JP2006225485A - Method for producing chondroitin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method whereby bone tissue can be easily separated even from conventionally discarded shark processing residues, and chondroitin sulfate can be produced from the separated bone tissue. <P>SOLUTION: By grinding shark processing residues (tissue aggregate) while keeping them frozen at -20°C and classifying the obtained group of ground particles, it is possible to easily separate bone tissue from the processing residues being a mixture of muscle tissue and bone tissue and to produce chondroitin sulfate from the separated bone tissue. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing chondroitin, and more particularly to a method for producing chondroitin from bone tissue separated from shark processing residues.

  In recent years, foods that are conscious of health have been actively developed. For example, chondroitin, which is abundant in shark cartilage, has been widely used as an internal medicine, injection, and eye drop since it was approved as a medical drug in the early 1960s. In particular, it has been attracting attention as a use for supplementing the action of articular cartilage against joint pain, shoulder periperitis and low back pain, and for protecting the surface layer of the ocular cornea.

  In addition to chondroitin, collagen, minerals, squalene, etc. are attracting attention as pharmaceuticals and health foods derived from shark cartilage and shark liver oil.

  However, at present, components derived from shark cartilage including chondroitin as described above (hereinafter sometimes referred to as bone tissue) depend on foreign countries. Specifically, most of the ingredients for shark-derived pharmaceuticals and health foods used in Japan are imported from Australia. In Australia, whole shark fish are used only to obtain shark bone tissue.

  The reason why shark-derived pharmaceuticals and health foods depend on imports from overseas is shark processing technology in Japan. Sharks are also caught in Japan, and tail fins, dorsal fins, breast fins, etc. are processed as shark fin ingredients, and the body is processed as fillets, but it is rich in useful ingredients including chondroitin The head and bone drop including bone tissue are incinerated as processing residues. The reason for this is that in the shark processing technology in Japan, the processed residue is a mixture of skin, bone and meat, and even if it is attempted to separate the bone tissue from the processed residue, much labor is required. It is difficult to improve productivity. Furthermore, since sharks contain a large amount of urea in their meat tissue, the urea is decomposed into ammonia by bacteria as the processing time elapses, causing a strong odor. This also makes it more difficult to separate (recover) the bone tissue from the processing residue.

  For the reasons as described above, it is difficult to recycle the processing residues of sharks discharged in Japan at present, and therefore, there is no choice but to incinerate. Therefore, as a matter of course, a technique for separating bone tissue from shark processing residues has not been established at all.

  Therefore, the present invention has been made in view of the above-mentioned problems, and the purpose of the present invention is to easily separate bone tissue even from shark processing residues that have been discarded until now. It is to provide a method capable of producing chondroitin from bone tissue.

  As a result of intensive studies in view of the above problems, the present inventors have found that bone tissue can be separated from processing residues by performing freeze-grinding and classification using a sieve, and to complete the present invention. It came.

  The inventors of the present application have so far used a variety of food materials to freeze them and have studied the difference in properties of the materials after freezing. In the course of the research, the inventors of the present application have found that the raw material pulverized after freezing causes a difference in particle diameter depending on the type. The inventors of the present application paid attention to this point, and intensively studied whether or not the type of the material can be classified by sieving and classifying the pulverized particles, and completed the present invention.

  That is, the chondroitin production method of the present invention is a chondroitin production method for producing chondroitin using a tissue assembly containing muscle tissue and bone tissue, and freezes and grinds the tissue assembly to obtain a pulverized particle group. And a classification step of classifying the pulverized particles composed of the bone tissue from the pulverized particle group obtained by the freeze pulverization step. In the classification step, pulverized particles having a predetermined particle diameter are obtained. It is characterized by classification.

  In the chondroitin production method, in the classification step, the pulverized particle group obtained in the freeze pulverization step is preferably classified using a plurality of sieves having different opening sizes.

  In the above chondroitin production method, the bone tissue is preferably shark cartilage.

  In the chondroitin production method, the tissue assembly is preferably frozen at −15 ° C. to −30 ° C. in the freeze pulverization step.

  In addition, the chondroitin production method described above is characterized in that the tissue aggregate is derived from shark and is discharged after collecting processing sites such as caudal fin, dorsal fin, pectoral fin, and body, and includes a processing residue including a head and a dropout. It is preferable that

  The method for producing chondroitin according to the present invention is a method for producing chondroitin using a tissue assembly including muscle tissue and bone tissue, and freezes and pulverizes the tissue assembly to obtain a pulverized particle group. Including a freeze pulverization step and a classification step of classifying the pulverized particles composed of the bone tissue from the pulverized particle group obtained by the freeze pulverization step, and in the classification step, pulverized particles having a predetermined particle diameter are classified. It is characterized by doing.

  This makes it possible to easily separate bone tissue from the processing residue even when using a shark processing residue (that is, tissue aggregate) that has been discarded until now, and the processing residue. Chondroitin can be easily produced from the bone tissue contained in.

  That is, conventionally, the problem in the processing residue as described above has made it difficult to separate the bone tissue from the processing residue, but according to the present invention, the classification step has a predetermined particle size. By classifying the pulverized particles, it is possible to mechanically classify the pulverized particles composed of bone tissue from the pulverized particles of the frozen and pulverized tissue aggregate.

  In particular, when using a shark processing residue, by setting the temperature of the tissue assembly in a range of −15 ° C. to −30 ° C., in the pulverized particle group obtained by the freeze pulverization step, pulverized particles made of muscle tissue The difference between the particle size of the crushed particles and the particle size of the pulverized particles made of bone tissue is the largest. This makes it possible to more accurately separate (classify) the pulverized particles made of bone tissue from the pulverized particle group obtained by the freeze pulverization step in the classification step.

  Therefore, according to the present invention, it is possible to mechanically separate the bone tissue from the processing residue even when using the processing residue of the shark processed in Japan. Can be easily separated and recovered.

  Therefore, according to the present invention, chondroitin can be produced from the bone tissue that has been easily separated and collected, and thus the production cost can be reduced.

  An embodiment of the present invention will be described. However, the present invention is not limited to this. In the present embodiment, the shark head is described as an example of a tissue assembly (processing residue) including muscle tissue and bone tissue, but the present invention is not limited to this, It can be applied to other parts of the shark as long as the part includes muscle tissue and bone tissue. Furthermore, the present invention can be applied not only to sharks but also to other living bodies containing complex polysaccharides such as chondroitin in bone tissue, such as fish and land animals. Further, in the following description, “chondroitin sulfate” will be described as an example of chondroitin of the present invention, but the present invention is not limited to this, and even in the case of complex polysaccharides such as chondroitin polysulfate and keratan sulfate, the present invention. Can be applied.

(1) Chondroitin sulfate Chondroitin sulfate is an acidic mucopolysaccharide (polysaccharide containing an amino group) having a molecular weight of 20,000 to 50,000 that is widely distributed in connective tissues such as cartilage, cornea, and blood vessel wall. N-acetyl-D-galactosamine and D -Consists of a disaccharide of glucuronic acid and a sulfate residue.

  Chondroitin sulfate is classified into types such as A, C, D, E, and K depending on the binding position of the sulfate group. In the case of a shark, chondroitin sulfate C (sulfate group is bonded to the C-6 position of D-galactosamine) and chondroitin sulfate D (C-6 position of D-galactosamine and D-glucuron to the cartilage of the skull, the middle bone and the shark It is known that a lot of sulfate groups are contained at the C-2 position of the acid. The chondroitin sulfate content in the tissue varies depending on the type of shark and is said to be around 10% per dry weight. Here, the structure of chondroitin sulfate C mainly contained in shark cartilage is shown in FIG.

  Shark cartilage is composed of mucopolysaccharide and a high molecular weight protein, and does not contain components that form a general bone nucleus such as calcium.

  In general, chondroitin sulfate exists as a chondroitin sulfate protein complex bound to protein in vivo, and chondroitin sulfate protein complex purified from shark cartilage is completely dissolved in water as a white powder. It is known that an aqueous solution exhibits weak acidity.

  In the present embodiment, a method for producing chondroitin sulfate, which is a useful component as described above, using shark cartilage (more specifically, shark processing residue), and an apparatus used for carrying out this method Will be described.

  The so-called processing residue after collecting caudal fins, dorsal fins, chest fins and the body for use in processing is in a state where skin, bones and meat are mixed. Therefore, it was very difficult to separate the bone tissue from the processing residue. In addition, since sharks contain a large amount of urea in their meat tissue, this urea is decomposed into ammonia by bacteria with a strong odor over the course of the processing time, and for processing dependents, bone tissue from processing residues The separation work was even more difficult. Under these circumstances, most of the raw materials for shark-derived pharmaceuticals and health foods used in Japan depend on imported products from Australia. In Australia, the whole fish body of shark is used only to obtain only the shark cartilage tissue in order to avoid the difficulty of work which has been a problem in shark processing in Japan. Thereby, although the difficulty of work is avoided, since only the cartilage tissue is collected and the other parts are disposed of, it is difficult to keep the price low. Therefore, in the present embodiment, the bone tissue can be separated by a simple method even when a processing residue in which the muscular tissue and the bone (cartilage) tissue are mixed is used. A production method capable of producing chondroitin sulfate from bone tissue and an apparatus used for carrying out this method will be described.

(2) Separation of bone tissue from tissue aggregate In the present embodiment, separation of the bone tissue from a tissue aggregate in which muscle tissue and bone tissue are mixed is based on the following basic principle.

(2-a) Basic Principle Even an elastic body such as rubber has the property of becoming very brittle when cooled below the embrittlement point. This property is generally called low temperature brittleness, and can be easily pulverized by cooling below the embrittlement point. Further, when the material is pulverized using this property, the size of the pulverized particles is different at the embrittlement point. That is, at a temperature lower than the embrittlement point, since the substance is in a brittle state (this is called a brittle fracture region), the pulverized particles become fine. On the other hand, at a temperature higher than the embrittlement point (this is referred to as a ductile fracture region), the material is difficult to break, so that the pulverized particles become coarse particles.

  Here, for example, a method of separating the substance A or the substance B from a binary mixture consisting of the substance A having a certain embrittlement point and the substance B having a lower embrittlement point than the substance A is considered. Considering the above-described embrittlement point, a temperature between the embrittlement point of the substance A and the embrittlement point of the substance B is selected, and the binary mixture is pulverized at that temperature, whereby the substances A and B Can be separated from each other. That is, assuming that the fracture frequency of both substances in the binary mixture is the same, the substance A is pulverized at a temperature below the embrittlement point (in the brittle fracture region), so that the pulverized particles are fine. Become particles. On the other hand, since the substance B is pulverized at a temperature higher than the embrittlement point (in the ductile fracture region), the pulverized particles become coarse particles. Therefore, by classifying the mixed pulverized particle group using, for example, a sieve having an appropriate opening, it is possible to separate the substance A and the substance B having different particle diameters.

  Using this principle, it was considered that the bone tissue from the processing residue could be separated and recovered in this embodiment. Taking shark processing residues with mixed heads and dropouts as examples, muscle tissue and bone tissue have different low-temperature vulnerabilities. Therefore, by freezing and pulverizing this processing residue at a certain optimal temperature, muscle tissue and bone tissue can be divided into pulverized particles having different particle sizes, and classified using a sieve with appropriate openings. Thus, the bone tissue can be separated and recovered from the processing residue (tissue aggregate).

  The production method and production apparatus of the present invention are intended to separate bone tissue (cartilage) from processing residues in which muscle tissue and bone (cartilage) tissue are mixed based on the basic principle as described above. .

(2-b) Freezing and grinding operation (freezing and grinding process)
2 (a) and 2 (b) are diagrams showing a configuration of a freeze pulverizing apparatus for freeze pulverizing a tissue aggregate in which muscle tissue and bone tissue are mixed in the present embodiment, and FIG. FIG. 2 is a side view of the freeze pulverization apparatus, and FIG. 2B is an enlarged plan view showing a configuration of a screen provided in the freeze pulverization apparatus shown in FIG. The freeze pulverization apparatus 1 is an apparatus for pulverizing (freezing and pulverizing) a tissue assembly frozen at a controlled temperature based on the basic principle described above. Therefore, the freeze pulverization apparatus 1 includes an input unit 2, a pulverization unit 3, a screen 4, a communication path 5, a thermostatic bath 6, and a freezing temperature control unit 7.

  The input part 2 is an input port for supplying a tissue aggregate mixed with muscular tissue and bone (cartilage) tissue, which is subjected to freeze pulverization. The input unit 2 is configured to input the tissue aggregate frozen by a freezing means (not shown) (hereinafter, the frozen tissue aggregate may be simply referred to as a tissue aggregate). In the charging unit 2, the freezing temperature of the frozen tissue aggregate is controlled by the freezing temperature control unit 7.

  In the freeze pulverization apparatus 1 of the present embodiment, the entire apparatus is temperature-controlled at a set temperature by the freezing temperature control unit 7. However, the present invention is not limited to this, and a structure may be used in which the tissue assembly is frozen in the charging unit 2 by using other freezing means. The apparatus 1 may be configured to be sufficiently cooled with liquid nitrogen in advance.

  Further, the freeze pulverization apparatus 1 is configured such that the tissue aggregate and / or the freeze pulverized particles move to the thermostat 6 according to gravity in the freeze pulverization apparatus 1, but the present invention is not limited to this. For example, the structure which a structure aggregate | tissue is sent to the grinding | pulverization part 3 by a belt conveyor may be sufficient.

  The pulverizing unit 3 is provided to actually freeze and pulverize the tissue aggregate charged into the input unit 2 from the outside. In the pulverizing unit 3, similarly to the charging unit 2, the temperature of the tissue aggregate and / or pulverized particles is controlled by the freezing temperature control unit 7. The crushing unit 3 is provided with a rotary blade 8 and a fixed blade 9 as illustrated. By the rotary blade 8 and the fixed blade 9, the sample put into the pulverizer is hit and destroyed by the fixed blade and the rotary blade in the pulverization section. Specifically, the sample is sandwiched between the fixed blade and the rotary blade, and the sample is broken by an impact force by the rotating blade rotating at high speed.

  The rotational speed of the rotary blade 8 is controlled by a rotation control unit (not shown). Specifically, it is preferably controlled in the range of 800 to 1200 rpm, more preferably in the range of 900 to 1100 rpm, and most preferably controlled to about 1000 rpm. When the rotational speed is less than 800 rpm, the “energy necessary for grinding” exerted on the tissue assembly by the rotary blade 8 is weak, so that the grinding is not sufficiently performed. Therefore, it is applied to the classification operation based on the basic principle described above. I can't. On the other hand, when the rotational speed exceeds 1200 rpm, the “energy required for grinding” exerted on the tissue assembly by the rotary blade 8 is strong and excessive grinding occurs. Therefore, this is applied to the classification operation based on the basic principle described above. I can't. Therefore, the rotational speed of the rotary blade 8 is preferably in the above range.

  In addition, an adjusting means (not shown) for adjusting the amount of the tissue aggregate introduced into the pulverizing unit 3 to a constant amount may be provided so that the pulverizing unit 3 can perform satisfactory pulverization.

  The screen 4 is provided below the pulverizing unit 3 and is arranged so that the pulverized particles pulverized by the pulverizing unit 3 pass therethrough. The screen 4 is also temperature-controlled by the freezing temperature control unit 7 in the same manner as the charging unit 2 and the pulverizing unit 3.

  As shown in FIG. 2B, the screen 4 is provided with an opening 4a having an opening diameter of 5 mm. Among the pulverized particles pulverized by the pulverization unit 3, pulverized particles having a particle diameter of less than 5 mm pass through the openings 4a and fall into the thermostat 6 through the communication passage 5 provided below the screen 4, The pulverized particles having a particle diameter of 5 mm or more are deposited on the screen 4 without passing through the openings 4a.

  The pulverized particles having a particle diameter of 5 mm or more deposited on the screen 4 can be put into the pulverization unit 3 and again subjected to freeze pulverization. In addition, when it is subjected to freeze pulverization again, the screen 4 may be removed from below the pulverization unit 3 and manually re-entered into the pulverization unit 3, or may be automatically re-introduced using other components. May be. Further, when the pulverized particles deposited on the screen 4 in this manner are reintroduced into the pulverizing unit 3, the configuration may be performed while maintaining the freezing temperature. The temperature management may be performed again by (and pulverizing unit 3).

  The communication path 5 is provided below the screen 4, and is provided to introduce the pulverized particles that have passed through the openings 4 a of the screen 4 into the thermostatic chamber 6. The temperature of the communication path 5 is also controlled by the freezing temperature control unit 7 in the same manner as the charging unit 2, the pulverizing unit 3, and the screen 4.

  The thermostatic chamber 6 is provided for accumulating the pulverized particle group frozen and pulverized by the pulverization unit 3 and is filled with liquid nitrogen. The pulverized particles that have been freeze-pulverized by the pulverization unit 3 and passed through the screen 4 and the communication path 5 fall into this liquid nitrogen. The thermostat 6 should just be comprised from the material which has a heat insulation effect, and can specifically be comprised with a polystyrene foam.

  By using the freeze pulverization apparatus 1 having the above-described configuration, freeze pulverization of a tissue aggregate (processing residue) in which muscle tissue and bone (cartilage) tissue are mixed is performed as follows.

  In addition, it is preferable to remove the skin from the tissue assembly in advance. Although it can be performed manually, besides this, it is also possible to remove the skin by freeze pulverization using a freeze pulverizer.

  As described above, the tissue assembly to be input to the input unit 2 of the freeze pulverization apparatus 1 may be configured to have an appropriate freezing temperature before being input to the input unit 2. It may be configured to. The appropriate freezing temperature is described in detail below. In the following description, this “appropriate freezing temperature” may be referred to as a separable temperature.

  The basic principle of the present invention proposed by the inventors of the present invention is that the substance A is made below the embrittlement point by freeze-grinding at a temperature between the embrittlement point of the substance A and the embrittlement point of the substance B. The material B becomes fine particles because it is pulverized at a temperature of 0.5 mm, and the substance B becomes coarse particles because it is pulverized at a temperature higher than the embrittlement point. Are to be separated. Accordingly, the inventors of the present application pay attention to the fact that the muscular tissue and bone tissue contained in the shark processing residue have different embrittlement points, and the bone tissue from the processing residue (tissue assembly) is removed by the above basic principle. The present invention was completed by thinking that it could be separated and recovered. Therefore, in order to separate and collect the bone tissue from the tissue aggregate, it is necessary to specify a separable temperature. Although details are described in the following examples, first, the inventors of the present application examined the pulverizability of each using muscular tissue and bone tissue alone. As a result, it was found that when freeze-pulverized in a temperature range of −15 ° C. to −30 ° C., the difference between the pulverized particle size of the bone tissue after pulverization and the pulverized particle size of the muscle tissue was increased. Specifically, when comparing the particle size distribution after freeze-grinding in muscle tissue and bone tissue in the above temperature range, muscle tissue has a smaller average particle size than bone tissue, and muscle tissue has fine particles. In addition, the bone tissue tended to be coarse particles.

  Therefore, the inventors of the present application next examined whether or not the above-described single pulverization study results can be applied to a tissue assembly (processing residue) in which muscle tissue and bone tissue are mixed. As described in the Examples, it was revealed that the pulverization property of the muscle tissue and the bone tissue in the tissue assembly was almost consistent with the pulverization property examination result of each tissue. That is, it was shown that bone tissue and muscle tissue can be separated by classification operation described later by freeze-pulverizing the shark processing residue in a temperature range of −15 ° C. to −30 ° C.

  In the present embodiment, the temperature range of −15 ° C. to −30 ° C. is preferably set as the separable temperature, but the temperature range of −18 ° C. to −22 ° C. is most preferably set as the separable temperature.

  That is, in the present embodiment, the tissue assembly may be frozen to the range of −15 ° C. to −30 ° C. before being put into the charging unit 2 of the freeze pulverization apparatus 1. The structure frozen in the range of -15 degreeC--30 degreeC may be sufficient.

As described above, in the freeze pulverization apparatus 1 of the present embodiment, the amount of the tissue aggregate introduced into the pulverization unit 3 is a certain amount so that the pulverization unit 3 can perform satisfactory pulverization. Adjusting means for adjusting to may be provided. Moreover, in order to implement favorable pulverization in the pulverization unit 3, the size of the tissue aggregate fed into the pulverization unit 3 is preferably 1 to 4 cm ³ . For example, it can be set to 1 × 1 × 1 cm ³ to 1 × 2 × 2 cm ³ . However, this size can be appropriately set depending on the size of the freeze pulverization apparatus 1, specifically the size of the connecting portion between the pulverization unit 3 and the charging unit 2, the size of the rotary blade 8 and the fixed blade 9, and the like. Therefore, the present invention is not limited to the above range.

  When the tissue aggregate frozen in the above temperature range is sent from the input unit 2 to the pulverizing unit 3, the tissue aggregate is obtained by the rotating blade 8 and the fixed blade 9 of the pulverizing unit 3 and the sample input to the pulverizer is In the crushing part, it is hit and destroyed by a fixed blade and a rotary blade. Specifically, the sample is sandwiched between the fixed blade and the rotary blade, and the sample is broken by an impact force by the rotating blade rotating at high speed.

  In order to classify the bone tissue well in the subsequent classification operation, it is preferable to adjust the amount of the tissue aggregate charged into the pulverizing unit 3 by the adjusting means described above. It is preferable that a new tissue aggregate is charged into the pulverizing unit 3 after all of the charged tissue aggregates have passed through the screen 4. Since all of the tissue aggregate put into the pulverizing unit 3 passes through the screen 4, the screen 4 is crushed particles accumulated on the screen 4 as described above (in this embodiment, pulverized particles having a particle diameter of 5 mm or more). The particles are again put into the pulverizing unit 3 and can be subjected to freeze pulverization. That is, the pulverizing unit 3 can freeze and pulverize the tissue aggregate and pulverize pulverized particles larger than the desired size in order to obtain pulverized particles having a desired size.

  The pulverized particle group that has been freeze-pulverized by the pulverization unit 3 and passed through the screen 4 and the communication path 5 falls into the liquid nitrogen in the thermostat 6. The pulverized particles in the liquid nitrogen in the thermostatic chamber 6 can be subjected to a subsequent classification operation by completely evaporating the liquid nitrogen in a freezer at about −30 ° C., for example. However, the temperature in the freezer is not limited to the above, and may be any temperature as long as the pulverized particles can maintain their shape.

(2-c) Classification operation (classification process)
FIG. 3 is a perspective view showing the configuration of the classification device 10 for classifying the pulverized particle group made of bone tissue from the pulverized particle group pulverized by the freeze pulverization apparatus 1 in the present embodiment. Therefore, the classifier 10 includes a sieve 11 (11a to 11g), a shaking means 12, a tray 13, and a lid 14.

  The sieve 11 is provided to classify the pulverized particle group pulverized by the freeze pulverization apparatus 1 according to the particle diameter. Each of the sieves 11a to 11g is the same as the screen 4 (FIGS. 2A and 2B) disposed in the freeze pulverization apparatus 1 (FIG. 2A) in the above-described (2-b) freeze pulverization operation. The openings of the sieves 11a to 11g are different from each other. For example, JIS standard sieves (openings: 0.355 mm, 0.50 mm, 0.71 mm, 1.00 mm, 1.41 mm, 2.00 mm) can be used as in Examples described later. And as shown in FIG. 3, it can be set as the structure which piled up in order from the sieve of a small opening, provided the saucer 13 in the lowest step, and provided the lid 14 in the uppermost step.

  The said shaking means 12 is provided in order to shake the sieve 11 so that the pulverized particle group thrown into the sieve 11a may be classified by each sieve 11a-11g. As the shaking means 12, a conventionally known shaker can be used.

  By using the classification device 10 having the above-described configuration, the classification of the pulverized particle group composed of bone tissue from the pulverized particle group pulverized by the freeze pulverization operation is performed as follows.

  When the pulverized particle group pulverized by the freeze pulverization apparatus 1 is put into the uppermost sieve 11a among the sieves 11, the pulverized particles having a diameter smaller than the diameter of the openings provided in the sieve 11a are related to the openings. And then drops onto the sieve 11b provided below the sieve 11a, and the pulverized particles having a diameter larger than the diameter of the mesh remain on the sieve 11a. On the other hand, the pulverized particles that have fallen on the sieve 11b are then passed to the sieve 11c provided below the sieve 11b through the pulverized particles having a diameter smaller than the diameter of the mesh provided on the sieve 11b. The pulverized particles that fall and have a diameter larger than the diameter of the openings remain on the sieve 11b. Thus, the pulverized particle group pulverized by the freeze pulverization apparatus 1 is classified according to the opening diameter of each sieve.

  In the present embodiment, seven types of sieves (sieves 11a to 11g) are used as the sieve 11, but the present invention is not limited to the number, and is appropriately set according to the diameter of the classified pulverized particles. It is possible.

(3) Production of chondroitin sulfate from bone tissue (extraction process)
Production (extraction) of chondroitin sulfate from bone tissue separated from a tissue aggregate (processing residue) by the above-described freeze-pulverization operation and classification operation is performed by the following method.

The separated bone tissue is dried, adjusted to pH 12.0 with an aqueous sodium hydroxide solution while adding 10% saline and stirring. This is stirred and extracted at room temperature for 2 hours. Furthermore, it stirs and extracts for 1 hour in a 55 degreeC water bath. This is then centrifuged and the residue is again extracted with stirring with 10% saline and centrifuged. Both supernatants are combined and adjusted to pH 2.0-2.5 with sulfuric acid. The precipitate produced at this time is removed by centrifugation. Distilled water is added to the supernatant, and a cationic surfactant is further added and stirred, and left for 1 hour. Next, a filter aid (Celite) is added, and suction filtration is performed with stirring. At this time, filtration is repeated until the filtrate becomes transparent. Wash the precipitate collected in Nutscheroth with distilled water. The precipitate is extracted with 10% brine and filtered. When ethyl alcohol is added to the filtrate, a precipitate is formed, which is collected by centrifugation and dried. By the above method, chondroitin sodium sulfate is extracted. (Refer to “Akira Itani, Tetsuo Shibata, Analysis of Sodium Chondroitin Sulfate in Foods, Journal of Japan Society for Food Hygiene, 5, 246-249 (1964)”)
As described above, the chondroitin sulfate production method according to the present invention is a chondroitin sulfate production method for producing chondroitin sulfate using a tissue assembly containing muscle tissue and bone tissue, and freezes and grinds the tissue assembly. Then, a freeze pulverization step for obtaining a pulverized particle group, a classification step for classifying the pulverized particles composed of the bone tissue from the pulverized particle group obtained by the freeze pulverization step, and the pulverization classified by the classification step An extraction step of extracting chondroitin sulfate from the particles, and the classification step is characterized by classifying pulverized particles having a predetermined particle diameter.

  This makes it possible to mechanically separate bone tissue even when using shark processing residues (ie, tissue aggregates) that have been discarded until now, and produce chondroitin sulfate from the bone tissue. can do.

  That is, conventionally, the problem in the processing residue as described above has made it difficult to separate the bone tissue from the processing residue, but according to the present invention, the classification step has a predetermined particle size. By classifying the pulverized particle group, the pulverized particle group composed of bone tissue can be classified from the pulverized particle group of the frozen and pulverized tissue aggregate.

  Specifically, by setting the temperature of the tissue aggregate in a range of −15 ° C. to −30 ° C., the particle size of the pulverized particles composed of muscle tissue in the pulverized particle group obtained by the freeze pulverization step The difference from the particle size of the pulverized particles made of bone tissue becomes the largest. Thereby, it becomes possible to carry out classification more accurately in the classification step.

  Therefore, according to the present invention, it is possible to mechanically separate the bone tissue from the processing residue even when using the processing residue of the shark processed in Japan. Can be easily separated and recovered.

  Therefore, according to the present invention, chondroitin sulfate can be produced from the bone tissue that has been easily separated and collected, and thus the production cost can be reduced.

  Note that the present invention is not limited to the above-described embodiment, and various modifications can be made within the scope of the claims, and the present invention can be obtained by appropriately combining the disclosed technical means. Included in the technical scope.

  Hereinafter, the features of the present invention will be described more specifically with reference to examples. In the present embodiment, as described above, the processing residue such as the shark's head or the middle drop is a tissue aggregate including muscle tissue and bone tissue. It was thought to be directly reflected in the grindability of the aggregate.

  Therefore, in this example, first, muscle tissue and bone tissue, which are constituent elements of shark processing residue (tissue aggregate), are used independently, and the freezing temperature of each tissue is changed to freeze at each freezing temperature. Pulverization was performed, and the relationship between the freezing temperature, the particle size distribution of the pulverized tissue, and chemical components was evaluated.

  Subsequently, in this example, based on the above evaluation results, the conditions of the freeze pulverization and classification operation most suitable for the separation and collection of the bone tissue from the tissue aggregate were examined. At the same time, chemical components of the pulverized particles after freeze pulverization were measured.

  In the following description, tissues when muscle tissue and bone tissue are used alone may be collectively referred to as a single tissue.

[Freeze pulverization of single structure, particle size distribution of pulverized particles after freeze pulverization, and dependence of chemical components on freeze pulverization temperature]
First, the influence of the tissue freezing temperature on the fracture behavior of the substance that is the focus of the present invention was examined. Specifically, in an actual freeze grinding operation, the influence of the freeze grinding temperature on the grindability of each tissue (muscle tissue and bone tissue) was examined.

As the muscular tissue and bone tissue, the head and the fall of the shark (Salmon shark, Lamna ditropis Hubbs et Follett) from Kesennuma, Miyagi Prefecture were used. These are stored in a freezer at about −30 ° C. as processing residues. The processing residue was thawed at room temperature and then manually divided into muscle tissue and bone tissue. Thereafter, each of the muscle tissue and the bone tissue is cut into 1 × 1 × 1 cm ³ to 1 × 2 × 2 cm ³ cut pieces by a cutter, and the temperature is respectively set to −80 ° C., −60 ° C., −40 ° C., and −20 ° C. A set stocker was prepared and each single tissue was frozen to a set temperature by storing a slice of muscle tissue and bone tissue in each stocker.

  The freeze pulverization operation was performed as follows.

  The freeze crushing operation was performed using the above-described freeze fracture apparatus 1 (FIG. 2). Specifically, as the freeze fracture apparatus 1, a cooling-type wheel pulverizer (manufactured by Yoshida Manufacturing Co., Ltd.) was used. The freeze breaking device 1 was sufficiently cooled with liquid nitrogen. The screen 4 of the freeze fracture apparatus 1 was a screen having an opening of 5 mm, and the rotational speed of the rotary blade 8 of the crushing unit 3 was set to 1000 rpm.

  The cut pieces of muscle tissue were freeze-pulverized at -80 ° C, -60 ° C, -40 ° C, and -20 ° C. In addition, the bone tissue cut pieces were freeze-ground at -60 ° C, -40 ° C, and -20 ° C. In the freeze pulverization in the pulverization unit 3, the amount of each single tissue introduced into the pulverization unit 3 (specifically, the input unit 2 and the pulverization unit 3) is 2 to 3 in terms of the above cut pieces. In addition, after the single structure once input to the pulverizing unit 3 was completely discharged from the screen 4, a new single structure was input.

  Each single-tissue ground particle group discharged from the screen 4 was received in a thermostatic bath 6 (made of expanded polystyrene) filled with liquid nitrogen.

  Each single tissue ground particle group accumulated in the thermostat 6 (total weight of bone tissue ground particle group: 250 g, total weight of muscle tissue ground particle group: 250 g) is stored in a freezer at about -30 ° C. The liquid nitrogen was completely evaporated and subjected to the subsequent classification operation.

  Classification operation was performed as follows.

  The classification operation was performed using the classification apparatus 10 (FIG. 3) described above. Specifically, the sieve 11 has an opening of 0.355 mm (sieve 11 f), 0.50 mm (sieve 11 e), 0.71 mm (sieve 11 d), 1.00 mm (sieve 11 c), 1.41 mm (sieve 11 b). A 2.00 mm (sieve 11a) JIS standard sieve was used. As shown in FIG. 3, a structure was adopted in which the sieve trays 13 were provided at the bottom of the screen, with the sieves having small openings being stacked in order. The classifier 10 was previously cooled in a low temperature room of about −20 ° C. Thus, in the present Example, it was set as the structure which classifies each single structure | tissue which carried out freeze grinding | pulverization into 7 fractions, respectively.

  Into the sieve 11a (aperture 2.00 mm), pulverized particles of a single structure obtained by the above-described freeze-grinding operation are put, and after the lid 14 is applied, the sieve 11 is shaken lightly manually, and then shaken. As the shaking means 12, an electromagnetic shaker (MVS-2000) manufactured by Hiraiko Seisakusho Co., Ltd. was used, and the sieve 11 was set on the shaking means 12 and shaken for 30 minutes. After shaking, the pulverized particles remaining on each sieve are transferred to sample bottles (polyethylene) that have been weighed in advance and cooled in a low-temperature chamber, and the weight of each sample bottle is measured again and weighed in advance. The difference in weight from the sample bottle was calculated. This difference is the weight of each fraction (ie, each particle size). From this, the grindability of each single structure at each freeze grinding temperature was evaluated.

  FIGS. 4 (a) and 4 (b) show the dependence of the particle size distribution on the freezing temperature when muscle tissue is freeze-ground at a freezing temperature of −80 ° C. to −20 ° C. FIG. In addition, FIGS. 5A and 5B show the freeze-pulverization temperature dependence of the particle size distribution when bone tissue is freeze-ground at a freezing temperature of −60 ° C. to −20 ° C. In FIGS. 4B and 5B, the horizontal axis represents the logarithmic scale particle diameter (mm), and the vertical axis represents the logarithmic scale integrated sieve bottom (%). Under integrated sieving, the total weight of all particles is taken as 100, and the weight ratio of each fraction is added in order from the fraction with the smallest particle size. Moreover, the fraction which remained in the saucer 13 (FIG. 3) after classification operation was shown on the graph as a particle diameter of 0 mm.

  In muscle tissue, the shape of the pulverized particles differs depending on the freezing temperature. When the freezing temperature is -80 ° C. to −40 ° C., the shape of the rice grains is changed. It became a shape. As shown in FIG. 4A, the fraction ratio with a particle diameter of 1.41 mm and 2.0 mm decreased as the freezing temperature increased, and the fraction ratio on the small particle side tended to increase. Further, from FIG. 4 (b), when the average particle size (particle size having a weight distribution of 50%) at each freezing temperature is compared, it is about 1.0 mm at −80 ° C. to −40 ° C., whereas −20 It became about 0.7 mm at ° C.

  Generally, when the pulverization is performed by changing the temperature of the substance, the pulverized particles become fine at a temperature lower than the embrittlement point, and the pulverized particles become coarse particles at a temperature higher than the embrittlement point. The lower the freezing temperature, the smaller the particles. However, when freeze-pulverizing shark muscle tissue, the particle size distribution shifted to the small particle side as the freezing temperature increased. This is considered to be caused by the fact that the crushing is performed by scraping from the periphery of the muscle tissue as a feature of the freeze crushing apparatus used in the present example. The freezing temperature of −20 ° C. is a temperature above the embrittlement point, and in the impact immediately after the sample is put into the pulverizer, it is difficult to break down below the size discharged from the screen and becomes coarse particles. Therefore, it takes a long time to be discharged, and during that period, the periphery of coarse particles is scraped off and pulverized into fine particles. This is a factor that decreases the average particle size at a freezing temperature of −20 ° C. It is thought that there is not.

  On the other hand, in the bone tissue, there was no difference in the shape of the pulverized particles depending on each freezing temperature, and it became a rice grain shape at any freezing temperature of −60 ° C. to −20 ° C. FIG. 5A shows that the fraction ratio with a particle diameter of 2.0 mm tends to decrease as the freezing temperature increases, but the fraction ratio on the large particle side tends to be high at any freezing temperature. Moreover, when the average particle diameter in each freezing temperature was compared from FIG.5 (b), it became the same value as about 1.41 mm in all the freezing temperatures of -60 degreeC--20 degreeC. These results suggest that the freezing temperature does not significantly affect the particle size distribution when freeze-pulverizing bone tissue.

  When a material is destroyed, the energy absorbed at the time of impact fracture changes abruptly at the embrittlement point where the brittle-ductile transition of the material occurs. However, in a brittle fracture region in a temperature zone lower than the embrittlement point, the absorbed energy at the time of fracture often hardly changes even when the temperature is lowered. In the pulverization operation, if the total energy per unit mass (or unit volume) absorbed by the particles at the time of destruction is considered as the energy required for pulverization, The smaller the particle size distribution, the smaller the energy per unit mass (or unit weight) that the particles absorb when breaking. Therefore, in the case of bone tissue, the absorption energy at the time of destruction is considered to be almost the same because the shape and particle size distribution of the pulverized particles did not change much when the freezing temperature was −60 ° C. to −20 ° C. It is done. From this, the temperature range of −60 ° C. to −20 ° C. in the bone tissue was considered to be the temperature zone of the brittle fracture region.

  As described above, when comparing the particle size distribution after freezing and pulverization in each of the muscle tissue and the bone tissue, the muscle tissue has a smaller average particle size than the bone tissue, the muscle tissue is a fine particle, the bone tissue is There was a tendency to become coarse particles. In addition, when comparing the particle size distribution of muscle tissue and bone tissue for each freezing temperature, the largest difference was observed in the grindability of each tissue at a freezing temperature of -20 ° C. And when separating bone, the freezing temperature -20 ° C was considered to be the optimum temperature.

  Next, the chemical components (moisture, ash, sodium chondroitin sulfate) of the crushed sample after single tissue pulverization are measured for each particle size, the characteristics of the chemical components in each tissue (muscle and bone), and the sample freezing temperature And the relationship between the chemical components of the ground sample was investigated.

  As a sample used for the measurement of chemical components, pulverized particles stored in an about −30 ° C. stocker were used after changing the freezing temperature, performing single-tissue grinding, classifying into 7 fractions.

  For determination of moisture and ash, pulverized particles of muscle tissue (freezing temperature: −80 ° C. to −20 ° C.) and pulverized particles of bone tissue (freezing temperature: −60 ° C. to −20 ° C.) were used. In addition, for quantification of chondroitin sulfate sodium, only pulverized particles of muscle and bone tissue that were subjected to single tissue pulverization at a freezing temperature of −20 ° C. where the greatest difference was observed in the pulverizability of each tissue (muscle and bone) were obtained. Using.

(A) Quantification of moisture The moisture content was measured by the atmospheric pressure heating and drying method (see Food Analysis Method, pp.3-8, Mitsuaki, Tokyo (1982). Edited by the Food Analytical Society of Japan). About 2 to 3 g of pulverized particles thawed at room temperature were put in a dish made of aluminum foil that had been weighed in advance and dried in an electric constant temperature dryer (105 ° C.). After drying, the sample was allowed to cool in a desiccator for 30 minutes, weighed, and the constant weight operation for drying again was repeated. The moisture was determined from the following formula (1).

Here, A is the weight (g) of the aluminum container, B is the weight (g) of the container and the pulverized particles before drying, and C is the total weight (g) of the container and the pulverized particles after drying.

(B) Determination of ash content Ash content was measured by a direct ashing method (see Food Analysis Method, pp.241-250, Mitsutoshi, Tokyo (1982), edited by the Japan Food Industry Association, Food Analysis Method Editorial Committee). As a preparatory experiment, the ashing container (crucible) was dried in an electric furnace at about 600 ° C. for 1 hour, then left in the electric furnace until it reached about 200 ° C., and allowed to cool in a desiccator for 30 minutes to measure the weight. did. This was taken as the weight of the ashing container that became a constant weight.

  About 2 to 3 g of the pulverized sample thawed at room temperature was transferred to an ashing container for which a constant weight had been obtained in advance, and dried in an electric constant temperature dryer (105 ° C.) for 20 hours. It gradually ashed until black smoke disappeared due to the weak flame of the burner and heated for 30 minutes. Then, it put in the electric furnace, kept at about 600 degreeC for 6 to 8 hours, and incinerated until it became white. After completion of ashing, it was left in an electric furnace until it reached about 200 ° C. The mixture was allowed to cool in a desiccator for 30 minutes and weighed. Ash content was calculated | required from following formula (2).

Here, A is the weight (g) of the ashing container that has become a constant weight, B is the weight (g) of the ashing container and sample after ashing, and C is the sample collection amount (g).

(C) Quantification of chondroitin sulfate sodium (ChSNa) ChsNa was quantified by the carbazole sulfate method (see T. Bitter and HM Muir, A modified uronic acid carbazole reaction, Analytical Biochemistry, 4, 330-334 (1962)). Alcian blue method (Yoshibe Yae, Takahiro Ninomiya, Hideaki Kaba, Takashi Sugano, Taro Okada, Analytical method of sodium chondroitin sulfate added to foods, Journal of Japan Society for Food Hygiene, 28, 13-18 (1987), Hattori Gaku, Arita Junya, Terato Teruto, Koike Shigeyuki, Nakamura Hiroshi, fractional determination of sodium chondroitin sulfate / hydroxypropyl methylcellulose mixture using Alcian Blue 8GX, analytical chemistry, 52, 259-263 (2003).) It was measured by.

  The carbazole sulfate method is a method in which a carbazole solution, which is a coloring pigment of glucuronic acid in ChSNa, is added and the absorbance is measured using a spectrophotometer. A calibration curve is prepared using a standard solution of glucuronic acid with a defined concentration, and the ChSNa content in the sample is determined. The carbazole sulfate method is a typical test method among several types of ChSNa measurement methods, but has a problem that it involves danger because the sample is decomposed by reacting concentrated sulfuric acid at a high temperature of 98 ° C. or higher. On the other hand, the Alcian blue method is a method in which Alcian blue, which is a coloring pigment of ChSNa, is added and the absorbance is measured using a spectrophotometer. When drawing a calibration curve, a ChSNa standard solution with a defined concentration is used. The Alcian Blue method is a safe and reproducible test method, but since a precise determination method for ChSNa has not been established at this stage, there is a problem that a ChSNa reagent with a specified content is not commercially available. there were. Therefore, in this example, first, the concentration of a commercially available ChSNa reagent (manufactured by Wako Pure Chemical Industries, Ltd., chondroitin sulfate C sodium reagent) is defined by the carbazole sulfate method, and this reagent is used as the ChSNa standard for performing the Alcian Blue method. Used as a solution, the ChSNa content in the ground sample was measured.

(C-1) Carbazole sulfate method 100 mg of chondroitin sulfate C sodium reagent (manufactured by Wako Pure Chemical Industries, Ltd.) was accurately weighed and then dissolved in distilled water to make a total amount of 100 ml. Next, 5 ml of this solution was accurately taken and dissolved in distilled water to make a total volume of 100 ml, which was used as a test solution.

  1 ml each of the test solution, glucuronolactone standard solution (manufactured by Wako Pure Chemical Industries, Ltd.) (10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml) and blank (distilled water) prepared in advance Each was weighed and injected into a test tube. Two samples were prepared for each. While the test tubes were cooled in ice water, 5 ml of sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) and borax solution (manufactured by Wako Pure Chemical Industries, Ltd.) were gradually added to each test tube and sufficiently stirred. The test tube was capped with a marble, heated for 10 minutes in a boiling water bath, and then cooled to room temperature in cooling water.

  0.2 ml of a carbazole solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred well, and then plugged again with a marble and heated in a boiling water bath for exactly 15 minutes to develop color. Then, it cooled to room temperature with running water.

  Absorbance was measured at 530 nm using a spectrophotometer Bio Spec-1600 (manufactured by Shimadzu Corporation). A calibration curve was prepared based on the absorbance of the standard solution, and the amount of glucuronolactone in the test solution was read from the calibration curve. Based on the glucuronolactone amount in the read test solution, the ChSNa content in the chondroitin sulfate C sodium reagent was calculated based on the following formulas (3) and (4).

(C-2) Alcian blue method 50 ml of diethyl ether was added to about 5 g of pulverized particles thawed at room temperature, and left for 60 minutes for degreasing. After adding 50 ml of water and homogenizing at 5000 rpm for 5 minutes, the mixture was centrifuged at 3000 rpm for 10 minutes to remove the upper layer of diethyl ether.

  After adjusting the pH to 7 with 1N sodium hydroxide solution, add 5 ml of Actinase E (Kaken Pharmaceutical Co., Ltd.) solution (100 mg / ml) and leave it in a 40 ° C water bath for 4 hours with occasional shaking to decompose the protein. did. Centrifugation was performed at 3000 rpm for 10 minutes, and the supernatant was filtered using glass fiber, and the total amount was made 100 ml with distilled water to obtain a test solution.

  To each 1 ml of the test solution and blank (distilled water), 0.2 ml of Alcian blue solution was added and allowed to stand for 20 minutes. Two samples were prepared for each. The deposited precipitate was centrifuged at 3000 rpm for 30 minutes, the supernatant was discarded, washed with 3 ml of water, and centrifuged again.

  The obtained precipitate was dissolved in 5 ml of monoethanolamine, and the absorbance at 615 nm (manufactured by Shimadzu Corporation, spectrophotometer Bio Spec-1600) was measured, and quantified by a calibration curve prepared in advance. A calibration curve was prepared by using a ChSNa standard solution (5 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml) in the same manner as the test solution. The ChsNa content in the test solution was determined from the following formula (5).

Here, 0.913 is the content correction term of the standard solution of ChSNa reagent obtained by the carbazole sulfate method.

  FIGS. 6 (a) and 6 (b) show the freezing temperature dependence of the water content for each particle diameter in the pulverized particles obtained by freeze-pulverizing muscle and bone tissues. FIGS. 7A and 7B show the dependence of the ash content for each particle diameter on the freeze pulverization temperature in the pulverized particles obtained by freeze pulverization of the muscle and bone tissues.

  From FIG. 6 (a) and FIG. 7 (a), regarding the muscle tissue, there was no difference in water and ash content at a freezing temperature of −80 ° C. to −40 ° C., but only at a freezing temperature of −20 ° C. A decrease in moisture and an increase in ash content were observed at a diameter of 0.71 to 2.0 mm. This is because the water content with a particle size of 0 to 0.355 mm is high (FIG. 6 (a)), and the proportion of the fraction collected in the saucer (particle size 0mm) compared to others at a freezing temperature of -20 ° C. From the fact that it is high (FIG. 4 (a)), it was considered that the moisture in the muscles became fine particles by pulverization and was screened out to a fraction with a small particle diameter. Also, at all freezing temperatures, on the small particle side with a particle size of 0.355-0.5 mm, the water content tends to be higher and the ash content is lower than on the large particle side, and the particle size is 0.71-2.0 mm. On the large particle side, the moisture and ash contents were almost constant regardless of the particle size. Compared with the freezing temperature of −80 ° C. to −40 ° C., the chemical components for each particle size changed at the freezing temperature of −20 ° C. Similarly, it was suggested that the freezing temperature affects the chemical composition of the pulverized particles at the embrittlement point.

  On the other hand, from FIG. 6 (b) and FIG. 7 (b), the moisture and ash contents were almost the same in the bone tissue at any freezing temperature of −60 ° C. to −20 ° C. The reason why the ash content at a freezing temperature of −60 ° C. was slightly higher than other freezing temperatures was considered to be due to insufficient ashing operation, and was set as a measurement error range. Also, at all freezing temperatures, the water content tends to be higher on the small particle side with a particle size of 0.355 to 0.5 mm than on the large particle side, and on the large particle side with a particle size of 0.71 to 2.0 mm. The water content was almost constant regardless of the particle size. From these facts, it was suggested that the freezing temperature does not affect the chemical components of the pulverized particles when freeze pulverization is performed at a tissue freezing temperature of −60 ° C. to −20 ° C. in bone tissue.

  8A and 8B show calibration curves obtained by the carbazole sulfate method and the Alcian blue method. The ChsNa content of the chondroitin sulfate C reagent determined by the carbazole sulfate method was 91.3%. FIG. 9 shows the ChSNa content for each particle size of the pulverized particles obtained by freeze-pulverizing muscle and bone tissue at a freezing temperature of −20 ° C.

  As shown in FIG. 9, in the muscle tissue, the ChSNa content showed a low value close to 0% in the particle diameter range of 0.355 to 1.41 mm, suggesting that the muscle tissue hardly contains ChsNa. It was done. On the other hand, the pulverized particles of muscle tissue showed a high ChsNa content value compared with other particle sizes at a particle size of 2.0 mm because the size of the pulverized sample was large, and the degreasing and proteolysis in the operation process were sufficiently It was considered that it was not carried out and precipitation other than ChsNa occurred.

  On the other hand, in the bone tissue, as shown in FIG. 9, the ChsNa content was higher on the small particle side with a particle diameter of 0.355 to 0.71 mm than on the large particle side. This is because the content is higher as the particle diameter is smaller in the particle diameter range of 0.355 to 0.71 mm, and the moisture content shows a constant content value on the large particle side with a particle diameter of 1.0 mm to 2.0 mm (Fig. 6 (b)), it was considered that the water-soluble ChSNa was dissolved in the water in the bone tissue and was screened down together with the water during the freeze-pulverization / classification operation. . Thereby, for example, when freeze-pulverized at −20 ° C. using only bone tissue, concentration of chondroitin sulfate from bone tissue can be realized by collecting pulverized particles having a particle size of 0.355 to 0.71 mm.

  When the chemical components in muscle and bone tissues were compared, there was no significant difference in water content between the tissues except for the freezing temperature of −20 ° C. In contrast, the ash content and the ChsNa content showed a tendency that the bone tissue had a significantly higher content than the muscle tissue. From this, it is possible to clarify the conditions under which each tissue is preferentially pulverized by measuring the ash content and ChsNa content of the pulverized sample when processing residue is used as a sample and freeze pulverization / classification operation is performed. Sex was suggested.

  Using shark processing residue divided into muscle tissue and bone tissue, and freeze-grinding with different freezing temperatures, and evaluating the relationship between the freezing temperature and the particle size distribution and chemical composition of the ground samples did.

  When comparing the particle size distribution after freeze-grinding in muscle tissue and bone tissue, muscle tissue has a smaller average particle size than bone tissue, muscle tissue tends to be fine particles, and bone tissue tends to be coarse particles The maximum difference was observed in the grindability of each tissue at a freezing temperature of −20 ° C. In addition, when chemical components were analyzed, the ash content and ChsNa content showed that bone tissue tended to be significantly higher than muscle tissue.

  These facts suggest that muscle and bone tissue can be separated by combining freeze-grinding operation and classification using a sieve. A temperature of −20 ° C. was considered the optimum temperature.

[Freeze pulverization of tissue aggregate, particle size distribution of pulverized particles after freeze pulverization, and dependence of chemical components on freeze pulverization temperature]
In the above, a single tissue of muscle and bone tissue was used, and after freeze pulverization / classification operations, the particle size distribution was measured and the distributions of both were compared. As a result, the muscle tissue tended to be fine particles and the bone tissue tended to be coarse particles, and the largest difference was observed in the grindability of each tissue at a freezing temperature of −20 ° C.

  Then, next, using a head (hereinafter referred to as a tissue assembly) as a tissue assembly assuming an actual processing residue, a pulverization operation was performed at a freezing temperature of −20 ° C., and the pulverization separability was examined. Then, a comparison was made between the pulverization separability predicted from the results of single tissue pulverization and the pulverization separability in the tissue aggregate. In addition, the particle size distribution of the ground sample was evaluated, and at the same time, chemical components (water, ash, and sodium chondroitin sulfate) of the ground sample were analyzed.

As the tissue assembly, the head of mouka shark from Kesennuma, Miyagi Prefecture was used as described above. This head was previously peeled manually. It can be regarded as a processing residue. After thawing this head (tissue assembly) stored in a freezer at about −30 ° C. at room temperature, it was cut into 1 × 1 × 1 cm ³ to 1 × 2 × 2 cm ³ pieces by a cutter, It preserve | saved in the stocker set to -20 degreeC temperature, and it was made to fully freeze to preset temperature.

  The freeze crushing operation was performed using the freeze fracture apparatus 1 (FIG. 2) (cooled wheeler pulverizer manufactured by Yoshida Manufacturing Co., Ltd.) that had been sufficiently cooled with liquid nitrogen, as described above. The screen 4 of the freeze fracture apparatus 1 was a screen having an opening of 5 mm, and the rotational speed of the rotary blade 8 of the crushing unit 3 was set to 1000 rpm.

  In the freeze pulverization in the pulverization unit 3, the input amount of the tissue assembly to the pulverization unit 3 (specifically, the input unit 2 and the pulverization unit 3) is 2 to 3 in terms of the above-mentioned cut pieces. Once the single structure that had been input to the crushing unit 3 was completely discharged from the screen 4, a new single structure was input.

  Each single-tissue ground particle group discharged from the screen 4 is received in a constant temperature bath 6 (made of Styrofoam) filled with liquid nitrogen, and the ground particle group (total weight 500 g) accumulated in the constant temperature bath 6 is about Liquid nitrogen was completely evaporated in a −30 ° C. freezer and subjected to subsequent classification operation.

  The classification operation was classified into 7 fractions as described above. That is, the openings are 0.355 mm (sieve 11 f), 0.50 mm (sieve 11 e), 0.71 mm (sieve 11 d), 1.00 mm (sieve 11 c), 1.41 mm (sieve 11 b), 2.00 mm (sieve 11 a). ) Using a classifier 10 (FIG. 3) equipped with a JIS standard sieve. Thereafter, the grindability was evaluated.

  FIGS. 10A and 10B show the particle size distribution of the pulverized particles obtained by freeze-pulverizing (−20 ° C.) the tissue assembly. FIG. 10 (a) shows the dependence of the particle size distribution on the freeze-grinding temperature, and FIG. 10 (b) shows the particle size (mm) on the horizontal axis of the logarithmic scale, and the integrated screen (%) is the vertical scale of the logarithmic scale. It is a particle size distribution of a pulverized particle group of a tissue aggregate when taken on the axis. For comparison, FIGS. 10A and 10B also show the particle size distributions of muscle and bone tissues when the above-mentioned single tissue is freeze-ground.

  From FIG. 10 (a), it was predicted that the pulverization separability of the muscle tissue did not change so much in the tissue aggregate, since a peak at a particle diameter of 0.5 mm appeared as in the case of a single muscle tissue. . In addition, the peak at a particle size of 1.41 mm, which was observed when bone was pulverized into a single tissue, appeared smaller when the tissue aggregate was pulverized. This is because, in FIG. 10B, the average particle diameter of the tissue aggregate was close to the average pulverized particle diameter of a single muscle tissue, and therefore the tissue aggregate contained more muscle tissue than bone tissue. This is considered to be because the influence of the pulverization property of the bone tissue does not appear so much.

  Next, the water content and the ash content of the pulverized particles obtained by freeze-pulverizing the tissue aggregate were calculated by the same method as described above. FIGS. 11A and 11B are graphs showing the moisture and ash content of the pulverized particles obtained by freeze-pulverizing the tissue assembly, respectively. In addition, in FIG. 11 (a) and (b), the analysis result of the chemical component of the muscle and bone tissue at the time of freeze-pulverizing the above-mentioned single tissue was also shown for comparison. As a result, from the graph of FIG. 11B, the ash content tends to increase as the particle diameter increases.

  Next, using the fact that there is a marked difference in the ash content between the muscle tissue and the bone tissue, the ratio of each of the muscle tissue and the bone tissue in the pulverized particle group of the tissue aggregate is expressed as the ash content in FIG. Based on. When the fraction of bone in the fraction is αi [−] and the ash content Ai [%] in the fraction, the following equation (6) is established.

Here, a _flesh is muscle ash (%), and a _bone is bone ash (%). When formula (6) is rearranged for αi, the following formula (7)

It becomes. Therefore, the weight ratio of the muscular tissue and the bone tissue of each fraction with respect to the total fraction weight was obtained from the following equations (8) and (9).

Here, Wi is a fraction weight (g). From formulas (6) and (7), the weight ratio of muscle and bone for each fraction when the total muscle mass and total bone mass contained in the tissue assembly are each 100 is given by the following formulas (10) and (11): can get.

  Based on the above formulas (10) and (11), the amount of muscle tissue and bone tissue in each fraction was calculated to determine the particle size distribution. FIGS. 12 (a) and 12 (b) show the particle size distributions of muscle and bone tissues in the tissue aggregate pulverization determined from the ash content. For comparison, the particle size distribution of muscle and bone tissue in single tissue grinding is also shown in the figure.

  From this, it became clear that the grindability of the muscle and bone tissues in the tissue aggregate was almost the same as the grindability of each tissue in a single tissue. That is, the muscle tissue has a smaller particle diameter as a whole, a peak appears at a particle diameter of 0.5 mm, and the bone tissue has a larger particle diameter as a whole, and the peak appears at a particle diameter of 1.41 mm, similar to single tissue grinding. The tendency was seen.

  Therefore, chondroitin sodium sulfate (ChsNa) contained in the pulverized particles having a particle diameter of 1.41 mm containing the largest amount of bone tissue was quantified. At that time, the amount of ChsNa contained in the pulverized particles of other fractions was compared. The quantitative method was measured by a method combining the carbazole sulfate method and the Alcian blue method. FIG. 13 is a graph showing the amount of ChsNa in each fraction. From FIG. 13, it was shown that the largest amount of sodium chondroitin sulfate was contained in the pulverized particles having a particle diameter of 1.41 mm.

  That is, in this example, the pulverized particles having a predetermined particle diameter (that is, the pulverized particles contained in the fraction having a particle diameter of 1.41 mm) are recovered from the freeze-pulverized pulverized particle group by the classification operation described above. With a simple configuration, bone tissue can be separated from the processing residue, and sodium chondroitin sulfate can be obtained from the recovered pulverized particles.

  As described above, according to the present invention, by classifying a pulverized particle group having a predetermined particle size, it is possible to classify a pulverized particle group made of bone tissue from a pulverized particle group of a frozen and pulverized tissue aggregate. Become. Thereby, the improvement of the production efficiency of chondroitin sulfate can be expected by simplifying the separation of the bone tissue from the processing residue, and the production cost can be reduced. Therefore, the present invention can be suitably used in other fishery processing industries.

It is a chemical structural formula of chondroitin sulfate C mainly contained in shark cartilage. 1 shows a configuration of a freeze pulverization apparatus for freeze pulverizing a tissue aggregate in which muscle tissue and bone (cartilage) tissue coexist in one embodiment according to the present invention, and (a) is a side view of the freeze pulverization apparatus. (B) is an enlarged plan view showing a configuration of a screen provided in the freeze pulverization apparatus shown in (a). FIG. 2 is a perspective view showing a configuration of a classification device for classifying a pulverized particle group composed of bone (cartilage) tissue from a pulverized particle group frozen and pulverized by the freeze pulverization apparatus shown in FIG. 2 in an embodiment according to the present invention. It is. (A) And (b) is a graph which showed the freezing temperature dependence of the particle diameter distribution at the time of freezing temperature -80 degreeC--20 degreeC when muscle tissue is freeze-pulverized. (A) and (b) are both graphs showing the freeze-pulverization temperature dependence of the particle size distribution when bone tissue is freeze-ground at a freezing temperature of −60 ° C. to −20 ° C. The freezing temperature dependence of the water content for each particle diameter in the classified ground particles is shown, (a) shows the water content of the ground particles made of muscle tissue, and (b) shows the water content of the ground particles made of bone tissue. It is the graph which showed. 2 shows the freezing temperature dependence of the ash content for each particle size in the classified pulverized particles, (a) shows the ash content of the pulverized particles made of muscle tissue, and (b) shows the ash content of the crushed particles made of bone tissue It is the graph which showed. (A) is a graph showing a calibration curve obtained by the carbazole sulfate method, and (b) is a graph showing a calibration curve obtained by the Alcian blue method. It is the graph which showed the ChSNa content for every particle diameter of the pulverized particle | grains which pulverized and classified the muscle and the bone tissue at freezing temperature-20 degreeC. The particle size distribution of a pulverized particle group obtained by freeze-grinding a tissue aggregate (−20 ° C.) is shown. (A) shows the freeze-grinding temperature dependence of the particle size distribution. 2 is a graph showing the particle size distribution of the pulverized particle group of the tissue assembly when the vertical axis of the logarithmic scale is taken under the integrated sieve. (A) is a graph showing the water content for each particle diameter in a pulverized particle group obtained by freeze-grinding a tissue aggregate, and (b) is a graph showing the ash content. Both (a) and (b) are graphs showing the particle size distribution of muscle and bone tissue in tissue aggregate grinding determined from the ash content. It is a graph which shows the amount of ChsNa for every particle diameter in the grinding | pulverization particle group which carried out the freezing grinding | pulverization (-20 degreeC) of the tissue assembly.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Freezing crushing apparatus 2 Input part 3 Crushing part 4 Screen 4a Opening 5 Communication path 6 Constant temperature bath 7 Freezing temperature control part 8 Rotary blade 9 Fixed blade 10 Classification apparatus 11a-g Sieve 12 Shaking means 13 Saucepan 14 Cover

Claims (5)

A method for producing chondroitin using chondroitin using a tissue assembly including muscle tissue and bone tissue,
Freezing and pulverizing the tissue assembly to obtain a pulverized particle group, and
A classifying step of classifying the pulverized particles composed of the bone tissue from the pulverized particle group obtained by the freeze pulverization step,
In the classifying step, a pulverized particle having a predetermined particle size is classified.   The chondroitin production according to claim 1, wherein in the classification step, the pulverized particle group obtained in the freeze pulverization step is classified using a plurality of sieves having different opening sizes. Method.   The method for producing chondroitin according to claim 1 or 2, wherein the bone tissue is shark cartilage.   The method for producing chondroitin according to any one of claims 1 to 3, wherein in the freeze pulverization step, the tissue aggregate is frozen at -15 ° C to -30 ° C.   The tissue assembly is derived from sharks and is a processing residue including a head and a fallen body, which is discharged after processing sites such as a caudal fin, a dorsal fin, a chest fin, and a body are collected. 5. The method for producing chondroitin according to any one of 1 to 4.

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