KR20140123131A - Antibiotic composition containing low molecular water soluble chitosan or derivative thereof - Google Patents

Abstract

The present invention relates to an antibacterial composition containing a low molecular water soluble β-chitosan or a derivative thereof as an active component. The antibacterial composition according to the present invention can be absorbed by a body rapidly by containing a low molecular water soluble β-chitosan of 500~20000 daltons derived from a natural substance, and does not perform antibody formation, and is safe to a human body with extremely low cytotoxicity. Also, because the antibacterial composition of the present invention has excellent antibacterial effect as to various pathogenic bacteria, the antibacterial composition can be used in various fields requiring antibacterial effect such as food additives, a bactericide, a disinfectant, detergent, deodorant and health functional food.

Description

[0001] The present invention relates to an antibiotic composition containing a low-molecular water-soluble? -Chitosan or a derivative thereof as an active ingredient,

The present invention relates to an antimicrobial composition containing a low-molecular water-soluble? -Chitosan or a derivative thereof as an active ingredient. Specifically, the low-molecular water-soluble? -Chitosan or derivative thereof of 500 Daltons to 20000 Daltons exhibits an excellent antibacterial activity against bacteria, Since it is in a low molecular form, it is advantageous to be absorbed advantageously, not only does it not form an antibody in the body, but also is safe from cytotoxicity, and thus can be effectively used as a composition for antimicrobial use which is safe for human body.

The term " antibacterial agent " refers generally to an antimicrobial agent, and particularly refers to a substance that has an antibacterial effect against bacteria, in particular, a substance that exhibits excellent antibacterial activity by inhibiting a system of bacteria synthesizing cell walls and proteins, it means. Antimicrobial agents are mainly extracted from fungi and are widely used today to treat diseases caused by bacterial infection. The most typical antimicrobial agent is penicillin manufactured by the British physician Alexander Fleming in 1928. Penicillin was the first antimicrobial agent produced by mankind to respond to bacteria in earnest. A representative antimicrobial agent developed after penicillin is methicillin, which is considered to be more effective than penicillin. Methicillin was made by partially modifying the chemical structure of penicillin.

Antimicrobial Resistant Bacteria refers to bacteria that are resistant to specific antimicrobial agents and do not respond to the antibiotics. For example, penicillin-resistant Staphylococcus aureus, which has no effect on penicillin as described above, is applicable. In addition, Methicillin-Resistant Staphylococcus aureus (MRSA) has been reported to academia for the first time in 1961 and has since become a major pathogenic bacterium worldwide.

In addition to the increased infection caused by pathogenic microorganisms resistant to the existing chemical-derived antimicrobial agents, recently, antimicrobial agents derived from existing chemical substances have been problematic in terms of side effects and resistance, and the demand for development of antimicrobial substances derived from natural materials has increased As a result, attempts have been actively made to develop various medicines or antimicrobial agents using natural products.

On the other hand, chitin exists in the upper part of insects such as crabs, shrimp, crickets of crickets, beetle of chrysanthemum, grasshoppers, squid bones, cell walls of bacteria and the like, and is a biopolymer compound rich in cellulose. Chitin and chitosan are classified as α-chitin, β-chitin and γ-chitin depending on the crystal structure. Α-chitin is commonly found in shells of crustaceans such as crabs, shrimp and crayfish, Chitin is found in squid cartilage, and its abundance is very small compared to that of α-chitin, while maintaining a parallel state between adjacent molecular chains (Non-Patent Document 1 and Non-Patent Document 2). The chitin of the structure is found in squid cartilage and the like, and its abundance is very small compared to the alpha structure, and the adjacent molecular chains are kept parallel to each other (Non-Patent Document 1 and Non-Patent Document 2). The chitin of the y-structure is mainly present in the carapace of insects, and its structure is not fully characterized, but consists of a structure in which one crossover and two parallelism exist (Non-Patent Document 3).

It is known that chitosan prepared by deacetylation of chitin has characteristics such as large molecular weight, positive charge existing in chitosan molecule, film forming ability, biocompatibility, biodegradability, heavy metal adsorption, gelation property, Research on the application of chitin and chitosan in various fields such as cosmetics, food industry and the like is actively proceeding (Non-Patent Documents 4 to 7).

chitin and β-chitosan are relatively small in quantity because chitin and chitosan produced in nature are mostly α-chitin and α-chitosan. Chitin and β-chitosan have a weak molecular interactions with α-chitin, so that β-chitin and deacetylated β-chitosan have a loose bond structure compared to α-chitin. Chitin. ≪ / RTI > Chitin and chitosan have been mainly studied due to low abundance and structural instability from the natural world, but it has been known that β-chitin and chitosan have superior physiological activities than α-chitin and chitosan. Research is also actively underway.

First, studies have been made on the properties of extracts of β-chitin and β-chitosan and their deacetylation (Non-Patent Document 8). In addition, studies on the physical properties such as reducing power, antioxidant ability and entrapping ability of the low molecular weight? -Chitosan extracted from squid, stability against the thermal stress of? -Chitosan derived from white squid and Taiwan squid, (Viscosity-average molecular weight), and optical properties, etc. (Non-Patent Document 9 and Non-Patent Document 10).

Accordingly, the inventors of the present invention have confirmed that antimicrobial activity against pathogenic bacteria is exhibited in the low-molecular water-soluble β-chitosan of 500 daltons to 20000 daltons, and that they are free from toxicity.

R. Minke, J. Blackwell., Journal of Molecular Biology (1978) 120, 167-181; K. H. Gardner, J. Blackwell., Biopolymers (1975) 14, 1581-1595; K. M. Rudall., Advances in Insect Physiology (1963) 1, 257-313; L. Illum., Pharmaceutical Research (1998) 15, 1326; Y. C. Chung, H. L. Wang, Y. M. Chen, S. L. Li., Bioresource Technology, (2003) 88, 179-184; F. Devlieghere, A. Vermeulen, J. Debevere., Food Microbiology, (2004) 21, 703-714; E. S. Abdou, K. S. A. Nagy, M. Z. Elsabee, Bioresource Technology (2008) 99, 1359-1367; J. Huang, D. Zhao, S. Hu, J. Mao, L. Mei., Carbohydrate Polymers (2012) 87, 2231-2236; A. Chandumpai, N. Singhpibulporn, D. Faroongsarng, P. Sornprasit., Carbohydrate Polymers (2004) 58, 467-474.

An object of the present invention is to provide an antimicrobial composition comprising a low-molecular water-soluble β-chitosan or a derivative thereof as an active ingredient.

Another object of the present invention is to provide a health functional food composition for preventing or ameliorating an infectious disease caused by a bacterium comprising an antimicrobial composition containing a low-molecular water-soluble? -Chitosan or a derivative thereof as an active ingredient.

In order to achieve the above object,

The present invention provides an antimicrobial composition comprising a low-molecular water-soluble? -Chitosan or a derivative thereof as an active ingredient.

The present invention also provides a health functional food composition for preventing or ameliorating an infectious disease caused by a bacterium, comprising an antimicrobial composition containing a low-molecular water-soluble β-chitosan or a derivative thereof as an active ingredient.

The antimicrobial composition according to the present invention contains a low molecular weight water-soluble β-chitosan or derivative thereof of 500 to 20,000 daltons derived from a natural substance as an active ingredient and has a rapid absorption into the body, no antibody formation, safe. In addition, since it has an excellent antibacterial effect against various pathogenic bacteria, it can be usefully used as an antibacterial composition used in various fields requiring antibacterial effects such as food additives, bactericides, disinfectants, detergents, deodorants, health functional foods and the like.

Figure 1 is a graph showing the processing time for bacterial kill kinetics Example 2 and Comparative Example 6, E. coli O-157 of the low-molecular-weight water-soluble amine chitosan (E. coli O-157) prepared in accordance with the present invention graph.
FIG. 2 is a graph showing the hemolytic action rate of the low-molecular water-soluble amine β-chitosan according to the present invention on human red blood cells (hRBC); A: the hemolysis rate according to the treatment concentration of chitosan; and B: the survival rate of human red blood cell (hRBC) according to the treatment concentration of chitosan.
FIG. 3 is a graph showing the degree of deformation of artificial cell membranes according to the pH of the low-molecular water-soluble amine chitosan prepared in Example 2 and Comparative Example 2 on the basis of fluorescence intensity change.
4 is a photograph of a modification of the E. coli cell membrane by the low-molecular water-soluble amine? -Chitosan of the present invention by scanning electron microscopy; A: E. coli not treated with low-molecular water-soluble β-chitosan ( E. Coli ), and B: E. coli treated with low-molecular water-soluble β-chitosan.
FIG. 5 is a graph showing the preform biofilm inhibition activity of Pseudomonas aeruginosa ATCC 27853 by the low-molecular water-soluble amine β-chitosan of the present invention measured by measuring light absorption at 595 nm for each concentration .
FIG. 6 is a graph showing the absorption spectrum of Pseudomonas aeruginosa BMP-Pa001 by the low-molecular water-soluble amine β-chitosan of the present invention measured at 595 nm for the concentration of the preform biofilm inhibitory activity to be.
FIG. 7 is a photograph of a change in the nude mouse infected with Staphylococcus aureus (CCARM 3087) according to Experimental Example 7 of the present invention over time after treatment with the low-molecular water-soluble amine β-chitosan of Example 2 ) And primary antibody anti-TNF-? (B) and anti-IL-1? (C) present in nude mouse skin tissue; Ⅰ: untreated group, Ⅱ: Example 2 water-soluble low molecular weight chitosan of amine β- (1 mg / mL) treated group, Ⅲ: Staphylococcus aureus (Staphylococcus aureus , CCARM 3087) Inoculation group, IV: Staphylococcus ( Staphylococcus aureus ) aureus , CCARM 3087) and the low-molecular water-soluble amine β-chitosan (0.5 mg / mL) administered in Example 2, V: Staphylococcus aureus , CCARM 3087) and low molecular weight soluble amine? -chitosan (1.0 mg / mL) of Example 2; 1: haematoxylin & eosin staining skin cells and 2: FITC-labeling secondary antibody treated skin cells.
8 is a photograph showing the degree of colonization of bacteria contained in a homogenate obtained by homogenizing lung tissue of an ICR mouse infected with Pseudomonas aeruginosa BMP-Pa001 according to Experimental Example 7 of the present invention.
9 is a photograph of skin tissue cells of an ICR mouse infected with Staphylococcus aureus (ATCC 25923) according to Experimental Example 7 of the present invention; A: untreated group, B: Pseudomonas aeruginosa BMP-Pa001 inoculation group, IV: Pseudomonas aeruginosa BMP-Pa001 inoculation and low molecular weight water soluble amine? -Chitosan (0.3 mg / mL) treated group, ⅴ: rugi labor (Pseudomonas Pseudomonas aeruginosa BMP-Pa001) and the low-molecular water-soluble amine? -Chitosan (0.6 mg / mL) of Example 2.

Hereinafter, the present invention will be described in detail.

The present invention provides an antimicrobial composition comprising a low-molecular water-soluble β-chitosan or a derivative thereof of 500 to 20,000 daltons as an active ingredient.

The low-molecular water-soluble β-chitosan or its derivative according to the present invention improves the binding force when β-chitosan obtained in nature is substituted with insoluble to water-soluble form and binds to the cell membrane of microorganism, .

The low-molecular water-soluble β-chitosan according to the present invention may be used in an amount of 500 to 20,000 daltons, preferably 5000 to 10,000 daltons.

The low molecular weight water-soluble β-chitosan of 500-20000 daltons according to the present invention is advantageous in that it is free from the absorption surface and does not form an antibody in the body as the polymer derived from a natural product is converted into a low molecular form. Particularly, the low-molecular water-soluble? -Chitosan of 5000 to 10,000 daltons according to the present invention has no cytotoxicity, and thus the possibility of causing side effects in the living body or human body of an animal is remarkably low and can be safely used.

In addition, the low-molecular water-soluble? -Chitosan derivative according to the present invention is characterized in that the 2-carbonamino group (-NH ₂ ) or the 6-carbon alcohol (-OH) group of glucose is a carboxymethyl, hydroxymethyl, hydroxypropyl or hydroxypropyl ether . ≪ / RTI >

The β-chitosan derivative can be produced by introducing a new chemical substituent into the free amino group (-NH ₂ ) or the glucose 6-carbon alcohol (-OH) group at the carbon atom in the glucose 2-position of the chitosan structure to reduce the molecular weight, solubility, the intrinsic properties of? -chitosan or? -chitosan derivatives do not change.

Furthermore, the antimicrobial composition according to the present invention is useful as a listeria monocytogenes monocytogenes , ATCC 19115), Staphylococcus ( Staphylococcus aureus) aureus , ATCC 25923), Bacillus subtilis (KTCT 1918), and Streptococcus epidermidis (KCTC 3096); Or Escherichia coli, ATCC 25922), E. coli O-157 (Escherichia coli O-157 , ATCC 43895), Vivrio vulnificus, ATCC 29307), rugi labor (Pseudomonas Pseudomonas aeruginosa , KCTC 1637) and Salmonella typhimurium , KCTC 1926). < / RTI >

As a result of the antibacterial activity of the low-molecular water-soluble β-chitosan according to the present invention, the low-molecular water-soluble amine β-chitosan according to the present invention exhibited an MIC value of about 9 μg / mL for pathogenic bacteria regardless of pH It showed a high antibacterial activity, rugi Pseudomonas having a drug-resistant industrial (Pseudomonas aeruginosa ) and Staphylococcus aureus (see Experimental Example 1 and Experimental Example 2). In addition, low-molecular water-soluble β-chitosan is not cytotoxic and does not cause hemolytic action of human erythrocytes (hRBC) (see Experimental Example 3), and has excellent effect of modifying bacterial cell membranes to cause bacterial death 4 to Experimental Example 6). Further, in vivo ( in vivo ) is excellent in antimicrobial effect against pathogenic bacteria (see Experimental Example 7).

Accordingly, the antimicrobial composition containing the low-molecular water-soluble? -Chitosan or its derivative according to the present invention as an active ingredient is useful as an antimicrobial composition used in various fields requiring antibacterial effects such as food additives, bactericides, disinfectants, detergents or deodorants Lt; / RTI >

The low-molecular water-soluble β-chitosan according to the present invention can be prepared from squid cartilage by the following production method:

Treating the squid cartilage with an aqueous solution of 1 M hydrochloric acid (HCl) to prepare an inorganic dehydrated solid (step 1);

Treating the solid produced in step 1 with a 2 M sodium hydroxide (NaOH) aqueous solution to prepare a protein-free solid (step 2);

Deacetylating the solid produced in step 2 to prepare insoluble? -Chitosan (step 3);

(4) preparing a salt by using an organic acid and an inorganic acid in the insoluble? -Chitosan prepared in the step 3, and then enzymatically decomposing the salt to prepare a chitosan polysaccharide;

Treating the solution of the organic acid or inorganic acid salt of the chitosan polysaccharide produced in step 4 with a base trialkylamine (step 5);

Adding an organic solvent to the mixed solution of step 5 to prepare a chitosan polysaccharide in the form of an organic acid or inorganic acid bound to the chitosan polysaccharide in the form of a trialkyl amine salt (step 6); And

The step of treating the inorganic acid-treated chitosan polysaccharide solution prepared in Step 6 with an activated carbon / ion exchange resin column to prepare a low-molecular water-soluble? -Chitosan (Step 7).

Hereinafter, a method for producing the low-molecular water-soluble? -Chitosan will be described in more detail.

First, in step 1 according to the present invention, squid cartilage is injected into a 1 M hydrochloric acid (HCl) aqueous solution and the mixture is stirred at room temperature for 24 hours to remove the inorganic substances. Then, the remaining solids are washed with distilled water, M hydrochloric acid (HCl) solution, and then stirred at 40 ° C for 8 hours to completely remove the inorganic substance remaining in the solid material, thereby preparing a solid material from which the inorganic material has been removed.

Next, in the step 2 according to the present invention, the inorganic matter-removed solid produced in the step 1 is added to an aqueous solution of 2 M sodium hydroxide (NaOH), stirred at room temperature for 24 hours to remove proteins, Washed with distilled water, and the washed solid was put back into an aqueous solution of 2 M sodium hydroxide (NaOH), and then heated for 5 hours and stirred to completely remove the protein remaining in the solid to prepare a protein-free solid .

Next, step 3 according to the present invention is a step of performing deacetylation by treating an aqueous solution of 40% sodium hydroxide (NaOH) with the solids from which the protein produced in step 2 is removed.

In step 4 according to the present invention, the insoluble chitosan extracted from chitin is dissolved in a salt form using an organic acid including lactic acid, acetic acid, propionic acid, formic acid, ascorbic acid, and tartaric acid and hydrochloric acid, nitric acid and sulfuric acid . The above-mentioned chitosan solution is enzymatically decomposed to obtain a chitosan polysaccharide.

At this time, the acid solution of the chitosan polysaccharide is a solution of an inorganic acid salt including an organic acid salt including lactic acid, acetic acid, propionic acid, formic acid, ascorbic acid, and tartaric acid and hydrochloric acid, nitric acid and sulfuric acid, buffered saline 7.0, 7.2 or 7.4 may be used, but is not limited thereto.

In order to effectively remove the form of the organic acid or inorganic acid salt contained in the chitosan polysaccharide in step 5 according to the present invention, the trialkylamine may be added in an amount of 2 to 3 equivalents based on 1 equivalent of the amine group of the chitosan polysaccharide , Preferably 2 equivalents may be added. Once the trialkylamine is added, the trialkylamine will attract H ⁺ from the amine groups of the strongly acidic chitosan polysaccharide and the electrostatic interactions between CH ₃ CHOHCOO ^- or CH ₃ COO ^- , Cl ^- and trialkylamines And the amine of the glucose 2 carbon atom can be obtained in the form of the free amine.

At this time, control of stoichiometric ratio of trialkyl amine to be added is in the of the organic or inorganic acid with an amine in the carbon number in a second glucose unit of chitosan and polysaccharide removal of a tri-alkylamine salts, glucose 6 carbon -CH ₂ It is necessary for the role to protect the OH group.

Further, in the trialkyl amine is trimethyl -C _{1 -4} may be used alkyl amines, can be preferably used a trimethyl amine, triethylamine, tripropylamine, tri-isopropyl ethyl amine, or tri-butylamine. More preferably, triamine can be used.

The step 6 according to the present invention is characterized in that the mixed reaction product formed by adding an organic solvent is reacted at room temperature for about 2 hours and an organic solvent selected from the group consisting of acetone, methanol, chloroform and dichloromethane is added and stirred, Thereby producing a chitosan polysaccharide from which an organic acid or an inorganic acid has been removed. After the above procedure, salts are removed by -CH ₂ OH group is 0.0005-0.010 N inorganic acid of the glucose 6 received protected by a trialkyl amine carburizing, where removal is _{(C 2 H 5) 3 NH} + and Cl ^- Can be removed in the form of a salt.

At this time, hydrochloric acid, nitric acid or sulfuric acid may be used as the inorganic acid. Preferably 0.001 N hydrochloric acid can be used.

Step 7 according to the present invention is a step of purifying a solution of chitosan polysaccharide from which an organic acid or inorganic acid has been removed with an activated carbon / ion exchange resin column to prepare pure low-molecular water-soluble? -Chitosan.

In addition, the antimicrobial composition containing the low-molecular water-soluble? -Chitosan or its derivative according to the present invention as an active ingredient can be used as a food additive, a bactericide, a disinfectant, a detergent or a deodorant.

At this time, the detergent generally includes, but is not limited to, a kitchen detergent, a laundry detergent, a vegetable / fruit detergent, a hand cleaner, etc., which are used in the home and require antibacterial activity.

The detergents of the present invention may comprise one or more surfactants. The surfactant may be anionic, non-ionic, cationic, amphoteric or zwitterionic type, or a mixture thereof. Representative examples of anionic surfactants include linear alkylbenzenesulfonic acid salts (LAS), alkylsulfuric acid salts (AS), alpha olefin sulfonic acid salts (AOS), alcohol ethoxy sulfate (AES) or alkali metal salts of natural fatty acids. Examples of non-ionic surfactants include alkylpolyethylene glycol ethers, nonylphenol polyethylene glycol ethers, fatty acid esters of sucrose and glucose, or esters of polyethoxylated alkyl glucosides.

In addition, the detergent of the present invention can be suitably used in various fields such as an abrasive, a bleaching agent, a surface active agent, a corrosion inhibitor, a metal blocker, a stain re-deposition inhibitor, a perfume, an enzyme and a bleach stabilizer, a formulation aid, , Fillers, fabric softeners, and the like, as well as other detergent ingredients known in the art. The detergent of the present invention may be formulated into any convenient form such as powder, liquid, and the like.

Furthermore, the present invention provides a health functional food composition for preventing or ameliorating an infectious disease caused by a bacterium, comprising an antimicrobial composition containing a low-molecular water-soluble? -Chitosan or a derivative thereof as an active ingredient.

The health functional food composition according to the present invention may be added to health functional foods such as foods, beverages and the like in order to prevent or ameliorate diseases caused by pathogenic bacteria.

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including dairy products, meat, sausage, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, Beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, all of which include health functional foods in a conventional sense.

The health functional food composition containing the low-molecular water-soluble? -Chitosan or derivative thereof of the present invention as an active ingredient can be used as it is, or can be used in combination with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). Generally, the amount of the composition in the health functional food may be 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention is not particularly limited to the other ingredients other than the above-mentioned compounds as essential ingredients in the indicated ratios and may contain various flavors or natural carbohydrates as additional ingredients such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 of the composition of the present invention.

In addition to the above, the health functional food composition containing the low-molecular water-soluble β-chitosan or its derivative of the present invention as an active ingredient may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, And may contain carbonating agents for use in foods (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, have. In addition, the health functional food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

These components may be used independently or in combination. Although the ratio of such additives is not so important, it is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the antimicrobial composition containing the low-molecular water-soluble? -Chitosan or its derivative of the present invention as an active ingredient.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following examples and experimental examples are intended to illustrate the present invention in detail, and the content of the present invention is not limited by the examples and the experimental examples.

< Example  1> Low molecule  Preparation of water-soluble amine? -Chitosan 1

Lactic acid is used as a solvent to prepare a 5% chitosan solution, respectively. A 5-unit chitosanase enzyme derived from Bacillus pumilus BN-262 was mixed with 100 ml of 5% chitosan solution (pH 5.0-5.5 ) and incubated at 40 ° C for 36 hours Lt; / RTI &gt; After completion of the reaction, pre-filtration was performed using a 1-μm prefilter, followed by re-filtration with a hollow filter having a molecular weight of 20,000. The filtrate obtained in the above step was concentrated using a nano filter system, sterilized and dried in an air spray dryer to prepare chitosan polysaccharide. The chitosan polysaccharide obtained above was dissolved in 1 L of PBS 7.0, and then 0.52 L of triethylamine was slowly dropped. At this time, 1 equivalent of the amine group of the chitosan polysaccharide is reacted with 2 equivalents of triethylamine. The reaction mixture was allowed to react at room temperature for about 2 hours, and then acetone was added thereto, followed by stirring and centrifugation. The above procedure was repeated 2 to 3 times, followed by air drying and lyophilization. At this time, centrifugation was carried out using Supra 30 K at 15,000 rpm at 4 ° C for 20 minutes. To the product obtained in the above procedure, 30-50 ml of 0.001 N HCl was added and reacted for about 2 hours. Acetone was added to the reaction mixture, followed by stirring under the same conditions as above. This process was repeated 2 to 3 times, followed by air drying and lyophilization. The dried product was dissolved in the secondary distilled water, purified by an activated carbon / ion exchange resin column, and the obtained aqueous solution was lyophilized to obtain a low-molecular water-soluble amine? -Chitolan of 5000 daltons in white color.

< Example  2> Low molecule  Preparation of water-soluble amine β-chitosan 2

In the same manner as in Example 1, except that the low-molecular water-soluble amine? -Chitosan of 5000 daltons was purified in Example 1, 10000 daltons low-molecular water-soluble amine? -Chitosan was obtained.

< Comparative Example  1> Low molecule  Preparation of water-soluble amine β-chitosan 3

Chitosan having a molecular weight of 1000 daltons was obtained in the same manner as in Example 1, except that 500,000 daltons of low-molecular water-soluble amine? -Chitosan was purified in Example 1.

< Comparative Example  2> Preparation of water-soluble amine β-chitosan from natural products 1

Chitosan having a low molecular weight of 15,000 daltons was obtained in the same manner as in Example 1, except that 15,000 daltons of the water-soluble amine? -Chitosan was purified in Example 1.

< Comparative Example  3> Preparation of water-soluble amine β-chitosan from natural products 2

In Example 1, 20000 daltons low-molecular water-soluble amine? -Chitosan was obtained by carrying out the same procedure as in Example 1, except that 20000 daltons of water-soluble amine? -Chitosan was purified.

< Comparative Example  4> Low molecule  Preparation of water-soluble amine a-chitosan 1

In the same manner as in Example 1, except for purifying 1000 daltons low-molecular water-soluble amine a-chitosan in Example 1, 1000 daltons low-molecular water-soluble amine a-chitosan was obtained.

< Comparative Example  5> Low molecule  Preparation of water-soluble amine a-chitosan 2

The low molecular weight water-soluble amine? -Chitosan of 5000 daltons was obtained in the same manner as in Example 1, except that the low-molecular water-soluble amine? -Chitosan of 5000 daltons was purified in Example 1.

< Comparative Example  6> Low molecule  Preparation of Water-Soluble Amine α-Chitosan 3

Chitosan having a molecular weight of 10,000 daltons was obtained in the same manner as in Example 1, except that the low molecular weight water-soluble amine? -Chitosan in Example 1 was purified.

< Experimental Example  1> Low molecule  Evaluation of Antimicrobial Activity and Drug Tolerance of Water-Soluble Amine β-Chitosan 1

In order to evaluate the antimicrobial activity and drug resistance of pathogenic bacteria of the low-molecular water-soluble amine β-chitosan according to the present invention, the following experiment was conducted.

The minimum growth inhibitory concentration (MIC) values of the low-molecular water-soluble chitosan prepared in Examples 1 to 2 and Comparative Examples 1 to 6 were measured.

First, the Gram-positive bacteria Listeria monocytogenes jeneseu (Listeria to the antimicrobial activity measurement monocytogenes , ATCC 19115), Staphylococcus ( Staphylococcus aureus) aureus , ATCC 25923), Bacillus subtilis (KTCT 1918), Streptococcus epidermidis (KCTC 3096) and Escherichia coli coli, ATCC 25922), E. coli O-157 (Escherichia coli O-157 , ATCC 43895), Vivrio vulnificus, ATCC 29307), Pseudomonas rugi labor (Pseudomonas aeruginosa, KCTC 1637) and Salmonella typhimurium (Salmonella typhimurium , KCTC 1926) were purchased from the genetic bank of the Institute of Biotechnology, and each strain was transfected in LB medium (10 g / L of bacto-trypsin, 5 g / L of yeast extract, 10 g / L of sodium chloride, pH 7.0) (SP) buffer solution (pH 5.4 or 7.4) containing 10% (v / v) growth medium was added to the culture medium to a mid-log phase (OD ₆₀₀ = 0.2-0.5) 10 ⁵ cells (CFU) / 1 mL, and inoculated into microtitre plate (Nunc). The low-molecular water-soluble amine chitosan prepared in the above Examples and Comparative Examples according to the present invention was diluted to a concentration of 5000, 2500, 1250, 625, 312.5, 156.3, 78.1, 39.1, 19.5, 9.5 and 4.9 μg / After incubation at 37 ° C for 18 hours, the absorbance was measured with a microtitre plate reader (Merck Elisa reader) at a wavelength of 620 nm to determine the MIC value of each strain. The results are shown in the following Tables 1 to 3 Respectively.

pH Gram-positive bacteria Gram-negative bacteria B. subtilis B.cereus B. megaterium S. aureus S. epidermidis E. coli E. coli O-157 P. aeruginosa S. typhimurium L. monocytogenes Example 1 7.4 0.018 0.009 0.009 0.018 0.018 0.018 0.009 0.009 0.018 0.009 5.4 0.009 0.009 0.009 0.009 0.009 0.009 0.009 0.009 0.009 0.009 Example 2 7.4 0.009 0.009 0.009 0.009 0.018 0.009 0.009 0.009 0.009 0.009 5.4 0.009 0.009 0.009 0.009 0.0045 0.009 0.009 0.009 0.009 0.009 Comparative Example 1 7.4 1.25 0.625 0.625 0.3125 0.3125 0.625 0.625 0.625 0.3125 0.3125 5.4 2.5 0.625 1.25 2.5 2.5 2.5 2.5 2.5 1.25 > 10 Comparative Example 2 7.4 0.009 0.009 0.009 0.009 0.018 0.009 0.009 0.018 0.009 0.018 5.4 0.009 0.009 0.009 0.009 0.0045 0.009 0.009 0.009 0.009 0.009 Comparative Example 3 7.4 0.018 0.018 0.018 0.009 0.018 0.009 0.009 0.018 0.018 0.018 5.4 0.009 0.009 0.009 0.009 0.0045 0.009 0.009 0.009 0.009 0.009 Comparative Example 4 7.4 > 10 > 10 2.5 > 10 5 > 10 > 10 > 10 > 10 > 10 5.4 0.039 0.018 0.018 0.039 0.009 0.156 0.009 0.156 0.0045 0.3125 Comparative Example 5 7.4 > 10 > 10 2.5 > 10 5 > 10 > 10 > 10 > 10 > 10 5.4 0.018 0.018 0.018 0.018 0.0045 0.0039 0.009 0.078 0.009 0.156 Comparative Example 6 7.4 0.009 0.0045 0.009 0.009 0.018 0.0045 0.009 0.009 0.0045 0.009 5.4 0.009 0.009 0.009 0.009 0.0045 0.009 0.009 0.009 0.0045 0.009

pH Pseudomonas aeruginosa ) ATCC 27853 BMP-Pa001 BMP-Pa002 BMP-Pa003 BMP-Pa004 BMP-Pa005 Example 1 7.4 0.009 <0.0045 <0.0045 <0.0045 <0.0045 <0.0045 5.4 0.009 0.009 0.009 0.009 0.009 0.009 Example 2 7.4 0.009 <0.0045 <0.0045 <0.0045 <0.0045 <0.0045 5.4 0.009 0.009 0.009 0.009 0.009 0.009 Comparative Example 1 7.4 0.625 0.625 0.625 1.25 1.25 1.25 5.4 2.5 5 5 5 5 5 Comparative Example 2 7.4 0.009 0.018 0.009 0.018 0.009 0.018 5.4 0.009 0.009 0.018 0.009 0.018 0.009 Comparative Example 3 7.4 0.018 0.009 0.018 0.009 0.018 0.009 5.4 0.018 0.018 0.018 0.018 0.018 0.018 Comparative Example 4 7.4 > 10 10 > 10 10 10 5 5.4 0.156 0.078 0.039 0.078 0.078 0.039 Comparative Example 5 7.4 > 10 10 10 10 10 5 5.4 0.078 0.039 0.039 0.039 0.018 0.018 Comparative Example 6 7.4 0.009 <0.0045 <0.0045 <0.0045 <0.0045 0.009 5.4 0.009 0.009 0.009 0.009 0.009 0.009

pH Staphylococcus ( Staphylococcus aureus) aureus ) ATCC 25923 BMP-Sa001 BMP-Sa002 BMP-Sa003 BMP-Sa004 BMP-Sa005 Example 1 7.4 0.18 <0.0045 0.039 0.039 0.018 0.039 5.4 0.009 0.009 0.0156 0.3125 0.039 0.078 Example 2 7.4 0.009 <0.0045 0.018 0.018 0.009 0.009 5.4 0.009 0.009 0.078 0.078 0.039 0.078 Comparative Example 1 7.4 1.25 2.5 1.25 1.25 1.25 2.5 5.4 2.5 2.5 2.5 2.5 1.25 1.25 Comparative Example 2 7.4 0.009 0.0045 0.018 0.018 0.018 0.009 5.4 0.018 0.009 0.078 0.078 0.009 0.078 Comparative Example 3 7.4 0.009 0.0045 0.018 0.018 0.018 0.009 5.4 0.039 0.018 0.078 0.078 0.039 0.078 Comparative Example 4 7.4 > 10 > 10 > 10 > 10 > 10 > 10 5.4 0.039 > 10 > 10 > 10 > 10 > 10 Comparative Example 5 7.4 > 10 > 10 > 10 > 10 > 10 > 10 5.4 0.018 > 10 > 10 > 10 > 10 > 10 Comparative Example 6 7.4 0.009 <0.0045 0.018 0.018 0.018 0.018 5.4 0.009 0.009 0.078 0.078 0.018 0.078

As shown in Table 1, it was confirmed that the low-molecular water-soluble amine β-chitosan according to the present invention had an excellent antibacterial effect against pathogenic bacteria regardless of pH. More specifically, most of the low-molecular water-soluble amine β-chitosan according to the present invention exhibited a MIC value of 9 μg / mL at pH 5.4 and 7.4. On the other hand, the MIC value of the low-molecular water-soluble amine α-chitosan was low only when the pH was 5.4 and the molecular weight was large. From this, it can be seen that the low-molecular water-soluble amine β-chitosan according to the present invention is not affected by the pH and has an excellent antibacterial effect against pathogenic bacteria as compared with the low-molecular water-soluble amine α-chitosan.

Further, in Table 2, and as it is shown in Table 3, the low molecular weight water-soluble amine in accordance with the present invention β- chitosan rugi to the pathogenic bacteria, Gram-negative bacteria with the drug-resistant Pseudomonas labor (Pseudomonas aeruginosa and gram-positive bacteria Staphylococcus aureus . More specifically, the low-molecular water-soluble amine β-chitosan according to the present invention has excellent antimicrobial activity irrespective of the pH, and in particular, Pseudomonas aeruginosa In the case of aeruginosa , the low-molecular water-soluble amine? -chitosan of Examples 1 and 2 was confirmed to have high antimicrobial activity without being influenced by the pH. In addition, the antimicrobial activity against Staphylococcus aureus was confirmed to be excellent when the pH was neutral. On the other hand, in the case of low-molecular water-soluble amine? -Chitosan, Pseudomonas The antimicrobial activity against aeruginosa and Staphylococcus aureus was similar to that of β-chitosan with a high molecular weight of 10,000 daltons. The lower the molecular weight, Respectively.

From this, it can be seen that the low-molecular water-soluble amine β-chitosan according to the present invention has an antimicrobial activity against pathogenic bacteria having drug resistance as compared with the low-molecular water-soluble amine α-chitosan.

Therefore, the low-molecular water-soluble? -Chitosan or its derivative according to the present invention has an excellent antimicrobial activity against pathogenic bacteria having drug resistance as compared with the low-molecular water-soluble? -Chitosan, It can be effectively used as an antimicrobial composition used in various fields requiring antimicrobial effect such as a bactericide, a disinfectant, a detergent, a deodorant, and a health functional food.

< Experimental Example  2> Low molecule  Evaluation of antimicrobial activity of water-soluble amine β-chitosan 2

In order to evaluate the antimicrobial activity of the low-molecular water-soluble amine β-chitosan according to the present invention against pathogenic bacteria, the following experiment was conducted.

Bacteriological kinetics of the low-molecular water-soluble amine? -Chitosan according to the present invention was measured using E. coli O-157 ( E. coli O-157 ). (PH 5.4 or 7.4) containing 10% (v / v) growth medium (2 x 10 ⁶ CFR / mL) and log-phase bacterium Chitosan was treated with 1X minimum growth inhibitory concentration (MIC) and 2X minimum growth inhibitory concentration (MIC), respectively. After 0, 2, 4, 6, 8, 10, 20, 30, 40, 60, And cultured at 37 DEG C for 16 hours to measure colloid formation units. At this time, the partial samples did not consider the fixed time interval, the degree of dissolution, and the degree of fixation on the agar plate. The above results are shown in Fig.

As shown in FIG. 1, the low-molecular water-soluble amine β-chitosan according to the present invention is excellent in bactericidal kinetics against pathogenic bacteria. More specifically, when the low-molecular water-soluble amine β-chitosan according to the present invention was treated with E. coli O-157 ( E. coli O-157 ) cultured at a minimum inhibitory concentration (MIC) of 1X, ( E. coli O-157 ) cultured with the minimum inhibitory concentration (MIC) of 2X was killed by 50%. After about 1 hour after the treatment, all of the bacteria were killed . On the other hand, the low-molecular water-soluble amine? -Chitosan prepared in Comparative Example 6 was found to kill all the bacteria in the same manner as the low-molecular water-soluble amine? -Chitosan prepared in Example 2 according to the present invention, But its functional group is different from the small-molecule water-soluble amine? -Chitosan according to the present invention.

From this, it can be seen that the low-molecular water-soluble amine β-chitosan according to the present invention is superior to the bacterium killing kinetics for killing bacteria against pathogenic bacteria.

Therefore, the low-molecular water-soluble β-chitosan or its derivative according to the present invention exhibits excellent cell killing kinetics against pathogenic bacteria and has excellent antimicrobial activity. Therefore, the antimicrobial composition containing the antibacterial composition as an active ingredient can be used as food additives, disinfectants, , A deodorant, a health functional food, and the like, which are used in various fields requiring an antimicrobial effect.

< Experimental Example  3> Low molecule  Evaluation of cytotoxicity of water-soluble amine β-chitosan

In order to evaluate the cytotoxicity of the low-molecular water-soluble amine β-chitosan according to the present invention, the hemolytic action of human red blood cells (hRBC) was performed as follows.

Hemolytic activity was tested using human red blood cells (hRBC) using heparin, an anticoagulant, provided by a healthy donor. Healthy human red blood cells (hRBC) were centrifuged (800 rpm) in phosphate buffered saline (PBS) for 10 minutes and then suspended again. (HRBC) dissolved in phosphate buffered saline (PBS) to chitosan prepared in Examples 1 to 2 and Comparative Examples 1 and 4 to 6 suspended in phosphate buffered saline (PBS) 100 μL of the solution was added. At this time, the chitosan concentration of the sample to which the standard solution in which the human erythrocyte cell (hRBC) was dissolved was 5, 10, 15 and 20 mg / mL, and the concentration of human erythrocyte (hRBC) was adjusted to be 8% v / v . Each sample was incubated at 37 ° C for 1 hour and then centrifuged (800 rpm) for 10 minutes. The centrifuged supernatant was measured for absorbance at 414 nm, and the human red blood cells (hRBC) suspended in phosphate buffer (PBS) to which 1% Triton X-100 was added were divided into the untreated group not treated with chitosan, The% hemolytic sample was also measured for absorbance, and the degree of collapse of the cell membrane due to hemolysis of human erythrocyte (hRBC) cells and the degree of preservation of the erythrocytes was confirmed. All the samples were subjected to three repeated experiments to measure the absorbance, and the results are shown in FIG. 2A.

In addition, cytotoxicity was measured using HEK293 (human fetal kidney cell), which is a normal cell line. More specifically, fetal kidney cells (HEK293) cultured in a DMEM medium containing 10% FBS (Fetal Bovine Serum) were dispensed into 96-well plates at 3 × 10 ³ cells / mL for 24 hours. Thereafter, Examples 1 to 2 and Comparative Examples 1 to 6 were treated for each concentration, and reacted in a 5% CO ₂ incubator for 24 hours. After completion of the reaction, MTT (Thiazolyl Blue Tetrazolium Bromide) solution (20 μl) dissolved in phosphate buffered saline (PBS) was added to each well at a concentration of 5 mg / ml and reacted for 4 hours. . 200 [mu] l of dimethylsulfoxide (DMSO) was injected into the supernatant-free residue, and the MTT crystals formed were dissolved to confirm the result at 560 nm. The results are shown in Fig. 2B.

As shown in FIG. 2, the low-molecular water-soluble amine-chitosan according to the present invention is not toxic to human red blood cells (hRBC) and fetal kidney cells. More specifically, the low-molecular water-soluble amine β-chitosan of Examples 1 and 2 having a molecular weight of 5000 Daltons to 10000 Daltons in FIG. 2 A was dissolved in erythrocyte cells (hRBC) hRBC) was found to be less than about 5%, whereas the low-molecular water-soluble amine β-chitosan of Comparative Example 2 and Comparative Example 3 having a molecular weight of more than 10000 daltons was found to be 20% It was confirmed that the hemolysis rate was 40%. In the case of human fetal kidney cells (HEK293) treated with low-molecular water-soluble amine? -Chitosan of Examples 1 and 2 having a molecular weight of 5000 Daltons to 10,000 Daltons in FIG. 2B, a high cell survival rate While the low-molecular-weight water-soluble amine β-chitosan of Comparative Example 2 and Comparative Example 3 having a molecular weight of more than 10000 daltons showed cell viability of 72% and 50%, respectively, when treated at a concentration of 10 mg / ml. In addition, in the case of the low-molecular water-soluble amine α-chitosan, hemolysis was not observed with respect to human red blood cells (hRBC). However, in the case of human fetal kidney cells (HEK293) .

From this, it can be seen that the low-molecular water-soluble amine β-chitosan according to the present invention has remarkably low cell cytotoxicity to human fetal kidney cells as compared with the low-molecular water-soluble amine α-chitosan, When the molecular weight exceeds 10000 Daltons, the cytotoxicity is markedly increased. On the other hand, the low-molecular-weight water-soluble amine β-chitosan having a molecular weight of 5000 Daltons to 10000 Daltons is not cytotoxic and is not likely to cause adverse effects on the living body or human body. .

Therefore, the low-molecular water-soluble β-chitosan or its derivative according to the present invention is remarkably superior in antimicrobial activity against pathogenic bacterium having general pathogenic bacteria and drug resistance, and also has excellent antibacterial activity against human red blood cell (hRBC) (HEK293) in comparison with a low-molecular water-soluble? -Chitosan without causing hemolysis and is safe for the human body. Therefore, the antimicrobial composition containing the antimicrobial composition as an active ingredient can be used as food additives, disinfectants, disinfectants, Deodorant, health functional food, and the like, which are used in various fields requiring antimicrobial effect.

< Experimental Example  4> Low molecule  Evaluation of cell membrane permeability of water-soluble amine β-chitosan

In order to evaluate the membrane permeability of the low-molecular water-soluble amine β-chitosan according to the present invention, the following experiment was conducted.

First, artificial liposomes (PE / PG = 7/3, w / w), which is a membrane lipid component of bacteria, were prepared and treated with chitosan prepared in Example 1 and Comparative Example 5 at pH 7.4 and 5.4, Activity levels. Chloroform solution (0.1 ml) in which lipid (PE / PG) was dissolved was injected into 10 ml of vial and chloroform was removed by N2 gas blowing for 2 hours or more. 1 ml of HEPES buffer solution (pH 5.4, 7.4, pH 7.4) containing 70 mM calcein was added to the lipid film coated on the glass wall, and the mixture was shaken vigorously. Then, a tip type sonicator Sonication was performed 20 times at intervals of 10 seconds. The calcein-bound liposomes were purified by gel filtration chromatography using a Sephadex G-50 column from glassy cutane. LUVs were extruded through a polycarbonate filter with a 0.2 m pore size of an Avanti Mini-Extruder (Avanti Polar Lipids Inc. Alabaster, AL). LUVs suspensions containing calcein with a lipid content of 20 M were then treated with various concentrations of chitosan of Example 1 and Comparative Example 5 dissolved in 10 mM HEPES at pH 5.4 or pH 7.4 and cultured. Thereafter, the fluorescence emitted by the calcine was measured at 480 nm for the excitation wavelength and at 520 nm for the fluorescence spectrometer (Perkin-Elmer LS55). Control of the dye emission completion was determined with Triton X-100. 100% dye release was completed by adding Triton X-100 to 0.1% of final concentration. All experiments were carried out at 25 캜, and the release rate was calculated according to the following equation 1. The results are shown in Fig.

F: untreated fluorescence intensity;

F ₀ : fluorescence intensity after chitosan treatment;

F _t : Fluorescence intensity of liposome.

As shown in FIG. 3, it was confirmed that the low-molecular water-soluble β-chitosan according to the present invention exhibited a high destructive power against an artificial liposome, which is an artificial cell membrane of bacteria. More specifically, the low molecular weight water-soluble β-chitosan of 5000 daltons according to the present invention exhibits a high fluorescence intensity at both pH 5.4 and pH 7.4, and it can be confirmed that it has high permeability to calcine-bound artificial liposome. On the other hand, when the low-molecular water-soluble α-chitosan had a pH of 7.4, the increase in the fluorescence intensity was insignificant, and the fluorescence intensity increased even at the pH of 5.4. Fluorescence was increased by 20%.

From this, it can be seen that the low-molecular water-soluble β-chitosan according to the present invention has excellent destructive power against the cell membrane, which is consistent with the result of Experimental Example 2. In addition, it can be seen that the cell membranes have significantly higher destructive power than the low-molecular water-soluble? -Chitosan.

< Experimental Example  5> Low molecule  Evaluation of cell membrane morphology change of water-soluble amine β-chitosan

The following experiment was conducted to observe the changes in cell membrane morphology of the low-molecular water-soluble amine β-chitosan according to the present invention.

First, E. coli was prepared at 10 ⁸ per 1 ml and suspended in Na-phosphate buffer (pH 7.4). Then, the chitosan prepared in Example 2 was treated with a minimal inhibitory concentration (MIC) for growth and reacted for 1 hour, and cells were fixed with 2% glutaraldehyde. Then, 10, 30, 50, 70 and 100% ethanol are sequentially treated and dehydration is carried out in the reverse order. Dehydrated bacteria were dried at critical point, coated with gold, and observed at 120 kV using a scanning electron microscope (HITHACHI S-2400, Japan). At this time, E. coli not treated with chitosan was also observed with a scanning electron microscope, and the results are shown in FIG.

As shown in Fig. 4, the low-molecular water-soluble amine? -Chitosan according to the present invention is found to modify the cell membrane of bacteria. More specifically, in the case of E. coli treated with the 10,000-daltons low-molecular water-soluble amine β-chitosan prepared in Example 2 according to the present invention, the cell membrane of E. coli was disrupted . On the other hand, E. coli , which is not treated with chitosan, shows that cell membrane deformation has not occurred.

From this, it can be seen that the low-molecular water-soluble amine β-chitosan according to the present invention is excellent in the effect of modifying the bacterial cell membrane, resulting in the death of bacteria.

Therefore, the low-molecular water-soluble β-chitosan or its derivative according to the present invention is excellent in the effect of killing bacteria by modifying the cell membrane of a bacterium. Therefore, the antimicrobial composition containing the antibacterial composition as an active ingredient can be used as food additives, disinfectants, , Health functional foods, and the like, which are used in various fields requiring antimicrobial effect.

< Experimental Example  6> Low molecule  Of water-soluble amine β-chitosan Antifibrotic membrane  evaluation

The following experiment was conducted to evaluate the antifouling film of the low-molecular water-soluble amine? -Chitosan according to the present invention.

The pseudomonas aeruginosa ( Pseudomonas sp. aeruginosa ATCC 27853 and BMP-Pa001) were measured. Pseudomonas aeruginosa ( Pseudomonas aeruginosa) aeruginosa ATCC 27853 and BMP-Pa001) were cultured at 37 占 폚. 1 x 10 &lt; ⁸ &gt; CFU / mL of the bacterium was injected into a 96-well plate, and the injected culture solution was shake-cultured at 37 DEG C and 50 rpm for 48 hours. The culture was then removed using a pipette and washed three times with 200 μL of phosphate buffered saline (PBS). After washing, the chitosan (200 [mu] L) prepared in Examples 1 to 2 and Comparative Examples 1 to 6 was added to a 96-well plate in which the biofilm was formed to a concentration of 18 to 50 mg / mL. The plate was incubated at 37 DEG C for 24 hours, and the cultured culture was removed using a pipette and washed three times with phosphate buffered saline (PBS). After washing, the biofilm was fixed to the plate at room temperature for 15 minutes with methanol and dried until all of the methanol was removed. To quantify the dried biofilm, 200 μL of 0.1% (w / v) crystal violet (Sigma-Aldrich) was added and stained for 5 to 10 minutes at room temperature. After staining, rinse three times to remove crystal violet left in phosphate buffered saline (PBS). In each well, Pseudomonas aeruginosa ATCC 27853 and BMP-Pa001) were allowed to solubilize in 95% ethanol and phosphate buffered saline (PBS, 200 μL) and incubated at room temperature for 30 min. Thereafter, the destructive activity of the preform biofilm was evaluated by measuring the optical absorption by a microplate veresa max ELISA at 595 nm, and the results are shown in FIG. 5 and FIG.

As shown in FIG. 5 and FIG. 6, it was confirmed that the low-molecular water-soluble amine β-chitosan according to the present invention had an antifungal effect. More specifically, the low-molecular water-soluble amine β-chitosan according to the present invention showed destructive activity in a concentration and molecular weight dependent manner when treated with Pseudomonas aeruginosa ATCC 27853 and BMP-Pa001 biofilm, And 10,000 daltons of low-molecular-weight water-soluble amine β-chitosan showed a preformed biofilm destruction activity of more than 50% at 3.12 mg / mL. On the other hand, low molecular weight water-soluble chitosan is α- rugi labor (Pseudomonas Pseudomonas aeruginosa bacterium BMP-Pa001 showed a remarkably low destructive activity, indicating that the preformed biofilm destruction activity was decreased according to the type of strain.

From this, it can be seen that the low-molecular water-soluble amine β-chitosan according to the present invention has a preformed biofilm destructive activity depending on the concentration and the molecular weight, and the preformed biofilm destructive activity regardless of the strain type It can be seen that it is excellent.

Therefore, the low-molecular water-soluble amine? -Chitosan or its derivative according to the present invention has excellent preformed biofilm destruction activity regardless of the strain type and thus has excellent effect of inhibiting biofilm formation. Therefore, Can be effectively used as an antimicrobial composition used in various fields requiring antibacterial effects such as food additives, bactericides, disinfectants, detergents, deodorants, health functional foods and the like.

< Experimental Example  7> Low molecule  Of the water-soluble amine? -Chitosan in vivo ( in vivo ) Evaluation of antibacterial effect

In the case of the low-molecular water-soluble amine β-chitosan according to the present invention ( in vivo &lt; / RTI &gt;

Drug resistance Staphylococcus Aureus ( Staphylococcus aureus )on  Antibacterial effect

Staphylococcus ( Staphylococcus aureus), a drug resistant substance in the back skin of nude mice, aureus , CCARM 3087, 5 x 10 ⁸ cells / mL). After 1 hour, the low-molecular water-soluble amine β-chitosan of Example 2 according to the present invention was inoculated at a concentration of 0.5 mg / mL and 1.0 mg / mL. After 7 days, the skin of the inoculated nude mice was collected and washed with phosphate buffered saline (PBS). The skin tissue of the washed nude mice was transferred to 4% paraformaldehyde for 24 hours, and 50% to 100% ethanol was dehydrated three times for 2 hours. Subsequently, the substrate was replaced with xylene for 1 hour, and the sample cut at 4 μm was treated with paraffin (Microtome, Thermo-scientific). Each sample was incubated for 30 minutes at room temperature in bovine serum albumin (BSA) containing primary anti-TNF-α, anti-IL-1β. The samples were washed with TBST buffer, treated with FITC-label secondary antibody, and subjected to hematoxylin and eosin staining (IX71, Olympus, Tokyo, Japan) simultaneously. At this time, bacteria not treated with chitosan as a control group were also measured, and the results are shown in FIG.

As shown in FIG. 7, the low-molecular water-soluble amine? -Chitosan according to the present invention is an in- vivo ). &lt; / RTI &gt; More specifically, pathogenic bacteria such as Staphylococcus ( Staphylococcus aureus) Inflammatory cytokines (anti-TNF-a and anti-IL-1 beta) present in the skin tissue of nude mice infected with the chitosan- aureus , CCARM 3087) (TNF-a and IL-l [beta]) are excessively secreted and strong fluorescence is emitted. In addition, it can be confirmed that the thickness of the epidermis infected with the pathogenic bacteria is increased through the hyaluronic acid and haematoxylin & eosin staining. When the low-molecular water-soluble amine β-chitosan according to the present invention is additionally administered, It can be understood that the thickness is restored. Furthermore, it can be visually confirmed that the skin inflammation of nude mice is alleviated by the low-molecular water-soluble amine? -Chitrosene of the present invention.

From this, it can be seen that the low-molecular water-soluble amine? -Chitosan according to the present invention has an excellent antibacterial effect in vivo .

Drug resistant Pseudomonas Eruginosa ( Pseudomonas aeruginosa )on  Antibacterial effect

( Pseudomonas aeruginosa BMP-Pa001, 1 x 10 &lt; ⁸ &gt; cells / mL) was inoculated to the epidermis of ICR mice and 10 mg / mL of low-molecular water-soluble amine? / mL and 40 mg / mL, respectively. Thereafter, the mice inoculated for 7 days were observed, and the lungs of the mice were excised and homogenized when the observation was completed. The homogenate was injected into NB + 0.5% sodium chloride agar plates to assess the degree of bacterial colonization of the homogenate. The results are shown in Fig.

As shown in FIG. 8, the low-molecular water-soluble amine? -Chitosan according to the present invention is an in- vivo ). More specifically, Pseudomonas In the homogenized homogenized homogenized lung tissue of the ICR mice inoculated with the aeruginosa BMP-Pa001, the bacterial colonies were inhibited when the low-molecular water-soluble amine? -chitosan of the present invention was treated, and the amount thereof was remarkably reduced.

From this, it can be seen that the low-molecular water-soluble amine? -Chitosan according to the present invention has an excellent antibacterial effect in vivo .

Non-drug resistance Staphylococcus Aureus ( Staphylococcus aureus )on  Antibacterial effect

Staphylococcus aureus (ATCC 25923, 5 x 10 &lt; ⁸ &gt; cells / mL) was inoculated to the epidermis of ICR mice and after 4 hours, the low-molecular water-soluble amine? Chitosan was inoculated at concentrations of 0.3 mg / mL and 0.6 mg / mL. After 10 days, the skin of the inoculated ICR mice was excised and washed with phosphate buffered saline (PBS). The skin tissue of the washed nude mice was transferred to 4% paraformaldehyde for 24 hours, and 50% to 100% ethanol was dehydrated three times for 2 hours. Thereafter, hematoxylin and eosin staining was performed and analyzed with a fluorescence analyzer (IX71, Olympus, Tokyo, Japan). At this time, bacteria not treated with chitosan as a control group were also measured, and the results are shown in FIG.

As shown in Fig. 9, the low-molecular water-soluble amine? -Chitosan according to the present invention is an in- vivo ). More specifically, when the low molecular weight water-soluble amine β-chitosan of the present invention was treated at a concentration of 0.3 mg / mL and 0.6 mg / mL in the skin tissue cells of an ICR mouse inoculated with Staphylococcus aureus , It can be seen that the skin tissue cells close to the non-treated group in which Staphylococcus aureus was not inoculated are identified.

From this, it can be seen that the low-molecular water-soluble amine? -Chitosan according to the present invention has an excellent antimicrobial activity in vivo .

Thus, the low molecular weight water-soluble β- chitosan or derivatives thereof according to the present invention in vivo (in vivo , the antimicrobial composition containing the antimicrobial composition as an effective ingredient is useful as an antimicrobial composition used in various fields requiring antibacterial effects such as food additives, bactericides, disinfectants, detergents, deodorants, health functional foods Can be used.

Meanwhile, the compounds of the present invention are capable of producing health functional foods in various forms depending on the purpose. Examples of the preparation of a health functional food for the composition of the present invention are shown below.

< Manufacturing example  1> Dairy products ( dairy products )

0.01 to 1 part by weight of the antibacterial composition of the present invention was added to milk and various dairy products such as butter and ice cream were prepared using the milk.

< Manufacturing example  2> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh. Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer. The antimicrobial composition of the present invention was concentrated under reduced pressure in a vacuum concentrator to obtain a dry powder.

The dry powders of the cereals, seeds and antimicrobial composition prepared above were blended in the following proportions.

Grain (34 parts by weight of brown rice, 19 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

(2 parts by weight),

(1.5 parts by weight), and

Rhizome (1.5 parts by weight).

< Manufacturing example  3> Manufacture of Health Functional Foods

100 mg of antibacterial composition

Vitamin mixture quantity

Vitamin A acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a component suitable for health functional food as a preferred embodiment, the compounding ratio may be arbitrarily changed, and the above components may be mixed , Granules may be prepared and used in the manufacture of health functional food compositions according to conventional methods.

< Manufacturing example  4> Manufacture of health functional drinks

       100 mg of antibacterial composition

       Citric acid 100 mg

       Oligosaccharide 100 mg

       Plum concentrate 2 mg

       Taurine 100 mg

Purified water was added to the whole 500 mL

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized container. The resulting solution was sealed and sterilized, &Lt; / RTI &gt;

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (7)

Chitosan or a derivative thereof of 500 to 20,000 daltons as an active ingredient.
The method according to claim 1,
Wherein the? -Chitric acid is 5000 to 10000 daltons.
The method according to claim 1,
Wherein said derivative is substituted with carboxymethyl, hydroxymethyl, hydroxypropyl or hydroxypropyl ether at the 2-carbon amino group (-NH ₂ ) or the 6-carbon alcohol (-OH) group of glucose.
The method according to claim 1,
The composition jeneseu L. monocytogenes (Listeria monocytogenes , ATCC 19115), Staphylococcus ( Staphylococcus aureus) aureus , ATCC 25923), Bacillus subtilis subtilis, KTCT 1918) and Streptococcus epidermidis (Streptococcus epidermidis , KCTC 3096); Or Escherichia E. coli , ATCC 25922), Escherichia coli O-157 (ATCC 43895), Vivrio vulnificus (ATCC 29307), Pseudomonas aeruginosa aeruginosa , KCTC 1637) and Salmonella typhimurium , KCTC 1926). &lt; / RTI &gt;
The method according to claim 1,
Wherein the antimicrobial composition is used as a food additive, a bactericide, a disinfectant, a detergent or a deodorant.
6. The method of claim 5,
Wherein the detergent is one selected from the group consisting of a kitchen detergent, a laundry detergent, a vegetable / fruit detergent, and a hand detergent.
A health-functional food composition for preventing or ameliorating an infectious disease caused by a bacterium containing the low-molecular water-soluble β-chitosan or a derivative thereof as an active ingredient.

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