JP2020127397A - Method for producing proteoglycan - Google Patents

Abstract

To produce proteoglycan having excellent physiological effect efficiently and high-purity from a cartilage tissue of an animal.SOLUTION: A method for producing proteoglycan includes: a process of extracting proteoglycan from a cartilage tissue of an animal in acid solution of pH5.5 or less in the presence or absence of 1 mass% or less of protease with respect to the total amount and obtaining crude extract containing proteoglycan; and a process of removing lipid and substance having the molecular weight of 50,000 or less from the crude extract to refine proteoglycan.SELECTED DRAWING: Figure 1

Description

TECHNICAL FIELD The present invention relates to a method for producing proteoglycan from animal cartilage tissue in a more efficient and highly pure manner.

Proteoglycan (hereinafter sometimes referred to as “PG”) is a glycoprotein obtained by covalently binding several to several tens of glycosaminoglycans such as chondroitin sulfate and keratan sulfate to one core protein, which is extracellular. As one of the matrices, it is widely distributed in the body such as skin and cartilage. PG in cartilage forms an aggregate together with collagen and hyaluronic acid, and a typical cartilage-type PG is called aggrecan. Aggrecan has a large amount of glycosaminoglycan sugar chains bound to the core protein, and has a binding region for hyaluronic acid and a link protein on its N-terminal side.

Glycosaminoglycans have a long straight chain structure without branching, have a large number of sulfate groups and carboxyl groups, and thus are negatively charged, and take an elongated shape due to their electric repulsive force. In addition, due to the water affinity of sugar, it retains a large amount of water and has the unique functions of cartilage, such as elasticity and resistance to impact. Furthermore, it has been clarified that PG has many physiological functions such as an anti-inflammatory action, a hyaluronic acid synthesis promoting action, and an epidermal growth factor (EGF)-like action, and it is expected to be applied to foods and cosmetics.

So far, methods for efficiently producing high-purity PG have been studied. For example, a method of extracting PG from salmon nasal cartilage using acetic acid (see Patent Document 1) and a method of extracting PG by controlling the extraction temperature and stirring speed of an acetic acid solution (see Patent Document 2) are known. There is. In addition, a method of extracting PG using an aqueous citric acid solution (see Patent Document 3), a method of immersing in an aqueous solution of saponin or sucrose fatty acid ester or a mixture thereof (see Patent Document 4), and the like have been reported. .. On the other hand, a low-molecular-weight PG obtained by treating crudely purified PG with a proteolytic enzyme has excellent collagen gel contraction promoting effect and anti-glycation effect, and has a skin-pulling effect (see Patent Document 5), and salmon nasal cartilage PG There is also a report of a tyrosinase activity inhibitor containing a low-molecular-weight PG obtained by digestion with proteolytic enzyme actinase E as an active ingredient (see Patent Document 6).

However, securing and preserving animal cartilage tissue, which is a raw material of PG, is not always easy, and a more efficient PG extraction and purification method than the conventional method is desired. In addition, PG existing in the extracellular matrix of cartilage and skin maintains tissues by forming an association structure with collagen and hyaluronic acid in vivo, but what kind of form is a huge PG extracted? It is difficult to analyze whether or not this is the case, and the relationship between the actual state of PG molecules purified by conventional methods and the various physiological functions of PG known so far has not been sufficiently clarified. For example, in Non-Patent Document 1, when PGs of different molecular weights extracted and fractionated from nasal cartilage of salmon were examined for physiological effects by oral administration, it was found that the molecular weight was 40 as compared with low molecular weight PGs degraded by protease. It has been reported that more than 10,000 high molecular weight PGs exhibit more effective anti-aging effects on the skin.

Goto, M.; , Et al. , Anti-agging effects of high molecular weight proteoglycan from salmon nasal cartridge in hairless mice. INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 29: 761-768, 2012

Japanese Patent No. 3731150 Japanese Patent No. 6317053 Japanese Patent No. 5749067 Japanese Patent No. 6006545 JP, 2017-155004, A JP, 2018-127423, A

The present invention has been made under such circumstances, and an object thereof is to provide a method for efficiently and highly producing proteoglycan having excellent physiological action from animal cartilage tissue. Is.

In order to solve the above problems, the present inventors, in the step of extracting proteoglycan from animal cartilage, as a result of examining the extraction efficiency using various acidic solutions, in the presence of a specific acidic solution or a predetermined concentration of protease It was found that proteoglycan can be efficiently and highly purified from cartilage tissue by performing extraction with the above-mentioned method, and the present invention has been completed.

That is, the present invention includes the following embodiments.
(1) A proteoglycan-containing crude extract is obtained by extracting proteoglycan from animal cartilage tissue in an acidic solution having a pH of 5.5 or less in the presence or absence of a protease of 1% by mass or less based on the total amount of the solution. A process for producing proteoglycan, which comprises the steps of: removing a lipid and a substance having a molecular weight of 50,000 or less (more preferably a molecular weight of 100,000 or less) from the crude extract to purify the proteoglycan.
(2) The method for producing proteoglycan according to (1), wherein the cartilage tissue is a nasal cartilage tissue of a salmon head.
(3) The method for producing a proteoglycan according to (1) or (2), wherein the protease is an acidic protease and the content in the acidic solution is 0.001% by mass to 0.5% by mass.
(4) The method for producing proteoglycan according to any one of (1) to (3), wherein the acidic solution is an aqueous solution containing at least one selected from the group consisting of acetic acid, citric acid, malic acid, and ascorbic acid. ..
(5) The acidic solution is acidic electrolyzed water or carbonated water, or an aqueous solution obtained by adding at least one selected from the group consisting of acetic acid, citric acid, malic acid, and ascorbic acid to these (1) to (3). The method for producing a proteoglycan according to any one of 1.
(6) The concentration of at least one selected from the group consisting of acetic acid, citric acid, malic acid, and ascorbic acid in the acidic solution is 0.1% by mass to 1% by mass (1) to (5). The method for producing a proteoglycan according to any one of 1.
(7) The method for producing a proteoglycan according to any one of (1) to (6), wherein the proteoglycan is an aggrecan having a mass average molecular weight of 400,000 to 1,000,000.

According to the method for producing proteoglycan of the present invention, proteoglycan having excellent physiological action can be efficiently and highly purified from animal cartilage tissue.

FIG. 1 shows the result of HPLC analysis of the proteoglycan obtained in Production Example 1. FIG. 2 shows the results of HPLC analysis of the proteoglycans obtained in Production Example 2.

In the present embodiment, as the material for extracting proteoglycan, for example, cartilage tissue, muscle tissue or skin tissue of fish, molluscs, birds or mammals can be used, and among them, cartilage tissue is preferable. The cartilage tissue used in this embodiment may be fish, molluscs, birds or mammals, especially their disposal site. In the present specification, the cartilage tissue is not limited to only cartilage, but may be a tissue around the cartilage, such as bone, muscle tissue, skin tissue, or the like.

In the present embodiment, from the viewpoint of availability, cost, and the like, for example, those derived from the nasal cartilage tissue contained in the head of salmonid fish are preferably used, for example, in the coast of Aomori prefecture or the coast of Hokkaido. The head that is ejected can be used when the caught salmon (mainly white salmon) is processed as various processed products.

In the present embodiment, in addition to salmon, trout, rays, sharks, fish-derived cartilage tissues such as cod, squid, mollusk-derived epidermis such as octopus, bird-derived cartilage tissue such as chicken, and mammals such as cows and whales. Derived cartilage tissue can also be used.

The acidic solution used in the present embodiment is acidic electrolyzed water, an acidic aqueous solution containing an inorganic acid such as carbonated water, or an acidic aqueous solution containing an organic acid such as acetic acid, citric acid, malic acid, and ascorbic acid. is there. The pH of the acidic aqueous solution is preferably 5.5 or less, more preferably the acidic aqueous solution having a pH of 2 to 4. In addition to these, the acidic solution may be an aqueous solution containing lactic acid, tartaric acid, succinic acid, phosphoric acid, gluconic acid, fumaric acid, glutaric acid, adipic acid, phytic acid, hydrochloric acid, sulfuric acid and the like. In a different embodiment, the acidic aqueous solution is a combination of two or more of these inorganic acids and organic acids. For example, it is a combination of acidic electrolyzed water and acetic acid or citric acid. The concentration of the organic acid in the acidic solution is appropriately selected depending on the purpose of use of the proteoglycan. For example, when acetic acid is used as the organic acid, the lower limit of the concentration is 0.01% by mass, preferably 0.1% by mass, and when acetic acid is used as the organic acid, the upper limit of the concentration is 10% by mass, preferably It is 5% by mass, more preferably 1% by mass.

The extraction process using these acidic solutions can be performed at a temperature of 1 to 60° C., for example. Since the extraction time can be further shortened and the extraction efficiency can be further increased by raising the temperature to some extent, the lower limit of the temperature relating to the extraction step using this acidic solution is preferably 4°C, more preferably 20°C. On the other hand, the upper limit of the temperature involved in the extraction step with this acidic solution is preferably 55° C., more preferably 50° C., so that proteoglycan can be efficiently extracted as a glycoprotein complex with almost no decomposition. And more preferably 40°C.

The extraction step is performed under the condition that the acidic solution is brought into fluid contact with the entire cartilage surface, and the flow rate of the acidic solution at this time is preferably 0.04 to 10.0 cm/sec. More preferably, the flow rate is 0.04 to 5.0 cm/sec. This flow is not only horizontal circular movement by a stirrer or mixer, but also vertical direction by a method of circulating a liquid, and a certain direction such as a liquid flowing from one side to the other side after placing cartilage tissue. However, the extraction efficiency can be improved by always convection of the liquid. The step in which the acidic solution is brought into fluid contact with the cartilage tissue may be performed for a sufficient time to extract the proteoglycan at a desired yield or yield, and the step may be performed intermittently at a predetermined time continuously during the extraction step. Or as part of the extraction process. This fluid contact time can be appropriately set according to the extraction temperature and the flow rate of the acidic solution. Since the solution after the extraction contains a large amount of the residue after extracting the proteoglycan, it is preferable to remove these by filtration, centrifugation or other methods.

In this extraction step, it is preferable that the acidic protease coexist at a concentration of 1% by mass or less with respect to the total amount of the acidic solution. If the concentration of the acidic protease in the acidic solution exceeds 1% by mass, the extracted proteoglycan molecule may be further decomposed to lower the molecular weight, and the production cost increases, which is not preferable. Therefore, the upper limit of the amount of acidic protease added to the acidic solution is more preferably 0.5% by mass, and even more preferably 0.1% by mass. On the other hand, the lower limit is preferably 0 mass% or more, more preferably 0.0001 mass% or more, and further preferably 0.001 mass% or more. The acidic protease in the present specification is a proteolytic enzyme originating from plants, animals or bacteria, and is particularly limited as long as it has an optimum pH for proteolytic activity (pH exhibiting maximum activity) of 6.0 or less. However, it is preferable that the optimum pH for proteolytic activity is 2.0 or more and 5.0 or less because it works in an acidic solution. Deactivation at a pH of 7.0 or higher is preferable for controlling the decomposition reaction. If the optimum pH for proteolytic activity is less than 2.0, there is a problem that the denaturation of the protein is large and it is difficult to control the decomposition reaction.

As these acidic proteases, acidic proteases such as endopeptidases are known, and particularly proteolytic enzymes originating from Aspergillus oryzae or Aspergillus niger are mentioned. As this proteolytic enzyme, a commercially available one can be used. Commercially available proteolytic enzymes include the product name "Nurase F3G" (manufactured by Amano Enzymes Inc.), the product name "Protease M "Amano" SD" (manufactured by Amano Enzymes Inc.), and the product name "Morcin F" (Kikkoman Bio). Chemifa), product name "Sumiteam AP" (manufactured by Shin Nippon Chemical Industry Co., Ltd.), product name "Denapsin 2P" (manufactured by Nagase Chemtex), product name "Grind Amill PR59" (manufactured by Danisco Japan), product name " Orientase AY" (manufactured by HBI), product name "Tetolase S" (manufactured by HBI), product name "Brewers Clarex" (manufactured by DSM Japan), product name "Protease YP-SS" ( Yakult Pharmaceutical Co., Ltd.) and other acidic proteases.

Next, the proteoglycan extract solution is simply adsorbed and removed by using powdered cellulose and/or an oil-absorbing mat or the like to remove lipid components which are considered to be mixed. The proteoglycan obtained from the liquid after lipid removal is subjected to solid-liquid separation with a separation membrane or the like having an appropriate molecular weight cutoff to recover the extract. In this operation, if a separation membrane of 50,000 or more is used, low molecular weight contaminants (collagen etc.) can be removed from the liquid phase and the purity of proteoglycan can be increased. It is preferable to use 100,000 or more separation membranes. Further, the obtained fractionated liquid may be made into a solid by using a vacuum freeze dryer. Alternatively, it can be dried with a spray dryer to obtain a powdery solid content.

The proteoglycan thus obtained is a typical proteoglycan derived from cartilage and is called aggrecan. Aggrecan binds with a large number of glycosaminoglycans and forms a macromolecule because it aggregates with hyaluronic acid and link protein, but the aggrecan obtained by the method of the present embodiment is 400,000 to 1,000,000 (preferably 400,000). To 900,000, more preferably 400,000 to 700,000, and even more preferably 400,000 to 600,000), it is an intact proteoglycan in which the core protein portion is not degraded and is not degraded. Conceivable. On the other hand, since the binding with other proteins and the aggregates are removed, the purity of proteoglycan is improved, and it is speculated that a complete proteoglycan molecule in which bioactivity is more easily expressed can be obtained. In the present specification, the mass average molecular weight refers to the pullulan-equivalent mass average molecular weight when performing gel filtration chromatography (also referred to as Gel Filtration Chromatography, GFC) analysis. The method for measuring the mass average molecular weight is not particularly limited, but as an example, pullulan, dextran, polyethylene glycol or the like having a known molecular weight is used, and the differential refraction detector is used at an arbitrary concentration of 2 or more points. Create a calibration curve. Then, the sample (concentration is arbitrary) is measured, and the relative molecular weight (pullulan, dextran, or polyethylene glycol-equivalent molecular weight) is generally calculated from the retention time using the above calibration curve.

Furthermore, using the proteoglycan obtained in the present invention, as a result of evaluating fibroblast proliferation action, collagen gel contraction promoting action and the like, in any test using conventional acetic acid dip-extracted proteoglycan at low temperature and It was confirmed that the same or higher effect was obtained, and that it had a higher purity than the proteoglycan obtained by the conventional method. Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

[Production Example 1]
400 g of nasal cartilage extracted from the head of a chum salmon frozen and stored at −30° C. was prepared as a starting material. 2 g of the starting material and Protease M (Amano) SD were added to 2000 g of a 0.4% by mass acetic acid aqueous solution, and the mixture was extracted at an extraction temperature of 50° C. for 24 hours.

The temperature of this extract was raised to 80° C. and heated for 30 minutes to inactivate the protease, and then the eluate was filtered through a stainless steel mesh (150 μm) to remove insoluble matter. Next, after the oil in the liquid upper layer was adsorbed with an oil absorption mat (made by Maeda Kosen Co., Ltd., trade name “Oil Adsorption Sheet SP-1300N(DX)”, powdered cellulose (made by Nippon Paper Industries Co., Ltd., trade name “KC Flock W” -400G") was added and the mixture was stirred for 30 minutes and then filtered. The filtrate was concentrated using a hollow fiber membrane with a molecular weight cut-off of 50,000 until the liquid volume was reduced to 1/10. Finally, 112 g of concentrated solution (pH 6 to 7) was obtained, and the obtained concentrated solution was freeze-dried to obtain about 8.6 g of proteoglycan.

[Production Example 2]
Extraction and purification were carried out under the same conditions as in Production Example 1 except that the concentration of the acetic acid aqueous solution used in Production Example 1 was changed to 4% by mass to obtain about 7.7 g of proteoglycan.

(Quantification of proteoglycan)
About 1 g of the freeze-dried product obtained in Production Examples 1 and 2 was precisely weighed, and a phosphate buffer (pH 6.8) was added to make exactly 10 mL, to give a sample solution. After passing each sample through a 0.45 μm membrane filter, HPLC was performed under the following operating conditions to determine the amount of proteoglycan from the calibration curve of the standard product. In addition, the preparation of the calibration curve is the same as the sample, since the proteoglycan standard product (derived from salmon nasal cartilage, manufactured by Wako Pure Chemical Industries, Ltd.) was dried for 3 hours with a room temperature vacuum desiccator (silica gel) and then sampled. A standard solution for preparing a calibration curve was prepared by dissolving in a phosphate buffer solution. Further, the molecular weight at the peak top was determined from a calibration curve prepared using Shodex STANDARD P-82 (manufactured by Showa Denko KK) as a molecular weight marker.

Operating conditions Analyzer: HPLC analyzer Detector: Differential refractive index detector (RID-10A manufactured by Shimadzu Corporation)
Column: Gel filtration column (TSKgel G5000PWXL manufactured by Tosoh Corporation)
Column temperature: 40°C
Sample injection volume: 50 μL
Mobile phase: phosphate buffer (pH 6.8)
Flow rate: 0.5 mL/min

The results are shown in FIGS. 1 and 2. 1 is a sample obtained in Production Example 1, and FIG. 2 is an analysis result of the sample obtained in Production Example 2. It is shown that all are highly pure proteoglycans consisting of a single peak. The molecular weight of the peak top calculated from the retention time of the column was about 460,000 in FIG. 1 and about 480,000 in FIG.

[Production Examples 3 to 12]
Approximately 40 g of a sliced salmon nasal cartilage similar to that used in Production Example 1 and the various extracts shown in Table 1 were placed in a 250 ml container and sealed. This was incubated in a 37° C. water bath for 72 hours with gentle agitation. After the completion of the extraction, each extract was filtered through a 100-mesh sieve, treated with an oil adsorption mat, and further subjected to KC floc filtration, and the filtrate was freeze-dried. The mass of the recovered freeze-dried powder and the amount of PG quantified by the same method as in Production Example 1 were measured to determine the PG yield and average molecular weight. The results are shown in Table 1. The electrolyzed water used as the extract was ORP water acidic ionized water (Baibai bacteria, manufactured by Yamashita Takaku Co., Ltd.), and the carbonated water was Iga natural water, carbonated water (manufactured by Japan Sangalia Co., Ltd.).

From the results of Table 1, it was found that PG of high purity can be obtained although the yield is slightly low even when only acidic electrolyzed water or only carbonated water is used as the extract. When 5% of citric acid was added to acidic electrolyzed water or carbonated water, the solid content yield increased but the purity of the obtained PG decreased. On the other hand, when malic acid and ascorbic acid were extracted alone or in combination with carbonated water, the yield and purity were excellent.

[Test Example 1] Collagen gel contraction test The proteoglycan obtained in Production Example 1 was evaluated for the effect of collagen gel contraction. The presence or absence of the effect was expressed by comparing with the effect exhibited by the standard product. In Test Example 1, salmon nasal cartilage-derived proteoglycan (Fujifilm Wako Pure Chemical Industries, Ltd. (product code: 162-22131, 168-22133)) was used as a standard product.

Collagen acidic solution (IPC-30, Koken Co., Ltd.), dilute hydrochloric acid (used for pH adjustment, pH 3.0), 10×MEM Hanks (Nitta Gelatin Co., Ltd.) and reconstitution buffer (Cellmatrix, Nitta Gelatin stock) Company) to prepare a collagen acidic solution prepared to have a concentration of 1.2 mg/ml under ice cooling. 0.4 ml of each well was poured into the prepared 24-well plate of the prepared collagen acidic solution. After the injection, collagen was gelated at 37°C. After the gelation, normal human skin fibroblasts (Kurabo) were seeded in each well so that the number of cells in each well was 0.6×10 ⁵ cells/well. On the day after the seeding (until the next day, the wells were allowed to stand in an environment of 37° C. and 5% CO ₂ ), the following samples (Sample 1 to Sample 5) were administered to each well, and the area around the gel was peeled off. Evaluation of gel contraction 48 hours after the addition of the sample was performed by measuring the area of the collagen gel using image processing software (ChemiDoc ^™ imaging system, BIORAD). The measurement was performed for 6 samples in each group (groups of Sample 1 to Sample 5). Table 2 below shows the results of calculating the average value of 6 samples in each group.

-Sample 1 group: A predetermined amount of purified water was administered as a control.
-Group of sample 2: The proteoglycan obtained in Production Example 1 was administered so that the final concentration was 1 mg/ml.
-Group of sample 3: The proteoglycan obtained in Production Example 1 was administered so that the final concentration was 0.5 mg/ml.
-Group of sample 4: administration of standard so that the final concentration is 1 mg/ml.
Group of sample 5: administration of standard product so that the final concentration was 0.5 mg/ml.

The evaluation results are shown in Table 2. As shown in Table 2, compared with the group of Sample 1, in the groups of Samples 2 and 3 (the proteoglycan administration group obtained in Production Example 1 which is one of the Examples according to the present invention), collagen gel It was confirmed that the area was reduced. The evaluation results of the groups of Sample 2 and Sample 3 were the same as those of the groups of Sample 4 and Sample 5 (administration group of the standard product).

[Test Example 2] Human fibroblast proliferation test The presence or absence of human fibroblast proliferation ability of the proteoglycan obtained in Production Example 1 was evaluated.
A 96-well plate in which a DMEM medium containing 5% FBS (manufactured by Thermo Trace) was present in each well was prepared. Normal human skin fibroblasts (Kurabo) were seeded on the prepared 96-well plate so that the number of cells in each well was 4×10 ³ cells/well. After this seeding, the normal human dermal fibroblasts were cultured in an environment of 37° C. and 5% CO ₂ for 24 hours. After that, the DMEM medium containing 5% FBS (manufactured by Thermo Trace) was replaced with the DMEM medium containing 0.25% FBS, and after replacement, the following sample was administered to each well, and the environment was kept at 37° C. and 5% CO ₂ . The normal human skin fibroblasts were cultured for 3 days under After culturing for 3 days, the number of normal human dermal fibroblasts in each group (sample 1 to sample 5 group) was measured using Cell Counting Kit-8 (DOJINDO). The measurement was performed for 10 samples in each group (sample 1 to sample 5 group). In Table 3 below, the following relative values are shown using the results of calculating the average value of 10 samples in each group.

-Sample 1 group: A predetermined amount of purified water was administered as a control.
-Sample 2 group: The proteoglycan obtained in Production Example 1 was administered so that the final concentration was 0.5 mg/ml.
-Group of sample 3: The proteoglycan obtained in Production Example 1 was administered so that the final concentration was 0.1 mg/ml.
-Sample 4 group: The same standard as in Test Example 1 was administered so that the final concentration was 0.5 mg/ml.
-Sample 5 group: The same standard product as in Test Example 1 was administered so that the final concentration was 0.1 mg/ml.

The measurement results are described below. The measurement results shown in Table 3 below show relative values in the groups of Sample 2 to Sample 5 as compared to the group of Sample 1, with the number of cells in the group of Sample 1 (control group) being 100. As shown in Table 3, compared with the group of Sample 1, in the groups of Samples 2 and 3 (the proteoglycan administration group obtained in Production Example 1 which is one of the Examples according to the present invention), Production Example 1 By administration of the proteoglycan obtained in 1., the proliferation of normal human dermal fibroblasts could be confirmed. The evaluation results of the groups of Sample 2 and Sample 3 were the same as those of the groups of Sample 4 and Sample 5 (administration group of the standard product).

Claims (7)

a step of extracting proteoglycan from cartilage tissue of an animal in an acidic solution having a pH of 5.5 or less in the presence or absence of a protease of 1% by mass or less based on the total amount to obtain a crude extract containing the proteoglycan. ,
A method for producing proteoglycan, comprising the step of purifying the proteoglycan by removing lipids and substances having a molecular weight of 50,000 or less from the crude extract. The method for producing proteoglycan according to claim 1, wherein the cartilage tissue is a nasal cartilage tissue of a salmon head. The method for producing proteoglycan according to claim 1, wherein the protease is an acidic protease, and the content in the acidic solution is 0.001% by mass to 0.5% by mass. The method for producing proteoglycan according to any one of claims 1 to 3, wherein the acidic solution is an aqueous solution containing at least one selected from the group consisting of acetic acid, citric acid, malic acid, and ascorbic acid. 4. The acidic solution is an acidic electrolyzed water or carbonated water, or an aqueous solution obtained by adding at least one selected from the group consisting of acetic acid, citric acid, malic acid, and ascorbic acid to these. The method for producing proteoglycan according to the item. The concentration of at least one selected from the group consisting of acetic acid, citric acid, malic acid, and ascorbic acid in the acidic solution is 0.1% by mass to 1% by mass. The method for producing proteoglycan according to the item. The method for producing proteoglycan according to any one of claims 1 to 6, wherein the proteoglycan is an aggrecan having a mass average molecular weight of 400,000 to 1,000,000.