Method for isolating and purifying chitin oligosaccharide formed of six to eight monosaccharide molecules, and chitin oligosaccharide containing high content of chitin oligosaccharide formed of six to eight monosaccharide molecules

Abstract

The present invention pertains to a method for isolating and purifying a chitin oligosaccharide formed of six to eight monosaccharide molecules, the method comprising: (A) a step for heating or increasing the temperature, to 40°C or higher, of an aqueous solution or an aqueous dispersion of a chitin oligosaccharide mixture to produce insoluble matter; (B) a step for washing the insoluble matter produced in step (A) by using a 30-90 vol% aqueous ethanol solution; and (C) a step for recovering the insoluble matter washed in step (B).　With the present invention, it is possible to provide: a method for isolating and purifying a chitin oligosaccharide formed of six to eight monosaccharide molecules from a chitin oligosaccharide mixture by a simple means; and a chitin oligosaccharide containing a high content of a chitin oligosaccharide formed of six to eight monosaccharide molecules.

Description

Method for isolating and purifying hexasaccharide to octasaccharide chitin oligosaccharides, and a method for containing hexasaccharide to octasaccharide chitin oligosaccharides in high content.

[0001] The present invention relates to a method for isolating and purifying hexasaccharide to octasaccharide chitin oligosaccharides, and to chitin oligosaccharides containing a high content of hexasaccharide to octasaccharide chitin oligosaccharides.

[0002] Chitin oligosaccharides have been reported to have various physiological activities, such as promoting the growth of Bifidobacterium and stimulating the immune system. In particular, chitin oligosaccharides with a high degree of polymerization of hexasaccharide or more have been reported to have higher antitumor activity compared to other oligosaccharides, and have shown growth inhibitory effects on solid tumors transplanted into mice (Non-Patent Document 1). Therefore, there is a need for a technology to produce chitin oligosaccharides with a high content of high-degree-of-polymerization chitin oligosaccharides. As such a technology, a method has been proposed to produce chitin oligosaccharides with a high content of high-degree-of-polymerization chitin oligosaccharides by providing a hydrolysis step in which chitin is hydrolyzed using concentrated hydrochloric acid of 30% or more at a reaction temperature of 5°C to 30°C, and setting the reaction time in the hydrolysis step in the range of 1 hour to 120 hours depending on the type of chitin hydrolysis product to be produced, thereby enabling the separate production of N-acetylglucosamine and chitin hydrolysis products containing chitin oligosaccharides (Patent Document 1). Furthermore, a method has been proposed for isolating and purifying chitin oligosaccharides having a degree of polymerization of 4 to 7 from a chitin oligosaccharide mixture, through the steps of preparing a high-concentration aqueous solution of a chitin oligosaccharide mixture, adding an organic solvent to the aqueous solution prepared in the first step to precipitate components insoluble in the mixed solvent of the organic solvent and water, and recovering the precipitated insoluble components (Patent Document 2).

[0003] However, in the chito-oligosaccharides obtained by the production method described in Patent Document 1, the content of chito-oligosaccharides of hexasaccharide or higher was about 10% by mass. Since the production method described in Patent Document 2 is a method of removing saccharides with a degree of polymerization of 1 to 3 and recovering oligosaccharides with a degree of polymerization of 4 to 7, due to the presence of tetrasaccharides and pentasaccharides, the content of chito-oligosaccharides of hexasaccharide or higher was limited to 30% to 40% by mass. Furthermore, in the production methods described in Patent Documents 1 and 2, since hydrochloric acid was used in both cases, it was necessary to perform neutralization and desalting in the purification process. Therefore, there is a need for a method to obtain chito-oligosaccharides containing a high content of chito-oligosaccharides of hexasaccharide or higher, mainly hexasaccharide to octasaccharide, which have various reported physiological activities, by a simple means.

[0004] Oga et al. dissolved chito-oligosaccharide trisaccharide as a starting material in an acetate buffer solution to which ammonium sulfate was added, added chicken egg white lysozyme, reacted it at 40 °C, recovered the precipitate formed, and isolated and purified it using an activated carbon - celite column. As a result of preparing chito-oligosaccharides of disaccharide to octasaccharide with high purity and performing physical property analysis, it was reported that only the chito-oligosaccharide hexasaccharide showed the unique thermoreversible responsiveness of a hydrogel, gelled when heated at 100 °C, and showed the property of redissolving when cooled to 4 °C (Non-Patent Document 2). The technique reported in Non-Patent Document 2 suggests the possibility of isolating only the chito-oligosaccharide hexasaccharide by gelling it by heating a solution containing chito-oligosaccharides with different degrees of polymerization. However, when a mixture containing monosaccharides and chito-oligosaccharides of disaccharide to pentasaccharide is used as the starting material, the present inventors have confirmed that about 10% by mass of monosaccharides and disaccharide to tetrasaccharides remain respectively in the method of heating, gelling, and insolubilizing reported in Non-Patent Document 2. Furthermore, it was also confirmed that the content of hexasaccharides levels off at about 30% by mass by this method.

[0005] Japanese Patent Application Laid-Open No. 2012 - 217396, Japanese Patent Application Laid-Open No. 2018 - 080124

[0006] A. Tokoro et al.; Chem. Pharm. Bull. 36 (2) 784-790 (1988) Ogata et al.; Chitin and Chitosan Research 18 (2) 152-153 (2012)

[0007] Therefore, the present invention aims to provide a method for isolating and purifying hexasaccharide to octasaccharide chitin oligosaccharides from a chitin oligosaccharide mixture by simple means, and to provide chitin oligosaccharides containing a high content of hexasaccharide to octasaccharide chitin oligosaccharides.

[0008] The present inventors, after diligently studying to solve the above problems, discovered that by heating or heating an aqueous solution or aqueous dispersion of a chitin oligosaccharide mixture to 40°C or higher to generate insoluble matter, then adding 30% to 90% by volume of an aqueous ethanol solution to the insoluble matter to suspend it, and recovering the insoluble matter from the suspension, it is possible to obtain chitin oligosaccharides containing a high content of hexasaccharide to octasaccharide chitin oligosaccharides. Further studies led to the completion of the present invention.

[0009] In other words, the present invention relates to the following: [1] Formula (I)

[0010]

[0011] A method for isolating and purifying chitin oligosaccharides having n1 of 6 to 8 from a chitin oligosaccharide mixture represented by the formula [wherein n1 is an integer from 1 to 8], the method comprising: (A) heating or heating an aqueous solution or aqueous dispersion of the chitin oligosaccharide mixture to 40°C or higher to generate insoluble matter; (B) washing the insoluble matter generated in step (A) with an aqueous ethanol solution of 30% to 90% by volume; and (C) recovering the insoluble matter washed in step (B). [2] The method according to [1], wherein the aqueous solution or aqueous dispersion of the chitin oligosaccharide mixture is an aqueous solution or aqueous dispersion containing 2% to 15% by mass of the chitin oligosaccharide mixture. [3] The method according to [1] or [2], wherein the step in (A) is heated or heated to 60°C or higher. [4] The method according to any one of [1] to [3], wherein the ethanol concentration of the aqueous ethanol solution in step (B) is 60% to 80% by volume. [5] The method according to any one of [1] to [4], wherein in step (B), the insoluble material produced in step (A) is washed with 20 mL to 120 mL of an ethanol aqueous solution per 1 g. [6] The method according to any one of [1] to [5], wherein the content of hexasaccharide chitin oligosaccharides in the isolated and purified chitin oligosaccharide is 60% by mass or more, and the content of hexasaccharide to octasaccharide chitin oligosaccharides is 75% by mass or more. [7] Chitin oligosaccharide containing 75% by mass or more of the chitin oligosaccharide represented by the following formula (II).

[0012]

[0013] [In the formula, n² represents an integer between 6 and 8.]

[0014] The present invention provides a simple method for isolating and purifying hexasaccharide to octasaccharide chitin oligosaccharides from a chitin oligosaccharide mixture. Furthermore, the present invention provides chitin oligosaccharides containing a high content of hexasaccharide to octasaccharide chitin oligosaccharides. The chitin oligosaccharides provided by the present invention contain a high content of hexasaccharide to octasaccharide chitin oligosaccharides, which have been reported to have various physiological activities, and can be used as active ingredients in pharmaceuticals and functional foods.

[0015] The present invention provides a method for isolating and purifying hexasaccharide to octasaccharide chitin oligosaccharides from a chitin oligosaccharide mixture (hereinafter also referred to as "the method of the present invention" in this specification). Here, "chitin oligosaccharide" is an oligomer formed by the β-1,4-linking of two or more N-acetylglucosamine molecules. In this specification, "chitin oligosaccharide mixture" is a mixture containing monosaccharides and disaccharide to octasaccharide chitin oligosaccharides, and is represented by the following formula (I).

[0016]

[0017] [In the formula, n1 represents an integer from 1 to 8.] Furthermore, "hexasaccharide to octasaccharide chitin oligosaccharides" refer to hexamers to octamers of N-acetylglucosamine, and correspond to chitin oligosaccharides in the above formula (I) where n1 is 6 to 8. Accordingly, the method of the present invention is a method for isolating and purifying chitin oligosaccharides in which n1 is 6 to 8 from a chitin oligosaccharide mixture represented by the above formula (I).

[0018] The method of the present invention includes the steps of (A) heating or warming an aqueous solution or aqueous dispersion of a chitin oligosaccharide mixture to 40°C or higher to generate insoluble matter, (B) washing the insoluble matter generated in the above step with a 30% to 90% by volume aqueous ethanol solution, and (C) recovering the insoluble matter washed in the above step.

[0019] In step (A) of the method of the present invention, "aqueous solution or aqueous dispersion of chitin oligosaccharide mixture" means an aqueous solution in which the chitin oligosaccharide mixture represented by formula (I) is dissolved in water, or an aqueous dispersion in which the chitin oligosaccharide mixture represented by formula (I) is contained in water beyond a saturation state, and undissolved chitin oligosaccharides are dispersed in water. In this step, the aqueous solution or aqueous dispersion of chitin oligosaccharide mixture can be prepared by dissolving or dispersing a commercially available product provided as "chitin oligosaccharide" by, for example, Koyo Chemical Co., Ltd. in water. The aqueous solution or aqueous dispersion of chitin oligosaccharide mixture is preferably an aqueous solution or aqueous dispersion of 2% to 30% by mass, more preferably an aqueous solution or aqueous dispersion of 2% to 15% by mass, and even more preferably an aqueous solution or aqueous dispersion of 6% to 15% by mass.

[0020] In the method of the present invention, in step (A), an aqueous solution or aqueous dispersion of the chitin oligosaccharide mixture is heated or warmed to generate insoluble matter. By heating or warming the aqueous solution or aqueous dispersion of the chitin oligosaccharide mixture, the high-polymerization chitin oligosaccharides with low solubility in water are rendered insoluble. Therefore, the insoluble matter produced in this step contains a higher content of hexasaccharide to octasaccharide chitin oligosaccharides compared to the aqueous solution or aqueous dispersion of the chitin oligosaccharide mixture. Even the insoluble matter initially present in the aqueous dispersion is composed of high-polymerization chitin oligosaccharides with low solubility in water that have been rendered insoluble. Therefore, within the above concentration range, it has little effect on increasing the content of hexasaccharide to octasaccharide chitin oligosaccharides and contributes to improving the yield. The heating or warming of the aqueous solution or aqueous dispersion of the chitin oligosaccharide mixture is carried out at 40°C or higher, preferably at 60°C or higher, and more preferably at 90°C or higher. Heating to 90°C or higher is preferable because it specifically gels and insolubilizes the hexasaccharide chitin oligosaccharide, resulting in a higher content of hexasaccharide chitin oligosaccharide in the resulting insoluble matter. The above heating or heating should be carried out for a time sufficient to make the high-polymerization chitin oligosaccharide supersaturated and sufficiently insolubilized, or for a time sufficient to gel the hexasaccharide chitin oligosaccharide, but it is preferable to carry it out for 1 to 3 hours. The insoluble matter produced by heating or heating can be recovered by conventional separation and recovery methods, such as filtration and centrifugation. Filtration can be carried out at 20°C to 90°C using filter paper (for example, Advantec No. 2, Advantec No. 5C (Advantec Toyo Co., Ltd.), etc.) with a particle size of approximately 1 μm to 5 μm that can retain 90% or more of the seven types of powders specified in Japanese Industrial Standard (JIS) Z 8901:2006 when naturally filtered. Furthermore, centrifugation is preferably performed for about 5 to 10 minutes under conditions of 20°C to 90°C and 3,500 rpm to 10,000 rpm. It is preferable to use centrifugation from the viewpoint of efficient recovery of insoluble matter generated by heating or warming.

[0021] In the method of the present invention, in step (B), the insoluble material produced in step (A) is washed with an aqueous ethanol solution. In this step, "washing with an aqueous ethanol solution" means that the monosaccharide (N-acetylglucosamine) and disaccharide to pentasaccharide chitin oligosaccharide present in the insoluble material produced in step (A), that is, the insoluble material with an increased content of hexasaccharide to octasaccharide chitin oligosaccharides, are solubilized and removed by adding an aqueous ethanol solution to the insoluble material and suspending it. The concentration of ethanol in the aqueous ethanol solution used in step (B) is 30% to 90% by volume, preferably 60% to 80% by volume, and particularly preferably 70% by volume. If the concentration of ethanol in the aqueous ethanol solution is less than 30% by volume, the hexasaccharide to octasaccharide chitin oligosaccharides are also solubilized, resulting in a decrease in yield. On the other hand, if the ethanol concentration in the ethanol aqueous solution exceeds 90% by volume, the solubility of monosaccharides and disaccharides to pentasaccharides in the ethanol aqueous solution decreases, and the efficiency of their removal decreases. The amount of ethanol aqueous solution added in step (B) is preferably 20 mL to 120 mL, and more preferably 20 mL to 70 mL, per 1 g (as dry weight) of insoluble matter produced in step (A). If the amount of ethanol aqueous solution added is less than 20 mL, the amount of monosaccharides and disaccharides to pentasaccharides remaining in the insoluble matter may increase, and if the amount of ethanol aqueous solution added exceeds 120 mL, the manufacturing cost of chitin oligosaccharides may increase due to the increased amount of ethanol used. Furthermore, in the method of the present invention, the temperature at which step (B) is performed is not particularly limited as long as the suspension treatment with an aqueous ethanol solution can be carried out. However, considering the boiling point of ethanol and from the viewpoint of ease of operation of the washing treatment, it is preferably -20°C to 78°C, and from the viewpoint of the effect of removing monosaccharides and disaccharides to pentasaccharides, it is more preferably 4°C to 30°C. In addition, in the method of the present invention, it is preferable to continue step (B), that is, the suspension with an aqueous ethanol solution to wash insoluble materials with an aqueous ethanol solution, for one hour or more.

[0022] In the method of the present invention, the insoluble material washed in step (B) is then recovered as step (C). As a means of recovering the washed insoluble material, filtration or centrifugation can be used, similar to when recovering the insoluble material generated in step (A). The conditions for filtration and centrifugation are as described above for step (A).

[0023] In the method of the present invention, the efficiency of removing monosaccharides and disaccharides to pentasaccharides of chitin oligosaccharides can be improved by repeating steps (A) to (C) above. In the present invention, considering the efficiency of removing monosaccharides and disaccharides to pentasaccharides of chitin oligosaccharides and the increase in cost due to the increased amount of ethanol used, it is preferable to repeat steps (A) to (C) up to about four times.

[0024] The method of the present invention may include, in addition to the above-described steps (A) to (C), general steps used in the manufacture of pharmaceuticals or food products, such as drying, grinding, and granulation, without impairing the features of the present invention. For example, the insoluble material recovered through steps (A) to (C) can be dried by means of freeze-drying, spray-drying, or the like. Furthermore, the grinding of the insoluble material recovered through steps (A) to (C) can be carried out under normal conditions using a commercially available grinder, and the granulation of the insoluble material recovered through steps (A) to (C) can also be carried out under normal conditions using a commercially available granulator, employing general granulation methods such as fluidized bed granulation, agitation granulation, tumbling granulation, compression granulation, or extrusion granulation.

[0025] The present invention allows for the isolation and purification of hexasaccharide to octasaccharide chitin oligosaccharides from a chitin oligosaccharide mixture by simple means of heating or warming an aqueous solution or aqueous dispersion containing the chitin oligosaccharide mixture and washing with an aqueous ethanol solution. Since the present invention does not require the addition of acid during the process, neutralization and desalting are not required. Furthermore, the present invention makes it possible to provide chitin oligosaccharides containing a high content of hexasaccharide to octasaccharide chitin oligosaccharides, and in one embodiment of the present invention, it is possible to provide chitin oligosaccharides containing a high content of 60% by mass or more of hexasaccharide chitin oligosaccharides and 75% by mass or more of hexasaccharide to octasaccharide chitin oligosaccharides.

[0026] Furthermore, the present invention provides a chitin oligosaccharide (hereinafter also referred to as "the chitin oligosaccharide of the present invention") containing 60% by mass or more of hexasaccharide chitin oligosaccharide and 75% by mass or more of hexasaccharide to octasaccharide chitin oligosaccharide. The hexasaccharide to octasaccharide chitin oligosaccharide contained in the chitin oligosaccharide of the present invention is represented by the following formula (II).

[0027]

[0028] [In the formula, n² represents an integer from 6 to 8.] In the chitin oligosaccharide of the present invention, the content of monosaccharides and disaccharides to pentasaccharides is significantly reduced, and hexasaccharides are present at a concentration of 60% by mass or more, and hexasaccharides to octasaccharides are present at a concentration of 75% by mass or more. Although the chitin oligosaccharide of the present invention may contain heptasaccharides and octasaccharides, chitin oligosaccharides of nusaccharides or higher are insoluble in water and are therefore hardly present in the aqueous solution or aqueous dispersion containing the chitin oligosaccharide mixture used as the starting material in the method of the present invention. Accordingly, the chitin oligosaccharide isolated and purified by the method of the present invention contains hardly any chitin oligosaccharides of nusaccharides or higher. Due to the difficulty in extraction, heptasaccharide and octasaccharide chitin oligosaccharides are rarely handled individually. For example, commercially available products offered as "chitin oligosaccharide" by companies such as Koyo Chemical Co., Ltd. may contain small amounts of heptasaccharide and octasaccharide chitin oligosaccharides. However, because there are no suitable standard samples for quantifying heptasaccharide and octasaccharide chitin oligosaccharides, their respective contents are currently estimated using standard samples of hexasaccharide chitin oligosaccharides. In terms of physiological activity such as antitumor activity, heptasaccharide and octasaccharide chitin oligosaccharides are treated similarly to hexasaccharide chitin oligosaccharides. Therefore, the presence of heptasaccharide and octasaccharide chitin oligosaccharides has little effect on the functionality of the chitin oligosaccharide of the present invention. The chitin oligosaccharide of the present invention can be isolated and purified from a chitin oligosaccharide mixture by the method of the present invention described above, and can be obtained simply and suitably.

[0029] The chitin oligosaccharide of the present invention contains a high content of 60% by mass or more of hexasaccharide chitin oligosaccharide, which has been reported to have antibacterial activity, antitumor activity, and immunostimulatory activity such as macrophage activation. Therefore, it is expected to exhibit these physiological functions well. For this reason, it can be suitably used as an active ingredient with the above physiological functions in functional foods and pharmaceuticals.

[0030] The present invention will be described in more detail below with reference to examples.

[0031] [Example 1] Isolation and Purification of Hexasaccharide to Octasaccharide Chitin Oligosaccharides As a chitin oligosaccharide mixture, 12.5 g of a commercially available product ("Koyo Chitin Oligosaccharide" (Lot No. 240423) (Koyo Chemical Co., Ltd.)) was added to 87.5 g of deionized water and stirred with a magnetic stirrer at room temperature for 1 hour to prepare a 12.5% ​​by mass aqueous dispersion of chitin oligosaccharides. The above aqueous dispersion was heated in a 90°C water bath for 3 hours, and the resulting insoluble matter was recovered as a precipitate by centrifugation (at 20°C and 3,500 rpm for 10 minutes). 130 mL of a 70% by volume aqueous ethanol solution was added to the recovered insoluble matter (dry weight = 2.2 g) at room temperature to suspend it and solubilize the monosaccharides and disaccharide to pentasaccharide chitin oligosaccharides in the insoluble matter. After continuing the suspension treatment with the ethanol aqueous solution for 3 hours, centrifugation (at 20°C and 3,500 rpm for 10 minutes) was performed, the supernatant was removed, and the precipitated insoluble material was collected and freeze-dried to obtain chitin oligosaccharides containing hexasaccharide to octasaccharide (dry weight = 1.0 g).

[0032] [Comparative Example 1] Isolation and Purification of Hexasaccharide-Octassaccharide Chitin Oligosaccharides by Insolubilization by Heating Only Similar to Example 1, a 12.5% ​​by mass aqueous dispersion of a chitin oligosaccharide mixture was prepared, heated to 90°C, and the resulting insoluble matter was centrifuged and recovered as a precipitate. This was then freeze-dried to obtain chitin oligosaccharides containing hexasaccharide-octasaccharide chitin oligosaccharides.

[0033] The chitin oligosaccharides isolated and purified in Example 1 and Comparative Example 1 were analyzed for composition by high-performance liquid chromatography (HPLC) under the measurement conditions shown below. The analysis results, along with the composition of the chitin oligosaccharide mixture used as raw material, are shown in Table 1. <Measurement Conditions> (i) Equipment used: Liquid chromatograph; LC-20A series (Shimadzu Corporation) (Online degasser; "DGU-20A", liquid transfer pump; "LC-20AB", autosampler; "SIL-20AC", column oven; "CTO-20AC", system controller; "CBM-20A") (ii) Column: "Shodex Asahipak NH2P-50 4E" (Shimadzu GLC Corporation) (iii) Elution solvent: Acetonitrile:Water = 7:3 (volume ratio) (iv) Flow rate: 0.8 mL / min (v) Detector: Ultraviolet-Vis (UV-VIS) detector; "SPD-20AV", Differential refractive index (RI) detector; "RID-10A" (Shimadzu Corporation) (vi) Column temperature: 27℃

[0034]

[0035] As shown in Table 1, the total content of monosaccharides (N-acetylglucosamine) and disaccharides to pentasaccharides in the chitin oligosaccharide isolated and purified in Example 1 was 24.0% by mass, which is significantly lower than that of the commercially available chitin oligosaccharide mixture ("Koyo Chitin Oligosaccharide") used as a raw material. Furthermore, in the chitin oligosaccharide isolated and purified in Example 1, the content of hexasaccharide chitin oligosaccharides increased to 61.8% by mass, and the content of hexasaccharides and heptasaccharide chitin oligosaccharides was 76.2% by mass. On the other hand, in Comparative Example 1, the chitin oligosaccharide was isolated and purified by only heating an aqueous dispersion of the chitin oligosaccharide mixture to generate insoluble matter, without washing with a 70% by volume ethanol aqueous solution. In this case, the content of monosaccharides (N-acetylglucosamine) and disaccharides to pentasaccharides was not sufficiently reduced, the content of hexasaccharides was less than 35% by mass, and the content of hexasaccharides and heptasaccharides was also less than 40% by mass. Furthermore, in the isolation and purification of Example 1 and Comparative Example 1, the content of octasaccharides in the chitin oligosaccharide mixture used as the starting material was below the detection limit, and octasaccharides were not detected in the chitin oligosaccharides isolated and purified in Example 1 and Comparative Example 1.

[0036] As detailed above, the present invention provides a method for isolating and purifying hexasaccharide to octasaccharide chitin oligosaccharides from a chitin oligosaccharide mixture by simple means, and provides chitin oligosaccharides containing 60% by mass or more of hexasaccharide chitin oligosaccharides and 75% by mass or more of hexasaccharide to octasaccharide chitin oligosaccharides. The chitin oligosaccharides provided by the present invention contain a high content of 60% by mass or more of hexasaccharide chitin oligosaccharides, which have been reported to have antibacterial activity, antitumor activity, and immunostimulatory activity such as macrophage activation, and are therefore useful as active ingredients that exert such physiological functions in functional foods and pharmaceuticals.

[0037] This application is based on Japanese Patent Application No. 2024-161410, which is entirely contained herein.

Claims

1. The following formula (I) A method for isolating and purifying chitin oligosaccharides having n1 of 6 to 8 from a chitin oligosaccharide mixture represented by the formula [wherein n1 represents an integer from 1 to 8], the method comprising: (A) heating or heating an aqueous solution or aqueous dispersion of the chitin oligosaccharide mixture to 40°C or higher to generate insoluble matter; (B) washing the insoluble matter generated in step (A) with a 30% to 90% by volume aqueous ethanol solution; and (C) recovering the insoluble matter washed in step (B).

2. The method according to claim 1, wherein the aqueous solution or aqueous dispersion of the chitin oligosaccharide mixture is an aqueous solution or aqueous dispersion containing 2% to 15% by mass of the chitin oligosaccharide mixture.

3. The method according to claim 1, wherein the step in (A) is heated to 60°C or higher.

4. The method according to claim 1, wherein the ethanol concentration of the aqueous ethanol solution in step (B) is 60% by volume to 80% by volume.

5. The method according to claim 1, wherein in step (B), the insoluble material generated in step (A) is washed with 20 mL to 120 mL of an ethanol aqueous solution per 1 g.

6. The method according to any one of claims 1 to 5, wherein the content of hexasaccharide chitin oligosaccharides in the isolated and purified chitin oligosaccharide is 60% by mass or more, and the content of hexasaccharide to octasaccharide chitin oligosaccharides is 75% by mass or more.

7. Chitin oligosaccharide containing 75% by mass or more of the chitin oligosaccharide represented by the following formula (II). [In the formula, n² represents an integer between 6 and 8.]

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