism, preferably 10 to 5,000 cfu, more preferably 10 to 1,000 cfu per IU of nisin. The contact time can be, for example, 10 to 180 seconds, preferably 10 to 60 seconds, and more preferably 20 to 30 seconds.

  In another aspect, the composition of the present invention may also be used in the first stage: nisin and microorganisms are contacted at a certain ratio for a predetermined time in a pH range suitable for sterilization of Gram positive bacteria, and then the second stage: Gram negative bacteria. It can be used in a method for sterilizing microorganisms by contacting nisin and microorganisms for a predetermined time in a pH range suitable for sterilization.

  In the first step, the ratio of nisin to microorganism can be, for example, 10 to 10,000 cfu, preferably 10 to 5,000 cfu, more preferably 10 to 1,000 cfu of gram-positive bacteria per IU of nisin. . The pH range can be, for example, pH 4.0 to 10.5, preferably 4.5 to 8.0, more preferably 5.0 to 7.5. The contact time can be, for example, 10 to 180 seconds, preferably 10 to 60 seconds, and more preferably 20 to 30 seconds.

  In the second stage, the pH range can be, for example, pH 4.0 to 8.0, preferably 4.5 to 7.0, more preferably 5.0 to 6.5. The contact time can be, for example, 10 to 180 seconds, preferably 10 to 60 seconds, and more preferably 20 to 30 seconds.

  Thus, by combining the method according to the first stage suitable for sterilization of gram-positive bacteria and the method according to the second stage suitable for sterilization of gram-negative bacteria, the microorganisms can be sterilized more efficiently. . Even if the order of the first stage and the second stage is changed, the same effect can be obtained.

  The composition of the present invention can be adjusted in advance to a component concentration and pH range suitable for sterilization. Furthermore, when it is considered convenient from the viewpoint of handling such as transportation and storage or storage stability, the concentration of components and pH range suitable for sterilization may be adjusted during use. An example of the latter form is a kit. In the kit, each component may exist independently, or may exist in a mixed state, and can be in any form such as powder, solid, liquid, and the like. For example, it may contain one or more premixes, solvents, and the like that contain each component independently or in combination in a preservatively effective form. The premix means a composition for use by dissolving, diluting, and / or mixing with an appropriate solvent at the time of use, in which each component according to the application is preliminarily prepared in a specific amount or mixing ratio.

  Another aspect of the present invention provides a composition having a pH of 2.0 to 5.0, more preferably a pH of 3.0 to 4.0. Nisin is known to be stable and highly soluble at acidic pH. For this reason, it can be said that it is preferable from the viewpoint of storage stability of nisin that it is stored in this pH range when not in use and adjusted to a pH suitable for sterilization of microorganisms immediately before use. Therefore, the composition adjusted to pH 2.0 to 5.0 can be used as a premix of a composition for sterilizing microorganisms. As another embodiment, when a composition containing nisin at a high concentration is used as a premix and diluted at the time of use, a diluted solution adjusted to a pH suitable for sterilization can be obtained. Such a use mode is preferable in practical use from the viewpoint of ease of use.

  In the following examples, specific examples of the present invention will be described, but the present invention is not limited thereto.

[Example 1] Examination of bactericidal activity with nisin A (1-1) Preparation of test solution Prepared nisin aqueous solution (2,000 IU / mL) by dissolving purified nisin A (Ohm Milk Industry Co., Ltd.) with sterilized purified water. The pH of the aqueous solution was adjusted to pH 3.4 with 0.1N hydrochloric acid. The aqueous solution was diluted five times with sterilized purified water to prepare test solutions having nisin activity shown in the following table. In addition, the pH of the diluted solution was measured (Table 1).

(1-2) Bactericidal activity test The caries-causing bacteria Streptococcus mutans Xc were cultured in an SCD bouillon medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 37 ° C for 24 hours. The culture solution was diluted with sterilized purified water to adjust the number of bacteria (10 ⁶ to 10 ⁷ cfu / mL). The diluted culture solution (100 μL) was added to each of the test solutions 1 to 6 (5 mL). After contact at room temperature for 30 seconds, the solution (100 μL) was added to and mixed with SCD broth medium (5 mL), and the mixture (100 μL) was immediately plated on SCD agar medium (Nissui Pharmaceutical Co., Ltd.). And cultured at 37 ° C. The number of viable bacteria after 48 hours of culture was counted (FIG. 1), and the bactericidal rate (%) was calculated based on the following formula.

Bactericidal rate (%) = [1− (number of surviving bacteria in test solution / number of test bacteria)] × 100
(1-3) Results The sterilization rate of each test solution against Streptococcus mutans Xc was 99.6% in test solution 3 (nisin activity 500 IU / mL, pH 4.7) (FIG. 1). The sterilization rates of Test Solution 1 (Nisin Activity 2,000 IU / mL, pH 3.4) and Test Solution 2 (Nisin Activity 1,000 IU / mL, pH 3.8), which have higher nisin activity than Test Solution 3, are 20% and 43%. On the other hand, test solution 4 (250 IU / mL, ≧ pH 6.9), test solution 5 (125 IU / mL, ≧ pH 6.9) and test solution 6 (63 IU / mL, ≧ pH 6.9) having lower nisin activity than test solution 3. ) Showed little bactericidal activity.

From this result, it was shown that the bactericidal activity of nisin A is affected by pH. This suggests that the bactericidal activity of nisin can be regulated, eg enhanced, by pH.
[Example 2] Effect of pH on bactericidal activity of nisin (2-1) Preparation of test solution Purified nisin A (produced by Ohm Dairy Co., Ltd.) was dissolved in sterilized purified water and nisin A aqueous solution (activity 4,000 IU / mL). ) And the pH of the aqueous solution was adjusted to pH 3.5 with hydrochloric acid (0.1N). The aqueous solution is diluted with sterilized purified water to adjust the nisin activity (100, 50, 10, 2 IU / mL), and the pH of each diluted solution is adjusted with hydrochloric acid (0.1N) or sodium hydroxide (0.1N). (PH 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0), and this was used as a test solution.

(2-2) Bactericidal activity test The test was conducted according to the method shown in Example 1 (1-2) (Fig. 2).
(2-3) Results The pH dependence of nisin activity against Streptococcus mutans Xc was most prominent in a test solution containing 50 IU / mL of nisin A. The bactericidal activity showed the maximum at pH 5.0. Furthermore, when the nisin activity was 50 IU / mL or more, it was shown that sufficient bactericidal activity was exhibited in the range of pH 4.5 to 7.0.

[Example 3] Effect of pH on bactericidal activity of nisin A or nisin Z (3-1) Preparation of test solution Nisin A and nisin Z (manufactured by Shanghai Nicechem) according to Example 2 (2-1) above, (4000 IU / mL, pH 3.5) was prepared. The aqueous solution is diluted with sterile purified water to adjust nisin activity (50 IU / mL), and the pH of the diluted solution is adjusted with hydrochloric acid (0.1N) or sodium hydroxide (0.1N) (pH 3.5, 4). 0.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0), and this was used as a test solution.

(3-2) Bactericidal activity test A test was performed according to the method described in Example 1 (1-2).
(3-3) Results The results are shown in FIG. It was suggested that the bactericidal activity of nisin A against Streptococcus mutans Xc was maximized around pH 5.0. On the other hand, it was shown that the bactericidal activity of nisin Z was maximized around pH 5.5. These results indicate that both nisin A and nisin Z have high bactericidal activity against Streptococcus mutans Xc at an optimum pH.

[Example 4] Effect of pH on bactericidal activity of nisin against gram-positive microorganisms (4-1) Preparation of test solution Nisin A was dissolved in sterilized purified water to adjust the pH to 2.8 to 10.5. Nisin aqueous solution (Nisin activity 0, 500, 4,000 IU / mL) was prepared. The pH was adjusted with hydrochloric acid (0.1N) or sodium hydroxide (0.1N).

(4-2) Bactericidal activity test Staphylococcus aureus IFO13276 was cultured in SCD bouillon medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at 37 ° C for 24 hours. The culture solution was diluted with sterilized purified water to adjust the number of bacteria (10 ⁷ cfu / mL). The diluted culture solution (100 μL) was added to the test solution (10 mL). After contact at room temperature for 30 seconds or 60 minutes, the solution (100 μL) is added to and mixed with SCDLP medium (10 mL) containing polysorbate 80 as an inactivating agent to prepare a dilution series of the mixture. Was mixed with SCD agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and cultured at 37 ° C. The number of viable bacteria after 48 hours of culture was counted, and the bactericidal rate (%) was calculated based on the following formula.

Bactericidal rate (%) = [1− (number of surviving bacteria in test solution / number of test bacteria)] × 100
(4-3) Results Nisin A also showed bactericidal activity against Staphylococcus aureus (FIG. 4). The test solution showed a sufficient bactericidal effect with a contact time of at least 60 minutes. Furthermore, at a contact time of 60 minutes, the test solution with nisin activity of 500 IU / mL showed a high bactericidal activity at a pH of 5.5 or higher and the test solution with nisin activity of 4,000 IU / mL at pH 5.0 or higher. At pH lower than this pH, the bactericidal activity was significantly reduced. On the other hand, at a contact time of 30 seconds, the test solution having a nisin activity of 4,000 IU / mL showed the maximum bactericidal activity near pH 7.5, and antibacterial activity was observed at pH 6.0 to 10.5. Outside this pH range, the bactericidal activity was significantly reduced.

From the above results, it was revealed that the bactericidal activity of nisin can be improved by raising the pH from an acidic region where nisin is stable.
[Example 5] Effect of pH on bactericidal activity of nisin against Gram-positive microorganisms (5-1) Preparation of test solution The test solution was prepared according to the method described in Example 4 (4-1).

(5-2) Bactericidal activity test A test was performed according to the method shown in Example 4 (4-2) except that Escherichia coli NBRC3972 was used as a test bacterium.

(5-3) Results Nisin A also showed a bactericidal effect against Escherichia coli, which is a gram-negative bacterium (FIG. 5). When the pH was out of the range of 5.1 to 6.6, the bactericidal activity rapidly decreased. On the other hand, the bactericidal activity was hardly confirmed in the test solution in the acidic region where nisin was stable.

In view of the conventional knowledge that nisin does not act against gram-negative bacteria, this effect was completely unexpected.
[Example 6] Effect of nisin activity on bactericidal activity of nisin against gram-positive microorganisms (6-1) Preparation of test solution Nisin A dissolved in sterilized purified water and adjusted to pH unadjusted or pH 5.0 An aqueous solution (Nisin activity: 0, 10, 50, 100, 250, 500, 750, 1,000, 1,500, 2,000, 4,000, 5,000 IU / mL) was prepared and used as a test solution. did. The pH was adjusted with sodium hydroxide (0.1N).

(6-2) Bactericidal activity test A test was carried out according to the method shown in Example 4 (4-2) except that Staphylococcus aureus ATCC31885 was used as a test bacterium.

(6-3) Results Bactericidal activity was observed only when the contact pH of Staphylococcus aureus ATCC31885 and nisin was adjusted to 5.0 (FIG. 6). A sterilization rate of 90% or more was achieved at least under the conditions of nisin 2,000 IU / mL or more and a contact time of 60 minutes. On the other hand, even in the presence of nisin (2,000 IU / mL or more), the bactericidal activity was hardly confirmed in the test solution without pH adjustment (pH 2.8 to 3.1).

[Example 7] Effect of nisin activity on bactericidal activity of nisin against gram-negative microorganisms (7-1) Preparation of test solution Nisin A dissolved in sterilized purified water and adjusted to pH unadjusted or pH 5.0 An aqueous solution (Nisin activity: 0, 500, 2500, 5,000, 10,000, 20,000, 40,000 IU / mL) was prepared and used as a test solution. The pH was adjusted with sodium hydroxide (0.1N).

(7-2) Bactericidal activity test The test was conducted according to the method shown in Example 4 (4-2) except that Escherichia coli NBRC3972 was used as a test bacterium and the contact time was 30 seconds or 15 minutes. went.

(7-3) Results The bactericidal activity was recognized only when the pH at the time of contact between Escherichia coli NBRC 3972 and nisin was adjusted to 5.0 (FIG. 7). A sterilization rate of 90% or more was achieved at least under conditions of nisin activity of 10,000 IU / mL or more and a contact time of 30 seconds or more. On the other hand, even in the presence of nisin (10,000 IU / mL or more), the bactericidal activity was hardly confirmed in the test solution without pH adjustment (pH 2.6 to 2.8).

[Example 8] Examination of contact time between nisin and gram-positive microorganism (8-1) Preparation of test solution Nisin A was dissolved in sterilized purified water to adjust the pH unadjusted (pH 2.6 to 3.0) or pH 5. Nisin aqueous solution adjusted to 0 (nisin activity 0, 1,000, 4,000 IU / mL) was prepared and used as a test solution. The pH was adjusted with sodium hydroxide (0.1N).

(8-2) Bactericidal activity test As described above, Staphylococcus aureus ATCC31885 was used as a test bacterium, except that the contact time was 0.5, 1, 2, 5, 10, 15, 30, 60 minutes. The test was conducted according to the method shown in Example 4 (4-2).

(8-3) Results Bactericidal activity was observed only when the pH at the time of contact between Staphylococcus aureus and nisin was adjusted to 5.0 (FIG. 8). A sterilization rate of 90% or more was achieved at least under conditions of nisin activity of 4,000 IU / mL or more and a contact time of 30 minutes or more.

[Example 9] Examination of contact time between nisin and gram-negative microorganism (9-1) Preparation of test solution Nisin A was dissolved in sterilized purified water, and pH was not adjusted (pH 3.0 to 3.2) or pH 5. A nisin aqueous solution adjusted to 0 (nisin activity: 0, 5,000, 20,000 IU / mL) was prepared and used as a test solution. The pH was adjusted with sodium hydroxide (0.1N).

(9-2) Bactericidal activity test Example 4 (4) except that Escherichia coli NBRC 3972 was used as a test bacterium and the contact time was 0.5, 1, 5, 10, 15, 30, 60 minutes. The test was performed according to the method shown in -2).

(9-3) Results The bactericidal activity was recognized only when the contact pH between Escherichia coli and nisin was adjusted to 5.0 (FIG. 9). A sterilization rate of 90% or more was achieved at least under conditions of nisin activity of 5,000 IU / mL or more and a contact time of 0.5 minutes or more.

[Example 10] Nisin-containing mouthwash (10-1) Preparation of nisin-containing mouthwash Purified nisin (manufactured by Ohm Dairy Co., Ltd.) was dissolved in sterilized purified water to prepare an aqueous nisin solution (10,000 IU / mL). Sorbitol (5% by weight, manufactured by Kao Corporation) and rosemary extract (0.1% by weight, manufactured by Koei Kogyo Co., Ltd.) are blended in the nisin aqueous solution, and the pH is adjusted to 3.5 with hydrochloric acid (0.1N). A mouthwash stock solution was prepared.

  The stock solution was diluted with sterilized purified water to adjust nisin activity (500 IU / mL), and the pH was adjusted to 3.0 or 5.0 with hydrochloric acid (0.1 N) or sodium hydroxide (0.1 N). The composition adjusted to pH 3.0 was used as a control mouthwash, and the composition adjusted to pH 5.0 was used as the mouthwash of the present invention for the following test.

(10-2) Bactericidal activity test Except that three types of caries-causing bacteria: Streptococcus mutans Xc, Streptococcus mutans JCM5707 and Streptococcus sobrinus AHT-K were used as test bacteria, Example 1 (1- The test was conducted according to the method shown in 2) (FIG. 10).

(10-3) Results The bactericidal rates against Streptococcus mutans Xc, Streptococcus mutans JCM5707 and Streptococcus sobrinus AHT-K were 29%, 63% and 24%, respectively, when treated with pH 3.0 control mouthwash. Met. On the other hand, the treatment with the mouthwash of the present invention at pH 5.0 showed extremely excellent bactericidal effects of 100%, 99.8%, and 98.9%, respectively.

From this result, it was revealed that the composition of the present invention is suitable for a mouthwash for preventing caries.
[Example 11] Effect of pH and nisin activity on the reaction between nisin and target cells (11-1) Preparation of test bacterial solution and nisin solution A test bacterial solution was prepared by the following procedure. Propionibacterium acnes ATCC 6919 is pre-cultured in an enhanced Clostridia medium (manufactured by OXOID), the culture solution (10 μL) is inoculated into an enhanced Clostridia medium (10 mL), and anaerobic environment at 37 ° C. for 4 days. Swirl culture (160 rpm) was performed. The anaerobic environment was adjusted by using an anero pack (Mitsubishi Gas Chemical Co., Ltd.). This culture solution was used as a test bacterial solution for the following antibacterial activity test.

  Nisin solution was prepared by the following procedure. Nisin A (Ohm Dairies Co., Ltd.) is dissolved in McIlvine buffer adjusted to pH 3.0 to 8.0 so that the nisin activity at the time of cell contact becomes 1,000 or 500 (IU / mL). Prepared.

(11-2) Antibacterial activity test Nisin and the test bacterium were brought into contact at room temperature (25 ° C) by adding the test bacterium solution (50 µL) to the nisin solution (450 µL). The liquid was used as a test liquid. On the other hand, a solution obtained by adding a test bacterial solution (50 μL) to a solution not containing nisin (450 μL) was used as a control test solution. Three minutes later, the amount of ATP present outside the cells of the test solution and the control test solution was measured as a relative light emission amount (RLU) derived from the reaction between ATP and luciferin. The test solution and the control test solution were diluted 100 times with the buffer used for the measurement and used for the measurement. Using a reagent kit for ATP measurement (Lucifer 250, manufactured by Kikkoman Co., Ltd.) and Lumitester C-100 (manufactured by Kikkoman Co., Ltd.), the relative luminescence was measured according to the description of the instruction manual attached to the product. A value obtained by subtracting the relative luminescence amount of the control test solution from the relative luminescence amount of the test solution was defined as the amount of ATP flowing out of the microbial cells (FIG. 11).

(11-3) Results Nisin acts by targeting the microbial cell membrane (Lipid II), forms pores in the cell membrane, and acts bactericidally. The holes formed by nisin allow not only ions but also ATP to flow out. Using the amount of ATP efflux from target cells (extracellular ATP concentration) as an index of bactericidal activity by nisin, the effects of pH and nisin activity (nisin action concentration) on bactericidal activity of nisin were evaluated.

  The amount of ATP efflux from Acne bacteria (Propionibacterium acnes) is 5.0 to 5.0 compared to the acidic region (pH 3.0 to 4.0) where nisin is stable and the weak alkaline region (pH 8.0). It increased remarkably at 6.0 and showed a maximum value around pH 6.0. From this result, the reaction between nisin and target cells (cell membrane pore formation) is significantly affected by the pH at the time of nisin action, and it is effective to act on acne bacteria at pH 5.0-6.0. Became clear. Further, the ATP efflux amount increased depending on nisin activity (nisin action concentration), but the degree of increase became significantly higher at pH 5.0 to 6.0.

  From these results, it was revealed that the composition of the present invention is effective also for acne prevention and treatment because it effectively acts against acne bacteria that are acne-causing bacteria.

Claims (6)

  Composition for sterilization of at least one gram-negative or gram-positive microorganism, adjusted to pH 4.0-10.5, containing nisin at 2 to 50,000 IU / mL, or a kit for its preparation .   The composition or kit according to claim 1, wherein the nisin is nisin A or nisin Z.   Nisin is nisin A, and the pH is adjusted to 5.0 to 6.5, for sterilization of at least one gram-positive microorganism and for sterilization of at least one gram-negative microorganism. The composition or kit according to claim 2.   Gram-positive bacteria are Streptococcus mutans, Streptococcus sobrinas, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermis ), Or Propionibacterium acnes, and the Gram-negative bacterium is selected from Escherichia coli.   The composition or kit according to claim 1, further comprising a surfactant.   A method for improving the sterilizing power of nisin, wherein the pH is adjusted to 5.0 to 7.0.

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