Method for separating proteoglycan

Abstract

To provide a method for separating proteoglycan present in compositions such as foods and cosmetics.SOLUTION: A method for separating proteoglycan from a composition containing proteoglycan, characterized by comprising the following sequential steps of: (1) treating the composition containing proteoglycan to prepare a solution having a pH of 1.4 or less; (2) passing the solution prepared in step (1) through an anion exchange resin equilibrated with an acidic solution having a pH of 1.4 or less; and (3) passing the acidic solution through the anion exchange resin. This can provide a method for separating proteoglycan contained in compositions such as foods and cosmetics.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for separating proteoglycans. [Background technology]

[0002] Proteoglycans have been reported to have various functions, including water retention, cell growth factor-like activity, hyaluronic acid production, and anti-allergic activity. In recent years, many products containing proteoglycans have been developed and sold. Furthermore, due to growing health consciousness among consumers, the demand for processed foods that claim health functionality is increasing. When claiming health functionality on the packaging of processed foods, it is important to accurately understand the amount and purity of the proteoglycans contained in the food. Therefore, a method is needed to analyze trace amounts of proteoglycans contained in foods that also contain organic compounds such as sugars, proteins, amino acids, and organic acids.

[0003] Methods for analyzing proteoglycans include methods using HPLC by size exclusion chromatography and methods using anion exchange resins (Patent Documents 1, 2, and 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2018-151207 A [Patent Document 2] JP 2020-134435 A [Patent Document 3] Patent Publication No. 2021-189178 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the method of Patent Document 1 has difficulty in separating proteoglycans, which have molecular weights of tens of thousands to hundreds of thousands, from polysaccharides and proteins, which have similar molecular weights. Furthermore, the methods of Patent Documents 2 and 3 require a large amount of resin when the composition contains a large amount of acidic sugars, which have carboxyl groups or other negatively charged groups similar to proteoglycans. Furthermore, while there are separation methods using ultrafiltration membranes or precipitation with organic solvents or salts to quantify proteoglycans, these methods are difficult when other polymeric compounds are present. Therefore, in order to solve the above problems, an object of the present invention is to provide a method for separating proteoglycans contained in compositions such as foods and cosmetics. [Means for solving the problem]

[0006] [1] A method for separating proteoglycans from a composition containing proteoglycans, comprising the following steps: (1) A step of adjusting a composition containing proteoglycan to a solution having a pH of 1.4 or less. (2) A step of passing the solution prepared in (1) through an anion exchange resin equilibrated with an acidic solution of pH 1.4 or less. (3) Passing the acidic solution through the anion exchange resin [2] The separation method according to [1], wherein the composition containing proteoglycan is a composition containing a polysaccharide having only carboxyl groups as acidic groups in its structure and a neutral sugar. [3] The separation method according to [1], wherein the anion exchange resin is a strong basic anion exchange resin. [Effects of the Invention]

[0007] The present invention can provide a method for separating proteoglycans contained in compositions such as foods and cosmetics. [Brief explanation of the drawings]

[0008] [Figure 1] This is a chromatogram of a 0.1 mg / mL proteoglycan solution. [Figure 2]This is a chromatogram of the elution fraction of honey-added apple cider vinegar to which proteoglycan has been added. [Figure 3] This is a chromatogram of the elution fraction of honey-added apple cider vinegar that does not contain proteoglycan. [Figure 4] 1 is a chromatogram of the elution fraction of agricultural product powder to which proteoglycan has been added. [Figure 5] 1 is a chromatogram of the elution fraction of agricultural product powder that does not contain proteoglycan. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention is a method for isolating proteoglycans from a composition containing proteoglycans, which comprises the following steps: (1) A step of adjusting a composition containing proteoglycan to a solution having a pH of 1.4 or less. (2) A step of passing the solution prepared in (1) through an anion exchange resin equilibrated with an acidic solution of pH 1.4 or less. (3) Passing the acidic solution through the anion exchange resin

[0010] Proteoglycans in the present invention are a type of glycoprotein. Found in animals and fish, they are natural polymeric compounds with molecular weights ranging from tens of thousands to millions, in which glycosaminoglycans such as chondroitin sulfate and keratan sulfate are covalently bonded to proteins. Depending on the organism of origin and the extraction and production conditions, the molecular weight, sugars contained (uronic acid, amino sugars, neutral sugars, etc.), and the types, amounts, and ratios of amino acids that make up the protein vary. However, the proteoglycans referred to in the present invention are not limited to the organism of origin or the extraction and production conditions.

[0011] The proteoglycan-containing compositions of the present invention include foods, beverages, supplements, and cosmetics. In addition to final products, proteoglycan-containing intermediate materials can also be used. Separation of proteoglycans from polysaccharides such as pectin, which have only carboxyl groups as acidic groups in their structures, is particularly difficult using known methods, and therefore, the method of the present invention is desirable.

[0012] In step (1), a solid or semi-solid composition is dissolved in water. It is desirable to perform a process such as pulverization as necessary. Next, the dissolved solid or semi-solid composition or liquid composition is adjusted to a pH of 1.4 or less. This pH adjustment can be performed using an inorganic acid such as hydrochloric acid or sulfuric acid, or an organic acid such as formic acid. It is also possible to use a buffer solution with a buffer capacity of around pH 1.4 when dissolving the composition. If solids remain in the solution after adjusting the pH, the liquid portion is recovered by solid-liquid separation. Methods for solid-liquid separation include filtration and centrifugation. To prevent decomposition of proteoglycans, it is desirable to carry out the procedure at 10°C or below, but this is not necessarily required.

[0013] In step (2), the anion exchange resin is equilibrated with an acidic solution of pH 1.4 or less. The acidic solution used can be prepared using the same method as in the preparation of the composition in step (1). It is desirable to pass the acidic solution in an amount approximately three times the volume of the resin. Next, the solution prepared in step (1) is passed through the equilibrated resin to adsorb the proteoglycan. While there is no problem if the flow rate of the solution passing through the resin is not adjusted, it is preferable to pass the solution through the resin with a peristaltic pump or the like, adjusting the flow rate to approximately 1 mL / min.

[0014] The anion exchange resin referred to in the present invention is a water-insoluble polymer resin having a positive charge. Anion exchange resins are classified into strong basic anion exchange resins having quaternary amino groups and weak basic anion exchange resins having one to three amino groups. While any basic anion exchange resin can be used in the present invention, strong basic anion exchange resins are preferred.

[0015] The resin can be packed in a column or a column packed with a commercially available resin can be used. A batch method in which the resin and the solution are stirred in a container can also be used, and the method used is not limited.

[0016] In step (3), the acidic solution is passed through the resin to wash away components other than proteoglycans remaining in the resin. It is desirable to pass about three times the volume of the solution through the resin. Furthermore, it is desirable to pass about three times the volume of pure water through the resin to wash away the acidic solution in the resin, but this is not necessarily required.

[0017] The proteoglycan adsorbed to the resin can be eluted and collected by a known method, such as passing a salt solution such as sodium chloride or an alkaline solution such as ammonia through the resin.

[0018] The recovered solution containing proteoglycan can be purified by known methods depending on the method used for elution from the resin. When a salt solution such as sodium chloride or an alkaline solution such as ammonia is used, desalting by ultrafiltration or dialysis can be used. Highly purified proteoglycan can also be obtained by removing the solvent using vacuum freeze-drying or an evaporator.

[0019] The obtained proteoglycans can be analyzed by known methods, such as liquid chromatography, which can measure absorbance at wavelengths from 200 to 290 nm, or a differential refractometer, or protein analysis by the Lowry or Bradford methods, the carbazole sulfate method, or a commercially available proteoglycan quantification kit.

[0020] The present invention will be specifically described below by showing examples, but these are given for the purpose of illustration only, and the present invention is not limited to these examples. [Example]

[0021] (Investigation of proteoglycan adsorption conditions) First, we confirmed the adsorption of proteoglycans to a strongly basic anion exchange resin under acidic conditions.

[0022] The proteoglycan used was salmon nasal cartilage-derived proteoglycan (manufactured by Kadohiro Proteoglycan Research Institute Co., Ltd.), which was dissolved in pure water to a concentration of 1 mg / mL to prepare a proteoglycan solution. Hydrochloric acid was added to 3 mL of the proteoglycan solution to a final concentration of 100 mM to prepare a sample solution. The pH of the sample solution was 1.0.

[0023] A glass column (20 mm inner diameter, equipped with a G2 glass filter) was packed with 10 mL of strong basic anion exchange resin (TOYOPEAL SuperQ-650C, Tosoh Corporation), and equilibrated with 30 mL of 100 mM hydrochloric acid solution. The sample solution was passed through the equilibrated resin, followed by 30 mL of the hydrochloric acid solution used for equilibration. The solution that passed through the resin was collected as the flow-through fraction. Next, 40 mL of pure water was passed through, and the solution that passed through the resin was collected as the wash fraction. Finally, 1.5 M sodium chloride solution was passed through, and the solution that passed through the resin was collected as the elution fraction.

[0024] The presence or absence of proteoglycan in each collected fraction was measured using the carbazole sulfate method. Carbazole sulfate is a colorimetric method for measuring the amount of uronic acid in a sample, and the presence of uronic acid in the sample will produce a red color. Since proteoglycans have uronic acid in their structure, the presence or absence of proteoglycan in a sample can be determined using carbazole sulfate.

[0025] To 0.1 mL of each fraction, 0.6 mL of concentrated sulfuric acid was added and stirred while cooling. Next, 0.02 mL of 0.125% carbazole solution was added and stirred, and the mixture was heated at 100°C for 15 minutes. After cooling in ice water, the mixture was left to stand at room temperature for at least 30 minutes. The color of the solution was visually confirmed to determine the presence or absence of proteoglycan.

[0026] Since no red coloration was observed in the flow-through and wash fractions, it was demonstrated that proteoglycan adsorbs to the strongly basic anion exchange resin even under the strongly acidic conditions of 100 mM hydrochloric acid and pH 1.0. At the same time, red coloration was confirmed in the elution fraction, demonstrating that the proteoglycan adsorbed to the resin can be recovered with a sodium chloride solution. The results of the presence or absence of proteoglycan in the elution fraction are shown in Table 1.

[0027] [Table 1] [Example]

[0028] (Study of pectin adsorption conditions) For polysaccharides that have only carboxyl groups as acidic groups in their structure, we used pectin, which is found in large quantities in agricultural products and is also used as a gelling agent, to examine the pH conditions under which separation from proteoglycans is possible.

[0029] Pectin was a commercially available reagent (Fujifilm Wako Pure Chemical Corporation) and dissolved in pure water to a concentration of 1 mg / mL to prepare a pectin solution. Hydrochloric acid was added to 3 mL of the pectin solution to final concentrations of 0.1 mM, 1 mM, 25 mM, 50 mM, 75 mM, or 100 mM to prepare sample solutions. The pH of each of the prepared sample solutions was also measured.

[0030] A glass column (20 mm internal diameter, equipped with a G2 glass filter) was packed with 10 mL of strong basic anion exchange resin (TOYOPEAL SuperQ-650C, Tosoh Corporation), and equilibrated with 30 mL of hydrochloric acid solution of the same concentration as the prepared pectin solution. The sample solution was passed through this equilibrated resin, followed by 30 mL of the hydrochloric acid solution used to equilibrate the resin. The solution that passed through the resin was collected as the flow-through fraction. Next, 30 mL of pure water was passed through, and the solution that passed through the resin was collected as the wash fraction. Finally, 1.5 M sodium chloride solution was passed through, and the solution that passed through the resin was collected as the elution fraction.

[0031] The presence or absence of pectin in each collected fraction was measured by the carbazole sulfate method shown in Example 1. Because pectin has uronic acid in its structure, the presence or absence of pectin in a sample can be determined using carbazole sulfate.

[0032] At hydrochloric acid concentrations of 0.1 mM and 1 mM, i.e., pH 3.3 or higher, red coloration was observed only in the eluted fraction. At a hydrochloric acid concentration of 25 mM, i.e., pH 1.6, red coloration was observed in both the flow-through and eluted fractions. At hydrochloric acid concentrations of 50 mM, 75 mM, and 100 mM, i.e., pH 1.4 or lower, red coloration was observed only in the flow-through fraction. This indicates that at pH 1.4 or lower, pectin does not adsorb to the resin and passes through in its entirety. Table 2 shows the presence or absence of pectin in each fraction when pectin was passed through a strongly basic anion exchange resin at different pH levels.

[0033] [Table 2] [Example]

[0034] (Separation of proteoglycans in pectin solution) Examples 1 and 2 showed that a pH of 1.4 or less is the condition for adsorbing only proteoglycan to the resin. Therefore, we investigated whether it was possible to separate proteoglycan from a pectin solution.

[0035] The same proteoglycan and pectin reagents as those used in Examples 1 and 2 were dissolved in pure water to a concentration of 1 mg / mL to prepare proteoglycan and pectin solutions. 3 mL of each solution was mixed, and 15 mL of potassium chloride-hydrochloric acid buffer (pH 1.2) prepared in accordance with "Japanese Industrial Standard K8001 General Rules for Testing Reagents" was added to prepare the sample solution.

[0036] A glass column (20 mm inner diameter, equipped with a G2 glass filter) was packed with 10 mL of strong basic anion exchange resin (TOYOPEAL SuperQ-650C, Tosoh Corporation), and equilibrated with 30 mL of potassium chloride-hydrochloric acid buffer (pH 1.2). The sample solution was passed through the equilibrated resin, and the flow-through fraction was collected. Another 30 mL of the potassium chloride-hydrochloric acid buffer (pH 1.2) used to equilibrate the resin was passed through, and this was collected as wash fraction 1. Next, 30 mL of pure water was passed through, and this was collected as wash fraction 2. Finally, 1.5 M sodium chloride solution was passed through, and the solution that passed through the resin was collected as the elution fraction.

[0037] The presence or absence of proteoglycan and pectin in each of the collected fractions was measured by the carbazole sulfate method shown in Example 1, and coloration was confirmed in the flow-through fraction, wash fraction 1, and elution fraction. Because both proteoglycan and pectin show a red coloration in the carbazole sulfate method, it is not possible to determine from this result alone whether proteoglycan has been separated.

[0038] The presence or absence of proteoglycan in each collected fraction was measured by the Lowry method. The Lowry method is a colorimetric method for measuring protein in a sample, and if protein is present in the sample, a blue color will appear. Since proteoglycans contain protein in their structure, while pectin does not, the Lowry method can be used to distinguish between proteoglycans and pectin.

[0039] For measurement by the Lowry method, each fraction was concentrated by ultrafiltration. Specifically, 10 mL of the fraction solution was placed in a hollow container with a molecular weight cutoff membrane (Amicon Ultra 15 30 kDa, Merck) and centrifuged. The concentrated solution remaining on the molecular weight cutoff membrane was collected and used as the measurement sample by the Lowry method.

[0040] To 0.2 mL of the sample, 1 mL of alkaline copper solution (a 50:1 mixture of 0.1 N sodium hydroxide solution containing 2% sodium carbonate and 1% sodium potassium tartrate solution containing 0.5% copper (II) sulfate pentahydrate) was added, stirred, and then allowed to stand at room temperature for 10 minutes. 0.1 mL of diluted Folin's reagent (a two-fold dilution of the commercially available reagent) was added, stirred, and then allowed to stand at room temperature for 30 minutes. The color of the solution was confirmed visually.

[0041] In the carbazole sulfate method, the flow-through fraction, wash fraction 1, and elution fraction all turned red, whereas in the Lowry method, only the elution fraction turned blue. This indicates that the entire amount of proteoglycan was adsorbed to the resin and then eluted with a 1.5 M sodium chloride solution. Example 2 demonstrates that pectin does not adsorb to the resin at pH 1.4 or below, passing through the entire resin and not being included in the elution fraction. These results demonstrate that proteoglycans can be separated by adsorbing only proteoglycans to the anion exchange resin and then passing polysaccharides, such as pectin, which have only carboxyl groups as acidic groups in their structure. Table 3 shows the coloration results of the carbazole sulfate method and the Lowry method for each fraction obtained by separating proteoglycans from a pectin solution using the resin, as well as the presence or absence of proteoglycans.

[0042] [Table 3] [Example]

[0043] (Separation of proteoglycans in hyaluronic acid solution) Examples 1, 2, and 3 demonstrate that proteoglycans can be separated from pectin solutions. In addition to pectin, hyaluronic acid is another polysaccharide that has only carboxyl groups as acidic groups in its structure. Therefore, we investigated whether proteoglycans can be separated from hyaluronic acid solutions.

[0044] The same reagent as in Example 1 was used for proteoglycan, and a commercially available reagent (Fujifilm Wako Pure Chemical Industries, Ltd.) was used for hyaluronic acid. Proteoglycan and hyaluronic acid were dissolved in pure water to a concentration of 1 mg / mL to prepare a proteoglycan solution and a hyaluronic acid solution. 5 mL of each solution was mixed, and the pH was adjusted to 1.3 using hydrochloric acid to prepare a sample solution. A solution containing only hyaluronic acid and no proteoglycan was also prepared in the same manner.

[0045] A glass column (20 mm inner diameter, equipped with a G2 glass filter) was packed with 10 mL of strong basic anion exchange resin (AP MP-1M, BIO-RAD), and equilibrated with 30 mL of hydrochloric acid solution adjusted to pH 1.3. The sample solution was passed through the equilibrated resin, and the flow-through fraction was collected. Another 50 mL of the hydrochloric acid solution used for equilibration was passed through the resin, and this was collected as wash fraction 1. Next, 50 mL of pure water was passed through, and this was collected as wash fraction 2. Finally, a 1.5 M sodium chloride solution was passed through the resin, and the solution that passed through the resin was collected as the elution fraction.

[0046] The presence or absence of proteoglycan and hyaluronic acid in each of the collected fractions was measured by the carbazole sulfate method shown in Example 1. When the sample solution containing proteoglycan was analyzed, coloration was confirmed in both the flow-through fraction and the eluted fraction. On the other hand, coloration was confirmed only in the flow-through fraction in the sample solution not containing proteoglycan.

[0047] The presence or absence of proteoglycan in each of the collected fractions was measured by the Lowry method shown in Example 3, and coloration was confirmed in the eluted fractions. On the other hand, no coloration was confirmed in any fraction of the sample solution that did not contain proteoglycan.

[0048] The results of the carbazole sulfate method and the Lowry method indicated that the entire amount of proteoglycan was adsorbed to the resin and then eluted with a 1.5 M sodium chloride solution, while hyaluronic acid, which contains uronic acid but no protein, did not adsorb to the resin and passed through in its entirety. These results demonstrate that it is possible to separate proteoglycans not only from pectin but also from hyaluronic acid. The results of the color development by the carbazole sulfate method and the Lowry method, as well as the presence or absence of proteoglycan, for each fraction obtained by separating proteoglycans from a hyaluronic acid solution using the resin, are shown in Table 4 for the hyaluronic acid solution containing proteoglycan, and in Table 5 for the hyaluronic acid solution without proteoglycan.

[0049] [Table 4]

[0050] [Table 5] [Example]

[0051] (Isolation of proteoglycans from apple cider vinegar with honey) We investigated whether it is possible to separate proteoglycans from honey-added apple cider vinegar, which contains a large amount of pectin and neutral sugars, which are difficult to separate from proteoglycans.

[0052] The same proteoglycan reagent as in Example 1 was used.

[0053] A 10 mL solution was prepared by adding proteoglycan to commercially available honey-added apple cider vinegar (Kanesho Co., Ltd.) to a concentration of 0.6 mg / mL, and the pH was adjusted to 1.3 using hydrochloric acid. In addition, commercially available apple cider vinegar without added proteoglycan was also similarly adjusted to pH 1.3 using hydrochloric acid.

[0054] Separation of proteoglycans using the resin was carried out in the same manner as in Example 1, and an eluted fraction was obtained.

[0055] Proteoglycan was qualitatively analyzed. Proteoglycan was dissolved in pure water to a concentration of 0.5 mg / mL, and then further diluted with pure water to a concentration of 0.1 mg / mL. This solution was injected into the high-performance liquid chromatograph described below, and a chromatogram of the proteoglycan was obtained. Equipment configuration: Pump (LC-20AD), degasser (DGU-20A3R), autosampler (SIL-20AHT), column oven (CTO-20A), photodiode array detector (SPD-M20A), system controller (CBM-20A). All manufactured by Shimadzu Corporation. Column: TSKgel Guardcolumn PWXL (Tosoh Corporation), TSKgel G5000PWXL (Tosoh Corporation) Column temperature: 40℃ Injection volume: 50μL Mobile phase: 50mM phosphate buffer (pH6.8) Flow rate: 0.5mL / min Detection wavelength: 203 nm

[0056] The chromatogram of 0.1 mg / mL proteoglycan is shown in Figure 1. The horizontal axis represents time (minutes) and the vertical axis represents absorbance.

[0057] The eluted fractions of apple cider vinegar containing and without proteoglycans were injected into a high performance liquid chromatograph, and chromatograms were obtained.

[0058] Figure 2 shows a chromatogram of the elution fraction from apple cider vinegar with added proteoglycan, while Figure 3 shows a chromatogram of the elution fraction from apple cider vinegar without added proteoglycan. The horizontal axis represents time (minutes), and the vertical axis represents absorbance. With apple cider vinegar with added proteoglycan, a peak was observed at the same position as in the chromatogram of the proteoglycan standard in Figure 1. On the other hand, no proteoglycan peak was observed with apple cider vinegar without added proteoglycan.

[0059] This indicates that it is possible to separate proteoglycans from honey-added apple vinegar, which contains a large amount of pectin and neutral sugars. [Example]

[0060] (Separation of proteoglycans from agricultural powders) We investigated whether it is possible to separate proteoglycans from agricultural powders that contain large amounts of pectin and other impurities, which are difficult to separate from proteoglycans.

[0061] The same proteoglycan reagent as in Example 1 was used.

[0062] Equal amounts of freeze-dried turnip greens, tomatoes, and edamame were ground and mixed to prepare a powder. 300 mg of powder was mixed with 5 mg of proteoglycan to prepare the analytical sample. 300 mg of powder without proteoglycan was also prepared in the same way.

[0063] 15 mL of 80% ethanol was added to the analytical sample, and after stirring, the mixture was centrifuged and the supernatant removed. This procedure was repeated three times. 15 mL of distilled water was added to the residue, and after stirring, the mixture was sonicated. The mixture was then centrifuged and the supernatant was collected. This procedure was repeated three times. The collected supernatants were combined and the volume was adjusted to 50 mL with distilled water. After the volume was adjusted, the mixture was filtered through a filter paper. 25 mL of the resulting filtrate was taken and adjusted to pH 1.3 with hydrochloric acid.

[0064] Separation of proteoglycans using the resin was carried out in the same manner as in Example 1, and an eluted fraction was obtained.

[0065] The proteoglycan was characterized in the same manner as in Example 5.

[0066] Figure 4 shows a chromatogram of the elution fraction of the agricultural product powder with added proteoglycan, and Figure 5 shows a chromatogram of the elution fraction of the agricultural product powder without added proteoglycan. The horizontal axis represents time (minutes), and the vertical axis represents absorbance. In the agricultural product powder with added proteoglycan, a peak was observed at a position similar to that of the proteoglycan standard in Figure 1. On the other hand, no proteoglycan peak was observed in the agricultural product powder without added proteoglycan.

[0067] This indicates that it is possible to separate proteoglycans from foods containing various contaminants, such as agricultural product powder. [Industrial Applicability]

[0068] The present invention makes it possible to separate proteoglycans contained in compositions such as foods and cosmetics without going through complicated steps, particularly from pectin and hyaluronic acid, which was difficult using conventional methods. Separating proteoglycans makes it possible to quantify them, which not only facilitates product quality control, but is also expected to increase the added value of products by labeling their health functionality on packaging, etc. as functional foods.

Claims

1. A method for isolating proteoglycans from a composition containing proteoglycans, comprising the following steps: (1) A step of adjusting a composition containing proteoglycan to a solution having a pH of 1.4 or less (2) A step of passing the solution prepared in (1) through an anion exchange resin equilibrated with an acidic solution of pH 1.4 or less. (3) Passing the acidic solution through the anion exchange resin

2. 2. The method for separating proteoglycans according to claim 1, wherein the composition containing proteoglycans is a composition containing polysaccharides having only carboxyl groups as acidic groups in their structures and neutral sugars.

3. 2. The separation method of claim 1, wherein the anion exchange resin is a strong base anion exchange resin.

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