Method for producing proteoglycan and chondroitin sulfate

Abstract

To provide a simple and efficient production method for proteoglycan and chondroitin sulfate.SOLUTION: A production method for proteoglycan and chondroitin sulfate includes the steps of: (a) extracting proteoglycan from a biomaterial to recover a soluble part; and (b) subjecting the extraction residue in the step (a) to a protease treatment, to give chondroitin sulfate.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing proteoglycan and chondroitin sulfate. [Background technology]

[0002] Proteoglycans are a type of polymer that constitutes the extracellular matrix of animals. They are known to have high water-retaining properties and diverse physiological functions, such as wound healing, anti-inflammatory, and cell proliferation-promoting properties. They are expected to be used in a wide range of applications, including pharmaceuticals, laboratory reagents, cosmetics, and food and beverages. Therefore, there is a need for a simple and efficient method for producing proteoglycans that can be used in these applications.

[0003] Chondroitin sulfate is known to have various physiological activities, such as neuronal elongation and chondrocyte differentiation. Therefore, chondroitin sulfate is desired for application in various fields, such as regenerative medicine, functionalization of pharmaceutical compounds, and treatment of joint diseases. Therefore, there is a need for the development of a simple and efficient method for producing chondroitin sulfate that can be used for these purposes. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-113382 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a simple and efficient method for producing proteoglycan and chondroitin sulfate. [Means for solving the problem]

[0006] When proteoglycan is obtained from biological raw materials, a method is adopted in which the biological raw materials are immersed in a solution containing an acid to extract the proteoglycan. For example, Patent Document 1 reports the use of umeboshi wastewater, which has long been used as a food preservative, as the extraction solution. On the other hand, when chondroitin sulfate is obtained from biological raw materials, a method is adopted in which the biological raw materials are treated with protease and chondroitin sulfate is obtained from the soluble matter. The extraction residue after proteoglycan extraction contains little chondroitin sulfate derived from proteoglycans, and therefore it has been thought that the total amount of chondroitin sulfate is also small.

[0007] The present inventors have conducted extensive research and have surprisingly found that the extraction residue after proteoglycan extraction also contains a large amount of chondroitin sulfate. Based on this finding, the present inventors have conducted further research and have found that both proteoglycan and chondroitin sulfate can be produced simply and efficiently by (a) extracting proteoglycan from a biological raw material and recovering the soluble portion, and (b) treating the extraction residue obtained in step (a) with a protease to obtain chondroitin sulfate. The present invention encompasses the following aspects.

[0008] Item 1. (a) A step of extracting proteoglycans from biological raw materials and recovering the soluble portion; (b) treating the extraction residue obtained in step (a) with a protease to obtain chondroitin sulfate; A method for producing proteoglycan and chondroitin sulfate, comprising:

[0009] Item 2. The production method according to Item 1, wherein in step (a), proteoglycans are extracted from the biological raw material with umeboshi wastewater (ume vinegar) or a solution containing magnesium sulfate.

[0010] Item 3. The production method according to Item 1 or 2, wherein in step (a), proteoglycans are extracted from biological raw materials using umeboshi plum wastewater (ume vinegar).

[0011] Item 4. The production method according to any one of Items 1 to 3, wherein the protease concentration in step (b) is 0.02 to 0.1% by mass.

[0012] Item 5. The production method according to any one of Items 1 to 4, wherein the organism from which the biological raw material is derived is at least one species selected from the group consisting of fish, mammals, mollusks, and echinoderms.

[0013] Item 6. The manufacturing method according to any one of Items 1 to 5, wherein the biological material is at least one selected from the group consisting of cartilage, connective tissue, bone, cornea, atrium, basement membrane, brain, skin, and processed products thereof.

[0014] Item 7. The production method according to any one of Items 1 to 6, wherein in step (b), a soluble portion is recovered from the treated product obtained by the protease treatment.

[0015] Item 8. A step of treating the proteoglycan extraction residue of a biological raw material with a protease to obtain chondroitin sulfate; A method for producing chondroitin sulfate, comprising:

[0016] Item 9. Chondroitin sulfate isolated from biological sources, having an average molecular weight of 80,000 or more.

[0017] Item 10. The chondroitin sulfate according to Item 9, which has an average molecular weight of 120,000 or more.

[0018] Item 11. (a) A feed containing an extraction residue obtained in the process of extracting proteoglycans from biological raw materials and recovering the soluble portion. [Effects of the Invention]

[0019] According to the present invention, a simple and efficient method for producing proteoglycan and chondroitin sulfate can be provided, and also according to the present invention, isolated chondroitin sulfate having a high molecular weight can be provided. [Brief explanation of the drawings]

[0020] [Figure 1] 1 shows the HPLC results of the proteoglycan obtained in Example 1 (Example 2). [Figure 2] HPLC results of the chondroitin sulfate obtained in Example 3 are shown (Example 4). DETAILED DESCRIPTION OF THE INVENTION

[0021] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0022] In one aspect, the present invention relates to a method for producing proteoglycans and chondroitin sulfate (sometimes referred to herein as the "production method of the present invention"), which comprises: (a) extracting proteoglycans from a biological raw material and recovering the soluble portion; and (b) treating the extraction residue obtained in step (a) with a protease to obtain chondroitin sulfate. This method is described below.

[0023] In step (a), proteoglycans are extracted from biological raw materials and the soluble portion is recovered. The biological raw material is not particularly limited as long as it is a biological tissue or a processed product thereof and contains proteoglycan and chondroitin sulfate.

[0024] The organisms from which the biological raw materials are derived are not particularly limited, and include a wide range of species, such as fish, mammals, mollusks such as sea hares, and echinoderms such as sea cucumbers. Of these, fish are preferred.

[0025] The fish is not particularly limited, and broad examples thereof include bony fish, cartilaginous fish, etc. Examples of bony fish include cod, tuna, salmon, trout, bonito, flounder, and yellowtail, while examples of cartilaginous fish include sharks and rays.

[0026] The biological tissue is not particularly limited, and examples thereof include cartilage, connective tissue, bone, cornea, atrium, basement membrane, brain, skin, etc. Among these, cartilage is preferred.

[0027] As the biological tissue of fish, fish cartilage is preferred. The cartilage is not particularly limited, but head cartilage, particularly nasal cartilage, is preferred. Furthermore, since the head is usually discarded when fish are processed into food products, head cartilage has the advantage of being inexpensive to obtain and can be supplied in large quantities stably.

[0028] The processed biological tissue is not particularly limited, and examples thereof include dried biological tissue, fragmented biological tissue, crushed biological tissue, etc. The processing method such as drying, fragmenting, crushing, etc. is not particularly limited, and known methods can be used.

[0029] From the viewpoint of extraction efficiency, etc., the biological raw material is preferably a processed product of biological tissue, more preferably a product processed so as to have a larger solvent contact area, and even more preferably a small fragment or crushed product.

[0030] The biological raw materials may be one type alone or a combination of two or more types.

[0031] In step (a), proteoglycans are typically extracted from biological raw materials using an extraction solution. The extraction solution is not particularly limited, and various solutions containing acids can be used. Specific examples of extraction methods include a method of extracting cartilage from fish such as salmon using a guanidine hydrochloride solution (JP 2001-172296 A), a method using an acetic acid solution as the extraction solution (JP 2002-069097 A), a method using pickled plum wastewater (plum vinegar) as the extraction solution (Patent Document 1: JP 2016-113382 A), and a method using a magnesium sulfate-containing solution as the extraction solution (JP 2020-063214 A). As the extraction solution, from the viewpoint of further reducing the unpleasant odor of the resulting proteoglycan and of more efficiently obtaining both proteoglycan and chondroitin sulfate, preferred examples include umeboshi waste liquid (ume vinegar) and a magnesium sulfate-containing solution, and particularly preferred is umeboshi waste liquid (ume vinegar).

[0032] The umeboshi waste liquid (umeboshi vinegar) is not particularly limited, and examples thereof include white umeboshi waste liquid, red umeboshi waste liquid, etc. Among these, white umeboshi waste liquid is preferred from the viewpoint of suppressing coloration of proteoglycans. White umeboshi waste liquid is typically obtained during the production of umeboshi by mixing ume and salt, placing a weight on top, and leaving the mixture for several days (e.g., 1 to 5 days). The pH of the umeboshi waste liquid is not particularly limited, but can be, for example, 1.0 to 4.0, preferably 1.5 to 3.0, and more preferably 1.5 to 2.5. The umeboshi waste liquid may be one type alone or a combination of two or more types.

[0033] The magnesium sulfate-containing solution is not particularly limited as long as it is a liquid that contains magnesium sulfate (MgSO4) and in which part or all of it is dissolved.

[0034] The solvent of the magnesium sulfate-containing solution is usually water. In addition to water, the solvent may contain alcohol such as ethanol. The content of the solvent other than water is, for example, 20% by mass or less, preferably 10% by mass or less, more preferably 3% by mass or less, even more preferably 1% by mass or less, still more preferably 0.3% by mass or less, and particularly preferably 0.1% by mass or less, relative to 100% by mass of the solvent. The lower limit of the content is, for example, 0%, 0.1%, 0.3%, 1%, or 3% by mass. The solvent may be used alone or in combination of two or more.

[0035] The magnesium sulfate concentration of the magnesium sulfate-containing solution is not particularly limited. The concentration is, for example, 0.5 to 5 M. From the viewpoints of extraction efficiency and suppression of protein contamination, the concentration is preferably 1 M or higher, more preferably 1.5 M or higher, even more preferably 2 M or higher, even more preferably 2.5 M or higher, and particularly preferably 2.8 M or higher. Furthermore, from the viewpoints of extraction costs and solution preparation efficiency, the concentration is preferably 4.5 M or lower, more preferably 4 M or lower, even more preferably 3.5 M or lower, and even more preferably 3.2 M or lower. From the above viewpoints, the concentration range is preferably 1.5 to 5 M, more preferably 2.5 to 5 M, even more preferably 2.5 to 3.5 M, and even more preferably 2.8 to 3.2 M.

[0036] The magnesium sulfate-containing solution may contain other components in addition to magnesium sulfate, as long as they do not significantly impair the extraction efficiency or degree of purification of proteoglycan. Examples of other components include additives that can be added to the extraction solvent (e.g., acids, bases, preservatives such as toluene, protease inhibitors, etc.). The other components may be present alone or in combination of two or more. The content of the other components is, for example, 20% by mass or less, 10% by mass or less, 5% by mass or less, or 1% by mass or less, relative to 100% by mass of the magnesium sulfate-containing solution. The lower limit of the content is, for example, 0% by mass, 0.1% by mass, 0.3% by mass, 1% by mass, or 3% by mass.

[0037] Proteoglycan extraction can be carried out according to known extraction methods, for example, by mixing the biological raw material with an extraction solution and then leaving it to stand (preferably while stirring).

[0038] The weight ratio of the biological raw material to the extraction solution is not particularly limited, but is preferably such that the biological raw material is immersed in the extraction solution. Specifically, the weight ratio (biological raw material:pickled plum waste liquid) can be, for example, 1:1 to 1:50, preferably 1:2 to 1:20, and more preferably 1:2.5 to 1:10.

[0039] The extraction time is not particularly limited as long as proteoglycans can be extracted. The extraction time can be, for example, 3 to 72 hours, preferably 8 to 48 hours, more preferably 12 to 36 hours, and even more preferably 18 to 30 hours. If the upper limit of the extraction time is within the above range, the proportion of proteoglycans in the extracted components can be further increased.

[0040] The extraction temperature is not particularly limited as long as it allows proteoglycan to be extracted. The extraction temperature can be, for example, 10 to 40°C, preferably 15 to 30°C.

[0041] The soluble portion obtained after the proteoglycan extraction procedure contains proteoglycan. Therefore, proteoglycan can be obtained by recovering the soluble portion. The method for recovering the soluble portion is not particularly limited as long as it is a method that allows solid-liquid separation, and known methods such as filtration and centrifugation can be used. This soluble portion can be used as "proteoglycan" as is, or the product obtained through the following steps can be used as "proteoglycan." The extraction residue remaining after recovering the soluble portion is used in step (b).

[0042] The proteoglycan extraction procedure may be performed once or multiple times. When multiple procedures are performed, proteoglycans are extracted from the extraction residue obtained in the first extraction procedure using an extraction solution that is the same as or different from the extraction solution used in the first extraction procedure. In this case, any of the extraction residues obtained in each extraction procedure can be used in step (b).

[0043] To further increase the purity of the resulting proteoglycan, step (a) preferably includes (a1) a step of purifying the proteoglycan. Examples of purification methods include alcohol precipitation, dialysis, column (preferably anion exchange column) chromatography, gel filtration chromatography, affinity column chromatography, ultrafiltration, and electrodialysis. These purification methods may be used alone or in combination.

[0044] In one embodiment of the present invention, ultrafiltration can be adopted as the purification method in step (a1) from the viewpoints of simplicity, cost, etc. Ultrafiltration can remove most of the solvent, salts, etc., and can purify substances with high molecular weights (proteoglycans).

[0045] Ultrafiltration can be carried out according to a known method. After ultrafiltration, it is preferable to dry the product (for example, freeze-drying).

[0046] In one embodiment of the present invention, alcohol precipitation can be employed as the purification method in step (a1).

[0047] Alcohol precipitation can be carried out according to a known method, typically by mixing the soluble fraction obtained in step (a) or a concentrate thereof obtained by ultrafiltration or the like with 1 to 5 volumes (preferably 2 to 4 volumes) of alcohol, leaving the mixture for a certain period of time to form a precipitate, and then centrifuging the mixture to recover the resulting pellet.

[0048] The alcohol is not particularly limited as long as it can precipitate proteoglycans. Examples of alcohol include ethanol and isopropanol, and among these, ethanol is preferred from the viewpoint of lower toxicity. The alcohol may be used alone or in combination of two or more.

[0049] The alcohol preferably contains a salt. The salt is not particularly limited, and examples thereof include common salts used in alcohol precipitation, such as sodium chloride, sodium acetate, ammonium acetate, lithium chloride, and magnesium chloride. The salt may be a single salt or a combination of two or more salts. The concentration of the salt in the alcohol is not particularly limited, and may be a common salt concentration used in alcohol precipitation, such as the saturation concentration of sodium chloride relative to the alcohol.

[0050] The temperature at which the mixture is left to form a precipitate is not particularly limited as long as it is capable of precipitating proteoglycans. The temperature can be, for example, from about -80°C to room temperature, and preferably from about 0 to 10°C.

[0051] The time for which the mixture is left to stand to form a precipitate is not particularly limited as long as it is capable of precipitating proteoglycan, and is appropriately set depending on the temperature. When the temperature is 0 to 10°C, the time can be, for example, 4 to 24 hours, preferably 8 to 16 hours.

[0052] The centrifugal force is not particularly limited as long as it can form a pellet, and can be, for example, 700 to 2500 g.

[0053] The type of proteoglycan obtained in step (a) depends on the raw material and is not particularly limited. The proteoglycan is not particularly limited as long as it is a compound in which chondroitin sulfate is bound to a core protein. Examples of proteoglycans include aggrecan, biglycan, versican, neurocan, decorin, biglycan, fibromodulin, lumican, perlecan, syndecan, serglycin, brevican, keratocan, mimecan, vermacan, agrin, etc., as well as degradation products thereof.

[0054] In step (a), by selecting the extraction solution, it is possible to further suppress proteoglycan degradation and obtain proteoglycans with higher molecular weights. From this perspective, the average molecular weight of the proteoglycans obtained by the production method of the present invention is preferably 250,000 or more, more preferably 300,000 or more, even more preferably 350,000 or more, and even more preferably 370,000 or more. The upper limit of the average molecular weight is not particularly limited and may be, for example, 5,000,000, 2,000,000, 1,000,000, 700,000, or 500,000. The average molecular weight of proteoglycans can be measured by fractionating them using chromatography and comparing the retention time with that of a molecular weight standard such as a pullulan standard, as in Example 2.

[0055] In step (b), the extraction residue obtained in step (a) is treated with a protease to obtain chondroitin sulfate. According to the production method of the present invention, chondroitin sulfate can be obtained unexpectedly and efficiently by treating the extraction residue from step (a) (particularly the extraction residue when pickled plum wastewater (plum vinegar) is used) with a protease.

[0056] The protease treatment is not particularly limited as long as it is a treatment that can decompose proteins that retain chondroitin sulfate. Examples of proteases that can be used include actinase (particularly actinase E), chymotrypsin, subtilisin, pepsin, cathepsin, HIV protease, thermolysin, papain, and caspase.

[0057] The protease treatment can be carried out according to a known method, for example, by mixing the extraction residue obtained in step (a) with a protease-containing solution and then subjecting the mixture to an enzymatic reaction.

[0058] The protease concentration in the protease-containing solution is, for example, 0.02 to 5% by mass. According to the production method of the present invention, by treating the extraction residue from step (a) (particularly the extraction residue when using umeboshi wastewater (ume vinegar)) with protease, chondroitin sulfate can be efficiently obtained even at a low protease concentration. From this perspective, the protease concentration is preferably 0.02 to 1% by mass, more preferably 0.02 to 0.5% by mass, even more preferably 0.02 to 0.2% by mass, and even more preferably 0.02 to 0.1% by mass.

[0059] In addition to the protease, the protease-containing solution preferably contains other components that are desirable for the protease to exhibit its enzymatic activity. Such components include, for example, a buffer, a cation source, etc. Examples of the cation source include divalent cation sources such as a calcium source (e.g., calcium chloride) and a magnesium source.

[0060] The weight ratio of the extraction residue to the protease-containing solution is not particularly limited, but is preferably such that the extraction residue is immersed in the protease-containing solution. Specifically, the weight ratio (biological raw material:protease-containing solution) can be, for example, 1:1 to 1:10, preferably 1:1.5 to 1:5, and more preferably 1:1.8 to 1:3.

[0061] The enzymatic reaction time is, for example, 8 to 72 hours, preferably 16 to 60 hours, and more preferably 36 to 56 hours.

[0062] The enzyme reaction temperature is, for example, 30 to 40°C, preferably 35 to 38°C.

[0063] The soluble portion obtained after the enzymatic reaction contains chondroitin sulfate. Therefore, chondroitin sulfate can be obtained by recovering the soluble portion. The method for recovering the soluble portion is not particularly limited as long as it is a method that allows solid-liquid separation, and known methods such as filtration and centrifugation can be used. This soluble portion can be used as "chondroitin sulfate" as is, or the product obtained through the following steps can be used as "chondroitin sulfate".

[0064] After the enzymatic reaction, if necessary, a treatment to stop the enzymatic reaction (protein denaturation treatment, for example, treatment with a strong acid such as trichloroacetic acid, heat treatment, etc.) can be carried out.

[0065] To further increase the purity of the resulting chondroitin sulfate, step (b) preferably includes (b1) a step of purifying the chondroitin sulfate. Examples of purification methods include alcohol precipitation, dialysis, column (preferably anion exchange column) chromatography, gel filtration chromatography, affinity column chromatography, ultrafiltration, and electrodialysis. These purification methods may be used alone or in combination of two or more.

[0066] According to the production method of the present invention, isolated chondroitin sulfate with a high molecular weight can be obtained. The average molecular weight of chondroitin sulfate obtained by the production method of the present invention is preferably 80,000 or more, more preferably 100,000 or more, even more preferably 120,000 or more, and even more preferably 130,000 or more. There is no particular upper limit to the average molecular weight, and it can be, for example, 400,000, 300,000, or 200,000. The average molecular weight of chondroitin sulfate can be measured by fractionating it using chromatography and comparing it with the retention time of a molecular weight standard such as a pullulan standard, as in Example 4. In one aspect, the present invention relates to chondroitin sulfate isolated from a biological source, which has an average molecular weight of 80,000 or more (more preferably 100,000 or more, even more preferably 120,000 or more, and even more preferably 130,000 or more).

[0067] The chondroitin sulfate obtained by the production method of the present invention includes not only those consisting of sugar chains alone, but also those in which amino acids or peptides (for example, peptides of 2 to 10 amino acid residues, or 2 to 4 amino acid residues) are attached to the sugar chains.

[0068] The uses of the proteoglycans and chondroitin sulfate obtained by the production method of the present invention are not particularly limited. For example, they can be preferably used as raw materials for foods, cosmetics, pharmaceuticals, and feed (e.g., pet and livestock feed), as well as biochemical and medical research reagents. Furthermore, since the extraction residue obtained in step (a) contains a large amount of chondroitin sulfate, the extraction residue can also be used as feed. Furthermore, medical applications are possible, taking advantage of the physiological activities of proteoglycans and chondroitin sulfate (wound healing, anti-inflammatory, and human epithelial cell proliferation). Furthermore, they can be used as cosmetic ingredients, taking advantage of their water-retaining properties. They can also be used as glycosylated supplements, the market for which has been expanding significantly in recent years.

[0069] The proteoglycan and chondroitin sulfate obtained by the production method of the present invention may be used in any manner without particular limitation. Because the proteoglycan and chondroitin sulfate have the effects described above, they are preferably used as topical or oral compositions. That is, they are preferably used as topical or oral compositions containing the proteoglycan and / or chondroitin sulfate obtained by the production method of the present invention. Furthermore, topical or oral compositions can be used as pharmaceutical compositions, quasi-drug compositions, cosmetic compositions, food compositions, and the like. These can be produced by conventional methods using the proteoglycan and / or chondroitin sulfate obtained by the production method of the present invention. These compositions are particularly suitable for use in the fields of cosmetics and food and beverages.

[0070] Cosmetic compositions containing proteoglycan and / or chondroitin sulfate obtained by the production method of the present invention (hereinafter sometimes referred to as "cosmetic compositions of the present invention") used in the cosmetics field may be the proteoglycan itself, or may be compositions produced by conventional methods by appropriately blending the proteoglycan with a cosmetically acceptable medium, base, carrier, additive, or other cosmetically acceptable ingredient or material. Specific examples include emulsions, lotions, creams, serums, foundations, packs, sunscreens, and the like, which contain the proteoglycan. Such cosmetic compositions of the present invention are preferably used for alleviating inflammation or anti-aging, and more specifically, for sunburn prevention, suncare, moisturizing, and anti-skin aging (e.g., prevention or improvement of dry skin, rough skin, wrinkles, sagging skin, etc.).

[0071] Food and beverage compositions containing proteoglycan and / or chondroitin sulfate obtained by the production method of the present invention (hereinafter sometimes referred to as "food and beverage compositions of the present invention") used in the food and beverage (beverage and food) field may be the proteoglycan and / or chondroitin sulfate itself, or may be a mixture of the proteoglycan and / or chondroitin sulfate with hygienically acceptable bases, carriers, additives, and other ingredients and materials that can be used in foods. Examples of such compositions include processed foods, beverages, health foods (nutrient functional foods, foods for specified health uses, etc.), supplements, beauty foods, and foods for medical use, all of which contain the proteoglycan and / or chondroitin sulfate and are used for moisturizing and anti-skin aging (e.g., for preventing or improving dry skin, rough skin, wrinkles, sagging skin, etc.). Furthermore, moisturizing agents and anti-skin aging agents made from such food and beverage compositions of the present invention are also encompassed by the present invention. The moisturizing agent and anti-skin aging agent can be supplied in the form of a drink, tablet, capsule, granule, jelly, lozenge, etc. for cosmetic or anti-skin aging purposes (for example, for preventing or improving dry skin, rough skin, wrinkles, sagging skin, etc.).

[0072] Although not particularly limited, the amount of the proteoglycan and / or chondroitin sulfate contained in the pharmaceutical composition, quasi-drug composition, cosmetic composition, or food and beverage composition according to the present invention is, for example, typically 0.001 to 100% by mass, preferably 0.01 to 95% by mass, of the total composition. [Example]

[0073] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0074] Reference Example 1. Preparation of Umeboshi Wastewater (Ume Vinegar) Fully ripe Nanko plums (3 kg) from Wakayama Prefecture were mixed with salt (360 g) and rubbed thoroughly. The mixture was bottled, a weight was placed on top, and left to stand in a cool place for 3 days. After standing, the plums were removed and the resulting solution was filtered to obtain 630 mL of umeboshi wastewater. The pH of this umeboshi wastewater was 2.1.

[0075] Example 1. Extraction of proteoglycans Fish nasal cartilage (100 g) sliced ​​into 3 cm squares was immersed in plum vinegar (300 mL), gently stirred at room temperature, and left for 24 hours. The resulting mixture was filtered through filter paper to recover the insoluble cartilage components. The resulting filtrate was ultrafiltered (MWCO 10,000). Ethanol saturated with salt (salt-saturated ethanol) (300 mL, 3 times the volume) was added to the resulting ultrafiltrate concentrate, and the mixture was left for 12 hours at 4°C. The resulting white precipitate was centrifuged (6,300 g × 10 min) to recover a white powder. The white powder was washed with ethanol (20 mL) and air-dried to obtain proteoglycan (2.7 g).

[0076] Example 2. Molecular weight measurement of proteoglycans The proteoglycan obtained in Example 1 was dissolved in water to prepare a 0.5% aqueous proteoglycan solution, and 30 μL of this solution was subjected to high performance liquid chromatography analysis under the following analytical conditions. Detector: Differential Refractometer (RI) 5450 RI Detector (Hitachi High-Technologies Corporation) Column: TOSOH TSK-gel G5000PWXL Eluent: 0.2M-NaCl The results are shown in Figure 1. As shown in Figure 1, a peak was observed at a retention time of 7.037 minutes, and its average molecular weight was calculated to be 400,000. The average molecular weight was calculated using a pullulan standard (Shodex, P-82).

[0077] Example 3. Extraction of chondroitin sulfate To the insoluble cartilage component (77.6 g) recovered in Example 1, 150 mL of 100 mM TrisHCl bf pH 8.0 containing 50 mM calcium chloride and actinase (100 mg) were added and incubated at 37°C for 48 hours. The mixture was then centrifuged (8000 g × 10 min) to collect the supernatant. Trichloroacetic acid (28 g) was added to the supernatant at 0°C and allowed to stand for 15 minutes. The mixture was then centrifuged (22300 g, 10 min, 4°C) to collect the supernatant. A three-fold volume of sodium chloride-saturated ethanol (450 mL) was added to the supernatant, and ethanol precipitation was performed to obtain chondroitin sulfate (2.6 g).

[0078] Example 4. Molecular weight measurement of chondroitin sulfate The chondroitin sulfate obtained in Example 2 was dissolved in water to prepare a 0.5% aqueous solution of chondroitin sulfate, and 30 μL of this solution was subjected to high performance liquid chromatography analysis under the following analytical conditions. Detector: Differential Refractometer (RI) 5450 RI Detector (Hitachi High-Technologies Corporation) Column: TOSOH TSK-gel G5000PWXL Eluent: 0.2M-NaCl The results are shown in Figure 2. As shown in Figure 2, a peak was observed at a retention time of 8,180, and its average molecular weight was calculated to be 150,000. The average molecular weight was calculated using a pullulan standard (Shodex, P-82).

[0079] Reference Example 1. Extraction of chondroitin sulfate from cartilage Fish nasal cartilage (100 g) sliced ​​into 3 cm squares was added to 150 mL of 100 mM TrisHCl (pH 8.0) containing 50 mM calcium chloride and actinase (650 mg) and incubated at 37°C for 48 hours. The mixture was then centrifuged (8000 g x 10 min) to collect the supernatant. Trichloroacetic acid (40 g) was added to the supernatant at 0°C and allowed to stand for 15 minutes. The supernatant was then centrifuged (22300 g, 10 min, 4°C) to collect the supernatant. Chondroitin sulfate (5.76 g) was precipitated by adding a three-fold volume of sodium chloride-saturated ethanol (450 mL). The molecular weight was measured using the same method as in Example 4 and was found to be similar to that of the chondroitin sulfate obtained in Example 2.

[0080] Thus, when raw cartilage that has not been subjected to proteoglycan extraction processing is used, it is necessary to use a large amount of actinase to destroy the tissue, and this method also decomposes the core protein of proteoglycan by actinase, making it impossible to obtain proteoglycan.

Claims

1. (a) extracting proteoglycans from a biological raw material with umeboshi wastewater (umeboshi vinegar) or a magnesium sulfate-containing solution, and recovering the soluble portion; and (b) treating the extraction residue obtained in step (a) with a protease to obtain chondroitin sulfate; and the protease concentration in step (b) is 0.02 to 0.1% by mass. Methods for producing proteoglycans and chondroitin sulfate.

2. 2. The method according to claim 1, wherein in step (a), proteoglycans are extracted from the biological raw material using umeboshi (pickled plum) wastewater (ume vinegar).

3. 3. The production method according to claim 1, wherein the organism from which the biological raw material is derived is at least one species selected from the group consisting of fish, mammals, mollusks, and echinoderms.

4. The method according to any one of claims 1 to 3, wherein the biological material is at least one selected from the group consisting of cartilage, connective tissue, bone, cornea, atrium, basement membrane, brain, skin, and processed products thereof.

5. The method according to any one of claims 1 to 4, wherein in step (b), a soluble portion is recovered from the treated product obtained by the protease treatment.

6. The proteoglycan extraction residue of biological raw materials extracted with umeboshi wastewater (umeboshi vinegar) or a magnesium sulfate-containing solution, a step of treating the mixture with a protease at a protease concentration of 0.02 to 0.1% by mass to obtain chondroitin sulfate; A method for producing chondroitin sulfate, comprising:

7. A method for producing feed, comprising the steps of extracting proteoglycan from biological raw materials and obtaining the extraction residue as feed.

8. A manufacturing method as described in claim 7, wherein in the step, proteoglycans are extracted from the biological raw material, which is cartilage, using pickled plum waste liquid (plum vinegar).

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