JP2006299108A - Ice crystal growth suppressing agent and its application - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop various applications utilizing antifreeze effects of antifreeze proteins without using the antifreeze proteins. <P>SOLUTION: The invention relates to the ice crystal growth suppressing agent based on the discovery of polymers having antifreeze effects excluding antifreeze proteins (for example an ammonium polyacrylate), wherein the substance which is precipitates in an aqueous solution with 10 mg/mL of concentration into non-flat disk-shaped ice crystals and the solution exhibits thermal hysteresis of ≥0.020°C, and the polymers can be used for various applications as a substitute for the antifreeze proteins (AFP). <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a synthetic substance, and more particularly to a substance exhibiting antifreeze activity as possessed by antifreeze proteins.

  The outline of the antifreeze activity of antifreeze protein is described in Non-Patent Document 1. In other words, antifreeze protein (sometimes referred to as Anti-Freeze Protein; AFP) is a special protein contained in fish, insects, plants, etc. that inhabit polar regions. For example, a normal body fluid of fish freezes at around −0.8 ° C., whereas a body fluid of fish having AFP in the body does not freeze even when it falls below −2 ° C. Since seawater freezes at about −1.9 ° C., fish that have AFP in their bodies can live without freezing body fluids.

  Freezing point depression is often explained by the molar freezing point depression law, where the freezing point depression temperature is directly proportional to the molar concentration of the solute. However, the molar concentration of AFP and the freezing point depression temperature are not directly proportional. In other words, AFP prevents the freezing of bodily fluids by an action different from the freezing point freezing point law. It specifically adsorbs to the growth surface of ice crystals grown in the living body and inhibits the growth of ice crystals by inhibiting the growth of ice crystals. Freezing is prevented.

  The growth of ice crystals will be described in more detail with reference to FIGS. 1, 2-a, and 2-b. FIG. 1 is a conceptual diagram showing the growth of ice crystals in the absence of AFP, and FIGS. 2-a and 2-b are conceptual diagrams showing an example of the growth of ice crystals in the presence of AFP. As shown in FIG. 1, generally, when a minimum nucleus of ice is formed, the minimum nucleus grows in both the a-axis direction and the c-axis direction. However, since the growth rate in the a-axis direction is about 100 times faster than the growth rate in the c-axis direction, a disk-shaped ice nucleus (ice crystal) 1 is formed. On the other hand, as shown in FIG. 2A, in the presence of AFP, as soon as the smallest ice nuclei are formed, they immediately adhere or adsorb to the surface in the a-axis direction (prism surface), and ice growth in the a-axis direction occurs. In order to suppress it, hexagonal ice crystals 2 are formed. And this hexagonal ice crystal 2 grows by stacking small hexagonal pillars in the c-axis direction as shown in FIG. AFP may inhibit the growth in the c-axis direction, and in this case, the AFP remains in a hexagonal crystal form (see FIG. 2A). In any case, if AFP is present, the ice crystals will have a different form from the normal (flat disk type).

  The antifreeze activity of AFP is not only characterized by the change in ice crystal morphology as described above, but also by the following points. That is, since crystal growth in the c-axis direction and the like is suppressed, coalescence of ice crystals is also suppressed. Therefore, suppression of the coarsening of ice crystals is one of the antifreeze activities.

  In addition, one of antifreeze activities is to show thermal hysteresis. That is, the aqueous solution in which AFP is dissolved is supercooled and completely frozen once, and then melts when the temperature of the system is gradually raised. If the temperature is slightly lower than this melting temperature (melting point) and left for a long period of time, freezing starts if normal (ie, the melting point and the freezing point coincide), but if AFP is present, freezing does not start. Freezing begins only when the temperature is further lowered. The difference between the melting temperature (melting point) and the freezing temperature (freezing point) again is called thermal hysteresis, and the presence of this thermal hysteresis is one of the requirements for certification of antifreeze activity.

  That is, the antifreeze activity means that 1) the ice crystal form changes (becomes a non-flat disk shape), 2) the coalescence of ice crystals is suppressed, and 3) there is thermal hysteresis. Note that 1) the change in ice crystal morphology and 2) the suppression of ice crystal coalescence are both caused by the suppression of ice crystal growth and can be identified. In this specification, it is assumed that 1) the ice crystal form changes (becomes a non-flat disk shape), and 2) there is antifreezing activity if there is thermal hysteresis.

  Various application developments have been studied for AFP having such antifreeze activity. For example, frozen food quality and texture (texture) improvement application (Patent Documents 1 to 14 etc.), biological tissue and body fluid freezing resistance improvement application (Patent Documents 15 to 17 etc.), ice heat storage system application (Patent Document 18 etc.) The development of is active. In addition to the above AFP, Japanese Patent Application No. 2003-362427 discloses that an aqueous solution of polyacrylamide changes the ice crystal morphology and has thermal hysteresis.

Shinichiro Nishimura, “Synthesis of Antifreeze Glycoproteins: Searching for Secrets of Antifreeze Fish”, Hyundai Chemistry, Tokyo Chemical Dojin, April 1999, No.337, pp.56-62 International Publication No. 96/39878 Pamphlet International Publication No. 96/11586 Pamphlet International Publication No. 98/4699 pamphlet WO 98/4147 pamphlet WO 98/4148 pamphlet JP 2000-157195 A International Publication No. 99/37164 Pamphlet WO99 / 37673 pamphlet International Publication No. 00/53025 Pamphlet International Publication No. 00/53026 Pamphlet International Publication No. 00/53027 Pamphlet International Publication No. 00/53028 Pamphlet International Publication No. 00/53029 Pamphlet WO99 / 37673 pamphlet WO91 / 10361 pamphlet International Publication No. 97/36547 Pamphlet International Publication No. 00/00512 Pamphlet JP-A-8-75328 Japanese Patent No. 3111219

  However, antifreeze proteins are extremely expensive and currently cost 1 million yen / g. Furthermore, it is easily denatured by heat and may cause an antigen-antibody reaction when applied to a living body.

  Patent Document 19 discloses a method for transporting cold heat using polyvinyl alcohol. In the column of Examples, it is shown that granular ice crystals are not recrystallized when polyvinyl alcohol is used. However, there is no indication as to how it behaves with respect to thermal hysteresis, an important requirement for antifreeze activity.

  The present invention has been made paying attention to the above-mentioned circumstances, and the object thereof is to achieve various application developments utilizing the antifreeze activity of the antifreeze protein without using the antifreeze protein. It is in.

  As a result of intensive studies to solve the above problems, the present inventors have found that a polymer having antifreeze activity (for example, an ammonium salt of polyacrylic acid) exists in addition to the protein system. Clarified that an aqueous solution having a concentration of 10 mg / ml causes precipitation of non-flat disc-shaped ice crystals, and that an aqueous solution having a concentration of 10 mg / ml exhibits a thermal hysteresis of 0.020 ° C. or higher, thereby completing the present invention. This polymer can be used in various applications in place of antifreeze protein (AFP).

  That is, the ice crystal growth inhibitor according to the present invention comprises a non-protein polymer in which an aqueous solution having a concentration of 10 mg / ml precipitates non-flat disk-type ice crystals. This ice crystal growth inhibitor can be added to the heat medium of the ice heat storage system and can be added to frozen foods.

  The ice crystal growth initiation temperature lowering agent according to the present invention comprises a non-protein polymer in which an aqueous solution having a concentration of 10 mg / ml exhibits a thermal hysteresis of 0.020 ° C. or higher. This ice crystal growth start temperature lowering agent can be sprayed or applied to the adhesion site in order to prevent the adhesion of ice. It can also be sprayed or applied to the ground or crops to prevent freezing or frost damage.

  The water coagulation control agent according to the present invention comprises a non-protein polymer in which an aqueous solution having a concentration of 10 mg / ml exhibits a thermal hysteresis of 0.020 ° C. or higher and precipitates non-flat disk-shaped ice crystals. . This water coagulation control agent can be injected into a living tissue or body fluid in order to prevent damage to the living tissue or freezing of the body fluid below freezing.

  The non-protein polymer is a polymer having a carbon chain as a main chain, for example.

  In the non-protein polymer of the present invention, an aqueous solution having a concentration of 10 mg / ml precipitates non-flat disk-type ice crystals and exhibits a thermal hysteresis of 0.020 ° C. or higher. It can be used for various applications utilizing antifreeze activity.

  The present invention relates to a non-protein polymer exhibiting antifreeze activity. When the polymer is an aqueous solution having a concentration of 10 mg / ml, the ice crystal form changes (becomes a non-flat disk shape) and exhibits thermal hysteresis.

  More specifically, the change in the ice crystal form is a flat disk-shaped ice crystal as shown in FIG. 1 when the non-protein polymer of the present invention is cooled as an aqueous solution having a concentration of 10 mg / ml. It means that it is not 1. For example, a hexagonal (flat hexagonal columnar) ice crystal 2 as shown in FIG. 2A or a bipyramidal ice crystal 3 as shown in FIG. The ice crystal shape changes in this way in a predetermined direction [for example, the a-axis direction (direction perpendicular to the prism surface) or both the a-axis direction and the c-axis direction (direction perpendicular to the base surface)]. This is because crystal growth is suppressed.

  The thermal hysteresis is a temperature defined as follows. That is, the target substance is an aqueous solution having a concentration of 10 mg / ml, and the aqueous solution is supercooled and completely frozen once. The temperature of the system is gradually increased to melt the ice, and a slight amount of ice crystals remains. (For example, at the stage where only one crystal having a size of about 0.08 to 0.1 mm per 0.1 mm × 0.1 mm is left), gradually cool again (for example, a cooling rate of about 1 ° C./min). Continue to grow ice crystals again. The temperature at which the ice crystals slightly remain (melting point) and the temperature at which ice crystal regrowth begins to be observed (freezing point) are measured, and the temperature difference (melting point-freezing point) is determined. The temperature difference (melting point−freezing point) inevitably includes an error, but when the temperature difference is 0.020 ° C. or more, it can be said that there is thermal hysteresis even when the error is taken into account. Therefore, the non-protein polymer of the present invention is a substance having a temperature difference (melting point−freezing point) of 0.020 ° C. or higher.

  It is considered that the larger the value of the thermal hysteresis is, the larger the interaction with ice is. Therefore, the value when measured using a 10 mg / ml aqueous solution is preferably 0.030 ° C. or higher.

  The non-protein polymer of the present invention is, for example, a polymer having a carbon chain as a main chain. The polymer can be obtained, for example, by homopolymerizing monomers as shown below or copolymerizing two or more monomers.

  Unsaturated carboxylic acids such as acrylic acid and methacrylic acid; unsaturated carboxylic acid esters such as methyl acrylate; unsaturated amides such as acrylamide; unsaturated carboxylic ammonium salts such as ammonium acrylate; [2- Unsaturated quaternary ammonium salts such as (acryloyloxy) ethyl] ammonium chloride; vinyl ethers such as methyl vinyl ether and ethyl vinyl ether; vinyl esters such as vinyl acetate; N-vinyl compounds such as N-vinyl pyrrolidone; Unsaturated alcohols such as methallyl alcohol; unsaturated amines such as allylamine; unsaturated sulfonic acids such as 2-acrylamido-2-methylpropanesulfonic acid.

  The polymerization method for obtaining the polymer is not particularly limited, but radical polymerization is preferred.

A polymer having a carbon chain as a main chain having the antifreeze activity (the property of changing the ice crystal form and the property of thermal hysteresis) has a functional group containing a nitrogen atom in the side chain. Examples of the functional group include an amino group and an ammonium group. The amino group includes a primary amino group in which active hydrogen remains, a secondary amino group or a tertiary amino group; an alkylamino group in which active hydrogen is substituted by an alkyl group; an alkanolamino group in which active hydrogen is substituted by an alkanol group For example. As also ammonium group, trimethyl ammonium chloride, triethylammonium chloride, ammonium group substituted by an alkyl group such as triethyl ammonium chloride; ammonium group substituted by an alkanol group such as tri-hydroxylethyl ammonium chloride; -CO ₂ ^- NR ¹ Examples thereof include a carboxyammonium group represented by R ² R ³ H ⁺ (wherein R ¹ , R ² and R ³ represent hydrogen, an alkyl group or an alkyl group partially substituted with a hydroxyl group). Suitable amino / ammonium _groups, ^-CO 2 - include _NH ^{4 +.} Moreover, as a suitable example of the polymer containing such an amino group and / or an ammonium group, polyacrylic acid ammonium etc. are mentioned.

  The molecular weight of the polymer of the present invention is not particularly limited, but is preferably in the range of 1,000 to 1,000,000 in terms of weight average molecular weight. If the molecular weight is less than 1,000, the coating area when adsorbed on ice crystals is small, so the effect as an ice crystal growth inhibitor may be reduced. Conversely, if the molecular weight exceeds 1,000,000, the ice crystals are dispersed. This is not preferable because the effect of reducing the concentration is rather small and there is a possibility of acting as an aggregating agent. A more preferable range is 3,000 to 500,000.

A polymer having antifreeze activity can be used for various purposes depending on the content of the antifreeze activity. For example, the property of changing the ice crystal morphology can be used as an ice crystal growth inhibitor. In this case, the coalescence of ice can be suppressed by suppressing the growth of ice. The ice crystal growth inhibitor may be added to, for example, a heat medium of an ice heat storage system, or may be added to a frozen food (for example, ice cream).
As an example of a method of using the polymer of the present invention for an ice heat storage system application, the liquid added with the polymer is cooled to generate an ice slurry, and the ice slurry is transported to a predetermined place by a pipe and a pump. Or storing the ice slurry obtained in the same manner to preserve the cold. If it is used for an ice heat storage system, it is possible to prevent the system from becoming inoperable due to ice coalescence when the heat medium deposits ice. In addition, since the quantity of ice itself does not change substantially, there is no possibility that the heat storage capacity of the system will decrease. In addition, since it is effective in a relatively small amount, it does not cause a great amount of supercooling. Furthermore, since the polymer of the present invention is chemically stable, it can be operated for a long time while maintaining the operation efficiency.
Moreover, if it adds to frozen food, it can prevent that the ice in a food is united and a food texture falls.

  On the other hand, the property of the polymer of the present invention showing thermal hysteresis can be used as an ice growth start temperature lowering agent. The ice crystal growth start temperature lowering agent may be sprayed or applied to an adhesion site (for example, a wing or an electric wire of an airplane, etc.) to prevent ice adhesion, and the ground (road surface, It may be sprayed or applied to soil). The polymer of the present invention is characterized by low corrosiveness to metals, and since it is effective when added in a small amount, the load on the environment is small.

  Moreover, when utilizing both the property of suppressing the growth of ice and the property of exhibiting thermal hysteresis, the polymer of the present invention can be used as a water coagulation control agent. The water coagulation control agent can be injected into a living tissue or body fluid, for example, to prevent damage to the living tissue or freezing of the body fluid under freezing. Specifically, it can be used for improving the freezing resistance of farmed fish, frozen storage of sperm and organs, cryosurgery and the like. Since the substance of the present invention is a non-protein system, there is no possibility of causing an antigen-antibody reaction even when used for organ preservation, for example.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention.

In the following experimental examples, the antifreeze activity of the following substances was examined.
Polyacrylic acid: manufactured by Nippon Shokubai Co., Ltd., trade name “AQUALIC HL-415”, molecular weight: 10,000
Sodium polyacrylate: neutralized polyacrylic acid of 1) above with a 30% aqueous sodium hydroxide solution to pH 7.0.
Polyethyleneimine: manufactured by Nippon Shokubai Co., Ltd., trade name “Epomin SP-200”, molecular weight: 10,000
Ammonium polyacrylate: A product obtained by neutralizing the polyacrylic acid of 1) above with 28% ammonia water (Wako Pure Chemical Industries, Ltd.) until pH 7.0.

Experimental Example An aqueous solution (concentration: 10 mg / ml) of each of the above substances was prepared. This aqueous solution was set on a freezing stage with temperature control (LK-600PM, manufactured by Linkham Co.), cooled to -30 ° C. and completely frozen, and then observed with a phase contrast microscope (magnification 100 times), about −1. The temperature was raised to 0 ° C. (temperature increase rate 100 ° C./min), and only one ice single crystal having a size of about 0.08 to 0.1 mm remained. The field of view was set to 0.1 mm × 0.1 mm (that is, the single crystal was enlarged and displayed on the screen), and from this state, it was slowly recooled (cooling rate 1 ° C./min), and the growth of ice crystals was observed. The difference (thermal hysteresis) between the temperature at which only one single crystal remained (melting point) and the temperature at which ice crystal growth was observed (freezing point) was determined. The morphology of ice grown by the above operation was also observed. The results are shown in Table 1 and the phase contrast micrographs of FIGS. FIG. 3 shows ice crystals in an aqueous solution of ammonium polyacrylate, FIG. 4 shows ice crystals when no agent is added, FIG. 5 shows ice crystals in an aqueous solution of polyacrylic acid, and FIG. 6 shows an aqueous solution of sodium polyacrylate. FIG. 7 shows ice crystals in a polyethyleneimine aqueous solution.

As is clear from Table 1 and FIGS. 3 to 6, unlike polyacrylic acid and sodium polyacrylate, which are acid forms, ammonium polyacrylate changed the ice crystal form and exhibited thermal hysteresis.
Moreover, it can be seen from Table 1 and FIG. 7 that polyethyleneimine having an amino group in the main chain does not change the ice crystal form and has small thermal hysteresis.
It can be seen that FIG. 3 shows a non-flat disk shape and FIGS. 4 to 7 show a flat disk shape.

  According to the data of the example of Japanese Patent Application No. 2003-362427, the thermal hysteresis of polyacrylamide is 0.021 ° C., and the ammonium polyacrylate of the present invention has a larger thermal hysteresis. It turns out that it is excellent as an inhibitor.

FIG. 1 is a conceptual diagram for explaining ice crystals precipitated from an aqueous solution of a substance that does not exhibit antifreeze activity. FIG. 2A is a first conceptual diagram for explaining ice crystals precipitated from an aqueous solution of a substance exhibiting antifreeze activity. FIG. 2B is a second conceptual diagram for explaining ice crystals precipitated from an aqueous solution of a substance exhibiting antifreeze activity. FIG. 3 is a photomicrograph showing ice crystals in an aqueous ammonium polyacrylate solution. FIG. 4 is a photomicrograph showing ice crystals when no agent is added. FIG. 5 is a photomicrograph showing ice crystals in an aqueous polyacrylic acid solution. FIG. 6 is a photomicrograph showing ice crystals in an aqueous sodium polyacrylate solution. FIG. 7 is a photomicrograph showing ice crystals in an aqueous polyethyleneimine solution.

Explanation of symbols

1 ... Flat disk-shaped ice crystals 2 ... Hexagonal ice crystals 3 ... Bipyramid ice crystals

Claims (12)

  An ice crystal growth inhibitor comprising a polymer having a carbon chain as a main chain and a functional group containing a nitrogen atom in a side chain. The ice crystal growth inhibitor according to claim 1, wherein the functional group is an amino group and / or an ammonium group.   The ice crystal growth inhibitor according to claim 1, wherein the polymer is a non-protein polymer.   The ice crystal growth inhibitor according to claim 1, which is added to a heat medium of an ice heat storage system.   A cold transport method comprising cooling the liquid to which the polymer according to any one of claims 1 to 3 is cooled to produce an ice slurry, and transporting the ice slurry.   A cold storage method, wherein the liquid to which the polymer according to any one of claims 1 to 3 is added is cooled to produce an ice slurry, and the cold heat of the ice slurry is stored.   The ice crystal growth inhibitor according to any one of claims 1 to 3, which is added to frozen food.   An aqueous solution having a concentration of 10 mg / ml is a polymer exhibiting thermal hysteresis of 0.020 ° C. or more, and the polymer includes a polymer having a carbon chain as a main chain and a functional group containing a nitrogen atom in a side chain. An ice crystal growth initiation temperature lowering agent. The ice crystal growth start temperature lowering agent according to claim 8, wherein the ice crystal growth start temperature lowering agent is sprayed or applied to an adhesion site to prevent ice adhesion.   The ice crystal growth start temperature lowering agent according to claim 8, wherein the ice crystal growth start temperature lowering agent is applied or applied to the ground or a crop to prevent freezing or frost damage.   An aqueous solution having a concentration of 10 mg / ml exhibits a thermal hysteresis of 0.020 ° C. or higher, and has a carbon chain on which a non-flat disc-shaped ice crystal is precipitated as a main chain and a functional group containing a nitrogen atom in the side chain. A water coagulation control agent comprising coalescence. The water coagulation control agent according to claim 11, which is injected into a living tissue or body fluid in order to prevent damage to the living tissue or freezing of the body fluid under freezing.

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