阅读说明：本技术 软骨机能改善用食品组合物 (Food composition for improving cartilage function ) 是由 加藤健 石田祐子 森田如一 和田政裕 中谷祥惠 上田博也 于 2017-02-03 设计创作，主要内容包括：提供的是：软骨机能改善用食品组合物,其对预防和治疗各种关节疾病有用；和软骨机能改善用饮食品、软骨机能改善用营养组合物和软骨机能改善用饲料,其各自包含软骨机能改善用食品组合物。所述软骨机能改善用食品组合物包含选自乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、和HMG样蛋白和/或其分解物的至少一种成分作为一种(或多种)有效成分。可以在日常的基础上长期摄取、并且发挥修正或再生变形或消失的软骨的显著效果的根据本发明的软骨机能改善用食品组合物,对预防或治疗各种关节疾病有用。(The method comprises the following steps: a food composition for improving cartilage function, which is useful for the prevention and treatment of various joint diseases; and a food or drink for improving cartilage function, a nutritional composition for improving cartilage function, and a feed for improving cartilage function, each of which comprises the food composition for improving cartilage function. The food composition for improving cartilage function comprises at least one component selected from lactoperoxidase, cystatin, and HMG-like protein and/or its decomposition product as one (or more) active ingredient(s). The food composition for improving cartilage function according to the present invention, which can be ingested on a daily basis for a long period of time and exhibits a significant effect of correcting or regenerating deformed or lost cartilage, is useful for the prevention or treatment of various joint diseases.)

1. Use of a food composition comprising cystatin and/or a decomposition product thereof as an active ingredient of the food composition for improving cartilage function.

2. Use of a food composition according to claim 1 wherein the cystatin is derived from mammalian milk.

3. Use of a food composition according to claim 1 or 2 wherein the cystatin is derived from bovine milk.

4. Use of a food composition according to claim 1 or 2, wherein the degradation product of cystatin is prepared by degrading cystatin in the presence of a protease.

5. Use of the food composition according to claim 4, wherein the protease is at least one protease selected from the group consisting of pepsin, trypsin, chymotrypsin, pancreatin and papain.

6. Use of a food composition comprising cystatin and/or a decomposition product thereof for use according to any one of claims 1 to 5 in the preparation of a food, drink, nutritional composition, or feed for improving cartilage function.

7. Use of a food composition comprising cystatin and/or a decomposition product thereof for use according to any one of claims 1 to 5 in the preparation of a food composition for improving cartilage function by a method of improving cartilage function by ingesting at least 1mg per day of cystatin and/or a decomposition product thereof for use according to any one of claims 1 to 5.

Technical Field

The present invention relates to a food composition for improving cartilage function. The composition has an effect of promoting the proliferation of chondrocytes and/or inhibiting the calcification of chondrocytes, has a remarkable effect of correcting or regenerating deformed or lost cartilage, and is useful in the prevention and treatment of various joint diseases. The present invention also relates to a food or drink for improving cartilage function, a nutritional composition for improving cartilage function, and a feed for improving cartilage function, which include the food composition for improving cartilage function.

Background

Japan has an average life of more than 80 years and has reached an ultra-advanced society, in which about one of four people is over 65 years old. In this aging situation, the prevalence of motor organ disorders has increased. In 2007, the japan society of orthopedics (japan orthopaedics Association Corporation) has proposed a new term "sports organ syndrome", thereby promoting consciousness reform toward national and medical practitioners who maintain and improve the health and care of sports organs. Motor organ syndrome represents a state of need of care caused by or at increased risk of dysfunction of motor organs. Motor organs generally include organs that support and move the body, such as bones, joints, ligaments, vertebrae, spinal cord, muscles, tendons, and peripheral nerves. Examples of representative diseases and dysfunctions observed in these locomotor apparatus include osteoporosis, sarcopenia (sarcopenia), and osteoarthritis.

Osteoarthritis is the most common joint disease and is expected to be found in 20% to 30% of people over the age of 50. In particular, the number of patients has increased, and a large number of patients having osteoarthritis are forced to suffer from inconvenience in daily life. The disease is thought to result from degeneration of joint-forming elements caused by aging and genetic factors, or load on joints resulting from obesity, labor, or exercise. The reduction of cartilage that occurs at the epiphyseal surface that supports the movement of the bone results in the deformation or disappearance of the cartilage. Such deformation or loss inhibits smooth movement of the joint, resulting in osteoarthritis.

Since cartilage has no blood vessels and nerve cells, it is considered that cartilage is difficult to recover spontaneously once it is damaged. For this reason, in the mild cases of osteoarthritis, physical therapy such as heat therapy or traction, or palliative therapy (palliative therapy) using analgesics or anti-inflammatory drugs is used, and in the severe cases, hyaluronic acid is injected into the joint or surgically replaced with an artificial joint. In order to improve quality of life (QOL) of patients by fundamentally treating osteoarthritis, patients should take food or effective ingredients contained in food daily and safely for a long time due to the nature of the disease, thereby repairing or regenerating deformed or lost cartilage of patients.

Cartilage is composed of chondrocytes differentiated from mesenchymal stem cells and a cartilage matrix of collagen, hyaluronic acid, and proteoglycan produced by the chondrocytes. Generally, cartilage is calcified after proliferation, maturation and hypertrophy of chondrocytes, and cartilage matrix is degenerated to be transformed into bone tissue. In joints and intervertebral discs, chondrocytes maintain their properties throughout life; cartilage thus provides smooth mobility to the body without converting to bone. Therefore, in order to repair or regenerate deformed or lost cartilage to treat osteoarthritis fundamentally, it is important to promote the proliferation of chondrocytes present in cartilage and the production of cartilage matrix in chondrocytes, and to prevent the conversion of chondrocytes into bone by inhibiting the calcification of chondrocytes.

Disclosed is a food ingredient for promoting the proliferation of chondrocytes present in cartilage and the production of cartilage matrix from chondrocytes, or for inhibiting the calcification of chondrocytes to thereby prevent the conversion of cartilage into bone: for example, glucosamine hydrochloride which promotes the production of cartilage matrix by chondrocytes and inhibits the calcification of chondrocytes (non-patent document 1), and a dipeptide derived from collagen, prolylhydroxyproline, which promotes the production of cartilage matrix by chondrocytes and delays the calcification of chondrocytes (non-patent document 2).

Milk and dairy products are food products that have been consumed since ancient times and can be ingested daily and safely for long periods of time. It is known that the production of cartilage matrix is promoted by a milk basic protein fraction (fraction) contained therein or a decomposition product thereof. Lactoferrin is also known to promote the proliferation of chondrocytes and inhibit the calcification of chondrocytes (patent documents 1 and 2, non-patent document 3).

Documents of the prior art

Patent document

Patent document 1: WO2010/058679

Patent document 2: WO2013/164992

Non-patent document

Non-patent document 1: nakatani et al, Biological & Pharmaceutical Bulletin,30:433-

Non-patent document 2: nakatani et al, Osteoarthitis and Cartilage,17:1620-

Non-patent document 3: takayama et al, Biometals,23:477-484(2010)

Disclosure of Invention

Problems to be solved by the invention

Lactoferrin is a food which can be ingested daily and safely for a long period of time, promotes the proliferation of chondrocytes, and inhibits the calcification of chondrocytes, but has disadvantages in terms of stability and flavor. For this reason, there has been a need for a cartilage function-improving food composition having improved properties to solve these problems.

The purpose of the present invention is to provide a food composition for improving cartilage function, which can be ingested daily and safely for a long period of time, has an effect of promoting the proliferation of chondrocytes and/or an effect of inhibiting the calcification of chondrocytes, has a significant effect of correcting or regenerating deformed or lost cartilage, and is useful for the prevention and treatment of various joint diseases. It is another object of the present invention to provide a food or drink for improving cartilage function, a nutritional composition for improving cartilage function, and a feed for improving cartilage function, which contain the food composition for improving cartilage function.

Means for solving the problems

The present invention relates to the following aspects:

aspect (1) is a cartilage function-improving food composition containing at least one active ingredient selected from the group consisting of lactoperoxidase, cystatin, High Mobility Group (HMG) -like protein, and degradation products thereof.

Aspect (2) the food composition for improving cartilage function according to aspect (1), wherein the lactoperoxidase, the cystatin, and the HMG-like protein are derived from mammalian milk.

Aspect (3) the cartilage function-improving food composition according to aspect (1) or (2), wherein the lactoperoxidase, the cystatin, and the HMG-like protein are derived from cow's milk.

Aspect (4) the food composition for improving cartilage function according to any one of aspects (1) to (3), wherein the lactoperoxidase, the cystatin, and the HMG-like protein are produced by decomposing the lactoperoxidase, the cystatin, and the HMG-like protein in the presence of a protease.

Aspect (5) the food composition for improving cartilage function according to aspect (4), wherein the protease is at least one protease selected from the group consisting of pepsin, trypsin, chymotrypsin, pancreatin and papain.

Aspect (6) is a food or drink for improving cartilage function, which comprises the food composition for improving cartilage function according to any one of aspects (1) to (5), a nutritional composition for improving cartilage function, which comprises the food composition for improving cartilage function according to any one of aspects (1) to (5), or a feed for improving cartilage function, which comprises the food composition for improving cartilage function according to any one of aspects (1) to (5).

Aspect (7) A method for improving cartilage function by daily intake of at least 1mg of lactoperoxidase, cystatin, HMG-like protein, and/or its decomposition product described in any one of aspects (1) to (5).

ADVANTAGEOUS EFFECTS OF INVENTION

The food composition for improving cartilage function according to the present invention is useful for the prevention and treatment of various joint diseases because of its effect of promoting the proliferation of chondrocytes and/or inhibiting the calcification of chondrocytes, and its significant effect of correcting, regenerating deformed or lost cartilage.

Drawings

FIG. 1 is a graph showing the thickness of the articular cartilage layer of the right hind limb of mice in six groups of mice which were fed with standard foods containing standard levels of phosphorus, high-phosphorus foods, and example products (innovative products), respectively.

FIG. 2 is a graph showing the number of chondrocytes present in the articular cartilage layer of the right hind limb of mice in six groups of mice fed for ten weeks with standard foods containing standard levels of phosphorus, high-phosphorus foods, and the preparations of examples, respectively.

Detailed Description

The present inventors have conducted intensive studies to solve the above problems, have found that proteins contained in milk and milk products, namely lactoperoxidase, cystatin, and high-speed swimming group (HMG) -like proteins and their decomposition products, promote the proliferation of chondrocytes and/or inhibit the calcification of chondrocytes, and correct or regenerate deformed or lost cartilage, and have completed the present invention.

It has not been found previously that proteins contained in milk and dairy products, i.e., lactoperoxidase, cystatin, HMG-like protein, and decomposition products thereof promote the proliferation of chondrocytes and/or inhibit the calcification of chondrocytes, and correct or regenerate deformed or lost cartilage.

The present invention is characterized by using, as an active ingredient, a protein contained in milk or a milk product, i.e., lactoperoxidase, cystatin, HMG-like protein, and/or a decomposition product thereof. Lactoperoxidase, cystatin, HMG-like protein used in the present invention may be those prepared from milk of mammals such as cow's milk, human milk, goat's milk, or sheep's milk, chemically synthesized products, those produced by genetic engineering techniques, and those purified from blood or organs. Commercially available reagents for purified lactoperoxidase, cystatin, HMG-like proteins may also be used. The decomposition products of lactoperoxidase, cystatin, and HMG-like proteins used in the present invention can be prepared from lactoperoxidase, cystatin, and HMG-like proteins treated with protease.

Lactoperoxidase, which is one of the active ingredients, is produced, for example, by a known method of purifying milk using a polysaccharide affinity carrier having sulfone groups introduced thereto (Japanese patent laid-open No. 3-109400). The decomposition product of lactoperoxidase can be prepared from lactoperoxidase in the presence of a protease such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease, and thus has a molecular weight of 10,000 or less, preferably 500 or more.

Another active ingredient, cystatin, is prepared by a known method for purifying milk by, for example, column chromatography (Japanese patent laid-open No. 2000-281587). The decomposition product of cystatin can be produced from cystatin in the presence of a protease such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease, and has a molecular weight of 8,000 or less, preferably 500 or more.

HMG-like protein as another active ingredient is produced by purifying milk by column chromatography, for example (Japanese patent laid-open No. 9-227403). The degradation product of the HMG-like protein can be prepared from the HMG-like protein in the presence of a protease such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease, and has a molecular weight of 6,000 or less, preferably 500 or more.

In the present invention, at least one active ingredient selected from the group consisting of lactoperoxidase, cystatin, HMG-like protein, and decomposition products thereof may be used as it is in the cartilage function-improving food composition according to the present invention. These proteins and their decomposition products can be formulated into a medicament in the form of a powder, granules, tablets, capsules, or liquid according to a usual method when necessary.

In the present invention, the active ingredient may be formulated as a medicament or added to a food or drink by any mixing or compounding process. For example, the active ingredients can be added and compounded in the following solutions: at least one active ingredient selected from the group consisting of lactoperoxidase, cystatin, HMG-like protein, and decomposition products thereof is suspended or dissolved in deionized water, and mixed with stirring. The solution is prepared into medicine or made into (finished) food, drink or feed for use.

The stirring and mixing can be carried out under any conditions that can uniformly mix the effective ingredients. For example, the active ingredients may be mixed with stirring using an ultrasonic disperser or a homomixer. When necessary, the solution of the composition may be used after desalting or concentrating or freeze-drying by a Reverse Osmosis (RO) or Ultrafiltration (UF) membrane, so that the composition solution is easily formulated into a medicament or used for food, drink or feed. The sterilization process generally used for the production of pharmaceuticals, foods, drinks, or feeds can be used in the present invention. The powder can also be sterilized by dry heat.

The cartilage function-improving food composition according to the present invention can be formed into various forms, such as liquid, gel, powder, and granule. The food composition for improving cartilage function according to the present invention after being formulated as a medicament may be compounded in foods, drinks and nutritional compositions such as nutritional supplements, yoghurts, milk drinks, and wafers (wafers).

The food composition for improving cartilage function according to the present invention contains at least one active ingredient selected from the group consisting of lactoperoxidase, cystatin, HMG-like protein, and decomposition products thereof as an active ingredient, and can be formulated into a medicament using a commonly used diluent or filler such as a filler, an extender, a binder, a disintegrant, a surfactant, and a lubricant. Examples of the filler include sucrose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicic acid, magnesium aluminate, synthetic aluminum silicate, magnesium aluminum metasilicate (magnesium aluminate), calcium carbonate, sodium bicarbonate, calcium hydrogen phosphate, and calcium carboxymethylcellulose. These fillers may be used alone or in combination.

The food composition for improving cartilage function according to the present invention may use stabilizers, saccharides, lipids, flavors (flavors), vitamins, minerals, flavonoids, and polyphenols in combination. These can be appropriately blended in the preparation of foods, drinks and feeds. These may also be used in combination with other ingredients that improve cartilage function such as glucosamine hydrochloride and lactoferrin.

As shown in the following experimental examples, in the food composition for improving cartilage function according to the present invention, at least one active ingredient selected from the group consisting of lactoperoxidase, cystatin, HMG-like protein, and decomposition products thereof can be orally administered to a mouse at 1mg/kg or more, thereby promoting the proliferation of chondrocytes and/or inhibiting the calcification of chondrocytes. The intake of the experimental animals corresponds to the intake of adults at blood concentration ("Yakkohouka (evaluation of Drug efficacy) Vol.8", Mitsuyoshi Nakashima (1993), Hirokawashoten, pp.2-18). In general, if an adult takes 1mg or more of at least one active ingredient selected from the group consisting of lactoperoxidase, cystatin, HMG-like protein, and decomposition products thereof per day, it is expected to improve the function of cartilage, and particularly to prevent and treat osteoarthritis.

Therefore, at least one active ingredient selected from the group consisting of lactoperoxidase, cystatin, HMG-like protein, and decomposition products thereof is formulated as a drug or compounded in a food or drink so that a necessary amount thereof can be secured. For example, in order to achieve 1mg or more of an active ingredient selected from the group consisting of lactoperoxidase, cystatin, HMG-like protein, and decomposition products thereof per day by an adult, the final product may contain at least one active ingredient selected from the group consisting of lactoperoxidase, cystatin, HMG-like protein, and decomposition products thereof in a proportion of 0.0005% to 5% (weight/weight), preferably 0.05 to 2.5% (weight/weight) relative to the total mass, depending on the form of the final product such as a medicine, food, drink, or feed.

The food composition for improving cartilage function may contain at least one active ingredient selected from the group consisting of lactoperoxidase, cystatin, HMG-like protein, and decomposition products thereof, or any combination of these ingredients. Specific examples thereof include one of the components; a combination of one of the three components with a decomposition product thereof; combinations of two of the three components, such as lactoperoxidase and cystatin, lactoperoxidase and HMG-like protein, and cystatin and HMG-like protein; a combination of one of the three components with a decomposition product thereof; a combination of lactoperoxidase, a cysteine protease inhibitor, and an HMG-like protein; and combinations of the three components, at least one of which is a decomposition product.

The food composition for improving cartilage function promotes the proliferation of chondrocytes and/or inhibits the calcification of chondrocytes. In other words, the food composition for improving cartilage function is useful for improving cartilage function due to its remarkable effect of correcting or regenerating cartilage that has been deformed or lost due to aging, trauma, or load (overweight) during exercise. Specifically, the food composition for improving cartilage function is useful for the prevention and treatment of various joint diseases such as osteoarthritis, particularly gonarthritis, and the like.

Examples of foods and drinks that may contain the food composition for improving cartilage function include, but should not be limited to, dairy products (e.g., cheese, cream (cream), butter (butter), anhydrous cream (butter oil), ice cream, fermented Milk, lactic acid bacteria beverage (fermented Milk), and Milk beverages) specified in article 2 of "ingredient Standards and the like on Milk and dairy products" in japan (minor organization on Milk and Milk products conceming. composite Standards, etc.), and foods and waffles based on these dairy products. Fermented milks include hard or set yogurts, soft or semi-fluid yogurts, and yoghurt drinks.

Examples and experimental examples of the present invention will be described in detail below. These are provided for illustrative purposes only and should not be construed as limiting the invention.

Examples

[ example 1]

(preparation of lactoperoxidase)

A column (diameter: 5cm, height: 30cm) packed with a cation exchange resin, sulfonated CHITO PEARL (manufactured by Fujibo Holdings, Inc.) (400g) was sufficiently washed with deionized water. Unsterilized skim milk (40L, pH: 6.7) was fed through the column at a flow rate of 25 ml/min. After the feeding, the column was sufficiently washed with deionized water, and elution was performed with 0.02M carbonate buffer (pH7.0) containing 2.0M sodium chloride. The eluted fraction containing lactoperoxidase was adsorbed on an S-Sepharose FF column (manufactured by GE Healthcare, Inc.), washed thoroughly with deionized water, and equilibrated with 10mM phosphate buffer (pH: 7.0). The adsorbed fraction was eluted by a linear gradient of 0 to 2.0M sodium chloride, thereby recovering the lactoperoxidase-containing fraction. The fractions were then subjected to gel filtration chromatography using HiLoad 16/60 Superdex 75 pg (manufactured by GE Healthcare, Inc.) to obtain lactoperoxidase (3.0 g). Lactoperoxidase had a purity of 94%. Lactoperoxidase can be used as it is in a food composition for improving cartilage function (cartilage function improving agent/example product 1).

[ example 2]

(preparation of lactoperoxidase decomposed product)

Lactoperoxidase (1g) prepared in example 1 was dissolved in water (200ml), and protease pancreatin (manufactured by Sigma-Aldrich Corporation) was added to make the final concentration 0.01 wt%. The solution was subjected to enzyme treatment at 37 ℃ for 5 hours. The enzyme was inactivated by heat treatment at 90 ℃ for 5 minutes. The solution was freeze-dried to obtain lactoperoxidase decomposed product (0.8 g). The lactoperoxidase decomposed product has a molecular weight of 10,000 or less. The product can be used as it is in a food composition for improving cartilage function (cartilage function improving agent/example product 2).

[ example 3]

(preparation of lactoperoxidase decomposed product)

Lactoperoxidase (1g) prepared in example 1 was dissolved in water (200ml), and protease trypsin (manufactured by Sigma-Aldrich Corporation) was added to make the final concentration 0.01 wt%. The solution was subjected to enzyme treatment at 37 ℃ for 5 hours. The enzyme was inactivated by heat treatment at 90 ℃ for 5 minutes, and freeze-dried, thereby obtaining lactoperoxidase decomposed product (0.9 g). The lactoperoxidase decomposed product has a molecular weight of 10,000 or less. The product can be used as it is in a food composition for improving cartilage function (cartilage function improving agent/example product 3).

[ example 4]

(preparation of cystatin)

The column packed with S Sepharose (3,000g) was washed thoroughly with deionized water. Skim milk (10,000L) was fed through the column. The column was washed thoroughly with deionized water and eluted with a linear gradient of 0.1 to 1.0M sodium chloride. The eluted fraction was subjected to heat treatment at 90 ℃ for 10 minutes, and centrifuged to remove the precipitate. The eluted fractions containing cystatin were re-fractionated by Mono S ion exchange chromatography. The fractions were sequentially treated by Mono Q ion exchange chromatography in a FPLC system followed by Superose 12 gel filtration chromatography and by hydroxyapatite chromatography in an HPLC system, and C4 reverse phase chromatography to give cystatin (58 mg). Cystatin can be used as such in a food composition for improving cartilage function (cartilage function improving agent/example product 4).

[ example 5]

(preparation of cystatin)

A5% whey protein solution (10,000L) was subjected to heat treatment at 90 ℃ for 10 minutes, and the precipitate was removed by centrifugation. The column was packed with a support in which carboxymethylated papain was bound to Tresyl-Toyopearl (manufactured by Tosoh Corporation). The system was equilibrated with 0.5M sodium chloride solution. The whey protein solution was fed through the column. After the feed, the column was washed with 0.5M sodium chloride solution followed by 0.5M sodium chloride solution containing 0.1% tween 20. In the next step, the cysteine protease was eluted with 20mM acetic acid-0.5M sodium chloride solution. The eluted fractions were rapidly neutralized with 1M sodium hydroxide solution and fractionated by sequential treatment by Mono S anion exchange chromatography and by hydroxyapatite chromatography and C4 reverse phase chromatography in an HPLC system to give cystatin (48 mg). Cystatin can be used as such in a food composition for improving cartilage function (cartilage function improving agent/example product 5).

[ example 6]

(preparation of cystatin degradation product)

Cystatin (25mg) prepared in example 4 was dissolved in water (100ml), and pancreatin (manufactured by Sigma-Aldrich Corporation) was added to make the final concentration 1%. The solution was subjected to enzyme treatment at 37 ℃ for 5 hours. The enzyme was inactivated by heat treatment at 90 ℃ for 5 minutes, and freeze-dried to obtain cystatin protease degradation product (example product 6A) (23 mg). Cystatin (25mg) prepared in example 5 was treated as described above to obtain cystatin degradation product (example product 6B) (24 mg). The cystatin has a molecular weight of 8,000 or less. The product can be used as it is in a food composition for improving cartilage function (cartilage function improving agent/example product 6A and example product 6B).

[ example 7]

(preparation of HMG-like protein)

The column packed with sulfonated CHITO PEARL (3,000g) (manufactured by Fujibo Holdings, Inc.) was thoroughly washed with deionized water. Skim milk (300L) was fed through the column. The column was washed thoroughly with deionized water. The basic protein was adsorbed onto the resin with 0.02M carbonate buffer (pH: 6.7) and recovered by elution with a linear gradient of 0.1M to 1.0M NaCl. In the next step, the eluted fraction containing HMG-like proteins was eluted by a linear gradient of 0.1M to 1.0M NaCl in S-Sepharose cation exchange chromatography with the system equilibrated with 0.1M phosphate buffer (pH: 6.5). The eluted fraction was subjected to heat treatment at 90 ℃ for 10 minutes, and centrifuged to remove the precipitate. The fractions were further fractionated by linear gradient of 0.1M to 1.0M NaCl in Mono Q ion exchange chromatography where the system was equilibrated with 0.1M phosphate buffer (pH: 6.5), and then by Sepharose 12 gel filtration chromatography where the system was equilibrated with 0.1M phosphate buffer (pH: 6.5). The fractions were finally subjected to high performance liquid chromatography using a C4 reverse phase column to thereby obtain HMG-like protein (135 mg). The HMG-like protein can be used as it is in a food composition for improving cartilage function (cartilage function improving agent/example product 7).

[ example 8]

(preparation of HMG-like protein degradation product)

HMG-like protein (25mg) prepared in example 7 was dissolved in water (100ml), and protease trypsin (manufactured by Sigma-Aldrich Corporation) was added so that the final concentration was 0.01% by weight. The solution was subjected to enzyme treatment at 37 ℃ for 5 hours. The enzyme was inactivated by heat treatment at 90 ℃ for 5 minutes. The solution was lyophilized to obtain HMG-like protein degradation product (24 mg). The lactoperoxidase decomposed product has a molecular weight of 6,000 or less. The product can be used as it is in a food composition for improving cartilage function (cartilage function improving agent/example product 8).

[ Experimental example 1]

(cell experiment)

The effect of promoting the proliferation of chondrocytes was evaluated with respect to lactoperoxidase of example product 1, lactoperoxidase-decomposed product of example product 2, cystatin of example product 4, cystatin-decomposed product of example product 6B, HMG-like protein of example product 7, HMG-like protein-decomposed product of example product 8, the mixture of example products 1 and 4, and the mixture of example products 5 and 7. In 96-well cell culture plates at 5X 10³Cell/well Density A mouse-derived chondroprogenitor cell line (ATDC5) was seeded and cultured in Dulbecco's Modified Eagle Medium/Ham's nutrient mixture F-12(Ham's nutrient mixture F-12) containing 5% fetal bovine serum at 37 ℃ in 5% CO₂The culture was carried out in an incubator under an atmosphere for 24 hours. The medium was then replaced entirely with fresh. Each of example products 1, 2, 4, 6B, 7, and 8, the mixture of example products 1 and 4, and the mixture of example products 5 and 7 dissolved in PBS (-) was added to the medium so that the final concentration was 1 μ g/mL, and then incubated for 48 hours. PBS (-) used as solvent was used as negative control. The medium was completely removed. The cell proliferation reagent WST-1 (manufactured by Roche Diagnostics GmbH) was added to the medium at a content of 1/10 in the medium, and the sample was cultured in the resulting medium for 6 hours. The absorbance of each sample was measured at 440nm using a plate reader. Because WST-1 is reduced to formazan dye by the metabolic activity of living cells, the amount of formazan dye is proportional to the number of metabolically active cells. Absorbance that reflects the amount of formazan dye is used as a fingerIndicates an index for promoting the proliferation of chondrocytes. The results are shown in table 1.

[ Table 1]

Values represent mean ± standard deviation (n ═ 5).

Indicates significant difference from PBS (-) (p < 0.05).

These results show that the number of chondroprogenitors significantly increased in the medium containing lactoperoxidase of example product 1, lactoperoxidase decomposed product of example product 2, cystatin of example product 4, cystatin decomposed product of example product 6B, HMG-like protein of example product 7, HMG-like protein decomposed product of example product 8, mixture of example products 1 and 4, and mixture of example products 5 and 7, as compared with the medium containing PBS (-). Therefore, lactoperoxidase, cystatin, HMG-like protein, and decomposition products thereof according to the present invention promote the proliferation of chondrocytes.

[ Experimental example 2]

(cell experiment)

The effect of inhibiting calcification of chondrocytes was evaluated with respect to lactoperoxidase of example product 1, lactoperoxidase-decomposed product of example product 3, cystatin of example product 5, cystatin-decomposed product of example product 6A, HMG-like protein of example product 7, HMG-like protein-decomposed product of example product 8, mixture of example products 1 and 7, and mixture of example products 4 and 8. 3X 10 in 96-well cell culture plates³Cell/well Density A mouse-derived chondroprogenitor cell line (ATDC5) was seeded and cultured in Dulbecco's modified Eagle Medium/Ham's nutrient mixture F-12 containing 5% fetal bovine serum at 37 ℃ in 5% CO₂The culture was carried out in an incubator under an atmosphere for 24 hours. The medium was then replaced entirely with fresh. Example preparations 1,3, 5, 6A, 7, and 8, example preparations 1 and 8 dissolved in PBS (-), were added7, and the mixtures of example articles 4 and 8 were each added to the medium to a final concentration of 1 μ g/mL. Cells were cultured for 7 days with daily medium changes. PBS (-) used as solvent was used as negative control. After the end of the incubation, the cells were fixed with 20% formalin solution for 20 minutes and washed with water. Water was added to the cells and the cells were allowed to stand for 15 minutes. Naphthol AS-BI phosphate and Fast Red Violet (LB) salt were dissolved in 0.05M 2-amino-2-methyl-1, 3-propanediol (AMP) solution to prepare alkaline phosphatase (ALP) staining solution (dying solution), and the cells were stained with the ALP staining solution at 37 ℃ for 20 minutes. After staining, cells were washed 5 times with water and air dried. From the image of the stained cells in the culture plate obtained using the scanner, the area of the stained cells was calculated with image processing software. The area of stained cells was expressed as a relative value with the value of negative control set to 1. ALP activity is an initial marker for calcification of chondrocytes, with smaller areas of stained cells indicating more inhibition of calcification. The results are shown in table 2.

[ Table 2]

Values represent mean ± standard deviation (n ═ 5).

Indicates significant difference from PBS (-) (p < 0.05).

The results showed that the area of ALP staining was significantly reduced in the medium containing lactoperoxidase of example product 1, lactoperoxidase decomposed product of example product 3, cystatin of example product 5, cystatin decomposed product of example product 6A, HMG-like protein of example product 7, HMG-like protein decomposed product of example product 8, mixture of example products 1 and 7, and mixture of example products 4 and 8. Therefore, lactoperoxidase, cystatin, HMG-like proteins, and degradation products thereof according to the present invention inhibit calcification of chondrocytes, as compared with a PBS (-) containing medium.

[ Experimental example 3]

(animal experiments)

The effects of lactoperoxidase of example product 1, lactoperoxidase-decomposed product of example product 3, cystatin of example product 4, and HMG-like protein of example product 7 on the initial denaturation of the articular cartilage layer were examined. 10-week-old C57BL/6J male mice were divided into 6 groups of 10 mice each, so as not to vary their weight between groups. Mice that ingested feed containing standard levels of phosphorus (0.2g/100g feed) without the example preparation (standard diet) were defined as group a, and mice that ingested feed containing large amounts of phosphorus (1.2g/100g feed) without the example preparation (high phosphorus diet) were defined as group B. A group that ingested the example article 1 was defined as group C, a group that ingested the example article 4 was defined as group D, a group that ingested the example article 7 was defined as group E, and a group that ingested the example article 3 was defined as group F. Each example preparation was blended with a high phosphorous diet such that the daily intake of the example preparation was 1mg/kg mouse. After ten weeks of rearing, the right hind limb was taken and dipped in a 10% neutral buffered formalin solution for fixation. Paraffin-embedded sections of the knee joints were prepared and hematoxylin-eosin staining was performed. Using the stained sample, the thickness of the articular cartilage layer and the number of chondrocytes present in the articular cartilage layer were measured, thereby evaluating the effect on the initial degeneration in the articular cartilage layer. The results are shown in fig. 1 and 2.

The results showed that in group B, which ingested the high-phosphorus diet without the example preparation, the thickness of the articular cartilage layer and the number of chondrocytes present in the articular cartilage layer were significantly reduced due to excessive ingestion of phosphorus, compared to group a, which ingested the standard diet without the example preparation. In contrast, in groups C to F taking example preparations 1,3, 4, and 7, the reduction in the thickness of the articular cartilage layer and the number of chondrocytes was significantly suppressed compared to group B. Therefore, lactoperoxidase, cystatin, and HMG-like protein, and decomposition products thereof according to the present invention inhibit initial degeneration of articular cartilage.

[ example 9]

(preparation of tablets for improving cartilage function)

The raw materials were mixed in the proportions shown in Table 3. The mixture was formed into 1g of a cartilage function-improving tablet by a usual method. The tablet (1g) contained 1mg of lactoperoxidase of example product 1.

[ Table 3]

[ example 10]

(preparation of nutrient composition for improving cartilage function)

Lactoperoxidase decomposition product (0.5g) of example product 3 was dissolved in deionized water (4,999.5g) and heated to 45 ℃. The raw materials were mixed with stirring at 6,000rpm for 30 minutes using a TK homomixer (TK ROBOMICS; Tokushu Kika Kogyo Co., manufactured by Ltd.) to prepare a solution containing 0.5g/5.0kg of the lactoperoxidase decomposed product of example product 3. The raw materials shown in table 4 were added to the solution (5.0kg), and further stirred and mixed for 10 minutes. The mixture was placed in a 200ml retort pouch (retort pouch) and sterilized with a sterilizing pot (type 1 pressure vessel, model: RCS-4 CRTGN, HISAKA WORKS, LTD. manufactured) at 121 ℃ for 20 minutes, thereby preparing 50kg of the liquid nutritional composition for improving cartilage function according to the present invention. The liquid nutritional composition for improving cartilage function contained 1mg of lactoperoxidase decomposed product of example product 3 per 100 g.

[ Table 4]

[ example 11]

(preparation of drink for improving cartilage function)

Skimmed milk powder (140g) was dissolved in deionized water (569.95 g). Cystatin (0.05g) of example product 4 was solubilized. The system was heated to 45 ℃ and stirred and mixed with an ultrasonic disperser (ULTRA-TURRAX T-25; manufactured by IKA Japan K.K) at 9,500rpm for 30 minutes. The raw materials shown in table 5 were added and further stirred and mixed for 15 minutes. The mixture was placed in a 100ml glass bottle and sterilized at 95 ℃ for 15 seconds. The bottles were sealed to prepare 10 bottles (100ml) of the cartilage function-improving beverage according to the present invention. The cartilage function-improving beverage contained 5mg of cystatin of example product 4 per 100 ml.

[ Table 5]

[ example 12]

(preparation of feed for improving cartilage function for dogs)

HMG-like protein of example product 7 (0.1g) was dissolved in deionized water (99.9 g). The system was heated to 45 ℃ and stirred and mixed with an ultrasonic disperser (ULTRA-TURRAX T-25; manufactured by IKA Japan K.K) at 9,500rpm for 40 minutes, thereby preparing a solution containing 0.1g/100g of HMG-like protein of example preparation 7. The raw materials shown in table 6 were added to the solution (100g), and further stirred and mixed for 40 minutes. The mixture was sterilized at 120 ℃ for 4 minutes, thereby preparing 1kg of the cartilage function-improving feed for dogs according to the present invention. The feed for improving cartilage function for dogs contained 10mg of HMG-like protein of example product 7 per 100 g.

[ Table 6]