JP5749067B2 - Proteoglycan production method - Google Patents

Description

  The present invention relates to a method for producing cartilage-type proteoglycans. More particularly, the present invention relates to a method for industrially producing a high-purity proteoglycan that can be efficiently applied to food, medicine, cosmetics, and the like from fish and the like in an environmentally friendly manner.

Proteoglycan is one of complex carbohydrates in which a long chain sulfated glycosaminoglycan is bound to a core protein. In glycoproteins, N-type sugar chains bound to asparagine, O-type sugar chains bound to threonine or serine are typical modifications of the protein, but in the case of proteoglycans, chondroitin sulfate, heparan sulfate, dermatan It is a complex biological material in which glycosaminoglycan sugar chains such as sulfuric acid are bound to proteins.
Until now, due to the complexity of its structure, a method for efficiently producing high-purity proteoglycans has not been established. A method for purifying cartilage-type proteoglycans using slightly acetic acid (Patent Document 1), using an alkaline solution However, only the extraction method (Patent Document 2) and the like have been reported.

However, since the method using acetic acid uses a large amount of 4% acetic acid solution and alcohol, many improvements are required for industrial application. In addition, there is a problem in that the possibility of obtaining a by-product bound with acetic acid is inevitable. Furthermore, this method does not always satisfy the extraction efficiency.
On the other hand, in the method using the above-mentioned alkali, in addition to protein denaturation / degradation, there is a concern about the detachment of proteoglycan sugar chains, and the amount of protein that is congested increases, so what is more suitable for obtaining high-purity proteoglycans? A step-by-step purification process is required.
In addition, in both methods, since there is no operation for removing contaminating lipids, the prepared proteoglycan preparation has a brown color, and there is a problem that a bad odor due to lipid oxidation occurs during storage.

JP 2002-069097 A International Publication No. 2007/094248 Pamphlet

  Therefore, an object of the present invention is to provide an environmentally friendly industrial production method that is more efficient than cartilage-type proteoglycans and that does not contaminate lipids and proteins.

  The present inventor studied the extraction of cartilage-type proteoglycan with various solutions to solve the above-mentioned problems, and found that proteoglycan can be efficiently extracted with a dilute solution of citric acid from salmon nasal cartilage and the like, and reached the present invention. .

That is, the present invention relates to the following invention.
(1) A step of immersing a fish sample containing a cartilage-type proteoglycan in a 0.05 % to 2 % by weight citric acid aqueous solution to extract the proteoglycan to obtain a proteoglycan extract, and the obtained proteoglycan extract A method for producing proteoglycan comprising a step of recovering proteoglycan.
(2) The method for producing proteoglycan according to (1), wherein the step of immersing and extracting is performed at room temperature.
(3) The method for producing proteoglycan according to (1) or (2), further comprising a step of removing the lipid by contacting the filtrate with a hydrophobic carrier or hydrophobic plastic beads after filtering and separating the proteoglycan extract.
(4) The method according to (3), further comprising the step of separating the proteoglycan extract containing a proteoglycan having a molecular weight of 1,000,000 or less and a proteoglycan of 100,000 or more after the step of removing the lipid by a molecular size selection membrane. Proteoglycan production method.
(5) The method for producing a proteoglycan according to (1) to (4) above, wherein the step of recovering proteoglycan from the proteoglycan extract includes a step of adding salt-saturated ethanol to the extract.
(6) The method for producing a proteoglycan according to (1) to (5) above, wherein the fish sample containing cartilage-type proteoglycan is a salmon head or a shark fin .
(7) Fish sample containing cartilage proteoglycan method of producing a proteoglycan according to the a salmon Hana軟bone (1) (5).

  The present invention provides an industrial production method capable of extracting and purifying proteoglycan in a very simple manner with a dilute citric acid solution in a high yield. Moreover, this invention provides the manufacturing method of the proteoglycan which removes a lipid efficiently by making an extraction filtrate contact a hydrophobic support | carrier or a hydrophobic plastic bead.

2 is a graph showing the change over time of the proteoglycan extract amount (citric acid 0.01 wt%) according to Example 1. 3 is a graph showing the change over time of the proteoglycan extract amount (citric acid 0.05 wt%) according to Example 1. 3 is a graph showing the change over time of the proteoglycan extract amount (citric acid 0.1 wt%) according to Example 1. 2 is a graph showing the change over time of the proteoglycan extraction amount (citric acid 0.5 wt%) according to Example 1. 2 is a graph showing the change over time of the proteoglycan extract amount (citric acid 1.0 wt%) according to Example 1. 2 is a graph showing the change over time of the proteoglycan extract amount (citric acid 2.0 wt%) according to Example 1. 3 is a graph showing the change with time of the proteoglycan extract amount (citric acid 4.0 wt%) according to Example 1. 4 is a graph showing the change over time in the amount of proteoglycan extracted by the acetic acid solution of Reference Example 1. 3 is an HPLC profile of proteoglycan obtained in Example 2. 2 is an electrophoresis profile of proteoglycan obtained in Example 2.

Hereinafter, the present invention will be described in detail.
The present invention includes a step of immersing a biological sample containing cartilage-type proteoglycan in an aqueous solution of 0.03% to 3% by weight of citric acid, extracting the proteoglycan to obtain a proteoglycan extract, and the obtained proteoglycan The present invention relates to a method for producing proteoglycan comprising a step of recovering proteoglycan from an extract.
That is, the first feature of the present invention is the extraction of proteoglycan from a biological sample containing cartilage-type proteoglycan with a dilute solution of citric acid.
Here, examples of biological samples containing cartilage-type proteoglycans include proteoglycans derived from cartilage tissues of fish, mollusks, birds and mammals. Among them, proteoglycans derived from the cartilage tissue of salmon heads and shark fins are preferably used, and particularly those derived from nasal cartilage tissue whose average weight is about 3% in the salmon head. Used.

  Here, the dilute solution of citric acid of the present invention refers to a 0.03% to 3% by weight aqueous solution of citric acid. When the citric acid concentration is less than 0.03% by weight, extraction takes time, and gelation, spoilage odor, and the like are likely to occur as the extraction time elapses. On the other hand, when the citric acid concentration exceeds 3% by weight, the extraction rate is rather slow. The citric acid concentration is preferably 0.05% to 2% by weight, more preferably 0.05% to 1% by weight, and still more preferably 0.1% to 0.5% by weight.

  In the extraction method using acetic acid described in Reference 1, an acetic acid concentration of 4% is optimal. However, in the present invention, a lower concentration of citric acid can be used, and the amount of alcohol used in the following recovery step It has the advantage that can be reduced.

  In the present invention, the extraction temperature may be from 0 ° C. to room temperature, but is preferably room temperature. Here, the room temperature is a temperature of 10 ° C. or higher and 30 ° C. or lower, preferably 15 ° C. to 25 ° C. In general, biological samples that are the subject of the present invention are processed at as low a temperature as possible because they are susceptible to spoilage. For example, in the extraction method using acetic acid described in Reference 1, extraction is performed at 4 ° C., and in the extraction using alkali metal described in Reference 2, an immersion temperature of 0 ° C. to 4 ° C. is more preferable. The present invention is epoch-making in that by using citric acid, immersion and extraction at room temperature are possible, and extraction efficiency can be increased.

  The extraction of proteoglycan with the citric acid solution of the present invention reaches saturation in about 40 hours, and is preferably 35 to 50 hours, but can be determined as appropriate. In addition, as the extraction method, a dipping method is used from the viewpoint of avoiding the narrow substances generated with stirring.

Next, in the present invention, by using the extracted proteoglycan with a hydrophobic carrier or hydrophobic plastic beads, it is possible to easily adsorb and remove lipid components that are considered to be mixed.
As the hydrophobic carrier, for example, a resin in which ODS (octadecylsilyl group) or octyl group is chemically bonded to silica or polymer can be used. The particle diameter of the carrier is preferably 5 μm to 10 μm. In addition, as the hydrophobic plastic beads, plastic beads such as polyethylene, polypropylene, polystyrene and the like can be used, and the size of the beads can be selected as appropriate, but generally those having an average diameter of 0.5 mm to 3 mm can be mentioned. Among the above, hydrophobic plastic beads can be suitably used from the standpoint of adsorption efficiency and economical viewpoint.

The proteoglycan extract obtained from the filtrate obtained by filtering and separating the hydrophobic carrier from which the lipid has been adsorbed and the hydrophobic beads usually contains about 2% to 4% of proteoglycan and its concentration is high. By precipitation, it can be recovered with good yield. Precipitation can be done efficiently by using centrifugation.
In addition, it is preferable to have the molecular weight adjustment and concentration step by the following ultrafiltration at least after the recovery step.

That is, the proteoglycan obtained from the above-described hydrophobic carrier from which the lipid has been adsorbed and the filtrate obtained by filtering off the hydrophobic beads is preferably made to have a molecular weight of 100,000 to 1,000,000 by molecular size selection membrane.
Specifically, proteoglycans precipitated by centrifugation are dissolved in distilled water, and giant proteoglycans and composites are removed through a membrane filter (removing molecules having a molecular weight of 1 million or more: 1000 k cut). Next, salt such as proteoglycan having a molecular weight of 100,000 or less, protein, and mineral is removed from the filtrate by a membrane filter having a cut of 100,000 (removing molecules having a molecular weight of 100,000 or less: 100 k cut).

Thereafter, the proteoglycan collected on the membrane is selectively collected by precipitation with the above-mentioned salt-saturated ethanol.
By the way, since proteoglycan and glycosaminoglycan sugar chains have negative charges of sulfate groups and carboxyl groups in the sugar chains, they repel each other and are difficult to precipitate. Therefore, when performing ethanol precipitation, it is possible to dramatically improve the efficiency of ethanol precipitation by adding sodium chloride, sodium acetate or the like to ethanol and neutralizing the negative charge of the sugar chain.
The proteoglycan thus obtained is washed several times with ethanol (preferably 100% ethanol) and then dried. Drying is performed by general vacuum drying or heat drying. When heating, a temperature of about 40 ° C. is preferably used.

  The proteoglycan can be purified by column chromatography using an anion exchange resin or gel filtration column.

  As described above, according to the present invention, proteoglycan can be extracted and purified in high yield from a biological sample such as salmon nasal cartilage by a very simple method. The advantages of the present invention are as follows.

According to the present invention, the conventional method of extracting at around 4 ° C. can be changed to room temperature, and extraction with a high yield can be performed in a shorter time. The capital investment for industrial production can be suppressed, and this can be reflected in the low price.
In the conventional extraction with acetic acid, acetic acid forms an ester with an ester bond with a carbohydrate and a hydroxyl group of protein, and this causes a return odor of acetic acid which becomes a problem after a lapse of time. Therefore, there is a problem in extraction at room temperature where the production of acetate ester is a concern and extraction for a long time.
Furthermore, in the present invention, the extraction efficiency is increased by changing the extraction method that has been conventionally stirred to a dipping method, and the separation operation of the impurities generated by the stirring can be omitted, which is a great merit in industrial operation.
In the conventional operation, there was a concern about lipid contamination until the end, but only the lipid can be easily adsorbed and removed easily by the treatment with the hydrophobic carrier or the hydrophobic plastic beads, and the subsequent operation can be performed smoothly.

The citric acid required for proteoglycan extraction can be reduced to about 1/3 of the volume of conventional acetic acid, and as a result, the proteoglycan is recovered by precipitation with about 1/5 of sodium chloride saturated ethanol compared to the acetic acid extraction method. It became possible. This is a great advantage both in terms of time, cost and environment.
Furthermore, it became possible to concentrate proteoglycan continuously by using a membrane filter having a molecular weight of 100,000 cut and 1 million cut.

In the present invention, in particular, since citric acid is used as an extraction solvent, there is no fear of the acetic acid odor (return odor), so the application of proteoglycans to foods and cosmetics has greatly expanded.
In addition, the preparation of purified proteoglycan by anion column chromatography makes it possible to prepare it as a uniform metal salt, which is important as a quality control method.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

1. HPLC analysis of proteoglycans:
The HPLC analysis conditions are as follows.
1) Column: TOSOH TSK-gel G5000PWXL + TSK-guard column PWXL
G5000PWXL: R0019
guard column PWXL: P0771 (manufactured by Tosoh Corporation)
2) Eluent: 0.2M NaCl
Flow rate: 0.5ml / min
Column temperature: 30 ° C
3) Detector: UV-VIS detector (215 nm)
4) Introduction volume: 50 μl (sample: 0.1% solution)

2. Ultrafiltration (molecular size selection membrane):
The ultrafiltration apparatus and filtration membrane are as follows.
1) Device name: Liquid pump MODEL7553 (Masterflex)
2) Ultrafiltration membrane
・ Million cut OMEGA Membrane Minimate TFF Capsule (Nippon Pole)
・ 100,000 cuts Omega Membrane Minimate TFF Capsule (Nippon Pole)

The nasal cartilage portion separated from the head of the chum salmon is stored frozen.
The frozen nasal cartilage is cut into 2 to 3 mm squares with a sharp blade. 0.01 wt%, 0.05 wt%, 0.1 wt%, 0.5 wt%, 1.0 wt%, 2.0 wt% and 4.0 wt% for 30 g of minced nasal cartilage 90 ml of each aqueous citric acid solution was used for immersion at 4 ° C. and room temperature (22-24 ° C.). 30 minutes later, 5 hours later, 12 hours later, 24 hours later, 36 hours later, 56 hours later and 72 hours later, uronic acid was quantified (carbazole sulfate method) by aliquoting the supernatant of each soaking solution and proteoglycan The extraction efficiency of was investigated. The results are shown in FIGS. 1-1 to 1-7.
As shown in FIG. 1-1, when the citric acid concentration was 0.01% by weight, the initial extraction efficiency was poor, and changes in physical properties such as gelation and turbidity of the extract were also observed. In addition, after 24 hours, obvious changes in physical properties such as generation of rot odor, color tone, and turbidity were observed.
On the other hand, as shown in FIG. 1-7, when the citric acid concentration was 4.0% by weight, the extraction efficiency was clearly reduced as compared to the case of citric acid concentration lower than that.
It can be seen that the extraction efficiency is better at room temperature than at low temperature (4 ° C.). Further, there was no difference in physical property change between the low temperature and room temperature.

Reference example 1

In Example 1, proteoglycan extraction efficiency was examined in the same manner as in Example 1 except that a 4.0 wt% acetic acid solution was used instead of the citric acid solution. The result is shown in FIG.
It can be seen that the citric acid solution can be treated at room temperature, and even a dilute solution of 0.05% by weight can achieve the same extraction rate as a 4% by weight acetic acid solution.

The salmon nasal cartilage cryopreserved in the same manner as in Example 1 was minced, and proteoglycan was extracted at room temperature with 60 ml of 0.1 wt% citric acid solution per 20 g of nasal cartilage.
24 hours later, after filtering through stain mesh (150 μm), filter paper (No. 2: pore size is 10 μm), filter paper (No. 5C: pore size is 1 μm) and glass fiber filter paper (GB-140: pore size is 0.4 μm) Then, 20 ml of polystyrene beads were added to the filtrate, and the mixture was gently stirred for about 30 minutes to adsorb the lipid, followed by filtration through a stainless mesh (150 μm).
The filtrate was applied to an ultrafilter equipped with a membrane having a molecular weight of 1 million cut to remove giant proteoglycan and its complex. Next, the filtered solution was applied to an ultrafilter equipped with a membrane having a molecular weight of 100,000 cut to obtain 5 ml of a concentrated liquid (residual liquid) from which small molecules were removed.
After adding 15 ml of salt saturated ethanol to the residual liquid and stirring for 30 minutes, the proteoglycan was separated by precipitation with a centrifuge (5 minutes, 1500 rpm).
After removing the supernatant, 5 ml of ethanol was added, stirred, and proteoglycan was precipitated with a centrifuge (1,500 rpm for 5 minutes). This washing operation was repeated twice. 50 ml of distilled water was added to the precipitated proteoglycan to obtain a clear solution. This solution was lyophilized to obtain 200 mg of proteoglycan fraction (Lot A).
FIG. 3 shows the extraction profile of the HPLC analysis of the proteoglycan sample obtained at this stage.

Next, the above operation was repeated to prepare lots B to D.
Each lot of proteoglycan was applied to a DEAE cellulose column, and extraction was performed with a concentration gradient of 0.2 to 1.0 M NaCl and 1.0 to 2.0 M NaCl. FIG. 4 shows the electrophoresis profile of each lot. The single peak obtained from the DEAE column was chondroitin sulfate, and the purified proteoglycan derived from salmon nasal cartilage was shown to be chondroitin sulfate proteoglycan. In the figure, HP represents heparin, ChS represents chondroitin sulfate, HS represents heparan sulfate, and HA represents hyaluronic acid.
The molecular weight was determined to be 450,000 from the maximum retention time of the HPLC peak using pullulan molecular markers.
As for the lipid component, proteoglycan was extracted and analyzed with a mixture of chloroform and methanol 2: 1 but could not be detected. The protein content was 0.51% by weight and the collagen content was 0.25% by weight.

50 g of dried whitefish shark fin cartilage was pulverized, 500 ml of 0.1 wt% citric acid was added, and the mixture was allowed to stand for 1 hour. The swollen fins were taken out, chopped, returned to the 0.1 wt% citric acid solution, and immersed and extracted for 18 hours.
After filtration through a nylon mesh (150 μm), the filtrate was No. 2, and no. Filtration through 5C filter paper and glass fiber filter paper (GB-140) gave 400 ml of filtrate. The filtrate was subjected to ultrafiltration of 1000 k cut, concentrated to 10 ml, and then concentrated by adding 30 ml of distilled water. This operation was repeated 3 times, and this was designated as 1000 k residual liquid (1).
The 1000 k filtrate was collected and collected to 450 ml. This was subjected to 100 k cut ultrafiltration. The operation of concentrating to about 10 ml, adding 30 ml of distilled water and filtering was repeated on the third floor to obtain 30 ml (2) of a 100 k concentrate. On the other hand, the 100 k filtrate was 500 ml (3).
Next, the residue of the previous 0.1% citric acid extract was extracted with 550 ml of 1% citric acid, and 25 ml of 1000 k residual liquid (4) and 1000 k filtrate were extracted in the same manner as the extraction with 0.1% citric acid. Yielded 26 ml (5) of 100k residual liquid and 600 ml (6) of 100k filtrate.
Three times the amount of salt-saturated ethanol was added to the fractions (1) to (6), and the mixture was allowed to stand at 4 ° C. for 24 hours. The precipitate was separated by centrifugation (10 minutes, 3000 rpm), and the collected precipitate was washed with 100% ethanol and then centrifuged (10 minutes, 3000 rpm). This washing and precipitation with ethanol was repeated three times. After these operations, the precipitate was collected, dried and weighed.
As a result, 385.9 mg ((1) + (4)) of proteoglycan having a molecular weight of 1000 k or more, 531.7 mg ((2) + (5)) of proteoglycan having a molecular weight of 100 to 1000 k, and 20.7 mg (( 3) + (6)) was obtained. The total proteoglycan having a molecular weight of 100 k or more was 917.6 mg, and the recovery rate from 50 g was 1.8%.

  INDUSTRIAL APPLICABILITY According to the present invention, there is provided an environmentally friendly industrial production method in which cartilage-type proteoglycan is more efficient and lipids and proteins are not congested. The proteoglycan obtained by the method of the present invention can be used as a raw material for health foods, supplements, pharmaceuticals and cosmetics.

Claims (7)

  A step of immersing a fish sample containing cartilage-type proteoglycan in a 0.05% to 2% by weight citric acid aqueous solution to extract the proteoglycan to obtain a proteoglycan extract, and recovering the proteoglycan from the obtained proteoglycan extract A process for producing a proteoglycan, comprising the step of:   The method for producing proteoglycan according to claim 1, wherein the step of immersing and extracting is performed at room temperature.   The method for producing a proteoglycan according to claim 1 or 2, further comprising a step of removing the lipid by contacting the filtrate with a hydrophobic carrier or hydrophobic plastic beads after filtering and separating the proteoglycan extract.   The method for producing a proteoglycan according to claim 3, further comprising a step of separating the proteoglycan extract containing a proteoglycan having a molecular weight of 1 million or less and a molecular weight of 100,000 or more after the step of removing the lipid by a molecular size selection membrane. . The method for producing a proteoglycan according to any one of claims 1 to 4 , wherein the step of recovering the proteoglycan from the proteoglycan extract includes a step of adding sodium chloride saturated ethanol to the extract. The method for producing a proteoglycan according to any one of claims 1 to 5 , wherein the fish sample containing a cartilage-type proteoglycan is a salmon head or a shark fin. The method for producing proteoglycan according to any one of claims 1 to 5, wherein the fish sample containing cartilage-type proteoglycan is salmon nasal cartilage.