Method for separating chitosan oligosaccharides

Abstract

To provide a method for conveniently obtaining chitosan oligosaccharides with a number of saccharides controlled in a predetermined range.SOLUTION: A chitosan oligosaccharide fractionating method comprises the steps of adding acid to aqueous solution of chitosan oligosaccharide, preparing into a predetermined concentration of acid solution and a predetermined concentration of chitosan oligosaccharide solution, thereby precipitating chitosan oligosaccharides with a number of saccharides in a predetermined range.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] The present invention relates to a technique for separating and isolating chitosan oligosaccharides (oligoglucosamines) having a specific range of sugar numbers. [Background technology]

[0002] Chitosan oligosaccharide is a sugar in which about 2 to 10 glucosamine units are linked together, and has immunostimulatory and anti-inflammatory effects, and therefore in recent years has been distributed mainly as a material for health foods. It can be produced by hydrolyzing chitosan with hydrochloric acid, or more generally, by dissolving chitosan in an organic acid such as hydrochloric acid, acetic acid, lactic acid, or citric acid, and then enzymatically decomposing it. In the case of enzymatic decomposition, chitosan oligosaccharide takes the form of an acid salt depending on the type of acid used for dissolution. Generally, most chitosan oligosaccharides are distributed in the form of chitosan oligosaccharide hydrochloride. The applicant has also separately developed a technology for high-content chitosan oligosaccharides that are inhibited from browning (Patent Document 3).

[0003] The applicants have also found that chitosan oligosaccharides are effective as a test agent for leaky gut syndrome (LGS), and molecular weight control is an important technical element in this technology (Patent Document 4). This is because chitosan oligosaccharides are derived from crab shells and are highly safe, and are not decomposed by human digestive enzymes, making it possible to detect the molecular weight of the chitosan that is leaking from the intestinal mucosa by blood measurement.

[0004] Therefore, there is a demand for a technique for easily separating chitosan oligosaccharides having an adjusted molecular weight, in other words, an adjusted number of sugars within a predetermined range, from a raw chitosan oligosaccharide material having a mixture of various molecular weights. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2002-011845 [Patent Document 2] Patent Publication No. 2003-212889 [Patent Document 3] Patent Publication No. 2013-079217 [Patent Document 4] WO2018 / 135543 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention has been made in view of the above, and an object of the present invention is to provide a method for simply obtaining chitosan oligosaccharide having a sugar number adjusted within a predetermined range. [Means for solving the problem]

[0007] The invention described in claim 1 is a method for isolating chitosan oligosaccharides, characterized in that hydrochloric acid in the range of 2 M to 6 M is added to an aqueous solution of chitosan oligosaccharides in the range of 3 w / v % to 20 w / v % to separate a precipitate fraction from a solution fraction, thereby fractionating chitosan oligosaccharides with different saccharide number distributions.

[0008] The invention described in claim 2 is a method for isolating chitosan oligosaccharides, characterized in that hydrochloric acid is added in the range of 2M to 6M to an aqueous solution of chitosan oligosaccharides in the range of 3 w / v % to 20 w / v %, to separate a precipitate fraction from a solution fraction, and hydrochloric acid is added to the solution fraction at a reduced acid concentration in the range of 2M to 6M to obtain a further precipitate fraction, or hydrochloric acid is added to the precipitate fraction at an increased acid concentration in the range of 2M to 6M to obtain a further solution fraction, by carrying out hydrochloric acid addition treatment multiple times, thereby fractionating chitosan oligosaccharides with different saccharide number distributions.

[0009] The invention described in claim 3 is the method for isolating the chitosan oligosaccharide described in claim 1 or 2, characterized in that the molecular weight of the chitosan oligosaccharide is controlled to be used as a test drug for leaky gut syndrome.

[0010] In addition, the present invention can also be said to be a method for isolating chitosan oligosaccharides, characterized in that (1) an acid is added to an aqueous chitosan oligosaccharide solution to prepare an acidic solution having a predetermined concentration and a chitosan oligosaccharide solution having a predetermined concentration, thereby precipitating chitosan oligosaccharides having a sugar number within a predetermined range. This method can also be expressed as an acid precipitation method for chitosan oligosaccharide. The present invention can also be said to be a method for isolating chitosan oligosaccharides according to claim 1, characterized in that (2) chitosan oligosaccharides are precipitated with different numbers of sugars by varying the concentration of an acid. The present invention can also be said to be a method for isolating chitosan oligosaccharide according to claim 1 or 2, characterized in that (3) hydrochloric acid is used at a concentration of 2M to 6M. The present invention can also be said to be a method for isolating chitosan oligosaccharide according to (1), (2) or (3), characterized in that the chitosan oligosaccharide concentration is set to any of the ranges of 3 w / v% to 20 w / v%. In terms of w / w %, the chitosan oligosaccharide concentration is preferably 2.4 w / w % to 15.9 w / w %. The present invention also provides (5) a method for isolating chitosan oligosaccharides according to (1), characterized in that chitosan oligosaccharides are precipitated by using hydrochloric acid to adjust the concentration to 2M to 6M, the solution fraction contains 50w / w% to 80w / w% of tetrasaccharides to hexasaccharides and 60w / w% to 95w / w% of tetrasaccharides to heptasaccharides, and the precipitate fraction contains 17w / w% or more of octasaccharides or more. In addition, the present invention also provides a method for precipitating chitosan oligosaccharides by using hydrochloric acid to adjust the concentration to 4M, and the solution fraction contains 55 w / w% to 80 w / w% tetrasaccharides to hexasaccharides and 70 w / w% tetrasaccharides to heptasaccharides. The method for isolating chitosan oligosaccharide according to (1), characterized in that the chitosan oligosaccharide contains 8 or more sugars at a maximum of 20 w / w% so that the total does not exceed 100 w / w%, and has a weight average molecular weight of 800 to 2000. The present invention also provides (7) a method for isolating chitosan oligosaccharides according to (1), characterized in that chitosan oligosaccharides are precipitated using hydrochloric acid to a concentration of 6 M, and the precipitated fraction contains monosaccharides to trisaccharides at 10 w / w% or less, tetrasaccharides to hexsaccharides at 20 w / w% or less, tetrasaccharides to heptasaccharides at 20 w / w% or less, and octosaccharides to 16saccharides at 5 w / w% or more, and has a weight average molecular weight of 5,000 or more. The present invention (8) is a method for isolating chitosan oligosaccharides according to (1), characterized in that chitosan oligosaccharides are fractionated by treating multiple times with hydrochloric acid at different concentrations within the range of 2M to 6M, and the resulting precipitate fraction or solution fraction contains 4-6 sugars at 15 w / w% or less, 4-7 sugars at 30 w / w% or less, and 8 or more sugars at 15 w / w% or more, with the total amount not exceeding 100 w / w% but 95 w / w% or less, and has a weight average molecular weight of 1000 to 5000.

[0015] In the above, the sugar number refers to the number of glucosamine bonds or the number of polymerizations. The sugar number being within a specified range means that the proportion of chitosan oligosaccharides with that sugar number is predominant, but does not mean that chitosan oligosaccharides with sugar numbers outside that range are not included at all. Also, since the raw chitosan oligosaccharide before being dissolved in water has a distribution of sugar numbers, the sugar number of the precipitated one does not necessarily contain one sugar, and conversely, the dissolved one contains an infinite number of sugars. In other words, in any case, most of the sugar numbers are distributed within a certain range. This is what is meant by being within a specified range. The precipitated chitosan oligosaccharide has a sugar number within a predetermined range, but the separation does not only focus on the precipitate but also includes the liquid phase soluble portion, because the sugar number of the liquid phase also falls within a separate predetermined range. Examples of the acid include hydrochloric acid, sulfuric acid, and nitric acid. The predetermined concentration refers to the final concentration. As the chitosan oligosaccharide concentration increases, it becomes more difficult to separate by filtration, so the preferred chitosan oligosaccharide concentration is approximately 7 w / v % to 15 w / v %. Effect of the Invention

[0016] According to the present invention, chitosan oligosaccharides having a sugar number within a predetermined range can be easily separated. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows an example of a fractionation procedure for chitosan oligosaccharides. [Diagram 2] 1 is a table showing the composition of the solution fraction (L fraction) and precipitate fraction (P fraction) of chitosan oligosaccharide depending on the hydrochloric acid concentration, and the respective yields and yields. [Diagram 3] 1 is a table showing the composition of the solution fraction (L fraction) and precipitate fraction (P fraction) and the respective yields and yields when the chitosan oligosaccharide concentration is changed as 4 M hydrochloric acid concentration. [Figure 4] FIG. 1 shows an example of a fractionation procedure for chitosan oligosaccharides. [Diagram 5] 1 is a table showing the yield and yield of the solution fraction (L fraction: 4ML) and the precipitate fraction (P fraction: 4-6MP and 6MP) based on the hydrochloric acid concentration. [Figure 6] 1 is a table showing the composition of chitosan oligosaccharide (raw COS) before fractionation by acid precipitation. [Figure 7] This is a table summarizing the composition, weight average molecular weight, yield, and yield rate of the 4ML fraction obtained by the acid precipitation method when the lot and concentration of raw chitosan oligosaccharide (raw COS) were changed and when the treatment procedure was changed. [Figure 8] This is a table summarizing the composition, weight average molecular weight, yield, and yield rate of the 4-6MP fraction obtained by the acid precipitation method when the lot and concentration of raw chitosan oligosaccharide (raw COS) were changed and when the treatment procedure was changed. [Figure 9] This is a table summarizing the composition, weight average molecular weight, yield, and yield rate of 6MP fractions obtained by acid precipitation method when the lot and concentration of raw chitosan oligosaccharide (raw COS) were changed and when the treatment procedure was changed. [Figure 10]FIG. 6 is a graph showing the average and standard deviation of the composition of each of the tables for the raw material chitosan oligosaccharide (raw material COS), the 4ML fraction, the 4-6MP fraction, and the 6MP fraction, which are summarized in FIG. 6, FIG. 7, FIG. 8, and FIG. 9. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings. Although chitosan oligosaccharides are water-soluble, we first investigated whether they would precipitate when acid was added and, if they did, how the sugar number distribution of the solid and liquid chitosan oligosaccharides would change. In the following, chitosan oligosaccharides will be referred to as COS. <Experiment 1> Hydrochloric acid was added to a 20 w / v% (weight / volume%) aqueous solution of COS derived from crab shells, which had been produced in-house, to prepare hydrochloric acid solutions of 2.0M, 3.0M, 4.0M, 4.5M, 5.0M, 5.5M, 6.0M, 8.0M, and 12M with a final COS concentration of 10 w / v%. The addition of hydrochloric acid produced precipitates, so the solution fraction (L fraction) and precipitate fraction (P fraction) were separated by filtration, deacidified, concentrated, and dried. Finally, the composition of the recovered chitosan oligosaccharides was analyzed. The processing procedure is shown in Figure 1.

[0019] Figure 2 is a table showing the yield and yield of the solution fraction (L fraction) and precipitate fraction (P fraction) of chitosan oligosaccharide as a function of hydrochloric acid concentration. Looking at the P fraction, the highest yield was obtained with 4M hydrochloric acid (12.9%). In addition, compared to the raw material without added hydrochloric acid (initial stage in Figure 2), the composition of the P fraction with 4M hydrochloric acid shows a large decrease in disaccharides to hexasaccharides and an increase in octasaccharides to decasaccharides.

[0020] (Sugar composition ratio measurement of chitosan oligosaccharide) The sugar composition ratio of chitosan oligosaccharide was measured or analyzed as a quantity of chitosan oligosaccharide hydrochloride according to Japanese Patent No. 6156829 (Example 3). The details are as follows. (1) Approximately 20 mg of chitosan oligosaccharides mixture (chitosan oligosaccharides hydrochloride, Seikagaku Bio) was weighed out, dissolved in 1 ml of water, and filtered through a 0.45 μm filter to prepare a standard solution. (2) 20 mg of each chitosan oligosaccharide sample was weighed out, dissolved in 1 ml of water, and filtered through a 0.45 μm filter (GLC TORAST Disc) to obtain a test solution. (3) HPLC analysis was performed under the following conditions. (Analysis conditions) Column: TSK-GEL AMIDE-80 4.6 x 250 mm Mobile phase: acetonitrile / 250mM phosphate buffer = 2 / 3 Flow rate: 0.7 mL / min Temperature: room temperature Injection volume: 20 μL Detector: Differential refractometer Equipment: LC-20A (Shimadzu Corporation) or liquid chromatography (JASCO) (4) The peak areas of chitosan oligosaccharide hydrochloride of each degree of polymerization in the test solution and the standard solution were measured, and the contents of each of 1 to 16 sugars relative to the total sugar were calculated using the following formula 1. The contents of each sugar were added together to obtain the content of chitosan oligosaccharide (hydrochloride reduced). The contents of 7 to 16 sugars were calculated from the standard peak of 6 sugars.

[0021]

number

[0022] (Molecular weight distribution measurement of chitosan oligosaccharide (GPC analysis)) Next, the molecular weight distribution was measured as follows. 2.5 mg of each chitosan oligosaccharide sample was dissolved in 5 mL of acetate buffer (0.5 M acetic acid / 0.5 M sodium acetate = 1:1) to prepare a test solution. Pullulan (STANDARD P-82, Shodex) was used as a standard reagent with molecular weights of 5,900 (P-5), 11,800 (P-10), 22,800 (P-20), 47,300 (P-50), 112,000 (P-100), 212,000 (P-200), and 788,000 (P-800). In addition, to improve the accuracy of the molecular weight measurements, a hexasaccharide of chitosan oligosaccharide (molecular weight: 1203, House Standard, Koyo Chemical) was used as a house standard. GPC analysis was carried out under the following quantitative conditions: The elution times of the standard reagent and each test solution were measured. Next, a calibration curve was created from the molecular weight and elution time of the standard reagent, and using this calibration curve, the weight average molecular weight (Mw), number average molecular weight (Mn), and dispersity (Mw / Mn) were calculated from the elution time of each test liquid. quantitative <Analysis conditions> Column: Shodex, OHpak SB-803HQ 8×300mm + SB-804HQ 8×300mm 2 connected Mobile phase: 0.5M acetic acid / 0.5M sodium acetate Flow rate: 1.0mL / min Temperature: 40℃ Injection volume: 200μL Detector: Differential refractometer Equipment: Liquid chromatograph manufactured by JASCO Analysis: JASCO ChromNAV GPC analysis software

[0023] <Experiment 2> Next, the relationship between the final COS concentration and the precipitation yield was determined. Hydrochloric acid was added to the COS aqueous solution so that the final COS concentrations were 5 w / v%, 10 w / v%, 20 w / v%, and 30 w / v%, and the final hydrochloric acid concentration was 4 M. The specific addition conditions were as follows. · 5w / v%COS: COS2.0g / water 20mL+8M HCl20mL ·10w / v%COS: COS2.0g / Water 10mL+8M HCl10mL ·20w / v%COS: COS2.0g / water 5mL+8M HCl 5mL ·30w / v%COS: COS3.0g / water 5mL+8M HCl 5mL A precipitate was obtained by adding hydrochloric acid, and the L and P fractions were separated by filtration, deacidified, concentrated, and dried. Finally, the composition of the recovered chitosan oligosaccharides was analyzed. The processing procedure was the same as that shown in Figure 1.

[0024] Figure 3 is a table showing the yield and yield of the solution fraction (L fraction) and precipitate fraction (P fraction) when the chitosan oligosaccharide concentration is changed as 4M hydrochloric acid concentration. Looking at the P fraction, the highest yield (25.7%) was obtained when the COS concentration was 30w / v%, but in reality, the concentration was too high and filtration separation was difficult. The next highest yield was obtained when the COS concentration was 10w / v%, at 16.3%.

[0025] From the results of Experiment 1 and Experiment 2, it was confirmed that filtration was easy and a large amount of precipitate was obtained when the final COS concentration was 10 w / v % and the amount of HCl added was about 4 M, resulting in a final concentration of 4 M. Although it depends on the type of specifications, generally, chitosan oligosaccharides having a sugar number within a predetermined range can be precipitated and separated by setting the chitosan oligosaccharide concentration in the range of 3 w / v % to 20 w / v % and adjusting the HCL concentration to 2 M to 6 M.

[0026] <Experiment 3> Next, we investigated whether a precipitate could be obtained from the L fraction by lowering the acid concentration. In the experiment, the P fraction was separated with a hydrochloric acid concentration of 6M (hereinafter referred to as 6MP), and the L fraction was diluted with hydrochloric acid to 4M, causing a precipitate (hereinafter referred to as 4-6MP and the liquid fraction as 4ML). The composition of these three fractions was analyzed. The processing procedure is shown in Figure 4.

[0027] FIG. 5 is a table showing the yields and yields of the solution fraction (L fraction: 4ML) and precipitate fraction (P fraction: 4-6MP and 6MP) according to the hydrochloric acid concentration. Compared to the initial stage, the 4-6MP fraction contained more 8-11 sugars. This was thought to be because the solubility of the 8-11 sugars dissolved in 6M hydrochloric acid was reduced by adjusting the concentration to 4M hydrochloric acid, resulting in the formation of precipitates of 8-11 sugars. Therefore, this method can be said to be useful for separating 8-11 sugars, and it was confirmed that by changing the final concentration of hydrochloric acid, it is possible to gradually separate COS with a specified range of sugar numbers (it can also be said that the COS in the solution fraction that is dissolved to the end can be separated as well).

[0028] Figure 6 is a table showing the composition of chitosan oligosaccharide (raw COS) before fractionation by acid precipitation. The average ± SD of the three lots was 14.5 ± 4.1 w / w% monosaccharides, 50.9 ± 7.4 w / w% tetrasaccharides, 60.8 ± 7.2 w / w% heptasaccharides, and 12.6 ± 2.7 w / w% octosaccharides.

[0029] Figure 7 is a table summarizing the composition, weight average molecular weight, yield, and yield of the 4ML fraction obtained by the acid precipitation method when the lot and concentration of the raw chitosan oligosaccharide (raw COS) were changed and when the processing procedure was changed. The 4ML fraction had 4-6 sugars at 66.2±5.2 w / w% (n=11, mean±SD, range: 57.9-73.1 w / w%), 4-7 sugars at 79.7±6.2 w / w% (n=11, mean±SD, range: 71.3-85.6 w / w%), and 8-13 sugars at 13.0±2.8 w / w% (n=11, mean±SD, range: 7.6-16.9 w / w%). ) and the contents of 4-6 sugars and 4-7 sugars were increased compared to the raw COS. The main components were tetrasaccharides to heptasaccharides, the weight-average molecular weight was 1480±160 (n=3, mean±SD), and the yield was 58.9±8.6w / w% (n=11, mean±SD). This data shows the compositions of the raw COS lot and concentration changed from 2.4 to 22.1w / w%, and when the treatment procedure was changed ((1) raw COS → 6M hydrochloric acid treatment → 6ML fraction → 4M hydrochloric acid treatment → 4ML fraction, (2) raw COS → 4M hydrochloric acid treatment → 4ML fraction). Therefore, it is considered that the 4ML fraction has a main composition of tetrasaccharides to heptasaccharides, regardless of the composition and concentration range of the raw COS and the procedure.

[0030] Figure 8 is a table summarizing the composition, weight average molecular weight, yield, and yield of the 4-6MP fraction obtained by the acid precipitation method when the lot and concentration of the raw chitosan oligosaccharide (raw COS) were changed and when the processing procedure was changed. The 4-6MP fraction contained 4-6 sugars to 6 sugars at 9.6 ± 5.3 w / w% (n = 5, mean ± SD, range: 3.8-14.3 w / w%), 4-6 sugars to 7 sugars at 16.5 ± 7.2 w / w% (n = 5, mean ± SD, range: 8.6-23.3 w / w%), and 8-13 sugars at 57.8 ± 24.0 w / w% (n = 5, mean ± SD, range: 22.5-87.4 w / w%). The contents of 4-6 sugars to 6 sugars and 4-7 sugars were lower than those of the raw COS, and the contents of 8-13 sugars were higher. The main composition was 8-13 sugars, the weight-average molecular weight was 4680±440 (n=2, mean±SD), and the yield was 5.7±1.6w / w% (n=5, mean±SD). This data shows the composition of the raw COS lot and concentration changed from 2.4 to 7.1w / w%, and the treatment procedure changed ((1) raw COS → 6M hydrochloric acid treatment → 6ML fraction → 4M hydrochloric acid treatment → 4-6MP fraction, (2) raw COS → 4M hydrochloric acid treatment → 4MP fraction → 6M hydrochloric acid treatment → 4-6MP fraction). Therefore, it is considered that the 4-6MP fraction has a main composition of 8-13 sugars, regardless of the composition and concentration range of the raw COS and the procedure.

[0031] Figure 9 is a table summarizing the composition, weight average molecular weight, yield, and yield of the 6MP fraction obtained by the acid precipitation method when the lot and concentration of the raw chitosan oligosaccharide (raw COS) were changed and when the processing procedure was changed. The 6MP fraction contained 10.8±5.7w / w% (n=7, mean±SD, range: 2.8-18.0w / w%) of 4-6 sugars, 14.3±7.1w / w% (n=7, mean±SD, range: 4.8-22.4w / w%) of 4-7 sugars, and 33.3±18.5w / w% (n=7, mean±SD, range: 10.9-57.9w / w%) of 8-13 sugars. The contents of 4-6 sugars and 4-7 sugars were lower than those of the raw COS, and the contents of 8-13 sugars were higher. The fractionation method to obtain the 6MP fraction is considered to be a useful method for efficiently reducing 8 sugars or less from the raw COS. On the other hand, the weight-average molecular weight was 5490 ± 240 (n = 4, mean ± SD), and the yield was 7.7 ± 3.3 w / w% (n = 7, mean ± SD). This data shows the composition of the raw COS lot and concentration changed from 4.4 to 8.9 w / w%, and the treatment procedure changed ((1) raw COS → 6M hydrochloric acid treatment → 6MP fraction, (2) raw COS → 4M hydrochloric acid treatment → 4MP fraction → 6M hydrochloric acid treatment → 6MP fraction). Therefore, it is considered that the 6MP fraction has a weight-average molecular weight of 5000 or more and has reduced 8 sugars or less, regardless of the procedure, within the composition and concentration range of the raw COS.

[0032] Figure 10 is a graph showing the average and standard deviation of the composition of each of the tables for the raw chitosan oligosaccharide (raw COS), 4ML fraction, 4-6MP fraction, and 6MP fraction summarized in Figures 6, 7, 8, and 9. The 4ML fraction is mainly composed of 4 to 7 sugars, and the 4-6MP fraction is mainly composed of 8 to 13 sugars. The 6MP fraction has a higher molecular weight than 4-6MP, and indicates that the amount of 8 sugars or less has been reduced.

[0033] The results of Experiment 3 lead us to the following conclusions. Note that the figures are based on data complementation or estimation. A: By adjusting the hydrochloric acid concentration to 2M to 6M, chitosan oligosaccharides can be precipitated, so that the solution fraction contains 50 w / w% to 80 w / w% of 4-6 sugars and 60 w / w% to 95 w / w% of 4-7 sugars, and the precipitate fraction contains 17 w / w% or more of 8 sugars or more. B: Chitosan oligosaccharides are precipitated by adjusting the hydrochloric acid concentration to 4M, and the solution fraction contains 55 w / w% to 80 w / w% 4-6 sugars, 70 w / w% to 95 w / w% 4-7 sugars, and a maximum of 20 w / w% 8 or more sugars so that the total does not exceed 100 w / w%, and the weight average molecular weight is 800 to 2000. C: Chitosan oligosaccharides are precipitated by adjusting the hydrochloric acid concentration to 6M, and the precipitated fraction contains monosaccharides to trisaccharides at 10 w / w% or less, tetrasaccharides to hexsaccharides at 20 w / w% or less, tetrasaccharides to heptasaccharides at 20 w / w% or less, and octosaccharides to 16saccharides at 5 w / w% or more, and has a weight average molecular weight of 5,000 or more. D: The chitosan oligosaccharide can be fractionated by treating multiple times with different hydrochloric acid concentrations within the range of 2M to 6M, so that the resulting precipitate fraction or solution fraction contains 4-6 sugars at 15 w / w% or less, 4-7 sugars at 30 w / w% or less, and 8 or more sugars at 15 w / w% or more, with the total content being 95 w / w% or less but not exceeding 100 w / w%, and has a weight average molecular weight of 1,000 to 5,000.

[0034] As described above, it was confirmed that it is possible to precipitate chitosan oligosaccharides having a sugar number within a predetermined range by preparing an acidic solution of a predetermined concentration and a chitosan oligosaccharide solution of a predetermined concentration. The sugar number in the liquid phase also becomes a range different from the above range accordingly. Therefore, it can be said that a technology has been provided that allows the sugar number of chitosan oligosaccharides to be adjusted and separated. In addition, after precipitation, water can be added to the liquid phase to cause further precipitation. This also provides a technology for adjusting the sugar number of chitosan oligosaccharide and isolating it. The dominant sugar number varies depending on the sugar number distribution and the degree of purification of the raw chitosan oligosaccharide. Similarly, the sugar number range may be appropriately designed depending on the type and concentration of the acid used, the extent of concentration reduction, and the application. [Industrial Applicability]

[0035] Using the present invention, LGS test agents and the like can be obtained.

Claims

1. A method for isolating chitosan oligosaccharides, comprising adding hydrochloric acid in the range of 2M to 6M to an aqueous solution of chitosan oligosaccharides in the range of 3 w / v % to 20 w / v %, and separating the chitosan oligosaccharides into a precipitate fraction and a solution fraction, thereby fractionating the chitosan oligosaccharides with different distributions of saccharide numbers.

2. A method for isolating chitosan oligosaccharides, comprising the steps of: adding hydrochloric acid in the range of 2M to 6M to an aqueous solution of chitosan oligosaccharides in the range of 3 w / v % to 20 w / v %, separating the solution fraction from a precipitate fraction; and adding hydrochloric acid to the solution fraction at a reduced acid concentration in the range of 2M to 6M to obtain a further precipitate fraction; or adding hydrochloric acid to the precipitate fraction at an increased acid concentration in the range of 2M to 6M to obtain a further solution fraction. By carrying out the hydrochloric acid addition treatment multiple times, chitosan oligosaccharides are fractionated with different saccharide number distributions.

3. 3. The method for isolating chitosan oligosaccharide according to claim 1 or 2, characterized in that the molecular weight of the chitosan oligosaccharide is controlled for use as a test drug for leaky gut syndrome.

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