JP2010124811A - Volume reduction extraction method of glycosaminoglycan and generation method of proteoglycan-containing precipitation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technology increasing workability and effectively extracting and isolating glycosaminoglycan and proteoglycan. <P>SOLUTION: There is provided a volume reduction extraction method of glycosaminoglycan, which comprises adding an alkaline aqueous solution to tissues derived from a living body to obtain an extraction liquid containing glycosaminoglycan, adding an acidic aqueous solution to the extraction liquid to generate precipitation to reduce volume, and exposing the precipitation to an acidic aqueous solution containing inorganic salts and adjusted its pH to be almost the pH in generation of the precipitation so as to elute glycosaminoglycan in the aqueous solution. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for reducing the volume of glycosaminoglycan and a method for producing a proteoglycan-containing precipitate, and more particularly to a technique for increasing extraction efficiency.

  Glycosaminoglycans (hereinafter referred to as GAG as appropriate) are also referred to as mucopolysaccharides, and those bound to the core protein are referred to as proteoglycans. Proteoglycans are widely present on the extracellular matrix and cell surface together with collagen and the like.

  In recent years, proteoglycans and glycosaminoglycans are substances that have been attracting attention in the fields of health foods, cosmetics, and medicine, and extraction techniques and purification techniques are being developed.

  However, the content of glycosaminoglycan varies depending on the organism and site, but 0.1 wt% to 0.05 wt% for the buns, 0.1 wt% to 0.05 wt% for the crow skin, 0 squid cartilage .4 wt% to 0.2 wt%, pork skin 0.3 wt% to 0.2 wt%, squid skin about 0.2 wt%, umbilical cord about 0.3 wt%, pig small intestine 0.2 wt% to 0.1 wt% %. In any case, since there is generally only a proportion of 0.05 wt% to 0.4 wt% of the living tissue, an efficient method for extracting proteoglycan and glycosaminoglycan is being sought. However, the nasal cartilage portion of the eyelid has a specific amount of 3 wt%.

  For example, in order to improve efficiency, a method using a raw material having a high content rate, specifically, a cow or fish cartilage as a raw material is known. And as the processing method, it passes through the extraction process by an alkali treatment, and the precipitation process by ethanol addition, and re-purifies as needed (patent documents 2 and 3). Moreover, about the proteoglycan, the technique of extracting and refine | purifying by an acid treatment is also developed (patent document 1).

JP 2001-172296 A Japanese Patent No. 3448710 JP 2002-69097 A

However, the conventional technique has the following problems.
Whether it is a glycosaminoglycan or a proteoglycan, it is finally precipitated with ethanol or the like, and repurified appropriately from this precipitate to increase the purity.

  Here, the precipitation treatment with ethanol generally requires an addition amount two to three times that of the target solution (extract solution in which glycosaminoglycan is dissolved). At this time, for example, when the raw material is extracted by acid treatment or alkali treatment, it is necessary to add 3 times to 10 times the weight or volume of the raw material acid or alkali. The amount of ethanol required for the above is 6 times to 30 times the amount based on the raw material, and there is a problem that the amount increases in a superimposed manner. In addition, since ethanol is not cheap, there is a demand to reduce the amount of use as much as possible.

  In addition, since glycosaminoglycan and proteoglycan are originally low in content, other proteins and amino acids are mixed in the precipitate, and multiple steps such as separation are required in the repurification (post-process) stage. There was also a problem. That is, conventionally, there is a problem that precipitation efficiency is low and a post process is complicated, and eventually purification efficiency is low.

  This invention is made | formed in view of the above, Comprising: It aims at providing the technique which isolate | separates and extracts glycosaminoglycan and proteoglycan efficiently, improving workability | operativity.

  In order to achieve the above-mentioned object, the volume reduction extraction method for glycosaminoglycan according to claim 1 obtains an extract containing glycosaminoglycan by adding an alkaline aqueous solution to a tissue derived from a living body, and this extraction By adding an acidic aqueous solution to the liquid to reduce the volume by producing a precipitate, and exposing the precipitate to an acidic aqueous solution containing an inorganic salt and adjusted to a pH approximately equal to the pH at the time of precipitation, glycosaminoglycan It is characterized by eluting in an aqueous solution.

  That is, since the invention according to claim 1 generates an intermediate precipitate and re-extracts glycosaminoglycan from this, ethanol and the like to be added thereafter can be greatly reduced, and the degree of purification can be significantly improved. It becomes. As will be described later, it is possible to reduce the volume of the intermediate precipitate to about ½ or less of the raw material.

  In the case of the conventional method in which a raw material (tissue derived from a living body) is treated with alkali and then a precipitate is immediately formed by adding ethanol, various proteins are entangled in the raw material, so the amount of alkali is inevitably It is necessary to add a large amount, for example, processing by adding 5 times or more of the raw material weight or volume, and 2 to 3 times the amount of this liquid, that is, 10 times to 15 times from the raw material. More ethanol is needed.

  On the other hand, when the glycosaminoglycan is purified from the intermediate precipitate, the processing efficiency is improved because it is once passed through a sieve, so that the treatment liquid is twice to three times the weight or volume of the intermediate precipitate (claims). In the case of the invention according to 1, an acidic aqueous solution in which an inorganic salt is dissolved may be added. Therefore, the amount of liquid of 2 to 3 times the amount of the precipitation that is less than half of the volume of the raw material is used as a reference, and it is only necessary to add 2 to 3 times the amount of ethanol. Then, purification becomes possible by adding about 2 to 5 times the amount of ethanol. In addition, since the pH is adjusted, the glycosaminoglycan can be released or extracted very efficiently by the action of the salt, and the purification efficiency is remarkably improved.

The tissue derived from a living body is derived from an animal and is preferably pretreated in a minced manner as appropriate in order to increase the processing efficiency. The addition of an alkaline aqueous solution means an alkali treatment, and the addition of an acidic aqueous solution means an acid treatment. Examples of the alkaline aqueous solution include aqueous solutions such as NaOH, Ca (OH) ₂ , Ba (OH) ₂ , KOH, Mg (OH) _{2 and the} like. Examples of the acidic aqueous solution include aqueous solutions of acetic acid, hydrochloric acid, nitric acid, perchloric acid, sulfuric acid, and the like. In addition, citric acid, hydroxycitric acid, galacturonic acid, malic acid, succinic acid, lactic acid, formic acid, fumaric acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid in aqueous solution can be mentioned. In addition, alkali concentration, acid concentration, and its addition amount can be adjusted suitably. Moreover, processing time, extraction time, and temperature shall be adjusted suitably. Moreover, it shall centrifuge or filter as needed, such as after producing | generating intermediate | middle precipitation.

  Further, including an inorganic salt means that the inorganic salt is dissolved, and the difference in pH is not large from an acidic aqueous solution adjusted to substantially the same pH, for example, pH is close to 7. For weak acids, 0.5 is not different, and where pH is close to 3, 1 or 2 is not different.

  Further, in the method for reducing the volume of glycosaminoglycan according to claim 2, an alkaline aqueous solution is added to a tissue derived from a living body to obtain an extract containing glycosaminoglycan, and a protease is added to the extract. Acidic aqueous solution in which protein is decomposed and then acidic protein is added, then an acidic aqueous solution is added to form a precipitate to reduce the volume, and this precipitate is adjusted to a pH substantially equal to that at the time of precipitation, including inorganic salts. The glycosaminoglycan is eluted in an aqueous solution by exposure to water.

  That is, the invention according to claim 2 is intermediate to the invention according to claim 1 by precipitating using a protein to which glycosaminoglycan easily binds or adheres after degrading the endogenous protein derived from living tissue. Further reduce the precipitation. As a result, the amount of ethanol required thereafter can be further reduced, and the degree of purification can be remarkably improved. As will be described later, the intermediate precipitation can be reduced to about 1/3 or less of the raw material.

  In addition, when adding protease, it shall carry out on optimal conditions suitably, and reaction time and a deactivation process shall be performed suitably. In addition, after protease treatment or after formation of an intermediate precipitate, centrifugation or filtration is performed as necessary. The acidic aqueous solution adjusted to substantially the same pH does not have a large difference in pH. For example, a weak acid having a pH close to 7 does not differ by 0.5, and the pH is close to 3. Means that 1 and 2 are not different.

  Further, the volume reduction extraction method for glycosaminoglycan according to claim 3 is the method for volume reduction extraction of glycosaminoglycan according to claim 2, wherein the acidic protein is soy protein, casein, albumin or gluten. It is characterized by being.

  That is, the invention according to claim 3 can provide a method using a protein that efficiently precipitates glycosaminoglycan.

  Further, the volume reduction extraction method of glycosaminoglycan according to claim 4 is the method of volume reduction extraction of glycosaminoglycan according to claim 1, 2 or 3, wherein the pH after adding the acidic aqueous solution is 5 It is characterized as follows.

  That is, the invention according to claim 4 can efficiently precipitate glycosaminoglycan having a large number of sulfate groups and carboxyl groups and being strongly negatively charged.

  Further, the volume reduction extraction method for glycosaminoglycan according to claim 5 is the volume reduction extraction method for glycosaminoglycan, wherein an alkaline aqueous solution is added to a tissue derived from a living body to obtain an extract containing glycosaminoglycan. Protease is added to the extract to degrade the protein, then basic protein is added to form a precipitate to reduce the volume, and the precipitate is exposed to an aqueous solution containing an inorganic salt, thereby allowing glycosami It is characterized in that noglycan is eluted in an aqueous solution.

  That is, the invention according to claim 5 is a glycosaminoglycan which is prepared by degrading endogenous protein derived from biological tissue and then precipitating it using a protein to which glycosaminoglycan easily binds or adheres, and does not adjust pH. Therefore, the intermediate precipitate can be further reduced in volume from the invention according to claim 1 and glycosaminoglycan can be easily re-extracted. As a result, it is possible to further reduce ethanol and the like necessary after simple pretreatment, and to significantly improve the degree of purification. As will be described later, the intermediate precipitation can be reduced to about 1/25 or less of the raw material. In addition, before adding basic protein, you may add suitably the process of adjusting pH and producing | generating precipitation, and removing this once.

  In addition, when adding protease, it shall carry out on optimal conditions suitably, and reaction time and a deactivation process shall be performed suitably. In addition, after protease treatment or after formation of an intermediate precipitate, centrifugation or filtration is performed as necessary.

  Further, in the volume reduction extraction method for glycosaminoglycan according to claim 6, an alkaline aqueous solution is added to a tissue derived from a living body to obtain an extract containing glycosaminoglycan, and an acidic aqueous solution is added to the extract. Removing the primary precipitate formed in the weakly acidic stage, adding basic protein to the residual liquid to reduce the volume by forming a precipitate, and exposing the precipitate to an aqueous solution containing an inorganic salt, The glycan is eluted in an aqueous solution.

  That is, the invention according to claim 6 can dramatically reduce the amount of intermediate precipitation with respect to the raw material to 1/40 or less, can extremely reduce the amount of ethanol required thereafter, and can significantly improve the degree of purification. In addition, it is not necessary to adjust the optimum conditions, and the working efficiency can be improved. The aqueous solution containing the inorganic salt is preferably acidic.

  The volume reduction extraction method of glycosaminoglycan according to claim 7 is the volume reduction extraction method of glycosaminoglycan according to claim 5 or 6, wherein the basic protein is protamine, polyarginine, polylysine, Or it is a histone, It is characterized by the above-mentioned.

  That is, the invention according to claim 7 can provide a method using a protein that efficiently precipitates glycosaminoglycan.

The volume reduction extraction method for glycosaminoglycan according to claim 8 is the volume reduction extraction method for glycosaminoglycan according to any one of claims 1 to 7, wherein the inorganic salt is NaCl, KCl. MgCl ₂ , CaCl ₂ .

That is, the invention according to claim 8 can be economically processed with an inexpensive raw material. In addition, since the inorganic salt is used, it is possible to suppress variation in isoelectric point, alteration of glycosaminoglycan, and mixing of impurities (miscellaneous components) as compared with the case of using an organic substance. In addition, Na ₂ SO ₄ , K ₂ SO ₄ , MgSO ₄ , or CaSO ₄ may be used. The addition amount is preferably 10 wt% or more in the case of an acidic aqueous solution, and preferably 5 wt% or more when a basic protein is used.

  Further, the volume reduction extraction method for glycosaminoglycan according to claim 9 is the volume reduction extraction method for glycosaminoglycan according to any one of claims 1 to 8, wherein the tissue derived from a living body is transformed into a fish. It is characterized by the residue generated during preparation.

  That is, the invention according to claim 9 uses not only the residue reduction but also the extraction of valuable materials because the raw material is the fish head, internal organs, skin and bones discarded at the fish processing plant. In addition, since it does not use cattle that are often shunned due to the BSE problem, it is also suitable for consumers.

  In the method for producing a proteoglycan-containing precipitate according to claim 10, an acidic aqueous solution is added to fish cartilage to obtain an extract containing proteoglycan, and a basic protein is added to the extract to obtain a precipitate containing proteoglycan. It is characterized by that.

  That is, the invention according to claim 10 generates an intermediate precipitate to enable efficient extraction of proteoglycan.

  The proteoglycan-containing precipitate generation method according to claim 11 is characterized in that, in the proteoglycan-containing precipitate generation method according to claim 10, the living body tissue is salmon nasal cartilage.

  In other words, the invention according to claim 11 uses shark nasal cartilage which is specifically large and has a high proteoglycan content (about 3 wt%) as a raw material, so that efficient proteoglycan purification is possible. Become.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide the technique which improves workability | operativity and isolate | separates and extracts glycosaminoglycan and proteoglycan efficiently.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In Embodiment 1, a technique for extracting and purifying glycosaminoglycan will be described, and in Embodiment 2, a technique for extracting proteoglycan will be described.

[Embodiment 1]
<Experimental example 1>
In Experimental Example 1, a method for extracting glycosaminoglycan from the heel head as a raw material will be described.
First, 670 g of the bun was minced with a food processor. To this, 3 liters of 0.5 M (mol / liter or less) NaOH aqueous solution was added and extracted overnight. The solution was filtered and acetic acid was added to the filtered alkaline extract until the final concentration was 5 vol% to form a composite precipitate. The precipitate was centrifuged to recover 350 g of intermediate precipitate.

  To the intermediate precipitate, 400 ml of a 5 vol% acetic acid solution containing 10 wt% NaCl and the same acid treatment as above was added, and glycosaminoglycan was extracted to the liquid side. Proteoglycans accompanied by glycansaminoglycans are considered to be present unless otherwise specified).

  Next, repurification was performed. Specifically, 800 ml of 2-fold amount of ethanol was added to the extracted liquid of glycosaminoglycan and left overnight at 4 ° C. to form a precipitate. The resulting precipitate was collected by centrifugation. This precipitate was washed with ethanol, ethanol was removed with a vacuum dryer, and finally 590 mg of a solid was obtained.

  Glycosaminoglycan has a repeating structure of disaccharides of uronic acid and amino sugar, and about 2 to 3 times the weight of uronic acid is the weight of glycosaminoglycan. Since the weight of uronic acid can be measured easily, it was decided to evaluate the purification of glycosaminoglycan with the weight of uronic acid. The amount of uronic acid in the recovered 590 mg of the solid was 246 mg. Therefore, it was possible to evaluate that the recovered 590 mg of the solid consisted almost of glycosaminoglycan.

  The re-purified weight (590 mg) is approximately 0.17 wt% of the intermediate precipitation amount (350 g). Considering that the glycosaminoglycan present in the raw material is 0.05 wt% to 0.1 wt%, it can be said that the purification efficiency is simply increased 2 to 3 times.

However, when an intermediate precipitate is generated, the following merits are also produced as compared with the conventional method.
First, in the conventional method, it is necessary to add 2 times the amount of ethanol to the approximately 5 times amount of alkaline aqueous solution (3 liters) added to the raw material (670 g), that is, 6 liters of ethanol. With the volume reduction effect of the intermediate precipitation (350 g), not only can the acidic aqueous solution to be originally added be reduced to about half, but so to speak, the primary purification is performed. Re-extraction is possible. That is, adding twice the amount of ethanol to this extract, ie, adding 800 ml of ethanol is sufficient. Therefore, in this method, the amount of ethanol can be reduced to 1/7 as compared with the conventional method.
Next, in the conventional method, other proteins are also precipitated, and it is necessary to further purify from the various precipitates. However, in this method, since the re-extraction is performed in the same manner as when the precipitate is formed, miscellaneous components other than the target are hardly mixed, and further purification treatment as in the conventional method becomes unnecessary as described above. .

  Therefore, as shown in Experimental Example 1, it can be said that when an intermediate precipitate is generated, the purification efficiency is increased in a superimposed manner.

<Experimental example 2>
Next, the pH dependence of glycosaminoglycan incorporated into the intermediate precipitate was examined.
First, the buns were minced and 10 g, in particular, 100 ml of 0.5N NaOH aqueous solution was added thereto, followed by refrigeration for 16 hours for extraction.
The solid was then filtered and the supernatant was removed by centrifugation. Acetic acid was added dropwise to the supernatant, and the change in the amount of precipitation was examined. FIG. 1 shows the relationship between the precipitation amount converted per 100 g of the bun and the pH. As shown in the figure, it was confirmed that precipitation suddenly occurred when acidified. When the pH is 4 or less, it can be confirmed that there is almost no change in the amount of precipitation and intermediate precipitation of about 30 mg occurs, so the volume reduction effect is about 1/3.

  Next, a solution obtained by re-extracting glycosaminoglycan from an intermediate precipitate in a solution in which NaCl was dissolved at 10 wt% at each pH was compared as an extraction amount from 100 g of buns using uronic acid as an index. The results are shown in FIG. The bar graph on the left side at each pH is obtained by using uronic acid as an index for glycosaminoglycan remaining in the supernatant at each pH when acetic acid is successively dropped into the alkaline extract to form an intermediate precipitate.

  As is apparent from the figure, when the pH is 5 or less, a large amount of glycosaminoglycan is contained in the precipitate, and when the pH is 4 or less, it can be confirmed that the pH is almost shifted to the intermediate precipitation side.

  From Experimental Example 1 and Experimental Example 2, it was confirmed that it was possible to improve the processing efficiency including subsequent purification by intermediate precipitation, and it was found that glycosaminoglycan could be almost extracted and purified. It was also confirmed that glycosaminoglycan has a property of binding or adhering to a protein that does not precipitate unless the pH is low. In addition, since glycosaminoglycan itself does not precipitate in an acidic aqueous solution, it can be said that the technique of selectively extracting by binding to or attaching to a protein is epoch-making.

<Experimental example 3>
Based on Experimental Example 1 and Experimental Example 2, it was studied to further improve the processing efficiency. In Experimental Example 1, the intermediate precipitation is ½ of the raw material, but the amount of precipitation is still large. Therefore, an attempt was made to decompose the extracted protein or the like with a protease, add an acidic protein, and then bind or attach a proteoglycan to the protein.

  First, the bun 150g was minced with a food processor. To this, 750 ml of 0.5 M NaOH aqueous solution was added and extracted overnight. The liquid was filtered to obtain 700 ml of an alkaline extract. The alkaline extract was neutralized with acetic acid, 100 ml was separated therefrom, 100 mg of actinase E (manufactured by Junsei Kagaku) was added, and the mixture was incubated at 60 ° C. for 24 hours for proteolysis. This reaction solution was treated at 100 ° C. for 10 minutes to inactivate the protease, and the supernatant was separated by centrifugation.

  To 7 ml of this supernatant, 87.5 mg of soy protein (manufactured by Wako Pure Chemical Industries, Ltd.) (previously dissolved in 2.5 ml of 0.1N NaOH aqueous solution) was added and stirred, and allowed to stand for 30 minutes. Thereafter, 500 μl of acetic acid was added to form a composite precipitate, which was further left for 30 minutes. This was centrifuged to recover 495 mg of intermediate precipitate. To this precipitate, 3 ml of a 5% acetic acid solution containing 10% NaCl was added to re-extract glycosaminoglycan.

  Two times the amount of ethanol was added to this extract and left overnight at 4 ° C., and the precipitate was collected by centrifugation. This precipitate was washed with ethanol, ethanol was removed by a vacuum dryer, and finally 2.7 mg of a solid substance was obtained. Since the uronic acid content of this solid was measured and found to be 812 μg, it was possible to evaluate that the obtained solid consisted almost of glycosaminoglycan.

  The re-purified weight (2.7 mg) is approximately 0.55 wt% of the intermediate precipitate (495 mg). Therefore, it can be said that the purification efficiency has increased by 6 to 11 times compared to the conventional method.

  In this case, since the intermediate precipitation (495 mg) is about 1/3 of the converted raw material (150 g × (100/700) × (7/100) = 1.5 g), it is more efficient than Experimental Example 1. It can be said that simple extraction has been realized. In addition, since protease is proteolyzed with protease, the precipitation of miscellaneous substances can be suppressed during precipitation with ethanol added thereafter.

<Experimental example 4>
Next, we decided to examine the types of acidic proteins other than soybean protein. Here, casein (isoelectric point: pH = about 4.6, manufactured by Wako Pure Chemical Industries, Ltd.), egg albumin (isoelectric point: pH = about 4.7, hereinafter referred to as albumin as appropriate), gluten (etc.). Electric point: pH = about 4-5, manufactured by Wako Pure Chemical Industries, Ltd.) and gelatin (isoelectric point: pH = about 4.5-7, manufactured by Wako Pure Chemical Industries, Ltd.) were examined. In addition, the isoelectric point of soybean protein (made by Wako Pure Chemical Industries, Ltd.) is pH = 4.2-4.5. For reference, basic proteins were also examined. Protamine (isoelectric point: pH = about 10-12) was used as the basic protein.

  After the same enzymatic decomposition treatment as in Experimental Example 3, the same amount (1 mg) of protein was added to the supernatant from which insolubles had been removed to obtain an intermediate precipitate, and glycosaminoglycan was added with an acetic acid aqueous solution containing 10% NaCl. The amount of uronic acid after extraction and ethanol treatment was measured. The results are shown in FIG. In the figure, the results when no protein was added are also shown.

  As is apparent from the figure, it was confirmed that the addition of acidic proteins such as casein, soy protein, albumin, and gluten has the effect of precipitating glycosaminoglycans together. Among these, it was confirmed that soy protein and albumin had high precipitation efficiency.

  In addition, when no protein was added, precipitation did not occur, and as a result, it was confirmed that the protein was properly decomposed by protease treatment, and that glycosaminoglycan was not precipitated alone in an acidic solution. In addition, when the protein is decomposed, precipitation occurs due to the added protein without mixing the impurities, leading to an increase in purification efficiency.

  Surprisingly, when protamine, a basic protein, was added, the amount of glycosaminoglycan extracted was higher than that of soybean protein, which had the highest extraction efficiency among acidic proteins. Therefore, in the following, first, the presence or absence of enzyme treatment and the optimum pH when protamine is added (Experimental Examples 5 and 6), comparison of the extraction efficiency of protamine and other basic proteins (Experimental Example 7), Comparison of extraction efficiency using an acidic protein and a basic protein under the optimum conditions (Experimental Example 9) was performed.

<Experimental example 5>
In Experimental Example 5, the alkaline extract was inactivated after protease treatment (pH was adjusted to remove the precipitate once), then protamine was added, and glycosaminoglycan was extracted from the obtained intermediate precipitate. Was evaluated by the amount of uronic acid. FIG. 4 is the amount of intermediate precipitate obtained from 80 mg of wharf. FIG. 5 is a diagram showing the relationship between the amount of uronic acid and pH when converted to 100 g of buns.

  As is clear from FIG. 4 and FIG. 5, it was found that when protamine is added, intermediate precipitation occurs to the same degree regardless of pH, and uronic acid to be re-extracted is also at the same level. Therefore, it has been shown that pH control is unnecessary when protamine is added, and operability can be improved. In particular, the amount of intermediate precipitation was 1/40 to 1/130, and it was confirmed that ethanol added thereafter could be dramatically reduced.

<Experimental example 6>
Next, the extraction result of glycosaminoglycan when protamine was added without protease treatment was examined. Acetic acid is added to the alkaline extract as in Experimental Example 2, and when it becomes acidic, many precipitates are generated, and in the case of adding protamine, it is difficult to depend on pH from the results of Experimental Example 5. It was decided to investigate when the pH was large (pH ≧ 6). FIG. 6 is the amount of intermediate precipitate obtained from 80 mg of wharf. FIG. 7 is a graph showing the relationship between the amount of uronic acid and pH when converted to 100 g of buns.

  First, as can be seen by comparing FIG. 6 and FIG. 4, it was found that the intermediate precipitation was reduced by protease treatment. Further, it was found that the re-extraction amount of glycosaminoglycan increases as the pH decreases even if the alkalinity is the same as a general tendency.

  From Experimental Example 5 and Experimental Example 6, it was found that when protamine was added after protease treatment, the volume reduction effect was extremely high, pH adjustment was unnecessary, and operability was improved.

<Experimental example 7>
Next, basic proteins other than protamine were examined. In the experiment, the alkaline extract of the bun was treated with protease (actinase E) under optimum conditions (optimum pH and optimum temperature), deactivated, and various basic proteins were then added to the supernatant from which insoluble matter was removed. Addition to form an intermediate precipitate. Thereafter, glycosaminoglycan was extracted from this intermediate precipitate with 10% NaCl water, and the amount of uronic acid was measured. The basic proteins used were polyarginine (isoelectric point: pH = 10.8, manufactured by MP Biomedical), polylysine (isoelectric point: pH = 9.8, manufactured by Wako Pure Chemical Industries), histone (isoelectric). Point: pH = about 11, manufactured by MP Biomedical Co., Ltd.), lysozyme (isoelectric point: pH = about 11, manufactured by Wako Pure Chemical Industries, Ltd.), and the added weight was the same amount.

  FIG. 8 is the amount of intermediate precipitate obtained from 40 mg of wharf. FIG. 9 is a diagram comparing the amount of uronic acid when converted to 100 g of wharf.

  As shown in FIG. 8, it was confirmed that the intermediate precipitation was caused by the addition of the basic protein, and the intermediate precipitation could be further reduced as compared with the case of the addition of the acidic protein. Further, as shown in FIG. 9, it was confirmed that approximately the same extent of glycosaminoglycan could be extracted.

<Experimental Example 8>
Next, experiments were conducted to change the type and concentration of inorganic salts.
In the experiment, first, the alkaline extract of the bun was treated with acetic acid in the same manner as in Experimental Example 1 to produce an intermediate precipitate, and this was treated with a solution having the same pH as that of the intermediate precipitate (approximately pH = 3.5). Thus, glycosaminoglycan was extracted with an aqueous solution of inorganic salt (NaCl, KCl, MgCl ₂ , CaCl ₂ ) having a predetermined concentration.

  FIG. 10 shows the relationship between the amount of glycosaminoglycan extracted in terms of the amount of uronic acid per 100 g of bun and the inorganic salt concentration. As is clear from the figure, it was confirmed that the extraction amount was almost constant at a concentration of 10 wt%, almost independent of the type of inorganic salt.

Next, in the same manner as in the case of using protamine in Experimental Example 3, the alkaline extract of the bun was treated with protease, and an intermediate precipitate was produced in the supernatant from which the insoluble matter was removed using protamine. Glycosaminoglycan was extracted with an aqueous solution of an inorganic salt (NaCl, KCl, MgCl ₂ , CaCl ₂ ) having a predetermined concentration in the same pH as the time (approximately pH = 7 because actinase E was used).

  FIG. 11 shows the relationship between the extracted amount of glycosaminoglycan converted to the amount of uronic acid per 100 g of bun and the inorganic salt concentration. As is clear from the figure, it was confirmed that the extraction amount was almost constant at a concentration of 5 wt%, almost independent of the type of inorganic salt.

<Experimental Example 9>
Next, we investigated the extraction efficiency when soy protein, albumin, and protamine were extracted to the maximum extent under the optimal conditions. That is, the alkaline extract is treated with protease and the insoluble matter is removed from the supernatant, and an optimal amount of protein is added. Soy protein and albumin are recovered by generating an intermediate precipitate at an optimum pH. The intermediate precipitate due to the addition was recovered and the glycosaminoglycan was re-extracted with a 10% NaCl solution at the pH at the time of precipitation. FIG. 12 is the amount of intermediate precipitate obtained from 80 mg of bun. FIG. 13 is a diagram comparing the amount of uronic acid when converted to 100 g of wharf.

  In FIG. 13, it can be said that the amount of glycosaminoglycan purified under the optimum conditions is the same level. However, when the intermediate precipitation in FIG. 12 is seen, the amount added is clearly smaller when the basic protein protamine is added. Moreover, it can be confirmed that the amount of intermediate precipitation is small and efficient.

  As described above, from the results of Experimental Examples 4 to 9, when protease treatment is performed, precipitation occurs when pH is not adjusted when basic protein is added, and the amount added is less than when acidic protein is used. In addition, it was confirmed that the amount of intermediate precipitation was small, so that it was efficient.

<Experimental example 10>
In the case of protease treatment, pH adjustment, temperature adjustment, treatment time adjustment, and deactivation treatment are required. Therefore, it was decided to seek an intermediate precipitation reduction method that is easier than protease treatment. As shown in FIG. 2, glycosaminoglycan is considered to adhere to or bind to the protein that precipitates in a strong acid atmosphere. Therefore, it was decided to generate an intermediate precipitate in two stages of weak acidity and strong acidity.

  First, 50 g of buns were minced with a food processor. To this, 250 ml of 0.5 M NaOH aqueous solution was added and extracted overnight. The liquid was filtered to obtain 240 ml of an alkaline extract. In particular, 24 ml of this alkaline extract was adjusted to pH 5.8 by adding acetic acid, and a precipitate was once generated at a weakly acidic stage. The solid part was removed by centrifugation, and a solution obtained by dissolving 62.5 mg of protamine in 1 ml of distilled water was added to form an intermediate precipitate, which was allowed to stand at room temperature for 30 minutes. Centrifugation was performed again to recover 113 mg of intermediate precipitate.

  To the intermediate precipitate, 5 ml of a 5 vol% acetic acid solution containing 10 wt% NaCl was added, and glycosaminoglycan was extracted to the liquid side.

  Next, repurification was performed. That is, 10 ml of 2-fold amount of ethanol was added and left overnight at 4 ° C. to form a precipitate. The resulting precipitate was collected by centrifugation. This precipitate was washed with ethanol, ethanol was removed by a vacuum dryer, and finally 5.6 mg of a solid substance was obtained. The uronic acid was measured and found to be 1.6 mg.

  In addition, since the intermediate | middle precipitation containing glycosaminoglycan is 113 mg and a raw material is 50 * 24/240 = 5g, the volume reduction effect is about 1/44. Therefore, this method can reduce the volume to the same extent as the volume reduction effect by protease, and can be said to be a method that substantially improves operability.

  In addition, although the example using a heel head was demonstrated in the above example, although it is not limited to this, glycosaminoglycan exists universally in a biological tissue although the content is small. Therefore, it can be said that it has become possible to provide a method capable of producing valuable materials using the head, bones, skin, and internal organs, which have been discarded as residues in the fish processing plant.

[Embodiment 2]
Next, a method for reducing the volume of proteoglycan will be described. In the experimental examples so far, it has been found that a basic protein typified by protamine is effective for extraction of glycosaminoglycan. Therefore, since glycosaminoglycan is bound to the core protein, proteoglycan was efficiently extracted using protamine. Here, the nasal cartilage of a frog with a track record of extraction was used.

<Example 11>
First, 16.3 g of vomeronasal cartilage was minced with a food processor. To this, 80 ml of 5% acetic acid was added and extracted for two days. When solid-liquid separation was performed, 67 ml of a colorless and transparent acetic acid extract was obtained. When 16.8 mg of protamine was added to 6.7 ml of this extract and stirred, a composite precipitate was obtained. After standing for 30 minutes, 104 mg of the precipitate was collected by centrifugation, and proteoglycan was extracted using 5 ml of 5% acetic acid solution containing 10% NaCl.

  Two times the amount of ethanol was added to this extract and left overnight at 4 ° C., and the precipitate was collected by centrifugation. The precipitate was washed with ethanol, and then the ethanol was removed with a vacuum dryer to obtain 16.7 mg of a solid. The uronic acid content of this solid was 6.7 mg.

  When protamine was used, precipitation occurred and the volume reduction effect was 0.104 / 1.63≈1 / 16, confirming that an efficient extraction process could be realized.

<Experimental example 12>
Next, the pH dependence of the precipitate formation when protamine was used was examined. Using acetic nasal cartilage as a raw material, an acetic acid extract was obtained in the same manner as in Experiment 11, and 200 μl of this solution was adjusted in pH and 500 μg of protamine was added to compare the amount of intermediate precipitation. The results are shown in FIG. In the figure, the amount of intermediate precipitation when 40 mg of nasal cartilage is used is shown. From the figure, it was confirmed that a pH of 8 or less is preferable because the amount of intermediate precipitate produced is reduced.

  In each pH, proteoglycan re-extracted with 300 μl of 10% NaCl solution was evaluated as the amount of uronic acid. The results are shown in FIG. As shown in the figure, it can be said that the extraction efficiency is better when the pH is smaller, but when protamine is used as in the first embodiment, the same result is obtained that the pH is not so much dependent.

<Experimental example 13>
Finally, using a protamine-like protein, that is, basic proteins polyarginine, polylysine, histone, and lysozyme, the amount of intermediate precipitate produced and the amount of proteoglycan extracted were compared.

  First, 200 .mu.l (supernatant) of acetic acid extract of vomeronasal cartilage was prepared, and 500 .mu.g of each protein was added thereto and left for 30 minutes. This was centrifuged, the intermediate precipitate was separated, and the weight was measured. Thereafter, proteoglycan was extracted with 300 μl of 10% NaCl aqueous solution, and the amount of uronic acid was measured. The results are shown in FIG. 16 and FIG. The results of intermediate precipitation with protamine under the same conditions are also shown.

  As is clear from FIG. 16, the amount of intermediate precipitate converted per 40 mg of nasal cartilage was almost the same, and showed the same tendency as the result of the wharf shown in FIG. 8 (alkali extraction). In addition, as shown in FIG. 17, it was confirmed that the amount of proteoglycan was also comparable except for the result of lysozyme. From the above, it was confirmed that the extraction of proteoglycan using a basic protein also confirmed the volume reduction effect, and that efficient extraction or purification was possible.

  According to the present invention, the volume can be reduced by the intermediate precipitation, and it can be said that the intermediate purification is performed, so that the workability is improved and the efficient extraction is possible. In addition, when extracting proteoglycan, sputum nasal cartilage is effective, but sputum nasal cartilage can also be used.

It is the figure which showed the amount of intermediate | middle precipitation accompanying the pH change by alkali extraction-> acid dripping using a bun. It is the figure which showed the re-extraction amount of the glycosaminoglycan by the inorganic salt solution in alkali extraction-> pH change by acid dripping-> intermediate precipitation-> acid treatment, and the same pH as a soot. It is the figure which showed the re-extraction amount of the glycosaminoglycan by the inorganic salt solution in the same pH as alkali extraction-> enzyme treatment-> acid protein addition-> acid treatment-> intermediate precipitation-> acid treatment using a bun. It is the figure which showed the amount of intermediate | middle precipitation obtained by adding protamine in alkaline extraction-> enzyme treatment-> each pH using a bun. It is the figure which showed the re-extraction amount of the glycosaminoglycan by the inorganic salt solution in the pH at the time of alkali extraction-> enzyme treatment-> protamine addition in each pH-> intermediate precipitation-> protamine addition using a bun. It is the figure which showed the amount of intermediate | middle precipitation obtained by adding protamine in alkaline extraction-> pH adjustment and precipitation removal-> each pH (base side) using a bun. Using buns, alkaline extraction → pH adjustment to remove precipitates → Protamine addition at each pH (base side) → Intermediate precipitation → Re-extraction amount of glycosaminoglycan by inorganic salt solution at pH at the time of protamine addition FIG. It is the figure which showed the amount of intermediate | middle precipitation obtained by adding alkali extraction-> enzyme treatment-> various protamine-like proteins using a bun. It is the figure which showed the re-extraction amount of the glycosaminoglycan by the inorganic salt solution in pH at the time of alkali extraction-> enzyme treatment-> addition of various protamine-like proteins-> intermediate precipitation-> protamine addition using a bun. It is the figure which evaluated the extraction amount by the inorganic salt density | concentration at the time of re-extracting glycosaminoglycan from the intermediate | middle precipitation in the case of alkali extraction-> acid treatment using a bun. It is the figure which evaluated the extraction amount by the inorganic salt density | concentration at the time of re-extracting glycosaminoglycan from the intermediate | middle precipitation in the case of alkali extraction-> enzyme treatment-> addition of protamine using a bun. It is the figure which compared the amount of intermediate | middle precipitation in optimal conditions, when adding protein after enzyme-treating an alkaline extract using a bun and obtaining intermediate | middle precipitation. It is the figure which compared the maximum extraction amount of the glycosaminoglycan in optimal conditions, when adding protein after enzyme-treating an alkaline extract using a wharf and obtaining intermediate | middle precipitation. It is the figure which showed the amount of intermediate | middle precipitation obtained by adding protamine in acid extraction-> each pH using vomeronasal cartilage. It is the figure which showed the amount of proteoglycans when re-extracting proteoglycan with the inorganic salt solution of the same pH from the intermediate | middle precipitation obtained by adding protamine in acid extraction-> each pH using nasal cartilage. It is the figure which showed the amount of intermediate | middle precipitation obtained by adding various basic proteins to an acid extraction solution using vomeronasal cartilage. It is the figure which showed the amount of proteoglycans at the time of re-extracting proteoglycan by adding inorganic salt solution from the intermediate | middle precipitation obtained by adding various basic proteins to an acid extraction solution using nasal cartilage.

Claims (11)

  An alkaline aqueous solution is added to a tissue derived from a living body to obtain an extract containing glycosaminoglycan, an acidic aqueous solution is added to the extract to generate a precipitate, and the volume is reduced. A method for volumetric extraction of glycosaminoglycan, wherein glycosaminoglycan is eluted in an aqueous solution by exposure to an acidic aqueous solution adjusted to substantially the same pH as that at the time of production.   An alkaline aqueous solution is added to a tissue derived from a living body to obtain an extract containing glycosaminoglycan, a protease is added to the extract to decompose the protein, and then an acidic protein is added, followed by addition of an acidic aqueous solution to precipitate. The glycosaminoglycan is eluted in an aqueous solution by exposing the precipitate to an acidic aqueous solution containing an inorganic salt and adjusted to a pH substantially equal to the pH at the time of precipitation. To reduce the volume of glycosaminoglycan.   The method for volumetric extraction of glycosaminoglycan according to claim 2, wherein the acidic protein is soy protein, casein, albumin, or gluten.   The method for volumetric extraction of glycosaminoglycan according to claim 1, 2 or 3, wherein the pH after addition of the acidic aqueous solution is 5 or less.   An alkaline aqueous solution is added to a tissue derived from a living body to obtain an extract containing glycosaminoglycan, protease is added to the extract to degrade the protein, and then basic protein is added to generate a precipitate to reduce the amount. A method for reducing the volume of glycosaminoglycan, wherein the precipitate is exposed to an aqueous solution containing an inorganic salt to elute the glycosaminoglycan into the aqueous solution.   An alkaline aqueous solution is added to a tissue derived from a living body to obtain an extract containing glycosaminoglycan, an acidic aqueous solution is added to the extract to remove a primary precipitate generated in a weakly acidic stage, and a basic protein is added to the remaining solution. The volume reduction of glycosaminoglycan is characterized in that the glycosaminoglycan is eluted in an aqueous solution by adding a solution to form a precipitate and reducing the volume and exposing the precipitate to an aqueous solution containing an inorganic salt. Extraction method.   The method for volumetric extraction of glycosaminoglycan according to claim 5 or 6, wherein the basic protein is protamine, polyarginine, polylysine, or histone. Inorganic salts, NaCl, _KCl, MgCl 2, glycosaminoglycans volume reduction extraction method glycans according to any one of claims 1 to 7, characterized in that the CaCl _2.   The method for volumetric extraction of glycosaminoglycan according to any one of claims 1 to 8, wherein the tissue derived from the living body is a residue generated when the fish is prepared.   A method for producing a proteoglycan-containing precipitate, wherein an acidic aqueous solution is added to fish cartilage to obtain an extract containing proteoglycan, and a precipitate containing proteoglycan is obtained by adding a basic protein to the extract.   The method for producing a proteoglycan-containing precipitate according to claim 10, wherein the living body tissue is salmon nasal cartilage.

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