JP3155746B1 - Method for producing type II collagen - Google Patents

Abstract

The present invention provides a method for extracting type II collagen from any raw material including cartilage without denaturation. SOLUTION: Impurities in a raw material are removed by using a high-pressure water stream, and high-molecular-weight and high-purity type II collagen is efficiently extracted.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing type II collagen, and a food product containing the type II collagen which is effective for the prevention and treatment of joint diseases.

[0002]

2. Description of the Related Art In recent years, functional foods and pharmaceuticals effective for joint diseases using type II collagen which is specifically present in cartilage tissue have been developed. Type II collagen is a component that exists specifically in cartilage tissues and the vitreous body of mammals, birds and fish, and has a small absolute amount as a raw material.
The use of type II collagen in food materials and pharmaceuticals has hardly been performed. On the other hand, collagen, which is widely used as food and food materials such as gelatin and sausage packaging materials, capsule materials such as pharmaceuticals, and cosmetic materials, is type I collagen (type III) contained in skin and bone. Collagen is included), and its production method is disclosed in Japanese Patent Publication No. 44-1175. On the other hand, cartilage has a small amount of type IX collagen and type XI collagen in addition to type II collagen.
Type collagen is substantially absent (Microscopy Resea
rch and Technique, 28, pp378-384, 1994). Therefore, highly purified type II collagen can be obtained by using a purified cartilage material.

[0003] However, cartilage is much softer than bone, and periosteum and tendons containing type I collagen penetrate into cartilage and are firmly bound, and meat is also attached to the surroundings. Unless impurities are removed, the conventional method for producing type I collagen cannot be applied as it is.
In addition, a method for producing type II collagen at the laboratory level is described in the literature (The Journal of Experimental Medicine, 146, pp.
857-868,1977, European Journal of Biochemistry, 18
1, pp. 159 to 173, 1989), which have already been established, but do not describe a method for purifying raw cartilage.
It is presumed that the type II collagen is probably purified by using a column or the like after removing the deposits by physical manual work and extracting the type II collagen. in this way,
It requires a great deal of labor and time, and it is impossible to process in large quantities. As described above, it is necessary to produce high-purity type II collagen in large quantities and at low cost because of low demand for type II collagen, few raw materials, and the need to develop special extraction technology. Was impossible.

As the only method for extracting a large amount of type II collagen, Japanese Patent Application Laid-Open No. 9-255581 discloses a method for producing a heat denatured type II collagen for treating rheumatoid arthritis using chicken rib cartilage as a raw material. . However, the type II collagen extracted by this production method is considered to contain the type I collagen and the like, and is gelatin whose tertiary structure has been modified by heating, and has a low molecular weight. In this patent, animal experiments show that low molecular weight denatured type II collagen is more effective than undenatured high molecular weight type II collagen that has not been thermally denatured. On the other hand, a report published in an American scientific journal (Science, 261, pp 1727 to 1730, 1993) states that native type II collagen with a molecular weight of about 100,000 was effective for rheumatoid arthritis. ,
The relationship between collagen molecular weight and morphology and its efficacy in rheumatoid arthritis is not clearly understood.

[0005] In addition, type II collagen in cartilage is surrounded by minerals such as calcium and proteoglycans such as chondroitin sulfate.
The hot water extraction described in US Pat. No. 55581 alone has a low extraction efficiency, and further contains proteins other than type II collagen and proteoglycans, and thus has a problem of low purification.

[0006] From the viewpoint of raw materials, Patent No. 9-25
Chicken costal cartilage used as a raw material in No. 5581 has a small absolute supply, and there is a concern that the raw material may be insufficient when industrially extracting type II collagen. Therefore, it is necessary to uniformly extract type II collagen from all raw materials containing cartilage such as scapular cartilage, costal cartilage, and bronchial cartilage of not only chickens but also large livestock animals such as fish, cows and pigs. In particular, the use of bovine scapular cartilage, which is currently discarded as industrial waste, has great industrial significance.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing high-purity, unmodified type II collagen in large quantities at low cost from cartilage materials of mammals, birds and fish.

[0008]

That is, the present invention provides a method for producing type II collagen, which comprises a step of injecting a high-pressure water stream into an animal tissue containing cartilage to remove impurities. Preferably, there is provided a method for producing type II collagen, which comprises the following steps (a) to (d). (A) a step of injecting a high-pressure water flow into an animal tissue containing cartilage to remove impurities, (b) a step of crushing under freezing,
(C) a step of removing calcium, and (d) a step of extracting type II collagen by pepsin digestion.

A method of separating bone and meat using a high-pressure water stream is disclosed in Japanese Patent Application Laid-Open No. 53-72879, a method of separating remaining meat and bone from the body of a chicken and collecting meat.
53 shows a method of injecting a high-pressure water stream after heat treatment in a method of separating fish bone and meat, but does not show an example in which a high-pressure water stream is used for purifying cartilage.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION In cartilage fragments generated during the slaughter process, residual meat and fat are firmly bound to the periosteum and tendons, and the periosteum and tendons are pierced and attached to the cartilage in the form of pinholes. In order to completely remove these deposits, if a treatment with an acid, an alkali or an enzyme is performed, the type II collagen in the cartilage is digested. Although it is possible to physically remove the cartilage using a brush or the like, the cartilage body is damaged. In the present invention, in order to remove the periosteum and tendons pierced into the minute holes, a high-pressure water flow is applied at a pressure of 5 to 150 MPa at a pressure of 20 to 5 per minute.
It is preferable to spray the cartilage surface with a water flow of 0 liter for several tens of seconds. However, it is natural that the condition of the high-pressure water flow is adjusted depending on the portion of the cartilage fragment used as a raw material. Further, the water temperature and the like are not particularly limited, and needless to say, unnecessary heating and cooling are not required.

Since the cartilage fragments are very soft compared to the bones and scatter when sprayed with a high-pressure water stream, it is naturally necessary to fix the cartilage fragments in advance with a wire mesh or the like, but the present invention is limited by the fixing method. It is of utmost importance to use a high pressure water stream instead.

The type II collagen can be extracted from the cartilage tissue having undergone the above pretreatment step in accordance with a conventional method.
One example is shown below. For example, the above raw material is -80 ° C
Freeze by freezing and storage at ~ -20 ° C, or instantaneously cool with liquid nitrogen, and further add liquid nitrogen and cool with a rotary cutter mill or the like while cooling.
Crush to 0 cm.

When the remaining fat is further removed from the pulverized cartilage pieces, the fat is removed by any of the following methods, but this step is not particularly required. Fat removal methods include (1) treatment with hexane or pentane, (2) treatment with alcohol such as ethanol, ketone or ether such as acetone or diethyl ether, (3) lipase treatment and dissolution.
(4) saponification and dissolution by an alkali metal hydroxide such as sodium hydroxide or an alkaline earth metal hydroxide such as calcium hydroxide; and (5) compression of fat by applying pressure.

[0014] Type II collagen in cartilage coexists with hydroxyapatite (calcium phosphate), and the extraction efficiency is reduced in its form. Therefore, calcium is removed by acidification using sodium citrate or hydrochloric acid. . The pH at this time is preferably from 1 to 4, and more preferably maintained at a pH of from 1.8 to 2.3.
Since the extraction efficiency of type II collagen changes depending on the degree of this decalcification, it is preferable that the initial calcium concentration be 90% by weight.
It is desirable to remove the above. The cartilage pieces excluding calcium are immersed in pepsin in an acidic aqueous solution to extract pepsin-soluble type II collagen.

The pepsin-soluble type II collagen solution is separated from the residue by physical means such as centrifugation. The step of precipitating and collecting type II collagen in the solution may be carried out by adding sodium chloride to increase the salt concentration or adjusting the pH to near neutrality. Further, the precipitated type II collagen is redissolved in an appropriate amount of an acidic solution, for example, an acetic acid solution, and dialyzed against a neutral solution such as a phosphate buffer (pH 6.5 to 8.0) or ion-exchanged water to obtain an acid. Recover soluble type II collagen. After collection, freeze-dry or
After mixing with an appropriate substance such as a dextrin (reduced maltose) solution and freeze-drying, it can be made to have a high purity of any concentration II.
A type collagen powder is obtained.

According to the method of the present invention, it is possible to efficiently remove the residual meat, fat, periosteum and tendon contained in cartilage tissue from any cartilage raw material and to extract type II collagen without denaturation.

The food containing type II collagen obtained by the present invention is particularly effective for rheumatoid arthritis, and has a synergistic effect when mixed with food materials effective for other joint diseases, so that it can be used widely. It can be used as a functional food. Examples of the food material include, but are not limited to, glucosamine hydrochloride, glucosamine sulfate, chondroitin sulfate, and plant extracts having an anti-inflammatory effect such as devil's claw and cat's claw. The amount of each compound is 0.1 to 2.0 g / day for glucosamine hydrochloride and glucosamine sulfate, 0.1 to 2.0 g / day for chondroitin sulfate, and 5 to 5 for type II collagen.
An amount that gives 200 μg / day is preferred.

[0018]

Next, the present invention will be described with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, all parts and percentages in this application are "parts by weight" and "% by weight", and temperatures are in degrees Celsius.

Example 1 (Production of Type II Collagen) 50 kg of bovine scapular cartilage, which had not been purified after collecting meat, was used as a starting material. The cartilage raw material for 50 kg of the starting material was treated for 60 seconds at a water temperature of 12 ° C. for 60 seconds under the conditions of a water pressure of 120 Mpa and a water flow of 40 liter / min for each piece of cartilage using a high-pressure water flow device. The total raw material weight after washing was 35 kg. Freezer (-25 ° C to -20 ° C)
With a rotary cutter mill equipped with a filtration net of 2 cm in diameter while flowing 500 ml of liquid nitrogen per minute at a rate of 1-2 cm in diameter.
Of cartilage (23 kg). The cartilage pieces were stirred in 50 liters of ethanol at 20 ° C. and a rotation speed of 5 rpm for 1 hour to remove residual fat. Thereafter, ethanol was replaced with 50 liters of water, and the mixture was further stirred for 10 minutes at a rotation speed of 5 rpm to perform washing. This operation was repeated three times.

The cartilage pieces were transferred to an acid-resistant kettle, and 50 liters of 0.1N hydrochloric acid was added thereto, and the pH was adjusted by dropping hydrochloric acid so as to maintain the pH between 1.8 and 2.3. Calcium was removed. Solid-liquid separation was performed, and the mixture was stirred for 30 minutes while flowing water at a flow rate of 10 L / min to wash the acid and calcium, and the decalcified cartilage powder was washed. Next, the demineralized cartilage was treated with a hydrochloric acid solution (p
H2.0-2.5) into 50 liters of the enzyme solution,
The mixture was treated at a rotation speed of 5 rpm at room temperature (15 to 20 ° C.) for 24 hours to extract type II collagen. Perform solid-liquid separation,
The type II collagen solution is collected and the phosphoric acid solution (pH 7.2)
7.5) for 2 hours to perform enzyme inactivation and collagen precipitation. The collagen solution recovered in the dialysis membrane was freeze-dried to obtain a type II collagen powder.

A part of the obtained powder was dissolved in a 0.1 M acetic acid solution so as to have a concentration of 1% (mass / volume), separated by electrophoresis, and silver-stained the protein. The main band was confirmed at the position, and by comparison with a commercially available bovine cartilage-derived type II collagen for research use as a standard substance,
It was confirmed that high-purity native type II collagen was obtained. Furthermore, as a result of performing immunostaining according to a conventional method using an anti-bovine cartilage type II collagen antibody and an anti-bovine type I collagen antibody, an antigen-antibody reaction was confirmed only in the former,
At the same time, it was confirmed that type I collagen was not mixed.

Example 2 (Measurement of Type II Collagen Recovery Rate) The collagen extracted in hot water and the collagen extracted in Example 1 were quantified using an antigen-antibody reaction. A commercially available type II collagen derived from bovine bronchial cartilage was used as a standard product, and an anti-bovine cartilage-derived type II collagen antibody was used as an antibody. In order to extract type II collagen from cartilage by hot water extraction, cartilage powder was prepared from 50 kg of bovine scapular cartilage in the same manner as in Example 1 according to the method of Example 1, and 50 liters of water was added and boiled for 30 minutes. . Solid-liquid separation was performed, and the collagen extract was freeze-dried to obtain a sample. As a result of the measurement, the amount of collagen obtained in Example 1 was 230 g, that obtained by hot water extraction was 180 g, and Example 1 was 27.8 compared with hot water extraction.
% Recovery was high, indicating high extraction efficiency.

Example 3 (Degree of Denaturation of Type II Collagen) 10 μg of each of the collagen obtained by the hot water extraction method obtained in Example 2 and the collagen obtained in Example 1 were dissolved in a 0.1 M acetic acid solution, and trypsin was dissolved. 0.2, 2.0, and 20.0 µg each were added, and digested for 1 hour at room temperature. The digested collagen is separated by electrophoresis according to a conventional method, and the digested protein is detected by silver staining. At this time, the collagen denatured in the extraction step is easily broken by digestive enzymes because the triple helical structure of the collagen is broken, and is likely to be a low molecular collagen fragment.
Therefore, the degree of collagen denaturation is compared using the remaining amount of high molecular weight collagen molecules having a molecular weight of about 100,000 as an index. However, the band concentration of untreated collagen that has not been subjected to the enzyme treatment is compared with a relative value with 100 as the band concentration. Table 1 shows the results. Although the band of type II collagen molecule having a molecular weight of 100,000 obtained in Example 1 was hardly reduced even when the trypsin was treated with 20 μg, the collagen extracted with hot water showed only about 2 μg of trypsin at around 100,000 even with trypsin amount of 2 μg.
No type I collagen band could be detected. This indicates that the type II collagen was denatured by hot water extraction and the triple helical structure of collagen was broken, making it more susceptible to enzyme digestion.

[0024]

[Table 1]

Example 4 (Reactivity with Rheumatoid Patient Sera) The proportion of autoimmune antibodies against type II collagen, which forms the cartilage skeleton of joints, is high in the sera of patients with rheumatoid arthritis. for that reason,
Joint cartilage is thought to be destroyed by its own immune system, causing rheumatism (The Lancet, 341, pp2
83-286, 1993; The Journal of Experimental Medicine,
146, pp85-868, 1977; Laboratory Investigation, 54,
pp26-31, 1986). Therefore, the effect of treatment with type II collagen on rheumatism depends on the reactivity of the patient with type II collagen antibody. Therefore, the reactivity of the serum prepared from the rheumatic patient with the collagen prepared in Example 1 is determined by the antigen-antibody reaction. Find out. Using various types of type II collagen, according to standard methods
ELISA method (Arthritis Rheumatism, 33, 10, pp1493-150
0, 1990), the anti-type II collagen antibody titer in the patient's serum was measured.

The types of type II collagen are as follows. Sample 1: Type II collagen powder extracted in Example 1
10 μg / ml sample 2: commercially available type II collagen derived from research bovine cartilage
10 μg / ml Sample 3: Diluent Only Serum used was as follows. Serum of patients with rheumatoid arthritis 1/10 with phosphate buffer
Diluted solution Serum from patients with non-rheumatic rheumatoid arthritis 1/1 with phosphate buffer
Table 2 shows the results of the diluted solution. Type II collagen extracted by this method was shown to have high reactivity with anti-type II collagen antibodies in patient serum. Furthermore, the same test was performed using non-patient serum, and as a result, the antigen-antibody reaction showed a very low value.

[0027]

[Table 2]

The following are examples of formulating functional foods using the type II collagen obtained by the present invention, but the present invention is not limited to these examples.

Formulation Example 1 (Manufacture of tablets) Ingredient composition (mass) Type II collagen powder 0.1 mg Corn starch 20.0 mg Lactose 169.3 mg Ascorbic acid 10.0 mg Rapeseed oil and fat powder 0.6 mg Total 200.0 mg According to a conventional method The mixture having the above composition was tableted to obtain tablets.

Formulation Example 2 (as a drink) Ingredient composition Type II collagen powder 0.1 mg Orange juice 100.0 ml Lemon juice 100.0 ml Total 200.0 ml After mixing the type II collagen powder with the above natural juice, a conventional method was used. And sterilized to produce a drink.

Formulation Example 3 (Functional food using glucosamine hydrochloride) Component composition (% by weight) Glucosamine hydrochloride 50.0% Corn starch 12.5% Lactose 35.0% Type II collagen powder 1.5% Rapeseed oil Powder 1.0% 100.0% The mixture having the above composition was tablet-molded according to a conventional method to obtain tablets.

Formulation Example 4 (Functional food using glucosamine hydrochloride and chondroitin sulfate) Component composition (% by weight) Glucosamine hydrochloride 50.0% Chondroitin sulfate 20.0% Erythritol 15.0% Starch hydrolyzate 12.0% Malic acid 1.0% Citric acid 1.0% Vegetable oil and fat powder 1.0% 100.0% According to a conventional method, the mixture having the above composition was tableted to obtain tablets.

[0033]

According to the present invention, high-purity high-molecular-weight type II collagen, which has not been achieved by the prior art, can be obtained from any cartilage tissue at a high yield and at low cost.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI A61P 19/02 A61K 37/12 (56) References JP-A-9-255581 (JP, A) (58) Fields investigated (Int. .Cl. ⁷ , DB name) A23J 1/10-3/34

Claims (6)

(57) [Claims] 1. A method for producing type II collagen, comprising the following steps (a) to (d). (A) a step of injecting a high-pressure water stream into an animal tissue containing cartilage to remove impurities; (b) a step of crushing under freezing; (c) a step of removing calcium; and (d) extracting type II collagen by pepsin digestion. Process. 2. The animal tissue is: scapular, costal or bronchial cartilage of livestock, bronchial or costal cartilage of poultry,
The production method according to claim 1 , wherein the production method is at least one of a nose column bone of a fish and a fin of a cartilage fish. 3. The condition of the high-pressure water flow is a water pressure of 5 MPa to 150 MPa.
The method according to claim 1 or 2, wherein the water is sprayed onto the surface of the cartilage with a water flow of 20 to 50 liters per minute. 4. A type II collagen produced by the method of any one of claims 1-3. 5. A food comprising the type II collagen according to claim 4 ( excluding foods containing a denatured type II collagen).
H) . 6. A food effective for joint diseases comprising the type II collagen according to claim 4 (heat denatured type II collagen).
) .