JP2023033802A - Method for producing high-sodium low-calcium proteoglycan - Google Patents

Abstract

To provide a method for producing proteoglycan derived from salmon, which contains sodium of 7 wt.% or more and calcium of 0.7 wt.% or less.SOLUTION: An aqueous solution of salmon-derived proteoglycan is brought into contact with 50 mM or more of sodium chloride solution through a semipermeable membrane.SELECTED DRAWING: None

Description

The present invention relates to a method for producing salmon-derived proteoglycan containing 7% by weight or more of sodium and 0.7% by weight or less of calcium.

Proteoglycans are widely present in extracellular matrices and body fluids of connective tissues of animals, and are known as water-retaining substances together with hyaluronic acid and collagen, which are also present in connective tissues. In recent years, in addition to water retention, EGF-like action and anti-allergic action have been clarified.

Proteoglycan is a general term for covalently bound products of glycosaminoglycan and protein, and is characterized by extremely high sugar content compared to general glycoproteins. Sugars in proteoglycans are mostly amino sugars and uronic acids, and less neutral sugars. Amino sugars are often sulfated. The molecular weight, the types and amounts of amino acids and sugars contained, and the ratio of sulfation also differ depending on the starting raw materials and extraction/manufacturing conditions, and there are various molecular species.

Among proteoglycans, proteoglycans containing a large amount of sodium and having a low calcium content have been reported to have higher water-retaining capacity and anti-allergic effects compared to proteoglycans containing a large amount of calcium and a low sodium content. Since proteoglycans contain a large number of functional groups such as sulfate groups and carboxyl groups, they contain a large amount of calcium as counter ions. Since proteoglycan is often extracted from cartilage, the calcium contained in proteoglycan is also derived from cartilage. As a method for adding sodium to proteoglycan, there is a method using a strongly acidic cation exchange resin (Patent Document 1), but there are drawbacks such as the amount of proteoglycan treated being limited to the amount of ion exchange resin, and it is not satisfactory. I didn't.

JP 2016-29150 A

In view of the above problems, an object of the present invention is to provide a novel method for producing salmon-derived proteoglycan containing 7% by weight or more of sodium and 0.7% by weight or less of calcium.

A method for producing a proteoglycan containing 7% by weight or more of sodium and 0.7% by weight or less of calcium for achieving the above object is to add salmon-derived proteoglycan to a sodium chloride solution of 50 mM or more through a semipermeable membrane. The gist is to make contact.

The present invention provides a method for producing proteoglycan containing 7% by weight or more of sodium and 0.7% by weight or less of calcium.

Hereinafter, embodiments will be described more specifically. It is an example of a method for embodying the technical idea of the present invention, and the technical idea of the present invention is not limited to the following. Various modifications can be made to the technical idea of the present invention within the technical scope defined by the claims.

The present invention is directed to salmon-derived proteoglycans. There are various extracting agents for proteoglycan, such as organic acids such as acetic acid, acids, alkalis, and guanidine hydrochloride. Proteoglycan is a type of glycoprotein, and is a natural polymer compound with a molecular weight of hundreds of thousands to millions, which is a covalent bond of glycosaminoglycan with a weight composition ratio of 70% or more and protein. Uronic acid and amino sugar account for the majority in approximately equal amounts, and the amount of protein is smaller than that of general glycoproteins. In addition, as a feature, the proportion of sulfate groups ester-bonded to hydroxy groups of glycosaminoglycans is high.

Specifically, the method for producing a proteoglycan containing 7% by weight or more of sodium and 0.7% by weight or less of calcium according to the present invention comprises preparing raw salmon-derived proteoglycan dissolved in water inside a semipermeable membrane, A sodium chloride solution of 50 mM or higher is prepared outside the semipermeable membrane, and both solutions are brought into contact with each other through the semipermeable membrane. A semipermeable membrane is a membrane that allows some components in a solution or dispersion system to pass through but does not allow other components to pass through. Materials for semipermeable membranes include cellulose, collodion, gelatin, acetylcellulose, polyacrylonitrile, and polysulfone. Many of the semipermeable membranes are tube-shaped, and it is easy to use by putting the raw salmon-derived proteoglycan dissolved in water inside the tube. In the present invention, an ultrafiltration membrane, which is a kind of semipermeable membrane and is a porous polymer membrane, can also be used, and in this case, the molecular weight cut off can be in the range of 3 kDa to 300 kDa.

The concentration of sodium chloride to be brought into contact with the proteoglycan through the semipermeable membrane must be 50 mM or higher. The amount of sodium chloride should be at least 35 times the amount of proteoglycan. For example, the amount of sodium prepared in an aqueous solution in which 1 g of proteoglycan is dissolved must be 35 g or more, which is 12 L or more when converted to 50 mM. Moreover, the contact time of the proteoglycan aqueous solution and the sodium chloride solution through the semipermeable membrane requires 5 hours or more. The temperature of the solution during contact is preferably a low temperature of 10° C. or less in order to prevent putrefaction. In addition, the sodium chloride solution is produced more efficiently by stirring.

After sodium substitution/addition to salmon-derived proteoglycans and reduction of calcium, excess sodium chloride in the proteoglycan solution is removed to complete the process. Methods for removing excess sodium chloride are conventional methods, such as dialysis against water, ultrafiltration, and reverse osmosis. In this case, the target proteoglycan is obtained in an aqueous solution state, but it can be dried and solidified by freeze drying, spray drying, or other means. It is also possible to precipitate, dry and solidify the proteoglycan obtained with an organic solvent such as ethanol or acetone. It is also possible to use a combination of these methods.

By the above production method, most of the calcium ions bound to the carboxyl groups and sulfate groups of proteoglycan are replaced with sodium ions. It is also conceivable that sodium ions are newly bound to free sulfate groups and carboxyl groups.

EXAMPLES The present invention will be specifically described below with reference to Examples, but these are merely for the purpose of illustration and the present invention is not limited to these Examples.

(Investigation of production method of high-sodium-low-calcium proteoglycan)
A commercially available proteoglycan (Kakuhiro Proteoglycan Research Institute Co., Ltd.) was purchased and used as the raw salmon-derived proteoglycan. 10 mg of proteoglycan was dissolved in 2 mL of distilled water, placed as a semipermeable membrane in a dialysis cellulose tube (circumference 5 cm×length 8 cm, EIDIA Co., Ltd.), and the upper and lower openings were sealed. 200 mL of an aqueous sodium chloride solution was placed in a 200 mL beaker, the cellulose tube containing proteoglycan was suspended in the aqueous sodium chloride solution, a stirrer was placed in the beaker, and the mixture was stirred with a stirrer. It was carried out in a cold room at 4°C. The concentrations of sodium chloride are 0M, 10mM, 20mM, 50mM, 100mM, 500mM, 2M, 4M.

After 6 hours, each cellulose tube containing proteoglycan was transferred to a beaker containing 200 mL of deionized water and dialyzed against water in a cold room at 4°C for 2 days. The deionized water in the beaker was changed three times a day. The liquid in the cellulose tube was collected, concentrated with a rotary evaporator while being heated to 40° C. in a water bath, and freeze-dried with a freeze dryer to obtain proteoglycan as a white cotton-like solid.

(measurement of proteoglycan sodium and calcium)
The sodium and calcium contents of the obtained proteoglycan were quantified using a capillary electrophoresis device (AgiLent 7100 capillary electrophoresis system, manufactured by Agilent Technologies). For the quantification method of each metal, by injecting the sample into a capillary column filled with a buffer solution having UV absorption and applying a voltage, each metal ion in the sample is separated and moved, and passes through the UV detection part. An indirect absorption method was employed in which detection was by the decrease in UV absorption over time. A fused silica capillary (inner diameter 50 μm, effective length 56 cm, manufactured by Agilent Technologies Inc.) is used for the column, and a cation analysis buffer (Part No. 5064-8203, manufactured by Agilent Technologies Inc.) is used as the buffer, UV wavelength Electrophoresis was performed at 310 nm, a migration temperature of 25° C., and a voltage of 30 kV. The sample injection conditions were 50 mbar and 4 seconds. To measure sodium and calcium concentrations, a calibration curve for sodium and calcium was prepared in advance. As sodium and calcium, sodium chloride (special grade, Wako Pure Chemical Industries, Ltd.) and calcium chloride (special grade, Wako Pure Chemical Industries, Ltd.) were used. It was created with an area ratio of As magnesium, magnesium chloride (special grade, Wako Pure Chemical Industries, Ltd.) was used. The resulting proteoglycan was adjusted to 300 ppm by adding 2 ppm of magnesium as an internal standard and subjected to electrophoresis. Since the lower measurement limit of sodium according to the prepared calibration curve was 2.5 ppm, the lower measurement limit concentration of sodium in the 300 ppm proteoglycan was 0.8% by weight. In addition, since the lower limit of calcium measurement according to the prepared calibration curve was 2 ppm, the lower limit of measurement concentration of calcium in the 300 ppm proteoglycan was 0.7% by weight.

When the concentrations of sodium chloride in the beaker were 0M, 10mM, 20mM, 50mM, 100mM, 500mM, 2M and 4M, the sodium content of the proteoglycans obtained was 0.8% by weight and 5.5% by weight, respectively. , 5.7% by weight, 7.3% by weight, 7.3% by weight, 8.6% by weight, 9.2% by weight and 8.8% by weight. In addition, the content of calcium is 4.5% by weight when the concentration of sodium chloride in the external solution is 0M, 1.1% by weight when 10mM, 1.0% by weight when 20mM, and all concentrations of 50mM or more are below the lower limit of measurement concentration. Met. When the salmon-derived proteoglycan was brought into contact with a sodium chloride solution of 50 mM or more through a semipermeable membrane, the sodium content of the proteoglycan was 7% by weight or more, and the calcium content was 0.7% by weight or less, which is below the lower limit concentration of measurement. rice field.

(Production of high-sodium-low-calcium proteoglycan)
A commercially available proteoglycan (Kakuhiro Proteoglycan Research Institute Co., Ltd.) was purchased and used as the raw salmon-derived proteoglycan. 400 mg of proteoglycan was dissolved in 50 mL of distilled water, placed as a semipermeable membrane in a dialysis cellulose tube (perimeter 9 cm×length 15 cm, EIDIA Co., Ltd.), and the upper and lower openings were sealed. 1 L of 500 mM sodium chloride aqueous solution was placed in a 1 L beaker, and the semipermeable membrane containing proteoglycan with the upper and lower openings closed was suspended in the sodium chloride aqueous solution. It was carried out in a cold room at 4°C.

After 16 hours, each semipermeable membrane containing proteoglycans was transferred to a beaker containing 1 L of deionized water and dialyzed against water in a cold room at 4°C for 2 days. The deionized water in the external liquid beaker was replaced three times a day. The liquid in the cellulose tube was collected, concentrated with a rotary evaporator while being heated to 40° C. in a water bath, and freeze-dried with a freeze dryer to obtain 366 mg of high-sodium-low-calcium proteoglycan as a white cotton-like solid.

(measurement of proteoglycan sodium and calcium)
The sodium and calcium contents of the obtained proteoglycan were measured in the same manner by preparing a calibration curve using the capillary electrophoresis apparatus described in Example 1.

As a result, the content of sodium was 7.2% by weight, and the content of calcium was 0.7% by weight or less, which is the lower limit concentration of measurement.

(Comparative experiment 1)
A commercially available proteoglycan (Kakuhiro Proteoglycan Research Institute Co., Ltd.) was purchased and used as the raw salmon-derived proteoglycan. 10 mg of proteoglycan was dissolved in 2 mL of a sodium chloride solution of a predetermined concentration and allowed to stand at 25° C. for 1 hour. The concentrations of sodium chloride are 10 mM, 20 mM, 50 mM, 100 mM, 500 mM, 2M, 4M. After that, in order to remove excess sodium chloride, this solution was placed in a dialysis cellulose tube (circumference 5 cm x length 8 cm, EIDIA Co., Ltd.), the upper and lower openings were sealed, and the solution was placed in a beaker containing 500 mL of deionized water. Float and dialyze against water for 2 days in a cold room at 4°C. The deionized water in the external liquid beaker was replaced three times a day. The liquid in the cellulose tube was collected, concentrated with a rotary evaporator while being heated to 40° C. in a water bath, and freeze-dried with a freeze dryer to obtain proteoglycan as a white cotton-like solid.

(measurement of proteoglycan sodium and calcium)
The sodium and calcium contents of the obtained proteoglycan were measured in the same manner by preparing a calibration curve using the capillary electrophoresis apparatus described in Example 1.

As a result, when the concentrations of sodium chloride in which proteoglycan was dissolved were 10 mM, 20 mM, 50 mM, 100 mM, 500 mM, 2 M, and 4 M, the sodium contents were 1.0% by weight, 1.2% by weight, and 2.1% by weight, respectively. %, 1.8%, 5.9%, 5.7% and 5.5% by weight. In addition, the calcium content was 3.1% by weight, 3.2% by weight, 2.6% by weight, 2.7% by weight, 0.8% by weight, and 0.8% by weight, respectively, and was below the lower limit concentration of measurement. rice field.

(Comparative experiment 2)
A commercially available proteoglycan (Kakuhiro Proteoglycan Research Institute Co., Ltd.) was purchased and used as the raw salmon-derived proteoglycan. 10 mg of proteoglycan was dissolved in 2 mL of distilled water, placed as a semipermeable membrane in a dialysis cellulose tube (circumference 5 cm×length 8 cm, EIDIA Co., Ltd.), and the upper and lower openings were sealed. Put 200 mL each of 100 mM sodium sulfate, 100 mM sodium nitrate, 100 mM disodium hydrogen phosphate, and 100 mM sodium acetate in a 200 mL beaker, float the cellulose tube containing proteoglycan in each sodium salt solution, and add a stirrer to the beaker. and stirred with a stirrer. It was carried out in a cold room at 4°C.

After 6 hours, the cellulose tubes containing proteoglycans were transferred to a beaker containing 200 mL of deionized water and dialyzed against water in a cold room at 4°C for 2 days. The deionized water in the external liquid beaker was replaced three times a day. The liquid in the cellulose tube was collected, concentrated with a rotary evaporator while being heated to 40° C. in a water bath, and freeze-dried with a freeze dryer to obtain proteoglycan as a white cotton-like solid.

(measurement of proteoglycan sodium and calcium)
The sodium and calcium contents of the obtained proteoglycan were measured in the same manner by preparing a calibration curve using the capillary electrophoresis apparatus described in Example 1.

As a result, when the aqueous solution in the beaker is sodium sulfate, sodium nitrate, disodium hydrogen phosphate, and sodium acetate, the sodium content is 4.3% by weight, 4.1% by weight, and 6.5% by weight, respectively. , 5.6% by weight. All calcium contents were below the lower limit of measurement.

By using a semipermeable membrane, sodium can be easily substituted and added to the salmon-derived proteoglycan, and calcium can be reduced, so that the obtained proteoglycan can be applied to cosmetics and foods.

Claims (1)

A method for producing proteoglycan containing 7% by weight or more of sodium and 0.7% by weight or less of calcium, comprising contacting salmon-derived proteoglycan with a sodium chloride solution of 50 mM or more through a semipermeable membrane.