JP2013014529A - Type ii collagen obtained from notochord of sturgeons by simple extraction method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for extracting a type II collagen from sturgeons' notochords, and to provide the type II collagen extracted from sturgeons' notochords.SOLUTION: The method for extracting the type II collagen from sturgeons' notochords includes: a step of obtaining notochords from sturgeons' spines; a step of chopping the obtained notochords to extract the type II collagen; and a step of collecting the collagen.

Description

  The present invention relates to a method of extracting type II collagen from sturgeon notochord, and further relates to type II collagen derived from sturgeon notochord.

  Type II collagen is a collagen composed of three α1 chains present in the cartilage tissue and notochord of mammals, birds and fish, and has various uses. For example, it attracts attention as a scaffold material for cartilage regeneration and the like, and there is a high demand as a research reagent as a medical material that can be used for treatment of joint diseases and the like. Furthermore, it can be expected to be used as a health food.

  However, since type II collagen is contained in cartilage, notochord, etc., it is difficult to extract, and only type II collagen derived from chicken cartilage is currently on the market.

In recent years, due to the problem of zoonotic disease, type I collagen is being replaced from type I collagen derived from mammals and birds with type I collagen derived from fish.
There is also a report that type II collagen is extracted from fish (see Non-Patent Documents 1 to 5).

  In the conventional method, in order to extract type II collagen from fish, a method comprising the following series of steps has been performed. (1) recover the rostral cartilage and spine from the fish, (2) fragment or grind the tissue, (3) extract and degrade proteoglycans and non-collagenous proteins, (4) extract collagen ( Acetic acid, salt, enzyme, etc. are used), (5) Collagen is recovered.

  However, for type II collagen, as described above, pretreatment is complicated and extraction is difficult, and at present, it is difficult to efficiently extract type II collagen from fish in large quantities. As a result, fish type II collagen has not yet been put on the market.

Miller and Mathews, Biochem Biophys Res Comm 60: 424-430, 1974 Sheren et al., CBP 85B: 5-14, 1986 Kimura and Kimura, CBP73B: 335-339, 1982 Rama and Chandrakasan, Connect Tissue Res 12: 111-118, 1984 Kittiphattanabawon et al., LWT-Food science and Technology, 43: 792-800, 2010

  An object of the present invention is to provide a method for extracting type II collagen from sturgeon notochord, and a type II collagen extracted from sturgeon notochord.

  As mentioned above, it has been widely known that fish contains type II collagen, but its extraction method requires complex pretreatment, etc., and efficiently produces type II collagen on an industrial scale. Was difficult.

  At present, sturgeon is being cultivated actively for caviar and fillets. The present inventor uses a sturgeon as a type II collagen supply source in order to explore the possibility of effective utilization of sturgeon other than as a source of caviar and fillet, and a method for efficiently extracting a large amount of type II collagen from sturgeon We conducted an intensive study. The inventor conducted analysis and characteristic evaluation of collagen in each part of sturgeon. As a result, we found that sturgeon notochord is a promising source of type II collagen. Generally, the notochord is inside the fish spine. The sturgeon has a unique shape in which the spine has no nodes and the shape of the notochord is a rod, and the notochord can be easily recovered. Furthermore, high-purity type II collagen could be efficiently extracted directly from the notochord without performing complicated pretreatment as in the conventional method of extracting type II collagen from fish. That is, (1) the notochord is recovered from the sturgeon spine, (2) the recovered notochord is cut, collagen is extracted (using acetic acid, salt, enzyme, etc.), and (3) the collagen is recovered to obtain high purity. The type II collagen could be obtained efficiently.

  Moreover, when the characteristic analysis of the obtained type II collagen derived from the sturgeon notochord was performed, the type II collagen derived from other fish species and other tissues of the sturgeon had different characteristics.

That is, the present invention is as follows.
[1] A method for extracting type II collagen from the sturgeon of sturgeon,
Collecting notochord from sturgeon vertebrae,
A method comprising: chopping the collected notochord and extracting type II collagen; and collecting the collagen.
[2] The method according to [1], further comprising collecting a notochord from the notochord and extracting type II collagen from the notochord.
[3] The method according to [1] or [2], wherein the collected notochord or chord sheath is not decalcified, degreased, or solubilized using guanidine hydrochloride.
[4] The method according to any one of [1] to [3], wherein the sturgeon is Vestel or Amur sturgeon.
[5] The process of extracting type II collagen
(i) a step of washing the chord or chordal sheath of shredded sturgeon,
(ii) treating with 0.1-1% (w / w) pepsin, based on the weight of the notochord or notochord used,
(iii) centrifuging, collecting the supernatant, and treating with 0.01-0.1% (w / w) pepsin based on the weight of the notochord or chord sheath used;
(iv) filtering to remove impurities,
(v) obtaining a type II collagen precipitate by salting out; and
(vi) Dialysis process,
Any one of the methods [1] to [4].
[6] The denaturation temperature measured by a circular dichroism (CD) spectrometer extracted by any one of the methods [1] to [5] is 33 ° C. or higher, and the imino acid residue in 1000 amino acid residues Type II collagen derived from sturgeon notochord having a base number of 200 or more and a hydroxylation rate of lysine of 57% or more.

  When type II collagen is extracted using sturgeon notochord as a raw material, type II collagen can be directly extracted from sturgeon nodule without complicated pretreatment due to its morphological characteristics. Therefore, highly purified type II collagen can be efficiently obtained at low cost from the sturgeon of sturgeon. Type II collagen obtained from sturgeon notochord may cause the contamination of prion causing BSE (bovine spongiform encephalopathy) like type II collagen derived from mammals, and the risk of contamination of avian influenza virus like type II collagen derived from birds Absent. Furthermore, type II collagen derived from sturgeon notochord has an advantage that the denaturation temperature is higher than collagen derived from other tissues.

It is a figure which shows the outline | summary of the method of extracting a type II collagen. It is a figure which shows the pre-processing method of the scale of sturgeon, skin, muscles, the rostral cartilage, and the spine. It is a figure which shows the extraction efficiency of the type II collagen extracted from each structure | tissue of each fish species, and the collection amount per 1 individual. It is a result which shows the SDS-PAGE image of the type II collagen extracted from each structure | tissue of each fish species. It is a figure which shows the amino acid composition of the type II collagen extracted from each structure | tissue of sturgeon (Vestel). It is a figure which shows the hydroxylation rate of Lys and Pro of type II collagen extracted from each structure | tissue of sturgeon (Vestel). It is a figure which shows the denaturation temperature of the type II collagen extracted from each structure | tissue of each fish species. It is a figure which shows the relationship between the denaturation temperature of type II collagen extracted from each structure | tissue of each fish species, and imino acid content. It is a figure which shows the extraction efficiency of the type II collagen extracted from Amur sturgeon. It is a figure which shows the result of the purity test of the type II collagen extracted from Amur sturgeon. It is a figure which shows the result of the amino acid analysis of the type II collagen extracted from Amur sturgeon. It is a figure which shows the result of the hydroxylation rate analysis of the type II collagen extracted from Amur sturgeon. It is a figure which shows the result of denaturation temperature measurement of type II collagen extracted from Amur sturgeon.

Hereinafter, the present invention will be described in detail.
In the present invention, sturgeons are used as a source of type II collagen. The sturgeon is a fish belonging to the teleost Acipenseriformes, and includes fish belonging to the Acipenseridae and the Polyodontidae. Specifically, sturgeon (Acipenser medirostris), sturgeon (Acipensor ruthenus), Amur sturgeon (Acipenser schrencki), white sturgeon (Acipenser transmontanus), Siberian sturgeon (Acipenser baeri), Russian sturgeon (Acepencer gueldencipi) sinensis), Yangtze sturgeon (Acipencer dabryanus), ship sturgeon (Acipenser nudiventris), sturgeon shark (Acipenser stellatus), giant sturgeon (Huso huso), etc. , Acipenser persicus, Acipenser sturio, Huso dauricus, Pseudoscaphirhynchus fedtschenkoi, Pseudoscaphirhynchus hermannii, Pseudoscaphirhynchus kaufmanni, Scaphirhynchus albus, Scaphirhynchus platorynchun, Scaphirsichu Also, hybrids of these species can be used. Examples of hybrids include, for example, Vaster, which is a hybrid of a female sturgeon (belgous sturgeon) female and a male sturgeon. Sturgeons can be captured and used wild, but sturgeons are actively cultivated to use eggs and fillets for food, and the rest of the cultured fish from which the eggs and fillets are collected is used. preferable.

  Tissues such as sturgeon scales, cartilage, and spine contain collagen, but most tissues contain type I collagen. The spine and rostral cartilage contain type II collagen, but the spine contains not only type II collagen but also type I collagen. In addition, since many tissues contain a large amount of calcium, fat, and the like, a complex pretreatment for decalcification and degreasing is essential when collagen is extracted from these tissues. For example, when scales are used, a large amount of calcium salt is included, so pretreatment for decalcification is necessary. When using skin, muscles, gastrointestinal tract, bladder, cartilage, spine, etc., a large amount of fat Therefore, a treatment for degreasing is necessary. Here, deashing was performed by pretreatment with EDTA, sodium citrate, or the like or alkaline solution treatment, and degreasing was performed by pretreatment with an alcohol such as ethanol or an organic solvent such as acetone, or pretreatment with lipase or the like. Furthermore, when using cartilage and spine that can contain type II collagen, it is necessary to extract fat, proteoglycan, and non-collagenous protein contained in the tissue by degreasing treatment and protein solubilization pretreatment using guanidine hydrochloride. Become.

  On the other hand, when sturgeon notochord is used, complicated pretreatment such as decalcification, degreasing, and solubilization is not required. In particular, it is not necessary to perform protein denaturation treatment (solubilization treatment) using guanidine hydrochloride. For this reason, the fall of the recovery rate by pre-processing can be prevented. Furthermore, since no pretreatment is required, type II collagen can be extracted with low cost and high efficiency. Further, since sturgeon notochord contains only type II collagen, it is not necessary to separate it from type I collagen. Therefore, in the present invention, sturgeon notochord is used as a source of type II collagen.

  That is, the present invention is a method of extracting type II collagen from the sturgeon's notochord, the step of collecting the notochord from the sturgeon spine, the step of chopping the collected notochord and extracting type II collagen, and the collagen And a recovery step, and does not require pretreatment steps such as decalcification, defatting, proteoglycan, and non-collagenous protein extraction.

  The chords of sturgeon are collected from the spine (spine) of sturgeon. The vertebrae of other vertebrates other than sturgeons are formed by connecting many vertebrae, and there are nodes, whereas the vertebrae of sturgeon exists in a state where the spine is not connected, so no nodes are formed. The notochord is rod-shaped. Usually, in vertebrates, the notochord is surrounded by surrounding bones and cartilage, leaving only traces in the spinal column. In sturgeons, the notochord remains in the spine for the rest of its life. . Therefore, in sturgeon, the spine can be easily removed from the spine by splitting the spine into 10 centimeters and then vertically dividing it, and then removing the notoch with the hand. It can use suitably as a supply source. The sturgeon's notochord consists of an outer chordal sheath and an inner spinal cord. Type II collagen is mainly contained in the notochord. In the present invention, when extracting type II collagen from the notochord of sturgeon, the notochord sheath may be collected from the notochord and the type II collagen may be extracted from the notochord sheath, or the notochord including the notochord sheath and the notochord spinal cord is left as it is. It may be used. In the case where the chorda sheath is collected from the notochord and the type II collagen is extracted from the notochord sheath, the type II collagen content per weight of the material used is increased, and the extraction efficiency is improved. In addition, when the chorda sheath is collected from the notochord and the type II collagen is extracted from the notochord sheath, the content of unnecessary organic substances contained in the waste liquid after the recovery of the type II collagen can be reduced. In the present invention, when the notochord is collected from the notochord and the type II collagen is extracted from the notochord, the extraction includes the type II collagen from the notochord.

  Shred the notochord into small pieces of appropriate size. The shredding may be performed to a size of about 1 cm × 1 cm, for example. Shredding can be performed using, for example, an industrial cutter or scissors. At this time, it may be shredded to the above size, and there is no need for grinding (grinding).

  The chopped notochord is washed with water for several hours to several days, preferably about one day. The washing is performed, for example, by immersing the cut notochord in deionized water and stirring using a stirrer, and changing the deionized water several times during that time. In order to avoid denaturation of type II collagen, the washing is preferably performed at a low temperature, for example, under refrigerated conditions of 2 to 15 ° C, preferably 2 to 8 ° C.

  Next, the washed notochord shredded material is treated with a protease (proteolytic enzyme) under acidic pH conditions to decompose the notochord tissue and facilitate extraction of type II collagen. At this time, pepsin, chymotrypsin, or the like, which is an acidic protease, is used as the protease. The pH may be adjusted to pH 1-6, preferably about pH 1-2 using an acid such as hydrochloric acid. For example, the notochord shredded material is immersed in a hydrochloric acid solution 3 to 10 times, preferably 4 to 8 times, more preferably 6 times the weight of the shredded material, and pepsin is added to 0.1 to 5% of the tissue weight ( w / w), preferably 0.1 to 1% (w / w), more preferably 0.5% (w / w), dissolved at low temperature, eg 2 to 15 ° C. for several hours to several days, For example, protease treatment (pepsin digestion) is performed by stirring for 1 day. Type II collagen is dissolved in the solution and extracted by protease treatment. Thereafter, the solution is centrifuged and the supernatant is recovered. Usually, the extracted collagen is subjected to alkali treatment using a strong alkaline solution such as 10M NaOH solution. However, when type II collagen is extracted using sturgeon notochord, alkali treatment after the extraction is not necessary.

  Centrifugation may be performed at a low temperature, for example, at 2 to 15 ° C., 1000 to 5000 g, preferably 1000 to 3000 g, for 5 to 60 minutes, preferably 10 to 30 minutes. The collected supernatant contains type II collagen, and then the protease treatment is performed again to atherotheize type II collagen. Atelolation refers to removal of telopeptides present at the ends of the helical structure of collagen. Since the collected supernatant is acidic, it is preferable to use an acidic protease such as pepsin or chymotrypsin as the protease. At this time, the protease may be used at a concentration of about 0.01% to 0.5% (w / w), preferably about 0.01% to 0.1% (w / w) based on the weight of the notochord used. Atelolation is carried out by allowing to stand at 10 ° C. to 30 ° C., preferably 15 ° C. to 25 ° C. below the denaturation temperature of type II collagen, for several hours to several days, for example, one day. Next, centrifugation is performed again to remove impurities, and the supernatant is recovered. Centrifugation at this time may be performed at a low temperature, for example, 2 to 15 ° C., 5000 to 50000 g, preferably 10,000 to 30000 g for 5 to 60 minutes, preferably 5 to 30 minutes.

  Impurities are further removed by filtering the collected supernatant. For the filtration, a membrane filter can be used. Examples of the membrane filter include a cellulose acetate type, a cellulose mixed ester type, and a polycarbonate type, and a cellulose acetate type is preferable. A membrane filter having a pore size of 0.2 μm to 5 μm may be appropriately combined, and filtration may be performed in the order from the largest pore size to the smallest pore size. For example, a filter having a pore size of 3.0 μm, 0.8 μm, and 0.45 μm may be used in order.

  After filtration, type II collagen is precipitated from the filtrate by salting out. Salting out can be performed by a known method using sodium chloride, ammonium sulfate or the like. For example, sodium chloride is added to the filtrate so as to be 1 M, and the mixture is allowed to stand overnight at a low temperature (2 to 15 ° C.). After that, the solution is centrifuged and the supernatant is removed. Centrifugation may be performed at a low temperature, for example, at 2 to 15 ° C., 1000 to 5000 g, preferably 1000 to 3000 g for several tens of minutes to several hours, preferably 60 to 180 minutes, preferably 60 to 120 minutes. The precipitate may be dissolved in hydrochloric acid having pH 1 to 4, preferably pH 2. Salting out, centrifuging and dissolving the precipitate are repeated several times, preferably three times.

  Furthermore, the dissolved precipitate is dialyzed against distilled water and concentrated to obtain type II collagen with high purity. The purity of the obtained type II collagen can be confirmed by HPLC or SDS-PAGE. Type II collagen preparations can be stored by lyophilization.

An outline of the type II collagen extraction method of the present invention is shown in FIG.
The purity of type II collagen obtained by the above method is 95% or more, preferably 99% or more as the purity estimated from the band density of the SDS-PAGE image.

  Further, the extraction efficiency of type II collagen from sturgeon notochord as a raw material is about 0.2 to 5%, preferably about 0.3 to 3%. About 100 to 500 mg of type II collagen can be recovered from the notochord of one sturgeon with an average weight of 2 kg.

Furthermore, type II collagen extracted from sturgeon notochord has the following characteristics.
Amino acid composition Type II collagen obtained from sturgeon notochord has Ser (serine) content, Ala (alanine) content, and Lys (lysine) content compared to type I collagen obtained from other tissues of sturgeon. Low, Glx (Gln (glutamine) and Glu (glutamic acid)) content, Leu (leucine) content, HyLys (lysine hydroxide), Pro (proline) content and imino acids (HyPro (hydroxyproline) and Pro (proline)) It is characterized by a high content.

The content of each amino acid is as follows. The content of each amino acid is indicated by the number of amino acid residues in 1000 amino acid residues.
Ser: 35 or less, preferably 33 or less, or preferably 30 to 33, more preferably 31 to 32
Ala: 100 or less, or 90-100
Lys: 20 or less, preferably 18 or less, more preferably 17 or less, or preferably 13 to 18, more preferably 13 to 17
Glx (Gln + Glu): 80 or more, preferably 85 or more, or 80 to 100, preferably 85 to 100
Leu: 25 or more, preferably 27 or more, or 25 to 35, preferably 27 to 33
HyLys: 20 or more, or 20-30, preferably 20-27
Pro: 115 or more, preferably 120 or more, or 115 to 145, preferably 120 to 140
Imino acid (Hypro + Pro): 200 or more, preferably 210 or more, or 200 to 250, preferably 210 to 250

Hydrolysis rate of lysine Type II collagen obtained from sturgeon notochord is expressed by Lys (lysine) hydroxylation rate (HyLys number of HyLys residues) compared to type I collagen obtained from other sturgeon tissues. The ratio of the number of residues to the total number of Lys residues) is high and is 55% or more, preferably 56% or more, more preferably 57% or more, still more preferably 58% or more, and particularly preferably 60% or more.

Denaturation temperature Type II collagen obtained from sturgeon notochord has a higher denaturation temperature than type I collagen obtained from other fish species and type I collagen obtained from other tissues of sturgeon at 30 ° C. Above, preferably 31 ° C. or higher, more preferably 32 ° C. or higher, particularly preferably 33 ° C. or higher.

  Here, the denaturation temperature is a temperature at which the triple helical structure of the collagen molecule is broken and the collagen molecule is denatured into gelatin, and is a value measured by a circular dichroism (CD) spectrometer.

  The type II collagen derived from sturgeon notochord extracted by the method of the present invention can be used as a medicine, food or medical material. The present invention also includes pharmaceutical compositions, foods and drinks, and medical materials containing type II collagen derived from sturgeon notochord. Examples thereof include pharmaceutical compositions for treating or preventing joint diseases, foods and drinks for treating or preventing joint diseases, and medical materials as scaffold materials for cartilage regeneration and the like. Foods and drinks containing type II collagen derived from sturgeon notochord can be used as health foods, functional foods, foods for specified health use, dietary supplements and the like effective for the treatment and prevention of joint diseases and the like. The food for specified health is a food that is ingested for the purpose of specific health in the diet, and indicates that the purpose of the health can be expected by the intake. These foods, for example, indicate that they are used to treat or prevent joint diseases, indicate that they are to relieve joint pain, or improve joint movement. It can be provided as a food or drink with a display such as an indication.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example 1 Extraction of Type II Collagen from Sturgeon (Vestel) Notochord Sturgeon (approximately 80 cm long) bred by aquaculture was used. At this time, as sturgeon, Vestel, which is a hybrid of a giant sturgeon (Huso huso) and a sturgeon (Acipenser ruthenus), was used.

  The spinal cord was divided into 10 centimeters along with the notochord, then vertically divided into two, and the notochord was removed from the spine by peeling the notochord with the hand. The weight of the collected sturgeon notochord was about 50 g.

  The collected sturgeon notochord was finely cut into a size of about 1.0 × 1.0 cm using scissors. The cut notochord was washed for 1 day by dipping in deionized water at 4 ° C. and stirring with a stirrer. During this time, deionized water was changed four times.

  The washed notochord shredded material is immersed in a hydrochloric acid solution of pH 2.0, 6 times the weight of shredded material, and pepsin (Wako Pure Chemical Industries, Ltd.) is added to 0.5% (w / w) of the tissue weight. The mixture was gently stirred at 10 ° C for 1 day. By this operation, type II collagen was extracted into hydrochloric acid.

  The hydrochloric acid solution from which type II collagen was extracted was centrifuged at 2000 g for 20 minutes at 4 ° C., and the supernatant was collected. To the collected supernatant, 0.05% (w / w) pepsin based on the weight of the notochord used as a raw material is added and left at 10 ° C. for 7 days to perform atelolation, and the telopeptide present at the end of collagen Was removed.

  The resulting solution containing type II collagen was centrifuged at 22000 g for 10 minutes at 4 ° C. to remove impurities, and the supernatant was collected. The collected supernatant was filtered stepwise using cellulose acetate membranes having different pore sizes (3.0, 0.8, and 0.45 μm: Toyo Filter Paper Co., Ltd.).

  Sodium chloride was added to the obtained filtrate to a final concentration of 1.0 M, and the mixture was allowed to stand at 10 ° C. overnight for salting out.

  Thereafter, the mixture was centrifuged at 2000 g for 90 minutes at 4 ° C., and the supernatant was removed. The precipitate was dissolved in hydrochloric acid at pH 2.0. Salting out and dissolution of the precipitate were repeated 3 times in total.

Thereafter, the dissolved product was dialyzed against distilled water, and the obtained type II collagen was lyophilized.
An outline of the extraction method is shown in FIG.

Comparative Example 1 Extraction of collagen from sturgeon scales, skin, muscle, bladder, rostral cartilage, vertebral cartilage Collagen was extracted from sturgeon scales, skin, muscle, bladder, rostral cartilage, and spinal cartilage.
The following pretreatment was performed for each tissue. An outline of the preprocessing method is shown in FIG.

Scale Sturgeon scales were collected and vigorously stirred in deionized water to remove attached skin. The skin that could not be removed by this operation was removed using tweezers and a scalpel. Thereafter, decalcification was performed in a 0.5 M EDTA solution at 4 ° C. for 3 days to soften the calcium salt by elution. Then, after confirming the degree of demineralization with 1% silver nitrate, the obtained one was used as a raw material as demineralized scale.

Skin and muscle Sturgeon skin or muscle was cut into pieces, degreased by soaking in 99.5% ethanol and treating at 4 ° C. for 2 days, and used as a raw material.

Stomach cartilage and vertebral cartilage Cartilage obtained from sturgeon snout or sturgeon vertebra is fragmented, degreased by soaking in 99.5% ethanol and treating at 4 ° C. for 2 days, and 50 mM Tris-HCl containing 4 M guanidine hydrochloride ( It was immersed in pH 7.5), treated at 4 ° C. overnight, and used as a raw material.

Gastrointestinal tract The sturgeon gastrointestinal tract was cut open and the contents were thoroughly scraped with a scalpel, followed by washing in deionized water at 4 ° C. for about 2 hours to remove blood and contents. Thereafter, it was cut into small pieces and used as a raw material.

Floating bag Sturgeon floating bag was cut into small pieces and used as a raw material.

  Extract collagen from decalcified scale, treated skin, muscle, rostral cartilage, vertebral cartilage, gastrointestinal tract, and bladder obtained by the above pretreatment by the same method as in Example 1 (outline is shown in FIG. 1) did. However, in the bag, the amount of the extract was 18 times, not 6 times the tissue weight. When extracting collagen from scales, rostral cartilage and vertebral cartilage, the extraction operation was repeated four times.

Comparative Example 2 Extraction method of collagen from each tissue of tilapia 5 tilapia individuals (about 26 cm fork length) were immersed in 0.1% 2-phenoxyethanol and anesthetized, then all scales except for side line scales were collected and descaled. Later skin, muscle, bladder and gastrointestinal tract were collected.

The scales were agitated in deionized water at 4 ° C. for 3 days to remove cells attached to the surface. Thereafter, it was decalcified for 1 day under the same conditions as sturgeon scales. Completion of decalcification was confirmed by subjecting some scales to silver nitrate staining. The obtained product was used as a raw material as demineralized scale.

After collecting the air bag, it was washed in deionized water at 4 ° C for about 2 hours to remove the adhering blood. Thereafter, it was cut into pieces and used as raw materials.

Gastrointestinal tract The gastrointestinal tract was cut open and the contents were removed with a scalpel, etc., and then washed in deionized water at 4 ° C. in the same manner as the float. Thereafter, it was cut into pieces and used as raw materials.

Skin and muscle Skin and muscle were cut into pieces and used as raw materials.
Collagen extraction and purification used the same method as Vestel's tissue. However, the extraction temperature was 20 ° C and the centrifugation was performed at 10 ° C. In addition, the amount of the extract of the floating bag was set to 6 times the tissue weight. The number of extractions was once for all tissues.

Example 2 Analysis of Type II Collagen Extraction Efficiency of Collagen and Amount Collected per Individual FIG. 3 shows the extraction efficiency (%) of collagen extracted from each tissue of sturgeon and each tissue of tilapia by the method described in Example 1 and Comparative Example. ) And the recovered amount (mg) per individual.
The extraction efficiency was expressed as a percentage (%) of the recovered collagen dry weight with respect to the wet weight of the raw material used.

  The figure also shows the extraction efficiency of goldfish, but the value of goldfish is quoted from Ogawa, 2010 (Ogawa Nobuhiro (2010) Research on early calcification mechanism of goldfish (Carassius auratus) regenerative scale, Hokkaido University dissertation). is there.

Determination of Purity of Extracted Collagen Collagen extracted from each tissue of sturgeon and each tissue of tilapia was analyzed by SDS-PAGE using 7.5% acrylamide gel.

  The results are shown in FIG. From the SDS-PAGE image, (1) the extracted collagen is highly purified, (2) each tissue of tilapia and sturgeon (Vestel) scales, skin, muscle, bladder, and digestive tract collagen are 100- Since it has two main bands around 120 kDa, it is type I collagen. (3) Vestel's notochord and rostral cartilage collagen has one main band around 130 kDa. As well as (4) Vester's vertebral cartilage collagen has three main bands, indicating that it is a mixture of type I and type II collagen.

Amino acid analysis of collagen extracted from each tissue of sturgeon Hydrolyzed collagen extracted from sturgeon scales, skin, muscles, bladder, digestive tract, notochord, rostral cartilage and vertebral cartilage and then fully automated amino acid analyzer (JLC- The amino acid composition was analyzed using 500V, JEOL). About the scale, the 1st extract and the 4th extract were used.

  The results are shown in FIG. In FIG. 5, the amino acid content is shown by the number of amino acid residues in 1000 amino acid residues. As shown in the figure, type II collagen extracted from sturgeon notochord has lower Ser (serine) content, Ala (alanine) content, and Lys (lysine) content than collagen obtained from other tissues of sturgeon. Glx (Gln (glutamine) and Glu (glutamic acid)) content, Leu (leucine) content, HyLys (hydroxylized lysine), Pro (proline) content and imino acid (HyPro (proline hydroxylated) and Pro (proline)) content It had the characteristic of being high.

Measurement of hydroxylation rate of Lys and Pro The hydroxylation rates of lysine (Lys) and proline (Pro) of collagen extracted from sturgeon scales, skin, muscle, bladder, digestive tract, notochord, rostral cartilage and spine were calculated. The hydroxylation rate was determined by the percentage of HyLys with respect to the value of HyLys + Lys in FIG. 5 and the percentage of Hypro with respect to the value of HyPro + Pro.

  The results are shown in FIG. Type II collagen extracted from sturgeon notochord has a higher hydroxylation rate of Lys than type I collagen extracted from other tissues of sturgeon, and the hydroxylation rate of Lys extracted from other tissues is 24.3% to 55.3% However, it was 61.3%.

Measurement of denaturation temperature of collagen extracted from various tissues Measurement of denaturation temperature of collagen extracted from sturgeon scale, skin, muscle, bladder, digestive tract and notochord, and tilapia scale, skin, muscle, bladder, and digestive tract . For the sturgeon scale, the first extract ((1) in FIG. 7) and the fourth extract ((4) in FIG. 7) were used. The measurement of the denaturation temperature of collagen is known in Ikoma et al. (2003) (Ikoma T, Kobayashi H, Tanaka J, Walsh D and Mann S (2003) Physical properties of type I collagen extracted from fish scales of Pagrus major and Oreochromis niloticas According to the method of International Journal of Biological Macromolecules 32: 199-204.

  The results are shown in FIG. As shown in the figure, the denaturation temperature of type II collagen extracted from sturgeon notochord was higher than that of collagen extracted from other tissues of sturgeon. Goldfish values are quoted from Ogawa (2010).

  Further, FIG. 8 shows a graph obtained by plotting the denaturation temperature and imino acid content of collagen extracted from various tissues. From this figure, it can be seen that the collagen denaturation temperatures of the various vestel and tilapia tissues obtained this time tend to increase as the imino acid content increases, as in other fish species and mammals. On the other hand, since the collagen denaturation temperatures of the various vestel and tilapia tissues obtained this time are not completely proportional to the imino acid content, there is another mechanism that produces a difference in collagen denaturation temperature for each tissue. I guess that.

Example 3 Collagen Extraction Method from Each Amur Sturgeon Tissue A sturgeon (about 80 cm long) bred by aquaculture was used. At this time, Amur sturgeon (Acipenser schrencki) was used as the sturgeon.

  The spinal cord was divided into 10 centimeters along with the notochord, then vertically divided into two, and the notochord was removed from the spine by peeling the notochord with the hand. The weight of the collected Amur sturgeon chord was about 24 g.

Only the notochord sheath was extracted from the collected notochord. The extracted chordal sheath was about 17 g.
The collected Amur sturgeon chordal sheath was finely cut into a size of about 1.0 × 1.0 cm using scissors. The chopped notochord was washed for 1 day by immersing in deionized water at 4 ° C. and stirring with a stirrer. During this time, deionized water was changed four times.

  The washed notochord sheath shredded material is immersed in a hydrochloric acid solution of pH 2.0, 10 times the weight of shredded material, and pepsin (Wako Pure Chemical Industries, Ltd.) is added to 1% (w / w) of the tissue weight. ) And gently stirred at 4 ° C. for 3 days. By this operation, type II collagen was extracted into hydrochloric acid.

  The hydrochloric acid solution from which type II collagen was extracted was centrifuged at 2000 g for 90 minutes at 4 ° C., and the supernatant was collected.

  The collected supernatant was filtered stepwise using cellulose acetate membranes having different pore sizes (3.0, 0.8, and 0.45 μm: Toyo Filter Paper Co., Ltd.).

  Sodium chloride was added to the obtained filtrate to a final concentration of 1.0 M, and the mixture was allowed to stand overnight at 4 ° C. for salting out.

Thereafter, the mixture was centrifuged at 2000 g for 90 minutes at 4 ° C., and the supernatant was removed. The precipitate was dissolved in hydrochloric acid at pH 2.0. Salting out and dissolution of the precipitate were repeated 3 times in total.
Thereafter, the dissolved product was dialyzed against distilled water, and the obtained type II collagen was lyophilized.

Comparative Example 3 Extraction of Collagen from Amur Sturgeon Skin and Bags Collagen was extracted from Amur sturgeon skin and bag.
The following pretreatment was performed for each tissue. An outline of the preprocessing method is shown in FIG.

Skin Amur sturgeon skin was cut into pieces, degreased by soaking in 99.5% ethanol and treating at 4 ° C. for 2 days, and used as a raw material.

Ukibu Amur sturgeon float was cut into small pieces and used as a raw material.
Collagen was extracted from the treated skin and float obtained by the above pretreatment by the same method as in Example 1 (an outline is shown in FIG. 1).

Example 4 Analysis of Amur Sturgeon Type II Collagen Collagen Extraction Efficiency and Amount Collected per Individual FIG. 9 shows the extraction efficiency (%) of collagen extracted from each tissue of Amur sturgeon by the method described in Example 3 and Comparative Example. ) And the recovered amount (mg) per individual.
The extraction efficiency was expressed as a percentage (%) of the recovered collagen dry weight with respect to the wet weight of the raw material used.

Determination of Purity of Extracted Collagen Collagen extracted from each tissue of Amur sturgeon was analyzed by SDS-PAGE using 7.5% acrylamide gel.

  The results are shown in FIG. From the SDS-PAGE image, (1) the extracted collagen is highly purified, and (2) the Amur sturgeon skin and bladder have two main bands near 100-120 kDa. (3) Collagen of the Amur sturgeon's notochord sheath has one main band around 130kDa, indicating that it is type II collagen.

Amino acid analysis of collagen extracted from each tissue of Amur sturgeon After hydrolyzing the collagen extracted from each tissue of Amur sturgeon, the amino acid composition was analyzed using a fully automatic amino acid analyzer (JLC-500V, JEOL).

  The results are shown in FIG. As shown in the figure, type II collagen extracted from Amur sturgeon chordal sheath has Ser (serine) content, Ala (alanine) content, and Lys (lysine) content compared to collagen obtained from other tissues of Amur sturgeon. Low, Glx (Gln (glutamine) and Glu (glutamic acid)) content, Leu (leucine) content, HyLys (lysine hydroxide), Pro (proline) content and imino acids (HyPro (hydroxyproline) and Pro (proline)) The content was high.

Measurement of hydroxylation rate of Lys and Pro The hydroxylation rate of lysine (Lys) and proline (Pro) of collagen extracted from Amur sturgeon skin, bladder, and chorda sheath was calculated. The hydroxylation rate was determined by the percentage of HyLys with respect to the value of HyLys + Lys in FIG. 12, and the percentage of Hypro with respect to the value of HyPro + Pro.

  The results are shown in FIG. Type II collagen extracted from Amur sturgeon notochord has a higher hydroxylation rate of Lys than type I collagen extracted from other tissues of Amur sturgeon, and the hydroxylation rate of Lys extracted from other tissues is 22.6% ~ It was 60% compared to 38.7%.

Measurement of Denaturation Temperature of Collagen Extracted from Each Amur Sturgeon Tissue The denaturation temperature of collagen extracted from Amur sturgeon skin, bladder, and notochord was measured. The measurement of the denaturation temperature of collagen is known in Ikoma et al. (2003) (Ikoma T, Kobayashi H, Tanaka J, Walsh D and Mann S (2003) Physical properties of type I collagen extracted from fish scales of Pagrus major and Oreochromis niloticas According to the method of International Journal of Biological Macromolecules 32: 199-204.

  The results are shown in FIG. As shown in the figure, the denaturation temperature of type II collagen extracted from Amur sturgeon notochord was higher than that of collagen extracted from other tissues of Amur sturgeon.

  Type II collagen extracted from sturgeon notochord by the method of the present invention can be used for various applications such as medical materials and raw materials for health foods.

Miller and Mathews, Biochem Biophys Res Comm 60: 424-430, 1974 Sheren et al., CBP 85B: 5-14, 1986 Kimura and Kamimura, CBP73B: 335-339, 1982 Rama and Chandrakasan, Connect Tissue Res 12: 111-118, 1984 Kittiphattanabawon et al., LWT-Food Science and Technology, 43: 792-800, 2010

Claims (6)

A method of extracting type II collagen from sturgeon notochord,
Collecting notochord from sturgeon vertebrae,
A method comprising: chopping the collected notochord and extracting type II collagen; and collecting the collagen.   The method according to claim 1, further comprising collecting a notochord from the notochord and extracting type II collagen from the notochord.   The method according to claim 1 or 2, wherein the collected notochord or chord sheath is not decalcified, degreased and solubilized using guanidine hydrochloride.   The method according to any one of claims 1 to 3, wherein the sturgeon is Wester or Amur sturgeon. The process of extracting type II collagen
(i) a step of washing the chord or chordal sheath of shredded sturgeon,
(ii) treating with 0.1-1% (w / w) pepsin, based on the weight of the notochord or notochord used,
(iii) centrifuging, collecting the supernatant, and treating with 0.01-0.1% (w / w) pepsin based on the weight of the notochord or chord sheath used;
(iv) filtering to remove impurities,
(v) obtaining a type II collagen precipitate by salting out; and
(vi) Dialysis process,
The method of any one of Claims 1-4 containing.   The denaturation temperature measured by a circular dichroism (CD) spectrometer extracted by the method according to any one of claims 1 to 5 is 33 ° C or higher, and an imino acid residue in 1000 amino acid residues Type II collagen derived from sturgeon notochord, having a number of 200 or more and a hydroxylation rate of lysine of 57% or more.

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