JP2004189825A - Method for producing sodium chondroitin sulfate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing sodium chondroitin sulfate, comprising a simple method. <P>SOLUTION: The method for producing sodium chondroitin sulfate comprises a process 2 for treating cartilage with an alkali and a process 4 for salting out the alkali-treated material. Consequently, polymer protein and lipid are separated and removed. The alkali is preferably NaOH and is preferably in a range of 0.1-2N. The alkali treatment is preferably carried out in the presence of NaCl. The salting out is preferably carried out with NaCl. NaCl is preferably in the range of 3-5M. After the process 4 of salting out, an alcohol is preferably added to a filtrate to salt out sodium chondroitin sulfate. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to sodium chondroitin sulfate. The present invention also relates to a method for producing sodium chondroitin sulfate.
[0002]
[Prior art]
Sodium chondroitin sulfate is based on a disaccharide repeating structure of glucoronic acid and N-acetylgalactosamine, and has a structure in which a sulfate group is bonded to N-acetylgalactosamine. At present, sodium chondroitin sulfate is produced mainly from shark fins and calf nasal septum cartilage. However, shark resources are low, and calf nasal septum cartilage has a problem with the safety of raw materials such as the risk of infectious disease (mad cow disease), and it is difficult to stably secure cheap raw materials in all cases. Situation. Thus, nasal cartilage obtained from salmon heads has recently attracted attention.
[0003]
It was known before the advent of the human immunodeficiency virus (HIV) that sodium chondroitin sulfate inhibits the infection of many viral cells, such as influenza virus and herpes simplex virus.
[0004]
As described above, since sodium chondroitin sulfate has various physiological activities and physical properties, it is used as cosmetics or eye drops as humectants, including pharmaceuticals such as anti-inflammatory agents.
[0005]
Various methods have been proposed for producing sodium chondroitin sulfate from cartilage. This is shown below.
[0006]
Nasal cartilage is collected from the salmon head and cut into small pieces. In order to decompose proteins contained in nasal cartilage, heat treatment is performed in an NaOH aqueous solution, and then a proteolytic enzyme is added and heat treatment is performed. After cooling, the mixture is centrifuged to obtain a supernatant containing low molecular weight contaminants generated by the decomposition treatment and sodium chondroitin sulfate. A method has been proposed in which the supernatant is subjected to continuous multi-stage ultrafiltration to simultaneously concentrate and purify sodium chondroitin sulfate (for example, see Patent Document 1).
[0007]
Crush the salmon nasal cartilage. This is heat-treated in an aqueous NaOH solution in order to decompose proteins, and then heat-treated by adding a protease. The digestion fluid is centrifuged and the supernatant is filtered. Add alcohol to the filtrate and collect the precipitate. There has been proposed a method in which a precipitate is dissolved in deionized water, treated with a cation exchange resin, and further subjected to dialysis (for example, see Patent Documents 2 and 3).
[0008]
[Patent Document 1]
JP 2000-273102 A
[Patent Document 2]
JP 2001-231497 A
[Patent Document 3]
JP 2001-247602 A
[0009]
[Problems to be solved by the invention]
However, in the above-mentioned conventional method for producing sodium chondroitin sulfate, in order to purify the sodium chondroitin sulfate, it is necessary to perform a continuous multi-stage ultrafiltration treatment or a cation exchange resin treatment, followed by dialysis. These methods have a problem that expensive equipment is required and a long time is required for performing complicated steps.
[0010]
The present invention has been made in view of such problems, and an object of the present invention is to provide a method for producing sodium chondroitin sulfate which is a simple method, and to provide sodium chondroitin sulfate obtained by the method.
[0011]
[Means for Solving the Problems]
The method for producing sodium chondroitin sulfate of the present invention is a method including the following steps. These are the step of treating cartilage with alkali and the step of salting out the cartilage. Thereby, the high molecular protein and the lipid can be separated and removed.
[0012]
Here, the cartilage is preferably fish cartilage. The cartilage is preferably salmon nasal cartilage. Preferably, the alkali is NaOH. Preferably, the alkali is in the range of 0.1-2N. The alkali treatment is preferably performed in the presence of NaCl. Preferably, NaCl is in the range of 1-2M. Preference is given to salting out with NaCl. Preferably, NaCl is in the range of 3-5M. After the salting out step, it is preferable to add alcohol to the filtrate to precipitate sodium chondroitin sulfate.
[0013]
The sodium chondroitin sulfate of the present invention is obtained by subjecting cartilage to alkali treatment and salting it out. As a result, high-molecular proteins and lipids are removed, and high-purity sodium chondroitin sulfate is obtained.
[0014]
Here, the cartilage is preferably fish cartilage. The cartilage is preferably salmon nasal cartilage. Preferably, the alkali is NaOH. Preference is given to salting out with NaCl.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described.
First, a method for producing sodium chondroitin sulfate will be described.
[0016]
FIG. 1 is a flowchart showing the steps of the method for producing sodium chondroitin sulfate of the present invention. The number assigned to each block is the number of the process. The description will be given in the order of the steps.
[0017]
In the first step, cartilage as a raw material is pulverized.
Salmon nasal cartilage is used as the raw material cartilage. Cartilage is not limited to this salmon nasal cartilage. In addition, all fish cartilage such as shark fin, shark spine cartilage, ray fin, ray head cartilage, other calf nasal septum cartilage, and all cartilage containing chondroitin sulfate can be used. .
[0018]
Salmon nasal cartilage is ground with a meat grinder. Grinding of the nasal cartilage is not limited to using a meat grinder. As the pulverizing method, in addition, stirring and grinding in an extraction pot can be employed.
[0019]
The nasal cartilage is preferably crushed to a size in the range of 1 to 10 mm. When the size is 1 mm or more, there is an advantage that the solution is improved. When the size is 10 mm or less, there is an advantage that the extraction time is shortened.
[0020]
In the second step, the pulverized cartilage is alkali-treated. The pulverized cartilage is stirred in a mixed aqueous solution of alkali and salt and heat-treated for a predetermined time.
[0021]
Caustic soda (NaOH) is used as the alkali. The alkali is not limited to NaOH. In addition, caustic potassium or the like can be used.
[0022]
The alkali concentration is preferably in the range of 0.1 to 2N. When the concentration of the alkali is 0.1 N or more, there is an advantage that the covalent bond between chondroitin sulfate and the protein is cleaved by β-elimination reaction. When the alkali concentration is 2N or less, excessive degradation of the protein can be prevented, and the chondroitin sulfate sodium and the protein can be cleaved.
[0023]
More preferably, the concentration of the alkali is in the range of 0.1 to 1N. When the alkali concentration is within this range, the above effects can be more remarkably exhibited.
[0024]
In the alkali treatment, salt (NaCl) coexists with NaOH. Preferably, the concentration of NaCl is in the range of 1-2M. When the concentration of NaCl is 1 M or more, there is an advantage that cleavage of chondroitin sulfate and proteins is accelerated. When the concentration of NaCl is 2M or less, there is an advantage that the decomposition of the protein is mild.
[0025]
More preferably, the concentration of NaCl is 2M. When the concentration of NaCl is this value, the above-described effect can be more remarkably exhibited.
[0026]
The temperature of the alkali treatment is preferably in the range of 10 to 50C. When the temperature is 10 ° C. or higher, there is an advantage that the alkali concentration can be increased and the processing time can be shortened. When the temperature is 50 ° C. or lower, there is an advantage that the alkali concentration can be reduced and the treatment time can be extended.
[0027]
The alkali treatment temperature is more preferably in the range of 20 to 50 ° C. When the temperature is within this range, the above effects can be more remarkably exhibited.
[0028]
The alkali treatment time is preferably in the range of 0.5 to 2 hours. When the time is 0.5 hours or more, there is an advantage that the alkali concentration can be increased and the temperature can be set low. When the time is 2 hours or less, there is an advantage that the alkali concentration can be reduced and the temperature can be set high.
[0029]
The alkali treatment time is more preferably in the range of 1 to 2 hours. When the time is within this range, the above-described effect can be more remarkably exhibited.
[0030]
In the above-described alkali treatment step, high-concentration salt and alkali (such as NaOH) are used in combination. This breaks the covalent bond between sodium chondroitin sulfate contained in cartilage and the protein, but suppresses the degradation of the protein itself. Degradation of the protein into peptides or amino acids is suppressed, and the protein remains in a macromolecular state.
[0031]
In the third step, neutralization is performed. An acid is added to the alkali-treated one while stirring to neutralize. Acetic acid is used as the acid.
[0032]
The acid is not limited to acetic acid. In addition, an organic acid such as citric acid and an inorganic acid such as hydrochloric acid can be used.
[0033]
The pH of the aqueous solution after neutralization is preferably in the range of 5.5 to 7.5. When the pH is 5.5 or more, there is an advantage that a dissolved protein is precipitated. When the pH is 7.5 or less, there is an advantage that a dissolved protein is easily salted out.
[0034]
In the fourth step, the product subjected to the alkali treatment is salted out. The high molecular weight protein is precipitated by increasing the NaCl concentration with stirring from the alkali treatment solution in which the protein remains in the polymer state without being decomposed.
[0035]
The salt used for salting out is not limited to NaCl. In addition, ammonium sulfate or the like can be used.
[0036]
Preferably, the salt concentration is in the range of 3-5M. When the salt concentration is 3M or more, there is an advantage that the precipitation of the protein is improved. When the salt concentration is 5 M or less, there is an advantage that NaCl completely precipitates the protein in a saturated solution.
[0037]
In the fifth step, adsorption is performed. By adding an adsorbent to the salted-out material while stirring, the precipitated proteins and lipids are adsorbed to the adsorbent. Lipids are saponified by NaOH and are easily adsorbed to the adsorbent. This fifth step can be skipped and proceed to the next step. The reason is that the adsorbent is used in the next step.
[0038]
Activated clay can be used as the adsorbent. The adsorbent is not limited to activated clay. In addition, activated carbon or the like can be employed.
[0039]
In the sixth step, filtration is performed. By adding a filter aid to the product obtained in the fourth or fifth step and filtering, high molecular proteins and lipids are separated and removed. The resulting filtrate is a clear aqueous solution containing chondroitin sulfate and sodium chloride.
[0040]
Filtration is performed by a filter press method. The filtration method is not limited to this filter press method. In addition, vacuum filtration, centrifugation and the like can be employed.
[0041]
Diatomaceous earth is used as a filter aid. The reason is that it is cheap and easily available.
[0042]
Filter aids are not limited to diatomaceous earth. In addition, paper pulp or the like can be used.
[0043]
In the seventh step, precipitation is performed. While stirring the filtrate, ethyl alcohol is added thereto to precipitate sodium chondroitin sulfate.
[0044]
Ethyl alcohol is used for the precipitation, but is not limited thereto. In addition, methyl alcohol or the like can be employed.
[0045]
The concentration of ethyl alcohol at the time of precipitation is preferably in the range of 30 to 70% by mass. When the concentration of ethyl alcohol is 30% by mass or more, there is an advantage that sodium chondroitin sulfate starts to precipitate. When the concentration of ethyl alcohol is 70% by mass or less, there is an advantage that sodium chondroitin sulfate is completely precipitated.
[0046]
In the eighth step, decanting and centrifugation are performed. When the precipitate is allowed to stand, sodium chondroitin sulfate precipitates and separates into two layers, a precipitate layer and a supernatant layer. The supernatant is removed by a decanter. The sediment of the sediment layer is introduced into a centrifuge and centrifuged. By separating and removing the solution, crude sodium chondroitin sulfate can be obtained.
[0047]
As the centrifuge, a top-discharge centrifuge is used, but is not limited thereto. In addition, a skimming type centrifugal separator, a reduced pressure filter, or the like can be employed.
[0048]
In the ninth step, dissolution is performed. Crystals of wet crude sodium chondroitin sulfate obtained by centrifugation are dissolved in a predetermined amount of warm water.
[0049]
Wet crystals contain NaCl. It is necessary to remove this NaCl. The required mass of water is determined as follows. The concentration of NaCl is determined from the mass of NaCl added in the alkali treatment (step 2) and salting out (step 4), the amount of water contained in the cartilage, and the mass of water added in the alkali treatment (step 2). The mass of NaCl in the filtrate is determined from the mass of the filtrate in the filtration (step 6). From the amount of liquid removed by the decanter / centrifugation (step 8) and the salt concentration of the liquid, the mass of NaCl removed is determined. By subtracting the mass of NaCl removed from the mass of NaCl in the filtrate, the mass of NaCl in the wet crystals is determined.
[0050]
In the later-described precipitation (step 12), precipitation is performed using a 30 to 70% by mass aqueous solution of ethyl alcohol. Set the concentration of ethyl alcohol within this range. Therefore, the mass of the aqueous ethyl alcohol solution (set concentration) required to dissolve the NaCl contained in the wet crystals is determined from the solubility curve (Chemical Handbook). From the obtained mass, the minimum required water mass is obtained. Thus, by calculating the minimum required mass of water, the mass of ethyl alcohol used in the precipitation (step 12) can be reduced.
[0051]
The mass of water required in the dissolving step 9 is not limited to this minimum required mass of water. The mass of water required for dissolution is preferably in the range of 1 to 2 times the minimum required mass of water. NaCl can be completely dissolved if it is at least one time the minimum required mass of water. There is an advantage that NaCl can be completely dissolved and removed when the mass of water is at least twice the minimum required mass.
[0052]
Upon dissolution, it is preferable to heat to 50 to 100 ° C. When the temperature is 50 ° C. or higher, there is an advantage that the dissolution of crude sodium chondroitin sulfate can be accelerated. When the temperature is 100 ° C. or lower, there is an advantage that dissolution can be performed in a shorter time.
[0053]
In the tenth step, adsorption is performed. By adding an adsorbent to the dissolved substance while stirring, lipids and remaining high molecular proteins are adsorbed to the adsorbent.
Activated clay can be used as the adsorbent. The adsorbent is not limited to activated clay, as described in Step 5.
[0054]
In the eleventh step, filtration is performed. By adding a filter aid to the product obtained in the adsorption step and performing filtration, lipids and high molecular proteins are separated and removed. A colorless and transparent aqueous solution of sodium chondroitin sulfate is obtained.
[0055]
Filtration is performed by a filter press method. Diatomaceous earth is used as a filter aid. The fact that the filtration method is not limited to the filter press method and that the filter aid is not limited to diatomaceous earth are the same as described in Step 6.
[0056]
In the twelfth step, precipitation is performed. While stirring the filtrate, a predetermined amount of ethyl alcohol is added to precipitate sodium chondroitin sulfate. In this step, the NaCl dissolved in the solution is kept in a dissolved state without being precipitated.
[0057]
The mass of ethyl alcohol to be added is determined from the ethyl alcohol concentration set in step 9 and the mass of water required for dissolution calculated in step 9. The set concentration of ethyl alcohol is preferably in the range of 30 to 70% by mass. This is for the reason explained in the step 7 of the deposition.
[0058]
In the thirteenth step, a decanter is performed. When the precipitate is allowed to stand, sodium chondroitin sulfate precipitates and separates into two layers, a precipitate layer and a supernatant layer. The supernatant is removed by a decanter.
[0059]
In the fourteenth step, temporary dehydration is performed. The mass of the precipitate obtained by the decanter is measured, and ethyl alcohol is added so that the concentration becomes 80% by mass, followed by stirring. By this step, the water and NaCl in the precipitate are diluted into an ethyl alcohol aqueous solution.
[0060]
The concentration of ethyl alcohol is not limited to 80% by mass. The concentration of ethyl alcohol is preferably in the range of 75 to 85% by mass. When the concentration is 75% by mass or more, there is an advantage that the wet precipitate of sodium chondroitin sulfate is powdered by stirring. When the concentration is 85% by mass or less, there is an advantage that NaCl does not precipitate.
[0061]
In the fifteenth step, centrifugation is performed. The temporarily dehydrated one is introduced into a centrifuge and centrifuged. Further, the remaining NaCl is removed by washing with an ethyl alcohol aqueous solution having the same concentration as used in the temporary dehydration step 14. By this step, crystals of sodium chondroitin sulfate can be obtained.
[0062]
In the sixteenth step, dehydration is performed. To the crystals of sodium chondroitin sulfate, 99% by mass of ethyl alcohol is added while stirring until the concentration becomes 95% by mass or more, and dehydration is performed. When the concentration of ethyl alcohol is 95% by mass or more, there is an advantage that water in sodium chondroitin sulfate is removed and powdered.
[0063]
In the seventeenth step, centrifugation is performed. The dehydrated one is introduced into a centrifuge and centrifuged. By this step, crystals of sodium chondroitin sulfate having low water content can be obtained.
[0064]
In step 18, drying is performed. The crystals of sodium chondroitin sulfate obtained by centrifugation are put into a drier and dried at a temperature of 50 to 80 ° C under reduced pressure. When the temperature is 50 ° C. or higher, there is an advantage that drying starts. When the temperature is 80 ° C. or lower, there is an advantage that sodium chondroitin sulfate is not changed over time.
[0065]
The method for producing sodium chondroitin sulfate has been described above with reference to steps 1 to 18. The method for producing sodium chondroitin sulfate is not limited to steps 1 to 18.
[0066]
Steps 8 to 18 are purification steps of the crude sodium chondroitin sulfate obtained in the step of Precipitation 7. Instead of Steps 8 to 18, a method by spray drying and desalting by dialysis after performing Steps 8 to 11 can be adopted.
[0067]
When sodium chondroitin sulfate having the required purity is obtained in step 7 of precipitation, steps 8 to 12 can be omitted.
[0068]
Next, the sodium chondroitin sulfate produced by the above method will be described.
[0069]
The obtained sodium chondroitin sulfate is white crystals.
As a result of elemental analysis, nitrogen was in the range of 2.6 to 2.8% by mass, and sulfur was in the range of 6.1 to 6.4% by mass. The protein content is 1% by mass or less. From these values, it was confirmed that the obtained sodium chondroitin sulfate had high purity.
[0070]
As described above, the method for producing sodium chondroitin sulfate according to the present embodiment is a method including the following steps. These are the step of treating cartilage with alkali and the step of salting out the cartilage. Thereby, the high molecular protein and the lipid can be separated and removed.
[0071]
The sodium chondroitin sulfate of the present embodiment is obtained by subjecting cartilage to alkali treatment and salting it out. As a result, sodium chondroitin sulfate having a low content of the high molecular protein and the lipid is obtained.
[0072]
As described above, according to the present embodiment, sodium chondroitin sulfate can be obtained by a simple method including a step of alkali-treating cartilage and a step of salting out the cartilage.
[0073]
Note that the present invention is not limited to the above-described embodiment, but can adopt various other configurations without departing from the gist of the present invention.
[0074]
【Example】
Next, examples according to the present invention will be specifically described. However, needless to say, the present invention is not limited to these examples.
[0075]
Example 1
Nasal cartilage is collected from the salmon head and stored frozen at -20 ° C. 1 kg of frozen nasal cartilage is left overnight at room temperature to thaw. The water content in nasal cartilage is 850 g. The thawed nasal cartilage is pulverized by a meat grinder, and 500 ml of water is added and dispersed by stirring. Next, 175.5 g of sodium chloride and 150 g of a 4% by mass aqueous solution of sodium hydroxide (NaOH) were added so that the final concentration was 2 M aqueous sodium chloride (NaCl) and 0.1 N aqueous sodium hydroxide (NaOH), and the temperature was 50 ± 2 ° C. For 2 hours. After completion of the alkali treatment, the pH is adjusted to 6 with acetic acid. Next, when 175.5 g of NaCl is added and dissolved so that the NaCl concentration becomes 4 M, the polymer protein is salted out. For the adsorption, 10 g of activated clay was added, and then diatomaceous earth was added and filtered to obtain 1500 ml of a transparent filtrate. To this filtrate (1500 ml), ethyl alcohol (2250 ml) is added to prepare a 55% by mass aqueous alcohol solution, and sodium chondroitin sulfate is separated and precipitated. After decanting, the precipitate was collected by centrifugation to obtain 150 g of crude sodium chondroitin sulfate. Since the obtained crude sodium chondroitin sulfate contains sodium chloride and a trace amount of oil, the crude sodium chondroitin sulfate is dissolved in 500 ml of 70 ° C. hot water for desalination and removal of a trace amount of oil, and 10 g of activated clay and diatomaceous earth are added. After filtration, 550 ml of a colorless and transparent aqueous solution of sodium chondroitin sulfate was obtained. Next, 700 ml of ethyl alcohol is added to this aqueous solution to form a 50% by mass aqueous solution of ethyl alcohol, and sodium chondroitin sulfate is separated and precipitated. NaCl is dissolved and desalted in a 50% by mass aqueous solution of ethyl alcohol. The precipitate is collected by a decanter, washed with 80% by mass of ethyl alcohol, and centrifuged.
Further, 99% by mass of ethyl alcohol is added while stirring until the concentration becomes 95% by mass, and dehydration washing is performed. When the precipitate obtained by centrifugation is dried under reduced pressure at a temperature of 60 to 70 ° C., 30 g of sodium chondroitin sulfate is obtained.
[0076]
The obtained sodium chondroitin sulfate was subjected to quantitative analysis of protein, nitrogen, and sulfur. Quantitative analysis of the protein was performed by the Picaulet method. The detection limit of this analytical method is 1% by mass.
[0077]
Quantitative analysis of nitrogen and sulfur was performed by the Kjeldahl method for nitrogen and the barium sulfate method for sulfur.
[0078]
The determination as to whether the obtained sodium chondroitin sulfate was a high-purity product was made according to the following criteria.
[0079]
The protein content was based on 1% by mass or less. The reason is that the protein was almost completely removed in the confirmation test.
[0080]
The nitrogen content was in the range of 2.6 to 2.8% by mass, and the sulfur content was in the range of 6.1 to 6.4% by mass. The molecular weight was calculated from the structural formula of sodium chondroitin sulfate, the theoretical value of 100% by mass of nitrogen and sulfur was determined, and those containing 90 to 100% by mass were regarded as high purity products.
[0081]
The analysis results of the obtained sodium chondroitin sulfate were as follows. The protein content was below the detection limit. Nitrogen was 2.7% by weight and sulfur was 6.3% by weight. From these analysis results, it was found that the chondroitin sulfate sodium obtained in this example was a highly purified product.
[0082]
Example 2
Nasal cartilage is collected from the salmon head and stored frozen at -20 ° C. 1 kg of frozen nasal cartilage is left overnight at room temperature to thaw. The thawed nasal cartilage is ground with a meat grinder. The water content in nasal cartilage is 850 g. 117 g of sodium chloride and 8 g of caustic soda (NaOH) were dissolved in 150 ml of water and added at a final temperature of 40 ± 2 ° C. for 2 hours so that the final concentration was 2 M aqueous sodium chloride (NaCl) and 0.2 N aqueous sodium hydroxide (NaOH). Treat with alkali. As a result, the liquid volume becomes 1000 ml, the amount of ethyl alcohol used later can be reduced, and the production cost can be reduced. After completion of the alkali treatment, the pH is adjusted to 6 with acetic acid. Next, when 117 g of NaCl is added and dissolved so that the NaCl concentration becomes 4 M, the high molecular protein is salted out. After addition of activated clay (10 g) for adsorption, diatomaceous earth was added and the mixture was filtered to obtain 1000 ml of a transparent filtrate. To 1000 ml of the filtrate, 1500 ml of ethyl alcohol is added to form a 55% by mass aqueous alcohol solution, and sodium chondroitin sulfate is separated and precipitated. After decanting, the precipitate was collected by centrifugation to obtain 135 g of crude sodium chondroitin sulfate. Since the obtained crude sodium chondroitin sulfate contains sodium chloride and a trace amount of oil, the crude sodium chondroitin sulfate is dissolved in 500 ml of 70 ° C. hot water for desalination and removal of a trace amount of oil, and 10 g of activated clay and diatomaceous earth are added. After filtration, 530 ml of a colorless and transparent aqueous solution of sodium chondroitin sulfate was obtained. Next, 650 ml of ethyl alcohol is added to this aqueous solution to prepare a 50% by mass aqueous solution of ethyl alcohol, and sodium chondroitin sulfate is separated and precipitated. NaCl is dissolved and desalted in a 50% by mass aqueous solution of ethyl alcohol. The precipitate is collected by a decanter, washed with 80% by mass of ethyl alcohol, and centrifuged. Further, 99% by mass of ethyl alcohol is added while stirring until the concentration becomes 95% by mass, and dehydration washing is performed. When the precipitate obtained by centrifugation is dried under reduced pressure at a temperature of 60 to 70 ° C., 28 g of sodium chondroitin sulfate is obtained.
[0083]
The obtained sodium chondroitin sulfate was subjected to quantitative analysis of protein, nitrogen, and sulfur. The quantitative analysis of protein and the quantitative analysis of nitrogen and sulfur were the same as in Example 1.
[0084]
The determination as to whether or not the obtained sodium chondroitin sulfate was a high-purity product was the same as in Example 1.
[0085]
The analysis results of the obtained sodium chondroitin sulfate were as follows. The protein content was below the detection limit. Nitrogen was 2.7% by weight and sulfur was 6.3% by weight. From these analysis results, it was found that the chondroitin sulfate sodium obtained in this example was a highly purified product.
[0086]
Example 3
Nasal cartilage is collected from the salmon head and stored frozen at -20 ° C. 1 kg of frozen nasal cartilage is left overnight at room temperature to thaw. The thawed nasal cartilage is ground with a meat grinder. The water content in nasal cartilage is 850 g. 117 g of sodium chloride and 40 g of caustic soda (NaOH) are dissolved in 150 ml of water so that the final concentration becomes 2M aqueous solution of sodium chloride (NaCl) and 1N aqueous solution of caustic soda (NaOH), and alkali treatment is performed at a temperature of 20 ± 2 ° C. for 1 hour. I do. After completion of the alkali treatment, the pH is adjusted to 6 with acetic acid. Next, when 117 g of NaCl is added and dissolved so that the NaCl concentration becomes 4 M, the high molecular protein is salted out. After addition of activated clay (10 g) for adsorption, diatomaceous earth was added and the mixture was filtered to obtain 1000 ml of a transparent filtrate. To 1000 ml of the filtrate, 1500 ml of ethyl alcohol is added to form a 55% by mass aqueous alcohol solution, and sodium chondroitin sulfate is separated and precipitated. After decanting, the precipitate was collected by centrifugation to obtain 140 g of crude sodium chondroitin sulfate. Since the obtained crude sodium chondroitin sulfate contains sodium chloride and a trace amount of oil, the crude sodium chondroitin sulfate is dissolved in 500 ml of 70 ° C. hot water for desalination and removal of a trace amount of oil, and 10 g of activated clay and diatomaceous earth are added. After filtration, 550 ml of a colorless and transparent aqueous solution of sodium chondroitin sulfate was obtained. Next, 700 ml of ethyl alcohol is added to this aqueous solution to form a 50% by mass aqueous solution of ethyl alcohol, and sodium chondroitin sulfate is separated and precipitated. NaCl is dissolved and desalted in a 50% by mass aqueous solution of ethyl alcohol. The precipitate is collected by a decanter, washed with 80% by mass of ethyl alcohol, and centrifuged. Further, 99% by mass of ethyl alcohol is added while stirring until the concentration becomes 95% by mass, and dehydration washing is performed. When the precipitate obtained by centrifugation is dried under reduced pressure at a temperature of 60 to 70 ° C., 25 g of sodium chondroitin sulfate is obtained.
[0087]
The obtained sodium chondroitin sulfate was subjected to quantitative analysis of protein, nitrogen, and sulfur. The quantitative analysis of protein and the quantitative analysis of nitrogen and sulfur were the same as in Example 1.
[0088]
The determination as to whether or not the obtained sodium chondroitin sulfate was a high-purity product was the same as in Example 1.
[0089]
The analysis results of the obtained sodium chondroitin sulfate were as follows. The protein content was below the detection limit. Nitrogen was 2.7% by weight and sulfur was 6.2% by weight. From these analysis results, it was found that the chondroitin sulfate sodium obtained in this example was a highly purified product.
[0090]
Example 4
Cartilage is collected from the shark spine and stored frozen at -20 ° C. 1 kg of frozen spinal cartilage is left at room temperature overnight to thaw. The thawed nasal cartilage is ground with a meat grinder. The water content in the spinal cartilage is 850 g. 117 g of sodium chloride and 8 g of caustic soda (NaOH) are dissolved in 150 ml of water and added at a final temperature of 40 ± 2 ° C. for 2 hours so that the final concentration becomes 2 M aqueous sodium chloride (NaCl) and 0.2 N aqueous sodium hydroxide (NaOH). Treat with alkali. After completion of the alkali treatment, the pH is adjusted to 6 with acetic acid. Next, when 117 g of NaCl is added and dissolved so that the NaCl concentration becomes 4 M, the high molecular protein is salted out. After addition of activated clay (10 g) for adsorption, diatomaceous earth was added and the mixture was filtered to obtain 1000 ml of a transparent filtrate. To 1000 ml of the filtrate, 1500 ml of ethyl alcohol is added to form a 55% by mass aqueous alcohol solution, and sodium chondroitin sulfate is separated and precipitated. After decanting, the precipitate was collected by centrifugation to obtain 150 g of crude sodium chondroitin sulfate. Since the obtained crude sodium chondroitin sulfate contains sodium chloride and a trace amount of oil, the crude sodium chondroitin sulfate is dissolved in 500 ml of 70 ° C. hot water for desalination and removal of a trace amount of oil, and 10 g of activated clay and diatomaceous earth are added. After filtration, 550 ml of a colorless and transparent aqueous solution of sodium chondroitin sulfate was obtained. Next, 700 ml of ethyl alcohol is added to this aqueous solution to form a 50% by mass aqueous solution of ethyl alcohol, and sodium chondroitin sulfate is separated and precipitated. NaCl is dissolved and desalted in a 50% by mass aqueous solution of ethyl alcohol. The precipitate is collected by a decanter, washed with 80% by mass of ethyl alcohol, and centrifuged. Further, 99% by mass of ethyl alcohol is added while stirring until the concentration becomes 95% by mass, and dehydration washing is performed. When the precipitate obtained by centrifugation is dried under reduced pressure at a temperature of 60 to 70 ° C., 15 g of sodium chondroitin sulfate is obtained.
[0091]
The obtained sodium chondroitin sulfate was subjected to quantitative analysis of protein, nitrogen, and sulfur. The quantitative analysis of protein and the quantitative analysis of nitrogen and sulfur were the same as in Example 1.
[0092]
The determination as to whether or not the obtained sodium chondroitin sulfate was a high-purity product was the same as in Example 1.
[0093]
The analysis results of the obtained sodium chondroitin sulfate were as follows. The protein content was below the detection limit. Nitrogen was 2.6% by weight and sulfur was 6.3% by weight. From these analysis results, it was found that the chondroitin sulfate sodium obtained in this example was a highly purified product.
[0094]
Example 5
Nasal cartilage is collected from the salmon head and stored frozen at -20 ° C. 1 kg of frozen nasal cartilage is left overnight at room temperature to thaw. The thawed nasal cartilage is ground with a meat grinder. The water content in nasal cartilage is 850 g. 117 g of sodium chloride and 8 g of caustic soda (NaOH) are dissolved in 150 ml of water and added at a final temperature of 40 ± 2 ° C. for 2 hours so that the final concentration becomes 2 M aqueous sodium chloride (NaCl) and 0.2 N aqueous sodium hydroxide (NaOH). Treat with alkali. After completion of the alkali treatment, the pH is adjusted to 6 with acetic acid. Next, when 58.5 g of NaCl is added and dissolved so that the NaCl concentration becomes 3 M, the polymer protein is salted out. After addition of activated clay (10 g) for adsorption, diatomaceous earth was added and the mixture was filtered to obtain 1000 ml of a transparent filtrate. To 1000 ml of the filtrate, 1500 ml of ethyl alcohol is added to form a 55% by mass aqueous alcohol solution, and sodium chondroitin sulfate is separated and precipitated. After decanting, the precipitate was collected by centrifugation to obtain 90 g of crude sodium chondroitin sulfate. Since the obtained crude sodium chondroitin sulfate contains salt and a trace amount of oil, it is dissolved in 400 ml of 70 ° C. hot water to remove salt and remove trace amount of oil, and 10 g of activated clay and diatomaceous earth are added. After filtration, 420 ml of a colorless and transparent aqueous solution of sodium chondroitin sulfate was obtained. Next, 530 ml of ethyl alcohol is added to this aqueous solution to obtain a 50% by mass aqueous solution of ethyl alcohol, and sodium chondroitin sulfate is separated and precipitated. NaCl is dissolved and desalted in a 50% by mass aqueous solution of ethyl alcohol. The precipitate is collected by a decanter, washed with 80% by mass of ethyl alcohol, and centrifuged. Further, 99% by mass of ethyl alcohol is added while stirring until the concentration becomes 95% by mass, and dehydration washing is performed. When the precipitate obtained by centrifugation is dried under reduced pressure at a temperature of 60 to 70 ° C., 28 g of sodium chondroitin sulfate is obtained.
[0095]
The obtained sodium chondroitin sulfate was subjected to quantitative analysis of protein, nitrogen, and sulfur. The quantitative analysis of protein and the quantitative analysis of nitrogen and sulfur were the same as in Example 1.
[0096]
The determination as to whether or not the obtained sodium chondroitin sulfate was a high-purity product was the same as in Example 1.
[0097]
The analysis results of the obtained sodium chondroitin sulfate were as follows. The protein content was 1% by mass. Nitrogen was 2.8% by weight and sulfur was 6.2% by weight. From these analysis results, it was found that the chondroitin sulfate sodium obtained in this example was a highly purified product.
[0098]
Example 6
Nasal cartilage is collected from the salmon head and stored frozen at -20 ° C. 1 kg of frozen nasal cartilage is left overnight at room temperature to thaw. The thawed nasal cartilage is ground with a meat grinder. The water content in nasal cartilage is 850 g. 117 g of sodium chloride and 8 g of caustic soda (NaOH) are dissolved in 150 ml of water and added at a final temperature of 40 ± 2 ° C. for 2 hours so that the final concentration becomes 2 M aqueous sodium chloride (NaCl) and 0.2 N aqueous sodium hydroxide (NaOH). Treat with alkali. After completion of the alkali treatment, the pH is adjusted to 6 with acetic acid. Next, when 175.5 g of NaCl is added and dissolved so that the NaCl concentration becomes 5 M, the polymer protein is salted out. After addition of activated clay (10 g) for adsorption, diatomaceous earth was added and the mixture was filtered to obtain 1000 ml of a transparent filtrate. To 1000 ml of the filtrate, 1500 ml of ethyl alcohol is added to form a 55% by mass aqueous alcohol solution, and sodium chondroitin sulfate is separated and precipitated. After decanting, the precipitate was collected by centrifugation to obtain 205 g of crude sodium chondroitin sulfate. Since the obtained crude sodium chondroitin sulfate contains salt and a small amount of oil, the crude sodium chondroitin sulfate is dissolved in 700 ml of 70 ° C. hot water for desalination and removal of a small amount of oil, and 10 g of activated clay and diatomaceous earth are added. After filtration, 800 ml of a colorless and transparent aqueous solution of sodium chondroitin sulfate was obtained. Next, 1000 ml of ethyl alcohol is added to this aqueous solution to prepare a 50% by mass aqueous solution of ethyl alcohol, and sodium chondroitin sulfate is separated and precipitated. NaCl is dissolved and desalted in a 50% by mass aqueous solution of ethyl alcohol. The precipitate is collected by a decanter, washed with 80% by mass of ethyl alcohol, and centrifuged. Further, 99% by mass of ethyl alcohol is added while stirring until the concentration becomes 95% by mass, and dehydration washing is performed. The precipitate obtained by centrifugation is dried under reduced pressure at a temperature of 60 to 70 ° C. to obtain 27 g of sodium chondroitin sulfate.
[0099]
The obtained sodium chondroitin sulfate was subjected to quantitative analysis of protein, nitrogen, and sulfur. The quantitative analysis of protein and the quantitative analysis of nitrogen and sulfur were the same as in Example 1.
[0100]
The determination as to whether or not the obtained sodium chondroitin sulfate was a high-purity product was the same as in Example 1.
[0101]
The analysis results of the obtained sodium chondroitin sulfate were as follows. The protein content was below the detection limit. Nitrogen was 2.6% by weight and sulfur was 6.3% by weight. From these analysis results, it was found that the chondroitin sulfate sodium obtained in this example was a highly purified product.
[0102]
From the above, according to the present embodiment, the following is achieved.
In a conventional method for producing sodium chondroitin sulfate, proteins are reduced to peptides or amino acids by using proteolytic enzymes after alkali treatment of cartilage. After the crude sodium chondroitin sulfate was prepared, cation exchange resin treatment and dialysis treatment or continuous multi-stage ultrafiltration treatment was performed to remove peptides, amino acids, and low-molecular-weight proteins for purification. As described above, in the conventional method, purification was performed by a complicated process in the latter half of the manufacturing process.
[0103]
On the other hand, in the present embodiment, purification can be performed in the first half of the manufacturing process. Therefore, complicated operations such as cation exchange resin treatment, dialysis treatment, and continuous multi-stage ultrafiltration treatment are not required, and expensive equipment for the treatment is not required.
[0104]
【The invention's effect】
The present invention has the following effects.
Sodium chondroitin sulfate can be obtained by a simple method including a step of treating cartilage with an alkali and a step of salting out the treated cartilage.
[Brief description of the drawings]
FIG. 1 is a flowchart showing steps of a method for producing sodium chondroitin sulfate of the present invention.

Claims (15)

A method for producing sodium chondroitin sulfate, comprising the following steps.
(A) Step of alkali-treating cartilage (b) Step of salting out the alkali-treated one The method for producing sodium chondroitin sulfate according to claim 1, wherein the cartilage is fish cartilage. The method for producing chondroitin sodium sulfate according to claim 1, wherein the cartilage is salmon nasal cartilage. The method for producing sodium chondroitin sulfate according to claim 1, wherein the alkali is NaOH. The method for producing sodium chondroitin sulfate according to claim 4, wherein the alkali is in a range of 0.1 to 2N. The method for producing sodium chondroitin sulfate according to claim 1, wherein the alkali treatment is performed in the presence of NaCl. The method for producing sodium chondroitin sulfate according to claim 6, wherein the NaCl is in a range of 1 to 2M. The method for producing sodium chondroitin sulfate according to claim 1, wherein the salting out is carried out by NaCl. The method for producing sodium chondroitin sulfate according to claim 8, wherein the NaCl is in a range of 3 to 5M. The method for producing sodium chondroitin sulfate according to claim 1, wherein after the salting-out step, alcohol is added to the filtrate to precipitate sodium chondroitin sulfate. Sodium chondroitin sulfate obtained by treating cartilage with alkali and salting out the same. The chondroitin sodium sulfate according to claim 11, wherein the cartilage is fish cartilage. The chondroitin sodium sulfate according to claim 11, wherein the cartilage is salmon nasal cartilage. The sodium chondroitin sulfate according to claim 11, wherein the alkali is NaOH. The chondroitin sodium sulfate according to claim 11, wherein the salting out is carried out by NaCl.

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