Methods for producing oligosaccharides, methods for analyzing oligosaccharides, methods for separating oligosaccharides, chitosan oligosaccharides, antibacterial agents and their use.

Abstract

This invention provides a safe, simple, and inexpensive method for producing oligosaccharides compared to conventional methods. [Solution] A method for producing oligosaccharides, comprising the steps of: dissolving a polysaccharide in an aqueous solution of a volatile strong acid to prepare an aqueous solution of the polysaccharide; and concentrating and drying the aqueous solution of the polysaccharide.

Description

Technical Field

[0001] The present invention relates to a method for producing oligosaccharides, a method for analyzing oligosaccharides, a method for separating oligosaccharides, chitosan oligosaccharides, antibacterial agents, and uses thereof.

Background Art

[0002] Oligosaccharides are molecules having a structure in which several to several tens of monosaccharides are linked by glycosidic bonds. Monosaccharide components include, for example, glucose, galactose, mannose, fructose, fucose, rhamnose, xylose, ribose, glucosamine, galactosamine, mannosamine, glucuronic acid, galacturonic acid, mannuronic acid, sialic acid, etc., which exist in nature, and those in which their hydroxyl groups, amino groups, carboxyl groups, etc. are modified with acyl groups, alkyl groups, phosphate groups, sulfate groups, amino acids, etc. There are also those in which the modification of the above monosaccharides is artificially chemically modified with respect to polysaccharides and is widespread as industrial products.

[0003] For the production of oligosaccharides, a method is used in which reagents such as acids and oxidants, enzymes, etc. are allowed to act to partially hydrolyze the glycosidic bonds between monosaccharides.

[0004] As a production method linked to the separation from the reagents and enzymes used in the production of oligosaccharides, for example, a method in which a polysaccharide is allowed to act on a reagent or enzyme immobilized on a solid phase, and the production and separation of oligosaccharides are linked by controlling the action time with the flow rate or the time until filtration, and a method in which ultrafiltration or a dialysis membrane is used and the production and separation of oligosaccharides are linked by utilizing the difference in molecular weight between the oligosaccharides and the reagents, enzymes, and polysaccharides used, etc. have been reported.

[0005] Sugars are generally non-volatile from polysaccharides to monosaccharides. On the other hand, solvents are generally volatile. Among the reagents used in oligosaccharide production, enzymes are generally non-volatile, but some low-molecular reagents are volatile.

[0006] Hydrogen chloride is a gas at room temperature, but it dissolves in water through hydration. Due to the relationship between the oxonium ion of water and the pKa of hydrogen chloride, a salt is formed between the oxonium ion of water and the chloride ion, and the solution exhibits acidity. As the water concentrates, this salt of oxonium ion and chloride ion volatilizes into hydrogen chloride and water. Therefore, hydrochloric acid is a volatile acidic solvent, and no salt of oxonium ion and chloride ion remains after concentration.

[0007] Hydrochloric acid is commonly used as an acid that can be separated by concentration due to its volatility, and in oligosaccharide production, hydrochloric acid treatment was performed, and the acid and the resulting oligosaccharides were separated by concentration. On the other hand, dilute hydrochloric acid has a low concentration of water oxonium ions, which are the rate-limiting factor in the hydrolysis of oligosaccharides, resulting in a low oligosaccharide production rate. Therefore, a mixture of highly concentrated hydrogen chloride and polysaccharides has been used in oligosaccharide production, and concentration has been used for its separation.

[0008] High concentrations of hydrochloric acid corrode reaction vessels and concentration equipment, and the hydrogen chloride generated during concentration, especially at high concentrations, exceeds the natural buffering capacity and poses a significant environmental burden. Therefore, since the generated hydrogen chloride needs to be diluted or neutralized, solid acids and enzymes, which have lower separation costs, have been widely used as oligosaccharide production methods, considering both environmental and manufacturing costs.

[0009] Chitosan oligosaccharides are oligosaccharides that have a structure in which several to tens of D-glucosamine (GlcN) molecules are linked together by β-1,4 bonds. Chitosan oligosaccharides can also be described as the deacetylated form of chitin oligosaccharides, which are oligosaccharides that have a structure in which several to tens of N-acetylglucosamine (GlcNAc) molecules are linked together by β-1,4 bonds.

[0010] Chitin oligosaccharides are produced by dissolving chitin in concentrated hydrochloric acid and performing partial hydrolysis at a temperature of 40-50°C for 5-6 hours. The chitin oligosaccharides prepared in this way are a mixture containing monosaccharides ranging from GlcNAc to hexasaccharides such as N-acetylchitohexose (NACOS-6). There is also a method of partially hydrolyzing chitin using chitinase, a chitin-degrading enzyme, but because chitin is insoluble in water, dilute acid, and dilute alkali, it is not easily subjected to enzymatic degradation, and most chitinases are disaccharide-producing types, so this method is not currently widely used as an industrial production method.

[0011] Chitosan oligosaccharides can be produced by either partial hydrolysis with concentrated hydrochloric acid (acid hydrolysis method), similar to chitin, or by partial hydrolysis with chitosanase (enzymatic hydrolysis method). Acid hydrolysis usually yields a mixture containing monosaccharides from GlcN to heptasaccharides (COS-7), while enzymatic hydrolysis produces almost no monosaccharides from GlcN, yielding a mixture containing disaccharides from chitobiose (COS-2) to COS-7.

[0012] Currently, the industrial production method for chitosan oligosaccharides predominantly employs enzymatic hydrolysis, which allows for safe partial hydrolysis under milder conditions, rather than acid hydrolysis using concentrated hydrochloric acid. Furthermore, the manufactured chitosan oligosaccharides are distributed in the form of hydrochloride salts.

[0013] Chitosan oligosaccharides exhibit superior solubility compared to chitosan and possess a variety of physiological activities, making them promising for use in various fields, including medicine. Furthermore, recent studies have revealed that their physiological activities differ depending on the degree of polymerization, necessitating the establishment of separation technologies for each degree of polymerization for industrial applications. In addition, while various chitosan oligosaccharide derivatives have been synthesized with the aim of improving specific functions, there are very few examples of detailed structural determination, including the degree of substitution, molecular weight, and substituent distribution.

[0014] Methods for separating chitosan oligosaccharides have been reported, including a method using chromatography (Patent Document 2), a method for separating oligosaccharide components using ultrafiltration (Patent Document 3), a method for obtaining an oligosaccharide fraction as a soluble component by adding a base to an acidic solution (Patent Document 4), a method for precipitating oligosaccharides by adding concentrated hydrochloric acid (Patent Document 5), a method for selectively precipitating oligosaccharides by adding an organic solvent (Patent Document 6), and a method for producing a precipitate by mixing with two oligosaccharide components (Patent Document 7).

[0015] Patent Document 1 describes a method for producing oligoglucosamine, comprising the steps of (1) preparing a 1-20% by weight chitosan dispersion; (2) adding a 5.0-40.0% acid to the solution from (1); (3) adding chitosanase to the solution from (2); (4) adjusting the pH of the solution from (3) to 3.5-4.5; (5) heating the solution from (4) to 50-85°C; and (6) drying the solution from (5).

[0016] Patent Document 2 describes a method for producing a novel monoacetyl chitooligosaccharide, characterized by reacting partially deacetylated chitin or partially N-acetylated chitosan with chitinase, and then reacting it with N-acetylhexosaminidase to produce a novel monoacetyl chitooligosaccharide of disaccharide or more in which only the reducing terminal sugar is N-acetylated.

[0017] Patent Document 3 describes a method for producing higher chitosan oligosaccharides, characterized by carrying out a chitosan decomposition reaction using an enzyme having chitosan decomposition activity in an ultrafilter with the membrane permeability adjusted to the maximum to produce a chitosan oligosaccharide mixture containing higher chitosan oligosaccharides, removing the chitosan oligosaccharide mixture from the ultrafilter, supplying a chitosan solution equivalent to the removed amount into the ultrafilter, and then continuously repeating the chitosan decomposition reaction, the removal of the chitosan oligosaccharide mixture, and the supply of the chitosan solution to produce higher chitosan oligosaccharides.

[0018] Patent Document 4 describes a method for fractionating chitosan oligomers, which involves dissolving chitosan in water using an acid, then mixing in an enzyme to hydrolyze the chitin region contained in the chitosan, stirring at 40-45°C for 9-10 hours, and then adding an alkaline substance to neutralize the mixture, thereby fractionating it into an aqueous solution of low-molecular-weight chitosan oligomers with a molecular weight of 2000 or less and a precipitate-type high-molecular-weight chitosan oligomer with a molecular weight of 8000 or more.

[0019] Patent Document 5 describes a method for separating chitosan oligosaccharides, characterized by precipitating chitosan oligosaccharides with a predetermined sugar content by adding an acid to an aqueous solution of chitosan oligosaccharides to prepare an acidic solution of a predetermined concentration and a chitosan oligosaccharide solution of a predetermined concentration.

[0020] Patent Document 6 describes a method for isolating and purifying chitosan oligosaccharides from a chitosan mixture in which n is an integer between 5 and 9, comprising the steps of (1) adding an amphiphilic anion to an aqueous solution of a chitosan mixture to form a precipitate; (2) dissolving the precipitate obtained in step (1) in an organic solvent and removing insoluble matter; (3) adding hydrochloric acid to the organic solution obtained in step (2) to form a precipitate; and (4) recovering the precipitate obtained in step (3).

[0021] Patent Document 7 describes a method for solubilizing a poorly soluble / insoluble active substance by forming an oligomer complex, characterized by comprising: a first step of mixing two types of oligomers derived from hydrophilic natural polymers and dissolving them in water to produce an oligomer complex with a cavity structure; and a second step of adding a poorly soluble / insoluble substance to the oligomer complex and encapsulating it in the hydrophobic cavity structure of the oligomer complex.

[0022] Non-patent document 1 describes how, when a hydrolyzed product obtained by partially decomposing chitosan with hydrochloric acid was fractionated by ion-exchange chromatography, five individual peaks were obtained, and analysis revealed that the substances in the peaks were glucosamine oligosaccharides, which eluted in order of decreasing molecular weight.

[0023] In the production methods of chitosan oligosaccharides described in Patent Documents 1 to 4, since enzymes are used, dedicated production equipment, its cleaning, strict management of culture conditions, etc. are required. In addition, the produced chitosan oligosaccharides are often a mixture of low-degree polymers with a degree of polymerization of 10 or less. In addition to this method, various chitosan oligosaccharide production methods have been reported. For example, there is a method of hydrolyzing chitosan heterogeneously in high-concentration hydrochloric acid to form oligosaccharides. However, this method has problems in terms of safety and mass production, and has not yet reached the production of chitosan oligosaccharides with a degree of polymerization exceeding 10 (Non-Patent Document 1).

Prior Art Documents

Patent Documents

[0024]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Non-Patent Documents

[0025]

Non-Patent Document 1

[0026] The synthesis of oligosaccharides by hydrolysis of polysaccharides is carried out by limited decomposition using acid treatment or enzymatic treatment, and generally, after producing the oligosaccharides, it is necessary to separate the chemical reagents or enzymes used.

[0027] Technologies that link the synthesis and separation methods of oligosaccharides have also been developed. For example, methods have been reported that link the production and separation of oligosaccharides by reacting polysaccharides with reagents or enzymes immobilized on a solid phase and controlling the reaction time by the flow rate or the time until filtration, and methods that link the production and separation of oligosaccharides by using ultrafiltration or dialysis membranes and utilizing the difference in molecular weight between the oligosaccharides and the reagents, enzymes, and polysaccharides used.

[0028] In the production of oligosaccharides by acid hydrolysis of polysaccharides, large excesses of acid or heating were required to ensure the reaction rate.

[0029] Methods for synthesizing partially acylated chitosan oligosaccharides have been reported, including methods that perform acylation after producing chitosan oligosaccharides, and methods that enzymatically hydrolyze residual N-acetyl groups from chitosan production (Patent Document 2). However, no simple method has been reported for efficiently obtaining partially acylated chitosan oligosaccharides from chitosan that has partial N-acyl groups other than acetyl groups.

[0030] One of the objectives of the present invention is to provide a method for producing oligosaccharides that is safer, simpler, and less expensive than conventional methods. [Means for solving the problem]

[0031] The inventors of the present invention conceived that when polysaccharides are concentrated in an acidic aqueous solution, the acid molecules accumulate on the sugar, which has many lone pairs of electrons that are non-volatile and can act as base sites, and exhibit hydrolytic activity. Furthermore, they observed that as the concentration progresses to a high degree, the acid that has become the site of hydrolysis volatilizes, and hydrolysis stops at the oligosaccharide level. Based on this observation, the inventors arrived at the present invention.

[0032] The present invention has the following aspects. [1] A step of preparing an aqueous solution of the polysaccharide by dissolving the polysaccharide in an aqueous solution of a volatile strong acid, and A process of concentrating and drying the aqueous solution of the above polysaccharide. A method for producing oligosaccharides, including [2] [1] By the method for producing oligosaccharides described above, an oligosaccharide mixture containing oligosaccharides with 10 or more sugar residues is produced, The above oligosaccharide mixture was subjected to mass spectrometry using a MALDI-TOF-MS (matrix-assisted laser desorption / ionization-time-of-flight) mass spectrometer. The degree of polymerization, type, and number of substituents of the oligosaccharides contained in the above oligosaccharide mixture are analyzed with high resolution and high sensitivity. Methods for analyzing oligosaccharides. [3] The method for producing oligosaccharides according to [1], wherein the polysaccharide is at least one selected from the group consisting of chitosan, chitin, cellulose, xylan, mannan, glucomannan, and starch. [4] The method for producing oligosaccharides according to any one of [1] to [3], wherein the oligosaccharide is a mixture of oligosaccharides with different degrees of polymerization. [5] A step of preparing an aqueous oligosaccharide solution containing a mixture of oligosaccharides with different degrees of polymerization, and The process involves adding alkali to the above-mentioned oligosaccharide aqueous solution to control the pH, and then precipitating the oligosaccharides from the above-mentioned oligosaccharide aqueous solution according to their degree of polymerization. A method for separating oligosaccharides, including [specific example]. [6] The above precipitation process is carried out in stages in the same container, A mixture of oligosaccharides with different degrees of polymerization is fractionated into oligosaccharides with a high degree of polymerization, oligosaccharides with a medium degree of polymerization, and oligosaccharides with a low degree of polymerization. The method for separating oligosaccharides described in [5]. [7] A step of preparing an oligosaccharide mixture or an aqueous solution thereof containing a mixture of oligosaccharides with different degrees of polymerization, The above oligosaccharide mixture or an aqueous solution thereof is mixed with a first organic solvent or a first mixed solvent containing the first organic solvent and water, and the mixture is separated into a first organic solvent extract and an insoluble residue of the first organic solvent. A method for separating oligosaccharides, including [specific example]. [8] A step of mixing the first organic solvent extract or the first organic solvent insoluble residue with a second organic solvent of a different type than the first organic solvent or a second mixed solvent containing the second organic solvent and water, and separating it into the second organic solvent extract and the second organic solvent insoluble residue. The method for separating oligosaccharides as described in [7], further comprising: [9] Chitosan oligosaccharide represented by the following formula (1). [ka] (In the formula, R is a hydrogen atom or an acyl group, m is an integer between 3 and 30, and n is an integer between 0 and 3.)

[10] The chitosan oligosaccharide described in [9], wherein R is an acyl group having 3 or more carbon atoms, n is an integer from 1 to 3, and m+n is an integer of 5 or more.

[11] The chitosan oligosaccharide described in

[10] , wherein m+n is an integer greater than or equal to 8. An antibacterial agent comprising at least one selected from the group consisting of chitosan oligosaccharides and their salts as described in any of

[12] [9] to

[11] as an active ingredient.

[13] Use of at least one selected from the group consisting of chitosan oligosaccharides and salts of any of [9] to

[11] in the manufacture of an antimicrobial agent. [Effects of the Invention]

[0033] The present invention provides a safer, simpler, and less expensive method for producing oligosaccharides compared to conventional methods. [Brief explanation of the drawing]

[0034] [Figure 1]This diagram illustrates the mechanism of glycosidic bond hydrolysis in polysaccharides. [Figure 2] This graph shows the mass spectrometry results of the chitosan oligosaccharide mixture prepared according to Experimental Example 1. The numbers at the top of the graph indicate the degree of polymerization. [Figure 3] This graph shows the mass spectrometry results of the agar oligosaccharide mixture prepared according to Experimental Example 1. [Figure 4] This graph shows the mass spectrometry results of the dextran oligosaccharide mixture prepared according to Experimental Example 1. [Figure 5] This graph shows the mass spectrometry results of the locust bean gum oligosaccharide mixture prepared according to Experimental Example 1. [Figure 6] This graph shows the mass spectrometry results of the starch oligosaccharide mixture prepared according to Experimental Example 1. [Figure 7] This graph shows the mass spectrometry results of chitosan oligosaccharides prepared, separated, and purified according to Experimental Example 2. From top to bottom, it shows the chitosan oligosaccharide mixture produced in Experimental Example 1, the precipitate from the first separation, the precipitate from the second separation, and the supernatant from the second separation, all of which were separated and purified in Experimental Example 2. The numbers at the top of the graph indicate the degree of polymerization. [Figure 8] This graph shows the mass spectrometry results of partially N-propanoylated chitosan oligosaccharides prepared, separated, and purified according to Experimental Example 3. From top to bottom, it shows the chitosan oligosaccharide mixture prepared in Experimental Example 1, the precipitate from the first separation, the precipitate from the second separation, and the supernatant from the second separation, all of which were separated and purified in Experimental Example 2. [DP,Pr] on the graph represents the degree of polymerization (DP) and the number of propanoyl groups (Pr) of the oligosaccharide. [Figure 9] This graph shows the mass spectrometry results of partially N-decanoylated chitosan oligosaccharides prepared according to Experimental Example 4. From top to bottom, it shows the chitosan oligosaccharide mixture produced in Experimental Example 1, the precipitate from the first separation, the precipitate from the second separation, and the supernatant from the second separation, all of which were separated and purified in Experimental Example 2. [DP,C10] on the graph represents the degree of polymerization (DP) and the number of decanoyl groups (C10) of the oligosaccharide. [Figure 10]This graph shows the mass spectrometry results of partially N-decanoylated chitosan oligosaccharides in Experimental Example 5. From top to bottom, it shows the product obtained by freeze-drying the chitosan solution remaining after preparative extraction, preparative (1), preparative (2), preparative (3), and preparative (4). [Figure 11] This graph shows the mass spectrometry results of partially N-decanoylated chitosan oligosaccharides in Experimental Example 6. From top to bottom, the graphs represent preparative samples (1), (2), (3), and (4). [Figure 12] This graph shows the MALDI-TOF / TOF measurement results for partially N-propanoylated chitosan oligosaccharides in Experimental Example 7. [Figure 13] This graph shows the MALDI-TOF / TOF measurement results for partially N-decanoylated chitosan oligosaccharides in Experimental Example 7. [Figure 14] This graph shows the antimicrobial test results for Bacillus atrophaeus ATCC 51189 in Experimental Example 8. [Figure 15] This graph shows the antibacterial test results for Staphyrococcus epidermidis ATCC 14490 in Experimental Example 8. [Figure 16] This graph shows the antimicrobial test results for Corynebacterium tuberculostearicum NBRC 113182 in Experimental Example 8. [Figure 17] This is a photographic representation of the results of the antimicrobial test of Prototheca zopfii NBRC 6998 in Experimental Example 8. [Figure 18] This graph shows the antimicrobial test results for Staphylococcus aureus ATCC 6538P in Experimental Example 8. [Figure 19] This graph shows the antimicrobial test results for Serratia marcescens ATCC 13380 in Experimental Example 8. [Figure 20] This diagram illustrates the acylation procedure of chitosan in Experimental Example 9. [Figure 21] This diagram illustrates the oligosaccharide treatment procedure of acylated chitosan in Experimental Example 9. [Figure 22] This graph shows the MALDI-TOF-MS measurement results for samples with 8 carbon atoms in the acyl group (C8_1, C8_2, C8_3) in Experimental Example 9. [Figure 23] This graph shows the MALDI-TOF-MS measurement results for samples with 10 carbon atoms in the acyl group (C10_1, C10_2, C10_3, C10_4) in Experimental Example 9. [Figure 24] This graph shows the MALDI-TOF-MS measurement results for samples with 12 carbon atoms in the acyl group (C12_1, C12_2, C12_3, C12_4) in Experimental Example 9. [Figure 25] This graph shows the results of the antibacterial activity evaluation against Bacillus atrophaeus (ATCC 51189) in Experiment Example 9. [Figure 26] This graph shows the results of the antibacterial activity evaluation against Staphylococcus epidermidis (ATCC 14990) in Experiment Example 9. [Figure 27] This graph shows the results of the antimicrobial activity evaluation against Enterococcus hirae (NBRC 113030) in Experiment Example 9. [Figure 28] This graph shows the results of the antimicrobial activity evaluation against Escherichia coli (ATCC 700728) in Experimental Example 9. [Figure 29] This diagram illustrates the preparation procedure for the C10 COS sample in Experimental Example 10. [Figure 30] This graph shows the MALDI-TOF-MS measurement results for the C10 COS sample in Experimental Example 10. [Figure 31] This graph shows the results of the antibacterial activity evaluation against Bacillus atrophaeus (ATCC 51189) in Experimental Example 10. [Figure 32] This graph shows the results of the antibacterial activity evaluation against Staphylococcus epidermidis (ATCC 14990) in Experimental Example 10. [Figure 33] This graph shows the results of the antibacterial activity evaluation against Staphylococcus aureus (ATCC 6538P) in Experimental Example 10. [Figure 34] This graph shows the results of the antimicrobial activity evaluation against Escherichia coli (ATCC 700728) in Experimental Example 10. [Figure 35] This diagram illustrates the procedure used in Experimental Example 11. [Figure 36] This graph shows the results of organic solvent fractionation using MeOH in Experimental Example 11. [Figure 37] This graph shows the results of organic solvent fractionation using Et:W(9:1) in Experimental Example 11. [Figure 38] This graph shows the results of organic solvent fractionation using Et:W(8:2) in Experimental Example 11. [Figure 39] This graph shows the results of organic solvent fractionation using IPA:W (8:2) in Experimental Example 11. [Figure 40] This diagram illustrates the operating procedure in Experimental Example 12. [Figure 41] This graph shows the MALDI-TOF-MS measurement results in Experiment Example 12. [Figure 42] This diagram illustrates the operating procedure in Experimental Example 13. [Figure 43] This graph shows the weight loss rate and solubilization yield of each raw material in Experimental Example 13. [Figure 44] This graph shows the MALDI-TOF-MS measurement results for a cotton-derived oligosaccharide mixture in Experimental Example 13. [Figure 45] This graph shows the MALDI-TOF-MS measurement results for a corrugated cardboard-derived oligosaccharide mixture in Experimental Example 13. [Figure 46]This graph shows the MALDI-TOF-MS measurement results for the oligosaccharide mixture derived from disposable chopsticks in Experimental Example 13. [Figure 47] This diagram illustrates the operating procedure in Experimental Example 14. [Figure 48] This graph shows the weight loss rate and solubilization yield of each raw material in Experimental Example 14. [Figure 49] This graph shows the MALDI-TOF-MS measurement results for the bamboo leaf-derived oligosaccharide mixture in Experimental Example 14. [Figure 50] This graph shows the MALDI-TOF-MS measurement results for the pine needle-derived oligosaccharide mixture in Experimental Example 14. [Figure 51] This graph shows the MALDI-TOF-MS measurement results for the pine cone-derived oligosaccharide mixture in Experimental Example 14. [Figure 52] This diagram illustrates the hot water extraction procedure for each raw material in Experimental Example 15. [Figure 53] This graph shows the yield of the hot water extract of each raw material in Experimental Example 15. [Figure 54] This diagram illustrates the procedure for oligosaccharide conversion of the hot water extraction residues of each raw material in Experimental Example 15. [Figure 55] This graph shows the extraction efficiency of solubilized substances from each raw material in Experimental Example 15. [Figure 56] This graph shows the MALDI-TOF-MS measurement results for the oligosaccharide mixture derived from Reishi mushroom (from China) in Experimental Example 15. [Figure 57] This graph shows the MALDI-TOF-MS measurement results for the oligosaccharide mixture derived from Reishi mushroom (Japanese origin) in Experimental Example 15. [Figure 58] This graph shows the MALDI-TOF-MS measurement results of the Tremella fuciformis-derived oligosaccharide mixture in Experimental Example 15. [Figure 59] This graph shows the MALDI-TOF-MS measurement results for the shiitake mushroom-derived oligosaccharide mixture in Experimental Example 15. [Figure 60] This graph shows the MALDI-TOF-MS measurement results for a green tea-derived oligosaccharide mixture in Experimental Example 15. [Figure 61] This graph shows the MALDI-TOF-MS measurement results for a coffee-derived oligosaccharide mixture in Experimental Example 15. [Figure 62] This diagram illustrates the operating procedure in Experimental Example 16. [Figure 63] This graph shows the weight loss rate of the raw materials and the yield of solubilized components for each HCl concentration in Experimental Example 16. [Figure 64] This graph shows the MALDI-TOF-MS measurement results for an oligosaccharide mixture (HCl concentration 0.1% by mass) derived from paper-based raw materials (Kimwipes) in Experimental Example 16. [Figure 65] This graph shows the MALDI-TOF-MS measurement results for an oligosaccharide mixture (HCl concentration 0.9% by mass) derived from paper-based raw materials (Kimwipes) in Experimental Example 16. [Modes for carrying out the invention]

[0035] The embodiments of the present invention will be described in detail below, but the present invention is not limited to the embodiments described later, and various modifications are possible as long as they do not depart from the spirit of the invention.

[0036] In this invention, "A~B" indicating a numerical range includes the numerical values ​​A and B written before and after the "~" as the lower limit and upper limit, or the upper limit and lower limit. For example, "1~10" means a numerical range of 1 to 10.

[0037] In this invention, "monomer" means a compound that can form a polymer by addition polymerization, ring-opening polymerization, condensation polymerization, etc. "Polymer" is a compound formed by the polymerization of two or more monomers. "Residue" is a general term for an atomic group directly formed by the polymerization of one monomer molecule, and an atomic stage obtained by chemically transforming a part of that atomic group. Residues based on specific monomers may be written by adding "residue" to the name or abbreviation of the specific monomer. For example, N-acetylglucosamine and glucosamine are monomers, chitin and chitosan are polymers, the residues constituting chitin include N-acetylglucosamine residues, and the residues constituting chitosan include glucosamine residues. Note that partially deacetylated chitin, obtained by partially deacetylating chitin, and partially acetylated chitosan, obtained by partially acetylating chitosan, may be the same compound.

[0038] The present inventors have succeeded in producing oligosaccharides from polysaccharides in a safe, simple, and inexpensive method compared to conventional methods, by dissolving the polysaccharide in an aqueous solution of a volatile strong acid to prepare an aqueous solution of the polysaccharide, and then concentrating and drying the aqueous solution of the polysaccharide. Furthermore, using the oligosaccharide production method of the present invention, they succeeded in producing an oligosaccharide mixture containing oligosaccharides with 10 or more sugar residues, and by performing mass spectrometry of the oligosaccharide mixture using MALDI-TOF / TOF-MS or MALDI-TOF-MS, they succeeded in analyzing with high sensitivity the degree of polymerization and the type and number of substituents of the oligosaccharides contained in the oligosaccharide mixture. In addition, they succeeded in separating and purifying oligosaccharides with different molecular weight distributions by gently neutralizing the oligosaccharides produced by the oligosaccharide production method of the present invention in an aqueous solution and stepwise precipitating and separating oligosaccharides with a predetermined degree of polymerization (hereinafter sometimes referred to as the "neutralization precipitation method"). Furthermore, by using a method of molecular weight fractionation with an organic solvent (hereinafter sometimes referred to as "organic solvent fractionation") to fractionate oligosaccharides produced by the present invention, we succeeded in separating and purifying oligosaccharides with different molecular weight distributions. It was shown that the chitosan oligosaccharide produced by the present invention is mainly composed of a single acyl group introduced to the reducing amino group. In addition, an antibacterial test was conducted on the chitosan oligosaccharide with a long-chain acyl group introduced, and it was revealed that it has a much higher antibacterial activity than conventional chitosan oligosaccharide.

[0039] Polysaccharides have numerous oxygen atoms that can act as hydrogen bond acceptors, and are expected to hold protons, which act as acid sites, more stably than the hydration effect of water. When polysaccharides are present in an aqueous solution, as shown in Figure 1, some of the hydrogen ions from strong acids such as hydrogen chloride move from the water molecule to the sugar hydroxyl group or acetal oxygen. The hydrogen ions that have moved onto the acetal oxygen function as acid sites for glycosidic bond hydrolysis, as shown in the reaction equation below, and hydrolysis proceeds via the sugar oxonium ion.

[0040] When hydrochloric acid is concentrated in the presence of polysaccharides in an aqueous solution, it is expected that complex salts of volatile acids such as hydrogen chloride will accumulate on the non-volatile sugars. However, to our knowledge, there are no methods for producing oligosaccharides that utilize this concentration effect.

[0041] The present inventors have developed a method for dissolving non-volatile polysaccharides in a volatile solvent containing a volatile strong acid. As a result, strong acids form complexes with more stable polysaccharides, and as the solvent concentrates, strong acids are produced on the polysaccharide. The acid becomes concentrated and exhibits hydrolytic activity, and this hydrolytic activity is stopped due to the volatility of the strong acid. And, they discovered that oligosaccharides are produced.

[0042] [Method for producing oligosaccharides] A method for producing oligosaccharides according to one aspect of the present invention (hereinafter also simply referred to as "the oligosaccharide production method of the present invention") includes a step of dissolving a polysaccharide in an aqueous solution of a volatile strong acid to prepare an aqueous solution of the polysaccharide (hereinafter also referred to as "polysaccharide aqueous solution") (hereinafter also referred to as "polysaccharide aqueous solution preparation step"), and a step of concentrating and drying the polysaccharide aqueous solution (hereinafter also referred to as "concentration and drying step").

[0043] The polysaccharide described above is not particularly limited as long as it is a polymer formed by linking 11 or more monosaccharides by glycosidic bonds. The glycosidic bonds may be either α-glycosidic bonds or β-glycosidic bonds, and α-glycosidic bonds and β-glycosidic bonds may be mixed within the polymer molecule. Furthermore, the polysaccharide may be linear or branched.

[0044] Examples of the above polysaccharides include agar (mainly composed of agarose), dextran (with glucose as its sole component and containing many α-1,6-glycosidic bonds), dextrin, locust bean gum (galactomannan with one galactose side chain for every four linearly linked mannose molecules), tara gum (galactomannan with one galactose side chain for every three linearly linked mannose molecules), and guar gum (with two linearly linked mannose molecules). Galactomannan (which has one galactose molecule as a side chain), tamarind seed gum (xyloglucan, which has a main chain made of glucose and side chains made of xylose and galactose), gum arabic (polyuronic acid, which has a main chain made of galactose and side chains made of galactose, arabinose, rhamnose, and glucuronic acid), starch (a polymer composed of glucose as a constituent monosaccharide, consisting of amylose, a linear polymer in which glucose is linked by α1→4 bonds, and amylopectin, a branched polymer linked by α1→6 bonds), cellulose (a polymer in which β-glucose is polymerized in a linear manner by glycosidic bonds), xylan (a heterosaccharide in which various side chains are linked to a main chain of xylose linked by β1-4 bonds), glucomannan (a polymer in which glucose and mannose are polymerized in a ratio of approximately 2:3 by β-1,4-glycosidic bonds), mannan (a polysaccharide made of mannose. Yeast-derived is linked by α-1,2, 1,3, 1,6 glycosidic bonds, coffee-derived is Examples of polysaccharides include, but are not limited to, those polymerized by β-1,4 glycosidic bonds, β-1,3-glucan (a polymer of glucose polymerized by β-1,3 bonds), pectin (polygalacturonic acid), carrageenan (a polysaccharide consisting of repeating units of D-galactose: kappa-carrageenan, iota-carrageenan, lambda-carrageenan), chitin (poly-β1-4-N-acetylglucosamine), and chitosan (poly-β1→4-glucosamine). As the above polysaccharides, at least one selected from the group consisting of chitin and chitosan is preferred, and chitosan is more preferred. Note that xylan, mannan, and glucomannan are sometimes collectively referred to as hemicellulose.

[0045] Chitosan is not particularly limited as long as it is composed of several to tens of monomeric glucosamine linked together by β1→4 bonds, and some of the glucosamine residues constituting the polymer chitosan may be N-acylated N-acylated glucosamine. When the total amount of glucosamine residues and N-acylated glucosamine residues constituting chitosan is 100 mol%, the proportion of N-acylated glucosamine residues (degree of acylation) is preferably 50 mol% or less, more preferably 40 mol% or less, and even more preferably 30 mol% or less. The lower limit of the degree of acylation is usually 0 mol%.

[0046] When chitosan contains N-acetylglucosamine as a polymer building block, it is sometimes specifically referred to as chitin. If the total amount of N-acetylglucosamine residues and glucosamine residues constituting chitin is 100 mol%, then the proportion of N-acetylglucosamine residues is greater than 0 mol% and less than or equal to 100 mol%.

[0047] The molecular weight and degree of polymerization of the above-mentioned chitosan and chitin are not particularly limited.

[0048] The origin of the above chitosan and chitin is not particularly limited and may be any of the following: the exoskeleton of crabs or shrimp, the backbone of squid, fungi (including molds, yeasts, and mushrooms, which mainly contain chitin in their cell walls), bacteria (which mainly contain N-acetylglucosamine as a component of peptidoglycan in their cell walls), or the exoskeleton of insects. As the chitosan mentioned above, you can also use one produced by deacetylating the chitin mentioned above. As a source of the chitosan or chitin mentioned above, food or food residue containing the chitosan or chitin may also be used.

[0049] The origin of the cellulose mentioned above is not particularly limited, and various materials such as paper, pulp, wood, bamboo, grass, vegetables, cotton, paper mulberry, mitsumata, etc. can be used. Furthermore, food or food residues containing cellulose can also be used as a source of the cellulose. When the polysaccharide is cellulose, the oligosaccharide production method of the present invention can be used to produce, for example, cellooligosaccharides.

[0050] The origin of the starch is not particularly limited, and various materials such as rice and potatoes can be used. Furthermore, starch-containing foods or food residues can also be used as the source of the starch. When the polysaccharide is starch, the oligosaccharide production method of the present invention can be used to produce, for example, maltooligosaccharide (starch oligosaccharide).

[0051] The origin of the above-mentioned xylan is not particularly limited, and various materials can be used, such as broad-leaved trees (glucuronoxylan), coniferous trees, and grasses (arabinoxylan). When the polysaccharide is xylan, the oligosaccharide production method of the present invention can be used to produce, for example, xylooligosaccharides.

[0052] The origin of the mannan mentioned above is not particularly limited; for example, beans such as coffee beans can also be used. When the polysaccharide is mannan, for example, manno-oligosaccharides can be produced by the oligosaccharide production method of the present invention.

[0053] The origin of the glucomannan mentioned above is not particularly limited; various materials can be used, such as coniferous trees and konjac potatoes. When the polysaccharide is glucomannan, the oligosaccharide production method of the present invention can be used to produce, for example, glucomannan oligosaccharide.

[0054] The origin of the above-mentioned β-1,3-glucan is not particularly limited, and for example, mushrooms (such as reishi, shiitake, and wood ear mushrooms) can be used. When the polysaccharide is β-1,3-glucan, the oligosaccharide production method of the present invention can be used to produce, for example, β-1,3-glucan oligosaccharide.

[0055] The above-mentioned volatile strong acid is not particularly limited, but it is preferably an acid that leaves no solid residue after evaporation of its aqueous solution, and has an acid dissociation constant pKa of 3.00 or less in water. The acid dissociation constant pKa of the above-mentioned volatile strong acid in water is preferably 2.00 or less, more preferably 1.00 or less, even more preferably -1.50 or less, and even more preferably -3.00 or less.

[0056] Examples of the volatile strong acids mentioned above include, but are not limited to, chloroacetic acid (CH2ClCOOH), hydrofluoric acid (HF), dichloroacetic acid (CHCl2COOH), trichloroacetic acid (CCl3COOH), nitric acid (HNO3), hydrochloric acid (HCl), hydrobromic acid (HBr), and perchloric acid (HClO4). The volatile strong acid is preferably one selected from the group consisting of nitric acid, hydrochloric acid, hydrobromic acid, and perchloric acid; more preferably one selected from the group consisting of nitric acid, hydrochloric acid, and hydrobromic acid; even more preferably hydrochloric acid or hydrobromic acid; and even more preferably hydrochloric acid.

[0057] In the above polysaccharide aqueous solution preparation step, the polysaccharide is dissolved in the aqueous solution of the above volatile strong acid to prepare the polysaccharide aqueous solution.

[0058] The method for dissolving the above polysaccharide in the aqueous solution of the above volatile strong acid is not particularly limited, and examples include mixing the above polysaccharide with the aqueous solution of the above volatile strong acid and stirring.

[0059] The temperature of the aqueous solution of the volatile strong acid used to dissolve the polysaccharide is not particularly limited, but is preferably 0 to 100°C, more preferably 5 to 95°C, even more preferably 5 to 80°C, and even more preferably 5 to 50°C.

[0060] The concentration of the volatile strong acid in the aqueous solution of the volatile strong acid is not particularly limited, but it is preferable to keep it low in order to dissolve polysaccharides such as chitosan and to avoid deterioration or corrosion of the equipment during concentration and drying. A concentration of 0.01 to 1% by mass is preferred, 0.1 to 1% by mass is more preferred, and 0.1 to 0.5% by mass is even more preferred.

[0061] Furthermore, the pH of the aqueous solution of the above-mentioned volatile strong acid at 25°C is not particularly limited, but is preferably 4.0 or less, and more preferably 2.0 or less, due to the high solubility of polysaccharides such as chitosan.

[0062] The concentration of the polysaccharide in the above-mentioned aqueous polysaccharide solution is not particularly limited, but considering the productivity of oligosaccharides and the decrease in operability due to the increase in viscosity after polysaccharide dissolution, 0.01 to 4.0% by mass is preferred, 0.05 to 2.0% by mass is more preferred, and 0.1 to 2.0% by mass is even more preferred.

[0063] In the above concentration and drying step, the polysaccharide aqueous solution prepared in the above polysaccharide aqueous solution preparation step is concentrated and dried. The method for concentrating and drying the polysaccharide aqueous solution is not particularly limited, and for example, natural drying, concentration and drying by blowing air or inert gas, concentration and drying by reduced pressure, concentration and drying by spray drying, concentration and drying by freeze-drying, etc., can be used.

[0064] When the above polysaccharide aqueous solution is concentrated and dried by freeze-drying, the freezing temperature is not particularly limited and should be any temperature at which the polysaccharide aqueous solution completely freezes. The temperature inside the container and trap during the freeze-drying process is not particularly limited and can be, for example, -20°C or lower. The pressure inside the container and trap during the freeze-drying process is also not particularly limited and can be, for example, 100 Pa or lower.

[0065] The oligosaccharides produced by the oligosaccharide production method of the present invention are usually a mixture of several types of oligosaccharides with different sugar residue counts, i.e., degrees of polymerization, and it is preferable that the mixture contains oligosaccharides with 10 or more sugar residue counts.

[0066] When the above polysaccharide is chitosan or chitin, and the above oligosaccharide is chitosan oligosaccharide, regioselectivity of the N-acylated glucosamine residue is observed in the resulting chitosan oligosaccharide.

[0067] In the oligosaccharide production method of the present invention, since the hydrolysis treatment is carried out under acidic conditions in the concentration and drying step described above, all free amino groups are protonated and generate a positive charge (quaternary ammonium cation; -NH3 + ). Due to this positive charge and hydrolysis, the acidic component, protonated water (oxonium ion, H3O + Because electrostatic repulsion occurs between the protons, the glycosidic bond of the N-acylated glucosamine residue preferentially attacks the proton nucleophilically, leading to hydrolysis (see formula below; where R represents the acyl group). Under concentrated hydrochloric acid conditions, the charge density is high, resulting in a smaller Debye length and lower selectivity, while under dilute hydrochloric acid conditions, the Debye length increases and selectivity improves. Under the conditions of the experimental examples described later, the regioselectivity of this hydrolysis is about 70%.

[0068] [ka]

[0069] [Methods for analyzing oligosaccharides] A method for analyzing oligosaccharides according to one embodiment of the present invention (hereinafter also simply referred to as "the analytical method of the present invention") includes the steps of: producing an oligosaccharide mixture containing oligosaccharides with 10 or more sugar residues by the oligosaccharide production method of the present invention described above (hereinafter also referred to as "oligosaccharide production step"); and performing mass analysis of the oligosaccharide mixture using a MALDI-TOF / TOF (matrix-assisted laser desorption / ionization-time-of-flight) mass spectrometer or a MALDI-TOF (matrix-assisted laser desorption / ionization-time-of-flight) mass spectrometer (hereinafter also referred to as "mass spectrometry step"), thereby analyzing the degree of polymerization and the type and number of substituents of the oligosaccharides contained in the oligosaccharide mixture with high resolution and high sensitivity.

[0070] Specific measurement conditions for the MALDI-TOF / TOF mass spectrometer or MALDI-TOF mass spectrometer in the mass spectrometry step of the analytical method of the present invention include, for example, dissolving the target to be measured in ion-exchanged water to a concentration of about 4 mg / mL and mixing it with an equal volume of BOA matrix (BOA / DHB / NaHCO3 (molar ratio 12:10:1)) or NMA matrix (NMA / DHB / NaHCO3 (molar ratio 12:10:1)). 1 μL of the mixed solution is dropped onto a MALDI-TOF / TOF-MS measurement plate or MALDI-TOF-MS measurement plate and allowed to stand at room temperature overnight or in a 60°C oven for about 30 minutes to completely evaporate the solvent. The MALDI-TOF / TOF-MS measurement plate or MALDI-TOF-MS measurement plate is inserted into the measuring device and MALDI-TOF / TOF-MS measurement or MALDI-TOF-MS measurement is performed in reflect mode / positive ion mode. For MALDI-TOF / TOF-MS or MALDI-TOF-MS measurements, the BOA matrix is ​​preferred over the NMA matrix because it yields more sensitive measurement results. On the other hand, when using the BOA matrix for MALDI-TOF / TOF or MALDI-TOF-MS measurements, the BOA tag initially fragments, which hinders the identification of the acyl group's position; therefore, the NMA matrix is ​​more preferable.

[0071] [Method for separating oligosaccharides - Neutralization precipitation method -] A method for separating oligosaccharides according to one aspect of the present invention (hereinafter also simply referred to as "the oligosaccharide separation method of the present invention" or "neutralization precipitation method") includes a step of preparing an aqueous oligosaccharide solution containing a mixture of oligosaccharides with different degrees of polymerization (hereinafter also referred to as the "preparation step"), and a step of adding alkali to the aqueous oligosaccharide solution to control the pH and precipitating the oligosaccharides from the aqueous oligosaccharide solution according to their degree of polymerization (hereinafter also referred to as the "precipitation step").

[0072] In the above preparation step, an aqueous oligosaccharide solution containing a mixture of oligosaccharides with different degrees of polymerization is prepared.

[0073] The above mixture of oligosaccharides with different degrees of polymerization can be obtained, for example, from polysaccharides by the oligosaccharide production method of the present invention described above.

[0074] An aqueous oligosaccharide solution is prepared by dissolving a mixture of oligosaccharides with different degrees of polymerization in water. For example, ion-exchanged water or deionized distilled water can be used as the water.

[0075] The concentration of the mixture of oligosaccharides with different degrees of polymerization in the above-mentioned aqueous oligosaccharide solution is not particularly limited, but if the amount of precipitate that precipitates due to the increase in solution viscosity and alkali addition becomes too large, uniform stirring becomes difficult. Therefore, 0.1 to 10% by mass is preferred, 0.5 to 2.0% by mass is more preferred, and 1.0 to 2.0% by mass is even more preferred.

[0076] After dissolving a mixture of oligosaccharides with different degrees of polymerization in water, alkali is gradually added to adjust the pH, causing the oligosaccharides to precipitate gradually. As the pH of the aqueous solution increases, oligosaccharides with higher degrees of polymerization precipitate first.

[0077] The alkali is not particularly limited as long as it can adjust the pH of the aqueous solution of the oligosaccharide mixture. For example, sodium hydroxide, potassium hydroxide, sodium bicarbonate, triethylamine, etc., can be used. Sodium hydroxide is particularly preferred from the viewpoint of neutralizing salts when the aqueous solution is acidic with hydrochloric acid.

[0078] The concentration of the alkali used to adjust the pH of the above oligosaccharide aqueous solution is not particularly limited, but 0.1 to 2.0 mol / L is preferred, and 0.5 to 1.0 mol / L is more preferred.

[0079] The pH range for stepwise precipitation of oligosaccharides with different degrees of polymerization from the above-mentioned oligosaccharide aqueous solution is not particularly limited, but 5 to 9 is preferred, and 6 to 8 is more preferred. The oligosaccharides precipitated from the aqueous solution are preferably separated by centrifuge or filter. The separation conditions are not particularly limited as long as they can completely separate the precipitate. After the first precipitation separation, it is preferable to further increase the pH by adding alkali to the supernatant or filtrate and precipitate the oligosaccharides again. The oligosaccharides precipitated again are separated by centrifuge or filter to obtain the second precipitate. Stepwise precipitation is obtained by repeating this process. Each precipitate, such as the first precipitate and the second precipitate, is dried, and the final supernatant or filtrate is concentrated and dried. The drying and concentration conditions are not particularly limited, but can be carried out in the same manner as the concentration and drying treatment method described above.

[0080] In the oligosaccharide separation method of the present invention, the above precipitation step is carried out stepwise in the same container, and a mixture of oligosaccharides with different degrees of polymerization can be fractionated into oligosaccharides with a high degree of polymerization, oligosaccharides with a medium degree of polymerization, and oligosaccharides with a low degree of polymerization.

[0081] [Method for separating oligosaccharides - Organic solvent fractionation method -] A method for separating oligosaccharides according to another aspect of the present invention (hereinafter also simply referred to as the "organic solvent fractionation method") includes the steps of: preparing a mixture of oligosaccharides with different degrees of polymerization or an aqueous solution thereof (hereinafter also referred to as the "first preparation step"); adding a first organic solvent or a first mixed solvent containing a first organic solvent and water to the oligosaccharide mixture or aqueous solution and mixing to prepare a first mixed solution (hereinafter also referred to as the "first organic solvent mixing step"); and separating the first mixed solution into a first organic solvent extract and a first organic solvent insoluble residue (hereinafter also referred to as the "first separation step").

[0082] In the first preparation step described above, a mixture of chitosan oligosaccharides with different degrees of polymerization or an aqueous solution thereof is prepared.

[0083] The above mixture of oligosaccharides with different degrees of polymerization can be obtained, for example, from polysaccharides by the oligosaccharide production method of the present invention described above.

[0084] The aqueous solutions of oligosaccharides with different degrees of polymerization described above are prepared by dissolving a mixture of oligosaccharides with different degrees of polymerization in water. For example, ion-exchanged water or deionized distilled water can be used as the water.

[0085] The concentration of the mixture of oligosaccharides with different degrees of polymerization in an aqueous solution of oligosaccharides with different degrees of polymerization is not particularly limited, but from the viewpoint of improving the yield of oligosaccharides, 0.1 to 10% by mass is preferred, 0.5 to 2.0% by mass is more preferred, and 1.0 to 2.0% by mass is even more preferred.

[0086] A first organic solvent or a first mixed solvent containing the first organic solvent and water is added to the mixture of oligosaccharides with different degrees of polymerization or an aqueous solution thereof to dissolve the oligosaccharides soluble in the first organic solvent in the first organic solvent. Then, the organic solvent containing the oligosaccharides soluble in the first organic solvent (first organic solvent extract) and the residue containing the oligosaccharides insoluble in the first organic solvent (first organic solvent insoluble residue) are separated. Separation of the first organic solvent extract (liquid) and the organic solvent insoluble residue is preferably done by centrifugation or filtration. When separating by centrifugation, it is preferable to recover the liquid phase and solid phase by means of decantation, suction, etc. Alternatively, the first organic solvent or the first mixed solvent containing the first organic solvent and water may be added directly to the mixture of oligosaccharides with different degrees of polymerization, or the first organic solvent or the first mixed solvent containing the first organic solvent and water may be added to an aqueous solution of the mixture of oligosaccharides with different degrees of polymerization. By drying the first organic solvent extract and the first organic solvent-insoluble residue, respectively, to remove the solvent, the oligosaccharides contained in the first organic solvent extract fraction and the oligosaccharides contained in the first organic solvent-insoluble residue fraction can be obtained as solids. The operation of further separating the first organic solvent extract and / or the first organic solvent-insoluble residue into a second organic solvent extract soluble in the second organic solvent or a second mixed solvent containing the second organic solvent and water, may be repeated using an organic solvent of a different type than the first organic solvent (second organic solvent) or a second mixed solvent containing the second organic solvent and water.

[0087] The above organic solvent is not particularly limited as long as it can dissolve oligosaccharides, but alcohols are preferred, and monohydric lower alcohols are preferred. The above monohydric lower alcohol is preferably an alcohol with 5 or fewer carbon atoms, and more preferably at least one selected from the group consisting of methanol (methyl alcohol), ethanol (ethyl alcohol), 1-propanol (n-propyl alcohol), 2-propanol (isopropyl alcohol), 1-butanol (n-butyl alcohol), 2-methyl-1-propanol (isobutyl alcohol), 2-butanol (sec-butyl alcohol), 2-methyl-2-propanol (tert-butyl alcohol), 1-pentanol (n-amyl alcohol), 2-pentanol (2-amyl alcohol), 3-pentanol, 2-methyl-1-butanol, 3-methyl-1-butanol (isoamyl alcohol), 2-methyl-2-butanol (tert-amyl alcohol), 3-methyl-2-butanol, and 2,2-dimethyl-1-propanol (neopentyl alcohol). Instead of the organic solvents mentioned above, a mixed solvent consisting of an organic solvent and water may be used. For example, a mixed solvent with a volume ratio of organic solvent to water in the range of 9:1 to 1:9 may be used.

[0088] The mixing ratio of the above organic solvent or the above mixed solvent to the aqueous solution of the mixture of oligosaccharides with different degrees of polymerization is not particularly limited and can be adjusted as appropriate depending on the organic solvent used. From the viewpoint of improving the yield of oligosaccharides, the amount of the above organic solvent or the above mixed solvent used is preferably in the range of 40 to 99% by volume, more preferably in the range of 60 to 95% by volume, and even more preferably in the range of 70 to 90% by volume, based on 100% by volume of the aqueous solution of the mixture of oligosaccharides with different degrees of polymerization.

[0089] The mixing ratio of the above organic solvent or the above mixed solvent to the mixture of oligosaccharides with different degrees of polymerization is not particularly limited and can be adjusted as appropriate depending on the organic solvent used. From the viewpoint of improving the yield of oligosaccharides, the amount of the above organic solvent or the above mixed solvent used is preferably in the range of 40 to 99% by mass, more preferably in the range of 60 to 95% by mass, and even more preferably in the range of 70 to 90% by mass, based on 100% by mass of the mixture of oligosaccharides with different degrees of polymerization.

[0090] The method for mixing the above-mentioned chitosan oligosaccharide aqueous solution with the organic solvent is not particularly limited, but examples include mixing by a shaker or stirrer.

[0091] The method for separating the above solution into an extract (organic solvent extract) and a residue (organic solvent insoluble matter) is not particularly limited, but examples include separation by centrifugation or filtration.

[0092] The method for drying the above extract and residue is not particularly limited, but examples include drying by heat drying, air drying, or freeze drying. The temperature for heat drying is not particularly limited, but can be in the range of 30 to 100°C, for example.

[0093] The above organic solvent fractionation method allows for the separation and purification of chitosan oligosaccharides with different molecular weight distributions by utilizing the differences in their solubility in organic solvents.

[0094] The above-described neutralization precipitation method and the above-described organic solvent fractionation method may be used individually or in combination as appropriate.

[0095] [Chitosan Oligosaccharide] A chitosan oligosaccharide according to one aspect of the present invention (hereinafter simply referred to as "the chitosan oligosaccharide of the present invention") is a compound represented by the following formula (1).

[0096] [ka]

[0097] In equation (1), R, m, and n have the following meanings. R is an acyl group, preferably an acyl group having 3 or more carbon atoms, more preferably an acyl group having 8 to 12 carbon atoms, and even more preferably an acyl group having 10 carbon atoms. An example of an acyl group with two carbon atoms is the ethanolyl group (acetyl group). Examples of acyl groups having three carbon atoms include the propanoyl group and the propa-2-enoyl group (acryloyl group). Examples of acyl groups with four carbon atoms include the butanoyl group, the isobutanoyl group, and the 2-methylpropa-2-enoyl group (methacryloyl group). Examples of acyl groups having 5 carbon atoms include the pentanoyl group, 2,2-dimethylpropanoyl group, 3-methylbutanoyl group, and (Z)-2-methylbuta-2-enoyl group. An example of an acyl group with six carbon atoms is the hexanoyl group. Examples of acyl groups with 7 carbon atoms include the heptanoyl group and the benzoyl group. An example of an acyl group with eight carbon atoms is the octanoyl group. An example of a carbon-9 acyl group is the nonanoyl group. An example of an acyl group with 10 carbon atoms is the decanoyl group. An example of an acyl group with 11 carbon atoms is the undecanoyl group. An example of an acyl group with 12 carbon atoms is dodecanoyl.

[0098] m is an integer between 2 and 30, preferably between 5 and 30, more preferably between 5 and 20, and even more preferably between 5 and 10. n is an integer between 1 and 3, preferably 1 or 2, and more preferably 1. m+n is an integer between 3 and 33, preferably between 3 and 12, more preferably between 5 and 12, even more preferably between 8 and 12, and even more preferably between 8 and 10.

[0099] If n is 2 or greater, the acyl groups of the N-acylated glucosamine residues may be different from each other.

[0100] The order in which glucosamine residues and N-acylated glucosamine residues are attached is not particularly limited. For example, the order in which glucosamine residues and N-acylated glucosamine residues are attached may be random, in tandem, or divided into blocks of glucosamine residues and blocks of N-acylated glucosamine residues.

[0101] The N-acylated glucosamine residue is preferably located at the terminal end of the chitosan oligosaccharide chain of the present invention. In particular, it is preferable that the carbon atom at position 1 of the N-acylated glucosamine residue does not participate in glycosidic bond formation.

[0102] [Antibacterial agent] An antibacterial agent according to one aspect of the present invention contains at least one selected from the group consisting of the chitosan oligosaccharides and salts of the present invention as described above, as an active ingredient.

[0103] The mechanism of action of the antibacterial agent of the present invention is not limited to that which may involve the positive charge derived from the amino group of chitosan oligosaccharide or its salt. Since the surface of microorganisms is negatively charged, it is thought that the interaction between chitosan oligosaccharide or its salt and the microorganism inhibits the growth of the microorganisms. Furthermore, when the acyl group of an acylated glucosamine residue is a decanoyl group, it exhibits stronger antibacterial activity compared to when the acyl group is an acetyl group. This mechanism of action is also thought to be related to the hydrophobicity of the acyl group, although it is not limited to this mechanism. Hydrophobic interactions occur due to the acyl group of an acylated glucosamine residue, and the decanoyl group, with 10 carbon atoms, is more likely to produce hydrophobic interactions than the acetyl group, with 3 carbon atoms. Furthermore, when acylated glucosamine residues are located at the end of the chitosan oligosaccharide, the terminal acyl group is more easily exposed, leading to even greater hydrophobic interactions. Additionally, because the acyl group does not disrupt the chitosan oligosaccharide's functional site, there is less interference with the chitosan oligosaccharide's antibacterial effect. Therefore, it can be expected that terminally modified chitosan oligosaccharides exhibit a dramatic improvement in their antibacterial effect.

[0104] Partially N-decanoylated chitosan oligosaccharides are preferred as the above-mentioned chitosan oligosaccharides. The degree of polymerization of the above-mentioned partial N-decanoylated chitosan oligosaccharide is not particularly limited, but is preferably 3 to 12, more preferably 3 to 9, and even more preferably 5 to 9. The number of N-decanoylglucosamine residues in the above-mentioned partial N-decanoylated chitosan oligosaccharide is not particularly limited, but 1 to 2 per molecule is preferred, and 1 per molecule is more preferred. The residues other than the N-decanoylglucosamine residue in the above-mentioned partially N-decanoylated chitosan oligosaccharide are preferably a glucosamine residue and at least one selected from N-acetylglucosamine residues and N-propanoylglucosamine residues, and more preferably a glucosamine residue. The above chitosan oligosaccharides are preferred in the form of salts, and more preferably in the form of salts of partially N-decanoylated chitosan oligosaccharides, because they have improved water solubility. As for the salts, hydrohalides are preferred, and hydrochlorides are more preferred.

[0105] The antibacterial agent of the present invention may contain at least one selected from the group consisting of chitosan oligosaccharides and their salts as an active ingredient, and there are no particular restrictions on the composition of the antibacterial agent, but it may also contain a resin, for example, from the viewpoint of supporting the antibacterial agent on a substrate.

[0106] The above resins are not particularly limited as long as the antibacterial properties of the antibacterial component obtained from the antibacterial agent are ensured, and examples include (meth)acrylic resins, polycarbonate resins, polyester resins, polypropylene resins, polystyrene resins, polyamide resins, urethane resins, epoxy resins, silicone resins, melamine resins, etc. Furthermore, various (meth)acrylate monomers can also be used. The above resins can be used individually or in combination of two or more types.

[0107] At least one compound selected from the group consisting of the above-mentioned chitosan oligosaccharide and its salts is preferably added in an amount of 0.1 to 30 parts by mass, more preferably 0.5 to 10 parts by mass, and even more preferably 1 to 5 parts by mass, per 100 parts by mass of the resin.

[0108] The antibacterial agent of the present invention may be used after being diluted with a solvent as needed. Examples of such solvents include water, alcohol-based solvents, carbitol-based solvents, ester-based solvents, ketone-based solvents, ether-based solvents, lactone-based solvents, unsaturated hydrocarbon-based solvents, cellosolve acetate-based solvents, carbitol acetate-based solvents, propylene glycol monomethyl ether acetate, and diethylene glycol dimethyl ether. These solvents can be used individually or in combination of two or more.

[0109] The antibacterial agent of the present invention may contain optional components other than those mentioned above, as long as they do not hinder the objective of the present invention. Examples of such optional components include leveling agents, dispersants, thickeners, defoaming agents, preservatives, coloring pigments, matting agents, and excipients.

[0110] The antibacterial agent of the present invention is preferably used as an antibacterial member obtained by coating it onto a substrate and drying and / or curing it.

[0111] The material of the above-mentioned substrate can be an organic polymer, ceramic, metal, glass, paper, decorative plywood, or a composite thereof. The shape of the substrate is not particularly limited and may be simple or complex in shape, such as a plate, sphere, cylinder, rod, prismatic, or hollow prismatic. The substrate may also be a porous material such as a filter. Among these, films made of transparent resins such as polyester resin, polycarbonate resin, triacetylcellulose resin, cycloolefin resin, acrylic resin, or styrene resin are preferred.

[0112] The above coating method is not particularly limited and includes coating methods such as spin coating, flow coating, spray coating, roll coating, dip coating, and inkjet printing, as well as printing methods such as relief printing, planographic printing, intaglio printing, and screen printing. Drying methods include, for example, heat drying, forced-air heat drying, and reduced-pressure drying. There are no particular restrictions on the method of heat drying, but examples include ovens, hot plates, infrared irradiation, and electromagnetic wave irradiation. Examples of forced-air heat drying methods include forced-air drying ovens.

[0113] Furthermore, the drying temperature and drying time can be set as appropriate during the drying process described above. For example, the drying temperature is preferably 60 to 120°C, and more preferably 70 to 100°C. The drying time is preferably 1 to 60 minutes, and more preferably 5 to 30 minutes. By setting the drying temperature and time within the above range, organic solvents can be sufficiently removed while suppressing the deterioration of the antibacterial agent's performance. If necessary, ultraviolet irradiation may be performed after drying the antibacterial component. This hardens the resulting coating film and increases its strength.

[0114] The dry film thickness of the coating containing the above antibacterial agent can be set appropriately depending on the application, but is preferably 0.1 to 1000 μm, more preferably 0.5 to 100 μm, and even more preferably 1 to 10 μm.

[0115] Furthermore, the antibacterial agent of the present invention may be used in any form, such as powder, granules, tablets, solutions, pastes, molded products, etc., in addition to the antibacterial components described above.

[0116] At least one selected from the group consisting of chitosan oligosaccharides and their salts can be used in the production of the antibacterial agent of the present invention as described above. [Examples]

[0117] The present invention will be explained in more detail below with experimental examples, but the present invention is not limited to the experimental examples described later.

[0118] [Experimental Example 1] Production of Oligosaccharides 10.0 g of chitosan (deacetylation degree: 100%, viscosity 40 mPa·s) was placed in a 5,000 mL beaker, and 0.3 mass% (≒0.084 mol / L) hydrochloric acid was added until the total volume reached 2,000.0 g. At room temperature, the mixture was stirred at 500 rpm using a magnetic stirrer with a polytetrafluoroethylene (hereinafter abbreviated as "PTFE") stirring bar to completely dissolve the chitosan. The resulting chitosan aqueous solution was poured into a tray and frozen overnight in a -30°C freezer. Then, freeze-drying was performed for 96 hours to obtain 10.2 g of chitosan oligosaccharide mixture.

[0119] The same method as described above was used to oligosaccharide conversion of polysaccharides. 100.0 mg of agar was placed in a 15 mL tube, and 0.3% by mass (≒0.084 mol / L) hydrochloric acid was added until the total volume was 10.0 g. The mixture was shaken at room temperature to completely dissolve the agar. The resulting agar aqueous solution was frozen overnight in a -30°C freezer. Freeze-drying was then performed for 24 hours to obtain an agar oligosaccharide mixture. Furthermore, the same method as described above was used to oligosaccharide conversion of agar with hydrochloric acid concentrations of 0.1% and 0.9% by mass. Furthermore, the same oligosaccharide conversion process was carried out for dextran, locust bean gum, and starch. Table 1 shows the yield and yield for oligosaccharide conversion of the above polysaccharides under different experimental conditions.

[0120] [Table 1]

[0121] The obtained oligosaccharide mixture was dissolved in ion-exchanged water to a concentration of 4 mg / mL and analyzed by MALDI-TOF-MS. A BOA matrix was used as the matrix. Tables 2 and 3 show the molecular weight of each chitosan oligosaccharide and the molecular weight of the oligosaccharides in each polysaccharide, respectively. Figure 2 shows the MALDI-TOF-MS analysis results of the chitosan oligosaccharide mixture. As shown in Figure 2, the chitosan oligosaccharide mixture prepared in Experimental Example 1 contained chitosan oligosaccharides with at least a degree of polymerization of 4 to 23, all of which were in the free form of amino groups, and the peak intensity decreased with increasing degree of polymerization. Figures 3-6 show the MALDI-TOF-MS analysis results for each oligosaccharide mixture of agar, dextran, locust bean gum, and starch. As shown in each figure, the oligosaccharide mixtures of each polysaccharide prepared in Experimental Example 1 contained oligosaccharides with a degree of polymerization of at least 2 to 10, and the peak intensity decreased with increasing degree of polymerization. Hydrochloric acid concentrations of 0.1% by mass, 0.3% by mass, and 0.9% by mass were set for oligosaccharide formation of each polysaccharide, and oligosaccharide formation was confirmed at all concentrations.

[0122] Analytical conditions for MALDI-TOF-MS • Equipment: Ultraflex-III (Bruker Daltonics) Conditions: Equal volumes of BOA matrix or NMA matrix were mixed with the aqueous solution of the substance to be measured (4 mg / mL), and 1 μL was dropped onto the measurement plate. After allowing the solvent to evaporate by standing at room temperature overnight or in a 60°C drying oven for 30 minutes, the measurement was performed in reflect mode / positive ion mode.

[0123] [Table 2]

[0124] In Table 2, DP represents the degree of polymerization, COS represents chitosan oligosaccharide, Ac represents (partially) N-acetylated COS, Pr(C3) represents (partially) N-propanoylated COS, and C10 represents (partially) N-decanoylated COS. The number below Ac represents the number of N-acetylglucosamine residues in COS, the number below Pr(C3) represents the number of N-propanoylglucosamine residues in COS, and the number below C10 represents the number of N-decanoylglucosamine residues in COS.

[0125] [Table 3]

[0126] In Table 3, DP represents the degree of polymerization.

[0127] [Experimental Example 2] Separation and Purification of Chitosan Oligosaccharides by Two-Step Precipitation Separation 30 mL of deionized water was poured into a 50 mL beaker, 150.8 mg of the chitosan oligosaccharide mixture prepared according to Experimental Example 1 was added, and the mixture was stirred at 500 rpm using a PTFE magnetic stirrer until completely dissolved. A 1 mol / L sodium hydroxide aqueous solution was gradually added to an aqueous solution of chitosan oligosaccharide mixture to adjust the pH to 6.70, and the chitosan oligosaccharide was precipitated. The pH-adjusted aqueous solution was centrifuged (18,000 × g, 15 minutes) to precipitate the chitosan oligosaccharide, and the precipitate (hereinafter also referred to as "first precipitate") and the supernatant were separated. A 1 mol / L sodium hydroxide solution was gradually added to the separated supernatant to adjust the pH to 7.76, and chitosan oligosaccharides were precipitated again. The pH-adjusted supernatant was centrifuged (18,000 × g, 15 minutes) to precipitate the chitosan oligosaccharides, and the precipitate (hereinafter also referred to as "second precipitate") and the supernatant (hereinafter also referred to as "second supernatant") were separated. Subsequently, the first precipitate, the second precipitate, and the second supernatant were each frozen overnight in a -30°C freezer, and then freeze-dried. The yields of the freeze-dried products from the first precipitate, the second precipitate, and the second supernatant were 83.1 mg, 22.2 mg, and 43.8 mg, respectively. Similar to Experimental Example 1, the results of product analysis by MALDI-TOF-MS are shown in Figure 7. In Figure 7, the first graph from the top shows the analysis results of the chitosan oligosaccharide mixture obtained in Experimental Example 1, the second graph shows the analysis results of the first precipitate, the third graph shows the analysis results of the second precipitate, and the fourth graph shows the analysis results of the second supernatant. As shown in Figure 7, the composition of chitosan oligosaccharides in the first precipitate, the second precipitate, and the second supernatant was, respectively, a degree of polymerization of 6 or higher (maximum degree of polymerization of 11), a degree of polymerization of 4 to 16 (maximum degree of polymerization of 10), and a degree of polymerization of 10 or less.

[0128] [Experimental Example 3] Production, separation, and purification of partially N-propanoylated chitosan oligosaccharides (1) Production of partially N-propanoylated chitosan 12 mL of 2% aqueous acetic acid solution was placed in a 20 mL beaker, 0.60 g (3.727 mmol) of chitosan (100% deacetylated, viscosity 40 mPa·s) was added, and the mixture was stirred at 500 rpm with a PTFE magnetic stirrer at room temperature until completely dissolved. After adding 12 mL of methanol to the chitosan aqueous solution and stirring further, 2.4 mL of 1.0% by mass propionic anhydride / methanol (0.186 mmol propionic anhydride) was added dropwise. The aqueous solution was stirred at room temperature for 2 hours. After stirring, a 1 mol / L sodium hydroxide solution was added dropwise to the aqueous solution to adjust the pH to 9.0, and partially N-propanoylated chitosan was precipitated. The pH-adjusted aqueous solution was centrifuged (10,000 × g, 10 minutes) to precipitate the precipitated partially N-propanoylated chitosan, and the supernatant was washed with deionized water until the pH was 7 or less. The precipitate was collected and freeze-dried to obtain 0.530 g of partially N-propanoylated chitosan. The product is partially N-propanoylated chitosan1 The degree of substitution was calculated from the area ratio of the proton at C2 in the glucosamine skeleton and the proton at the terminal methyl group of the propanoyl group in the 1H NMR spectrum, and it was found to be approximately 0.03.

[0129] (2) Production and separation / purification of chitosan oligosaccharides The obtained partially N-propanoylated chitosan was then separated and purified as an oligosaccharide mixture using the same method as in Experimental Example 1, and then separated and purified using the same method as in Experimental Example 2. Compared to 152.6 mg of the sample before separation, the yields of the lyophilized products from the first precipitate, the second precipitate, and the second supernatant were 52.3 mg, 43.5 mg, and 16.9 mg, respectively. Similar to Experimental Example 2, the results of product analysis by MALDI-TOF-MS are shown in Figure 8. In Figure 8, the first graph from the top shows the analysis results of the N-propanoylated chitosan oligosaccharide mixture (pre-separation product), the second graph shows the analysis results of the first precipitate, the third graph shows the analysis results of the second precipitate, and the fourth graph shows the analysis results of the second supernatant. The analysis results showed that only one propanoyl group was attached to all samples, i.e., mo The peak pattern of the substituted compound was primarily observed. As shown in Figure 8, the pre-separation sample showed mono-substituted compounds with a degree of polymerization higher than 8, and the peak intensity decreased with increasing degree of polymerization. The composition of partially N-propanoylated chitosan oligosaccharides in the first precipitation, the second precipitation, and the second supernatant was, respectively, a degree of polymerization of 8 or higher (maximum at 15), a degree of polymerization of 8-18 (maximum at 12), and a degree of polymerization of 10 or lower. In addition, peaks of di- and tri-substituted compounds were also observed in the second supernatant.

[0130] [Experimental Example 4] Production, separation, and purification of partially N-decanoylated chitosan oligosaccharides (1) Production of partially N-decanoylated chitosan Partially N-decanoylated chitosan was prepared in the same manner as in Experimental Example 3, using decanoic acid anhydride instead of propionic acid anhydride as the acylating agent. The amount of decanoic acid anhydride used was such that the molar ratio to the glucosamine residues of chitosan was 0.15, and the reaction conditions were 40°C for 24 hours. The yield of partially N-decanoylated chitosan was 874.7 mg for 905.2 mg of chitosan. The degree of substitution was approximately 0.10.

[0131] (2) Production and separation / purification of chitosan oligosaccharides The obtained partially N-decanoylated chitosan was then used to obtain a partially N-decanoylated chitosan mixture as an oligosaccharide using the same method as in Experimental Example 1, and subsequently separated and purified using the same method as in Experimental Example 2. For a sample size of 150.1 mg before separation, the yields of the freeze-dried products from the first precipitate, the second precipitate, and the second supernatant were 41.6 mg, 47.8 mg, and 34.5 mg, respectively. Similar to Experimental Example 2, the results of product analysis by MALDI-TOF-MS are shown in Figure 9. In Figure 9, the first graph from the top shows the analysis results of the N-decanoylated chitosan oligosaccharide mixture (pre-separation product), the second graph shows the analysis results of the first precipitate, the third graph shows the analysis results of the second precipitate, and the fourth graph shows the analysis results of the second supernatant. The analysis results primarily showed peaks for the monodecanoylated and completely free forms. As shown in Figure 9, no clear peak indicating chitosan oligosaccharides was observed in the first precipitate. The second precipitate contained mono- and di-substituted compounds with a degree of polymerization of 3 or higher (maximum degree of polymerization of 9), while the second supernatant contained mono-substituted compounds with a degree of polymerization of 12 or lower.

[0132] [Experimental Example 5] Verification of the concentration effect by freezing 104.7 mg of partially N-decanoylated chitosan prepared in Experimental Example 4 was placed in a 50 mL beaker, and 20 mL of 0.3% by mass (≒0.084 mol / L) hydrochloric acid was added. At room temperature, the mixture was stirred at 500 rpm using a magnetic stirrer with a PTFE stirring bar to completely dissolve the partially N-decanoylated chitosan. The resulting chitosan aqueous solution was divided into four 1.5 mL tubes, each containing 1 mL, and processed as follows. Preparative sample (1): Frozen overnight in a -30°C freezer, then freeze-dried. Preparative sample (2): After freezing overnight in a -30°C freezer, it was left to stand at room temperature to thaw. Preparative sample (3): Freezed using liquid nitrogen (liquid N2), then freeze-dried. Preparative sample (4): After freezing with liquid nitrogen (liquid N2), it was allowed to stand at room temperature to thaw.

[0133] The partially N-decanoylated chitosan aqueous solution remaining after separation was frozen overnight in a -30°C freezer and then freeze-dried to obtain 106.9 mg of a partially N-decanoylated chitosan oligosaccharide mixture. The product and preparatives (1) to (4) were analyzed by MALDI-TOF-MS. The product, preparative (1), and preparative (3) were dissolved in deionized water to a concentration of 5 mg / mL, while the solutions obtained after melting preparatives (2) and (4) were used directly for analysis. As a result of the analysis, as shown in Figure 10, peak patterns of monosubstituted products were mainly observed in the product, preparative (1), and preparative (3), respectively, but no chitosan oligosaccharide peaks were observed in preparative (2) and preparative (4).

[0134] [Experiment Example 6] Verification of drying and concentration effects 104.3 mg of partially N-decanoylated chitosan prepared in Experimental Example 4 was placed in a 50 mL beaker, and 20 mL of 0.3% by mass (≒0.084 mol / L) hydrochloric acid was added. At room temperature, the mixture was stirred at 500 rpm using a magnetic stirrer with a PTFE stirring bar to completely dissolve the partially N-decanoylated chitosan. The resulting chitosan aqueous solution was divided into four 1.5 mL tubes, each containing 1 mL, and processed as follows. Preparative sample (1): Frozen overnight in a -30°C freezer, then freeze-dried. Preparative sample (2): Freezed using liquid nitrogen (liquid N2), then freeze-dried. Preparative sample (3): Dried by blowing dry nitrogen gas. Preparative sample (4): Centrifuged and dried under reduced pressure. The product and preparatives (1) to (4) were dissolved in deionized water to a concentration of 5 mg / mL and analyzed by MALDI-TOF-MS. As a result of the analysis, as shown in Figure 11, the peak pattern of the monosubstituted product was mainly observed in all of the preparatives (1) to (4).

[0135] [Experimental Example 7] Detailed structural determination using MALDI-TOF / TOF (1) Analysis results of partially N-propanoylated chitosan oligosaccharides MALDI-TOF / TOF measurements were performed on the partially N-propanoylated chitosan oligosaccharides prepared in Experimental Example 3. The measurement results are shown in Figure 12. MALDI-TOF-MS measurements were performed using an NMA matrix, and the peak corresponding to the monosubstituted compound with a degree of polymerization of 5 was used as the parent peak for LIFT-TOF / TOF mode measurements from the obtained spectrum. During TOF / TOF fragmentation, the m / z decrease differed between cases where the reducing end was substituted and cases where it was not, being 235.11 and 179.08, respectively. The percentage of cases where the amino group at the reducing end was substituted was determined to be 77.7% from the fragment peak intensity ratio of the TOF / TOF spectrum.

[0136] (2) Analysis results of partially N-decanoylated chitosan oligosaccharides. Using the same method as in (1), MALDI-TOF / TOF measurements were performed on the partially N-decanoylated chitosan oligosaccharides prepared in Experimental Example 4. The measurement results are shown in Figure 13. The percentage of molecules with substituted amino groups at the reducing end was found to be 75.7%.

[0137] [Experimental Example 8] Antibacterial Test Antimicrobial activity tests were conducted on various microbial strains using chitosan oligosaccharides. The chitosan oligosaccharides used were those obtained by oligosaccharide conversion of chitosan with a 90% degree of deacetylation (i.e., containing 10% acetyl groups) using the method described in Experimental Example 1 (hereinafter referred to as "COS10(Ac)") and partially N-decanoylated chitosan oligosaccharides prepared in Experimental Example 4 (hereinafter referred to as "COS10(C10)"). The Microbial Viability Assay Kit-WST (manufactured by Dojin Chemical Co., Ltd.) was used for the tests, and the procedures followed the instructions in the kit's manual.

[0138] The scientific names and strain numbers of the microorganisms used in the antimicrobial tests are as follows: Bacillus atrophaeus (ATCC 51189) Staphyrococcus epidermidis ATCC 14490 Corynebacterium tuberculostearicum NBRC 113182 Prototheca zopfii NBRC 6998 Staphylococcus aureus ATCC 6538P Serratia marcescens ATCC 13380

[0139] The sources for obtaining the microbial strains are as follows: ATCC:American Type Culture Collection(10801 University Blvd,Manassas,VA 20110-2209,USA) NBRC: NITE Biological Resource Center (National Institute of Technology and Evaluation Biotechnology Center, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture)

[0140] The graph in Figure 14 shows the antimicrobial test results for Bacillus atrophaeus ATCC 51189. Figure 15 shows the antimicrobial test results for Staphyrococcus epidermidis ATCC 14490. Figure 16 shows the antimicrobial test results for Corynebacterium tuberculostearicum NBRC 113182. Figure 17, a substitute photograph, shows the antimicrobial test results for Prototheca zopfii NBRC 6998. Figure 18 shows the antimicrobial test results for Staphylococcus aureus ATCC 6538P. Figure 19 shows the antimicrobial test results for Serratia marcescens ATCC 13380.

[0141] Table 4 summarizes the results of the antimicrobial activity tests on these microbial strains.

[0142] [Table 4]

[0143] Compared to COS10(Ac), COS10(C10) showed approximately 16 to 64 times greater antibacterial activity against all types of bacteria. In particular, IC showed greater antibacterial activity against Corynebacterium tuberculostearicum. 50 It showed a value 500 times greater.

[0144] [Experimental Example 9] Evaluation of antibacterial activity of partially N-acylated chitosan oligosaccharides ~Optimization of the number of carbon atoms in the acyl group and the degree of acylation~ A library of partially N-acylated chitosan oligosaccharides (COS) was synthesized, their antibacterial activity was evaluated, and the carbon number of the acyl group and the degree of acylation that exhibited the highest antibacterial activity were determined.

[0145] <Production of partially N-acylated COS> In this experimental example, the production of partially N-acylated COS was carried out by performing a step to produce acylated chitosan by acyling chitosan (process and conditions are shown in Figure 20), and a step to produce acylated COS from acylated chitosan (process and conditions are shown in Figure 21). Table 5 shows the N-acylated COS with 8, 10, and 12 carbon atoms in the acyl group (C8_1~C8_4, C10_1~C10_4, and C12_1~C10_4, respectively).

[0146] <Degree of substitution of partially N-acylated COS> The degree of substitution of the fabricated partially N-acylated COS was measured by NMR. Table 5 shows the results of the degree of substitution measurement for each sample.

[0147] [Table 5]

[0148] <Evaluation of acylation degree by degree of polymerization> Mass spectrometry was performed on partially N-acylated COS using MALDI-TOF (matrix-assisted laser desorption / ionization-time-of-flight)-MS (mass spectrometer) to evaluate the degree of acylation according to the degree of polymerization.

[0149] Figure 22 shows the MALDI-TOF-MS measurement results for samples with 8 carbon atoms in the acyl group (C8_1, C8_2, C8_3). The numbers in parentheses next to the sample names represent the degree of substitution (shown in Table 5). The degree of acylation of chitosan increased, leading to an increase in the degree of acylation for each degree of polymerization (DP).

[0150] Figure 23 shows the MALDI-TOF-MS measurement results for samples with 10 carbon atoms in the acyl group (C10_1, C10_2, C10_3, C10_4). The numbers in parentheses next to the sample names represent the degree of substitution (shown in Table 5). The degree of acylation of chitosan increased, leading to an increase in the degree of acylation for each degree of polymerization (DP).

[0151] Figure 24 shows the MALDI-TOF-MS measurement results for samples with 12 carbon atoms in the acyl group (C12_1, C12_2, C12_3, C12_4). The numbers in parentheses next to the sample names represent the degree of substitution (shown in Table 5). Unlike the samples with 8 and 10 carbon atoms in the acyl group, no acylation degree based on the degree of polymerization (DP) was observed as the degree of acylation of chitosan increased.

[0152] <Antibacterial evaluation test> Target bacteria: Bacillus subtilis (Bacillus atrophaeus, ATCC 51189) Staphylococcus epidermidis (ATCC 14490) Enterococcus hirae (NBRC 113030) Escherichia coli (ATCC 43888) Note that ATCC is an acronym for American Type Culture Collection, and NBRC is an acronym for NITE Biological Resource Center.

[0153] Partially N-acylated COS sample: C8_1~C8_3 (as mentioned above) C10_1~C10_4 (as mentioned above) C12_4 (as mentioned above) C10 COS (Degree of substitution (DS) = 0.10) (Manufactured separately) For each of the nine samples, a sample dilution series (eight levels: 1024 μg / mL, 128 μg / mL, 16 μg / mL, 2 μg / mL, 0.25 μg / mL, and 0.031 μg / mL) was prepared using MHB medium (Müller-Hinton broth).

[0154] operation: (1) Dispense the sample dilution series into a 96-well plate at a rate of 180 μL / well. (2) Dispense 10 μL / well of bacterial suspension (OD550=0.125 diluted 10-fold). (3) After incubation at 37°C for 2 hours, dispense 10 μL / well of chromogenic reagent. (4) Absorbance was measured over time at a wavelength of 450 nm (at 15-minute intervals, 37°C).

[0155] Antimicrobial activity evaluation results against Bacillus atrophaeus (ATCC 51189): Figure 25 shows the antibacterial activity at negative control saturation (8 hours after the start of absorbance measurement).

[0156] Antimicrobial activity evaluation results against Staphylococcus epidermidis (ATCC 14990): Figure 26 shows the antibacterial activity at negative control saturation (14 hours after the start of absorbance measurement).

[0157] Antimicrobial activity evaluation results against Enterococcus hirae (NBRC 113030): Figure 27 shows the antibacterial activity at negative control saturation (9.5 hours after the start of absorbance measurement).

[0158] Antimicrobial activity evaluation results against Escherichia coli (ATCC 700728): Figure 28 shows the antibacterial activity when the negative control is saturated (5 hours after the start of absorbance measurement).

[0159] Table 6 shows the antibacterial evaluation results (summary) of partially N-acylated COS.

[0160]

Table 6

[0161] [Experimental Example 10] Antibacterial Evaluation of C10 COS ~ Comparison of Fractions by Neutralization Precipitation Method ~ <Preparation of C10 COS Sample> C10 COS was fractionated by the neutralization precipitation method, and the antibacterial properties of each fraction were evaluated. The operation procedure is shown in Fig. 29, and the MALDI-TOF-MS measurement results are shown in Fig. 30. In the low molecular weight region, those with a high degree of acylation (high hydrophobicity) precipitated (Precipitate A, Precipitate B).

[0162] <Antibacterial Evaluation> Target bacteria: Bacillus atrophaeus, ATCC 51189 Staphylococcus epidermidis, ATCC 14490 Staphylococcus aureus, ATCC 6538P Eschelichia coli, ATCC 43888

[0163] Samples: Before fractionation of C10 COS C10 COS Precipitate A C10 COS Precipitate B C10 COS Supernatant Dilution series were prepared for each sample in MHB medium (4 steps: 1024 μg / mL, 256 μg / mL, 64 μg / mL, 16 μg / mL, 4 μg / mL, 1 μg / mL, 0.25 μg / mL) Precipitate A and Precipitate B were dissolved in 1% acetic acid to a concentration of 1% by mass, and then dilution series were prepared in MHB medium

[0164] Operation: <00008(1) Dispense 180 μL / well of the sample dilution series into a 96-well plate (2) Dispense 10 μL / well of the bacterial solution (10-fold dilution of OD550 = 0.125) (3) After culturing at 37 °C for 2 hours, dispense 10 μL / well of the chromogenic reagent (4) Measure the absorbance over time at a wavelength of 450 nm (at intervals of 15 minutes, at 37 °C)

[0165] Antibacterial evaluation results against Bacillus atrophaeus (ATCC 51189): The antibacterial activity at the time of negative control saturation (14.5 hours after the start of absorbance measurement) is shown in Fig. 31. The antibacterial activity of the supernatant (low molecular weight fraction) was lower compared to precipitates A and B.

[0166] Antibacterial evaluation results against Staphylococcus epidermidis (ATCC 14990): The antibacterial activity at the time of negative control saturation (10.5 hours after the start of absorbance measurement) is shown in Fig. 32. Precipitate B showed the highest antibacterial activity.

[0167] Antibacterial evaluation results against Staphylococcus aureus (ATCC 6538P): The antibacterial activity at the time of negative control saturation (10 hours after the start of absorbance measurement) is shown in Fig. 33. Precipitate B showed the highest antibacterial activity.

[0168] Antibacterial evaluation results against Escherichia coli (ATCC 700728): The antibacterial activity at the time of negative control saturation (10.5 hours after the start of absorbance measurement) is shown in Fig. 34. Precipitates A and B showed the highest antibacterial activity.

[0169] The antibacterial evaluation results (summary) of C10 COS are shown in Table 7.

[0170]

Table 7

[0171] [Experimental Example 11] Fractionation of C10 COS organic solvents Molecular weight fractionation was performed by extracting the solubilized components in methanol, ethanol, etc. Organic solvents: methanol (MeOH) Ethanol + Water (volume ratio 9:1) Et:W(9:1) Ethanol + Water (volume ratio 8:2) Et:W(8:2) 2-Propanol + Water (volume ratio 8:2) IPA:W (8:2)

[0172] operation: The operating procedure is shown in Figure 35.

[0173] result: Figure 36 shows the results of organic solvent fractionation using MeOH. Medium to high molecular weight molecules were extracted.

[0174] Figure 37 shows the results of organic solvent fractionation using Et:W(9:1). The medium molecular weight region was extracted.

[0175] Figure 38 shows the results of organic solvent fractionation using Et:W(8:2). The medium molecular weight region was extracted.

[0176] Figure 39 shows the results of organic solvent fractionation using IPA:W(8:2). The medium molecular weight region was extracted.

[0177] Table 8 shows the results of the C10 COS organic solvent fractionation (summary). The extraction pattern could be controlled by the combination of organic solvents.

[0178] [Table 8]

[0179] [Experimental Example 12] Oligosaccharide production from paper-based raw materials An examination was conducted to determine whether the oligosaccharide production method of the present invention can be applied to paper-based raw materials.

[0180] Paper-based raw materials: Paper (Kimwipe (registered trademark), manufactured by Nippon Paper Crecia Co., Ltd.)

[0181] Operation: The operation procedure is shown in Fig. 40. After treating the paper-based raw material (150 mg) with hydrochloric acid (concentrations of 0.1 wt%, 0.3 wt%, 0.9 wt%), it was frozen with liquid nitrogen and freeze-dried to prepare an oligosaccharide mixture (Kimwipe OS). Furthermore, each Kimwipe OS was immersed in ion-exchanged water (4 mL, 2 mL, 2 mL) for 30 minutes, and the soluble components were extracted by pressing filtration and then freeze-dried. MALDI-TOF-MS measurement was performed on the soluble components.

[0182] Results: The MALDI-TOF-MS measurement results are shown in Fig. 41. A peak pattern of 162 was observed, indicating the oligosaccharification of the paper material.

[0183] [Experimental Example 13] Oligosaccharide production from paper-based raw materials An examination was conducted to determine whether the oligosaccharide production method of the present invention can be applied to paper-based raw materials.

[0184] Paper-based raw materials: Cotton (absorbent cotton) Corrugated cardboard Skewers

[0185] Operation: An oligosaccharide mixture was prepared from the paper-based raw material according to the operation procedure shown in Fig. 42. (1) Each 550 mg of paper-based raw materials (cotton, corrugated cardboard, and skewers) was prepared. (2) The prepared paper-based raw materials were immersed in hydrochloric acid (0.9 mass%, 3 mL) for hydrolysis of the paper-based raw materials. (3) The paper-based raw materials after hydrolysis were dried under reduced pressure at room temperature. (4) A mixture of the paper-based raw material after vacuum drying and 3 mL of ion-exchanged water was prepared and shaken for 15 minutes. (5) After shaking, the mixture was filtered to separate it into a solid phase (insoluble matter) and a liquid phase (solubilized matter). (6) The solid phase (insoluble matter) and liquid phase (solubilized matter) obtained by solid-liquid separation were freeze-dried to obtain a mixture of oligosaccharides derived from paper raw materials. (7) The weight loss rate of paper-based raw materials and the yield of solubilized components were calculated. (8) MALDI-TOF-MS analysis was performed on an oligosaccharide mixture derived from paper-based raw materials.

[0186] result: Figure 43 shows the weight loss rate and solubilization yield of various paper-based raw materials. The MALDI-TOF-MS measurement results for oligosaccharide mixtures derived from paper-based raw materials are shown in Figure 44 (cotton-derived oligosaccharide mixture), Figure 45 (corrugated cardboard-derived oligosaccharide mixture), and Figure 46 (disposable chopstick-derived oligosaccharide mixture). The results shown in Figures 44-46 demonstrate that the oligosaccharide production method of the present invention can be applied to paper-based raw materials (cotton, corrugated cardboard, disposable chopsticks) to obtain an oligosaccharide mixture derived from paper-based raw materials.

[0187] [Experimental Example 14] Oligosaccharide production from plant raw materials We investigated whether the oligosaccharide production method of the present invention can be applied to plant-based raw materials.

[0188] Plant material: bamboo leaves Pine needles Pine cone (pine cone)

[0189] operation: An oligosaccharide mixture was prepared from plant materials according to the procedure shown in Figure 47. (1) 550 mg each of plant materials (bamboo leaves, pine needles, and pine cones) were prepared. (2) The prepared plant material was immersed in hydrochloric acid (0.9% by mass, 3 mL) to hydrolyze the plant material. (3) The plant material after hydrolysis was freeze-dried. (4) A mixture of freeze-dried plant material and 3 mL of deionized water was prepared and shaken for 15 minutes. (5) After shaking, the mixture was filtered to separate it into a solid phase (insoluble matter) and a liquid phase (solubilized matter). (6) The solid phase (insoluble matter) and liquid phase (solubilized matter) obtained by solid-liquid separation were freeze-dried to obtain an oligosaccharide mixture derived from plant raw materials. (7) The weight loss rate of the plant material and the yield of the solubilized component were calculated. (8) MALDI-TOF-MS analysis was performed on a mixture of oligosaccharides derived from plant materials.

[0190] result: Figure 48 shows the weight loss rate and solubilization yield of various plant materials. The MALDI-TOF-MS measurement results for plant-derived oligosaccharide mixtures are shown in Figure 49 (bamboo leaf-derived oligosaccharide mixture), Figure 50 (pine needle-derived oligosaccharide mixture), and Figure 51 (pine cone-derived oligosaccharide mixture). The results shown in Figures 49-51 demonstrate that the oligosaccharide production method of the present invention can be applied to plant materials (bamboo leaves, pine needles, pine cones) to obtain a mixture of oligosaccharides derived from plant materials.

[0191] [Experimental Example 15] Oligosaccharide Production from Food Raw Materials We investigated whether the oligosaccharide production method of the present invention can be applied to food ingredients.

[0192] Food ingredients: Shiitake mushrooms Wood ear mushrooms White fungus Reishi mushroom (from China) Reishi mushroom (Japanese origin) Green tea (used tea leaves) Coffee (coffee grounds)

[0193] operation: (a) Hot water extraction Hot water extraction of food ingredients was performed according to the procedure shown in Figure 52. (1) 500 mg each of the following food ingredients were prepared: shiitake mushrooms, wood ear mushrooms, white wood ear mushrooms, reishi mushrooms (from Japan), reishi mushrooms (from China), green tea, and coffee. (2) The prepared food ingredients were mixed with 25 mL of 90°C hot water to prepare a mixture, and hot water extraction was performed for 1 hour. (3) The mixture was filtered to separate it into a filtrate (extract) and a precipitate (residue). (4) The filtrate and precipitate were freeze-dried to obtain the extract and residue, respectively. (5) The yield of the hot water extract of each food ingredient was calculated.

[0194] (b) Oligosaccharide conversion to mass spectrometry An oligosaccharide mixture was prepared from the hot water extraction residue according to the procedure shown in Figure 54. (6) 4 mg each of the residue obtained by hot water extraction was mixed with 4 mL of hydrochloric acid (HCl concentration: 0.1% or 0.5% by mass) or water (HCl concentration: 0% by mass) to prepare a mixture. (7) The mixture was shaken at room temperature for 1 hour. (8) After shaking, the mixture was freeze-dried. (9) The freeze-dried product was mixed with 1 mg of deionized water and filtered. (10) After filtration, the obtained filtrate (solubilized material) was subjected to MALDI-TOF-MS measurement.

[0195] result: The yield of the hot water extract is shown in Figure 53. Figure 55 shows the extraction efficiency of the solubilized substance. The MALDI-TOF-MS measurement results for oligosaccharide mixtures derived from food ingredients are shown in Figure 56 (Reishi mushroom (from China), HCl concentration 0.1% by mass, 0.5% by mass), Figure 57 (Reishi mushroom (from Japan), HCl concentration 0.1% by mass, 0.5% by mass), Figure 58 (Tremella fuciformis, HCl concentration 0.1% by mass, 0.5% by mass), Figure 59 (Shiitake mushroom, HCl concentration 0.1% by mass, 0.5% by mass), Figure 60 (Green tea, HCl concentration 0% by mass (water extract), 0.5% by mass), and Figure 61 (Coffee, HCl concentration 0% by mass (water extract), 0.5% by mass). The results shown in Figures 56-61 demonstrate that the oligosaccharide production method of the present invention can be applied to food ingredients (Reishi mushroom (from China), Reishi mushroom (from Japan), Tremella fuciformis, Shiitake mushroom, green tea, and coffee) to obtain a mixture of oligosaccharides derived from food ingredients.

[0196] [Experimental Example 16] Investigation of the amount of oligosaccharides produced and the differences in the oligosaccharides produced due to differences in hydrochloric acid concentration of paper-based raw materials. Paper raw materials: Paper (Kimwipes®, manufactured by Nippon Crecia Co., Ltd.) operation: The operating procedure is shown in Figure 62. Paper-based raw material (1 Kimwipe sheet: 150 mg) was treated with 3 mL of hydrochloric acid (concentrations 0% by mass, 0.1% by mass, 0.3% by mass, 0.6% by mass, 0.9% by mass, 1.2% by mass, 1.5% by mass, 1.8% by mass, 2.7% by mass). After hydrochloric acid treatment, the treatment solution was freeze-dried. After freeze-drying, the freeze-dried material was mixed with 3 mL of deionized water. The mixture was shaken at room temperature for 15 minutes. After shaking, the mixture was filtered to separate the filtrate and precipitate. The filtrate and precipitate were both freeze-dried. The water-soluble components obtained by freeze-drying the filtrate were subjected to MALDI-TOF-MS analysis. Figure 63 shows the weight loss rate of precipitate and the yield of solubilized components for each hydrochloric acid concentration. Figure 64 shows the MALDI-TOF-MS measurement results when treated with hydrochloric acid (HCl concentration 0.1% by mass). Figure 65 shows the MALDI-TOF-MS measurement results when treated with hydrochloric acid (HCl concentration 0.9 mass%). These results indicate that increasing the HCl concentration to 1% by mass or more does not improve the oligosaccharide production efficiency. [Industrial applicability]

[0197] In this invention, chitosan oligosaccharides can be produced safely, simply, and inexpensively compared to conventional methods. Furthermore, chitosan oligosaccharides with a higher degree of polymerization can be separated and purified from a mixture of chitosan oligosaccharides with a wide molecular weight distribution compared to conventional methods. In addition, the chitosan oligosaccharides obtained by this invention have high antibacterial properties and are expected to be used in topical medicines and other applications.

Claims

1. A step of preparing an aqueous solution of polysaccharide by dissolving a polysaccharide in an aqueous solution of a volatile strong acid, and A step of concentrating and drying the aqueous solution of the polysaccharide. A method for producing oligosaccharides, including

2. An oligosaccharide mixture containing an oligosaccharide with 10 or more sugar residues is produced by the method for producing oligosaccharides described in claim 1. The oligosaccharide mixture was subjected to mass spectrometry using a MALDI-TOF-MS (matrix-assisted laser desorption / ionization-time-of-flight) mass spectrometer. The degree of polymerization, type, and number of substituents of the oligosaccharides contained in the oligosaccharide mixture are analyzed with high resolution and high sensitivity. Methods for analyzing oligosaccharides.

3. The method for producing an oligosaccharide according to claim 1, wherein the polysaccharide is at least one selected from the group consisting of chitosan, chitin, cellulose, xylan, mannan, glucomannan, and starch.

4. The method for producing an oligosaccharide according to claim 3, wherein the oligosaccharide is a mixture of oligosaccharides with different degrees of polymerization.

5. A step of preparing an aqueous oligosaccharide solution containing a mixture of oligosaccharides with different degrees of polymerization, and The process involves adding alkali to the oligosaccharide aqueous solution to control the pH, and then precipitating the oligosaccharides from the oligosaccharide aqueous solution according to their degree of polymerization. A method for separating oligosaccharides, including [specific example].

6. The aforementioned precipitation process is carried out in stages within the same container. A mixture of oligosaccharides with different degrees of polymerization is fractionated into oligosaccharides with a high degree of polymerization, oligosaccharides with a medium degree of polymerization, and oligosaccharides with a low degree of polymerization. The method for separating oligosaccharides according to claim 5.

7. A step of preparing an oligosaccharide mixture or an aqueous solution thereof containing a mixture of oligosaccharides with different degrees of polymerization, and The process involves adding a first organic solvent or a first mixed solvent containing the first organic solvent and water to the oligosaccharide mixture or an aqueous solution thereof and mixing, and separating it into a first organic solvent extract and an insoluble residue of the first organic solvent. A method for separating oligosaccharides, including [specific example].

8. A step of mixing the first organic solvent extract or the first organic solvent insoluble residue with a second organic solvent of a different type than the first organic solvent, or a second mixed solvent containing the second organic solvent and water, and separating it into the second organic solvent extract and the second organic solvent insoluble residue. The method for separating oligosaccharides according to claim 7, further comprising:

9. Chitosan oligosaccharide, represented by the following formula (1). 【Chemistry 1】 (In the formula, R is a hydrogen atom or an acyl group, m is an integer from 3 to 30, and n is an integer from 0 to 3.)

10. The chitosan oligosaccharide according to claim 9, wherein R is an acyl group having 3 or more carbon atoms, n is an integer from 1 to 3, and m+n is an integer of 5 or more.

11. The chitosan oligosaccharide according to claim 10, wherein m + n is an integer of 8 or more.

12. An antibacterial agent comprising at least one selected from the group consisting of chitosan oligosaccharides and salts thereof as described in any one of claims 9 to 11 as an active ingredient.

13. The use of at least one selected from the group consisting of chitosan oligosaccharides and salts thereof as described in any one of claims 9 to 11 in the manufacture of an antimicrobial agent.

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