Method for analysis

Abstract

To provide a method for analyzing the content of proteoglycan in a gel composition.SOLUTION: The present disclosure relates to a method for analyzing proteoglycan content in a gel composition, the method including: a degradation step for degrading the gel composition; and an analysis step for analyzing proteoglycan in a degradation product obtained in the degradation step.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to analytical methods. [Background technology]

[0002] Proteoglycans are molecules that constitute the extracellular matrix together with collagen, hyaluronic acid, etc. Proteoglycans are known to have excellent water-retaining properties and various physiological functions, such as anti-inflammatory effects, hyaluronic acid synthesis promotion effects, and cell proliferation promotion effects. For this reason, various studies have been conducted on the use of proteoglycans as functional ingredients in foods, etc.

[0003] Foods containing the functional ingredient can claim functionality by submitting a notification to the Commissioner of the Consumer Affairs Agency before sale. The notification requires the submission of analytical test results and documents describing analytical methods showing that the food contains the functional ingredient as an active ingredient, in order to guarantee its effectiveness based on scientific evidence (Non-Patent Document 1). Therefore, methods for analyzing foods containing proteoglycan to confirm that the food contains proteoglycan are being explored (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2022-177785 [Non-patent literature]

[0005] [Non-Patent Document 1] [For food-related businesses] Notification of functional food claims: Consumer Affairs Agency [Retrieved February 20, 2024], Internet <https: / / www.caa.go.jp / policies / policy / food_labeling / foods_with_function_claims / notice / #guideline> Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, an object of the present disclosure is to provide, for example, a method capable of analyzing the proteoglycan content contained in a gel composition. [Means for solving the problem]

[0007] In order to achieve the above-mentioned object, the analytical method of the present disclosure is a method for analyzing the proteoglycan content contained in a gel composition, and includes a decomposition step of decomposing the gel composition and an analytical step of analyzing the proteoglycans in the obtained decomposition product. [Effects of the Invention]

[0008] According to the present disclosure, for example, it is possible to provide a method capable of analyzing the proteoglycan content contained in a gel composition. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows the main disaccharide structures of each chondroitin sulfate. DETAILED DESCRIPTION OF THE INVENTION

[0010] <Definition> As used herein, "gel" refers to a substance in which a solvent is incorporated into a polymer having a three-dimensional network structure, and which is in a state intermediate between a solid and a liquid. Examples of compositions containing the gel (gel compositions) include jelly-like confectioneries. Examples of jelly-like confectioneries include gummy candy, jelly beans, jelly, pudding, panna cotta, bavarois, and mousse.

[0011] As used herein, the term "protease" refers to a hydrolase that catalyzes the breakdown of proteins into polypeptides and amino acids.

[0012] As used herein, "proteoglycan" refers to a molecule (glycoprotein) in which a protein (core protein) is covalently bonded to a glycosaminoglycan (GAG, also called a "polysaccharide" or "sugar chain"). The proteoglycan exists as an extracellular matrix in, for example, skin, organs, and cartilage. The glycosaminoglycan is generally known as a sugar chain having a long chain structure without a branched structure. Examples of the proteoglycans include aggrecan, versican, decorin, testican, brevican, biglycan, serglycin, syndecan, perlecan, dystroglycan, agrin, claustrin, glypican, lumican, keratocan, and neurocan. The proteoglycans can be classified into chondroitin sulfate proteoglycans, dermatan sulfate proteoglycans, heparan sulfate proteoglycans, and keratan sulfate proteoglycans, depending on the type of GAG bound to the protein.

[0013] Examples of the GAG ​​include chondroitin, chondroitin sulfate, dermatan sulfate (chondroitin sulfate B), heparan sulfate, heparin, and keratan sulfate. Examples of the chondroitin include O-type sugar chains whose main disaccharide structure is a disaccharide structure of glucuronic acid and acetylgalactosamine, and iO-type sugar chains whose main structure is a disaccharide structure of iduronic acid and acetylgalactosamine (hereinafter referred to as "chondroitin sulfate O" and "chondroitin sulfate iO," respectively). The chondroitin sulfate has a structure in which a sulfate group is added to a sugar chain in which the disaccharide structure of glucuronic acid and acetylgalactosamine is repeated. Examples of the chondroitin sulfate include chondroitin sulfate A (A-type), whose main disaccharide structure is a disaccharide structure of glucuronic acid and acetylgalactosamine tetrasulfate; chondroitin sulfate iA (iA-type), whose main disaccharide structure is a disaccharide structure of iduronic acid and acetylgalactosamine tetrasulfate; chondroitin sulfate C (C-type), whose main disaccharide structure is a disaccharide structure of glucuronic acid and acetylgalactosamine hexasulfate; and chondroitin sulfate iC (iC-type), whose main disaccharide structure is a disaccharide structure of iduronic acid and acetylgalactosamine hexasulfate. Each chondroitin sulfate has, for example, the disaccharide structure shown in FIG. 1 as its main disaccharide structure. Note that in FIG. 1, the sulfate group (sulfo group) is bonded to a hydrogen atom, but the present disclosure is not limited thereto. For example, the sulfate group of the GAG ​​may be ionized by elimination of the hydrogen atom, or may form a salt.

[0014] As used herein, "crosslinking" refers to the linking of multiple molecules by chemical bonds, which may be non-covalent bonds such as ionic interactions (ionic bonds), hydrophobic interactions (hydrophobic bonds), hydrogen bonds, and / or coordinate bonds, or covalent bonds.

[0015] As used herein, "chelation" refers to the formation of multiple coordinate bonds between a ligand, such as a chelating agent, and a metal ion, forming a complex with the metal ion.

[0016] The present disclosure will be described below using examples, but the present disclosure is not limited to the following examples and can be implemented with any modifications. Furthermore, the descriptions in this disclosure are mutually applicable unless otherwise specified. In this specification, the expression "to" is used to include the numerical or physical values ​​before and after it. In this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B."

[0017] <Analysis method> In one aspect, the present disclosure provides a method for analyzing the proteoglycan content of a gel composition. The method includes a degradation step of degrading proteins contained in the gel composition and an analysis step of analyzing the proteoglycans in the resulting degradation product. The analytical method of the present disclosure allows the proteoglycan content of a gel composition to be analyzed.

[0018] As a result of extensive research, the present inventors attempted to quantify the proteoglycans contained in gel compositions using known methods (e.g., References 1 to 3 below), but found that none of these methods allowed for quantitative determination of the proteoglycans contained in gel compositions. Therefore, as a result of further research, the present inventors discovered that the proteoglycans contained in gel compositions can be analyzed by decomposing a gel composition and analyzing the proteoglycans in the resulting decomposition product, thereby establishing the present disclosure. In this disclosure, it is presumed that the proteoglycans contained in the gel composition can be more accurately quantified by the mechanism described below. However, the present disclosure is not limited in any way by the mechanism described below. The proteoglycans contain many functional groups, such as sulfate groups and carboxyl groups, and many metal ions, such as calcium ions, as counterions. Furthermore, since the gelling agents generally contain functional groups, such as hydroxyl groups and carboxyl groups, they are thought to be capable of chelating metal ions via these functional groups. Therefore, it is presumed that in the gel composition, the metal ions are chelated to the gelator contained in the gel composition and the proteoglycan, thereby crosslinking them (physical crosslinks). Furthermore, it is presumed that the presence of the crosslinks in the gel composition causes proteoglycans to remain crosslinked with the gelator even when the gel composition is dispersed in a solvent such as a buffer solution or water, making it difficult to accurately quantify the proteoglycans. On the other hand, in the present disclosure, the proteoglycans can be released from the gelator by decomposing the gel composition and cleaving the crosslinks or the crosslinked structures formed by the crosslinks, thereby presumed to enable more accurate quantification of the proteoglycans. Specifically, the present disclosure is presumed to enable more accurate quantification of the proteoglycans by, for example, chelating the metal ions forming the crosslinks between the gelator and the proteoglycan, and then directly cleaving the crosslinks between the gelator and the proteoglycan, thereby releasing the proteoglycans.Furthermore, when the gel composition contains a protein as a gelling agent, the present disclosure is believed to enable more accurate quantification of the proteoglycan by, for example, decomposing the protein as the gelling agent, thereby preventing the gelling agent contained in the gel composition from forming a crosslinked structure with the proteoglycan, thereby enabling the proteoglycan content in the gel composition to be analyzed. Reference 1: JP 2018-151207 A Reference 2: JP 2021-189178 A Reference 3: JP 2022-177785 A

[0019] The proteoglycan may be, for example, an animal proteoglycan. The animal is not particularly limited, and examples thereof include mammals (mammals) such as pigs, cows, and whales; birds (birds) such as chickens; fish such as flatfish (e.g., sole), salmonids (e.g., chum salmon, Atlantic salmon), rays (including, for example, skimmer); and mollusks such as squid. The animal is preferably salmon, pig, chicken, flatfish, or rays.

[0020] The animal tissue is, for example, a tissue containing proteoglycan and / or collagen, and examples thereof include epithelial tissue such as skin, cartilaginous tissue such as cartilage, digestive organs, circulatory organs, respiratory organs, and placenta, etc. Specific examples of the animal tissue include cartilage, fins, digestive organs, circulatory organs, respiratory organs, and ears.

[0021] Examples of the sugar chain of the proteoglycan include chondroitin, chondroitin sulfate, dermatan sulfate (chondroitin sulfate B), heparan sulfate, heparin, and keratan sulfate. The sugar chain is preferably chondroitin sulfate.

[0022] The gel composition contains, as a gelling agent, for example, collagen, gelatin, pectin, carrageenan, locust bean gum, guar gum, agar, deacylated gellan gum, native gellan gum, glucomannan, xanthan gum, tamarind seed gum, alginate, etc. The gelling agent is preferably collagen or gelatin.

[0023] The gel composition contains, for example, 1 to 20 mass %, or 3 to 15 mass %, of the gelling agent based on the total mass of the gel composition.

[0024] The gel composition may contain, for example, sugars. Examples of the sugars include sucrose, glucose, fructose, galactose, maltose, trehalose, maltitol, lactitol, sorbitol, mannitol, xylitol, erythritol, reduced starch hydrolysates, reduced xylooligosaccharides, palatinit, reduced branched oligosaccharides, reduced starch syrup, reduced maltose, starch syrup, and reduced maltose starch syrup. The gel composition may contain one type of sugar or several types of sugars.

[0025] When the gel composition contains the sugar, the gel composition may contain, for example, 50 to 100% by mass, 50 to 96% by mass, 55 to 95% by mass, 60 to 93% by mass, or 60 to 90% by mass of the sugar, based on the total mass of the gel composition.

[0026] The analytical method of the present disclosure may include a dispersion step of dispersing the gel composition prior to the decomposition step. The dispersion step may also be referred to as dissolution, for example. The dispersion step is not particularly limited as long as it can disperse the gel composition, and can be determined depending on the gelling agent, for example. Specific examples include heat treatment. Examples of the heat treatment include a water bath and heating. The dispersion step may be performed in the presence of a solvent, for example. The solvent is preferably an aqueous solvent, and specific examples include Tris-HCl buffer solution.

[0027] In the dispersing step, the dispersion temperature is, for example, 20 to 50°C, preferably 30 to 40°C, and more preferably 37°C.

[0028] In the dispersing step, the dispersing time is not particularly limited as long as the gel composition can be dispersed, and is preferably 2 minutes or more.

[0029] In the dispersion step, the pH of the solvent is not particularly limited as long as the gel composition can be dispersed therein. The pH is preferably such that no pH adjustment is required in the decomposition step described below.

[0030] In the dispersion step, for example, sugars may be added to the gel composition because this can improve the recovery rate of proteoglycans contained in the gel composition. The concentration of the added sugars is, for example, 50 to 100% by mass, 50 to 96% by mass, 55 to 95% by mass, 60 to 93% by mass, or 60 to 90% by mass, based on the total mass of the gel composition after the addition of the sugars.

[0031] In the analysis method of the present disclosure, as described above, the gel composition is decomposed in the decomposition step. The decomposition may be, for example, decomposition by cleavage of crosslinks between the gelator and the proteoglycan contained in the gel composition, or decomposition by cleavage of covalent bonds of the gelator. Decomposition by cleavage of covalent bonds can also be considered, for example, as chemical decomposition of the crosslinked structure formed between the gelator and the proteoglycan.

[0032] When the degradation is due to cleavage of the crosslinks, the degradation step may involve, for example, contacting the gel composition with a chelating agent capable of cleaving metal ion crosslinks between the gelator and the proteoglycan contained in the gel composition. The degradation is due to cleavage of the crosslinks between the proteoglycan and the gelator, for example, when the metal ions are chelated by the chelating agent, rendering the proteoglycan unable to form crosslinks with the gelator. The contact between the gel composition and the chelating agent can be carried out appropriately, for example, depending on the state of the gel composition and the chelating agent. The gel composition and the chelating agent to be contacted may be, for example, solid or liquid. When the gel composition and the chelating agent are solid, it is preferable that the gel composition and the chelating agent be dispersed in a solvent in advance and then contacted. When one of the gel composition and the chelating agent is solid and the other is liquid, the contact can be carried out, for example, by a known solid-liquid contact method. Specifically, the contact can be carried out by mixing or blending (hereinafter collectively referred to as "blending") the gel composition and the chelating agent. When the gel composition and the chelating agent are solids, the contact can be carried out, for example, by adding the gel composition and the chelating agent to a solvent, and the solvent can be, for example, a Tris-HCl buffer solution.

[0033] In the decomposition step, for example, after the gel composition and the chelating agent are brought into contact with each other, further mixing may be performed using a stirring device such as a vortex mixer, by inversion mixing, or by ultrasonic treatment.

[0034] The chelating agent is not particularly limited as long as it is a chelating agent (a substance that forms a complex with the metal ion) that can cleave the crosslink between the gelling agent contained in the gel composition and the proteoglycan, and can be appropriately selected depending on the metal ion contained in the proteoglycan. Examples of the chelating agent include chitin, chitosan, ethylenediaminetetraacetic acid (EDTA), organic acids such as citric acid, phytic acid, gluconic acid, glucuronic acid, lactic acid, and malic acid, and salts thereof, and sugars such as disaccharides such as sucrose.

[0035] In the decomposition step, the concentration of the chelating agent is 0.05 mol / L or more, 0.1 mol / L or more, 0.15 mol / L or more, or 0.2 mol / L or more.

[0036] The temperature in the decomposition step is not particularly limited as long as the chelating agent can cleave the crosslink between the gelling agent and the proteoglycan, and examples thereof include 15 to 100°C and 30 to 70°C.

[0037] The pH in the decomposition step is not particularly limited, as long as the pH is such that the chelating agent can cleave the crosslink between the gelling agent and the proteoglycan.

[0038] The time period in the decomposition step is not particularly limited as long as it is a time period that allows the chelating agent to cleave the crosslink between the gelling agent and the proteoglycan, and is, for example, 1 to 60 minutes, 1 to 30 minutes, 1 to 20 minutes, or 10 minutes.

[0039] When the degradation is caused by cleavage of covalent bonds in the gelator, the degradation step may degrade the gel composition by, for example, degrading the gelator constituting the gel composition. The degradation can be carried out, for example, by using a degrading enzyme, heat treatment, and / or pressure treatment, depending on the type of gelator. Examples of the degrading enzyme include protease (e.g., protease); peptide-degrading enzyme (e.g., peptidase); and polysaccharide-degrading enzyme (e.g., cellulase, glycosidase, agarase). When the gelator is a protein, the analytical method of the present disclosure can be carried out, for example, by contacting the gel composition with a protease capable of degrading the protein contained in the gel composition. This allows the protein contained in the gel composition to be degraded and the proteoglycan to be dissociated from the gel. The contact between the gel composition and the protease can be carried out appropriately, for example, depending on the state of the gel composition and the protease. The gel composition and the protease to be contacted may be, for example, solid or liquid. When the gel composition and the protease are solid, they are preferably dispersed in a solvent before contacting each other. When one of the gel composition and the protease is solid and the other is liquid, the contact can be carried out, for example, by a known solid-liquid contact method. Specifically, the contact can be carried out by mixing or blending (hereinafter collectively referred to as "blending") the gel composition and the protease. When the gel composition and the protease are solid, the contact can be carried out, for example, by adding the gel composition and the protease to a solvent. Examples of the solvent include Tris-HCl buffer. When the protease is collagenase, as described below, the solvent preferably contains calcium, such as calcium chloride, to maximize the catalytic activity of the collagenase.

[0040] In the decomposition step, for example, after the gel composition and the protease are brought into contact with each other, further mixing may be performed using a stirring device such as a vortex mixer, by inversion mixing, or by ultrasonic treatment.

[0041] The protease is not particularly limited as long as it is an enzyme that can decompose the protein contained in the gel composition, and examples thereof include collagenase, gelatinase, papain, bromelain, and actinidin.

[0042] In the decomposition step, the mass of the protease is 0.01 to 10 mass %, or 0.01 to 2 mass %, based on the total mass of the gel composition.

[0043] The temperature in the degradation step can be set, for example, at a temperature at which the protease exhibits activity, preferably at the optimal temperature for the protease. When the protease is collagenase, the temperature is, for example, 30 to 70°C.

[0044] The pH in the degradation step can be set, for example, to a pH at which the protease exhibits activity, preferably to the optimal pH for the protease. When the protease is collagenase, the pH is, for example, pH 5 to pH 10.

[0045] The time period for the decomposition step is not particularly limited as long as it is a time period that allows the protein contained in the gel composition to be decomposed, and is, for example, 4.5 to 20 hours, or 17 to 20 hours.

[0046] In the decomposition step, after the protein contained in the gel composition is decomposed, the protease may be inactivated. The method for inactivating the protease is not particularly limited as long as it can inactivate the protease, and can be carried out, for example, by heat treatment, addition of a protease inhibitor, etc. Examples of the heat treatment include heating.

[0047] The inactivation temperature of the protease is not particularly limited as long as it is a temperature that can inactivate the protease. When the protease is collagenase, the inactivation temperature is preferably 60°C or higher, 70°C or higher, or 80°C or higher.

[0048] Next, as described above, in the analysis method of the present disclosure, the proteoglycans in the degradation products obtained in the degradation process are analyzed in the analysis process. The analysis process first involves measuring the proteoglycan concentration in the degradation products (measurement process). The measurement process is not particularly limited as long as it is a method capable of measuring the proteoglycan concentration, and specific examples include colorimetry, HPLC, electrophoresis, etc.

[0049] After the measurement step, the proteoglycan content of the gel composition is calculated from the proteoglycan concentration obtained in the measurement step. The calculation can be performed by a known calculation method. [Example]

[0050] Next, examples of the present disclosure will be described. However, the present disclosure is not limited to the following examples. Commercially available reagents were used according to their protocols unless otherwise specified. Note that "mol / l" may also be abbreviated as "M."

[0051] [Example 1] The analytical method of the present disclosure enabled the analysis of the proteoglycan content contained in the gel composition.

[0052] (1) Preparation of gel composition containing proteoglycan A commercially available gel composition (gummy candy (containing gelatin)) was dissolved in a water bath at 80°C. After dissolution, 5 mg of Proteoglycan F (manufactured by Ichimaru Pharcos Co., Ltd.) was added to 6.2 g of the gel composition and mixed thoroughly. The mixture was then poured into a mold and cooled in a refrigerator to harden, yielding a gel composition containing proteoglycan.

[0053] (2) Examination of the proteoglycan content in the gel composition We investigated whether the proteoglycan content in a gel composition could be measured. Specifically, 0.5 g of the gel composition obtained in Example 1(1) was weighed out and 2.5 ml of 50 mmol / L Tris-HCl buffer (containing calcium chloride) was added. After the addition, the gel composition was dissolved in a water bath at 37°C. Next, 4 mg of collagenase (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 200 units / mg) was dissolved in 15 ml of the buffer, and 1 ml of the solution was added to the dissolved gel composition. After the addition, the mixture was left to stand in a water bath at 37°C for 18 hours. After the standing, the collagenase was inactivated by heat treatment at 95°C for 2 minutes. After the inactivation, 10 ml of the dissolved gel composition was recovered and diluted 2-fold with purified water to obtain a measurement sample. Next, 2 mg of proteoglycan was weighed out and phosphate buffer (pH 6.8) was added to prepare a proteoglycan solution to a total volume of 10 ml. After the preparation, the proteoglycan solution was diluted 4 / 10, 2 / 10, 1 / 10, 1 / 20, and 1 / 40 to prepare standard solutions. The proteoglycan content in the measurement samples was then measured using a DMMB colorimetric assay based on the obtained calibration curve. For the DMMB colorimetric assay, a Blyscan™ Glycosaminoglycan Assay Kit (manufactured by Biocolor) was used. Furthermore, the proteoglycan content (recovered proteoglycan amount) and proteoglycan recovery rate in 0.5 g of the gel composition were calculated based on the measurement results of the collected sample volume and the collected proteoglycan volume (calculated value contained in the collected measurement sample). These results are shown in Table 1 below.

[0054] [Table 1]

[0055] As shown in Table 1 above, as a result of calculating the recovery rates of the proteoglycans contained in Gel Compositions 1 to 3, they were 100.0%, 96.5%, and 100.0% respectively. From these results, it was found that according to the analysis method of the present disclosure, the content of the proteoglycan contained in the gel composition can be analyzed.

[0056] [Comparative Example 1-1] Regarding the gel composition obtained in Example 1(1) above, an amount was weighed to be 1 g, and 7 ml of purified water was added. After the addition, extraction was performed by ultrasonic treatment for 15 minutes, and the supernatant was recovered by centrifugation. After the recovery, 7 ml of purified water was added to the precipitate after the centrifugation. After the addition, extraction was performed by ultrasonic treatment for 15 minutes, and the supernatant was recovered by centrifugation. The obtained supernatants were combined and adjusted with purified water so that the total volume became 20 ml. After the adjustment, 5 ml of the adjusted solution was added to Amicon (registered trademark) Ultra-15, Ultracel 100k (manufactured by Millipore), and centrifugation was performed under the conditions of 3500 rpm for 30 minutes. After the centrifugation, 15 ml of purified water was added to the supernatant, and centrifugation was performed under the conditions of 3500 rpm for 30 minutes for washing with water. The washing with water was performed a total of 5 times, and the supernatant after the washing with water was used as a measurement sample. Regarding the sample, HPLC was performed under the following conditions, and the content of the proteoglycan contained in the gel composition was measured. As a result, no peak was observed at the retention time at which the proteoglycan should be detected in both the differential refractive index detector and the UV detector. <HPLC Conditions> Column: TSKgel G5000PWXL (manufactured by Tosoh Corporation) Column temperature: 40 °C Sample injection volume: 50 μL Mobile phase: Phosphate buffer (pH 6.8) Flow rate: 0.5 mL / min Detector: UV detector (SPD-20A manufactured by Shimadzu Corporation) Detection wavelength = 215 nm, Differential refractive index detector (RID-10A manufactured by Shimadzu Corporation)

[0057] [Comparative Example 1-2] The same method as in Comparative Example 1-1 was used, except that Vivaspin (registered trademark) 15 Turbo MWCO 100000 (manufactured by Sartorius) was used instead of Amicon (registered trademark) Ultra-15, Ultracel 100k (manufactured by Millipore). As a result, no peak was observed at the retention time at which proteoglycan should be detected, either with the differential refractive index detector or the UV detector.

[0058] [Comparative Example 1-3] The same procedure as in Comparative Example 1-1 was carried out except that 10 mmol / L PIPES-NaOH buffer was used instead of purified water. As a result, no peak was observed at the retention time at which proteoglycan should be detected, either with the differential refractive index detector or the UV detector.

[0059] [Comparative Example 1-4] The same method as in Comparative Example 1-1 was used, except that a 4 mol / L guanidine hydrochloride solution was used instead of purified water, and Vivaspin® 15 Turbo MWCO 100000 (manufactured by Sartorius) was used instead of Amicon® Ultra-15 and Ultracel 100k (manufactured by Millipore). As a result, no peak was observed at the retention time at which proteoglycan should be detected, using either the differential refractive index detector or the UV detector.

[0060] [Comparative Example 1-5] The gel composition obtained in Example 1(1) was analyzed for the proteoglycan content using the method described in JP 2021-189178 A. As a result, no peak was observed at the retention time at which proteoglycan should be detected using either the differential refractive index detector or the UV detector.

[0061] [Comparative Example 1-6] The gel composition obtained in Example 1 (1) was analyzed for the content of proteoglycan contained in the gel composition using the method described in JP 2022-177785 A. The recovery rate of the proteoglycan was calculated from the content of the proteoglycan contained in the gel composition. The results are shown in Table 2 below.

[0062] [Table 2]

[0063] As shown in Table 2 above, the recovery rate of proteoglycan in Sample 1 was 1.5%, and the recovery rate of proteoglycan in Sample 2 was 3.8%.

[0064] [Example 2] We investigated whether the proteoglycan content in a gel composition could be measured. Specifically, 5.6 ml of purified water was added to 2.0 g of commercially available gelatin and heated in a microwave oven for 40 seconds to dissolve. After dissolution, 5.31 mg of proteoglycan was added to a solution of Proteoglycan F (manufactured by Ichimaru Pharcos Co., Ltd.) and mixed thoroughly. After mixing, the mixture was poured into a mold and cooled in a refrigerator to harden, yielding 4.36 g of a sugar-free gel composition. A 0.45 g portion of the gel composition was weighed out, and 2.5 g of sucrose (manufactured by Kishida Chemical Co., Ltd.) and 2.5 ml of 50 mmol / L Tris-HCl buffer (containing calcium chloride) were added. After the addition, the gel composition was dissolved in a 37°C water bath. Next, 4 mg of collagenase (Fujifilm Wako Pure Chemical Industries, Ltd., 200 units / mg) was dissolved in 15 ml of buffer solution, and 2.5 ml of the solution was added to the dissolved gel composition. After the addition, the mixture was stirred for 18 hours in a water bath at 37°C. After the stirring, the collagenase was inactivated by heat treatment at 95°C for 2 minutes. After the inactivation, 10 ml of the dissolved gel composition was recovered and diluted 2-fold with purified water to obtain a measurement sample (Example 2).

[0065] [Example 3] A 0.45 g portion of the gel composition obtained in Example 2 was weighed out and 2.5 ml of 50 mmol / L Tris-HCl buffer (containing calcium chloride) was added. After the addition, the gel composition was dissolved in a water bath at 37°C. Next, 4 mg of collagenase (200 units / mg, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 15 ml of the buffer, and 2.5 ml of the solution was added to the dissolved gel composition. After the addition, the mixture was stirred for 18 hours in a water bath at 37°C. After the stirring, 2 ml of 0.5 mol / L disodium ethylenediaminetetraacetate dihydrate (EDTA·2Na, manufactured by Kishida Chemical Co., Ltd.) was added. After the addition, the mixture was heat-treated at 95°C for 2 minutes to inactivate the collagenase. Then, 10 ml of the dissolved gel composition was recovered and diluted 2-fold with purified water to obtain a measurement sample (Example 3).

[0066] [Comparative Example 2-1] A measurement sample (Comparative Example 2-1) was obtained in the same manner as in Example 2, except that sucrose and collagenase were not added.

[0067] [Comparative Example 2-2] A measurement sample (Comparative Example 2-2) was obtained in the same manner as in Example 2, except that collagenase was not added.

[0068] [Comparative Example 2-3] A measurement sample (Comparative Example 2-3) was obtained in the same manner as in Example 2, except that sucrose was not added.

[0069] [Comparative Example 2-4] A measurement sample (Comparative Example 2-4) was obtained in the same manner as in Example 3, except that collagenase was not added.

[0070] [Test example] The proteoglycan content in the measurement sample was measured using the same method as in Example 1(2). The proteoglycan content in the gel composition (recovered proteoglycan amount) and the proteoglycan recovery rate were calculated. The results are shown in Table 3 below.

[0071] [Table 3]

[0072] As shown in Table 3, the recovery rates of proteoglycans contained in the measurement samples were calculated as follows: Example 2: 100.0%, Example 3: 100.0%, Comparative Example 2-1: not detectable, Comparative Example 2-2: not detectable, Comparative Example 2-3: 91.0%, and Comparative Example 2-4: 32.1%. These results demonstrate that the analytical method of the present disclosure can be used to analyze the content of proteoglycans contained in a gel composition.

[0073] [Example 4] (1) Preparation of gel composition containing proteoglycan 2.0 g of commercially available gelatin was added to 5.6 ml of purified water and heated in a microwave oven for 40 seconds to dissolve. After dissolution, 5.35 mg of proteoglycan was added to a solution of Proteoglycan F (manufactured by Ichimaru Pharcos Co., Ltd.). The mixture was mixed well and poured into a mold. The mixture was cooled and solidified in a refrigerator to obtain 4.62 g of a gel composition containing no sugars (a proteoglycan-containing gel composition). A control gel composition without Proteoglycan F (a proteoglycan-free gel composition) was also prepared in the same manner.

[0074] (2) Examination of the proteoglycan content in the gel composition We investigated whether the proteoglycan content in a gel composition could be measured. Specifically, 0.45 g of the proteoglycan-containing gel composition obtained in Example 4(1) was weighed out and 3 ml of 50 mmol / L Tris-HCl buffer (containing calcium chloride) was added. After the addition, the gel composition was dissolved in a 37°C water bath. Next, 4 ml of 0.5 mol / L disodium ethylenediaminetetraacetate dihydrate (EDTA, manufactured by Kishida Chemical Co., Ltd.) was added and stirred for 10 minutes. After the stirring, 10 ml of the dissolved gel composition was recovered and diluted 2-fold with purified water to obtain a measurement sample (proteoglycan-containing sample). A measurement sample (proteoglycan-free sample) was also obtained using the proteoglycan-free gel composition obtained in Example 4(1) by the same method. The proteoglycan content in the measurement sample was measured by the same method as in Example 1(2). The content of proteoglycan in the gel composition (recovered amount of proteoglycan) and the recovery rate of proteoglycan were calculated. The results are shown in Table 4 below.

[0075] [Table 4]

[0076] As shown in Table 4, the recovery rate of proteoglycan in the proteoglycan-containing composition was 100.0%. These results demonstrate that the analytical method of the present disclosure can be used to analyze the content of proteoglycan contained in a gel composition.

[0077] Although the present disclosure has been described above with reference to the embodiments and examples, the present disclosure is not limited to the above-described embodiments and examples. Various modifications that can be understood by a person skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0078] The patents, patent applications, and publications cited herein are incorporated by reference into this specification in their entirety as if the contents were specifically set forth herein.

[0079] This application claims priority based on Japanese Patent Application No. 2024-023597 filed on February 20, 2024, and incorporates herein all of its disclosures.

[0080] <Supplementary Note> Some or all of the above-described embodiments and examples may be described as follows, but are not limited thereto. <Analysis Method> (Supplementary Note 1) <PG Analysis Method> An analysis method for the proteoglycan content contained in a gel composition, comprising: a decomposition step of decomposing the gel composition; an analysis step of analyzing the proteoglycan in the obtained decomposition product; and the analysis method. (Supplementary Note 2) In the decomposition step, the crosslinking by metal ions between the gelling agent contained in the gel composition and the proteoglycan is cleaved by chelating the metal ions with a chelating agent, and the gel composition is decomposed. The analysis method according to Supplementary Note 1. (Supplementary Note 3) The chelating agent is ethylenediaminetetraacetic acid (EDTA) and / or a disaccharide. The analysis method according to Supplementary Note 1 or 2. (Supplementary Note 4) The concentration of the chelating agent is 0.2 mol / l or more. The analysis method according to any one of Supplementary Notes 1 to 3. (Supplementary Note ⑤) In the decomposition step, the protein contained in the composition is decomposed. The analysis method according to any one of Supplementary Notes 1 to 4. (Supplementary Note ⑥) The decomposition is decomposition by a proteolytic enzyme. The analysis method according to Supplementary Note 5. (Supplementary Note ⑦) The proteolytic enzyme is collagenase. The analysis method according to Supplementary Note 6. (Supplementary Note ⑧) 8. The analytical method according to any one of claims 1 to 7, wherein the gel composition contains at least one of collagen and gelatin as a gelling agent. (Appendix 9) 9. The analytical method according to claim 8, wherein the gel composition contains 1 to 20% by mass of the gelling agent based on the total mass of the gel composition. (Appendix 10) The analytical method according to any one of Appendices 1 to 9, wherein the decomposition step is carried out at 30 to 70°C. (Appendix 11) The analytical method according to any one of appendices 1 to 10, wherein the decomposition step is carried out at a pH of 5 to 10. (Appendix 12) 12. The analytical method according to any one of claims 1 to 11, wherein the gel composition contains a sugar. (Appendix 13) 13. The analytical method according to claim 12, wherein the gel composition contains 50 to 96% by mass of the sugars based on the total mass of the gel composition. (Appendix 14) 14. The analytical method according to any one of claims 6 to 13, wherein in the decomposition step, the mass of the protease is 0.01 to 2 mass % based on the total mass of the gel composition. (Appendix 15) An analytical method described in any one of Appendices 1 to 114, wherein a dispersion step of dispersing the gel composition is carried out prior to the decomposition step. (Appendix 16) The analyzing step a measuring step of measuring the proteoglycan concentration of the degradation product; A calculation step of calculating the proteoglycan content contained in the gel composition from the obtained proteoglycan concentration; 116. The analytical method of any one of appendices 1 to 115, comprising: [Industrial Applicability]

[0081] As described above, the present disclosure can provide a method capable of analyzing the content of proteoglycan in a gel composition, etc. Therefore, the present disclosure can be said to be extremely useful, for example, in the food industry.

Claims

1. A method for analyzing the content of proteoglycan contained in a gel composition, comprising: a decomposition step of decomposing the gel composition; an analytical step of analyzing proteoglycans in the obtained degradation product; Analytical methods, including:

2. The analytical method according to claim 1, wherein in the decomposition step, cross-links formed by metal ions between the gelling agent contained in the gel composition and the proteoglycan are broken by chelating the metal ions using a chelating agent, thereby decomposing the gel composition.

3. The analytical method according to claim 1 or 2, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA) and / or a disaccharide.

4. 3. The analytical method according to claim 1, wherein the concentration of the chelating agent is 0.2 mol / L or more.

5. The analytical method according to claim 1 or 2, wherein the decomposition step decomposes proteins contained in the gel composition.

6. The analytical method according to claim 5 , wherein the degradation is performed by a proteolytic enzyme.

7. The analytical method according to claim 6 , wherein the protease is collagenase.

8. The analysis method according to claim 1 or 2, wherein the gel composition contains at least one of collagen and gelatin as a gelling agent.

9. The analytical method according to claim 8, wherein the gel composition contains 1 to 20 mass % of the gelling agent based on the total mass of the gel composition.

10. The analytical method according to claim 1 or 2, wherein the decomposition step is carried out at 30 to 70°C.

11. The analytical method according to claim 1 or 2, wherein the decomposition step is carried out at a pH of 5 to 10.

12. The analytical method according to claim 1 or 2, wherein the gel composition contains a sugar.

13. The analysis method according to claim 12, wherein the gel composition contains 50 to 96% by mass of the sugars based on the total mass of the gel composition.

14. 7. The analysis method according to claim 6, wherein in the decomposition step, the mass of the protease is 0.01 to 2 mass % based on the total mass of the gel composition.

15. The analytical method according to claim 1 or 2, further comprising the step of dispersing the gel composition prior to the step of decomposing the gel composition.

16. The analyzing step a measuring step of measuring the proteoglycan concentration of the degradation product; A calculation step of calculating the proteoglycan content contained in the gel composition from the obtained proteoglycan concentration; The analytical method according to claim 1 or 2, comprising:

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