阅读说明：本技术 抗菌性组合物 (Antibacterial composition ) 是由 宫崎泰成 影岛宏纪 齐藤章 于 2020-05-27 设计创作，主要内容包括：本发明提供一种针对不动杆菌的抗菌性组合物。即,提供含有使溶菌酶与壳聚糖结合而得到的复合物且对不动杆菌具有抗菌性的抗菌性组合物,以及含有使溶菌酶与壳聚糖结合而得到的复合物且用于治疗或预防不动杆菌感染症的医药组合物。(The invention provides an antibacterial composition against acinetobacter. Specifically, an antibacterial composition containing a complex obtained by binding lysozyme to chitosan and having antibacterial activity against Acinetobacter, and a pharmaceutical composition containing a complex obtained by binding lysozyme to chitosan and used for treating or preventing Acinetobacter infection are provided.)

1. An antibacterial composition contains a complex obtained by binding lysozyme to chitosan, and has antibacterial activity against Acinetobacter.

2. The antibacterial composition according to claim 1, wherein the acinetobacter is multidrug-resistant acinetobacter.

3. The antibacterial composition according to claim 1 or 2, wherein the concentration of the complex in 1mL of the antibacterial composition is 200 μ g/mL or more.

4. The antibacterial composition according to any one of claims 1 to 3, wherein the antibacterial property is accompanied by bactericidal property.

5. A cleaning water containing the antibacterial composition according to any one of claims 1 to 4.

6. A pharmaceutical composition comprising a complex obtained by binding lysozyme to chitosan, and used for treating or preventing Acinetobacter infection.

7. The pharmaceutical composition of claim 6, wherein the Acinetobacter infection is multi-drug resistant Acinetobacter infection.

8. Use of a complex obtained by binding lysozyme to chitosan for the manufacture of a pharmaceutical composition for the treatment or prevention of acinetobacter infection.

Technical Field

The present invention relates to an antibacterial composition having an antibacterial activity against Acinetobacter (Acinetobacter) and the like.

Background

Nontuberculous mycobacterium such as Pseudomonas aeruginosa or MRSA is an infectious bacterium causing respiratory tract infection. This infection is regarded as a problem due to its intractable nature. Recently, it has been clarified that a complex obtained by binding lysozyme to chitosan is effective against pseudomonas aeruginosa and MRSA (patent document 1). On the other hand, acinetobacter which is considered to be an important bacterium in severe pneumonia has some effective agents such as colistin and polymyxin B, but these are not approved in japan for the reason of side effects of nephrotoxicity, and even if general antibacterial agents are used, acinetobacter infection is difficult to treat (non-patent document 1). Furthermore, no effective drug has been found for drug-resistant acinetobacter (multi-drug resistant acinetobacter infection), and a new drug is expected to be invented.

Documents of the prior art

Patent document

Patent document 1 International publication No. 2017/138476 pamphlet

Non-patent document

Non-patent document 1 homepage of information center for infection of national institute of infection, multidrug-resistant Acinetobacter infection Q & A [ http:// idsc. nih. go. jp/disease/MDRA/QA01.html ]

Disclosure of Invention

The purpose of the present invention is to provide an antibacterial composition against Acinetobacter, particularly against multidrug-resistant Acinetobacter. The present invention also provides an antibacterial composition having an effective therapeutic and prophylactic effect against acinetobacter infection or multi-drug resistant acinetobacter infection, a therapeutic and prophylactic method for acinetobacter infection or multi-drug resistant acinetobacter infection, and a method of using the antibacterial composition in the production of an antibacterial agent for acinetobacter infection or multi-drug resistant acinetobacter infection.

The present inventors have made a review of the technical common knowledge that the general antibacterial agents are ineffective against acinetobacter as described above, and have verified the antibacterial activity against acinetobacter by using a complex of lysozyme and chitosan as a natural food additive having high safety, and have found that the antibacterial agent exhibits an excellent antibacterial effect against acinetobacter, thereby completing the present invention.

More specifically, the present invention may be in the following manner.

[1 ] an antibacterial composition comprising a complex obtained by binding lysozyme to chitosan and having antibacterial activity against Acinetobacter.

The antibacterial composition according to [1 ] above, wherein said Acinetobacter is multidrug-resistant Acinetobacter.

The antibacterial composition according to [1 ] or [ 2 ] above, wherein the concentration of the complex in 1mL of the antibacterial composition is 200. mu.g/mL or more.

The antibacterial composition according to any one of [1 ] to [ 3 ], wherein the antibacterial property is accompanied by bactericidal property.

[5 ] washing water containing the antibacterial composition according to any one of [1 ] to [ 4 ] above.

[ 6 ] A pharmaceutical composition comprising a complex obtained by binding lysozyme to chitosan, and used for the treatment or prevention of Acinetobacter infection.

The pharmaceutical composition according to [ 6 ] above, wherein the Acinetobacter infection is multi-drug resistant Acinetobacter infection.

Use of a complex obtained by binding lysozyme to chitosan for the manufacture of a pharmaceutical composition for the treatment or prevention of Acinetobacter infection.

According to the present invention, there can be obtained an effect of sterilizing, inhibiting bacteria or bacteria of acinetobacter and an effect of inhibiting the proliferation thereof. In particular, the complex obtained by binding lysozyme and chitosan according to the present invention not only inhibits the proliferation of acinetobacter, but also has a significant killing effect. Therefore, according to the present invention, acinetobacter infection, multi-drug resistant acinetobacter infection can be cured and/or prevented. In addition, lysozyme is widely used as a natural food additive with high safety, and an antibacterial composition using a complex of lysozyme and chitosan can reassure the patient who is using it and reduce the burden of the patient.

Drawings

FIG. 1 is a schematic diagram of a reaction in synthesizing a complex obtained by binding lysozyme to chitosan.

FIG. 2A is a graph showing the bactericidal effect of Pseudomonas aeruginosa (NBRC13275) in a standard medium by using a lysozyme-chitosan complex (LYZOX (registered trademark)) and a mixture of lysozyme and chitosan.

FIG. 2B is a graph showing the bactericidal effect of Pseudomonas aeruginosa (PAO1) in a standard medium using lysozyme-chitosan complex (LYZOX (registered trademark)) and a mixture of lysozyme and chitosan.

Fig. 2C is a graph showing the bactericidal effect of acinetobacter (JCM6841) in a standard medium using lysozyme-chitosan complex (LYZOX (registered trademark)) and a mixture of lysozyme and chitosan.

FIG. 2D is a graph showing the bactericidal effect of Pseudomonas aeruginosa (NBRC13275) against lysozyme-chitosan complex (LYZOX (registered trademark)) at various concentrations.

FIG. 3A is a graph showing the proliferation inhibitory effect of Pseudomonas aeruginosa (NBRC13275) in a standard medium by using a lysozyme-chitosan complex (LYZOX (registered trademark)), a lysozyme monomer, a chitosan monomer, and a mixture of lysozyme and chitosan.

FIG. 3B is a graph showing the proliferation inhibitory effect of Pseudomonas aeruginosa (PAO1) in a standard medium by using a lysozyme-chitosan complex (LYZOX (registered trademark)), a lysozyme monomer, a chitosan monomer, and a mixture of lysozyme and chitosan.

Fig. 3C is a graph showing the proliferation inhibitory effect of acinetobacter (JCM6841) in a standard medium using lysozyme-chitosan complex (LYZOX (registered trademark)), lysozyme monomer, chitosan monomer, and a mixture of lysozyme and chitosan.

FIG. 3D is a graph showing the growth inhibitory effect of Pseudomonas aeruginosa (NBRC13275) against lysozyme-chitosan complex (LYZOX (registered trademark)) at various concentrations.

Fig. 4A is a graph showing the results of measuring the integrity of the cell membrane of pseudomonas aeruginosa (NBRC13275) by absorbance when contacted with lysozyme-chitosan complex (LYZOX (registered trademark)) at various concentrations.

Fig. 4B is a graph showing the results of measuring the integrity of the cell membrane of acinetobacter (JCM6841) by absorbance when contacted with lysozyme-chitosan complex (LYZOX (registered trademark)) at each concentration.

Fig. 5A is a graph showing the results of measuring cell membrane damage of pseudomonas aeruginosa (NBRC13275) by the NPN test (outer membrane permeability test) when contacted with lysozyme-chitosan complex (LYZOX (registered trademark)) at various concentrations.

Fig. 5B is a graph showing the results of measuring damage of the cell membrane of acinetobacter (JCM6841) by the NPN test (outer membrane permeability test) when contacted with lysozyme-chitosan complex (LYZOX (registered trademark)) at each concentration.

Fig. 6A is a graph showing the results of measuring cell membrane damage of pseudomonas aeruginosa (NBRC13275) by the ONPG test (intracellular membrane permeability test) when contacted with lysozyme-chitosan complex (LYZOX (registered trademark)) at various concentrations.

Fig. 6B is a graph showing the results of measuring cell membrane damage of acinetobacter (JCM6841) by the ONPG test (intracellular membrane permeability test) when contacted with lysozyme-chitosan complex (LYZOX (registered trademark)) at various concentrations.

Fig. 7A is an image showing the results of measurement by visualizing the cell survival rate of pseudomonas aeruginosa (NBRC13275) by Confocal Laser Scanning Microscope (CLSM) when contacted with lysozyme-chitosan complex (LYZOX (registered trademark)) at various concentrations.

Fig. 7B is an image showing the results of measurement of cell survival rate of acinetobacter (JCM6841) by Confocal Laser Scanning Microscope (CLSM) when contacted with lysozyme-chitosan complex (LYZOX (registered trademark)) at each concentration.

Fig. 8A is an image showing morphological changes of pseudomonas aeruginosa (NBRC13275) obtained by scanning electron microscopy in the case where a lysozyme-chitosan complex (LYZOX (registered trademark)), a lysozyme monomer, a chitosan monomer, and a mixture of lysozyme and chitosan were added, respectively.

Fig. 8B is an image showing morphological changes of acinetobacter (JCM6841) obtained by scanning electron microscope in the case where lysozyme-chitosan complex (LYZOX (registered trademark)), lysozyme monomer, chitosan monomer, and a mixture of lysozyme and chitosan were added, respectively.

Fig. 9A is an image showing morphological changes of pseudomonas aeruginosa (NBRC13275) obtained by transmission electron microscopy in the case where a lysozyme-chitosan complex (LYZOX (registered trademark)), a lysozyme monomer, a chitosan monomer, and a mixture of lysozyme and chitosan were added, respectively.

Fig. 9B is an image showing morphological changes of acinetobacter (JCM6841) obtained by transmission electron microscopy in the case where a lysozyme-chitosan complex (LYZOX (registered trademark)), a lysozyme monomer, a chitosan monomer, and a mixture of lysozyme and chitosan were added, respectively.

FIG. 10A is a graph showing the effect of the resistance acquisition test of Pseudomonas aeruginosa (NBRC13275) in a standard medium using a lysozyme-chitosan complex (LYZOX (registered trademark)) and a mixture of lysozyme and chitosan.

FIG. 10B is a graph showing the effect of the resistance acquisition test of Pseudomonas aeruginosa (PAO1) in a standard medium using lysozyme-chitosan complex (LYZOX (registered trademark)) and a mixture of lysozyme and chitosan.

Fig. 10C is a graph showing the effect of the resistance acquisition test of acinetobacter (JCM6841) in a standard medium using lysozyme-chitosan complex (LYZOX (registered trademark)) and a mixture of lysozyme and chitosan.

Detailed Description

Hereinafter, one embodiment of the present invention will be described in detail.

< antimicrobial composition >

The present invention relates to an antibacterial composition containing a complex obtained by binding lysozyme to chitosan and having antibacterial activity against acinetobacter. The present invention will be specifically described below.

The "complex obtained by binding lysozyme to chitosan" refers to a complex in which lysozyme and chitosan are bound by, for example, a maillard reaction or the like (see fig. 1). By binding lysozyme to water-soluble chitosan through a Maillard reaction, most or all of the antigenic structures in lysozyme are masked, and thus have a characteristic of being less likely to cause allergy even if human beings ingest the lysozyme-chitosan complex. Alternatively, the complex may be obtained by covalently bonding lysozyme to chitosan using a crosslinking agent.

Here, the "lysozyme" is an enzyme that hydrolyzes mucopolysaccharides, and chicken-derived lysozyme and human-derived lysozyme can be preferably used.

The upper limit of the molecular weight of lysozyme is, for example, 30000Da or less, more preferably 25000Da or less, and may be 20000Da or less, 18000Da or less, or 15000Da or less. The lower limit of the molecular weight of lysozyme is not particularly limited, but is, for example, 1000Da or more, preferably 5000Da or more, and may be 10000Da or more and 12000Da or more. The range of the molecular weight of the lysozyme may be between any of the above upper and lower limits, for example, 1000Da to 30000Da, preferably 5000Da to 20000Da, and more preferably 10000Da to 15000 Da.

"Chitosan" is poly-. beta.1 → 4-aminoglucose ((C) represented by the following formula (I)₆H₁₁NO₄)_nCAS registry number 9012-76-4).

The chitosan is water soluble. The upper limit of the molecular weight of chitosan is, for example, 30000Da or less, more preferably 20000Da or less, and may be 15000Da or less, 10000Da or less, 7000Da or less. The lower limit of the molecular weight of chitosan is not particularly limited, but is, for example, 300Da or more, preferably 500Da or more, and may be 1000Da or more and 3000Da or more. The molecular weight of the chitosan may be in a range between any of the above upper and lower limits, for example, 300Da to 30000Da, preferably 500Da to 15000Da, more preferably 1000Da to 10000Da, and particularly preferably 3000Da to 7000 Da. When the antibacterial property is taken into consideration, chitosan having a large molecular weight is more advantageous, and when the ease of production is taken into consideration, chitosan having a small molecular weight is more excellent in solubility and stability, and chitosan having a low molecular weight is more advantageous.

The chitosan mentioned herein may include chitosan oligosaccharide and glucosamine in addition to the chitosan described above. Chitosan oligosaccharide is a substance formed by linking several D glucosamine represented by the above formula (I) and is a low molecular weight chitosan or a substance formed by hydrolyzing chitosan in a narrow sense with hydrochloric acid or an enzyme.

Examples of the crosslinking agent include an amine-reactive crosslinking agent (e.g., alkoxyamine), a carbonyl-reactive crosslinking agent (e.g., hydrazine compound), and a mercapto-reactive crosslinking agent.

The mass ratio of lysozyme to chitosan is, for example, 99/1 to 1/99, preferably 90/10 to 10/90, more preferably 80/20 to 20/80, still more preferably 60/40 to 40/60, and particularly preferably 50/50.

Specific examples of the method for producing a complex in which lysozyme is bound to chitosan include the following methods. First, the lysozyme and the chitosan in the above mass ratio are mixed and dissolved in water, and the aqueous solution is prepared so that the total mass of the lysozyme and the chitosan is 5 to 30 mass%. The resulting aqueous solution was lyophilized and powdered. The complex of lysozyme and chitosan of the present invention can be produced by subjecting the obtained powder to Maillard reaction for 2 to 20 days, more preferably 7 to 14 days, for example, at a temperature of 50 to 80 ℃, preferably 55 to 65 ℃, and a relative humidity of 50 to 80%, preferably 60 to 70%, for example.

Whether the lysozyme-chitosan complex of the present embodiment is formed or not can be confirmed by various known methods, and for example, the formation of a high molecular substance as a protein-chitosan complex can be confirmed by staining a plate obtained by SDS (sodium dodecyl sulfate) or SDS-PAGE (sodium dodecyl sulfate-polyacrylamide) polyacrylamide electrophoresis.

Acinetobacter (Acinetobacter) is a kind of aerobic gram-negative bacteria, a kind of eubacteria of gram-negative bacilli, and a kind of bacteria of the genus Acinetobacter. Acinetobacter baumannii (JCM6841) is known as a causative bacterium of infectious diseases, particularly respiratory infectious diseases, such as Acinetobacter calcoaceticus (Acinetobacter calcoaceticus), Acinetobacter iwoffii (Acinetobacter lwoffii) and Acinetobacter baumannii (Acinetobacter baumannii). The invention has the following characteristics: particularly, it is effective against Acinetobacter, and further multi-drug resistant Acinetobacter which does not acquire drug resistance.

The invention has antibacterial property to acinetobacter and multi-drug resistant acinetobacter. Here, the antibacterial property broadly includes bacteria elimination, sterilization, antibacterial property, and the like, and means all of the states in which bacteria are not at least proliferated when the number of bacteria is equal to or less than the same. The term "sterilization" means all the meanings of removing bacteria, including sterilization. Sterilization means the killing of at least a portion of the bacteria. Therefore, the antibacterial composition in the present invention is meant to include a bactericidal composition, a bactericide and an antibacterial agent.

The present invention is further effective for the treatment and prevention of acinetobacter infection and multi-drug resistant acinetobacter infection. Here, "treatment" includes not only complete cure of the inflammation to be the subject of the present invention, but also suppression of inflammation to reduce its severity. "prevention" includes, in addition to cases of medical history in which inflammation is not the subject of the present invention, prevention of recurrence of inflammation after the inflammation is cured.

The antibacterial composition of the present invention may optionally contain 1 or more kinds of other active ingredients in addition to the above-mentioned complex. The other active ingredient includes, in addition to the ingredient which itself functions as an active ingredient, an ingredient (auxiliary agent) which does not function as an active ingredient but exerts an effect by combining with the above-mentioned complex as an active ingredient of the present invention. Examples of the other active ingredient include terpene alcohols, fatty acids, and/or salts of the fatty acids. By adding these terpene alcohols, fatty acids, and/or salts of the fatty acids, additive and synergistic effects can be obtained by combination with the complex of the present invention. Examples of the terpene alcohol include terpinen-4-ol, hinokitiol, geraniol and menthol. The fatty acid includes, for example, a fatty acid having 8 to 12 carbon atoms, preferably capric acid (capric acid) or lauric acid. Examples of the salt of the fatty acid include sodium salt, potassium salt, magnesium salt, and calcium salt. In addition, essential oils derived from lemon grass, spearmint, geranium, perilla, etc., which are known to have an effect on inflammation caused by neutrophils, and glucosamine may be added as the other active ingredients.

The antibacterial composition of the present invention may optionally contain 1 or more of other components in addition to the above-mentioned complex. As other components, additives such as excipients, binders, emulsifiers, solvents, binders, disintegrants, thickeners, lubricants, colorants, flow agents, and humectants may be added.

Examples of the humectant include glycerin, butylene glycol, collagen, hyaluronic acid, and ceramide.

Examples of the binder include cellulose, methyl cellulose, hydroxyethyl cellulose, and sodium carboxymethyl cellulose.

Examples of the emulsifier include lecithin, Polyethylene Glycol (PG), PG-hydrogenated castor oil, glycerin fatty acid ester, sorbitan fatty acid ester, Ceteth, and the like.

Examples of the solvent include water, ethanol, 1-butanol, 2-butanol, 1-propanol, 2-propanol, and 1-pentanol.

The antibacterial composition of the present invention suitably contains, for example, 10. mu.g/mL to 200000. mu.g/mL, preferably 100. mu.g/mL to 100000. mu.g/mL, more preferably 50. mu.g/mL to 50000. mu.g/mL, further preferably 100. mu.g/mL to 10000. mu.g/mL, particularly preferably 200. mu.g/mL to 5000. mu.g/mL, of the lysozyme-chitosan complex of the present invention in 1 mL. The antibacterial composition of the present invention preferably contains other active ingredients by, for example, 0.001 to 5.0% by mass, preferably 0.002 to 0.2% by mass, more preferably 0.005 to 0.1% by mass, still more preferably 0.01 to 0.05% by mass, and particularly preferably 0.02 to 0.04% by mass. Further, the content of the other components may be appropriately adjusted depending on the respective components, and is, for example, 0.1 to 90% by mass, preferably 1 to 70% by mass, and more preferably 10 to 50% by mass. The antibacterial composition of the present invention may be a solvent such as water, and for example, the antibacterial composition of the present invention contains 95% by mass or more of the solvent, preferably 98% by mass or more, more preferably 99% by mass or more, and still more preferably 99.5% by mass or more. The antibacterial composition of the present invention preferably contains a fatty acid having 8 to 12 carbon atoms such as lauric acid and capric acid (capric acid) or a salt of such a fatty acid in an amount of 0.001 to 5% by mass, preferably 0.005 to 1% by mass, more preferably 0.01 to 0.5% by mass, and particularly preferably 0.04 to 0.2% by mass.

< method for treatment/prevention and pharmaceutical composition for use therein >

The present invention may comprise: a method for treating, inhibiting or preventing Acinetobacter infection, preferably multi-drug resistant Acinetobacter infection, by administering to a subject a pharmaceutical composition containing a complex obtained by binding lysozyme to chitosan, and a pharmaceutical composition for use therein. It is considered that the therapeutic, inhibitory or prophylactic effect is derived from the antibacterial property of the complex obtained by binding lysozyme to chitosan, and is particularly effective for respiratory diseases. The pharmaceutical composition includes the above-mentioned antibacterial composition, and is all the same as the above-mentioned antibacterial composition in terms of the content, and the like, and other active ingredients may be contained.

Here, the "subject" includes mammals such as cats, dogs, monkeys, cows, and horses, in addition to humans.

The definition of each of lysozyme, chitosan and the like, other effective ingredients, other ingredients, the amount to be administered, the mode of administration, the method of administration and the like are as described above.

[ dosage, administration method ]

The antibacterial composition and the pharmaceutical composition of the present invention vary depending on the dosage form, the subject to be administered, the route of administration, the target disease, the symptoms, and the like, and in the case of a spray system for spraying human skin, for example, the amount to be administered is, for example, 0.1 to 20mg/kg body weight, preferably 0.2 to 10mg/kg body weight, and more preferably 0.5 to 10mg/kg body weight per day, and the amount is preferably administered 1 to several times (for example, 2 times, 3 times, 4 times, or 8 times) per day. The dose can be applied to other preparations such as a cream described below, in addition to a spray.

The antibacterial composition and the pharmaceutical composition of the present invention may be administered orally or parenterally, and their formulation does not require any special technique and can be formulated by a general technique. Examples of administration forms include creams, ointments, pastes, liquids, sprays, gels, injections, tablets, suppositories, capsules, granules, powders, eye drops, eye ointments and the like, and creams, ointments, pastes, liquids and sprays are particularly preferable.

The antibacterial composition and the pharmaceutical composition of the present invention can be administered by a suitable method in combination with the above-described administration method. The administration method may be a known administration method, and in the case of a cream, for example, the pharmaceutical composition of the present invention may be applied to an inflammation site in the above-mentioned administration amount.

< use >)

In addition, the present invention may comprise: use of a complex obtained by binding lysozyme to chitosan for the manufacture of a pharmaceutical composition for the treatment or prevention of multi-drug resistant acinetobacter infection.

In the manufacture of a pharmaceutical composition for treating or preventing acinetobacter infection, preferably multidrug-resistant acinetobacter infection, a complex obtained by binding lysozyme to chitosan may be mixed with a component of the pharmaceutical composition other than the complex to obtain the pharmaceutical composition. As the mixing method, a known method can be used, and for example, in the case of a liquid agent, a complex obtained by binding lysozyme and chitosan and any of the other components described above are added to a solvent such as water, mixed, and added with an emulsifier as necessary to be mixed to prepare a dispersant or an emulsion, thereby preparing a liquid agent.

The antibacterial composition can be made into washing water for sprayer. Thus, the antibacterial composition can be directly sprayed onto the infected site to inhibit the growth of infectious bacteria.

The antibacterial composition and the pharmaceutical composition of the present embodiment have an antibacterial effect against acinetobacter, preferably multi-drug resistant acinetobacter, an inhibitory effect on growth of bacteria, and a therapeutic, inhibitory, and prophylactic effect against infectious diseases caused by the bacteria, and also have an effect of preventing acquisition of drug resistance.

The present invention will be described in further detail below with reference to examples, but the present invention is not limited to these examples. In the present specification and drawings, the complex obtained by binding lysozyme to chitosan is referred to as a lysozyme-chitosan complex or LYZOX (registered trademark of Wako Filter Technology co., ltd.).

Examples

[ preparation of sample ]

Lysozyme-chitosan complex (LYZOX (registered trademark)) solution

As a solution of a complex (lysozyme-chitosan complex) obtained by binding lysozyme to chitosan, commercially available LYZOX (registered trademark, Wako Filter Technology co., ltd.) was used. Commercially available LYZOX (registered trademark) is a powder comprising lysozyme from chicken (about 14000Da) and water-soluble chitosan (chitosan oligosaccharide) of about 5000Da in a mass ratio of 1: 1. Specifically, the lysozyme and the water-soluble chitosan were mixed and dissolved in water, and then lyophilized to form a powder, and further subjected to maillard reaction at a temperature, humidity and number of days sufficient for the maillard reaction to completely end, to obtain a lysozyme-chitosan complex (LYZOX (registered trademark)) used in the present invention. The lysozyme-chitosan complex (LYZOX (registered trademark)) was mixed with a prescribed solvent in such a manner that the lysozyme-chitosan complex became a desired concentration in each experimental example, to prepare a target lysozyme-chitosan complex (LYZOX (registered trademark)) solution.

Solutions of lysozyme monomers

As for the solution of the lysozyme monomer, lysozyme (Japan Biocon co., ltd., adelomik) was mixed with a predetermined solvent so that the concentration of the lysozyme was a desired concentration in each experimental example, to prepare a solution of the target lysozyme monomer.

Solutions of chitosan monomers

As for the solution of chitosan monomer, chitosan oligosaccharide (KimikA chitosan oligosaccharide COS-A manufactured by ltd.) was used as chitosan, and the chitosan oligosaccharide was mixed with A predetermined solvent so that the concentration of the chitosan oligosaccharide became A desired concentration in each experimental example to prepare A solution of the target chitosan monomer.

Solutions of mixtures of lysozyme and chitosan

The mixture of lysozyme and chitosan was prepared by mixing the lysozyme monomer and the chitosan monomer in a mass ratio of 1:1, and mixing a predetermined solvent so that the mixture, lysozyme and chitosan had a desired concentration.

Acinetobacter bacterial suspension

If not otherwise stated, Acinetobacter baumannii (JCM6841) was proliferated in Tryptic Soy Broth (TSB) at 37 ℃ until evening, centrifuged at the logarithmic proliferation phase, and subjected to colonization. The obtained bacteria were mixed with a predetermined amount of each solvent in each test to obtain Acinetobacter bacterial suspensions.

Pseudomonas aeruginosa bacterial suspension

Bacterial suspensions were prepared in the same manner as the above Acinetobacter baumannii bacterial suspensions, except that Pseudomonas aeruginosa (NBRC13275 or PAO1) was used in place of Acinetobacter baumannii (JCM6841), unless otherwise specified.

The details of each experimental example are as follows.

Measurement of absorbance

Unless otherwise specified, a spectrophotometer ASV11D manufactured by AS ONE co was used for the measurement of absorbance in this example. In the actual measurement of absorbance, the absorbance that can be measured by the spectrophotometer is considered, and the sample is diluted and measured as necessary, and the absorbance is calculated.

EXAMPLE 1 (Sterilization test)

In comparison with a mixture of lysozyme and chitosan, etc., the bactericidal activity of the lysozyme-chitosan complex (LYZOX (registered trademark)) against Acinetobacter, etc. was measured.

Specifically, as to the above lysozyme-chitosan complex (LYZOX (registered trademark)) solution (2000. mu.g/mL, using physiological saline as a solvent), and a solution of a mixture of lysozyme and chitosan (a solution of lysozyme monomer [ 1000. mu.g/mL ]]And chitosan monomerSolution [ 1000. mu.g/mL ]]And using physiological saline as a solvent), 30. mu.L of 1.0M phosphate buffer (pH7.2) was added to each 1.0mL of the solution, and the pH was adjusted to neutral pH (7.0 to 7.3) to prepare each solution. As a control, physiological saline (0.9% NaCl solution) was used. Acinetobacter bacterial suspensions or Pseudomonas aeruginosa bacterial suspensions were prepared as follows: proliferating Pseudomonas aeruginosa (NBRC13275 or PAO1) or Acinetobacter baumannii (JCM6841) in Tryptic Soy Broth (TSB) at 37 deg.C for one night, centrifuging at logarithmic proliferation stage, collecting bacteria, resuspending the bacteria with physiological saline (0.9% NaCl) to obtain final suspension concentration of 1.0 (10) at 600nm absorbance⁸～10⁹CFU/mL)。

To 10mL of each solution of the above lysozyme-chitosan complex or the like was added 0.1mL of each of the above Acinetobacter bacterium suspension or Pseudomonas aeruginosa bacterium suspension (1X 10)⁷-10⁸CFU), incubation was performed at 37 ℃ using a water bath. After 0 minute, 30 minutes, 60 minutes, and 120 minutes from the start of incubation, 0.1mL of each solution was added to a solid medium (Mueller-Hinton medium) at a time, and after proliferation, the number of bacteria was measured. Each bacterial suspension was tested 3 times against each solution and measured. The results are shown in FIGS. 2A to C.

As a result, in acinetobacter (fig. 2C), the number of bacteria was significantly reduced at 60 minutes and 120 minutes by the lysozyme-chitosan complex compared to the control.

EXAMPLE 2 (Sterilization test)

The bactericidal effect of pseudomonas aeruginosa (NBRC13275) was measured for each concentration of lysozyme-chitosan complex (LYZOX (registered trademark)). Specifically, as the lysozyme-chitosan complex (LYZOX (registered trademark)) solution, solutions (200. mu.g/mL, 2000. mu.g/mL, and 10000. mu.g/mL) having different concentrations were prepared using a physiological saline solution (0.9% NaCl solution) as a solvent, and the experiment was repeated in the same manner as in Experimental example 1 to measure the number of bacteria of Pseudomonas aeruginosa (NBRC 13275). The results are shown in FIG. 2D. As shown in fig. 2D, the lysozyme-chitosan complex showed concentration-dependent bactericidal activity.

EXAMPLE 3 (growth inhibition test)

In comparison with a lysozyme monomer, a chitosan monomer, and a mixture of lysozyme and chitosan, the proliferation inhibitory effect of a lysozyme-chitosan complex (LYZOX (registered trademark)) on acinetobacter and the like was determined.

Specifically, the above lysozyme-chitosan complex (LYZOX (registered trademark)) solution (2000. mu.g/mL), lysozyme monomer solution (1000. mu.g/mL), chitosan monomer solution (1000. mu.g/mL), and lysozyme and chitosan mixture solution (lysozyme monomer solution [ 1000. mu.g/mL ] (. mu.g/mL)) were prepared using a Trypsin Soybean Broth (TSB) solution (Japan Becton Dickinson Co., Ltd.) as a solvent]And chitosan monomer solution [ 1000. mu.g/mL]) Each solution was prepared by adding 10. mu.L of 1.0M phosphate buffer (pH7.2) to 1.0mL of each solution and adjusting the pH to a neutral pH (6.8 to 7.3). As a control, TSB medium without addition was used. Acinetobacter bacterial suspension or Pseudomonas aeruginosa bacterial suspension was prepared in the same manner as in Experimental example 1. To 10mL of each solution of the above lysozyme-chitosan complex or the like was added 50. mu.L (2.5X 10) of the above Acinetobacter bacterium suspension or Pseudomonas aeruginosa bacterium suspension further diluted 2000 times, respectively³～10⁴CFU), incubated at 37 ℃ for 6 hours. After 6 hours, 0.1mL of each solution was added to the solid medium (Mueller-Hinton medium) and the number of cells was measured after proliferation. Each bacterial suspension was tested against each solution by 18 separate experiments with pseudomonas aeruginosa (NBRC13275), pseudomonas aeruginosa (PAO1) and acinetobacter, in duplicate (mean of 3 determinations and SEM (standard deviation of sample mean)). The results are shown in FIGS. 3A to C. Note that, in order to make the mass concentration consistent with each constituent component (lysozyme, chitosan), the lysozyme monomer and chitosan monomer were compared with the lysozyme-chitosan complex at a concentration 2 times that of the lysozyme monomer and chitosan monomer to compare the antibacterial effects.

As a result, in acinetobacter (fig. 3C), the lysozyme-chitosan complex significantly decreased the number of bacteria compared to the control.

EXAMPLE 4 (growth inhibition test)

The proliferation inhibitory effect test of Pseudomonas aeruginosa (NBRC13275) was conducted for each concentration of lysozyme-chitosan complex (LYZOX (registered trademark)). Specifically, as the lysozyme-chitosan complex (LYZOX (registered trademark)) solution, solutions (2000. mu.g/mL, 5000. mu.g/mL, and 10000. mu.g/mL) having different concentrations were prepared using TSB as a solvent, and the number of Pseudomonas aeruginosa (NBRC13275) cells was measured by repeating the experiment in the same manner as in Experimental example 3. The results are shown in FIG. 3D. As shown in fig. 3D, the lysozyme-chitosan complex showed a concentration-dependent proliferation inhibitory effect.

EXAMPLE 5 (test for integrity of cell Membrane)

By passing at 260nm (A)_260nm) The absorbance of the cells was measured, and the integrity of the cell membrane was evaluated by estimating the amount of nucleic acid released from the cytoplasm. If the cell membrane of bacteria is damaged by the antibacterial agent, intracellular components are released outside the cells. Measurement of absorbance at 260nm can be used to predict the amount of DNA and RNA released from the cytoplasm.

Specifically, whether or not a substance having an absorption at 260nm is released from the bacteria was evaluated by measuring the absorption at 260nm using Thermo Fisher Science Co., Ltd., ND-1000 manufactured by Ltd. As the lysozyme-chitosan complex (LYZOX (registered trademark)) solution, solutions having different concentrations (4000. mu.g/mL, 10000. mu.g/mL, and 20000. mu.g/mL (final concentrations after mixing with the bacterial suspension are 2000. mu.g/mL, 5000. mu.g/mL, and 10000. mu.g/mL)) were prepared using a 0.5% NaCl solution as a solvent, and 60. mu.L of 1.0M phosphate buffer (pH7.2) was added to each 1.0mL of the solution to adjust the pH to a neutral pH (6.6 to 7.1) to prepare each solution. As a control, 0.5% NaCl solution was used. The Pseudomonas aeruginosa bacterial suspension and Acinetobacter bacterial suspension prepared in the same manner as in Experimental example 1 were resuspended in a 0.5% NaCl solution so that the final suspension concentration became an absorbance of 0.7 at 420 nm. To 10mL of each solution of the above lysozyme-chitosan complex or the like, the above Acinetobacter bacterium suspension or Pseudomonas aeruginosa bacterium suspension was added, and incubation was performed at 37 ℃ using a water bath. After 20 minutes and 40 minutes from the start of incubation,At 60 minutes, 80 minutes, 100 minutes and 120 minutes, bacteria were removed using a 0.22 μm syringe filter for each solution, and 260nm (A) was measured_260nm) Absorbance of (b) in (c). The absorbance was expressed as the mean value of 3 measurements and SEM (standard deviation of the mean value of samples), and the results are shown in FIGS. 4A and B.

As a result, the absorbance at 260nm showed a concentration-dependent increase (FIG. 4). In the case of treating each bacterial suspension with each concentration of the complex obtained by binding lysozyme to chitosan, the absorbance at 260nm sharply increased for the first 20 minutes, and then the rate of increase decreased up to 120 minutes. Therefore, the lysozyme-chitosan complex compared to the control showed: higher absorbance was shown in acinetobacter.

EXAMPLE 6(NPN test)

The extracellular membrane permeability was determined by the NPN test (1-N-phenylnaphthylamine (NPN) uptake assay). 1-N-phenylnaphthylamine can strongly emit fluorescence under phospholipid environment and can weakly emit fluorescence under aqueous environment. This property can be used to evaluate the permeability of the outer cell membrane of bacteria, i.e., membrane damage.

Specifically, the fluorescence intensities at an excitation wavelength of 350nm and an emission wavelength of 420nm were measured with a fluorescence spectrophotometer (manufactured by Shimadzu corporation, RF-6000), and evaluated. As the lysozyme-chitosan complex (LYZOX (registered trademark)) solution, solutions having different concentrations (20. mu.g/mL and 40. mu.g/mL (final concentrations after mixing with the bacterial suspension are 10. mu.g/mL and 20. mu.g/mL)) were prepared using a 0.5% NaCl solution as a solvent, and 60. mu.L of 1.0M phosphate buffer (pH7.2) was added to each 1.0mL of the solution, and the pH was adjusted to a neutral pH (7.2 to 7.3) to prepare each solution. As a control, 0.5% NaCl solution was used. To these solutions was added 30. mu.L of a 1.0mM NPN solution. The Pseudomonas aeruginosa bacterial suspension and Acinetobacter bacterial suspension prepared in the same manner as in Experimental example 1 were resuspended in a 0.5% NaCl solution so that the final suspension concentration became an absorbance of 1.0 at 420 nm. The above Acinetobacter bacteria suspension or Pseudomonas aeruginosa bacteria suspension was added to each solution obtained by adding an NPN solution to the above lysozyme-chitosan complex or the like, incubated in an indoor atmosphere, and the fluorescence intensity at an excitation wavelength of 350nm and an emission wavelength of 420nm was measured for each solution using the above fluorescence spectrophotometer for 10 minutes. The measurement was performed 3 times, and the fluorescence intensity of the solution containing no bacteria was subtracted from the fluorescence intensity of the solution containing bacteria to obtain the result as a Relative Fluorescence Unit (RFU). The results are shown in FIGS. 5A and B.

In various bacteria, the fluorescence intensity increased after 1 minute, and then stayed at the same level. The lysozyme-chitosan complex showed a concentration-dependent proliferation inhibitory effect. The lysozyme-chitosan complex showed an increase in the outer membrane permeability within 1 minute from contact with acinetobacter. From this result, it was found that the lysozyme-chitosan complex increases the permeability of the outer cell membrane within 1 minute from the contact with Acinetobacter.

EXAMPLE 7(ONPG test)

Intracellular membrane permeability was determined by the ONPG assay (o-nitrophenyl- β -D-galactopyranoside (ONPG) assay). If the cell membrane is damaged, intracellular enzymes containing β -galactosidase leak out of the cell. ONPG is generally colorless, but is hydrolyzed by β -galactosidase to galactose and o-nitrophenol, with an increase in absorbance at 420 nm. Intracellular membrane permeability of bacteria can be determined by measuring the activity of cytoplasmic β -galactosidase released from the bacteria using ONPG as a substrate.

Specifically, whether or not a substance having an absorption at 420nm was released was evaluated by measuring the absorption at 420nm using a spectrophotometer ASV 11D. As the lysozyme-chitosan complex (LYZOX (registered trademark)) solution, solutions having different concentrations (400. mu.g/mL, 4000. mu.g/mL and 10000. mu.g/mL (final concentrations after mixing with the bacterial suspension are 200. mu.g/mL, 2000. mu.g/mL and 5000. mu.g/mL)) were prepared using a 0.5% NaCl solution as a solvent, and 60. mu.L of 1.0M phosphate buffer (pH7.2) was added to each 1.0mL of the solution to adjust the pH to neutral (6.8 to 7.3) to prepare each solution. As a control, 0.5% NaCl solution was used. The Pseudomonas aeruginosa bacterial suspension and Acinetobacter bacterial suspension prepared in the same manner as in Experimental example 1 were resuspended in a 0.5% NaCl solution toThe final suspension concentration was prepared so that the absorbance at 420nm became 1.2. 1.6mL of each solution of the lysozyme-chitosan complex and the like, 150. mu.L of a 30mM ONPG solution, and 1.6mL of the Acinetobacter bacteria suspension or Pseudomonas aeruginosa bacteria suspension were mixed, incubated in a room atmosphere, and the absorbance was measured every 10 minutes until 100 minutes from the start. 420nm (A)_420nm) The increase in absorbance under (b) was evaluated by 3 measurements. The results are shown in FIGS. 6A and B.

For Acinetobacter, the absorbance at 420nm with lysozyme-chitosan complexes (2000. mu.g/mL and 5000. mu.g/mL) increased sharply initially, followed by a slow increase until 100 minutes. In Acinetobacter treated with lysozyme-chitosan complex (200. mu.g/mL), the absorbance at 420nm increased for the first 10 minutes, after which it was constant. In various bacteria, the absorbance at 420nm increased concentration-dependently. Intracellular enzymes leak out of the cell concentration-dependently within 10 minutes after contact. It is known that lysozyme-chitosan complex destroys the inner membrane of acinetobacter.

EXAMPLE 8 (confocal laser scanning microscope observation)

In order to visualize damage to the cell membrane of the bacteria treated with the lysozyme-chitosan complex, observation was performed with a Confocal Laser Scanning Microscope (CLSM) (scale bar 25 μm in fig. 7).

Specifically, a solution of the lysozyme-chitosan complex (LYZOX (registered trademark)) at 2000. mu.g/mL (final concentration of 1000. mu.g/mL after mixing with the bacterial suspension) was prepared using physiological saline (0.9% NaCl solution) as a solvent, and 60. mu.L of 1.0M phosphate buffer (pH7.2) was added to each 1.0mL of the solution to adjust the pH to neutral (pH 7.0 to 7.1) to prepare a lysozyme-chitosan complex solution. As a control, physiological saline (0.9% NaCl solution) was used. To 500. mu.L of these solutions, 500. mu.L of the Pseudomonas aeruginosa bacterial suspension and Acinetobacter bacterial suspension prepared in the same manner as in Experimental example 1 above were added, and incubated at 37 ℃ for 2 hours. Thereafter, 3. mu.L of LIVE/DEAD Baclight reagent (a mixture of SYTO 9 staining nucleic acids and Propidium Iodide (PI)) was added and further incubated at room temperature for 15 minutes. SYTO 9 with green fluorescence will label all bacteria, but PI with red fluorescence will only penetrate into bacteria with damaged cytoplasmic membranes, and SYTO 9 fluorescence will be reduced if both pigments are present. The excitation wavelength/emission wavelength was observed at 488/495-515 nm in SYTO 9 and 488/635-700 nm in PI (FIGS. 7A and B).

In the control, almost all bacteria were non-wounded (green fluorescence), and a few bacteria had a wounded membrane (red fluorescence). In each sample treated with the lysozyme-chitosan complex, the bacterial cells having the damaged membrane were more numerous (state in which red fluorescence was predominant) than the corresponding control. In Pseudomonas aeruginosa treated with lysozyme-chitosan complex, intact bacterial cells and bacterial cells with damaged membranes agglutinate to form clusters. In contrast, in acinetobacter treated with lysozyme-chitosan complex, bacterial cells agglutinated less compared to pseudomonas aeruginosa. The observed results of CLSM are consistent with the results of the membrane damage test thus far.

EXAMPLE 9 (Observation with scanning Electron microscope)

Morphological changes of pseudomonas aeruginosa and acinetobacter caused by the lysozyme-chitosan complex were observed by Scanning Electron Microscopy (SEM) (photographs were taken at 2 ten thousand times magnification, and the scale bar in fig. 8 was 1.5 μm).

Specifically, the above lysozyme-chitosan complex (LYZOX (registered trademark)) solution (1000. mu.g/mL), lysozyme monomer solution (500. mu.g/mL), chitosan monomer solution (500. mu.g/mL), and a mixture solution of lysozyme and chitosan (lysozyme monomer solution [ 500. mu.g/mL ] and chitosan monomer solution [ 500. mu.g/mL ]) were prepared using physiological saline (0.9% NaCl solution) as a solvent. As a control, physiological saline (0.9% NaCl solution) was used. The supernatants of the Pseudomonas aeruginosa bacterial suspension and Acinetobacter bacterial suspension prepared in the same manner as in Experimental example 1 were all removed except that the absorbance was adjusted to 2.0, and 1mL of each solution of the lysozyme-chitosan complex and the like was added to the resultant mixture, followed by incubation at 37 ℃ for 2 hours. Thereafter, centrifugation was carried out, and the resulting pellet was washed with physiological saline (0.9% NaCl) and fixed with a fixing solution (2.5% glutaraldehyde buffered in 0.1M phosphate buffer) at 4 ℃ for 2 hours. Afterwards, it was incubated in 0.1M phosphate buffer-o-evening.

Thereafter, each bacterium was immobilized in 1.0% buffered osmium tetroxide in 0.1M phosphate buffer for 1 hour, followed by dehydration with ethanol, and then drying using a critical point drying apparatus (HCP-2). After coating with about 20nm of platinum/palladium, the sample was observed with a scanning electron microscope (S-4500) (FIGS. 8A and B).

Observation of SEM of untreated pseudomonas aeruginosa (NBRC13275) (fig. 8A) and acinetobacter baumannii (fig. 8B) showed smooth surfaces (fig. 8A (a) and fig. 8B (f)). Pseudomonas aeruginosa and Acinetobacter partially formed spherical cells, i.e., spheroplasts, after treatment with lysozyme monomers (FIG. 8A (c) and FIG. 8B (h)). In both P.aeruginosa and A.acinetobacter, the samples treated with lysozyme-chitosan complex (FIG. 8A (B) and FIG. 8B (g)) were able to observe a large number of vesicles as compared with the samples treated with chitosan monomers and mixtures ((d) and (e) of FIG. 8A, and (i) and (j) of FIG. 8B.) the P.aeruginosa and A.acinetobacter treated with lysozyme-chitosan complex and mixtures lost their integrity of surface structure and were partially spherical (FIG. 8A (B) and (e), and FIG. 8B (g) and (j)). after treatment with each solution, in particular with lysozyme-chitosan complex, extracellular filamentous structures appear around the cells (fig. 8A (B) to (e) and fig. 8B (g) to (j)).

EXAMPLE 10 (Transmission electron microscope observation)

Morphological changes of pseudomonas aeruginosa and acinetobacter caused by the lysozyme-chitosan complex were observed by a Transmission Electron Microscope (TEM) (photographs were taken at 50000 times magnification, and the scale bar in fig. 9 was 0.2 μm).

Specifically, the above lysozyme-chitosan complex (LYZOX (registered trademark)) solution (1000. mu.g/mL), lysozyme monomer solution (500. mu.g/mL), chitosan monomer solution (500. mu.g/mL), and a mixture solution of lysozyme and chitosan (lysozyme monomer solution [ 500. mu.g/mL ] and chitosan monomer solution [ 500. mu.g/mL ]) were prepared using physiological saline (0.9% NaCl solution) as a solvent. As a control, physiological saline (0.9% NaCl solution) was used.

Among these solutions, 1mL of each solution of the lysozyme-chitosan complex and the like was added and incubated at 37 ℃ for 2 hours, to which 1mL of each solution was prepared, by diluting the Pseudomonas aeruginosa bacterial suspension and the Acinetobacter bacteria suspension prepared in the same manner as in Experimental example 1 with physiological saline (0.9% NaCl), preparing the suspension so that the final suspension concentration became absorbance at 600nm of 4.0, and removing the whole supernatant. After that, the resulting pellet was centrifuged, washed with physiological saline (0.9% NaCl), fixed in 0.1M phosphate buffer solution in 1.0% buffered osmium tetroxide buffer solution for 1 hour, embedded in 2.0% agar, dehydrated with ethanol, and then embedded in Epon 812(TAAB Laboratories Equipment Ltd.). Ultrathin sections at 70nm were collected on a copper mesh, double stained with uranyl acetate and lead citrate, and observed using a transmission electron microscope (H-7100) at 75 kV.

In TEM observation of untreated pseudomonas aeruginosa and acinetobacter, normal surface structure of the bacteria was shown, and smooth and lamellar structures were seen (fig. 9A (a), fig. 9B (f)). If treated with lysozyme monomers, both pseudomonas aeruginosa and acinetobacter will form spheroplasts (fig. 9A (c), fig. 9B (h)), and if treated with chitosan monomers, these bacteria will form a large number of vesicles on their surfaces (fig. 9A (d), fig. 9B (i)). Vesicles were produced from the outer membrane, which were consistent with the vesicles observed with SEM of each bacterium. In TEM observations of pseudomonas aeruginosa and acinetobacter baumannii after treatment with lysozyme-chitosan complex or mixture, globular cells with outer membrane vesicles were shown, defining the morphological changes resulting from both lysozyme monomer and chitosan monomer treatments (fig. 9A (B) and (e), fig. 9B (g) and (j)). Further, the cell wall of the bacterium is broken, and the intracellular contents are released from the cell.

EXAMPLE 11 (acquisition test of drug resistance)

To evaluate the acquisition of drug resistance, pseudomonas aeruginosa (NBRC13275 and PAO1) and acinetobacter baumannii were repeatedly subcultured in LB medium containing lysozyme-chitosan complex, and the change in sensitivity to lysozyme-chitosan complex was evaluated.

Specifically, for the MIC of lysozyme-chitosan complex (minimum development preventing concentration: minimum amount of agent necessary for preventing proliferation of bacteria), Pseudomonas aeruginosa (NBRC13275 and PAO1) or Acinetobacter baumannii was adjusted in such a manner that the absorbance at 600nm was 1.0 using physiological saline (NaCl 0.9%) as a solvent, and 60. mu.L of the adjusted bacterial suspension was added to 140. mu.L of LB medium in 96-well microwell plates, finally prepared so that the concentration of bacteria in 1 well was 10⁴Incubation was performed at 35 ℃. Evaluation was performed after 18 hours and 24 hours, and the minimum concentration of the agent at which turbidity could not be visually observed was defined as MIC. Bacteria were subcultured repeatedly (10 times) at a concentration of lysozyme-chitosan complex which was half the MIC, i.e., 1/2MIC, and the respective MICs were evaluated (LYZOX, passage in fig. 10A and B). For comparison, the same test as that for the lysozyme-chitosan complex was performed using the above mixture of lysozyme and chitosan (control in fig. 10A and B) (passage in fig. 10A and B). Each bacterium without subculture was evaluated as a control (CTL in fig. 10A and B). The MIC assay was repeated 10 times to evaluate the expression of drug resistance in these bacteria.

The resulting resistance test is shown in fig. 10. After 10 subcultures, pseudomonas aeruginosa and acinetobacter did not acquire resistance to lysozyme-chitosan complexes and mixtures thereof.

EXAMPLE 12 (hemolytic toxicity test)

To confirm the safety of the lysozyme-chitosan complex, a hemolytic toxicity test was performed.

Specifically, 400 μ L of rabbit defibrinated blood (obtained from Cosmo Bio co., ltd.) was diluted using Phosphate Buffered Saline (PBS) as a solvent to prepare lysozyme-chitosan complex (LYZOX (registered trademark)) solutions of 20 μmul mL, 200 μ g/mL, 2000 μ g/mL, and 20000 μ g/mL (concentrations in the hemolytic toxicity evaluation test were 10 μ g/mL, 100 μ g/mL, 1000 μ g/mL, and 10000 μ g/mL, respectively, as shown in table 1 (table 1)). Further, 500. mu.L of defibrinated blood of the above rabbits diluted with the above PBS was prepared, and 500. mu.L of each lysozyme-chitosan complex (LYZOX (registered trademark)) solution, PBS (negative control), and 0.1% Triton-X100 solution (positive control) were added. Each of the resulting mixtures was incubated at 37 ℃ for 1 hour, centrifuged at 1500rpm for 10 minutes, and the supernatant was collected. 750 μ L of each supernatant was added to 750 μ L of the above PBS, and 750 μ L of a control (negative control or positive control) was added to 750 μ L of each lysozyme-chitosan complex at the corresponding concentration. In order to calculate the hemolysis rate by using the following formula, each absorbance (OD) was measured at 545 nm. The absorbance at 545nm is expressed as the mean and SEM (standard deviation of the mean of the samples) of 3 measurements.

Hemolysis Rate (HR) (%) (absorbance of sample [ AS ] -negative control absorbance [ AN ])/(absorbance of positive control [ AP ] - [ AN ]) × 100 corresponding to lysozyme-chitosan complex concentration.

TABLE 1

TABLE 1 hemolytic toxicity test

As shown in Table 1, rabbit erythrocytes were hardly hemolyzed after incubation of lysozyme-chitosan complex at 37 ℃ for 1 hour. The hemolysis rate of the lysozyme-chitosan complex is less than 5% in a concentration ranging from 10 μ g/mL to 10000 μ sec mL. It should be noted that a hemolysis rate of 5% or less is reported to be acceptable and safe in a living material for clinical use [ Dcng JD, He B, He DH, Chen zf. a potential biological: chemical composition, antibacterial and lipophilic activities of leave kind of infection oil from Alpinia guinanensis Ind Crop prod.2016; 94:281-7.].

Industrial applicability

According to the present invention, a novel antibacterial composition, a pharmaceutical composition, and the like can be provided to acinetobacter.