JP2012176926A - Method for producing unmodified type ii collagen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently and inexpensively recovering unmodified type II collagen from natural resources.SOLUTION: The method for producing unmodified type II collagen is characterized by extracting the unmodified type II collagen from an unmodified type II collagen-containing biological sample with an aqueous solution containing an oxygen-based microbicide such as peracetic acid as an extractant. In the method, the unmodified type II collagen-containing biological sample is cartilaginous tissue or skin of fishes, molluscs, birds or mammals.

Description

  The present invention relates to biological samples containing non-denatured type II collagen useful as pharmaceutical raw materials, medical supplies materials, cosmetic raw materials, food materials, industrial materials, etc., such as fish, mollusks, birds and mammalian cartilage tissues. It relates to a method of extracting and manufacturing.

  Collagen is the most abundant protein in mammals, accounting for about 30% of body protein and about 6% of body weight, and about 80% of dry weight in skin. Non-denatured type II collagen is a kind of protein mainly contained in cartilage, notochord, intervertebral disc and the like. It is an extracellular matrix having a triple helix structure, in which three α1 chains are intertwined in a spiral, called a super helix, and has a molecular weight of about 300,000.

  As a general method for extracting collagen, there is a method in which proteoglycan existing simultaneously with collagen as an extracellular matrix is removed in advance. This is because collagen cannot be extracted efficiently if proteoglycan is present during collagen extraction. A number of techniques for extracting proteoglycans have been reported as pretreatment methods. In cartilage tissue, non-denatured type II collagen does not exist alone, but exists in a form in which proteoglycan, which is a glycoprotein complex, and hyaluronic acid coexist. Extracting non-denatured type II collagen as it is, this proteoglycan became an interfering substance and became a bottleneck for collagen extraction. Several methods for producing non-denatured type II collagen from cartilage tissue that also serve as an effective use of waste have been proposed. For example, there is a method in which an acidic solution containing an acidic protease is brought into contact with cartilage (see Patent Document 1).

Japanese Patent No. 3935938

  An object of the present invention is to develop a method for producing non-denatured type II collagen that can be taken orally at low cost from fish, mollusks, birds or mammals, particularly from their disposal sites.

The present inventors use a peracetic acid preparation, which is an oxygen-based bactericidal agent, under certain conditions, thereby efficiently converting non-denatured type II collagen, which is a protein, into cartilage tissue or other non-denatured type II collagen. The inventors have found that it can be recovered from a biological sample, and completed the following inventions.
(1) A method for producing non-denatured type II collagen, comprising a step of immersing a biological sample containing non-denatured type II collagen in an oxygen-based disinfectant and a step of recovering the solid matter after the immersion.
(2) The production method according to (1), further comprising a step of separating, purifying, dehydrating and drying the recovered non-denatured type II collagen.
(3) The production method according to (1) or (2), wherein the oxygen-based disinfectant is a solution of a peracetic acid preparation.
(4) Composition of peracetic acid formulation solution, peracetic acid 5 × ¹⁰ -5 to 1 wt%, hydrogen peroxide 5 × ¹⁰ -5 to 1 wt%, acetic acid 3 × ¹⁰ -5 to 2% by weight (1 ) To (3).
(5) The production method according to any one of (1) to (3), wherein the biological sample containing non-denatured type II collagen is fish, mollusk, avian or mammalian cartilage tissue, or skin.
(6) The production method according to (4), wherein the biological sample containing non-denatured type II collagen is fish cartilage tissue.

  The present invention is a method for producing non-denatured type II collagen, which is generally heat labile, using a peracetic acid preparation. Non-denatured type II collagen is a protein. In general, proteins are unstable to heat and have the property of being easily denatured and decomposed. From this property, it is not known that high-purity non-denatured type II collagen can be produced with peracetic acid while preventing denaturation of the protein portion of non-denatured type II collagen.

  The present invention can be applied to biological samples containing non-denatured type II collagen, such as fish, mollusk, avian or mammalian cartilage tissue, muscle fibers or skin, but application to cartilage tissue is preferred. . As the cartilage tissue used in the present invention, any of fish, birds and mammals, particularly those waste sites can be used. In the present invention, the cartilage tissue means either cartilage alone or a tissue including a peripheral part of the cartilage, such as bone, muscle fiber, and skin.

  In the present invention, it is particularly preferable to use nasal cartilage tissue, which is generally called an ice head, and is contained in the salmon head in an average weight of about 6%. When salmon caught on the coast of Hokkaido (mostly salmon) are processed as various processed products, their heads are often unnecessary, so some of the cut heads Although processed into fish meal and used, most of it is disposed of as industrial waste. Ice heads can be obtained simply, inexpensively and stably from such waste.

  In the present invention, in addition to ice head, fish-derived cartilage tissue such as ray cartilage tissue, shark cartilage tissue, avian cartilage tissue such as chicken cartilage tissue, and mammals such as bovine throat cartilage, bronchial cartilage and whale cartilage Animal-derived cartilage tissue can also be used.

  Many of the biological samples containing the above-mentioned non-denatured type II collagen are also industrial waste and are easily available. These raw materials are preferably crushed as finely as possible in order to increase the surface area and increase the extraction amount of non-denatured type II collagen before immersion in an alkaline solution described below.

  The immersion of the cartilage tissue in the peracetic acid preparation is preferably about 0 ° C. to 15 ° C., and the non-denatured type II collagen is hardly decomposed and can be extracted as a high molecular protein maintaining a super (triple helix) helix structure. it can. Immersion may be performed using 2 to 15 parts by weight, preferably 4 to 12 parts by weight, more preferably 6 to 12 parts by weight of peracetic acid preparation solution per 1 part by weight of cartilage. Preferably, it is immersed while stirring using a mixer or a stirrer.

Extraction of non-denatured type II collagen from cartilage tissue includes, for example, a method in which hydroproline is quantified by, for example, an automatic amino acid analysis method and is calculated using a collagen conversion count for each source.
The calculation formula is type II collagen (%) = hydroxyproline (g / 100 g) × 12.51, where 12.51 is a conversion factor for fish collagen.

  The peracetic acid preparation solution that has been soaked is a solution from which proteoglycan has been extracted, and non-denatured type II collagen is contained on the solid side. These are preferably removed by filtration, centrifugation or other methods. The solid containing non-denatured type II collagen is preferably purified by an appropriate method after the pH adjustment to the purity required for various uses.

  In the present invention, a method for purifying non-denatured type II collagen is not particularly required, but a preferable method is a centrifugation method. A solid matter can be precipitated by centrifugal separation, and can be easily solid-liquid separated from the aqueous solution side.

  Furthermore, by putting the obtained solid into an organic solvent such as ethanol or acetone, it is possible to easily remove the fish-like odor and moisture derived from the slightly remaining lipid of non-denatured type II collagen. This non-denatured type II collagen can be made into a solid using a vacuum freeze dryer when it is not treated with an organic solvent. In addition, when an organic solvent treatment is performed, it is preferable to use a solvent recovery type vacuum dryer.

  Peracetic acid has already been approved as a food additive in foreign countries, especially in North America, and it is directly applied to vegetables, fruits and meat, and there are cases where it is regularly required for microbial control. There are various sterilization methods for sterilization methods such as manufacturing equipment, manufacturing environment and containers and packaging. Among them, the method using oxygen-based disinfectants such as hydrogen peroxide and peracetic acid preparations is extremely effective for sterilizing microorganisms. It is characterized by being strong against. In particular, peracetic acid exhibits a broad bactericidal property against bacteria, fungi (molds, yeasts) and viruses including budding bacteria, and it is said that it has a bactericidal power 100 to 200 times that of hydrogen peroxide.

  The production process of non-denatured type II collagen cannot include a heat sterilization step in order to prevent protein denaturation. For this reason, how to make the extraction raw material aseptic is an important point in the hygiene management of the process. If peracetic acid is used as an extraction solvent, the extraction process can serve as a sterilization process, and if a non-denatured type II collagen can be produced efficiently without denaturing the protein, a heat sterilization process is usually used. It is possible to easily maintain hygienic management of the production process of non-denatured type II collagen that cannot be performed.

  Further, compared to conventional extraction methods, non-denatured type II collagen can be easily recovered in an undenatured and undegraded state, and the production cost of non-denatured type II collagen can be greatly reduced. INDUSTRIAL APPLICABILITY The method of the present invention can recover non-denatured type II collagen that is highly industrially useful from disposal sites such as fish, birds and mammals that have been originally disposed of, and can effectively utilize industrial waste and industrial waste. It can also contribute to its own weight reduction.

  By using peracetic acid as an extraction solvent, the extraction step serves as a sterilization step, and hygiene management of the production process of non-denatured type II collagen can be easily maintained.

  Non-denatured type II collagen can be easily recovered in an undegraded state from a biological sample containing non-denatured type II collagen.

  EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples.

  100 g of minced pulverized nasal cartilage extracted from the head of chum salmon stored frozen at −40 ° C. was prepared as a starting material. Distilled water (799.92 g) that had been cooled to 4 ° C. in advance was placed in a 2 L extraction container, and 0.08 g of a peracetic acid preparation was added to prepare a total amount of 800 g (0.01%) of peracetic acid aqueous solution. 100.00 g of starting material was put into this extraction container and immersed for 24 hours while stirring with a stirrer.

  After the immersion, centrifuge at 7000 rpm for 15 minutes using a centrifuge of himac CF7D2 to collect the solid content (SS content) containing non-denatured type II collagen, about 10 times the weight of the solid content. After stirring and washing with distilled water, it was dehydrated and freeze-dried. As a result, 3.5 g of dry solids corresponding to 29.2% in terms of conversion value could be obtained from 12 g of starting material.

  As a result of quantifying the amount of amino acid in the dry solid using an automatic amino acid analyzer (manufactured by Hitachi, Ltd., L-8500 Amino Acid Analyzer) and calculating the amount of collagen, the amount of collagen in the solid was 82.1%. (Hydroxyproline g / 100 g × 12.1 (conversion factor of fish collagen)).

  When the heat denaturation temperature of the protein was measured by DSC (differential scanning calorimetry) of the obtained collagen sample, an endothermic reaction peak was obtained at 45 ° C., so that non-denatured type II collagen maintaining a triple helical structure was obtained. I found out that

  In addition, as a result of microbial examination of the collected collagen, general bacteria: 100 cells / g or less, coliform group: negative.

  The nasal cartilage extracted from the head of a salmon salmon frozen at −40 ° C. was finely crushed with an electric meat chopper and minced, and immersed in acetone to degrease and dehydrate the nasal cartilage. 12.00 g of the nasal cartilage that had been treated and dried under reduced pressure was used as the starting material. 1678.3 g of distilled water previously cooled to 10 ° C. was put into a 3 L extraction container, and 1.68 g of a liquid peracetic acid preparation (Mitsubishi Gas Chemical: Diamond Power) was added, for a total amount of 1680 g (0.1 %) Peracetic acid preparation solution. 12.00 g of starting material was put into this extraction container and immersed for 48 hours while stirring with a stirrer.

After the immersion, centrifugation was performed at 7000 rpm for 15 minutes using a himac CF7D2 type centrifuge, and the solid content (SS content) was recovered.
The solid content separated into solid and liquid as an extraction residue by centrifugation was collected, washed with distilled water about 10 times the weight of the solid content, then dehydrated and freeze-dried. As a result, 3.6 g of dry solid content corresponding to 30.0% of the converted value was obtained from 12 g of starting material.

  As a result of quantifying the amount of amino acid in the dry solid using an automatic amino acid analyzer (manufactured by Hitachi, Ltd., L-8500 Amino Acid Analyzer) and calculating the amount of collagen, the amount of collagen in the solid was 79.3%. (Hydroxyproline g / 100 g × 12.1 (conversion factor of fish collagen)).

  When the heat denaturation temperature of the protein was measured by DSC (differential scanning calorimetry) of the obtained collagen sample, an endothermic reaction peak was obtained at 45 ° C., so that non-denatured type II collagen maintaining a triple helical structure was obtained. I found out that In the case of denatured collagen, there is no endothermic reaction peak, or the peak becomes broad and the denaturation temperature cannot be determined.

  In addition, as a result of microbial examination of the collected collagen, general bacteria: 100 cells / g or less, coliform group: negative.

  The nasal cartilage extracted from the head of chum salmon stored frozen at −40 ° C. was finely crushed with an electric meat chopper and minced, and immersed in ethanol to degrease and dehydrate the nasal cartilage. 12.00 g of the nasal cartilage that had been treated and dried under reduced pressure was used as the starting material. Into a 3 L extraction vessel was placed 1678.82 g of distilled water that had been cooled to 9 ° C. in advance, and 1.18 g of a liquid peracetic acid preparation (Mitsubishi Gas Chemical: Diamond Power) was added to a total amount of 1680 g (0.07 %) Peracetic acid preparation solution. 12.00 g of starting material was put into this extraction container and immersed for 48 hours while stirring with a stirrer.

After the immersion, centrifugation was performed at 7000 rpm for 15 minutes using a himac CF7D2 type centrifuge, and the liquid phase containing the solid content (SS content) was recovered.
The solid content separated into solid and liquid as an extraction residue by centrifugation was collected, washed with distilled water about 10 times the weight of the solid content, then dehydrated and freeze-dried. As a result, 3.9 g of dry solid content corresponding to 32.5% in terms of conversion value could be obtained from 12 g of starting material.

  As a result of quantifying the amount of amino acid of the dry solid using an automatic amino acid analyzer (manufactured by Hitachi, Ltd., L-8500 Amino Acid Analyzer) and calculating the amount of collagen, the amount of collagen in the solid was 77.9%. (Hydroxyproline g / 100 g × 12.1 (conversion factor of fish collagen)).

  When the heat denaturation temperature of the protein was measured by DSC (differential scanning calorimetry) of the obtained collagen sample, an endothermic reaction peak was obtained at 44 ° C., so that non-denatured type II collagen maintaining a triple helical structure was obtained. I found out that In the case of denatured collagen, there is no endothermic reaction peak, or the peak becomes broad and the denaturation temperature cannot be determined.

  In addition, as a result of microbial examination of the collected collagen, general bacteria: 100 cells / g or less, coliform group: negative.

  The nasal cartilage extracted from the head of a salmon salmon frozen at −40 ° C. was finely crushed with an electric meat chopper and minced, and immersed in acetone to degrease and dehydrate the nasal cartilage. 12.00 g of the nasal cartilage that had been treated and dried under reduced pressure was used as the starting material. 1677.9 g of distilled water previously cooled to 6 ° C. was placed in a 3 L extraction vessel, and 2.02 g of a liquid peracetic acid preparation (Mitsubishi Gas Chemical: Diamond Power) was added, for a total amount of 1680 g (0.12 %) Peracetic acid preparation solution. 12.00 g of starting material was put into this extraction container and immersed for 24 hours while stirring with a stirrer.

After the immersion, centrifugation was performed at 7000 rpm for 15 minutes using a himac CF7D2 type centrifuge, and the liquid phase containing the solid content (SS content) was recovered.
The solid content separated into solid and liquid as an extraction residue by centrifugation was collected, washed with stirring with distilled water about 8 times the weight of the solid content, then centrifuged and dehydrated, and 3 times the amount of ethanol was added to the dehydrated solid content. .
The mixture was stirred and mixed for 0.5 hour, and solid-liquid separation was performed by filtration. The solid matter after filtration was dried under reduced pressure, the solid content was dried without heating, and the dry solid content was recovered.
As a result, 3.2 g of dry solids corresponding to 26.7% of the converted value was obtained from 12 g of the starting material.

  As a result of quantifying the amount of amino acid in the dry solid using an automatic amino acid analyzer (L-8500 Amino Acid Analyzer manufactured by Hitachi, Ltd.) and calculating the amount of collagen, the amount of collagen in the solid was 84.5%. (Hydroxyproline g / 100 g × 12.1 (conversion factor of fish collagen)).

  The obtained collagen sample was subjected to DSC (differential scanning calorimetry) to measure the heat denaturation temperature of the protein. As a result, an endothermic reaction peak was obtained at 43 ° C., so that non-denatured type II collagen maintaining a triple helical structure was obtained. I found out that In the case of denatured collagen, there is no endothermic reaction peak, or the peak becomes broad and the denaturation temperature cannot be determined.

  In addition, as a result of microbial examination of the collected collagen, general bacteria: 100 cells / g or less, coliform group: negative.

  After the meat pieces were manually removed from domestic chicken bean cartilage, the minced material was finely crushed with an electric meat chopper and immersed in ethanol to degrease and dehydrate the chicken bean cartilage. 12.0 g of the treated cartilage dried under reduced pressure was used as a starting material. Add 1678.3 g of distilled water that has been cooled to 12 ° C. in a 3 liter extraction container, add 1.18 g of liquid peracetic acid preparation, and add 1680 g (0.07%) peracetic acid aqueous solution in total. Got ready. 12.0 g of starting material was put into this extraction container and immersed for 24 hours while stirring with a stirrer.

After the immersion, centrifugation was performed at 7000 rpm for 15 minutes using a himac CF7D2 type centrifuge, and the solid content (SS content) was recovered.
Further, the solid content separated into solid and liquid as an extraction residue by centrifugation was collected, washed with distilled water about 10 times the weight of the solid content, then dehydrated and freeze-dried. As a result, 3.1 g of dry solid content corresponding to 25.8% of the converted value was obtained from 12 g of starting material.

  The amount of amino acid was quantified for this dry solid using an automatic amino acid analyzer (manufactured by Hitachi, Ltd., L-8500 Amino Acid Analyzer), and the amount of collagen was calculated. As a result, the amount of collagen in the solid was 79.7%. (Hydroxyproline g / 100 g × 11.1 (conversion coefficient of porcine collagen: food analysis method (Koji))).

  When the heat denaturation temperature of the protein was measured by DSC (differential scanning calorimetry) of the obtained collagen sample, an endothermic reaction peak was obtained at 63 ° C., so that non-denatured type II collagen maintaining a triple helical structure was obtained. I found out that In the case of denatured collagen, there is no endothermic reaction peak, or the peak becomes broad and the denaturation temperature cannot be determined.

  In addition, as a result of microbial examination of the collected collagen, general bacteria: 100 cells / g or less, coliform group: negative.

  The cartilage manually extracted from the shark was finely crushed with an electric meat chopper and minced, and immersed in acetone for degreasing and dehydration. 12.00 g of the cartilage after drying under reduced pressure was used as a starting material. Add 1678.74 g of distilled water that has been cooled to 5 ° C. in a 3 liter extraction container, and then add 2.02 g of a liquid peracetic acid preparation to a total amount of 1680.00 g (0.15%) peracetic acid. An aqueous solution was prepared. 12.00 g of starting material was put into this extraction container and immersed for 8 hours while stirring with a stirrer.

After the immersion, centrifugation was performed at 7000 rpm for 15 minutes using a himac CF7D2 type centrifuge, and the liquid phase containing the solid content (SS content) was recovered.
The solid content separated into solid and liquid as an extraction residue by centrifugation was collected, washed with distilled water about 10 times the weight of the solid content, then dehydrated and freeze-dried. As a result, 2.7 g of dry solid content corresponding to 22.5% in terms of conversion value could be obtained from 12 g of starting material.

  As a result of quantifying the amount of amino acid in the dry solid using an automatic amino acid analyzer (manufactured by Hitachi, Ltd., L-8500 Amino Acid Analyzer) and calculating the amount of collagen, the amount of collagen in the solid was 77.5%. (Hydroxyproline g / 100 g × 12.1 (conversion coefficient of fish collagen).

  In addition, as a result of microbial examination of the collected collagen, general bacteria: 100 cells / g or less, coliform group: negative.

  According to the present invention, the sterilization step and the extraction step can be carried out simultaneously, and the type II collagen can be extracted undegraded without being denatured, and the type II collagen can be undenatured from various biological samples. Applicable to separation and purification.

Claims (6)

  A method for producing non-denatured type II collagen, comprising a step of immersing a biological sample containing non-denatured type II collagen in an oxygen-based disinfectant and a step of recovering the solid matter after immersion.   The production method according to claim 1, further comprising a step of separating, purifying, dehydrating and drying the recovered non-denatured type II collagen.   The manufacturing method of Claim 1 or Claim 2 whose oxygen type germicide is a solution of a peracetic acid formulation. The composition of the peracetic acid formulation solution, peracetic acid 5 × ¹⁰ -5 to 1 wt%, hydrogen peroxide 5 × ¹⁰ -5 to 1 wt%, claims 1 acetate 3 × ¹⁰ -5 to 2 wt% Item 4. The manufacturing method according to Item 3.   The method according to any one of claims 1 to 3, wherein the biological sample containing non-denatured type II collagen is fish, mollusk, avian or mammalian cartilage tissue, or skin.   The production method according to claim 4, wherein the biological sample containing non-denatured type II collagen is fish cartilage tissue.