Method for quantifying chitosan and method for preparing sample used for quantitative analysis of chitosan by high performance liquid chromatography

Abstract

To provide a method for quantifying chitosan and a method for preparing a sample used for quantitative analysis of chitosan by high performance liquid chromatography, which enable accurate quantification of chitosan content in food through simple operation.SOLUTION: A method for quantifying chitosan comprises a dissolution step in which chitosan contained in a food is dissolved in an acidic aqueous solution, an enzymatic degradation step of enzymatically degrading the chitosan using chitosanase, an acid degradation step of degrading the chitosan into glucosamine using an acid, and a quantification step of quantifying the glucosamine by high performance liquid chromatography to quantify the chitosan content.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for quantifying chitosan and a method for preparing a sample for use in the quantitative analysis of chitosan by high performance liquid chromatography. [Background technology]

[0002] Chitosan is a deacetylated chitin that is widely found in crustaceans, insects, and fungi, and is classified as a polysaccharide. Chitosan is soluble in dilute acids, and is expected to have the effect of dietary fiber due to its indigestibility. In particular, chitosan has many amino groups, so it adsorbs bile acids in the digestive tract and inhibits the absorption of cholesterol and the reabsorption of bile acids, thereby exhibiting an excellent effect of lowering serum cholesterol.

[0003] Chitosan, an excellent dietary fiber material, exists naturally, but foods designed to be added to foods to exert its effects are considered important in today's world where dietary fiber intake is insufficient.Accurate quantification of chitosan content is therefore important from a nutritional standpoint, and against this background, methods for quantifying chitosan in foods have been proposed (e.g., Patent Documents 1 and 2).

[0004] Patent Document 1 discloses a method for quantifying chitosan by using digestive enzymes to remove components other than chitosan from a sample (Prosky method), hydrolyzing the sample with acid to decompose the components into glucosamine, and then quantifying the chitosan by colorimetry using the indole hydrochloric acid method. Figure 1 is a flow chart of the conventional method for quantifying chitosan described in Patent Document 1. Patent Document 2 discloses a method for quantifying chitosan by the indole hydrochloric acid method, which includes a step of recovering dietary fiber containing chitosan by the Prosky method using a phosphate-free Tris-acetate buffer, and a step of recovering the dietary fiber on a glass filter without Celite, measuring a constant weight, and calculating the amount of dietary fiber.

[0005] However, in the indole hydrochloric acid method used in the conventional technology, not only glucosamine, which is a decomposition product of chitosan, but also amino sugars belonging to hexosamine develop color, so that the quantitative value obtained is not an accurate chitosan content, which is a problem. In addition, the conventional technology has many steps and is complicated in operation, so that the reproducibility of the quantitative value is low. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 2885353 [Patent Document 2] JP 2004-20266 A Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a method for quantitatively determining chitosan, which is simple in operation and enables accurate quantification of the chitosan content in food, and a method for preparing samples to be used in quantitative analysis of chitosan by high performance liquid chromatography. [Means for solving the problem]

[0008] The means for solving the above problems are as follows. <1> A dissolving step of dissolving chitosan in a food containing chitosan with an acidic aqueous solution; an enzymatic degradation step of enzymatically decomposing the polymer using chitosanase; an acid decomposition step of decomposing the glucosamine using an acid; A quantitative determination step of quantifying the glucosamine by high performance liquid chromatography to quantify the chitosan content. <2> The acidic aqueous solution used in the dissolving step is an aqueous solution of acetic acid. <1> This is a method for quantifying chitosan as described in the above. <3> The acid used in the acid decomposition step is hydrochloric acid. <1> from <2> The present invention relates to a method for quantifying chitosan. <4> The acid decomposition step is followed by an activated carbon treatment step. <1> from <3> The present invention relates to a method for quantifying chitosan. <5> A method for preparing a sample for quantitative analysis of chitosan by high performance liquid chromatography, comprising the steps of: A dissolving step of dissolving chitosan in a food containing chitosan with an acidic aqueous solution; an enzymatic degradation step of enzymatically decomposing the polymer using chitosanase; and an acid decomposition step of decomposing the glucosamine into glucosamine using an acid. <6> The acidic aqueous solution used in the dissolving step is an aqueous solution of acetic acid. <5> This is a method according to the present invention. <7> The acid used in the acid decomposition step is hydrochloric acid. <5> from <6> The method according to any one of the above items. <8> The acid decomposition step is followed by an activated carbon treatment step. <5> from <7> The method according to any one of the above items. Effect of the Invention

[0009] According to the present invention, there are provided a method for quantifying chitosan which is simple in operation and enables accurate quantification of the chitosan content in food, and a method for preparing a sample to be used in quantitative analysis of chitosan by high performance liquid chromatography. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a flow chart showing a conventional method for quantifying chitosan. [Diagram 2] FIG. 2 is a flowchart showing a method for quantifying chitosan according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, an embodiment of the present invention will be described. Note that the present invention is not limited to the embodiment described below, and can be modified as appropriate without departing from the gist of the present invention.

[0012] (Method of quantification of chitosan) The method for quantifying chitosan of the present invention is characterized by comprising a dissolving step of dissolving chitosan in a food containing chitosan in an acidic aqueous solution, an enzymatic decomposition step of enzymatically decomposing chitosan using chitosanase, an acid decomposition step of decomposing chitosan into glucosamine using an acid, and a quantitative determination step of quantifying the glucosamine by high performance liquid chromatography to determine the chitosan content, and further comprises other steps as necessary, such as an activated carbon treatment step of performing activated carbon treatment after the acid decomposition step to remove impurities. A flowchart of a method for quantifying chitosan according to one embodiment of the present invention is shown in Figure 2. Compared with the flowchart of the conventional method for quantifying chitosan shown in Figure 1, this method is characterized by having fewer steps and being simpler in operation.

[0013] <Dissolution process> In the dissolution step, chitosan in a food product containing chitosan is dissolved in an acidic aqueous solution.

[0014] -Chitosan- "Chitosan" is a 1,4-polymer of glucosamine or a 1,4-copolymer (random copolymer) of glucosamine and N-acetylglucosamine, and is a linear polysaccharide. Industrially, chitosan is obtained by extracting chitin (a 1,4-polymer of N-acetylglucosamine) contained in large quantities in the shells of crabs, shrimp, squid, insects, etc., and then hydrolyzing and deacetylating it in an alkali. Commercially available chitosan products with various physical properties and qualities, such as molecular weight, percentage of deacetylation (%DA), and viscosity, are available. The structural formulas of chitin and chitosan are shown below.

[0015] [ka] Here, m and n are integers.

[0016] The deacetylation percentage (% DA) of the chitosan is not particularly limited as long as it is soluble in an acidic aqueous solution and can be appropriately selected depending on the purpose, but is preferably 60% or more and 100% or less, and more preferably 80% or more and 100% or less. The percentage of deacetylation (% DA) corresponds to m / (m+n)×100 in the structural formula of chitosan.

[0017] -Foods containing chitosan- The food containing chitosan is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include supplements, etc. The food containing chitosan may consist of chitosan alone or may contain other ingredients. The form of the food containing chitosan is not particularly limited and can be appropriately selected depending on the purpose. Examples of the form include tablets, granules, powder, and the like.

[0018] - Acidic aqueous solution - In the present invention, an acidic aqueous solution is used to dissolve chitosan, and as will be described later, acidic conditions are also necessary for chitosanase to exhibit its enzymatic activity. The acidic aqueous solution is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include aqueous solutions of hydrochloric acid, sulfuric acid, acetic acid, etc. Among them, an aqueous solution of acetic acid is preferred from the viewpoints of the enzymatic activity of chitosanase and the inhibition of excessive decomposition of chitosan. The amount of the acidic aqueous solution used is not particularly limited and can be appropriately selected, but is preferably 0.5 mL to 1.3 mL, more preferably 0.8 mL to 1.0 mL, per 1 g of chitosan. An amount of 0.5 mL or more is preferable in terms of sufficient dissolution of chitosan, and an amount of 1.3 mL or less is preferable in terms of the optimum pH of the enzyme in the enzymatic decomposition step described below.

[0019] The temperature of the aqueous solution in the dissolving step is not particularly limited and may be appropriately selected depending on the purpose, but is preferably from 65° C. to 95° C., more preferably from 70° C. to 90° C., and particularly preferably from 75° C. to 85° C. A temperature of 65° C. or higher is preferable in that chitosan is completely dissolved, and a temperature of 95° C. or lower is preferable in that side reactions and excessive decomposition can be suppressed.

[0020] The time of the dissolving step is not particularly limited and can be appropriately selected depending on the conditions. For example, when an aqueous acetic acid solution is used as the acidic aqueous solution and the temperature of the dissolving step is 80°C, the time is preferably more than 10 minutes and less than 60 minutes, more preferably 15 minutes or more and 45 minutes or less, and particularly preferably 20 minutes or more and 30 minutes or less. If the time is 20 minutes or more, it is preferable in that chitosan is completely dissolved, and if it is 30 minutes or less, it is preferable in that side reactions and excessive decomposition can be suppressed.

[0021] The stirring conditions in the dissolving step are not particularly limited and can be appropriately selected according to the purpose. For example, when stirring is performed by a magnetic stirrer, the stirring speed is preferably 400 rpm or more, more preferably 450 rpm or more, and particularly preferably 500 rpm or more. A stirring speed of 500 rpm or more is preferable in that the time required to dissolve chitosan can be shortened.

[0022] It is preferable to wash the chitosan-containing food with water before the dissolving step to remove water-soluble substances in advance, which is preferable in that side reactions can be suppressed.

[0023] <Enzyme decomposition process> In the enzymatic decomposition step, chitosan dissolved in an acidic aqueous solution is enzymatically decomposed using chitosanase to obtain an enzymatic decomposition solution. The enzymatic decomposition step advances hydrolysis of the glycosidic bonds in the main chain of chitosan, producing oligomers of glucosamine ranging from dimers to hexamers. The chitosan quantification method of the present invention has the advantage that, by including the enzymatic decomposition step, the acid decomposition step described below can be carried out under milder conditions, thereby suppressing a decrease in the recovery rate due to overdecomposition.

[0024] -Chitosanase- In the present invention, chitosanase refers to any enzyme having a function of catalyzing the hydrolysis of the main chain glycosidic bond of chitosan (hereinafter referred to as chitosanase activity), and is not limited to a specific enzyme. The chitosanase is not particularly limited as long as it has chitosanase activity, and can be appropriately selected depending on the purpose. Commercially available chitosanase products include, for example, Chitosanase L (manufactured by HI Inc.) and Chitosanase C9830 (manufactured by Sigma-Aldrich). The amount of chitosanase used is not particularly limited as long as the enzymatic decomposition proceeds, and can be appropriately selected depending on the purpose, but is preferably 0.4 mL to 1.0 mL, more preferably 0.4 mL to 0.8 mL, per 1 g of food containing chitosan. An amount of 0.4 mL or more is preferable in that the enzymatic decomposition proceeds sufficiently, and an amount of 0.8 mL or less is preferable in that the analysis cost can be suppressed.

[0025] The temperature of the enzymatic decomposition step is set within the temperature range in which chitosanase exhibits its chitosanase activity. For example, the temperature range in which chitosanase L exhibits its chitosanase activity is 30°C or higher and 60°C or lower.

[0026] The time of the enzymatic decomposition step is not particularly limited and can be appropriately selected depending on the conditions. For example, when chitosanase L is used and the temperature of the enzymatic decomposition step is 40°C, it is preferably more than 2 hours and less than 6 hours, more preferably 2.5 hours or more and 5 hours or less, and particularly preferably 3 hours or more and 4 hours or less. A time of 3 hours or more is preferable in that the enzymatic decomposition proceeds sufficiently, and a time of 4 hours or less is preferable in that excessive decomposition can be suppressed.

[0027] The stirring conditions of the enzymatic decomposition step are not particularly limited and can be appropriately selected according to the purpose.For example, when stirring is performed by a magnetic stirrer, the stirring speed is preferably 400 rpm or more, more preferably 450 rpm or more, and particularly preferably 500 rpm or more.A stirring speed of 500 rpm or more is preferable in that the time required for completing the enzymatic decomposition can be shortened.

[0028] <Acid decomposition process> In the acid decomposition step, an acid is added to the enzymatic decomposition solution obtained in the enzymatic decomposition step to obtain an acid decomposition solution, in which chitosan oligomers are decomposed into monomers.

[0029] -acid- In the present invention, an acid is used to decompose chitosan oligomers into monomers. The acid is not particularly limited as long as it can decompose chitosan oligomers into monomers, and can be appropriately selected depending on the purpose, and examples thereof include hydrochloric acid, sulfuric acid, etc. Among them, hydrochloric acid is preferred from the viewpoint of suppressing excessive decomposition. The amount of acid used is not particularly limited and can be appropriately selected depending on the conditions. For example, when 35% hydrochloric acid is used as the acid, it is preferable to use 1 mL of the acid per 1 g of the enzymatic decomposition solution.

[0030] The temperature in the acid decomposition step is not particularly limited and can be appropriately selected depending on the purpose, but for example, when 35% hydrochloric acid is used as the acid, the temperature is preferably 100° C. or higher, more preferably 110° C. or higher, and particularly preferably 121° C. or higher. A temperature of 121° C. or higher is preferable in that the acid decomposition proceeds sufficiently.

[0031] The time for the acid decomposition step is not particularly limited and can be appropriately selected depending on the purpose, but for example, when 35% hydrochloric acid is used as the acid and the temperature for the acid decomposition step is 121° C., it is preferably more than 1 hour and less than 4 hours, more preferably 1.5 hours or more and 3.5 hours or less, and particularly preferably 2 hours or more and 3 hours or less. A time of 2 hours or more is preferable in that the acid decomposition proceeds sufficiently, and a time of 3 hours or less is preferable in that excessive decomposition can be suppressed.

[0032] <Quantitative process> In the quantitative determination process, the chitosan monomers produced by the acid decomposition process are quantified by high performance liquid chromatography (HPLC) to determine the chitosan content. Compared to the conventional quantitative method using the indole hydrochloric acid method, HPLC measurement can detect and quantify only glucosamine, resulting in more accurate quantitative results.

[0033] In the quantification step, the acid decomposition liquid itself may be quantified as a specimen, or a dried product obtained by removing the solvent from the acid decomposition liquid may be quantified as a specimen.

[0034] The conditions of the HPLC measurement in the quantitative determination step are not particularly limited as long as glucosamine can be detected, but if compounds other than glucosamine are detected at the same time, the measurement conditions are appropriately adjusted so that the peaks do not overlap with those of glucosamine. Adjustable measurement conditions include the type of HPLC column, the composition of the eluent, the flow rate of the eluent, and the column temperature.

[0035] There are no particular limitations on the glucosamine detector as long as it can detect glucosamine, but from the viewpoint of peak intensity, an RI (differential refractometer) detector is preferred.

[0036] In quantitative engineering, the amount of glucosamine contained in the sample is quantified. From the quantified glucosamine content, the chitosan content can be quantified by the following calculation formula. Chitosan content (g / mL) = Glucosamine content (g / mL) × {(179 - 18) / 179}

[0037] <Other processes> As the other processes, there is no particular limitation as long as the effects of the present invention are not impaired, and they can be appropriately selected according to the purpose. For example, an activated carbon treatment process, a drying process, etc. can be mentioned.

[0038] <<Activated carbon treatment process>> In the present invention, an activated carbon treatment process may be performed after the acid decomposition treatment process. In the activated carbon treatment process, activated carbon is added to the acid decomposition solution obtained in the acid decomposition process, stirred or allowed to stand, and then filtered to obtain an activated carbon-treated solution as a filtrate. At this time, the acid decomposition solution may be diluted with water. By performing the activated carbon treatment process, it is possible to suppress the adhesion of glucosamine to impurities and the resulting decrease in the recovery rate.

[0039] - Activated carbon - There is no particular limitation on the activated carbon used in the activated carbon treatment process, and it can be appropriately selected according to the purpose. For example, Purified Egret (manufactured by Osaka Gas Chemical Co., Ltd.), Taiko K Type M (manufactured by Fudamura Chemical Co., Ltd.), etc. can be mentioned. The amount of activated carbon used is not particularly limited and can be appropriately selected according to the purpose. However, it is preferably 0.25% by mass or more and 0.45% by mass or less, and more preferably 0.25% by mass or more and 0.35% by mass or less, with respect to the acid decomposition solution. When the amount of activated carbon used is 0.25% by mass or more, it is preferable in that impurities can be sufficiently removed. When it is 0.35% by mass or less, it is preferable in that it can suppress the decrease in the recovery rate due to the adsorption of glucosamine by the activated carbon.

[0040] <<Drying process>> The drying step is a step of drying the material after the acid decomposition step or the activated carbon treatment step. The drying method is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include natural drying, hot air drying, reduced pressure drying, freeze-drying, etc., among which freeze-drying is preferred. These drying methods may be used alone or in combination of two or more.

[0041] (Preparation of samples for quantitative analysis of chitosan) The method for preparing a sample used in the quantitative analysis of chitosan of the present invention is characterized by including a dissolution step of dissolving chitosan in a food containing chitosan with an acidic aqueous solution, an enzymatic decomposition step of enzymatically decomposing chitosan using chitosanase, and an acid decomposition step of decomposing chitosan into glucosamine using an acid, and may further include other steps as necessary, such as an activated carbon treatment step in which activated carbon treatment is performed after the acid decomposition step to remove impurities. The dissolution step, enzymatic decomposition step, acid decomposition step and other steps in the method for preparing a sample used in the quantitative analysis of chitosan of the present invention can be suitably carried out in the same manner as described in the method for quantitatively analyzing chitosan of the present invention. EXAMPLES

[0042] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0043] (Example) -Dissolution process- The chitosan used was chitosamine (deacetylation degree 83%, manufactured by Nippon Kayaku Food Techno Co., Ltd.), which is used as a raw material for FOSHU. 0.25 g of chitosamine was precisely weighed and placed in a 100 mL glass bottle with a screw cap, 40 mL of distilled water was added, and then 0.2 mL of acetic acid was added, the bottle was capped with a screw cap, and the bottle was stirred at 500 rpm for 30 minutes in an 80°C water bath. -Enzyme decomposition process- After cooling to room temperature in running water, 9.9 mL of distilled water and 0.1 mL of chitosanase (Chitosanase L, manufactured by HBI Inc.) were added and stirred at 40° C. and 500 rpm for 3 hours to obtain an [enzymatic hydrolysis solution]. -Acid decomposition process- 2 g of the [enzymatic decomposition solution] was weighed out and placed in a 15 mL test tube, to which 2 mL of 35% hydrochloric acid was added. The tube was then sealed with a rubber stopper equipped with a glass cooling tube, and heated in a heat block set at 121°C for 3 hours to obtain the [acid decomposition solution]. -Activated carbon treatment process- Wash out the [acid digestion solution] in the test tube four times with 4 mL of distilled water, then place in a centrifuge tube with a screw cap, Powdered activated carbon (Seijin Shirasagi, manufactured by Osaka Gas Chemicals Co., Ltd.) was added in an amount of 0.25% by mass of the liquid volume. After standing for 30 minutes, the mixture was filtered using a suction filter, and the filtrate was collected in a 100 mL eggplant flask to obtain [activated carbon-treated liquid]. -Drying process- The [activated carbon-treated liquid] was pre-frozen in a freeze dryer, and then dried in a freeze dryer to obtain a [dried product]. -Quantitative process- 1 mL of distilled water was added to the [dried product] to dissolve it, and then 1 mL of acetonitrile was added to obtain [acetonitrile water solution]. The [acetonitrile water solution] was filtered through a 0.45 μm filter, and the filtrate was used as a sample for HPLC measurement. The HPLC measurement conditions are as follows. Guard column: Shodex Asahipak NH2P-50G 4A (4.6mm x 10mm) Column: Shodex Asahipak NH2P-50 4E (4.6mm x 250mm) Mobile phase: acetonitrile / water = 70 / 30 Flow rate: 0.8mL / min Temperature: 28℃ Detector: RI (differential refraction) Using a calibration curve prepared by measuring standard glucosamine hydrochloride (D(+)-glucosamine hydrochloride, Kanto Chemical Co., Ltd.) by HPLC, the measured value of the glucosamine concentration in the sample was calculated from the peak area of ​​glucosamine, and the chitosan content was calculated. Meanwhile, the theoretical value of the chitosan content in the sample was calculated from the degree of deacetylation of chitosan and the weight of the chitosan when it was precisely weighed. The recovery rate was calculated by dividing the measured value of the chitosan content by the theoretical value. The closer the recovery rate is to 100%, the more suitable it is for quantifying chitosan.

[0044] (Comparison of dissolution process conditions) Table 1 shows the results of comparing the recovery rates when only the dissolution process time was changed.

[0045] [Table 1]

[0046] As can be seen from Table 1, good recovery rates were obtained when the dissolution step time was 20 and 30 minutes. When the dissolution step time was 10 minutes, chitosan was not sufficiently dissolved, and enzymatic degradation did not proceed sufficiently in the next enzymatic degradation step, which is thought to have reduced the recovery rate. When the dissolution step time was 60 minutes or longer, chitosan crosslinked and precipitated, and enzymatic degradation did not proceed sufficiently in the next enzymatic degradation step, which is thought to have reduced the recovery rate.

[0047] (Comparison of enzymatic hydrolysis process conditions) Table 2 shows the results of comparing the recovery rates when only the time of the enzymatic hydrolysis step was changed.

[0048] [Table 2]

[0049] From Table 2, good recovery rates were obtained when the enzymatic hydrolysis process time was 3 and 4 hours. When the enzymatic hydrolysis process time was 2 hours, the recovery rate is thought to have decreased because chitosan was not decomposed sufficiently. When the enzymatic hydrolysis process time was 6 hours or more, the recovery rate is thought to have decreased because over-decomposition progressed.

[0050] (Comparison of acid decomposition process conditions) Table 3 shows the results of comparing the recovery rates when only the acid decomposition step time was changed.

[0051] [Table 3]

[0052] From Table 3, good recovery rates were obtained when the acid decomposition process time was 2 and 3 hours. When the acid decomposition process time was 1 hour, it is believed that the recovery rate decreased due to insufficient acid decomposition. When the acid decomposition process time was 4 hours or more, it is believed that the recovery rate decreased due to excessive decomposition.

[0053] (Comparison with and without activated carbon treatment process) Table 4 shows the results of comparing the recovery rates with and without the activated carbon treatment process.

[0054] [Table 4]

[0055] As can be seen from Table 4, a good recovery rate was obtained when the activated carbon treatment step was performed. It is believed that the recovery rate decreased when the activated carbon treatment step was not performed because glucosamine adhered to impurities.

[0056] (Comparison based on the degree of deacetylation) Chitosan with different deacetylation degrees (deacetylation degree: 72.3%, prototype, manufactured by Nippon Kayaku Food Techno Co., Ltd.) was quantified using the chitosan quantification method described in the Examples (dissolution step: 30 minutes, enzymatic decomposition step: 3 hours, acid decomposition step: 3 hours, activated carbon treatment: yes). The theoretical chitosan content was 0.573%, while the measured chitosan content was 0.565%, with a recovery rate of 98.6%. This shows that the quantitative results are not affected even when samples with different deacetylation degrees are used.

[0057] (Comparison between the conventional method and the present invention) Table 5 shows the results of a comparison of the recovery rates between the conventional chitosan quantification method described in Patent Document 1 and the chitosan quantification method described in the Examples (dissolution step: 30 minutes, enzymatic decomposition step: 3 hours, acid decomposition step: 3 hours, activated carbon treatment: yes). Each measurement was performed 10 times, and the average value and standard deviation were calculated. Two types of samples were used: chitosan powder (Chitosamine, manufactured by Nippon Kayaku Food Techno Co., Ltd.) and chitosan-containing tablets (Calolimit, manufactured by FANCL Corporation).

[0058] [Table 5]

[0059] The conventional method for quantifying chitosan is shown below. 0.1 g of sample was weighed out and placed in a 400 mL tall beaker, 50 mL of 0.05 mol / L phosphate buffer (pH 6) was added, and 0.1 mL of heat-resistant α-amylase solution (A3306, Sigma-Aldrich) was added. The beaker was covered with aluminum foil and heated in a boiling water bath for 30 minutes. During heating, the beaker was gently stirred every 5 minutes. After cooling to room temperature, 0.171 N sodium hydroxide solution was added to adjust the pH to 7.5 ± 0.1. 100 μL of a solution prepared by diluting 5 mL of heat-stable protease (P5380, Sigma-Aldrich) with 100 μL of 0.05 mol / L phosphate buffer was added, the solution was covered with aluminum foil, and the solution was shaken in a 60°C water bath for 30 minutes. After cooling to room temperature, 0.205 mol / L phosphoric acid was added to adjust the pH to 4.5±0.2, 0.3 mL of amyloglucosidase solution (A9913, Sigma-Aldrich) was added, the mixture was covered with aluminum foil, and shaken in a 60°C water bath for 30 minutes. 280mL of 95% ethanol preheated to 60℃ was added, and the mixture was left to stand at room temperature for 45 minutes, after which it was suction filtered using a glass filter (Prosity-2, pore size: 40-90μm) with 0.5g of acid-washed celite evenly spread over it. After filtration, the chitosan remaining in the tall beaker was washed twice with 20mL of 78% ethanol onto the glass filter while suctioning, and then the chitosan on the glass filter was washed twice with 10mL of 95% ethanol and twice with 10mL of acetone, and dried overnight in a dryer at 105℃. The dried chitosan on the glass filter was scraped together with the Celite and the entire amount was placed in a 50 mL centrifuge tube. 1 mL of 12 mol / L sulfuric acid was added and the mixture was stirred well with a glass rod, and then the mixture was left at room temperature for 2 hours while being kneaded every 10 minutes. Thereafter, 32.6 mL of deionized water was added while washing the chitosan adhering to the glass rod, and a cooling tube was attached and hydrolysis was carried out at 100° C. for 6 hours. Then, without cooling, the tube was centrifuged to precipitate the celite, and the supernatant was collected. Furthermore, 10 mL of 0.358 mol / L sulfuric acid was added to the celite remaining in the centrifuge tube, and the tube was stirred thoroughly, heated to 100°C, centrifuged, and the supernatant was collected. This procedure was repeated twice. The resulting supernatants were combined, and the volume was adjusted with deionized water to a chitosan concentration of 0.02 to 0.4 mg / mL to prepare a test solution. 0.5 mL of the test solution was placed in a test tube and 0.5 mL of 5% sodium nitrite aqueous solution was added. For the blank test, 0.5 mL of deionized water was added instead of the 5% sodium nitrite aqueous solution. 0.5 mL of 33% acetic acid was added, mixed, and left at room temperature for 10 minutes. 0.5 mL of 12.5% ​​aqueous ammonium sulfamate solution was added, and the mixture was allowed to stand for 30 minutes with stirring every 5 minutes. After adding 2 mL of 5% hydrochloric acid, 0.2 mL of 1% indole ethanol solution was added and mixed, and the mixture was heated in a boiling water bath for 5 minutes. After adding 2 mL of 99.5% ethanol, the mixture was cooled and the absorbance at 490 nm and 520 nm was immediately measured. The optical density (OD value) was calculated from a calibration curve prepared using an aqueous solution of glucosamine hydrochloride, and the recovery rate was calculated.

[0060] As can be seen from Table 5, when chitosan powder was used, the conventional method had a low recovery rate and a large error in the quantitative value, whereas the method of the present invention had a high recovery rate and a small error in the quantitative value. Furthermore, when chitosan-containing tablets were used, the recovery rate significantly exceeded 100% in the conventional method, whereas the recovery rate did not significantly exceed 100% in the method of the present invention.

[0061] (Enzymatic decomposition other than by chitosanase) The enzymatic degradation of chitosan was investigated using enzymes other than chitosanase. The experimental and measurement methods are shown below.

[0062] -Experimental Method- 19 g of water was added to 1 g of chitosan and stirred to prepare a suspension with a chitosan concentration of 5%. 1 g of the enzyme shown in Table 6 was added to the suspension, and enzymatic hydrolysis was carried out for 48 hours with stirring in a thermostatic chamber. The temperature of the thermostatic chamber was set to the optimal temperature for the added enzyme. For comparison, a control was prepared by adding 1 g of distilled water instead of 1 g of enzyme. The enzyme was then inactivated by heating at 80°C for 20 minutes. The enzymatic decomposition product was centrifuged (3000 rpm, 5 minutes) to precipitate the solid matter, and the supernatant was filtered through filter paper, and the resulting filtrate was used as the measurement sample. The presence of low molecular weight water-soluble chitosan decomposed by the enzyme in the measurement sample was measured by cationization.

[0063] -Measurement method- A white test cotton cloth (4 cm x 4 cm) was soaked in 1 mL of the test specimen liquid, and water-soluble chitosan with cationic properties was attached to the test cotton cloth. After immersing the cloth in 50 mL of 0.01% Direct Blue aqueous solution with anionic properties that binds to the cationic chitosan for 10 minutes, the cloth was washed with 50 mL of distilled water to remove the Direct Blue liquid that was not bound to the chitosan. Washing with 50 mL of distilled water was performed three times in total. The test cotton cloth was dried in a hot air dryer at 50°C for 15 minutes. The presence or absence of low molecular weight water-soluble chitosan in the test specimen liquid was evaluated based on whether the white test cotton cloth turned blue with Direct Blue, and the b* value (degree of blueness) of the test cotton cloth was measured with a color difference meter. The results are shown in Table 6.

[0064] [Table 6] Proteax: Manufactured by Amano Enzyme Co., Ltd. Neurase F3G: Amano Enzyme Co., Ltd. Pancreatin F: Amano Enzyme Co., Ltd. Protease A "Amano" SD: Amano Enzyme Co., Ltd. Protease M "Amano" SD: Amano Enzyme Co., Ltd. Protease P "Amano" SD: Amano Enzyme Co., Ltd. Peptidase R: Amano Enzyme Co., Ltd. Samoase PC10F: Amano Enzyme Co., Ltd. Protin SD-AY10: Amano Enzyme Co., Ltd. Protin SD-NY10: Amano Enzyme Co., Ltd.

[0065] The b* value (blue color) of the sample treated at 40°C with the enzyme "Neurase F3G" was good, indicating that a large amount of water-soluble chitosan was obtained.

[0066] (Enzymatic digestion using Neurase F3G) -Experimental Method- 19 g of water was added to 1 g of chitosan and stirred to prepare a suspension with a chitosan concentration of 5%. Neurase F3G was added to the suspension in the amount shown in Table 7, and enzymatic hydrolysis was carried out for 48 hours with stirring in a thermostatic chamber at 40°C. The enzyme was then inactivated by heating at 80°C for 20 minutes. The enzymatic decomposition product was centrifuged (3000 rpm, 5 minutes) to precipitate the solid matter, and the supernatant was filtered through filter paper, and the resulting filtrate was used as a measurement sample. The amount of chitosan contained in the measurement sample was measured by the indole hydrochloric acid method.

[0067] -Measurement method- 0.5 mL of the test solution was placed in a test tube and 0.5 mL of 5% sodium nitrite aqueous solution was added. For the blank test, 0.5 mL of deionized water was added instead of the 5% sodium nitrite aqueous solution. 0.5 mL of 33% acetic acid was added, mixed, and left at room temperature for 10 minutes. 0.5 mL of 12.5% ​​aqueous ammonium sulfamate solution was added, and the mixture was allowed to stand for 30 minutes with stirring every 5 minutes. After adding 2 mL of 5% hydrochloric acid, 0.2 mL of 1% indole ethanol solution was added and mixed, and the mixture was heated in a boiling water bath for 5 minutes. After adding 2 mL of 99.5% ethanol, the mixture was cooled and the absorbance at 490 nm and 520 nm was immediately measured. The optical density (OD value) was calculated from a calibration curve prepared using an aqueous solution of glucosamine hydrochloride, and the recovery rate was calculated. The results are shown in Table 7.

[0068] [Table 7]

[0069] From Table 7, it was found that the chitosan concentration increased with increasing amounts of Neurase F3G, but the amount of water-soluble chitosan obtained dropped off sharply when the amount added was about 1 g. The chitosan recovery rate was 70% at an addition amount of 2 g, and it was found that a high recovery rate could not be obtained even when using cheap food enzymes in double the amount of chitosan. From the above, it was found that chitosanase is suitable as the enzyme to be used in the enzymatic decomposition step of the present invention.

Claims

1. A dissolving step of dissolving chitosan in a food containing chitosan with an acidic aqueous solution; an enzymatic degradation step of enzymatically decomposing the polymer using chitosanase; an acid decomposition step of decomposing the glucosamine using an acid; A method for quantifying chitosan, comprising the steps of quantifying the glucosamine by high performance liquid chromatography and quantifying the chitosan content.

2. 2. The method for quantifying chitosan according to claim 1, wherein the acidic aqueous solution used in the dissolving step is an aqueous solution of acetic acid.

3. 2. The method for quantifying chitosan according to claim 1, wherein the acid used in the acid decomposition step is hydrochloric acid.

4. The method for quantifying chitosan according to claim 1 , further comprising an activated carbon treatment step of performing activated carbon treatment after the acid decomposition step.

5. A method for preparing a sample for quantitative analysis of chitosan by high performance liquid chromatography, comprising the steps of: A dissolving step of dissolving chitosan in a food containing chitosan with an acidic aqueous solution; an enzymatic degradation step of enzymatically decomposing the polymer using chitosanase; and an acid decomposition step of decomposing the glucosamine using an acid.

6. 6. The method according to claim 5, wherein the acidic aqueous solution used in the dissolving step is an aqueous solution of acetic acid.

7. The method according to claim 5, wherein the acid used in the acid decomposition step is hydrochloric acid.

8. The method according to any one of claims 5 to 7, further comprising an activated carbon treatment step of performing an activated carbon treatment after the acid decomposition step.

Images